Anti-Carbamylated Protein Antibodies in ACPA-Negative and ACPA-Positive Patients with Rheumatoid Arthritis.

The objective of this study was to assess the level of antibodies to carbamylated proteins and analyze the clinical and immunological associations in patients with ACPA-negative and ACPA-positive variants of rheumatoid arthritis. MATERIALS AND METHODS: . The study involved 150 patients with a reliable diagnosis of rheumatoid arthritis and 25 patients as healthy controls. Depending on ACPA values, two groups of patients were recruited: ACPA-positive (n = 75) and ACPA-negative (n = 75). RA activity was assessed by the DAS28 index. Determination of antibodies to carbamylated proteins was performed by enzyme-linked immunosorbent assay (BlueGene Biotech, China). Quantitative determination of ACPA in serum was performed by enzyme immunoassay using a commercial reagent kit (AxisShield, UK; upper limit of normal 5.0 U/mL; Orgentec, Germany; upper limit of normal 20.0 U/mL). RESULTS AND DISCUSSION: . Median anti-CarP in patients with RA was 126.2 [100.83; 157.41] ng/mL and was statistically significantly higher (p < 0.001) than in healthy controls (88.89 [70.53; 107.75] ng/mL). Among all patients with RA, 50 (33.3%) were anti-Carp-positive (22 (29.3%) in the ACPA(+) group and 28 (37.3%) in the ACPA(-) group), and one (2%) volunteer from healthy controls was anti-CarP(+) (p = 0.002). In ROC analysis performed to assess the diagnostic significance of anti-CarP for RA for all patients with RA, the area under the curve was 0.783 ± 0.047 with 95% CI: 0.691-0.874 (p < 0.001), with a cut-off point of 143 ng/mL, specificity 96%, sensitivity 36.7%. In the ACPA(+) RA group, the erosion count was statistically significantly higher (p = 0.044) in anti-CarP(+) patients than in anti-CarP(-) patients. A weak direct correlation between anti-CarP and DAS28 was found in the ACPA(-) RA group. CONCLUSIONS: . We studied the predictive value of anti-CarP as an auxiliary biomarker in ACPA(+) and ACPA(-) subtypes of RA. ACPA(+) anti-CarP(+) patients have a more "erosive" subtype of the disease than ACPA(+) anti-CarP(-) patients. In ACPA(-) patients, anti-CarP helps to identify a more erosive subtype of the disease, and among ACPA(-) patients it helps to reduce the proportion of seronegative patients. Further studies are required to determine the optimal standards for the laboratory diagnosis of anti-CarP and to clarify the diagnostic potential of these ABs as part of the differential diagnosis of arthritis in other rheumatic diseases.

Anti-carbamylated protein antibodies drive AEC II toward a profibrotic phenotype by interacting with carbamylated TLR5.

OBJECTIVES: This study was to explore the role of Anti-carbamylated protein (Anti-CarP) antibodies in contributing to lung fibrosis in connective tissue disease (CTD)-associated interstitial lung disease (ILD) in an autoantigen-dependent manner. METHODS: ELISA tested serum samples, including 89 of CTD-ILD group and 170 of non-ILD CTD, for the anti-CarP levels. Male C57BL/6 mice were used for pulmonary fibrosis model and anti-CarP treatment in vivo (n = 5), and patient serum-derived or commercialized anti-CarP for cell treatment. We identified the carbamylated membrane protein via immunofluorescence (IF) and coimmunoprecipitation followed by mass spectrometry (MS) analysis. RT-qPCR, IF and western blot were performed to explore the antigen-dependent role of anti-CarP. Native electrophoretic mobility shift assay and MS analysis were used to verify direct interaction and carbamylation sites. RESULTS: A significantly higher serum anti-CarP level was observed in CTD with ILD than without ILD. In vivo, intrapulmonary delivery of anti-CarP induces epithelial-mesenchymal transition (EMT) and micro fibrotic foci. Carbamylation was enriched in type II alveolar epithelial cells (AEC II). A novel carbamylated membrane receptor, specifically recognized by anti-CarP, was identified as toll-like receptor 5 (TLR5). We found anti-CarP induces the nuclear translocation of NF-κB and downstream events, including EMT and expression of inflammatory cytokines in AEC II, which were reversed by TLR5 blocking or TLR5 knockdown. Moreover, up to 12 lysine carbamylation sites were found in TLR5 ectodomain, allowing the interaction of anti-CarP with carbamylated TLR5. CONCLUSIONS: Overall, we found anti-CarP drives aberrant AEC II activation by interacting with carbamylated TLR5 to promote ILD progress.

Influence of anti-carbamylated protein antibodies on disease activity and joint erosions in seronegative and seropositive rheumatoid arthritis.

Anti-carbamylated protein (anti-CarP) antibodies are promising biomarkers in rheumatoid arthritis (RA), although their significance in seronegative disease (SNRA) remains uncertain. To assess the influence of anti-CarP antibodies on disease activity and erosive joint damage in SNRA patients. In RA patients, rheumatoid factor (RF), anti-citrullinated protein antibodies, and anti-CarP antibodies were measured. Disease activity was assessed using DAS28-CRP and SDAI indices, while musculoskeletal ultrasound identified bone erosions. A total of 77 patients were enrolled, comprising 49 with seropositive RA (SPRA) and 28 with SNRA. Notably, 28% of SPRA and 10% of SNRA patients were positive to anti-CarP antibodies. Anti-CarP-positive patients exhibited elevated C-reactive protein (median 10.6, interquartile range 4.6-20.0 vs. 3.4, 1.7-9.9 mg/L; p = 0.005), erythrocyte sedimentation rate (34, 19-46 vs. 16, 7-25 mm/h; p = 0.002), DAS28-CRP (3.2, 2.6-4.2 vs. 2.6, 1.9-3.5; p = 0.048), and SDAI (19.9, 6.3-32.1 vs. 10.9, 5.5-18.1; p = 0.034) indices. Multivariate analysis revealed RF positivity as the sole predictor for anti-CarP antibodies (odds ratio [OR] = 5.9). Musculoskeletal ultrasound revealed bone erosions in 36% of RA patients; 35% among anti-CarP-negative patients and 40% among anti-CarP-positive patients. Notably, RF presence (OR = 44.3) and DAS28-CRP index (OR = 2.4) emerged as predictors of musculoskeletal ultrasound-confirmed erosive joint disease. Anti-CarP antibodies are detected at similar frequencies among both SPRA and SNRA patients. While associated with increased disease activity, these antibodies did not correlate with increased erosive joint damage.

Anti-carbamylated protein antibodies in systemic sclerosis.

BACKGROUND: To investigate the presence of different isotypes of anti-carbamylated protein (CarP) antibodies in systemic sclerosis (SSc) patients and its association with skin involvement. METHODS: Sera of 194 SSc patients from the Leiden CCISS cohort, fulfilling ACR/EULAR 2013 criteria and a clinical diagnosis of SSc, 83 patients with other connective tissue diseases/Raynaud's Phenomenon, 24 rheumatoid arthritis patients and 98 age and sex-matched healthy controls were tested for the presence of anti-CarP IgG, IgA and IgM, determined by ELISA. Clinical characteristics, that were evaluated in SSc patients, included age, anti-topoisomerase antibodies (ATA), anti-centromere antibodies (ACA) and modified Rodnan Skin Score (mRSS). RESULTS: The SSc patients were 55 (SD:13) years and 155 (80%) were female. Forty-four (23%) patients tested positive for ATA, and 80 (42%) ACA. The median mRSS was 2 (range: 0; 47). Prevalence of anti-CarP IgG was higher in SSc patients than in healthy controls (8% vs 3%, p = 0.007. Prevalence of anti-CarP IgA and IgM and levels of anti-CarP isotypes were comparable between SSc patients and healthy controls. Fifteen (8%) SSc patients tested positive for anti-CarP IgG, 16 (8%) for anti-CarP IgA, and 36 (19%) for anti-CarP IgM. There were no significant correlations between age and levels of anti-CarP isotypes. No correlation between anti-CarP IgG levels and mRSS was found (r = 0.141, p = 0.049), nor for anti-CarP IgM and IgA levels. Anti-CarP IgA levels were higher in ATA compared to ACA positive SSc patients (ATA: 616 aU/ml [359; 1103]; ACA: 424 aU/ml [300; 673], p = 0.015). CONCLUSION: SSc patients can test positive for Anti-CarP IgG, IgA and IgM. We do not observe a relevant clinical association between anti-CarP antibody response and skin involvement in SSc.

Association of serum anti-carbamylated protein antibodies with disease activity and bone loss in rheumatoid arthritis.

BACKGROUND: We investigated the association of serum anti-carbamylated protein (anti-CarP) antibodies with disease activity and bone loss in rheumatoid arthritis (RA). METHODS: The serum anti-CarP antibody concentrations of RA and non-RA patients and healthy controls were determined by enzyme-linked immunosorbent assay (ELISA) and then compared. The diagnostic value of anti-CarP antibodies in RA was determined by the receiver operating characteristic curve. Patients with RA and bone erosions were evaluated using ultrasound examinations. Ultrasonography was performed using a semiquantitative scale. The serum receptor activator of nuclear factor Κ-Β ligand (RANKL) concentrations were measured by ELISA to focus on bone loss. RESULTS: Peripheral serum anti-CarP antibody concentrations in patients with RA were significantly higher than those in patients without RA and in healthy controls and were positively correlated with disease activity. Anti-CarP antibody concentrations were significantly increased in patients with anti-CCP-positive RA. Positive correlation were found between anti-CarP and RANKL. Increased serum anti-CarP antibodies in women with postmenopausal osteoporosis (OP). CONCLUSIONS: Anti-CarP antibodies are associated with RA disease activity and may play an important role in bone loss associated with RA. The concentration of anti-CarP antibodies may be beneficial in the early diagnosis of RA, thus supporting its potential as a novel disease biomarker.

Evolution of anti-modified protein antibody responses can be driven by consecutive exposure to different post-translational modifications.

BACKGROUND: Besides anti-citrullinated protein antibodies (ACPA), rheumatoid arthritis patients (RA) often display autoantibody reactivities against other post-translationally modified (PTM) proteins, more specifically carbamylated and acetylated proteins. Immunizing mice with one particular PTM results in an anti-modified protein antibody (AMPA) response recognizing different PTM-antigens. Furthermore, human AMPA, isolated based on their reactivity to one PTM, cross-react with other PTMs. However, it is unclear whether the AMPA-reactivity profile is "fixed" in time or whether consecutive exposure to different PTMs can shape the evolving AMPA response towards a particular PTM. METHODS: Longitudinally collected serum samples of 8 human individuals at risk of RA and 5 with early RA were tested with ELISA, and titers were analyzed to investigate the evolution of the AMPA responses over time. Mice (13 per immunization group in total) were immunized with acetylated (or carbamylated) protein (ovalbumin) twice or cross-immunized with an acetylated and then a carbamylated protein (or vice versa) and their serum was analyzed for AMPA responses. RESULTS: Human data illustrated dynamic changes in AMPA-reactivity profiles in both individuals at risk of RA and in early RA patients. Mice immunized with either solely acetylated or carbamylated ovalbumin (AcOVA or CaOVA) developed reactivity against both acetylated and carbamylated antigens. Irrespective of the PTM-antigen used for the first immunization, a booster immunization with an antigen bearing the other PTM resulted in increased titers to the second/booster PTM. Furthermore, cross-immunization skewed the overall AMPA-response profile towards a relatively higher reactivity against the "booster" PTM. CONCLUSIONS: The relationship between different reactivities within the AMPA response is dynamic. The initial exposure to a PTM-antigen induces cross-reactive responses that can be boosted by an antigen bearing this or other PTMs, indicating the formation of cross-reactive immunological memory. Upon subsequent exposure to an antigen bearing another type of PTM, the overall reactivity pattern can be skewed towards better recognition of the later encountered PTM. These data might explain temporal differences in the AMPA-response profile and point to the possibility that the PTM responsible for the initiation of the AMPA response may differ from the PTM predominantly recognized later in time.

Anti-Carbamylated Fibrinogen Antibodies Might Be Associated With a Specific Rheumatoid Phenotype and Include a Subset Recognizing In Vivo Epitopes of Its γ Chain One of Which Is Not Cross Reactive With Anti-Citrullinated Protein Antibodies.

To identify the targets recognized by anti-carbamylated protein antibodies (anti-CarP) in patients with early Rheumatoid Arthritis (RA), to study the cross-reactivity between anti-CarP and anti-citrullinated protein antibodies (ACPA) and to evaluate their prognostic value. 331 patients (184 RA and 147 other rheumatisms) from the Very Early Arthritis (VErA) French cohort were analyzed. We performed mass spectrometry analysis of RA sera displaying anti-CarP activity and epitope mapping of the carbamylated fibrinogen γ chain to identify immunodominant peptides. The specificity of these targets was studied using competition assays with the major antigens recognized by ACPA. The prognostic value of anti-carbamylated fibrinogen IgG antibodies (ACa-Fib IgG) was compared to that of anti-cyclic citrullinated peptide antibodies (anti-CCP) and anti-CarP using an in-house ELISA. Besides the α chain, the γ chain of fibrinogen, particularly one immunodominant epitope that has a specific reactivity, was identified as a circulating carbamylated target in sera. The prevalence of ACa-Fib was 37% at baseline and 10.9% for anti-CCP-negative RA. In anti-CCP-negative patients, ACa-Fib positivity was associated with a more inflammatory and erosive disease at baseline but not with rapid radiological progression, which remains strongly related to anti-CCP antibodies. Fibrinogen seems to be one of the antigens recognized in vivo by the anti-CarP response, particularly 2 epitopes of the γ chain, one of which is not cross reactive with ACPA. This specificity might be associated with a distinct clinical phenotype since ACa-Fib IgG were shown to be linked to systemic inflammation in very early RA but not to rapid radiological progression.

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.

From Rheumatoid Factor to Anti-Citrullinated Protein Antibodies and Anti-Carbamylated Protein Antibodies for Diagnosis and Prognosis Prediction in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease mainly involving synovial inflammation and articular bone destruction. RA is a heterogeneous disease with diverse clinical presentations, prognoses and therapeutic responses. Following the first discovery of rheumatoid factors (RFs) 80 years ago, the identification of both anti-citrullinated protein antibodies (ACPAs) and anti-carbamylated protein antibodies (anti-CarP Abs) has greatly facilitated approaches toward RA, especially in the fields of early diagnosis and prognosis prediction of the disease. Although these antibodies share many common features and can function synergistically to promote disease progression, they differ mechanistically and have unique clinical relevance. Specifically, these three RA associating auto-antibodies (autoAbs) all precede the development of RA by years. However, while the current evidence suggests a synergic effect of RF and ACPA in predicting the development of RA and an erosive phenotype, controversies exist regarding the additive value of anti-CarP Abs. In the present review, we critically summarize the characteristics of these autoantibodies and focus on their distinct clinical applications in the early identification, clinical manifestations and prognosis prediction of RA. With the advancement of treatment options in the era of biologics, we also discuss the relevance of these autoantibodies in association with RA patient response to therapy.

The effect of sex on immune responses to a homocitrullinated peptide in the DR4-transgenic mouse model of Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) is more common and severe in women compared to men. Both women and men with RA express autoantibodies to post-translationally modified antigens, including citrullinated and homocitrullinated proteins or peptides. These autoantibodies are strongly linked with the HLA-DR4 gene. The objective of this study was to determine sex differences in immune responses to homocitrullinated antigens. We used a humanized animal model of RA, DR4-transgenic mice and immunized them with a homocitrullinated peptide called HomoCitJED. Immune responses in these mice were measured for splenocyte proliferation by tritiated thymidine incorporation, serum autoantibody production by ELISA and cytokine levels by multiplex. We found that T cell and antibody responses to homocitrullinated antigens were similar in male and female mice. However, we found sex differences in serum cytokine profiles with female mice having higher ratio of IL-1α to IL-5, suggesting imbalances in immune regulation. This is the first study to report that immune responses to homocitrullinated antigens can be differentiated by sex.

Presence of autoantibodies in "seronegative" rheumatoid arthritis associates with classical risk factors and high disease activity.

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.

Activated neutrophil carbamylates albumin via the release of myeloperoxidase and reactive oxygen species regardless of NETosis.

Objective: An anti-carbamylated albumin antibody was detected in rheumatoid arthritis (RA) patients, and its presence was associated with serum myeloperoxidase (MPO) levels, as we reported previously. Since MPO is a key enzyme for carbamylation and is released by neutrophil extracellular traps (NETs), we aimed to demonstrate that NETosis induces carbamylation.Methods: Human neutrophils were isolated from a healthy donor, pre-treated with or without diphenyleneiodonium (DPI, an inhibitor for the generation of reactive oxygen species (ROS)), Cl-amidine (a peptidylarginine deiminase inhibitor), 4-aminobenzoic acid hydrazide (4-ABAH, an MPO inhibitor), or GW311616A (a neutrophil elastase (NE) inhibitor), and incubated for 8 h with or without phorbol 12-myristate 13-acetate (PMA). Proteins in the medium were collected and the carbamylation of albumin was evaluated by Western blotting.Results: The carbamylation of albumin was detected in the culture medium of activated neutrophils. NETosis was observed under the stimulation by PMA. DPI and 4-ABAH inhibited the carbamylation of albumin and NETosis. GW311616A inhibited NETosis, but not carbamylation. Neither carbamylation nor NETosis was inhibited by Cl-amidine.Conclusion: Activated neutrophils may carbamylate ambient albumin, and this is dependent on ROS and MPO, but does not require NETosis.

Improved RA classification among early arthritis patients with the concordant presence of three RA autoantibodies: analysis in two early arthritis clinics.

BACKGROUND: The patients with RA benefit from early identification soon after the first clinical symptoms appear. The 2010 ACR/EULAR classification criteria were developed to fulfill this need and their application has been demonstrated to be effective. However, there is still room for improvement. Therefore, we aimed to evaluate the potential of the concordant presence of RF, anti-CCP and anti-carbamylated protein antibodies to improve current RA classification among early arthritis (EA) patients. METHODS: Data from the first visit of 1057 patients in two EA prospective cohorts were used. The serological scores from the 2010 ACR/EULAR criteria and the concordant presence of the three RA autoantibodies were assessed relative to a gold standard consisting of the RA classification with the 1987 ACR criteria at the 2 years of follow-up. RESULTS: The concordant presence of three antibodies showed predictive characteristics allowing for direct classification as RA (positive predictive value = 96.1% and OR = 80.9). They were significantly better than the corresponding to the high antibody titers defined as in the 2010 classification criteria (PPV = 88.8%, OR = 26.1). In addition, the concordant presence of two antibodies was also very informative (PPV = 82.3%, OR = 15.1). These results allowed devising a scoring system based only on antibody concordance that displayed similar overall performance as the serological scoring system of the 2010 criteria. However, the best classification was obtained combining the concordance and 2010 serological systems, a combination with a significant contribution from each of the two systems. DISCUSSION: The concordant presence of RA autoantibodies showed an independent contribution to the classification of EA patients that permitted increased discrimination and precision.

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.

Autoantibody and metalloproteinase activity in early arthritis.

OBJECTIVES: The aim of the study was to evaluate the frequency of anti-mutated citrullinated vimentin antibodies (a-Sa), anti-citrullinated α-enolase peptide 1 antibodies (a-CEP-1), anti-filaggrin antibodies (AFAs), heterogeneous nuclear ribonucleoprotein compies/anti-RA33-antibodies (a-hnRNP/RA33), anti-carbamylated protein antibodies (a-CarP), and metalloproteinase (MMPs) activity in patients with early inflammatory arthritis (EIA). METHODS: Seventy-four patients with EIA: 51 diagnosed with RA (rheumatoid arthritis) and 23 with UA (undifferentiated arthritis), and 20 healthy volunteers were enrolled to the study. Inflammatory markers, rheumatoid factor (RF), and antibodies mentioned above were assessed in all patients. RESULTS: In the EIA group, we observed significantly higher concentration of a-CEP-1 (65.8 ± 111.6 RU/mL) than in controls (2.0 ± 0.0 RU/mL). In RF(+) RA patients, we observed higher concentration of a-Sa and a-CEP-1 than in other groups. A-Sa were positive in 69% of RF(+) RA, 37% of RF(-) RA, 26% of UA patients and in 10% of controls. A-CEP-1 were positive in 77% of RF(+) RA patients, in 56% of RF(-) RA patients, in 8.7% of UA patients, but they were negative in controls. In patients with RF(+) RA, positive a-CarP were present statistically significantly more often than in RF (-) RA patients. No statistically significant difference in frequency of a-hnRNP/RA33 and AFA between RF(+) RA, RF(-) RA, and UA was observed. CONCLUSIONS: Our results suggest that a-CEP-1 may help in differentiation between RF(-) RA and UA. a-CEP-1 and a-Sa may be useful while diagnosing EIA. a-CarP may be used in differentiation of RA RF(-) and UA. However, a follow-up study is needed to evaluate the prognostic value of analyzed antibodies.

Low mortality rate in Italian rheumatoid arthritis patients from a tertiary center: putative implication of a low anti-carbamylated protein antibodies prevalence.

BACKGROUND AND OBJECTIVE: Anti-carbamylated protein antibodies (anti-CarP Ab) represent a novel kind of autoantibodies specificity detectable in the sera of patients with rheumatoid arthritis (RA). They have been recently reported to be associated with increased mortality in Spanish patients with RA. The aim of our study was to compare the incidence mortality rates (IMRs) detected in RA patients from a tertiary Italian center with those reported in other European tertiary centers and to evaluate the putative role of anti-CarP Ab in modulating the low IMR detected in our patients. METHODS: Clinical charts of patients consecutively admitted to our center, from January 1, 2008, to December 31, 2014, were retrospectively reviewed. The mortality rate (expressed as the number of deaths in the cohort divided by the number of years of IMR follow-up) and causes of death were assessed at December 31, 2015. Sera of 61 patients, representative of the whole cohort, collected at the time of admission to our center were investigated for the presence and the level of anti-CarP Ab. Demographic and clinical features, mortality rates and prevalence of anti-CarP Ab in our series were compared with those reported in other European cohorts. RESULTS: We observed 608 patients for a median of 3.51 years. All-cause and cause-specific IMRs in our cohort were significantly lower than the Better Anti-rheumatic Farmaco-therapy and the Spanish cohort, while only all-cause and cardiovascular IMRs were significantly lower in our series with respect to the Leiden Early Arthritis Clinic cohort. Anti-CarP Ab prevalence was significantly lower in our series than in any other European cohorts. CONCLUSION: We confirm that the mortality rate is lower in our Italian RA cohort with respect to other European cohorts. Whether the low prevalence of anti-CarP Ab might be responsible for this result awaits to be furtherly investigated.

Clinical and etiological meaning of anti-carbamylated protein antibodies in rheumatoid arthritis.

Several autoantibodies against proteins with post-translational modifications have been detected in patients with rheumatoid arthritis (RA) and are called anti-modified protein antibodies (AMPAs). Anti-carbamylated protein antibodies (Anti-CarP Ab) are the second most vigorously researched AMPAs following anti-citrullinated protein/peptide antibodies (ACPA). Anti-CarP Ab and ACPA show cross-reactivity to some extent and frequently co-exist with each other in RA, but are two distinct antibodies. Although the diagnostic efficacy of anti-CarP Ab is inferior to that of ACPA, the diagnostic specificity of RA may improve when used in combination with ACPA and rheumatoid factor. Anti-CarP Ab and ACPA are also useful for identifying patients at high risk of more severe joint destruction and cardiovascular diseases. The high prevalence of the co-existence of both antibodies suggests a common factor in their production, and this is important for the development of RA because both antibodies emerge before the onset of clinical symptoms. Neutrophils may also be crucially involved. It is important to distinguish citrullinated antigens from carbamylated antigens because the methods commonly used to detect the former are now known to be cross-reactive with the latter. Research on anti-CarP Ab will provide novel insights into the pathology and etiology of RA.

Autoantibodies in the Pathogenesis, Diagnosis, and Prognosis of Juvenile Idiopathic Arthritis.

Autoantibody production occurs in juvenile idiopathic arthritis (JIA) and numerous other autoimmune diseases. In some conditions, the autoantibodies are clearly pathogenic, whereas in others the roles are less defined. Here we review various autoantibodies associated with JIA, with a particular focus on antinuclear antibodies and antibodies recognizing citrullinated self-antigens. We explore potential mechanisms that lead to the development of autoantibodies and the use of autoantibody testing in diagnosis and prognosis. Finally, we compare and contrast JIA-associated autoantibodies with those found in adults with rheumatoid arthritis (RA).

Pitfalls in the detection of citrullination and carbamylation.

Carbamylation and citrullination are both post-translational modifications against which (auto)antibodies can be detected in sera of rheumatoid arthritis (RA) patients. Carbamylation is the chemical modification of a lysine into a homocitrulline, whereas citrullination is an enzymatic conversion of an arginine into a citrulline. It is difficult to distinguish between the two resulting amino acids due to similarities in structure. However, differentiation between citrulline and homocitrulline is important to understand the antigens that induce antibody production and to determine which modified antigens are present in target tissues. We have observed in literature that conclusions are frequently drawn regarding the citrullination or carbamylation of proteins based on reagents that are not able to distinguish between these two modifications. Therefore, we have analyzed a wide spectrum of methods and describe here which method we consider most optimal to distinguish between citrulline and homocitrulline. We have produced several carbamylated and citrullinated proteins and investigated the specificity of (commercial) antibodies by both ELISA and western blot. Furthermore, detection methods based on chemical modifications, such as the anti-modified citrulline-"Senshu" method and also mass spectrometry were investigated for their capacity to distinguish between carbamylation and citrullination. We observed that some antibodies are able to distinguish between carbamylation and citrullination, but an overlap in reactivity is often present in the commercially available anti-citrulline antibodies. Finally, we conclude that the use of mass spectrometry is currently essential to differentiate between citrullinated and carbamylated proteins present in complex biological samples.

Assessment of anti-CarP antibodies, disease activity and quality of life in rheumatoid arthritis patients on conventional and biological disease-modifying antirheumatic drugs.

OBJECTIVE: Good biomarkers are important to guide decisions in the clinical management of rheumatoid arthritis (RA). RA patients harbor antibodies directed against carbamylated proteins which may predict joint damage. This study investigated whether antibodies against carbamylated proteins (anti-CarP) may serve as surrogate prognostic markers. MATERIAL AND METHODS: Fifty-three patients with a diagnosis of rheumatoid arthritis according to ACR 1987 criteria were included. Blood samples were analyzed for CarP antibody levels using the ELISA method. Quality of life (QoL) was assessed by the WHO SF-36 questionnaire, and disease activity was assessed using the DAS28 calculator. Newly diagnosed patients were assessed at the first visit and at 12 weeks of treatment, while a single assessment was made for patients already on maintenance therapy. RESULTS: Out of 53 patients, 22 had titers of anti-CarP above the cut-off range and considered as positive for anti-CarP antibodies. Anti-CarP antibody serum level was significantly higher in patients with deformity of joints and with erosions in comparison to those without any destructive changes (p < 0.05). There was a weak positive correlation between anti-CarP and DAS 28 (p > 0.05). Also there was a weak negative correlation in all domains of quality of life with anti-CarP antibody titers (p > 0.05). There was no significant correlation between titers of anti-CarP antibodies and presence or absence of rheumatoid factor. CONCLUSIONS: Serum levels of anti-CarP antibodies in RA patients with joint erosions/deformities were much higher than in those without any joint damage. Anti-CarP antibodies may have good prognostic value in RA patients with erosions. Disease activity and QoL of RA patients improved during treatment, but no correlation was found between DAS 28/QoL and anti-CarP antibody serum levels.