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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.)

Competing Interests: Competing interests: None declared.

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

Introduction: The second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients., Methods: We investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array., Results: The prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset., Conclusions: Multiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement., Competing Interests: Competing interests: The project is part of the Innovative Medicines Initiative (IMI) project Be The Cure where Karolinska Institutet is a scientific partner and Thermo Fisher Scientific is a commercial partner. The project follows the rules for IMI projects. JR has obtained reagents from Thermo Fisher Scientific for the investigation on other rheumatology cohorts. LM-A is employed by Thermo Fisher Scientific. LK and RH were cofounders of a company, Curara AB, which has previously collaborated with Thermo Fisher Scientific concerning certain technical aspects of the multiplex assay. This development is done with partial support from an ERC Proof of Concept Grant (pRActice) to LK. RH is a coinventor of a patent (US Patent 7148020) protecting the use of the CitC1 and C1 peptides. KS is coinventor of the patents US 13/141,960 and EP 09799354.7 describing the diagnostic use of the hnRNP-A3 peptide epitopes. GS is coinventor of several international patents about ACPA antigens held by BioMérieux Cy and licenced to Eurodiagnostica Cy and Axis-Shield Cy for commercialisation of the CCP2 assays; according to French laws, he receives a part of the royalties paid to the Toulouse III University and the University Hospital of Toulouse. KL is coinventor of patent US12/524,465, describing the diagnostic use of the CEP-1 epitope. The other authors declare that they have no competing interests., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objectives: To study the effects of abatacept on disease activity and on muscle biopsy features of adult patients with dermatomyositis (DM) or polymyositis (PM)., Methods: Twenty patients with DM (n=9) or PM (n=11) with refractory disease were enrolled in a randomised treatment delayed-start trial to receive either immediate active treatment with intravenous abatacept or a 3 month delayed-start. The primary endpoint was number of responders, defined by the International Myositis Assessment and Clinical Studies Group definition of improvement (DOI), after 6 months of treatment. Secondary endpoints included number of responders in the early treatment arm compared with the delayed treatment arm at 3 months. Repeated muscle biopsies were investigated for cellular markers and cytokines., Results: 8/19 patients included in the analyses achieved the DOI at 6 months. At 3 months of study, five (50%) patients were responders after active treatment but only one (11%) patient in the delayed treatment arm. Eight adverse events (AEs) were regarded as related to the drug, four mild and four moderate, and three serious AEs, none related to the drug. There was a significant increase in regulatory T cells (Tregs), whereas other markers were unchanged in repeated muscle biopsies., Conclusions: In this pilot study, treatment of patients with DM and PM with abatacept resulted in lower disease activity in nearly half of the patients. In patients with repeat muscle biopsies, an increased frequency of Foxp3 + Tregs suggests a positive effect of treatment in muscle tissue., Competing Interests: Competing interests: IEL: research grant and advisory board consultant for BMS, research grant from AstraZeneca, consultant for MedImmune, aTyr and IDERA. RA: research grant from BMS., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objectives: Immunological events in the lungs might trigger production of anti-citrullinated protein antibodies during early rheumatoid arthritis (RA). We investigated the presence of shared immunological citrullinated targets in joints and lungs of patients with RA., Patients and Methods: Proteins extracted from bronchial (n=6) and synovial (n=7) biopsy specimens from patients with RA were investigated by mass spectrometry-based proteomics. One candidate peptide was synthesised and used to investigate by ELISA the presence of antibodies in patients with RA (n=393), healthy controls (n=152) and disease controls (n=236). HLA-DRB1 shared epitope (SE) alleles were detected in patients with RA., Results: Ten citrullinated peptides belonging to seven proteins were identified, with two peptides shared between the synovial and bronchial biopsy samples. Further analysis, using accurate mass and retention time, enabled detection of eight citrullinated peptides in synovial and seven in bronchial biopsy specimens, with five peptides shared between the synovial and bronchial biopsy specimens. Two citrullinated vimentin (cit-vim) peptides were detected in the majority of synovial and lung tissues. Antibodies to a synthesised cit-vim peptide candidate (covering both cit-vim peptides identified in vivo) were present in 1.8% of healthy controls, 15% of patients with RA, and 3.4% of disease controls. Antibodies to cit-vim peptide were associated with the presence of the SE alleles in RA., Conclusions: Identical citrullinated peptides are present in bronchial and synovial tissues, which may be used as immunological targets for antibodies of patients with RA. The data provide further support for a link between lungs and joints in RA and identify potential targets for immunity that may mediate this link., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: Smoking can induce autoantibodies in persons who are genetically predisposed to rheumatoid arthritis. We investigated the association between smoking and antiphospholipid antibodies (aPL) in systemic lupus erythematosus (SLE), a question not previously addressed. Further, we explored the relationship between smoking, aPL and vascular events (arterial and venous, VE)., Methods: In this cross-sectional study, clinical evaluation and questionnaire data were collected from 367 prevalent SLE patients. At the same time, we measured aPL (anticardiolipin (aCL), anti-β2 glycoprotein-1 (aβ2GP1) antibodies IgG/IgM/IgA, and lupus anticoagulant (LA)), and a large set of other SLE-associated autoantibodies for comparison. Association analyses using logistic regression models with smoking, (ever, former and current with never as reference) and antibody status as outcome variable were performed. As a secondary outcome, we investigated the associations between aPL, smoking and VE., Results: In multivariable-adjusted models ever, and in particular former, cigarette smoking was associated with the most pathogenic aPL; LA, aCL IgG and aβ2GP1 IgG. Other SLE-associated autoantibodies were not associated with smoking. The combination of smoking and aPL was strongly associated with VE. We noted a positive interaction between smoking-LA and smoking-'triple aPL' positivity for previous VE., Conclusions: We investigated a large set of commonly occurring autoantibodies in SLE, but only aPL were positively associated with a history of smoking. This association was especially apparent in former smokers. Among ever regular smokers who were aPL positive, we observed a strikingly high frequency of former VE. The underlying mechanisms and temporality between smoking, aPL and VE need further investigations., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1-13), anti-double stranded DNA antibodies (14-18), specific antibodies (19-23) and validation of methods (24-25) were created. Significant differences between experts were observed regarding recommendations 24-25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

Background: Rheumatoid factors (RFs) and antibodies against cyclic citrullinated peptides (CCPs) of IgG, IgA and IgM isotype have been shown to precede disease onset by years., Objective: To evaluate serological risk markers in first-degree relatives from multicase families in relation to genetic and environmental risk factors., Methods: 51 multicase families consisting of 163 individuals with rheumatoid arthritis (RA) (mean±SD age, 60±14 years; disease duration 21 years; 71.8% female) and with 157 first-degree relatives unaffected by RA (54±17 years; 59.9% female) were recruited. Isotypes of antibodies against CCPs (IgG, IgA and IgM) and RFs (IgM and IgA) were determined using automated enzyme immunoassays. Cut-off levels were established using receiver operating characteristic curves based on values for 100 unrelated healthy controls., Results: The concentrations and frequencies of all anti-CCP and RF isotypes were significantly increased in first-degree relatives and patients with RA compared with unrelated healthy controls. The relative distribution of IgA and IgM isotypes was higher than IgG in the relatives, whereas the IgG isotype dominated in patients with RA. The patients carried human leucocyte antigen-shared epitope (HLA-SE) significantly more often than the relatives (71.4% vs 53.9%, p=0.01), while the frequency of the PTPN22 T variant was similar. HLA-SE, combined with smoking, was significantly related to all combinations of anti-CCP and RF isotypes in patients with RA. No such relationships were found for the first-degree relatives., Conclusions: All anti-CCP and RF isotypes analysed occurred more commonly in unaffected first-degree relatives from multicase families than in controls, but with different isotype distribution from patients with RA.

Background: Earlier studies have demonstrated that smoking and genetic risk factors interact in providing an increased risk of rheumatoid arthritis (RA). Less is known on how smoking contributes to RA in the context of genetic variability, and what proportion of RA may be caused by smoking., Objectives: To determine the association between the amount of smoking and risk of RA in the context of different HLA-DRB1 shared epitope (SE) alleles, and to estimate proportions of RA cases attributed to smoking., Design: Setting and Participants Data from the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA) case-control study encompassing 1204 cases and 871 controls were analysed. Main Outcome Measure Estimated OR to develop RA and excess fraction of cases attributable to smoking according to the amount of smoking and genotype., Results: Smoking was estimated to be responsible for 35% of anticitrullinated protein/peptide antibody (ACPA)-positive cases. For each HLA-DRB1 SE genotype, smoking was dose-dependently associated with an increased risk of ACPA-positive RA (p trend <0.001). In individuals carrying two copies of the HLA-DRB1 SE, 55% of ACPA-positive RA was attributable to smoking., Conclusions: Smoking is a preventable risk factor for RA. The increased risk due to smoking is dependent on the amount of smoking and genotype.

Background: Rheumatoid arthritis (RA) can be divided into two major subsets based on the presence or absence of antibodies to citrullinated peptide antigens (ACPA). Until now, data from genome-wide association studies (GWAS) have only been published from ACPA-positive subsets of RA or from studies that have not separated the two subsets. The aim of the current study is to provide and compare GWAS data for both subsets., Methods and Results: GWAS using the Illumina 300K chip was performed for 774 ACPA-negative patients with RA, 1147 ACPA-positive patients with RA and 1079 controls from the Swedish population-based case-control study EIRA. Imputation was performed which allowed comparisons using 1,723,056 single nucleotide polymorphisms (SNPs). No SNP achieved genome-wide significance (2.9 × 10⁻⁸) in the comparison between ACPA-negative RA and controls. A case-case association study was then performed between ACPA-negative and ACPA-positive RA groups. The major difference in this analysis was in the HLA region where 768 HLA SNPs passed the threshold for genome-wide significance whereas additional contrasting SNPs did not reach genome-wide significance. However, one SNP close to the RPS12P4 locus in chromosome 2 reached a p value of 2 × 10⁶ and this locus can thus be considered as a tentative candidate locus for ACPA-negative RA., Conclusions: ACPA-positive and ACPA-negative RA display significant risk allele frequency differences which are mainly confined to the HLA region. The data provide further support for distinct genetic aetiologies of RA subsets and emphasise the need to consider them separately in genetic as well as functional studies of this disease.

Objective: To study the association between silica exposure, separately as well as combined with smoking, and the risk of developing rheumatoid arthritis (RA) with or without the presence of antibodies against citrullinated peptide antigens (ACPA)., Methods: This Swedish population based case-control study analysed 577 incident RA cases and 659 randomly selected controls, all men aged 18-70 years, included during May 1996 to May 2006. Self-reported silica exposure, defined as exposure to stone dust, rock drilling or stone crushing and cigarette smoking was registered. ACPA status among cases was analysed., Results: Silica-exposed subjects were found to have a moderately increased risk of ACPA-positive RA (odds ratio (OR) adjusted for age and residency=1.67 (95% CI 1.13 to 2.48), but not of ACPA-negative RA (OR=0.98 (95% CI 0.57 to 1.66)), compared with subjects unexposed to silica., Subjects: exposed to rock drilling were found to have a somewhat more markedly increased risk of ACPA-positive RA (OR=2.34 (95% CI 1.17 to 4.68)). A high risk of developing ACPA-positive RA was observed among silica-exposed current smokers (OR=7.36 (95% CI 3.31 to 16.38)), exceeding the risk expected from the separate effects of silica exposure and current smoking, indicating an interaction between these exposures (attributable proportion due to interaction=0.60 (95% CI 0.26 to 0.95))., Conclusion: Silica exposure combined with smoking among men is associated with an increased risk of developing ACPA-positive RA. These results suggest that different inhalation exposures may interact in the aetiology of ACPA-positive RA.

Objective: To investigate whether the cytokine-inducing properties of surface-bound collagen type II (CII)-containing immune complexes (IC), which were reported earlier, have any clinical impact., Methods: Anti-CII serology was analysed in 274 patients with early rheumatoid arthritis (RA). Patients with increased levels of anti-CII were followed serially for 1-5 years with regard to anti-CII IC-induced levels of tumour necrosis factor (TNF)alpha, interleukin (IL)1beta and IL8. Levels of antibodies and IC-induced cytokines were compared with clinical indices over 5 years of follow-up., Results: 5/100 healthy controls and 24/274 (8.8%) patients with RA exhibited increased levels (>29 arbitrary units (AU)/ml) of anti-native CII antibodies, a non-significant difference. 9/274 (3.3%) patients with RA and no controls comprised a discrete group with high anti-CII levels>450 AU/ml. These high anti-CII level sera were associated with induction of pro-inflammatory cytokines by anti-CII-containing IC formed in vitro. 8/9 patients with high baseline anti-CII levels exhibited a parallel decline in antibody levels, IC-induced cytokines, C reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Anti-CII-positive patients had significantly increased levels of CRP and ESR at baseline, but not later during the follow-up., Conclusions: Anti-native CII-positive patients with RA have a distinct clinical phenotype characterised by an early acute phase response that might be driven by anti-CII-containing IC in joint cartilage.