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Because rheumatoid arthritis (RA) is a heterogeneous autoimmune disease in terms of disease manifestations, clinical outcomes, and therapeutic responses, we developed and applied a novel antigen microarray technology to identify distinct serum antibody profiles in patients with RA.Synovial proteome microarrays, containing 225 peptides and proteins that represent candidate and control antigens, were developed. These arrays were used to profile autoantibodies in randomly selected sera from 2 different cohorts of patients: the Stanford Arthritis Center inception cohort, comprising 18 patients with established RA and 38 controls, and the Arthritis, Rheumatism, and Aging Medical Information System cohort, comprising 58 patients with a clinical diagnosis of RA of <6 months duration. Data were analyzed using the significance analysis of microarrays algorithm, the prediction analysis of microarrays algorithm, and Cluster software.Antigen microarrays demonstrated that autoreactive B cell responses targeting citrullinated epitopes were present in a subset of patients with early RA with features predictive of the development of severe RA. In contrast, autoimmune targeting of the native epitopes contained on synovial arrays, including several human cartilage gp39 peptides and type II collagen, were associated with features predictive of less severe RA.Proteomic analysis of autoantibody reactivities provides diagnostic information and allows stratification of patients with early RA into clinically relevant disease subsets. [ABSTRACT FROM AUTHOR]

Antibodies directed toward citrullinated proteins (e.g., anti–cyclic citrullinated peptide antibodies) are highly specific for rheumatoid arthritis (RA) and are produced locally at the site of inflammation. Although the presence of citrullinated proteins in rheumatoid synovium has been described in the literature, it is uncertain whether their presence is specific for RA. The present study was undertaken to investigate this.The local production of the anti–citrullinated protein antibodies was investigated by comparing the concentration of the antibodies (corrected for the total amount of IgG present) in paired samples of serum and synovial fluid from RA patients. The presence of citrullinated proteins in the synovial tissue was investigated by immunohistochemical analysis of synovial tissue from RA patients and from patients with other arthropathies, using a variety of specific antibodies to citrullinated proteins.In RA patients, anti–citrullinated protein antibodies constituted a 1.4?fold higher proportion of IgG in synovial fluid compared with serum, which is indicative of a local production of the antibodies. Immunohistochemical staining of citrullinated proteins was observed in the lining layer, the sublining layer, and in extravascular fibrin deposits in inflamed synovial tissue from RA as well as non?RA patients.The presence of citrullinated proteins in the inflamed synovium is not specific for RA, but rather, it may be an inflammation?associated phenomenon. The high specificity of the anti–citrullinated protein antibodies is, therefore, most likely the result of an abnormal humoral response to these proteins. [ABSTRACT FROM AUTHOR]

The functional role of HLA class II molecules in the pathogenesis of rheumatoid arthritis (RA) is unclear. HLA class II molecules are involved in the interaction between T and B lymphocytes required for long‐lived B cell responses and generation of high‐affinity IgG antibodies. We undertook this study to investigate the relationship between HLA class II gene polymorphisms and RA‐specific IgG antibodies against cyclic citrullinated peptides (anti‐CCP antibodies). High‐resolution HLA–DR and DQ typing and anti–CCP‐2 antibody testing were performed on 268 RA patients from the Early Arthritis Clinic cohort at the Department of Rheumatology of the Leiden University Medical Center. The presence of anti‐CCP antibodies was analyzed in carriers of the different DR and DQ alleles. Disease progression was measured over a period of 4 years by scoring radiographs of the hands and feet using the Sharp/van der Heijde method.Carriership of the individual alleles HLA–DRB1*0401, DRB1*1001, DQB1*0302, and DQB1*0501 was associated with the presence of anti‐CCP antibodies. Carriers of DQ–DR genotypes containing proposed RA susceptibility alleles were significantly more often anti‐CCP antibody positive. Carriership of one or two HLA–DRB1 shared epitope (SE) alleles was significantly associated with production of anti‐CCP antibodies (odds ratio [OR] 3.3, 95% confidence interval [95% CI] 1.8–6.0 and OR 13.3, 95% CI 4.6–40.4, respectively). An increased rate of joint destruction was observed in SE+, anti‐CCP+ patients (mean Sharp score 7.6 points per year) compared with that in SE−, anti‐CCP+ patients (2.4 points per year) (P = 0.04), SE+, anti‐CCP− patients (1.6 points per year) (P < 0.001), and SE−, anti‐CCP− patients (1.6 points per year) (P < 0.001). HLA class II RA susceptibility alleles are associated with production of anti‐CCP antibodies. Moreover, more severe disease progression is found in RA patients with both anti‐CCP antibodies and SE alleles. [ABSTRACT FROM AUTHOR]

To compare the autoantigenicity of the recently described N-terminally elongated PM–Scl-75 protein with that of PM–Scl-100 and the originally defined PM–Scl-75 polypeptide, and to determine its value for analyzing sera from patients with the polymyositis (PM)/scleroderma overlap syndrome. Serum samples obtained from patients with the PM/scleroderma overlap syndrome and from patients with several other diseases were analyzed for the presence of autoantibodies reactive with recombinant PM–Scl-100 and PM–Scl-75 (both the original and the longer form) proteins, in an enzyme-linked immunosorbent assay (ELISA). Autoantibodies recognizing the longer PM–Scl-75 protein isoform were detected in 28% of the patients with PM/scleroderma. This percentage is slightly higher than that for PM–Scl-100 (25%) and is significantly higher than that for the previously defined PM–Scl-75 protein (11%). In addition, we identified a significant number of patients who had anti–PM–Scl-75 but not anti–PM–Scl-100 antibodies. This finding contrasts with what has been previously reported for the shorter version of the PM–Scl-75 protein. Our data indicate that use of the long PM–Scl-75 isoform in addition to PM–Scl-100 in ELISAs significantly increases the number of patients in whom anti–PM-Scl autoantibodies can be detected. [ABSTRACT FROM AUTHOR]

To evaluate the prevalence and predictive value of anti–cyclic citrullinated peptide (anti-CCP) antibodies in individuals who subsequently developed rheumatoid arthritis (RA) and to determine the relationship to rheumatoid factor (RF) of any isotype. A case–control study was nested within the Northern Sweden Health and Disease Study and the Maternity cohorts of Northern Sweden. Patients with RA were identified among blood donors whose samples had been taken years before the onset of symptoms. Control subjects matched for age, sex, date of sampling, and residential area were selected randomly from the same cohorts. Anti-CCP antibody and RFs were determined using enzyme immunoassays. Eighty-three individuals with RA were identified as having donated blood before presenting with any symptoms of joint disease (median 2.5 years [interquartile range 1.1–4.7] before RA). In samples obtained before the onset of RA, the prevalence of autoantibodies was 33.7% for anti-CCP, 16.9% for IgG-RF, 19.3% for IgM-RF, and 33.7% for IgA-RF (all highly significant compared with controls). The sensitivities for detecting these autoantibodies >1.5 years and ≤1.5 years before the appearance of any RA symptoms were 25% and 52% for anti-CCP, 15% and 30% for IgM-RF, 12% and 27% for IgG-RF, and 29% and 39% for IgA-RF. In conditional logistic regression models, anti-CCP antibody and IgA-RF were found to be significant predictors of RA. Anti-CCP antibody and RFs of all isotypes predated the onset of RA by several years. The presence of anti-CCP and IgA-RF predicted the development of RA, with anti-CCP antibody having the highest predictive value. This indicates that citrullination and the production of anti-CCP and RF autoantibodies are early processes in RA. [ABSTRACT FROM AUTHOR]

Antibodies directed to citrulline-containing proteins are highly specific for rheumatoid arthritis (RA) and can be detected in up to 80% of patients with RA. Citrulline is a nonstandard amino acid that can be incorporated into proteins only by posttranslational modification of arginine by peptidylarginine deiminase (PAD) enzymes. The objective of this study was to investigate the presence of anticitrulline antibodies, PAD enzymes, and citrullinated antigens in mouse models of both acute and chronic destructive arthritis: streptococcal cell wall (SCW)–induced arthritis and collagen-induced arthritis (CIA), respectively. Synovial tissue biopsy specimens were obtained from naive mice, mice with CIA, and mice with SCW-induced arthritis. The expression of messenger RNA (mRNA) for PAD enzymes was analyzed by reverse transcriptase–polymerase chain reaction; the presence of PAD proteins and their products (citrullinated proteins) was analyzed by Western blotting and by immunolocalization. The presence of anticitrullinated protein antibodies was investigated by an anti–cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) and an ELISA using in vitro citrullinated fibrinogen. In both mouse models, PAD type 2 (PAD2) mRNA was present in the synovium but was not translated into PAD2 protein. In contrast, PAD4 mRNA, although absent from healthy synovium, was readily transcribed and translated by polymorphonuclear neutrophils infiltrating the synovial tissue during inflammation. As a consequence, several synovial proteins were subjected to citrullination. One of these proteins was identified as fibrin, which has been reported to be citrullinated also in synovium of patients with RA. Although generation of citrullinated antigens during synovial inflammation in the mice was eminent, no anti-CCP antibodies could be detected. Citrullination of synovial antigens is an active process during joint inflammation in both mice and humans, but the induction of autoantibodies directed to these proteins is a more specific phenomenon, detectable only in human RA patients. [ABSTRACT FROM AUTHOR]