Geo-epidemiology of autoantibodies in rheumatoid arthritis: comparison between four ethnically diverse populations.

BACKGROUND: Rheumatoid arthritis (RA) occurs across the globe in different ethnic populations. Most RA patients harbor anti-modified protein antibodies (AMPA); however, it is unclear whether differences exist in autoantibody responses at different geographic locations and between different ethnic groups, which could provide new clues regarding factors underlying autoantibody development. We therefore investigated AMPA prevalence and association with HLA DRB1 alleles and smoking in four ethnically diverse populations on four different continents. METHODS: Anti-carbamylated (anti-CarP), anti-malondialdehyde acetaldehyde (anti-MAA), and anti-acetylated protein antibodies (anti-AcVim) IgG were determined in anti-citrullinated protein antibody-positive Dutch (NL, n = 103), Japanese (JP, n = 174), First Nations Peoples in Canada (FN, n = 100), and black South African (SA, n = 67) RA patients. Ethnicity-matched local healthy controls were used to calculate cut-offs. Risk factors associated with AMPA seropositivity in each cohort were identified using logistic regression. RESULTS: Median AMPA levels were higher in First Nations Peoples in Canada and especially South African patients, as reflected by percentage seropositivity: NL, JP, FN, and SA: anti-CarP: 47%, 43%, 58%, and 76% (p < 0.001); anti-MAA: 29%, 22%, 29%, and 53% (p < 0.001); and anti-AcVim: 20%, 17%, 38%, and 28% (p < 0.001). Total IgG levels also differed markedly, and when autoantibody levels were normalized to total IgG, differences between cohorts became less pronounced. Although there were some associations with AMPA and HLA risk alleles and smoking, none was consistent across all four cohorts. CONCLUSIONS: AMPA against various post-translational modifications could consistently be detected on different continents across ethnically diverse RA populations. Differences in AMPA levels corresponded to differences in total serum IgG levels. This suggests that, despite differences in risk factors, a common pathway may be involved in AMPA development across geographic locations and ethnicities.

Antibodies against citrullinated proteins of IgA isotype are associated with progression to rheumatoid arthritis in individuals at-risk.

OBJECTIVE: Events triggering disease outbreak in individuals at-risk for rheumatoid arthritis (RA at-risk) remain unclear, and the role of the various anticitrullinated protein antibody (ACPA) isotypes in this process is still to be established. We aimed to investigate the prevalence of IgA ACPA in RA at-risk individuals, their role in the transition from the RA at-risk status to RA and their dynamics during this transition. METHODS: Cross-sectional measurement of serum IgA1 and IgA2 ACPA levels was conducted in healthy controls, RA at-risk individuals and patients with RA and compared with the frequency of RA development in at risk individuals during a follow-up of 14 months. In addition, longitudinal measurements of serum IgA1 and IgA2 ACPA levels prior to, at and after the onset of RA were performed. RESULTS: Approximately two-thirds of RA at-risk individuals were positive for serum IgA1 and IgA2 ACPA in levels comparable to IgG ACPA positive patients with RA. IgA1, but not IgA2 ACPA positivity was associated with the transition from the RA at-risk state to RA within the following 14 months. Interestingly, during this transition process, IgA1 ACPA levels declined at RA onset and also thereafter during the early phase of RA. This decline was confirmed in a second, independent cohort. CONCLUSION: Both IgA1 and IgA2 ACPA are present in RA at-risk individuals, but only IgA1 ACPA are associated with the progression to RA. The observed decline in serum IgA1 ACPA levels before the onset of RA might indicate starting barrier leakiness prior to disease outbreak.

A new platform for autoimmune diseases. Inducing tolerance with liposomes encapsulating autoantigens.

Autoimmune diseases (AIDs) are caused by the loss of self-tolerance and destruction of tissues by the host's immune system. Several antigen-specific immunotherapies, focused on arresting the autoimmune attack, have been tested in clinical trials with discouraging results. Therefore, there is a need for innovative strategies to restore self-tolerance safely and definitively in AIDs. We previously demonstrated the therapeutic efficacy of phosphatidylserine (PS)-liposomes encapsulating autoantigens in experimental type 1 diabetes and multiple sclerosis. Here, we show that PS-liposomes can be adapted to other autoimmune diseases by simply replacing the encapsulated autoantigen. After administration, they are distributed to target organs, captured by phagocytes and interact with several immune cells, thus exerting a tolerogenic and immunoregulatory effect. Specific PS-liposomes demonstrate great preventive and therapeutic efficacy in rheumatoid arthritis and myasthenia gravis. Thus, this work highlights the therapeutic potential of a platform for several autoimmunity settings, which is specific, safe, and with long-term effects.

The association between autoantibodies and risk for venous thromboembolic events among patients with rheumatoid arthritis.

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

Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a life-threatening autoimmune disease characterized by adaptive immune system activation, formation of double-stranded DNA autoantibodies and organ inflammation. Five patients with SLE (four women and one man) with a median (range) age of 22 (6) years, median (range) disease duration of 4 (8) years and active disease (median (range) SLE disease activity index Systemic Lupus Erythematosus Disease Activity Index: 16 (8)) refractory to several immunosuppressive drug treatments were enrolled in a compassionate-use chimeric antigen receptor (CAR) T cell program. Autologous T cells from patients with SLE were transduced with a lentiviral anti-CD19 CAR vector, expanded and reinfused at a dose of 1 × 106 CAR T cells per kg body weight into the patients after lymphodepletion with fludarabine and cyclophosphamide. CAR T cells expanded in vivo, led to deep depletion of B cells, improvement of clinical symptoms and normalization of laboratory parameters including seroconversion of anti-double-stranded DNA antibodies. Remission of SLE according to DORIS criteria was achieved in all five patients after 3 months and the median (range) Systemic Lupus Erythematosus Disease Activity Index score after 3 months was 0 (2). Drug-free remission was maintained during longer follow-up (median (range) of 8 (12) months after CAR T cell administration) and even after the reappearance of B cells, which was observed after a mean (±s.d.) of 110 ± 32 d after CAR T cell treatment. Reappearing B cells were naïve and showed non-class-switched B cell receptors. CAR T cell treatment was well tolerated with only mild cytokine-release syndrome. These data suggest that CD19 CAR T cell transfer is feasible, tolerable and highly effective in SLE.

Microstructural Bone Changes Are Associated With Broad-Spectrum Autoimmunity and Predict the Onset of Rheumatoid Arthritis.

OBJECTIVE: To assess if microstructural bone lesions in individuals at risk of developing rheumatoid arthritis (RA) are related to the spectrum of anti-modified protein antibodies (AMPAs) and affect the risk of developing RA. METHODS: Cortical microchannels as well as cortical and trabecular bone mineral density (BMD) volumes (expressed as mg hydroxyapatite/cm3 ) were analyzed by high-resolution peripheral quantitative computed tomography of the hand joints of individuals at risk of RA. AMPA response was profiled, including reactivities against citrullinated proteins (vimentin, enolase, and fibrinogen) as well as carbamylated and acetylated vimentin. All subjects were followed up for the development of RA. RESULTS: Subjects at risk of developing RA (n = 75) who had broad-spectrum AMPAs (6-8 reactivities) had significantly more severe microstructural changes, including a higher mean ± SD number of cortical microchannels per joint (95 ± 3) and lower total volumetric BMD (vBMD; 265 ± 45), trabecular vBMD (176 ± 42), and cortical vBMD (585 ± 138), than those with moderate AMPA reactivity (3-5 reactivities) (number of cortical microchannels, 79 ± 30; total vBMD, 293 ± 33; trabecular vBMD, 195 ± 32; and cortical vBMD, 627 ± 91) and those with narrow AMPA reactivity (1-2 reactivities) (number of cortical microchannels, 47 ± 20; total vBMD, 311 ± 34; trabecular vBMD, 211 ± 30; and cortical vBMD, 674 ± 56). Progressors to RA had significantly higher numbers of cortical microchannels (103 ± 30 versus 71 ± 35) and lower bone volume (258 ± 37 versus 295 ± 34) compared to nonprogressors. Furthermore, rate of progression to RA was high in subjects with broad AMPA reactivity (48%) versus those with medium AMPA reactivity (26%) or narrow AMPA reactivity (0%), as well as in those with a high number of cortical microchannels (44%) versus those with a low number of cortical microchannels (10%). CONCLUSION: Microstructural changes in individuals at risk of RA are associated with broad-spectrum autoimmunity and predict the onset of RA. These data support the concept of structural priming of joints by autoimmunity before the onset of the inflammatory phase of the disease.

IgA anti-citrullinated protein antibodies are associated with flares during DMARD tapering in rheumatoid arthritis.

OBJECTIVES: A substantial proportion of RA patients flare upon withdrawal of DMARDs, and thus the definition of prognostic markers is crucial. ACPA positivity has been identified as a risk factor for flare. However, only the role of IgG ACPA is established in this context, while the role of IgA ACPA is poorly defined. We thus aimed to investigate the role of IgA ACPA in flaring of RA. METHODS: Serum levels of IgA1 and IgA2 ACPA at baseline and after 12 months were measured in 108 patients from the randomized controlled RETRO study. RA patients in stable remission for at least 6 months at study recruitment were assigned to either one of the DMARD tapering arms or to continuation of DMARDs. RESULTS: In patients remaining in remission but not in the ones who flared, IgA2 ACPA levels and proportion of IgA2 in ACPA (IgA2% ACPA) significantly declined (median of 17.5%; P < 0.0001). This seemed to be independent of the treatment choice, as there was no difference in IgA2 ACPA dynamics between the study arms. IgA2% ACPA was associated with disease activity (DAS28) at flare (r = 0.36; P = 0.046). IgA and IgG ACPA showed a tendency towards independent contribution to the risk of flare with the highest risk if a patient had both antibody classes. CONCLUSION: In this study, IgA ACPA was identified as a risk factor for flare in combination with IgG ACPA. IgA2 ACPA levels were associated with flare severity and declined in patients in stable remission.

The impact of autoantibodies against citrullinated, carbamylated, and acetylated peptides on radiographic progression in patients with new-onset rheumatoid arthritis: an observational cohort study.

BACKGROUND: A range of anti-modified protein antibodies (AMPAs) are associated with rheumatoid arthritis. We aimed to assess the relationship between AMPA profiles and radiographic progression in patients with new-onset rheumatoid arthritis. METHODS: In this cohort study, we obtained samples and data from the Scottish Early Rheumatoid Arthritis (SERA) inception cohort and biobank, which recruited patients with new-onset rheumatoid arthritis or undifferentiated arthritis who had at least one swollen joint from 20 hospitals across Scotland. AMPAs in plasma samples were measured by ELISAs at baseline. Paired radiographs of the hands and feet were taken at baseline and at 1 year and were scored with the Sharp-van der Heijde (SvH) method. We calculated differences in radiographic progression using estimated marginal mean changes between baseline and 1 year, with the baseline values of radiographic variables, rheumatoid factor, sex, age at recruitment, symptom duration, and Disease Activity Score 28 with C-reactive protein included as covariates. FINDINGS: Between March 1, 2011, and April, 30, 2015, 1073 patients were recruited to the SERA study. 362 patients with rheumatoid arthritis were included in our study and had their AMPA profiles determined. Patients were grouped into four main autoantibody profiles by reactivities to post-translational modifications: single positivity for anti-citrullinated peptide antibodies (ACPAs; 73 [20%]); double positivity for ACPAs and anti-acetylated peptide antibodies (AAPAs; 45 [12%]); triple positivity for ACPAs, AAPAs, and anti-carbamylated peptide antibodies (151 [42%]); and AMPA negativity (74 [20%]). 19 (5%) patients were in one of the minor autoantibody groups. Of the 233 patients with both antibody data and radiographs of sufficient quality, triple-positive patients had more radiographic progression between baseline and 12 months (estimated mean change in total SvH score 1·8, 95% CI 0·9-2·6, SE 0·4) than did single-positive patients (0·5, 0·1-1·0, 0·2; estimated mean difference in the total change in SvH score 1·2, 95% CI 0·1-2·4, SE 0·5). There was no difference in radiographic progression between single positive patients and AMPA negative patients (estimated mean change in total SvH score 0·7, 95% CI 0·1-1·4, SE 0·3; estimated mean difference in the total change in SvH score -0·2, 95% CI -1·1 to 0·7, SE 0·4). INTERPRETATION: This study suggests that the optimal prediction of future rates of radiographic progression in patients with rheumatoid arthritis will require an assessment of autoantibodies against multiple post-translationally modified proteins or peptides. FUNDING: The EU FP7 HEALTH programme, the Scottish Translational Medicine Research Collaboration, and the Chief Scientist Office Scotland.

Presence of anti-acetylated peptide antibodies (AAPA) in inflammatory arthritis and other rheumatic diseases suggests discriminative diagnostic capacity towards early rheumatoid arthritis.

AIMS: To determine the diagnostic value of anti-acetylated peptide antibodies (AAPA) in patients with rheumatoid arthritis (RA). METHODS: Three acetylated peptides (ac-lysine, ac-lysine.inv and ac-ornithine) derived from vimentin were employed to measure AAPA by enzyme-linked immunosorbent assay (ELISA) in sera of 120 patients with early RA (eRA), 195 patients with established RA (est RA), 99 healthy controls (HC), and 216 patients with other inflammatory rheumatic diseases. A carbamylated and a citrullinated version of the vimentin peptide were used additionally. Receiver operating characteristics and logistic regression analyses were used to assess the discriminative capacity of AAPA. RESULTS: AAPA were detected in 60% of eRA and 68.7% of estRA patients, 22.2% of HC, and 7.1- 30.6% of patients with other rheumatic diseases. Importantly, AAPA were also present in 40% of seronegative RA patients, while antibodies to the carbamylated peptide were detected less frequently. Diagnostic sensitivity of individual peptides for eRA was 28.3%, 35.8%, and 34% for ac-lysine, ac-ornithine, and ac-lysine.inv, respectively. Positive likelihood ratios (LR+) for eRA versus HC were 14.0, 7.1, and 2.1. While the presence of a single AAPA showed varying specificity (range: 84-98%), the presence of two AAPA increased specificity considerably since 26.7% of eRA, as compared with 6% of disease controls, were double positive. Thus, double positivity discriminated eRA from axial spondyloarthritis with a LR+ of 18.3. Remarkably, triple positivity was 100% specific for RA, being observed in 10% of eRA and 21.5% of estRA patients, even in the absence of RF and ACPA. CONCLUSION: AAPA are highly prevalent in early RA and occur also independently of RF and ACPA, thereby reducing the gap of seronegativity. Furthermore, multiple AAPA reactivity increased the specificity for RA, suggesting high diagnostic value of AAPA testing.

Cross-reactivity of IgM anti-modified protein antibodies in rheumatoid arthritis despite limited mutational load.

BACKGROUND: Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms. METHODS: Polyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation. RESULTS: Pentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG. CONCLUSIONS: We provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation.

A Comprehensive Evaluation of the Relationship Between Different IgG and IgA Anti-Modified Protein Autoantibodies in Rheumatoid Arthritis.

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.

Improved classification of rheumatoid arthritis with a score including anti-acetylated ornithine antibodies.

The presence of rheumatoid factor (RF) or anti-cyclic citrullinated peptide (anti-CCP) autoantibodies contributes to the current rheumatoid arthritis (RA) classification criteria. These criteria involve stratification on antibody levels, which limits reproducibility, and underperform in the RA patients without RF and anti-CCP. Here, we have explored if two anti-acetylated peptide antibodies (AAPA), anti-acetylated lysine (AcLys) and anti-acetylated ornithine (AcOrn), could improve the performance of the current criteria. The analysis was done in 1062 prospectively-followed early arthritis (EA) patients. The anti-AcOrn were more informative than the anti-AcLys, the conventional RA antibodies and the anti-carbamylated protein antibodies. The anti-AcOrn produced a classification that did not require antibody levels and showed improved specificity (77.6% vs. 72.6%, p = 0.003) and accuracy (79.0% vs. 75.8%, p = 0.002) over the current criteria. These improvements were obtained with a scoring system that values concordance between anti-AcOrn, RF and anti-CCP. No significant gain was obtained in sensitivity (80.2% vs. 78.8%, p = 0.25) or in improving the classification of the RA patients lacking RF and anti-CCP, although the anti-AcOrn ranked first among the analysed new antibodies. Therefore, the anti-AcOrn antibodies could contribute to the improvement of RA classification criteria by exploiting antibody concordance.

Dietary Short-Term Fiber Interventions in Arthritis Patients Increase Systemic SCFA Levels and Regulate Inflammation.

Chronic inflammatory diseases are often initiated and guided by the release of proinflammatory mediators. Rheumatoid arthritis (RA) is caused by an imbalance between the pro- and anti-inflammatory mediators in the joints, thereby favoring chronic inflammation and joint damage. Here, we investigate if short-term high-fiber dietary intervention shifts this towards anti-inflammatory mediators. Healthy controls (n = 10) and RA patients (n = 29) under routine care received daily high-fiber bars for 15 or 30 days, respectively. Stool and sera were analyzed for pro- and anti-inflammatory mediators. A high-fiber dietary intervention resulted in increased anti-inflammatory short-chain fatty acids (SCFA), decreased proarthritic cytokine concentrations, along with a durable shift in the Firmicutes-to-Bacteroidetes ratio. Together, these results further strengthen high-fiber dietary interventions as a practical approach complementing existing pharmacological therapies.

Assessment of the association of baseline anti-CarbV and anti-MCV antibodies with response to treatment and radiographic progression in an RA population treated with either methotrexate or baricitinib: post-hoc analyses from RA-BEGIN.

BACKGROUND: The development of autoantibodies in patients with rheumatoid arthritis (RA) has potential as a marker of treatment response. This analysis assessed the association of an autoantibody response to carbamylated vimentin (anti-CarbV) and to vimentin modified by citrullination (anti-MCV) with response to treatment and structural damage progression in the phase III study RA-BEGIN. METHODS: Data from patients in the modified intent-to-treat population of RA-BEGIN were included for analysis; these patients received methotrexate (MTX), baricitinib 4 mg once daily, or baricitinib plus MTX during the 52-week study period. Endpoints analyzed were clinical response to treatment, assessed using change from baseline (CFB) in Simplified Disease Activity Index (SDAI) and Disease Activity Score for 28-joint count with serum high-sensitivity C-reactive protein (DAS28-hsCRP), and structural damage progression, assessed using CFB greater than the smallest detectable change in the van der Heijde-modified Total Sharp Score. The anti-CarbV and anti-MCV isotypes assessed were immunoglobulin (Ig) A, IgG, and IgM. Multivariable mixed-effect models for repeated measures (MMRMs) were used for the longitudinal analysis of treatment response, and multivariable logistic regression models were used for the analysis of structural damage progression at week 52. RESULTS: Analysis of the association between autoantibodies and treatment response showed that high titers of anti-CarbV (IgA and IgG) were associated with a greater clinical response as measured by SDAI and DAS28-hsCRP. Anti-CarbV IgA and IgG, but not IgM, demonstrated an association after adjustment for other factors included in the MMRMs. High titers of anti-CarbV IgM were associated with a poor response to MTX monotherapy, whereas a nonsignificant trend toward a better response to baricitinib and baricitinib plus MTX was observed. There was no association between anti-MCV antibodies and treatment response. High titers of anti-CarbV IgA were associated with a greater probability of radiographic progression, but no association between anti-MCV antibodies and radiographic progression was observed. CONCLUSIONS: High titers of anti-CarbV IgA and IgG isotypes, but not anti-MCV isotypes, may be useful prognostic biomarkers for identifying the likelihood of the response to treatment and structural damage progression in patients with RA.

Automated Fecal Biomarker Profiling - a Convenient Procedure to Support Diagnosis for Patients with Inflammatory Bowel Diseases.

BACKGROUND: Fecal calprotectin is a valuable non-invasive marker for intestinal inflammation and contributes to the selection of patients with suspected inflammatory bowel disease (IBD) for endoscopy. The aim of this study was to evaluate the performance of three automated immunoassays for fecal calprotectin (FC), fecal lactoferrin (FL) and fecal alpha-1-antitrypsin (A1AT) for diagnosis and follow-up of IBD, to investigate if automated analysis of this biomarker profile may further improve the diagnostic process, and to compare them to manual ELISA tests from different manufacturers. METHODS: Stool samples from 72 patients with Crohn's disease (42), ulcerative colitis (17), irritable bowel syndrome (5), other gastrointestinal inflammation (8), and 72 healthy controls were analyzed for FC, FL, and A1AT on the automated Alegria® system (ORGENTEC Diagnostika, Germany). The results were verified by commercially available manual ELISA tests and discrepancies were further analyzed by immunoblotting. The fecal test results were correlated with the patients' endoscopic findings and with disease activity. RESULTS: The automated assays FC and FL for Alegria® detected endoscopically active intestinal inflammation with a sensitivity and specificity of 74% and 87% (FC) and 62% and 87% (FL), respectively, and efficiently discriminated IBD from non-IBD samples in the patient cohort. Healthy controls tested negative in all assays. The results of the automated biomarker assays significantly correlated to those of the manual ELISAs. Alegria® results for FC were confirmed by immunoblotting in 7 discrepant samples. Levels of A1AT out of the normal range were detectable in a substantial number of IBD and irritable bowel syndrome (IBS) samples: 50% Crohn's disease, 35% ulcerative colitis, and 60% IBS, reflecting the disease-related changes of intestinal permeability in these patients. In IBD patients, elevated levels of A1AT correlated with relapsing disease. CONCLUSIONS: Measurement of FC concentrations with Alegria® is a convenient, promising, and useful tool for improving laboratory diagnostic accuracy and accelerating the diagnostic process and helps to identify those patients in whom endoscopy may be avoided. Automated analysis of a comprehensive profile of fecal biomarkers with Alegria®, including A1AT, provides further substantial benefit for laboratory diagnostics of IBD by improving stratification of patients for treatment and care.

IgA subclasses have different effector functions associated with distinct glycosylation profiles.

Monomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that the two IgA subclasses (IgA1 and IgA2) differ in their effect on immune cells due to distinct binding and signaling properties. Whereas IgA2 acts pro-inflammatory on neutrophils and macrophages, IgA1 does not have pronounced effects. Moreover, IgA1 and IgA2 have different glycosylation profiles, with IgA1 possessing more sialic acid than IgA2. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Of note, disease-specific autoantibodies in patients with rheumatoid arthritis display a shift toward the pro-inflammatory IgA2 subclass, which is associated with higher disease activity. Taken together, these data demonstrate that IgA effector functions depend on subclass and glycosylation, and that disturbances in subclass balance are associated with autoimmune disease.

The Role of Dietary Fiber in Rheumatoid Arthritis Patients: A Feasibility Study.

Short-chain fatty acids are microbial metabolites that have been shown to be key regulators of the gut-joint axis in animal models. In humans, microbial dysbiosis was observed in rheumatoid arthritis (RA) patients as well as in those at-risk to develop RA, and is thought to be an environmental trigger for the development of clinical disease. At the same time, diet has a proven impact on maintaining intestinal microbial homeostasis. Given this association, we performed a feasibility study in RA patients using high-fiber dietary supplementation with the objective to restore microbial homeostasis and promote the secretion of beneficial immunomodulatory microbial metabolites. RA patients (n = 36) under routine care received daily high-fiber bars or cereals for 28 days. Clinical assessments and laboratory analysis of immune parameters in blood and stool samples from RA patients were done before and after the high-fiber dietary supplementation. We observed an increase in circulating regulatory T cell numbers, favorable Th1/Th17 ratios, as well as decreased markers of bone erosion in RA patients after 28 days of dietary intervention. Furthermore, patient-related outcomes of RA improved. Based on these results, we conclude that controlled clinical studies of high-fiber dietary interventions could be a viable approach to supplement or complement current pharmacological treatment strategies.

Different classes of anti-modified protein antibodies are induced on exposure to antigens expressing only one type of modification.

OBJECTIVES: Autoantibodies against post-translationally modified proteins (anti-modified protein antibodies or AMPAs) are a hallmark of rheumatoid arthritis (RA). A variety of classes of AMPAs against different modifications on proteins, such as citrullination, carbamylation and acetylation, have now been described in RA. At present, there is no conceptual framework explaining the concurrent presence or mutual relationship of different AMPA responses in RA. Here, we aimed to gain understanding of the co-occurrence of AMPA by postulating that the AMPA response shares a common 'background' that can evolve into different classes of AMPAs. METHODS: Mice were immunised with modified antigens and analysed for AMPA responses. In addition, reactivity of AMPA purified from patients with RA towards differently modified antigens was determined. RESULTS: Immunisation with carbamylated proteins induced AMPAs recognising carbamylated proteins and also acetylated proteins. Similarly, acetylated proteins generated (autoreactive) AMPAs against other modifications as well. Analysis of anti-citrullinated protein antibodies from patients with RA revealed that these also display reactivity to acetylated and carbamylated antigens. Similarly, anti-carbamylated protein antibodies showed cross-reactivity against all three post-translational modifications. CONCLUSIONS: Different AMPA responses can emerge from exposure to only a single type of modified protein. These findings indicate that different AMPA responses can originate from a common B-cell response that diversifies into multiple distinct AMPA responses and explain the presence of multiple AMPAs in RA, one of the hallmarks of the disease.

In rheumatoid arthritis, changes in autoantibody levels reflect intensity of immunosuppression, not subsequent treatment response.

BACKGROUND: Rheumatoid arthritis (RA) is characterized by the presence of autoantibodies like rheumatoid factor (RF), anti-cyclic citrullinated peptide-2 (anti-CCP2), and anti-carbamylated protein (anti-CarP) antibodies. It is currently unclear whether changes in autoantibody levels are associated with disease activity/treatment outcomes and whether they are modified by treatment intensity. Therefore, we determined longitudinal changes in RA-autoantibody levels, the association between these changes and activity score (DAS) and treatment outcomes, and the effect of intensity of immunosuppressive treatment on levels. METHODS: In 381 seropositive RA patients from the IMPROVED study, we measured IgG, IgM, and IgA of anti-CCP2 and anti-CarP; IgM and IgA of RF; and IgG against four citrullinated and two acetylated peptides at 4-month intervals over the first year of treatment. Following initial prednisone and methotrexate (MTX), treatment was changed every 4 months aiming for DAS < 1.6. We investigated changes in autoantibody levels following treatment escalation versus tapering, and the association of levels with DAS over time, EULAR response, and drug-free remission (DFR) ≥ 1 year. RESULTS: For all 14 autoantibodies, levels decreased from 0 to 4 months and then rose until 12 months. Following treatment escalation, autoantibody levels dropped markedly, while they rose following tapering: RF IgM levels, a representative autoantibody, dropped 10% after restarting prednisone and rose 15% aU/mL after tapering MTX (p < 0.0001). There was no association between autoantibody levels and DAS over time or EULAR response. Greater relative changes between 0 and 12 months did not predict DFR (0-12-month relative change RF IgM, - 39% for no DFR (n = 126) and - 16% for DFR (n = 18)). CONCLUSIONS: Changes in RA-autoantibody levels are not associated with DAS or long-term treatment response, but reflect intensity of immunosuppression. This suggests that autoantibody levels are modifiable by current therapies, but that modifying levels is in itself of limited clinical relevance. TRIAL REGISTRATION: ISRCTN11916566 . Registered on 7 November 2006.