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BackgroundThere 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.MethodsUsing 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. ResultshnRNP-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-CCP2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. This CNDL (Citrullinated-Native-hnRNP-DL)-index identified and the bioinformatic value was explored, as a new value for an “individual window of treatment success” in early RA and for the detection of RF-IgM/α-CCP2 seronegative RA patients (24-46%). Negative CNDL-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 CNDL-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.ConclusionCNDL-index defines patients is a possible biomarker for develop RA and the “window of treatment success” thereby potentially closing the sensitivity gap.

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.

Supplemental material, sj-pdf-1-lup-10.1177_09612033211014570 for Associations with thrombosis are stronger for antiphosphatidylserine/prothrombin antibodies than for the Sydney criteria antiphospholipid antibody tests in SLE by Sahwa Elbagir, Giorgia Grosso, NasrEldeen A Mohammed, Amir I Elshafie, Elnour M Elagib, Agneta Zickert, Vivek Anand Manivel, Eleftheria Pertsinidou, Musa A. M Nur, Iva Gunnarsson, Johan Rönnelid and Elisabet Svenungsson in Lupus

Supplemental material, sj-pdf-2-lup-10.1177_09612033211014570 for Associations with thrombosis are stronger for antiphosphatidylserine/prothrombin antibodies than for the Sydney criteria antiphospholipid antibody tests in SLE by Sahwa Elbagir, Giorgia Grosso, NasrEldeen A Mohammed, Amir I Elshafie, Elnour M Elagib, Agneta Zickert, Vivek Anand Manivel, Eleftheria Pertsinidou, Musa A. M Nur, Iva Gunnarsson, Johan Rönnelid and Elisabet Svenungsson in Lupus

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.

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)-a2 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-a2, 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. Funding agencies: The Swedish Research Council; Swedish RheumatismAssociation (R-859621; R-862161; R-844801); Professor Nanna Svartz Foundation (2018-00250); Ulla and Roland Gustafsson Foundation (2019-12); King Gustaf V’s 80-year Foundation; Swedish Society of Medicine; Ingegerd Johansson Donation; King Gustaf V and Queen Victoria’s Foundation of Freemasons; Region Östergötland (ALF grants); Region Stockholm; Region Uppsala; Region Skåne; Faculty of Medicine, Lund University; AlfredÖsterlund’s Foundation; Anna-Greta Crafoord Foundation; Greta and Johan Kock’s Foundation; Skåne UniversityHospital; and the Karolinska Institutet Foundations.

Supplemental material, sj-pdf-1-lup-10.1177_0961203320945387 for High IgA antiphospholipid autoantibodies in healthy Sudanese explain the increased prevalence among Sudanese compared to Swedish systemic lupus erythematosus patients by Sahwa Elbagir, Amir I Elshafie, Elnour M Elagib, NasrEldeen A Mohammed, Mawahib IE Aledrissy, Vivek Anand Manivel, Eleftheria Pertsinidou, Musa AM Nur, Iva Gunnarsson, Elisabet Svenungsson and Johan Rönnelid in Lupus

Additional file 3: Supplementary Table 3. Baseline characteristics in CCP2-positive and -negative RA. The table show baseline characteristics for the EIRA patients included in the study, based on anti-CCP2 IgG status; p-values indicate differences between subsets with respect to: age, female-to-male ratio, number of smokers, HLA-DRB1 SE-positivity, PTPN22-positivity, DAS28 and CRP.

Additional file 7: Supplementary Table 6. Associations between different ACPA fine-specificities and HLA-DRB1 SE, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between HLA-DRB1 SE and presence/absence of 19 different ACPA fine-specificities, in anti-CCP2-positive and -negative RA: p-values indicate differences in ORs between ACPA fine-specificity-positive and -negative subsets in the anti-CCP2-positive subset.

Additional file 6: Supplementary Table 5. Associations between different autoantibodies and PTPN22 polymorphism, in anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between PTPN22 polymorphism and presence/absence of ACPA, RF, other autoantibodies, or "any autoantibody" in anti-CCP2-negative RA.

Additional file 4: Supplementary Figure 1. ACPA fine-specificity levels in anti-CCP2-negative RA and controls. ACPA levels for 19 individual ACPA fine-specificities are shown in separate graphs with AU values on the Y-axes. Only ACPA levels above cutoff for positivity are shown.

Additional file 1.

Additional file 8: Supplementary Table 7. Associations between RF isotypes and smoking, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between smoking and presence/absence of IgM, IgG or IgA RF, in anti-CCP2-positive and -negative RA; p-values indicate differences in ORs between RF isotype-positive and -negative subsets.