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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.

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. 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. 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 pvalues 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). 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.

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&lt; 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.

Autoimmune polyendocrine syndrome type 1 (APS1) is a monogenic disorder that features autoimmune Addison disease as a major component. Although APS1 accounts for only a small fraction of all patients with Addison disease, early identification of these individuals is vital to prevent the potentially lethal complications of APS1.

Studies of the clinical and immunological features of autoimmune Addison disease (AAD) are needed to understand the disease burden and increased mortality.

The presence of rheumatoid factor (RF), anti-carbamylated protein antibodies (anti-CarP) and antibodies against citrullinated protein and peptides (ACPA) precedes the onset of symptoms of rheumatoid arthritis (RA) by several years. Relationships between the development of these antibodies are not obvious. Three isotypes [immunoglobulin A (IgA), IgG and IgM) of RF were analysed in 321 pre-symptomatic individuals who provided 598 samples collected a median of 6.2 (interquartile range 7.2) years before the onset of symptoms, and in 492 population control subjects. All samples were donated to the Biobank of Northern Sweden. RF isotypes were analysed using the EliA system (Phadia GmbH, Freiburg, Germany) with 96 % specificity according to receiver operating characteristic curves. Ten ACPA specificities were analysed using the ImmunoCAP ISAC system, and anti-CCP2 and anti-CarP antibodies were evaluated using enzyme-linked immunosorbent assays. The frequencies of RF isotypes in pre-symptomatic individuals were significantly increased compared with control subjects (p< 0.0001). In samples collected ≥15 years before the onset of symptoms, the IgA-RF isotype was significantly more prevalent than the most frequent ACPAs. Combinations of IgM- and IgA-RF isotypes with ACPA specificities [α-enolase (CEP-1/Eno5–21)], fibrinogen (Fib)β36–52, Fibα580–600, filaggrin (CCP-1/Fil307–324) and anti-CCP2 antibodies were associated with a significantly shorter time to onset of symptoms (p< 0.001–0.05). Using conditional inference tree analysis, anti-CCP2 in combination with anti-filaggrin antibodies gave the highest probability, 97.5 %, for disease development. RF isotypes predicted the development of RA, particularly in combination with ACPA, anti-CCP2 or anti-CarP antibodies. The highest probability for disease development was the presence of anti-CCP2 and anti-filaggrin antibodies.

Antibodies against citrullinated proteins (ACPA) are common in patients with rheumatoid arthritis (RA). ACPA can appear before disease onset and target many self-antigens. Citrullinated fibrin/fibrinogen represents a classical ACPA target antigen, and mass spectrometry of RA synovial fluid reveals elevated citrullinated (cit) fibrinogen (Fib) peptides compared to non-RA controls. We investigated the extent to which these less-studied peptides represent autoantibody targets and sought to visualize the corresponding cit-Fib-reactive B cells in RA patients. An in-house ELISA was established against four cit-Fib α-subunit peptides (cit-Fib α-35; cit-Fib α-216,218; cit-Fib α-263,271 and cit-Fib α-425,426) and serum from patients with established RA (n= 347) and disease controls with psoriatic arthritis (PsA) or ankylosing spondylitis (AS) (n= 236) were analyzed. RA patients were genotyped for HLA-DR alleles, PTPN22 R620W and screened for anti-CCP2 and cit-Fib protein antibodies. The cit-Fib peptides were also used to assemble antigen tetramers to identify cit-Fib-reactive B cells in peripheral blood by flow cytometry. The frequencies of autoantibodies against different cit-Fib epitopes in RA patients compared to PsA/AS patients were: cit-Fib α-35 (RA 20%, vs PsA/AS 1%); cit-Fib α-216,218 (13% vs 0.5%); cit-Fib α-263,271 (21% vs 0.5%) and cit-Fib α-425,426 (17% vs 1%). The presence of autoantibodies against these peptides was associated with presence of anti-CCP2 and anti-cit-Fib protein antibodies. No association was found between HLA-DR shared epitope and antibodies to the different cit-Fib peptides. However, association was observed between the PTPN22 risk allele and positivity to cit-Fib α-35 and cit-Fib α-263,271. B cells carrying surface Ig reactive to these cit-Fib peptides were found in RA peripheral blood and these tend to be more common in PTPN22 risk allele carriers. Our data show that several cit-Fib peptides are targeted by autoantibodies in RA, but not in PsA/AS, implicating that these are not due to arthritis but more specific for RA etiology. The RA-associated anti-cit protein response is broad with many parallel immune responses. The association between cit-Fib autoantibodies and the PTPN22 R620W risk allele supports the hypothesis of altered B cell regulation, such as autoreactive B cells evading tolerance checkpoints.

In addition to anti-citrullinated protein antibodies (ACPAs), antibodies targeting carbamylated (i.e., homocitrullinated) proteins (anti-CarP antibodies) have been described in rheumatoid arthritis (RA). However, the extent to which anti-CarP antibodies are truly distinct from ACPA remains unclear, and few studies have focused on specific autoantigens. Here, we examine cross-reactivity between ACPA and anti-CarP antibodies, in the context of the candidate autoantigen α-enolase. Cross-reactivity was examined by immunoblotting of citrullinated and carbamylated proteins using purified ACPA; and by peptide absorption experiments, using the citrullinated α-enolase peptide CEP-1 and a homocitrulline-containing version (carb-CEP-1) in ELISA. The population-based case-control cohort EIRA (n = 2836 RA; 373 controls) was screened for reactivity with CEP-1 and carb-CEP-1, using the ISAC multiplex array. Associations between anti-CarP antibodies, smoking and genetic risk factors were analysed using unconditional logistic regression models. Differences in antibody levels were investigated using the Mann-Whitney Utest. Affinity-purified ACPA was found to bind carbamylated proteins and homocitrulline-containing peptides, demonstrating definitive cross-reactivity between ACPA and anti-CarP antibodies. Anti-carb-CEP-1 reactivity in EIRA was almost exclusively confined to the CEP-1-positive subset, and this group of RA patients (21 %) displayed a particularly strong ACPA response with marked epitope spreading. The small RA subset (3 %) with homocitrulline reactivity in the absence of citrulline reactivity did not associate with smoking or risk genes, and importantly had significantly lower anti-carb-CEP-1 antibody levels. Our data presented herein cast doubt on the specificity of anti-CarP antibodies in RA, which we posit may be a subset of cross-reactive ACPA.

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&lt; 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.

The presence of a new autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, has been identified in rheumatoid arthritis (RA). The presence of anti-CarP antibodies was evaluated in samples taken from individuals who subsequently developed RA before and after onset of symptoms and related to previously analysed antibodies against citrullinated peptides (ACPA specificities) and anti-CCP2. A total of 252 individuals, with 423 samples from before onset of symptoms of RA, and 197 population controls were identified as donors to the Medical Biobank of Northern Sweden; 192 of them were also sampled at the time of diagnosis. All samples were analysed for anti-CarP IgG and anti-CCP2 antibodies using ELISAs. Ten different antibody reactivities against citrullinated antigens (ACPA specificities) were analysed using a custom-made microarray based on the ImmunoCAP ISAC system (Phadia). The concentration of anti-CarP antibodies was significantly increased in the pre-symptomatic individuals compared with controls (P<0.001) and also increased significantly after disease onset (P<0.001). The sensitivity for anti-CarP antibodies in the pre-symptomatic individuals was 13.9% (95% CI: 11 to 17.6) and 42.2% (95% CI: 35.4 to 49.3) following development of RA. Anti-CarP antibody positivity was found in 5.1% to 13.3% of individuals negative for anti-CCP2 or ACPA specificities. Presence of anti-CarP antibodies was significantly related to radiological destruction at baseline, at 24 months and also to radiological change (P<0.05, all). The results indicate that anti-CarP antibodies are associated with disease development, even after adjusting for the presence of different ACPA fine specificities, and in anti-CCP2 negative individuals and contribute to the identification of a subset of patients with worse radiological progression of the disease independent of ACPA.

Rheumatoid arthritis (RA) patients with autoantibodies against collagen type II (CII) are characterized by acute RA onset with elevated inflammatory measures and early joint erosions as well as increased production of tumor necrosis factor-α (ΤΝF-α) by peripheral blood mononuclear cells (PBMC) stimulated by anti-CII immune complexes (IC) in vitro. Polymorphonuclear granulocytes (PMN) are abundant in RA synovial fluids, where they might interact directly with anti-CII IC in the articular cartilage, but no studies have investigated PMN responses towards anti-CII IC. The aim was to investigate whether PMN react towards anti-CII IC, and to what extent such reactivity might relate to the clinical acute onset RA phenotype associated with elevated levels of anti-CII. PMN and PBMC isolated from healthy donors were stimulated with IC made with a set of 72 baseline patient sera (24 anti-CII positive, 48 anti-CII negative) chosen from a clinically well-characterized RA cohort with two-year radiological follow-up with Larsen scoring. PMN expression of cluster of differentiation (CD)11b, CD66b, CD16 and CD32 was measured by flow cytometry, whereas PMN production of myeloperoxidase (MPO) and interleukin (IL)-17, and PBMC production of ΤΝF-α was measured with enzyme linked immunosorbent assay. PMN expression of CD11b, CD66b and MPO, and PBMC production of ΤΝF-α were upregulated whereas PMN expression of CD16 and CD32 were downregulated by anti-CII IC. CD16, CD66b, and MPO production correlated to serum anti-CII levels (Spearman’s ρ = 0.315, 0.675 and 0.253, respectively). CD16 was associated with early joint erosions (P= 0.024, 0.034, 0.046 at baseline, one and two years) and CD66b was associated with changes in joint erosions (P= 0.017 and 0.016, at one and two years compared to baseline, respectively). CD66b was associated with baseline C-reactive protein and PBMC production of ΤΝF-α was associated with baseline erythrocyte sedimentation rate, in accordance with our earlier findings. No clinical associations were observed for MPO or IL-17. PMN responses against anti-CII IC are more closely associated with early joint erosions than are PBMC cytokine responses. PMN reactivity against anti-CII IC may contribute to joint destruction in newly diagnosed RA patients with high levels of anti-CII.

We have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA). The autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC® system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP)2 using the CCPlus® ELISA kit. Two peptides derived from the fibrinogen α chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen β chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65 %, 15 %, 35 %, and 53 % of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5 % (Cit573), 6 % (Cit591), 8 % (Cit72), and 4 % (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84 % and 63 % respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency. Here we demonstrate extensive autoantibody reactivity against in vivocitrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.

It has previously been shown that an increased number of antibodies against citrullinated peptides/proteins (ACPA) predate the onset of rheumatoid arthritis (RA). Over time antibody positivity expands, involving more specific responses when approaching the onset of symptoms. We investigated the impact of human leukocyte antigen-shared epitope (HLA-SE) alleles and smoking on the development of ACPA, as well as in combination with ACPA during the state of quiescent autoimmunity (before the onset of symptoms), on the development of RA. Blood samples donated to the Medical Biobank of Northern Sweden from individuals prior to the onset of symptoms of RA (n = 370) and after onset (n = 203) and from population-based controls (n = 585) were used. Antibodies against 10 citrullinated peptides, fibrinogen (Fibα561-583, α580-600, ß62-81a, ß62-81b, ß36-52), vimentin (Vim2-17, 60-75), filaggrin (CCP-1/Fil307-324), α-enolase (CEP-1/Eno5-21), collagen type II (citC1359-369), and anti-cyclic citrullinated peptide (CCP)2 antibodies were analysed. HLA-SE-positive individuals were more frequently positive for ACPA compared with HLA-SE-negative individuals prior to the onset of symptoms of RA, particularly for antibodies against CEP-1 and Fibß62-81a (72). Smoking was associated with antibodies against Vim2-17 and citC1359-369. HLA-SE and smoking showed increasing association to the presence of the antibodies closer to disease onset. The highest odds ratio (OR) for development of RA was for the combination of HLA-SE alleles and ACPA positivity, especially for antibodies against Fibß62-81b, CCP-1/Fil307-324, and Fibβ36-52. A gene-environment additive interaction between smoking and HLA-SE alleles for the risk of disease development was found, with the highest OR for individuals positive for antibodies against Fibβ36-52, CEP-1, and Fibα580-600. The relationships between antibodies against the different ACPA specificities, HLA-SE, and smoking showed a variable pattern in individuals prior to the onset of RA. The combination of smoking and HLA-SE alleles was significantly associated with the development of some of the antibody specificities closer to onset of symptoms, and these associations remained significant at diagnosis. An additive gene-environment interaction was found for several of the antibodies for the development of RA.

Objective.Analysis of antibodies against dsDNA is an important diagnostic tool for systemic lupus erythematosus (SLE), and changes in anti-dsDNA antibody levels are also used to assess disease activity. Herein, 4 assays were compared with regard to SLE specificity, sensitivity, and association with disease activity variables.Methods.Cross-sectional sera from 178 patients with SLE, of which 11 were followed consecutively, from a regional Swedish SLE register were analyzed for immunoglobulin G (IgG) anti-dsDNA by bead-based multiplex assay (FIDIS; Theradig), fluoroenzyme-immunoassay (EliA; Phadia/Thermo Fisher Scientific), Crithidia luciliaeimmunofluorescence test (CLIFT; ImmunoConcepts), and line blot (EUROLINE; Euroimmun). All patients with SLE fulfilled the 1982 American College of Rheumatology and/or the 2012 Systemic Lupus International Collaborating Clinics (SLICC-12) classification criteria. Healthy individuals (n = 100), patients with rheumatoid arthritis (n = 95), and patients with primary Sjögren syndrome (n = 54) served as controls.Results.CLIFT had the highest SLE specificity (98%) whereas EliA had the highest sensitivity (35%). When cutoff levels for FIDIS, EliA, and EUROLINE were adjusted according to SLICC-12 (i.e., double the reference limit when using ELISA), the specificity and sensitivity of FIDIS was comparable to CLIFT. FIDIS and CLIFT also showed the highest concordance (84%). FIDIS performed best regarding association with disease activity in cross-sectional and consecutive samples. Fisher’s exact test revealed striking differences between methods regarding associations with certain disease phenotypes.Conclusion.CLIFT remains a good choice for diagnostic purposes, but FIDIS performs equally well when the cutoff is adjusted according to SLICC-12. Based on results from cross-sectional and consecutive analyses, FIDIS can also be recommended to monitor disease activity.

Objective.To investigate biomarker patterns in rheumatoid arthritis (RA) with extraarticular manifestations.Methods.Cartilage oligomeric matrix protein (COMP), COMP-C3b, and soluble terminal complement complexes (sTCC) were measured by ELISA.Results.COMP-C3b levels were higher in patients with RA than in healthy controls and lower in extraarticular RA (ExRA) than in RA controls. In patients with ExRA, sTCC levels were higher than in RA controls, and correlated inversely with serum COMP-C3b levels in the ExRA group.Conclusion.Patients with ExRA had lower levels of COMP-C3b. This may be a consequence of complement consumption or a lower potential for COMP from these patients to activate complement.

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.

The purpose of this review is to describe how the current knowledge of antibodies to citrullinated protein antigens in rheumatoid arthritis and related conditions emerged; to discuss the diagnostic and prognostic value associated with antibodies to citrullinated protein antigens as a biomarker; and most importantly for this review, to discuss the potential pathogenetic significance of these antibodies.

OBJECTIVE: Intramuscular gold is a well documented treatment in rheumatoid arthritis (RA), but its mechanism of action is still poorly understood. From an observation that gold sodium thiomalate (GSTM) induces monocyte-derived interleukin 6 (IL-6) and IL-10 production in vitro, a hypothesis has been proposed that gold exerts its action mainly as a selective immunostimulator rather than as a general immunosuppressant. In this prospective study we investigated cytokine production in peripheral blood from patients with RA during treatment with GSTM. METHODS: A total of 20 patients with RA were treated with GSTM for at least 3 months. Disease activity was recorded at baseline, 12, 20, and 28 weeks. The ELISPOT method was used to measure spontaneous production of IL-6, IL-10, and interferon-g (IFN-g) from peripheral blood mononuclear cells (PBMC) at baseline and 4 and 12 weeks and production after incubation with GSTM in vitro, at different concentrations (0, 3, 12.5, 40 µg/ml) at baseline. IL-6 and IL-10 concentrations in serum were measured with ELISA. RESULTS: The numbers of IL-10-producing cells were increased after 4 weeks' treatment with GSTM (p < 0.01). The numbers of cells spontaneously producing IL-6 were increased after 4 weeks (p < 0.01) and 12 weeks (p < 0.01). The numbers of IFN-g-producing cells were increased after 4 weeks (p < 0.01). Serum concentrations of IL-10 were increased after 4 weeks (p < 0.01). Serum concentrations of IL-6 were not changed at any timepoint. The in vitro effect of GSTM on IL-10 production from PBMC at baseline predicted development of skin reactions during GSTM treatment, with lack of skin reactions being associated with high gold induced IL-10 production (p < 0.05). There was no correlation between clinical response and cytokine production. CONCLUSION: This study indicates an immunostimulatory effect of GSTM treatment in patients with RA. The increase in IL-10 production during GSTM treatment may contribute to the positive effects of gold in RA.

OBJECTIVE: To investigate the effects of gold salt on the differential production of proinflammatory and antiinflammatory cytokines in vitro. METHODS: Heparinized blood from 10 blood donors and 10 patients with polyarthritis was density separated and incubated with various concentrations of gold salt [Myocrisin gold sodium thiomalate (GSTM) plus phenyl mercury nitrate]. Cytokine production was measured after incubation for 16-20 h using an Elispot method detecting interleukin 10 (IL-10), IL-6, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) producing cells. In addition, parallel cell culture supernatants were collected and analyzed by ELISA for IL-10, IL-6, TNF-alpha, IFN-gamma, and IL-2. In some cultures phytohemagglutinin A (PHA) was added in predefined concentrations. RESULTS: GSTM increased the number of cells producing IL-6 and IL-10 in a dose dependent manner, both with and without simultaneous addition of PHA. These effects were seen in samples from both healthy blood donors and patients with polyarthritis. The increase in IL-10 production was inhibited when monocytes were depleted. No effects of GSTM were seen on IFN-gamma or TNF-alpha producing cells. Parallel supernatant cultures displayed a GSTM dose dependent decrease in IFN-gamma levels after mitogen stimulation, whereas no changes were seen in IL-6 or TNF-alpha levels. CONCLUSION: The differential effects of gold salt on cytokine production, with a marked stimulatory effect on IL- 10 and IL-6, indicate that gold salt may act as a relatively selective immunostimulator rather than as a general immunosuppressant.

It is controversial whether mixed connective tissue disease (MCTD) should be regarded as a distinct disease entity. In the present study, we investigated immunological parameters in patients with MCTD by studying serum levels of immunoglobulins, C-reactive protein (CRP) and cytokines and compared the results to the corresponding values in systemic lupus erythematosus (SLE), in rheumatoid arthritis (RA) patients and in healthy controls. Using the ELISA technique, serum levels of the cytokines interferon-γ (IFN-γ), interleukin-10 (IL-10) and tumour necrosis factor-α (TNF-α) were investigated. Cytokine levels in SLE and MCTD were correlated to disease activity as assessed by systemic lupus activity measure (SLAM). They were also correlated to serum levels of CRP, IgG, IgA and IgM in the three patient groups. The MCTD patients had the highest levels of immunoglobulins, followed by the SLE patients. In contrast, the highest CRP levels were observed in RA patients, followed by the MCTD patients. The MCTD patients had the highest serum levels of IL-10, but also had elevated IFN-γ and TNF-α levels similar to the RA patients. There was no correlation between the investigated cytokine levels and disease activity, as assessed by SLAM. We conclude that MCTD patients have high immunoglobulin levels as well as high CRP levels and that this situation is compatible with the observed increase in both type 1 and type 2 cytokine levels. The findings imply that MCTD shares some distinct immunological properties with both RA and SLE and that MCTD may also be considered as a separate disease entity according to these properties.

The simultaneous appearance of autoantibodies with either a functional or structural relationship to anti-C1q antibodies (anti-C1q) was investigated in 39 systemic lupus erythematosus (SLE) patients and in 28 rheumatoid arthritis (RA) patients, in both cross-sectional and longitudinal design. Levels of anti-C1q showed an isotype-specific correlation to levels of immune con-glutinin (IK) in SLE patients, whereas no correlation was evident to levels of antibodies to the structurally related antigen type II collagen (anti-CII) in SLE or RA patients. IgG anti-C1q levels correlated with serum levels of the terminal complement complex (sC5b-9) in SLE patients. In two longi-tudinally followed patients, the IK response preceded the anti-C1q response. Possibilities for regulation of the humoral anti-complement response are discussed.

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.

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.

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.

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.

Autoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA.

We have previously reported that high levels of antibodies specific for native human type II collagen (anti-CII) at the time of RA diagnosis were associated with concurrent but not later signs of inflammation. This was associated with CII/anti-CII immune complex (IC)-induced production of pro-inflammatory cytokines in vitro. In contrast, anti-cyclic citrullinated peptide antibodies (anti-CCP) were associated both with late inflammation and late radiological destruction in the same RA cohort. We therefore hypothesized that anti-CII are also associated with early erosions.

We and others have previously shown that antibodies against cyclic citrullinated proteins (anti-CCP) precede the development of rheumatoid arthritis (RA) and in a more recent study we reported that individuals who subsequently developed RA had increased concentrations of several cytokines and chemokines years before the onset of symptoms of joint disease. Here we aimed to evaluate the prevalence and predictive values of anti-CCP antibodies of IgG, IgM and IgA isotype in individuals who subsequently developed RA and also to relate these to cytokines and chemokines, smoking, genetic factors and radiographic score.

IFN-γ was measured in supernatants after in vitrostimulation of peripheral blood mononuclear cells with collagen type II (CII), purified protein derivative or influenza virus. IFN-γ production in response to CII was similar in rheumatoid arthritis (RA) patients and healthy control individuals. The IFN-γ response to purified protein derivative and influenza virus was lower in RA patients, reflecting a general T-cell hyporesponsiveness in RA. After recalculating the response to CII taking this hyporesponsiveness into account the CII response was higher in RA patients, and was associated with human leucocyte antigen (HLA)-DRB1*0401 and HLA-DQA1*0301-DQB1*0302 (HLA-DQ8). Rheumatoid arthritis patients with elevated serum levels of immunoglobulin (Ig)G anti-CII antibodies had lower CII-induced IFN-γ production than patients with low anti-CII levels. The relative increase in CII-reactivity in RA patients as compared with healthy control individuals, and the association of a higher response with RA-associated HLA haplotypes, suggest the existence of a potentially pathogenic cellular reactivity against CII in RA.