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Patients with rheumatoid arthritis (RA) have altered levels of exhaled nitric oxide (NO) compared with healthy controls. Here, we investigated whether the clinical features of and immunological factors in RA pathogenesis could be linked to the NO lung dynamics in early disease. A total of 44 patients with early RA and anti-citrullinated peptide antibodies (ACPAs), specified as cyclic citrullinated peptide 2 (CCP2), were included. Their exhaled NO levels were measured, and the alveolar concentration, the airway compartment diffusing capacity and the airway wall concentration of NO were estimated using the Högman-Meriläinen algorithm. The disease activity was measured using the Disease Activity Score for 28 joints. Serum samples were analysed for anti-CCP2, rheumatoid factor, free secretory component, secretory component containing ACPAs, antibodies against Porphyromonas gingivalis (Rgp) and total levels of IgA, IgA1 and IgA2. Significant negative correlations were found between the airway wall concentration of NO and the number of swollen joints (Rho -0.48, p = 0.004), between the airway wall concentration of NO and IgA rheumatoid factor (Rho -0.41, p = 0.017), between the alveolar concentration and free secretory component (Rho -0.35, p = 0.023) and between the alveolar concentration and C-reactive protein (Rho -0.36, p = 0.016), but none were found for anti-CCP2, IgM rheumatoid factor or the anti-Rgp levels. In conclusion, altered NO levels, particularly its production in the airway walls, may have a role in the pathogenesis of ACPA-positive RA.

Scleritis is a severe and painful ophthalmic disorder, in which a pathogenic role for collagen-directed autoimmunity was repeatedly suggested. We evaluated the presence of sclera-specific antibodies in a large cohort of patients with non-infectious scleritis. Therefore, we prospectively collected serum samples from 121 patients with non-infectious scleritis in a multicenter cohort study in the Netherlands. In addition, healthy (n = 39) and uveitis controls (n = 48) were included. Serum samples were tested for anti-native human type II collagen antibodies using a validated enzyme-linked immunosorbent assay (ELISA). Further, sclera-specific antibodies were determined using indirect immunofluorescence (IIF) on primate retinal/scleral cryosections. Lastly, human leukocyte antigen (HLA) typing was performed in 111 patients with scleritis. Anti-type II collagen antibodies were found in 13% of scleritis patients, in 10% of healthy controls and in 11% of uveitis controls (p = 0.91). A specific reaction to scleral nerve tissue on IIF was observed in 33% of patients with scleritis, which was higher than in healthy controls (11%; p = 0.01), but similar to uveitis controls (25%; p = 0.36). Reactivity to the scleral nerve tissue was significantly associated with earlier onset of scleritis (48 versus 56 years; p < 0.001), bilateral involvement (65% versus 42%; p = 0.01), and less frequent development of scleral necrosis (5% versus 22%; p = 0.02). HLA-B27 was found to be twice as prevalent in patients with scleritis (15.3%) compared to a healthy population (7.2%). In conclusion, scleral nerve autoantibody reactivity was more common in scleritis and uveitis patients in contrast to healthy controls. Further research is needed to characterize these scleral-nerve directed antibodies and assess their clinical value., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: In SLE, anti-dsDNA can co-occur with autoantibodies against other chromatin components, like histones and nucleosomes. These antibodies induce type-1 interferon production, a hallmark of SLE. We measured antinuclear antibody (ANA) sub-specificities and investigated their associations to inflammatory biomarkers including interferon-regulated chemokines., Methods: We included 93 Sudanese and 480 Swedish SLE patients and matched controls (N = 104 + 192). Autoantibodies targeting ANA-subspecificites: dsDNA, Sm, Sm/U1RNPcomplex, U1RNP, SSA/Ro52, SSA/Ro60, SSB/La, ribosomal P, PCNA and histones were quantified in all subjects, anti-nucleosome only in the Swedish patients, with a bead-based multiplex immunoassay. Levels of 72 plasma biomarkers were determined with Proximity Extension Assay technique or ELISA., Results: Among Sudanese patients, the investigated antibodies significantly associated with 9/72 biomarkers. Anti-histone antibodies showed the strongest positive correlations with MCP-3 and S100A12 as well as with interferon I-inducible factors MCP-1 and CXCL10. Anti-dsDNA antibodies associated with CXCL10 and S100A12, but in multivariate analyses, unlike anti-histone, associations lost significance.Among Swedish patients, MCP-1, CXCL10, SA100A12 also demonstrated stronger associations to anti-histone and anti-nucleosome antibodies, compared with anti-dsDNA and other ANA sub-specificities. In multiple regression models, anti-histone/nucleosome retained the strongest associations. When excluding anti-histone or anti-nucleosome positive patients, the associations between MCP-1/CXCL10 and anti-dsDNA were lost. In contrast, when excluding anti-dsDNA positive patients, associations with anti-histone and anti-nucleosome remained significant., Conclusion: In two cohorts of different ethnical origin, autoantibodies targeting chromatin correlate stronger with IFN-induced inflammatory biomarkers than anti-dsDNA or other ANA sub-specificities. Our results suggest that anti-histone/nucleosome autoantibodies may be main drivers of type-1 interferon activity in SLE., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Objectives: To investigate how individual rheumatoid arthritis (RA) autoantibodies associate with individual signs and symptoms at the time of RA diagnosis., Methods: IgA, IgG, IgM rheumatoid factor (RF), antibodies against cyclic citrullinated peptide version 2 (anti-CCP2) and 16 individual antibodies against citrullinated protein (ACPA) reactivities were analysed centrally in baseline sera from 1600 patients with RA classified according to the 1987 American College of Rheumatology (ACR) criteria. These results were related to C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), number of swollen and tender joints (SJC and TJC), 28-joint disease activity scores (DAS28 and DAS28CRP), global disease activity evaluated by the patients and Health Assessment Questionnaire, all obtained at baseline., Results: Individually, all autoantibodies except immunoglobulin G (IgG) RF associated with low SJC and TJC and with high ESR. In IgM RF-negative patients, ACPA associated strictly with low number of swollen and tender joints. This association persisted in multiple regression and stratified analyses where IgM and IgA RF instead associated with inflammation expressed as ESR. Among subjects without any ACPA peptide reactivity, there was no association between RF isotypes and ESR. The effect of RF on ESR increased with the number of ACPA reactivities, especially for IgM RF. In patients fulfilling the 1987 ACR criteria without taking RF into account, associations between IgM RF and high ESR, as well as between ACPA and low joint counts, remained., Conclusion: Whereas ACPA associate with low counts of affected joints in early RA, RF associates with elevated measures of systemic inflammation in an ACPA-dependent manner. This latter finding corroborates in vitro models of ACPA and RF in immune complex-induced inflammation. These phenotypic associations are independent of classification criteria., Competing Interests: Competing interests: EP and LM-A are employees at Thermo Fisher Scientific. JA has had or have research agreements with AbbVie, BMS, Eli Lilly, Galapagos, MSD, Pfizer, Roche, Samsung Bioepis and Sanofi, mainly for the national safety monitoring of rheumatology immunomodulators in Sweden (ARTIS). JR has been a member of the Scientific Advisory Board for Thermo Fisher Scientific and for Inova/Werfen and has received consulting fees, speaking fees and/ or honoraria by Thermo Fisher Scientific. RH has received consulting fees from Regor, Lipum AB and Cyxone AB and is the founder of Vacara AB. LK has been a co-ordinator of several IMI-funded projects which included collaborations with Janssen, Pfizer, Sanofi, UCB, GSK and Eli-Lilly. This work has been presented as a poster in a preliminary form at EULAR (June 2019) and to the European Workshop for Rheumatology Research (March 2023)., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: The aim of the present study was to evaluate performance of serum and synovial fluid levels of the granulocyte protein calprotectin as inflammatory biomarker in rheumatoid arthritis (RA) patients with knee synovitis., Methods: 76 RA patients with ongoing knee synovitis were included. Data on disease activity score with 28 joints and their subcomponents and radiological destruction of the affected knee were collected. White blood cell count, C-reactive protein, anti-citrullinated peptide antibodies (ACPA) against cyclic citrullinated peptide version 2 (anti-CCP2), IgM rheumatoid factor (RF) and calprotectin were analysed in parallel in circulation and in synovial fluid (SF). Counts of polynuclear and mononuclear cells were measured in SF., Results: Serum (S) calprotectin correlated stronger than SF-calprotectin with inflammatory markers and disease activity. Instead, SF-calprotectin showed a strong correlation to SF counts of white blood cells, and especially to polymorphonuclear cell counts (Spearman's rho = 0.72, p< 0.001). S-calprotectin showed markedly stronger correlation with inflammatory markers and disease activity in ACPA positive as compared with ACPA negative RA patients; a similar difference was observed for patients with and without IgM RF., Conclusion: The particularly strong association between circulating calprotectin and inflammation in ACPA positive RA is a new argument for a specific role for polymorphonuclear granulocytes/neutrophils in this RA subset. Measurement of calprotectin in SF does not convey any additional benefit compared with measurement in the circulation in RA patients with knee synovitis., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Background: Interstitial lung disease (ILD) is the most common cause of death in patients with systemic sclerosis (SSc). Prognostic biomarkers are needed to identify SSc-ILD patients at risk for progressive pulmonary fibrosis. This study investigates autoantibodies measured in bronchoalveolar lavage (BAL) fluid and in serum in reference to the clinical disease course of SSc-ILD., Methods: Fifteen patients with new onset SSc-ILD underwent bronchoscopy. Autoantibody levels were analyzed using addressable laser bead immunoassay from BAL fluid and the serum. In a separate longitudinal cohort of 43 patients with early SSc-ILD, autoantibodies in serum were measured at baseline and pulmonary function tests were performed at least 2 times over the course of at least 2 or more years. Linear mixed effect models were created to investigate the relationship between specific autoantibodies and progression of SSc-ILD. Finally, lung tissue from healthy controls and from subjects with SSc was analyzed for the presence of the Ro52 antigen using immunohistochemistry., Results: Among SSc-ILD patients who were positive for anti-Ro52 (N = 5), 3 (60%) had enrichment of anti-Ro52 in BAL fluid at a ratio exceeding 50x. In the longitudinal cohort, 10/43 patients (23%) were anti-Ro52 positive and 16/43 (37%) were anti-scl-70 positive. Presence of anti-Scl-70 was associated with a lower vital capacity (VC) at baseline (-12.6% predicted VC [%pVC]; 95%CI: -25.0, -0.29; p = 0.045), but was not significantly associated with loss of lung function over time (-1.07%pVC/year; 95%CI: -2.86, 0.71; p = 0.230). The presence of anti-Ro52 was significantly associated with the loss of lung function over time (-2.41%pVC/year; 95% CI: -4.28, -0.54; p = 0.013). Rate of loss of lung function increased linearly with increasing anti-Ro52 antibody levels (-0.03%pVC per arbitrary units/mL and year; 95%CI: -0.05, -0.02; p < 0.001). Immunohistochemical staining localized the Ro52 antigen to alveolar M2 macrophages in peripheral lung tissue both in subjects with and without SSc., Conclusions: This study suggests that antibodies targeting Ro52 are enriched in the lungs of patients with new-onset SSc-ILD, linking Ro52 autoimmunity to the pulmonary pathology of SSc. Clinical and immunohistochemical data corroborates these findings and suggest that anti-Ro52 may serve as a potential biomarker of progressive SSc-ILD., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Objectives: To assess the association between venous thromboembolic (VTE) events and autoantibodies, following patients from RA diagnosis, measuring occurrence, levels and collective load of different autoantibodies against post-translational protein modifications, in particular recognizing citrullination (e.g. citrullinated fibrinogen) and RF by isotype., Methods: A cohort of 2814 patients with newly diagnosed RA were followed for incident VTE through register linkages. Sera from RA diagnosis were centrally analysed for antibodies to second generation cyclic citrullinated peptides (anti-CCP2), 20 anti-citrullinated protein antibody (ACPA) fine-specificities, antibodies to additional protein modifications (carbamylation and acetylation) and RF by isotype. Association between baseline serology status and future VTE was analysed using Cox regression adjusted for age, sex and calendar period of RA diagnosis, overall and stratified by anti-CCP2 and RF positivity., Results: During a median 16 years of follow-up, 213 first-ever VTE events were registered (5.0/1000 person-years). IgG anti-CCP2 (present in 65% of cohort) associated with VTE (hazard ratio [HR] = 1.33, 95% CI: 1.00, 1.78), in a dose-response manner. The risk of VTE increased with number of ACPA fine-specificities. IgM RF, but no other RF isotypes, associated with VTE (HR = 1.38, 95% CI: 1.04, 1.82). The associations were independent from smoking and HLA-DRB1 shared epitope alleles. None of the carbamylated or acetylated antibody reactivities associated with VTE., Conclusion: Anti-CCP2, load of ACPA fine-specificities and IgM RF at RA diagnosis are associated with an increased risk of future VTE in RA. Antibodies to citrullinated fibrinogen did not differ substantially from other ACPA fine-specificities. Autoreactivity to other post-translational modifications was not associated with VTE risk., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Objectives: Emerging evidence demonstrates that aPS-PT associate with thrombotic events. Genetic predisposition, including HLA-DRB1 alleles, is known to contribute to the occurrence of conventional aPL [anti-β2glycoprotein-I (anti-β2GPI) and aCL]. We investigated associations between aPS-PT and HLA-DRB1* alleles and thrombosis in SLE. Conventional aPL were included for comparison., Methods: We included 341 consecutive SLE patients, with information on general cardiovascular risk factors, including blood lipids, LA and thrombotic events. aPS/PT, anti-β2GPI and aCL of IgA/G/M isotypes and LA were quantified., Results: aPS/PT antibodies associated positively with HLA-DRB1*13 [odds ratio (OR) 2.7, P = 0.002], whereas anti-β2GPI and aCL antibodies associated primarily with HLA-DRB1*04 (OR 2.5, P = 0.0005). These associations remained after adjustment for age, gender and other HLA-DRB1* alleles. HLA-DRB1*13, but not DRB1*04, remained as an independent risk factor for thrombosis and APS after adjustment for aPL and cardiovascular risk factors. The association between DRB1*13 and thrombosis was mediated by aPS-PT positivity. HLA-DRB1*03, on the other hand, associated negatively with thrombotic events as well as all aPL using both uni- and multivariate analyses. HLA-DRB1*03 had a thrombo-protective effect in aPL-positive patients. Additionally, HLA-DRB1*03 was associated with a favourable lipid profile regarding high-density lipoprotein and triglycerides., Conclusions: HLA-DRB1*13 confers risk for both aPS-PT and thrombotic events in lupus. The association between HLA-DRB1*13 and thrombosis is largely, but not totally, mediated through aPS-PT. HLA-DRB1*03 was negatively associated with aPL and positively with favourable lipid levels. Thus, HLA-DRB1*03 seems to identify a subgroup of SLE patients with reduced vascular risk., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Objectives: Anti-TIF1-gamma autoantibodies can be detected with immunoprecipitation (IP), line blot (LB) and ELISA. We compared assay performance in patients with DM and the potential of these assays to detect anti-TIF1-gamma positive cancer-associated DM (CADM)., Methods: We included sera from 131 patients with DM followed at Karolinska University Hospital, Stockholm, Sweden and 82 healthy controls. Serum samples taken at DM diagnosis were tested for anti-TIF1-gamma autoantibodies with IP, two ELISAs (in-house and commercial) and LB. Cancer diagnosis and dates were obtained from the Swedish national cancer register. CADM was defined as a malignancy that developed within 3 years of DM diagnosis., Results: Anti-TIF1-gamma autoantibodies were detected in 19/101 (18.8%), 15/113 (13.2%), 34/131 (26%) and 45/131 (34.4%) of the patients with IP, LB, in-house and commercial ELISA, respectively. The anti-TIF1-gamma results from the in-house ELISA were confirmed with IP in 93 of 101 (92%) cases, κ = 0.76, with a commercial ELISA in 110 of 131 (84%) cases, κ = 0.63, and with LB in 101 of 113 (89.3%) cases, κ = 0.67. Anti-TIF1-gamma results with IP were confirmed with LB in 85 of 92 (92.4%) cases, κ = 0.73. For detecting CADM, the anti-TIF1-gamma in-house ELISA had a sensitivity of 58% and specificity of 86%, the commercial ELISA had a sensitivity of 63% and specificity of 82%, IP had a sensitivity of 52% and specificity of 92%, LB had a sensitivity of 40% and specificity of 96%., Conclusion: The two anti-TIF1-gamma ELISA assays had advantages both for autoantibody detection and to identify anti-TIF1-gamma-positive CADM., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Objectives: Pulmonary manifestations in RA are common comorbidities, but the underlying mechanisms are largely unknown. The added value of a multiplex of ACPA and genetic risk markers was evaluated for the development of pulmonary fibrosis (PF) in an inception cohort., Methods: A total of 1184 patients with early RA were consecutively included and followed prospectively from the index date until death or 31 December 2016. The presence of 21 ACPA fine specificities was analysed using a custom-made microarray chip (Thermo Fisher Scientific, Uppsala, Sweden). Three SNPs, previously found related to PF were evaluated, rs2609255 (FAM13A), rs111521887 (TOLLIP) and rs35705950 (MUC5B). ACPA and genetic data were available for 841 RA patients, of whom 50 developed radiologically defined PF., Results: In unadjusted analyses, 11 ACPA specificities were associated with PF development. In multiple variable analyses, six ACPA specificities were associated with increased risk of PF: vimentin (Vim)60-75, fibrinogen (Fib)β62-78 (72), Fibα621-635, Bla26, collagen (C)II359-369 and F4-CIT-R (P < 0.01 to P < 0.05). The number of ACPA specificities was also related to PF development (P < 0.05 crude and adjusted models). In multiple variable models respectively adjusted for each of the SNPs, the number of ACPA specificities (P < 0.05 in all models), anti-Vim60-75 (P < 0.05, in all models), anti-Fibβ62-78 (72) (P < 0.001 to P < 0.05), anti-CII359-369 (P < 0.05 in all models) and anti-F4-CIT-R AQ4 (P < 0.01 to P < 0.05), anti-Fibα621-635 (P < 0.05 in one) and anti-Bla26 (P < 0.05 in two) were significantly associated with PF development., Conclusion: The development of PF in an inception cohort of RA patients was associated with both presence of certain ACPA and the number of ACPA specificities and risk genes., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Interstitial lung disease can be the first sign of systemic autoimmune disease. If associated with myositis, interstitial lung disease may be the only symptom, with no presence of muscular weakness or other extramuscular manifestations. ANA-testing performed with indirect immune fluorescence may be negative. Testing for myositis antibodies should be considered as a step in the diagnostic process when strong clinical suspicion of interstitial lung disease of unknown origin is present. If interstitial lung disease is suspected, the patients should be referred to a specialist clinic for further investigation and, if possible, for discussion within a multidisciplinary team. Early suspicion of systemic inflammatory disease, rapid diagnosis and early start of treatment are crucial for future prognosis, quality of life and survival.

Autoantibodies constitute important tools for diagnosing the autoimmune liver diseases (AILD) autoimmune hepatitis and primary biliary cholangitis. The EUROLINE immunoblot assay, detecting multiple specificities, is widely used, but the clinical importance of weakly positive findings is unclear. The manufacturer's recommended cut-off was evaluated by investigating AILD-associated autoantibodies in 825 blood donors and 60 confirmed AILD cases. Positive findings were followed up with immunofluorescence microscopy on rat tissue, anti-M2-ELISA, alternative immunoblot assay, and liver function tests. Thirty-six (4.4%) blood donors were positive with EUROLINE. The most common specificities were LC-1 (1.6%), gp210 (1.3%), and AMA-M2 (1.1%). In general, the positive results were higher in patients than in blood donors, whereas anti-LC-1 was higher in blood donors. The liver function tests were slightly elevated in 2 of the 36 immunoblot positive blood donors. The majority of the positive EUROLINE findings could not be confirmed with the follow-up tests. The EUROLINE-Autoimmune Liver Diseases-(IgG) immunoblot detected autoantibodies in 4.4% of blood donors without signs of AILD. Our findings indicate that the recommended cut-off can be raised for most specificities without loss of diagnostic sensitivity. The prevalence of anti-LC-1 among blood donors indicates a problem with the antigen source.

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a large production of autoantibodies and deficient clearance of cellular waste. The disease typically oscillates between episodes of elevated disease activity and quiescent disease. C-reactive protein (CRP) is a pentameric acute-phase protein usually reflecting inflammation and tissue damage. However, despite increased inflammation and elevated interleukin-6, the levels of CRP typically remain low or only slightly raised in SLE. Under certain conditions, pentameric CRP (pCRP) can dissociate into its monomeric isoform (mCRP), which mainly has been ascribed pro-inflammatory properties. The present study aims to investigate the potential relationship between pCRP and mCRP, respectively, with disease activity and clinical features of SLE., Methods: The levels of pCRP and mCRP were measured, by turbidimetry (high-sensitive) and sandwich enzyme-linked immunosorbent assay (ELISA) respectively, in serum samples from 160 patients with SLE and 30 patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Twenty-two of the SLE cases were selected for analysis at two time-points; quiescent disease and active disease. The two CRP isoforms were evaluated in relation to disease activity and clinical features in the two diseases., Results: Levels of pCRP and mCRP were significantly lower in SLE than AAV (p < 0.001) and the ratio of mCRP/pCRP was higher in SLE compared to AAV. The mCRP/pCRP ratio was higher for patients in remission and able to significantly separate between active/quiescent disease in paired, but not in non-paired, samples from patients with SLE. Significant correlations were observed with SLICC/ACR damage index for pCRP levels as well as inversely with the mCRP/pCRP ratio. Lower mCRP levels associated with malar rash., Conclusion: As the interrelationship between the two isoforms appear to (a) discriminate between quiescent and active SLE and (b) differ between SLE and AAV, our data indicates that the two CRP isoforms could exert contrasting immunological effects and/or reflect different milieus. Given the biological effects of mCRP, it is possible that altered levels may indicate increased opsonization of immune complexes and apoptotic debris, and thereby prevent their deposition outside the reticuloendothelial system and manifestations such as lupus nephritis and lupus-related skin disease., (© 2022. The Author(s).)

Objective: To investigate levels and possible extramucosal formation of secretory Ig, including anti-citrullinated protein antibodies (ACPAs), in rheumatoid arthritis (RA)., Methods: Three patient groups were studied: 1) ACPA-positive patients with musculoskeletal pain without clinical arthritis, 2) patients with recent-onset RA, and 3) patients with established RA. In baseline serum samples (groups 1 and 2) and paired synovial fluid samples (group 3), we analyzed total secretory IgA, total secretory IgM, free secretory component (SC), and SC-containing ACPA. Extramucosal formation of SC-containing ACPA was investigated by preincubating RA sera and affinity-purified ACPA with recombinant free SC., Results: Compared to healthy controls, serum levels of total secretory IgA and total secretory IgM were increased both in patients with early RA and at-risk patients (P < 0.05). Patients with early RA with elevated total secretory Ig had significantly higher disease activity during the 3-year follow-up period compared to those without increased levels. At-risk patients who developed arthritis during follow-up (39 of 82) had higher baseline total secretory IgA levels compared to those who did not (P = 0.041). In established RA, total secretory IgA and total secretory IgM levels were higher in serum than in synovial fluid (P < 0.0001), but SC-containing ACPAs adjusted for total secretory Ig concentration were higher in synovial fluid (P < 0.0001). Preincubation with recombinant free SC yielded increased SC-containing ACPA reactivity in sera as well as in affinity-purified IgA and IgM ACPA preparations., Conclusion: Circulating secretory Ig are elevated before and at RA onset. In the presence of free SC, secretory Ig may form outside the mucosa, and SC-containing ACPAs are enriched in RA joints. These findings shed important new light on the mucosal connection in RA development., (© 2021 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Patients with established rheumatoid arthritis (RA) and disease modifying treatments have lower nitric oxide (NO) levels in the alveolar compartment (C A NO) and in the airway wall (C aw NO), but also higher diffusion capacities for NO in the airways (D aw NO) compared to matched controls. The aim of the present study was to investigate the NO lung dynamics in patients with recent onset RA before and after immune suppression with methotrexate therapy. Patients with early RA and antibodies against anticitrullinated peptides (ACPA) were recruited. Measurement of exhaled NO and inflammatory markers in serum were performed. Clinical disease activity was evaluated with Disease Activity Score for 28 joints. Healthy individuals were used as matched controls. Data are presented as median (lower quartile, upper quartile) values. RA patients (n = 44) had lower exhaled NO (F E NO 50 ) 16 (10-24) ppb compared to controls 21 (15, 29) ppb, p = 0.013. In NO-dynamics, C A NO was lower in RA patients 1.6 (1.0, 2.2) ppb compared to the control subjects 2.3 (1.3, 3.1) ppb, p = 0.007. C aw NO was also lower in the RA patients 55 (24, 106) ppb compared to control subjects 124 (110, 170) ppb, p < 0.001, but D aw NO was higher 17 (8, 30) mL/s and 9 (5, 11) mL/s respectively, p < 0.001. Methotrexate treatment for three months reduced disease activity, but did not change the NO dynamics. In conclusion, the altered NO dynamics of the lung in ACPA-positive RA patients are already present in the early stages of the disease before any treatments and do not change after methotrexate therapy suggesting a role in the pathogenesis., (© 2022. The Author(s).)

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium Porphyromonas gingivalis to citrullinate proteins, it has been suggested that production of anti-citrullinated protein antibodies (ACPA), which are present in a majority of RA patients, may be triggered in the gum mucosa. To address this hypothesis, we investigated the antibody response to a citrullinated P. gingivalis peptide in relation to the autoimmune ACPA response in early RA, and examined citrulline-reactivity in monoclonal antibodies derived from human gingival B cells. Antibodies to a citrullinated peptide derived from P. gingivalis (denoted CPP3) and human citrullinated peptides were analyzed by multiplex array in 2,807 RA patients and 372 controls; associations with RA risk factors and clinical features were examined. B cells from inflamed gingival tissue were single-cell sorted, and immunoglobulin (Ig) genes were amplified, sequenced, cloned and expressed (n=63) as recombinant monoclonal antibodies, and assayed for citrulline-reactivities by enzyme-linked immunosorbent assay. Additionally, affinity-purified polyclonal anti-cyclic-citrullinated peptide (CCP2) IgG, and monoclonal antibodies derived from RA blood and synovial fluid B cells (n=175), were screened for CPP3-reactivity. Elevated anti-CPP3 antibody levels were detected in RA (11%), mainly CCP2+ RA, compared to controls (2%), p<0.0001, with a significant association to HLA-DRB1 shared epitope alleles, smoking and baseline pain, but with low correlation to autoimmune ACPA fine-specificities. Monoclonal antibodies derived from gingival B cells showed cross-reactivity between P. gingivalis CPP3 and human citrullinated peptides, and a CPP3+/CCP2+ clone, derived from an RA blood memory B cell, was identified. Our data support the possibility that immunity to P. gingivalis derived citrullinated antigens, triggered in the inflamed gum mucosa, may contribute to the presence of ACPA in RA patients, through mechanisms of molecular mimicry., Competing Interests: Karolinska Institutet has been a partner with Thermo Fisher Scientific within the Innovative Medicines Initiative BTCure, a public–private partnership between the EU and the European Federation of Pharmaceutical Industries (www.BTcure.eu). Thermo Fisher Scientific has contributed to this consortium with in-kind contributions for the development of the multiplex assay used in the study. LM-A is employed by Thermo Fischer Scientific. JR is member of the Thermo Fisher Scientific advisory board. KL is co-inventor of patent: US12/524,465, describing the diagnostic use of the CEP-1 epitope. RH is a co-inventor of patent: US Patent 7 148 020, protecting the use of the CitC1 peptide. GJ was employed by Praktikertjänst AB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sherina, de Vries, Kharlamova, Sippl, Jiang, Brynedal, Kindstedt, Hansson, Mathsson-Alm, Israelsson, Stålesen, Saevarsdottir, Holmdahl, Hensvold, Johannsen, Eriksson, Sallusto, Catrina, Rönnelid, Grönwall, Yucel-Lindberg, Alfredsson, Klareskog, Piccoli, Malmström, Amara and Lundberg.)

Rheumatoid arthritis (RA) is the most common systemic autoimmune disease and also the most severe arthritic disorder. The measurement of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) in serum supports the diagnosis of RA, which gained increasing significance over the last 65 years. However, a high variability between RF and ACPA methods has been described, impacting the diagnostic performance of the current ACR/EULAR RA classification criteria. The great number of commercially available assays, often lacking traceability to an international standard, is a major factor attributing to this in-between assay variability. The adoption of an international standard for ACPA, as is since long available for rheumatoid factor, is therefore highly desirable. Further harmonization in clinical interpretation of RF/ACPA assays could be obtained by harmonization of the cut-offs, for both the low and high antibody levels, based on predefined specificity in disease controls. Reporting test result specific likelihood ratios (LR) adds value in the interpretation of autoantibody tests. However, a good understanding of the control population used to define antibody test result interval-associated LRs is crucial in defining the diagnostic performance characteristics of antibody serology. Finally, specificity in RA classification can be improved by refining serological weight scoring taking into account the nature of the antibody, the antibody level and double RF + ACPA positivity., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Background: There is a need for biomarker to identify patients "at risk" for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers., Methods: Using protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry., Results: HnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP-2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. The citrullinated/native index of autoantibodies against hnRNP-DL (CN DL -Index) was identified as a new value for an "individual window of treatment success" in early RA and for the detection of RF IgM/α-CCP-2 seronegative RA patients (24-46%). Negative CN DL -index was found in SLE patients, risk-RA and early RA cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CN DL -values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients., Conclusion: CN DL -index defines people at risk to develop RA and the "window of treatment success" thereby closing the sensitivity gap in RA., (© 2021. The Author(s).)

Objectives: Antiphosphatidylserine/prothrombin complex antibodies (aPS/PT) are risk factors for thrombosis, yet further validation of their clinical relevance in different ethnic groups is required. We investigated the performance of aPS/PT of IgA/G/M isotypes among Sudanese and Swedish systemic lupus erythematosus (SLE) patients., Methods: Consecutive SLE patients/matched controls from Sudan (n = 91/102) and Sweden (n = 332/163) were included. All patients fulfilled the 1982 ACR SLE classification criteria. IgA/G/M of aPS/PT, anti-cardiolipin and anti-β 2 glycoprotein I (anti-β 2 GPI) were tested in both cohorts, and lupus anticoagulant (LA) also in the Swedish cohort. Clinical antiphospholipid syndrome-related events and atherosclerosis, measured as carotid plaques were assessed for associations. Univariate and multivariate analyses adjusting for cardiovascular risk factors were performed., Results: Sudanese SLE patients had higher levels of IgM aPS/PT, but using national cut-offs, the frequency of positivity was similar to Swedish patients for all isotypes. Among Swedish patients, all isotypes of aPS/PT associated with venous thromboembolism (VTE), while only IgA aPS/PT associated with arterial thrombosis (AT). aPS/PT antibodies associated strongly with LA and they were, independently, the best predictor for VTE. Double positivity for aPS/PT and anti-β 2 GPI associated with higher VTE risk than the conventional triple positivity. Carotid plaques did not associate with any antiphospholipid antibody., Conclusions: IgA aPS/PT associated with AT, and the association of IgG/M aPS/PT with VTE outperforms LA and criteria antiphospholipid antibodies in Swedish SLE patients. Furthermore, double positivity for aPS/PT and anti-β 2 GPI performed better than conventional triple positivity. Future studies need to address if aPS/PT can replace LA, as this would simplify clinical procedures.

Objectives: To examine the association between individual rheumatoid arthritis (RA) autoantibodies, sex and age at RA onset., Methods: Anti-CCP2, IgA-, IgG- and IgM-RF were analysed centrally in baseline sera from 1600 RA patients diagnosed within one year of RA symptom onset. Cut-offs for RF isotypes were determined at the 98th percentile based on RA-free controls, close to the 98.4% anti-CCP2 specificity., Results: Anti-CCP2 was found in 1020 patients (64%), IgA RF in 692 (43%), IgG RF in 529 (33%) and IgM RF in 916 (57%) of the patients. When assessed one by one, anti-CCP2 and IgM RF were both associated with lower age at RA diagnosis. When assessed in one joint model, the association to IgM RF weakened and a strong association between IgA RF and higher age at RA diagnosis appeared. IgA RF and IgG RF associated with male sex, and IgM RF with female sex, with no difference for anti-CCP2. When the model was adjusted for sex, the association between IgM RF and age disappeared, whereas the strong associations between IgA RF and high age and between anti-CCP2 and low age at diagnosis remained. Further adjustments for smoking, shared epitope and inclusion year did not change the outcome. Univariate analyses stratified on anti-CCP2 and IgA RF status confirmed the findings., Conclusions: Anti-CCP associate with low, and IgA RF with high age at RA onset. RFs and anti-CCP2 display opposing association with sex. These results underscore that studies on RA phenotypes in relation to autoantibodies should accommodate age and sex.

Competing Interests: Competing interests: None declared.

Seropositive rheumatoid arthritis (RA) is characterized by the presence of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) with different fine-specificities. Yet, other serum anti-modified protein autoantibodies (AMPA), e.g. anti-carbamylated (Carb), -acetylated (KAc), and malondialdehyde acetaldehyde (MAA) modified protein antibodies, have been described. In this comprehensive study, we analyze 30 different IgG and IgA AMPA reactivities to Cit, Carb, KAc, and MAA antigens detected by ELISA and autoantigen arrays in N=1985 newly diagnosed RA patients. Association with patient characteristics such as smoking and disease activity were explored. Carb and KAc reactivities by different assays were primarily seen in patients also positive for anti-citrulline reactivity. Modified vimentin (mod-Vim) peptides were used for direct comparison of different AMPA reactivities, revealing that IgA AMPA recognizing mod-Vim was mainly detected in subsets of patients with high IgG anti-Cit-Vim levels and a history of smoking. IgG reactivity to acetylation was mainly detected in a subset of patients with Cit and Carb reactivity. Anti-acetylated histone reactivity was RA-specific and associated with high anti-CCP2 IgG levels, multiple ACPA fine-specificities, and smoking status. This reactivity was also found to be present in CCP2+ RA-risk individuals without arthritis. Our data further demonstrate that IgG autoreactivity to MAA was increased in RA compared to controls with highest levels in CCP2+ RA, but was not RA-specific, and showed low correlation with other AMPA. Anti-MAA was instead associated with disease activity and was not significantly increased in CCP2+ individuals at risk of RA. Notably, RA patients could be subdivided into four different subsets based on their AMPA IgG and IgA reactivity profiles. Our serology results were complemented by screening of monoclonal antibodies derived from single B cells from RA patients for the same antigens as the RA cohort. Certain CCP2+ clones had Carb or Carb+KAc+ multireactivity, while such reactivities were not found in CCP2- clones. We conclude that autoantibodies exhibiting different patterns of ACPA fine-specificities as well as Carb and KAc reactivity are present in RA and may be derived from multireactive B-cell clones. Carb and KAc could be considered reactivities within the "Cit-umbrella" similar to ACPA fine-specificities, while MAA reactivity is distinctly different., Competing Interests: HB is an employee at Orgentec Diagnostika and LM-A is an employee at Thermo Fisher Scientific. KL is co-inventor of patent: US12/524,465, describing the diagnostic use of the CEP-1 epitope. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Grönwall, Liljefors, Bang, Hensvold, Hansson, Mathsson-Alm, Israelsson, Joshua, Svärd, Stålesen, Titcombe, Steen, Piccoli, Sherina, Clavel, Svenungsson, Gunnarsson, Saevarsdottir, Kastbom, Serre, Alfredsson, Malmström, Rönnelid, Catrina, Lundberg and Klareskog.)

Measurement of two groups of autoantibodies, rheumatoid factor (RF) and anti-citrullinated protein/peptide antibodies (ACPA) have gained increasing significance in the diagnosis and classification of rheumatoid arthritis (RA) over the last 65 years. Despite this rising importance of autoimmune serology in RA, there is a palpable lack of harmonization between different commercial RF and ACPA tests. While a minimal diagnostic specificity has been defined for RF tests, which almost always are related to an international reference preparation, neither of this applies to ACPA. Especially assays with low diagnostic specificity are associated with very low positive predictive values or post-test probabilities in real world settings. In this review we focus on issues of practical bearing for the clinical physician diagnosing patients who potentially have RA, or treating patients diagnosed with RA. We advocate that all clinically used assays for RF and ACPA should be aligned to a common diagnostic specificity of 98-99% compared to healthy controls. This high and rather narrow interval corresponds to the diagnostic specificity seen for many commercial ACPA tests, and represents a specificity that is higher than what is customary for most RF assays. Data on antibody occurrence harmonized in this way should be accompanied by test result-specific likelihood ratios for the target diagnosis RA on an ordinal or interval scale, which will provide the clinical physician with more granular and richer information than merely relating numerical values to a single cut-off point. As many physicians today are used to evaluate autoantibodies as positive or negative on a nominal scale, the introduction of test result-specific likelihood ratios will require a change in clinical mindset. We also discuss the use of autoantibodies to prognosticate future arthritis development in at-risk patients as well as predict severe disease course and outcome of pharmacological treatment., Competing Interests: JR has been a member of the scientific advisory board for Thermo Fisher Scientific, and has research collaboration with the diagnostic companies Thermo Fischer Scientific, Inova Diagnostics, Euroimmun and Theradiag. CT has received a research grant from Bristol-Myers Squibb, consultancy fees from Roche, and speaker’s honoraria from Abbvie, Bristol-Myers Squibb, Nordic Drugs, Pfizer and Roche. AK has received speaker’s honoraria from Werfen and was previously employed by Sanofi. The handling editor declared a past co-authorship with one of the authors, JR., (Copyright © 2021 Rönnelid, Turesson and Kastbom.)

Patients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless they tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays using samples from patients with chronic inflammatory diseases collected prior to April 2019, thus defined as negative. Samples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and systemic lupus erythematosus (SLE, n=10) with or without RF, were analyzed for SARS-CoV-2 antibodies using 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed IgG multiplex bead-based assay. Six LFA and the in-house validated IgG assay correctly produced negative results for all samples. However, the majority of assays (n=13), gave false positive signal for samples from patients with RA and SLE. This was most notable in samples from RF positive RA patients. No false positive samples were detected in any assay using samples from patients with MS. Poor specificity of commercial serological assays could possibly be, at least partly, due to interfering antibodies in samples from patients with chronic inflammatory diseases. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kharlamova, Dunn, Bedri, Jerling, Almgren, Faustini, Gunnarsson, Rönnelid, Pullerits, Gjertsson, Lundberg, Månberg, Pin, Nilsson, Hober, Fink and Fogdell-Hahn.)

Objective: To assess clinical phenotypes of anti-aminoacyl-transfer RNA synthetases (aaRS) autoantibodies in Vietnamese patients of Kinh ethnicity with idiopathic inflammatory myopathies (IIM)., Methods: In a cross-sectional study 23 patients with anti-aaRS autoantibodies were compared to 36 patients with other myositis-specific antibodies and to 69 seronegative patients with IIM. Assessments included muscle performance, extra-muscular involvement, and disease activity according to the International Myositis Assessment and Clinical Studies (IMACS). Sera were tested by a line immunoassay (Euroline Myositis Profile 4)., Results: The frequency of anti-Jo-1 antibodies was 56.5%, anti-EJ antibodies 26.1%, and anti-PL-7 antibodies 17.4%, while anti-PL-12 and anti-OJ antibodies were not present in any case. All patients with anti-aaRS autoantibodies had signs of myositis. At time of investigation 22/23 patients had muscle weakness, 52.2% arthritis, 34.8% Raynaud's phenomenon, 73.9% fever, 14.3% mechanic's hands and 56.5% dysphagia. Interstitial lung disease was present in 52.2%, and pulmonary hypertension in 56.5%. The anti-aaRS autoantibody positive group had higher disease activity in the domains of skin and pulmonary disease compared to the seronegative group and had lower disease activity in skeletal disease compared to the anti-melanoma differentiation-associated protein 5-positive patients. The clinical presentation of antisynthetase syndrome was similar between the aaRS autoantibody specificities with the exception of more frequent pulmonary hypertension in anti-Jo-1 positive patients., Conclusions: Different aaRS autoantibody specificities may vary between different ethnic populations for reasons that still need to be clarified. Furthermore, the high frequency of pulmonary hypertension is noteworthy but otherwise clinical manifestations associated with aaRS autoantibodies did not differ from other ethnic populations., (© 2021 The Authors. International Journal of Rheumatic Diseases published by Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

The role of anti-nuclear autoantibody (ANA) specificities in immune complexes (IC) formation has been studied to a limited extent in SLE, and not at all in African SLE patients. We compared ANA in IC from Sudanese and Swedish SLE patients. We included 93 Sudanese and 332 Swedish SLE patients fulfilling the 1982 ACR criteria. IC were captured using C1q-coated beads. ANA specificities were quantified in sera and IC. Results were related to modified SLEDAI. Whereas serum levels of anti-Sm, anti-dsDNA and anti-ribosomal P were higher in Swedish patients, IC levels of most ANA specificities were higher among Sudanese patients. This difference was especially prominent for anti-chromatin antibodies, which remained after adjustment for age, disease duration and treatment. Total levels of C1q-binding IC correlated with levels of specific ANA in IC, with highest correlations for anti-chromatin antibodies among Sudanese patients. Whereas occurrence of anti- SSA/Ro60, anti-histone and anti-U1RNP in both serum and IC associated with high SLEDAI score, anti-dsDNA in IC but not in serum associated with high SLEDAI. ANA, especially antibodies targeting chromatin, accumulate more in IC from Sudanese SLE patients. If the autoantibody fraction forming IC is pathogenically important, this might explain the generally described severe SLE in black populations.

Competing Interests: Competing interests: No, there are no competing interests for any author.

Objectives: IgA antiphospholipid antibodies (aPL) are prevalent in systemic lupus erythematosus (SLE) patients of African American, Afro-Caribbean and South African origin. Nevertheless, data from North Africa are lacking, and most studies use manufacturer-suggested cut-offs based on Caucasian controls. Therefore, we compared aPL isotypes in Sudanese and Swedish SLE patients using nation-based cut-offs., Methods: Consecutive SLE patients and age- and sex-matched controls from Sudan ( N  = 115/106) and Sweden ( N  = 340/318) were included. All patients fulfilled the 1982 American College of Rheumatology SLE classification criteria. Antiphospholipid syndrome-related events were obtained from patients' records. IgA/G/M anticardiolipin and anti-β 2 glycoprotein I (β 2 GPI) were analysed with two independent assays. IgA anti-β 2 GPI domain 1 (D1) was also investigated. Manufacturers' cut-offs and the 95th and 99th percentile cut-offs based on national controls were used., Results: Sudanese patients and controls had higher levels and were more often positive for IgA aPL than Swedes when using manufacturers' cut-offs. In contrast, using national cut-offs, the increase in IgA aPL among Sudanese patients was lost. Occurrence of IgA anti-D1 did not differ between the countries. Venous thromboses were less common among Sudanese patients and did not associate with aPL. No clinical associations were observed with IgA anti-β 2 GPI in Sudanese patients. Thromboses in Swedes were associated with IgG/M aPL. Fetal loss was associated with aPL in both cohorts., Conclusions: IgA anti-β 2 GPI prevalence was higher among Sudanese compared to Swedish patients when manufacturers' cut-offs were used. This situation was reversed when applying national cut-offs. Anti-D1 was not increased in Sudanese patients. Previous studies on populations of African origin, which demonstrate a high prevalence of IgA aPL positivity, should be re-evaluated using a similar cut-off approach.

Objective: To investigate the relationship between anti-citrullinated protein antibodies (ACPAs), specific ACPA subspecificities, rheumatoid factor (RF) isotypes, and incident cardiovascular (CV) events in patients with rheumatoid arthritis (RA)., Methods: Serum samples from Swedish patients with new-onset RA (diagnosed within 1 year of symptom onset between 1996 and 2009) were centrally typed for anti-cyclic citrullinated peptide 2 (anti-CCP2) antibodies, 20 ACPA subspecificities, and RF isotypes. Patients were followed up longitudinally in nationwide registers to monitor the occurrence of acute coronary syndrome (ACS), stroke, CV-related death, and major adverse CV events (MACE). The association between each serologic marker and CV outcome, and the impact of adjustment for the Disease Activity Score in 28 joints (DAS28), smoking status, and income at baseline, were assessed using Cox proportional hazards models. In addition, associations of serologic markers with all-cause mortality were explored., Results: In total, 2,814 patients with RA were included in the study. The median follow-up was 13 years, during which the CV end points of ACS, stroke, or CV-related death were reported to occur in 375 patients. Occurrence and/or levels of anti-CCP2 were associated with risk of incident ACS (hazard ratio [HR] 1.46, 95% confidence interval [95% CI] 1.03-2.06), stroke (HR 1.47, 95% CI 1.03-2.10), CV-related death (P = 0.024 for association with anti-CCP2 levels), and MACE (HR 1.34, 95% CI 1.06-1.70). Similarly, an association with the number of ACPA subspecificities was observed; however, this could not be attributed to any individual or group of ACPA subspecificities. Presence of IgM-RF was associated with all CV end points except ACS, and IgA-RF was exclusively associated with CV-related death. Adjustment for smoking status, income, and DAS28 scores decreased most of the HRs, whereas IgA-RF remained associated with CV-related death (HR 1.61, 95% CI 1.05-2.48). All of the assessed serologic makers were associated with all-cause mortality., Conclusion: RF isotypes and ACPAs are associated with future CV events in patients with RA. ACPA levels and number of subspecificities seem more important than the occurrence of particular subspecificities, and these associations were not explained by a history of ever smoking., (© 2020 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Special conditions associated with laboratory autoimmune testing are not well compatible with recent developments in regulatory frameworks such as EN/ISO 15189 accreditation or in vitro diagnostic medical device regulation (IVD-R). In addition, international recommendations, guidelines and disease criteria are poorly defined with respect to requirements on autoantibody testing. Laboratory specialists from Austria, Belgium, Croatia, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Norway, Poland, Portugal, South Africa, Spain, Sweden, Switzerland, and The Netherlands collected information, reported national experience, and identified quality issues in relation to autoantibody testing that require consensus on interpretation of the regulatory frameworks and guidelines. This process has been organized by the European Autoimmunity Standardisation Initiative (EASI). By identifying the critical items and looking for a consensus, our objective was to define a framework for, in particular, EN/ISO accreditation purposes. Here, we present a review of current publications and guidelines in this field to unify national guidelines and deliver in this way a European handout on quality control and accreditation for laboratories involved in autoantibody testing. We focus on quality items that can be checked during accreditation visits. Despite various local varieties, we encountered an overwhelming dedication to quality assurance in all contributing countries.

Background: Rheumatoid arthritis (RA) is classified as seropositive or seronegative, depending on the presence/absence of rheumatoid factor (RF), primarily IgM RF, and/or anti-citrullinated protein antibodies (ACPA), commonly detected using anti-cyclic citrullinated peptide (CCP) assays. Known risk factors associate with the more severe seropositive form of RA; less is known about seronegative RA. Here, we examine risk factors and clinical phenotypes in relation to presence of autoantibodies in the RA subset that is traditionally defined as seronegative., Methods: Anti-CCP2 IgG, 19 ACPA fine-specificities, IgM/IgG/IgA RF, anti-carbamylated-protein (CarP) antibodies, and 17 other autoantibodies, were analysed in 2755 RA patients and 370 controls. Antibody prevalence, levels, and co-occurrence were examined, and associations with risk factors and disease activity during 5 years were investigated for different antibody-defined RA subsets., Results: Autoantibodies were detected in a substantial proportion of the traditionally defined seronegative RA subset, with ACPA fine-specificities found in 30%, IgA/IgG RF in 9.4%, and anti-CarP antibodies in 16%, with a 9.6% co-occurrence of at least two types of RA-associated autoantibodies. HLA-DRB1 shared epitope (SE) associated with the presence of ACPA in anti-CCP2-negative RA; in anti-CCP2-positive RA, the SE association was defined by six ACPA fine-specificities with high co-occurrence. Smoking associated with RF, but not with ACPA, in anti-CCP2-negative RA. Presence of ACPA and RF, but not anti-CarP antibodies, in conventionally defined "seronegative" RA, associated with worse clinical outcome., Conclusions: "Seronegative" RA is not truly a seronegative disease subset. Additional screening for ACPA fine-specificities and IgA/IgG RF defines a group of patients that resembles seropositive patients with respect to risk factors and clinical picture and may contribute to earlier diagnosis for a subset of anti-CCP2-/IgM RF- patients with a high need for active treatment.

Objective: An often-neglected subset of patients with systemic lupus erythematosus (SLE) is those with secondary Sjögren syndrome (SLE-sSS). Further, primary SS overlaps and can be difficult to delineate from SLE. To shed light on the SLE-sSS subset, we investigated a large and well-characterized SLE cohort, comparing patients with SLE-sSS and SLE patients without SS (SLE-nonsSS) and controls., Methods: We included 504 consecutive patients with SLE, fulfilling the 1982 revised American College of Rheumatology criteria, and 319 controls from the general population, matched for age and sex to the first 319 patients. SLE-sSS was defined according to the American-European Consensus Criteria (AECC). A thorough clinical examination, including subjective and objective quantifications of sicca symptoms, was performed in all participants. Autoantibodies and 20 selected cytokines were measured by luminex and multiplex analysis, respectively., Results: SLE-sSS, as defined by AECC, occurred in 23% of the patients with SLE. In comparison to SLE-nonsSS, the SLE-sSS group was older and more frequently female. Leukopenia and peripheral neuropathy were more frequent and nephritis less frequent. Circulating levels of 6/20 investigated proinflammatory cytokines [tumor necrosis factor-α, interleukin (IL) 6, monocyte chemoattractant protein 4, macrophage inflammatory protein 1β, IL-12/IL-23p40, and interferon γ-induced protein 10], total IgG, anti-SSA/Ro52, anti-SSA/Ro60, anti-SSB/La antibodies, and rheumatoid factor (IgM and IgA) were higher in the SLE-sSS group (p < 0.05 for all comparisons)., Conclusion: The frequency of SLE-sSS increased with age and affected roughly one-quarter of all patients with SLE. Despite less internal organ involvement, a systemic inflammatory state with high levels of proinflammatory cytokines is present in the SLE-sSS subgroup. This is a novel observation that may affect future understanding and treatment of the SLE-sSS subset.

We investigated whether belimumab treatment impacts on levels of autoantibodies and cytokines of interest in systemic lupus erythematosus (SLE). Longitudinally collected serum samples from 78 belimumab-treated Swedish SLE patients were analysed. Serum cytokine levels were determined using Luminex xMAP technology, and nuclear antigen autoantibody specificities using addressable laser bead immunoassay. In patients with detectable levels at baseline, interferon (IFN)-α2 levels were lower at month 6 (median; interquartile range (IQR): 8.9; 1.5-54.9 pg/mL) versus baseline (28.4; 20.9-100.3 pg/mL; p = 0.043). Interleukin (IL)-6 (baseline: 7.1; 2.9-16.1 pg/mL) decreased from month 6 (0.5; 0.5-6.3 pg/mL; p = 0.018) and throughout a 24 month follow-up. IL-10 (baseline: 12.6; 2.8-29.7 pg/mL) showed more rapid decreases from month 3 (1.8; 0.6-9.1 pg/mL; p = 0.003). Levels of anti-dsDNA ( p < 0.001), anti-Smith antigen (Sm) ( p = 0.002), anti-U1 small nuclear ribonucleoprotein (U1RNP) ( p < 0.001), anti-Sm-U1RNP complex ( p = 0.028), and anti-ribosomal P ( p = 0.012) antibodies decreased from month 3 and remained decreased. Anti-Sm positivity at baseline was associated with higher probability and/or shorter time to achieve sustained SLE responder index-4 response (hazard ratio (HR): 2.52; 95% CI: 1.20-5.29; p = 0.015), independently of other factors. Decline of IL-6 levels through month 3 was greater in responders. In summary, belimumab treatment lowered IFN-α2, IL-6, and IL-10 levels, as well as levels of multiple autoantibodies, however after different time spans. Notably, anti-Sm positivity and early decline in IL-6 levels were associated with favorable treatment outcome., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Objective: SLE is known to have an aggressive phenotype in black populations, but data from African cohorts are largely lacking. We therefore compared immunological and clinical profiles between Sudanese and Swedish patients using similar tools., Methods: Consecutive SLE patients from Sudan (n = 115) and Sweden (n = 340) and from 106 Sudanese and 318 Swedish age- and sex-matched controls were included. All patients fulfilled the 1982 ACR classification criteria for SLE. Ten ANA-associated specificities and C1q-binding immune complexes (CICs) were measured. Cut-offs were established based on Sudanese and Swedish controls, respectively. Disease activity was measured with a modified SLEDAI and organ damage with the SLICC Damage Index. In a nested case-control design, Swedish and Sudanese patients were matched for age and disease duration., Results: Females constituted 95.6% and 88.1% of Sudanese and Swedish patients, respectively (P = 0.02), with younger age at inclusion (33 vs 47.7 years; P < 0.0001) and shorter disease duration (5 vs 14 years; P < 0.0001) among Sudanese patients. Anti-Sm antibodies were more frequent in Sudanese patients, whereas anti-dsDNA, anti-histone and CICs were higher in Swedish patients. In the matched analyses, there was a trend for higher SLEDAI among Swedes. However, Sudanese patients had more damage, solely attributed to high frequencies of cranial/peripheral neuropathy and diabetes., Conclusion: While anti-Sm is more common in Sudan than in Sweden, the opposite is found for anti-dsDNA. Sudanese patients had higher damage scores, mainly because of neuropathy and diabetes. Sudanese patients were younger, with a shorter SLE duration, possibly indicating a more severe disease course with impact on survival rates., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Introduction: Antibodies against double-stranded DNA (anti-dsDNA) are a specific biomarker for systemic lupus erythematosus (SLE). The first WHO International Standard (IS) for anti-dsDNA (established in 1985), which was used to assign units to diagnostic tests, was exhausted over a decade ago., Methods: Plasma from a patient with SLE was first evaluated in 42 European laboratories. The plasma was thereafter used by the National Institute for Biological Standards and Control to prepare a candidate WHO reference preparation for lupus (anti-dsDNA) antibodies. That preparation, coded 15/174, was subjected to an international collaborative study, including 36 laboratories from 17 countries., Results: The plasma mainly contained anti-dsDNA, other anti-chromatin antibodies and anti-Ku. The international collaborative study showed that the field would benefit from 15/174 as a common reference reagent improving differences in performance between different assays. However, no statistically meaningful overall potency or assay parallelism and commutability could be shown., Conclusion: 15/174 cannot be considered equivalent to the first IS for anti-dsDNA (Wo/80) and was established as a WHO Reference Reagent for lupus (oligo-specific) anti-dsDNA antibodies with a nominal value of 100 units/ampoule. This preparation is intended to be used to align test methods quantifying levels of anti-dsDNA antibodies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Immune complexes are of importance in systemic lupus erythematosus pathogenesis, and autoantibodies are believed to participate in immune complex formation. Quantification of autoantibody levels in circulating IC might be of prognostic value., Methods: A C1q-binding-eluting technique was applied to purify immune complexes from 55 belimumab-treated systemic lupus erythematosus patients during a 24-month follow-up. Autoantibodies in serum and in solubilized immune complexes were quantified using addressable laser bead immunoassay. We investigated whether levels of autoantibodies in immune complexes associate with disease activity and response to belimumab treatment., Results: High baseline anti-double-stranded DNA and anti-histone levels in immune complexes associated with attainment of zero scores in clinical systemic lupus erythematosus disease activity index 2000 during the 24-month follow-up (p = 0.003 and p = 0.048, respectively). Low complement levels associated with high serum anti-double-stranded DNA and anti-ribosomal P levels (p = 0.003 and p = 0.008, respectively) and high anti-double-stranded DNA (p = 0.002) but not anti-ribosomal P levels in immune complexes. Anti-SSA/SSB serum levels were lower in patients attaining lupus low disease activity state at month 6; these associations were stronger for corresponding immune complex levels. Serum levels of most autoantibodies had declined at month 3, whereas autoantibody levels in immune complexes, except for anti-double-stranded DNA, showed a more gradual decline over 1-2 years. Serum anti-double-stranded DNA levels decreased in all patients irrespective of systemic lupus erythematosus disease activity index 2000=0 attainment, whereas immune complex levels decreased only in achievers., Conclusion: Immune complex levels of autoantibodies against double-stranded DNA and the SSA/SSB complex show more specific associations with treatment outcome compared with serum levels in belimumab-treated systemic lupus erythematosus patients. Characterization of autoantibody content in circulating immune complexes could prove useful in treatment evaluation in systemic lupus erythematosus and other immune complex-associated diseases.

Background International autoantibody standards, traditionally based on material obtained from plasmapheresis of single subjects, represent individual immune response and may not comprehend the heterogeneity of the general population. The anti-DFS70 autoantibody yields a characteristic dense fine speckled (DFS) nuclear pattern on indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and speaks against autoimmunity. We propose a novel strategy for developing autoantibody reference standards, based on stepwise pooling of serum samples from hundreds of individuals with anti-DFS70 antibodies. Methods Within a 2-year period, serum samples were selected from routine HEp-2 IFA according to the following criteria: DFS HEp-2 IFA pattern at titer ≥1:640; anti-DFS70 reactivity in three analyte-specific tests (Western blot [WB], enzyme-linked immunosorbent assay [ELISA] and chemiluminescent immunoassay [CLIA]). Aliquots of individual samples were combined into progressively larger pools with stepwise validation of intermediary pools as for individual samples. Validated intermediary pools were merged into a final pool for lyophilization. Results A total of 741 validated samples yielded a 750 mL final pool that was lyophilized into thousands of 200 μL-aliquots. Reconstituted aliquots yielded the expected anti-DFS70 reactivity in ELISA, CLIA and WB, as well as high-titer DFS HEp-2 IFA pattern. The appropriate anti-DFS70 reactivity of the lyophilized pool was confirmed by seven international expert centers, using HEp-2 IFA, ELISA, WB and immunoprecipitation. Conclusions This proof-of-concept study provides an innovative and efficient strategy to build serum reference standards for autoantibody testing. The anti-DFS70 standard will integrate the panel of standards of Autoantibody Standardization Committee (ASC, www.autoab.org), contributing to education for proper assay validation and interpretation of the DFS pattern and other HEp-2 IFA patterns.

Rheumatoid arthritis (RA) is the most common immune-mediated arthritis. Anti-citrullinated peptide antibodies (ACPA) are highly specific to RA and assayed with the commercial CCP2 assay. Genetic drivers of RA within the MHC are different for CCP2-positive and -negative subsets of RA, particularly at HLA-DRB1. However, aspartic acid at amino acid position 9 in HLA-B (B pos-9 ) increases risk to both RA subsets. Here we explore how individual serologies associated with RA drive associations within the MHC. To define MHC differences for specific ACPA serologies, we quantified a total of 19 separate ACPAs in RA-affected case subjects from four cohorts (n = 6,805). We found a cluster of tightly co-occurring antibodies (canonical serologies, containing CCP2), along with several independently expressed antibodies (non-canonical serologies). After imputing HLA variants into 6,805 case subjects and 13,467 control subjects, we tested associations between the HLA region and RA subgroups based on the presence of canonical and/or non-canonical serologies. We examined CCP2(+) and CCP2(-) RA-affected case subjects separately. In CCP2(-) RA, we observed that the association between CCP2(-) RA and B pos-9 was derived from individuals who were positive for non-canonical serologies (omnibus&#95;p = 9.2 × 10 -17 ). Similarly, we observed in CCP2(+) RA that associations between subsets of CCP2(+) RA and B pos-9 were negatively correlated with the number of positive canonical serologies (p = 0.0096). These findings suggest unique genetic characteristics underlying fine-specific ACPAs, suggesting that RA may be further subdivided beyond simply seropositive and seronegative., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Anticitrullinated peptide antibody (ACPA) responses for 22 citrullinated peptides in patients with early rheumatoid arthritis (RA) were analysed and related to radiological and clinical outcome during the first 2 years in a prospective inception cohort., Methods: The ACPA reactivities were assessed in 1022 patients with early RA (symptoms <12 months) using the custom-made microarray chip (Thermo Fisher Scientific, Uppsala, Sweden) in a prospective longitudinal study of observational assessments of Disease Activity Score (DAS28 and its components) and radiology during the first 24 months, accounting for the treatment., Results: Frequency of ACPA reactivities varied between 13.3% and 63.1%. Of the anticyclic citrullinated peptide-2 (anti-CCP2) antibody-negative patients, ACPA reactivities were positive in 32.6%. Smoking, human leucocyte antigen-shared epitope (HLA-SE), anti-CCP2/rheumatoid factor, protein tyrosine phosphatase non-receptor type 22 (1858C/T) and DAS28 were significantly associated with number of ACPA reactivities. The ACPA reactivities modified differently the development of DAS28 over 24 months (identified using trajectories). Anti-Filaggrin307-324, anti-hnRNP (Peptide)-Z1 and anti-F4-CIT-R antibodies anticipated lower DAS28 values (p<0.01-0.05), while positivity for anti-Fibrinogen(Fib)β62-78(74), and anti-Fibα563-583 predicted higher DAS28 (p<0.01 both). Interaction between anti-Fibß36-52, anti-Pept-5 and anti-Bla-26 antibodies, respectively, and DAS28 during 24 months decreased significantly the DAS28 values (p<0.01-0.05). Corticosteroids and biologicals were related to DAS28-area under the curve and Larsen score 24 months. Anti-vimentin2-17 antibodies remained significantly associated with Larsen score at baseline and 24 months, respectively, and radiological progression, besides biologicals at 24 months adjusted for sex and age., Conclusions: Several ACPA reactivities modified significantly the DAS28 development during the first 24 months and were significantly associated with Larsen score at baseline, 24 months and radiological progression., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Smoking is associated with an increased risk of rheumatoid arthritis (RA) in subsets of patients defined according to the presence or absence of anti-citrullinated protein antibodies (ACPAs) and rheumatoid factors (RFs). Moreover, an interaction between smoking and the HLA-DRB1 shared epitope (SE) has been demonstrated to be a risk factor for seropositive RA. The aim of this study was to investigate the interplay between smoking and the HLA-DRB1 SE with regard to risk of RA in different patient subsets based on ACPA and RF status., Methods: Incident cases of RA (3,645 cases, 5,883 matched controls) were divided into 4 subgroups based on the presence or absence of RF and anti-cyclic citrullinated peptide 2 (anti-CCP2) antibodies. The influence of smoking on the risk of disease was determined in each RA subgroup, using logistic regression models with calculation of odds ratios and 95% confidence intervals (95% CIs). The potential interaction between smoking and HLA-DRB1 SE genes was evaluated by calculating the attributable proportion due to interaction (AP)., Results: In the RF+/anti-CCP2+ subset of RA patients, both smoking and the presence of the HLA-DRB1 SE conferred independent disease risks, and there was a strong interaction between the 2 risk factors (AP 0.4, 95% CI 0.3, 0.5). In the RF-/anti-CCP2+ patient subset, the HLA-DRB1 SE conferred an increased risk of RA, whereas the independent influence of smoking was limited. However, there was a significant interaction between the HLA-DRB1 SE and smoking (AP 0.2, 95% CI 0.02, 0.5). In the RF+/anti-CCP2- patient subset, there was an increased risk of disease among smokers, which was only marginally affected by the presence of the HLA-DRB1 SE, and no interaction between the 2 factors was observed (AP 0.002, 95% CI -0.3, 0.3). In the RF-/anti-CCP2- patient subset, neither smoking nor the presence of the HLA-DRB1 SE conferred an increased risk of RA., Conclusion: These findings demonstrate different effects of smoking and HLA-DRB1 in the 4 serologically defined RA subsets., (© 2019 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.)