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Background: Trigger finger, or stenosing tendovaginitis, is one of the most common causes of hand disability, where a finger or thumb painfully snaps and locks due to a tendon-sheath size mismatch at the A1 pulley. The exact aetiology of trigger finger is unknown, though it is associated with factors like diabetes, rheumatic disease and carpal tunnel syndrome. The main purpose of this prospective study was to explore clinical characteristics and comorbidities in a cohort of 139 patients who underwent surgery for trigger finger and find factors of importance for the outcome 1 year postoperatively. Methods: Pain, range of motion, hand function evaluated by the Disabilities of the Arm Shoulder and Hand questionnaire as well as Quinnell grade of triggering were examined preoperatively. Symptom duration, working status, medical history and comorbidities at baseline were also noted. Further, range of motion was evaluated 3 months after surgery, pain and hand function were evaluated 3 and 12 months after surgery. An outcome scale with three levels was defined. The development of any new comorbidities was monitored during an extended postoperative observation period, with a mean duration of 70 months (range: 56–88 months). Results: Poor outcome was strongly associated with younger age (P = 0.0009), a high level of preoperative pain in the operated hand (P = 0.0027), psoriatic arthritis (P = 0.021) and atopic disease (P = 0.028; odds ratio [OR]: 3.87, 95% confidence interval [CI]: 1.15–13.04). A low range of motion preoperatively did not affect the outcome. Carpal tunnel syndrome was the most common comorbidity but did not affect the outcome. A good preoperative range of motion, good hand function and less pain were associated with better outcomes. Conclusion: Younger age, a high level of preoperative pain, psoriatic arthritis and atopic disease were factors associated with a worse outcome of trigger finger surgery. Pain and disability decreased 3 months postoperatively and continued to decrease between 3 and 12 months.

BackgroundAnti-nuclear antibodies (ANA) assessed by immunofluorescence (IF) microscopy are associated with systemic autoimmune rheumatic diseases (SARD) and can be detected years before onset of clinical symptoms. Recent data indicate dysregulation of the immune system with increased levels of proinflammatory cytokines, including type I interferons (IFN), in ANA-positive versus ANA-negative individuals. Herein, the aims were to investigate IF-ANA, ANA fine specificities, and IFN-α protein levels in relation to self-reported symptoms, as well as clinical signs, of SARD in a large group of healthy blood donors (HBD).MethodsSera from 825 HBD (48.8% females) were included. IF-ANA was assessed, using HEp-2 cells, according to the routine at the accredited laboratory of Clinical Immunology, Linköping University Hospital. All samples were analyzed for IgG-ANA fine specificities using addressable laser bead assay (ALBIA) at the same laboratory. IFN-α was determined using ELISA. Antibody-positive individuals, and their sex- and age-matched antibody-negative controls, were asked to fill a questionnaire regarding symptoms associated with SARD.ResultsIn total, 130 HBD (15.8%) were positive with IF-ANA and/or ALBIA. Anti-U1RNP was significantly more common among women. Generally, self-reported symptoms correlated poorly with IF-ANA and/or ALBIA results. Two females with high levels of Ro60/SSA, Ro52/SSA and IFN-α reported mild sicca symptoms and were diagnosed with Sjögren’s disease after clinical evaluation.ConclusionA considerable proportion of apparently HBD are autoantibody positive, but without clear association to self-reported symptoms. Nevertheless, the combination of autoantibodies, relevant symptoms and high IFN-α levels identified the small proportion of individuals with SARD in the study population.

Abstract 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

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.

Immunological mechanisms, both alone and in combination, are associated with a broad range of psychiatric disorders encompassing autoimmune, autoinflammatory disorders but also genetic, metabolic, or other immunological disorders. Early treatment improves the outcome for autoimmune disorders, but early diagnosis is more difficult when isolated psychiatric symptoms are manifestations of autoimmunity. Treatment of these cases must encompass integrated models of disease, as both systemic autoimmunity and psychological processes influence mental health. Several challenges need to be overcome to efficiently merge psychiatric and somatic disease paradigms and medical care ranging from language and conceptual barriers to organizational barriers. Since 2015, the Immunopsychiatry team at Uppsala University has developed a collaborative multidisciplinary approach to improve and integrate care for patients with moderate to severe psychiatric disorders. Based on this experience, we have outlined the obstacles to be overcome in taking steps forward to achieve the long-term goal of understanding and early detection and identification of treatable immunological conditions within the psychiatric patient population; the described framework of evaluations and work-flow may serve as a model for other centers.

Abstract 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

Abstract Patients with established rheumatoid arthritis (RA) and disease modifying treatments have lower nitric oxide (NO) levels in the alveolar compartment (CANO) and in the airway wall (CawNO), but also higher diffusion capacities for NO in the airways (DawNO) 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 (FENO50) 16 (10–24) ppb compared to controls 21 (15, 29) ppb, p = 0.013. In NO-dynamics, CANO 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. CawNO was also lower in the RA patients 55 (24, 106) ppb compared to control subjects 124 (110, 170) ppb, p

Abstract 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 (CNDL-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 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. Conclusion CNDL-index defines people at risk to develop RA and the “window of treatment success” thereby closing the sensitivity gap in RA.

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

Abstract 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.

Abstract Background A myositis-specific autoantibody can now be identified in the majority of patients with myositis. They identify homogeneous patient subgroups and are key tools in developing a personalized approach to disease management. There is substantial clinical interest in exploiting myositis autoantibodies as biomarkers, and consequently, a large number of commercial assays have been developed for their detection. These assays are already in widespread clinical use. In order to better understand perceived concerns from the international myositis community in relation to the reliability of these assays and how they are being used, we conducted a survey of international myositis experts, all of whom were members of the International Myositis Assessment and Clinical Studies group. Results We collected data on the types of assay used, manufacturers, and the nature of the report provided by different laboratories and received 111 complete responses. Respondents also provided information on how they used the different assays, their confidence in the results, and how this influenced their clinical practice. Enzyme immunoassay/ELISA was the most popular assay method used worldwide followed by line blot. Line blot was the most popular method used in Europe. Despite concerns from over 80% of respondents regarding false-positive and false-negative results with the assay used by their laboratory, over 80% reported that the identification of a myositis autoantibody influenced their diagnostic confidence, the information they provided to a patient, and their recommended treatment. Conclusions In spite of ongoing concerns from the majority of users regarding the reliability of the results, myositis-specific autoantibody testing, using commercial immunoassays, is being used globally to inform clinical decision-making. These findings highlight the need for urgent guidance on the use of myositis autoantibody testing and on the interpretation of results. Knowledge of the reliability of currently available assays is essential given the importance already placed on myositis-specific autoantibodies as clinical decision-making tools.

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.

Abstract 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.

Abstract Background Previous studies and own clinical observations of patients with systemic lupus erythematosus (SLE) suggest that SLE harbors distinct immunophenotypes. This heterogeneity might result in differences in response to treatment in different subgroups and obstruct clinical trials. Our aim was to understand how SLE subgroups may differ regarding underlying pathophysiology and characteristic biomarkers. Methods In a cross-sectional study, including 378 well-characterized SLE patients and 316 individually matched population controls, we defined subgroups based on the patients’ autoantibody profile at inclusion. We selected a core of an antiphospholipid syndrome-like SLE (aPL+ group; positive in the lupus anticoagulant (LA) test and negative for all three of SSA (Ro52 and Ro60) and SSB antibodies) and a Sjögren’s syndrome-like SLE (SSA/SSB+ group; positive for all three of SSA (Ro52 and Ro60) and SSB antibodies but negative in the LA test). We applied affinity-based proteomics, targeting 281 proteins, together with well-established clinical biomarkers and complementary immunoassays to explore the difference between the two predefined SLE subgroups. Results The aPL+ group comprised 66 and the SSA/SSB+ group 63 patients. The protein with the highest prediction power (receiver operating characteristic (ROC) area under the curve = 0.89) for separating the aPL+ and SSA/SSB+ SLE subgroups was integrin beta-1 (ITGB1), with higher levels present in the SSA/SSB+ subgroup. Proteins with the lowest p values comparing the two SLE subgroups were ITGB1, SLC13A3, and CERS5. These three proteins, rheumatoid factor, and immunoglobulin G (IgG) were all increased in the SSA/SSB+ subgroup. This subgroup was also characterized by a possible activation of the interferon system as measured by high KRT7, TYK2, and ETV7 in plasma. In the aPL+ subgroup, complement activation was more pronounced together with several biomarkers associated with systemic inflammation (fibrinogen, α-1 antitrypsin, neutrophils, and triglycerides). Conclusions Our observations indicate underlying pathogenic differences between the SSA/SSB+ and the aPL+ SLE subgroups, suggesting that the SSA/SSB+ subgroup may benefit from IFN-blocking therapies while the aPL+ subgroup is more likely to have an effect from drugs targeting the complement system. Stratifying SLE patients based on an autoantibody profile could be a way forward to understand underlying pathophysiology and to improve selection of patients for clinical trials of targeted treatments.

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.

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.

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.

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.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10−9, 3 × 10−6, and 5 × 10−6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal ACPA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NET reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.

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.

Antibodies to citrullinated proteins, common in rheumatoid arthritis (RA) patients, are strongly associated to a specific set of HLA-DR alleles including HLA-DRB1*04:01, *04:04, and *01:01. Here, we first demonstrate that autoantibody levels toward the dominant citrullinated B cell epitope from α-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified α-enolase-derived T cell epitopes and demonstrated that native and citrullinated versions of several peptides bind with different affinities to HLA-DRB1*04:01, *04:04, and *01:01. The citrulline residues in the eight identified peptides are distributed throughout the entire length of the presented epitopes and more specifically, localized at peptide positions p-2, p2, p4, p6, p7, p10, and p11. Importantly, in contrast to its native version peptide 26 (TSKGLFRAAVPSGAS), the HLA-DRB1*04:01-restricted citrullinated peptide Cit26 (TSKGLFCitAAVPSGAS) elicited significant functional T cell responses in primary cells from RA patients. Comparative analysis of the crystal structures of HLA-DRB1*04:01 in complex with peptide 26 or Cit26 demonstrated that the posttranslational modification did not alter the conformation of the peptide. And since citrullination is the only structural difference between the two complexes, this indicates that the neo-antigen Cit26 is recognized by T cells with high specificity to the citrulline residue.