Objective. To generate a catalog of citrullinated proteins that are present in the synovia of patients with rheumatoid arthritis (RA) and to elucidate their relevance for the anti-citrullinated protein antibody response in RA.Methods. Polypeptides isolated from the synovial fluid of patients with RA were identified by mass spectrometry. Three proteins (apolipoprotein E [Apo E], myeloid nuclear differentiation antigen [MNDA], and ␤-actin) were studied in more detail, using immunoprecipitation and Western blotting. The presence of autoantibodies to synthetic peptides derived from these proteins in sera from patients with RA, sera from patients with other diseases, and sera from healthy control subjects was studied by enzyme-linked immunosorbent assay (ELISA).Results. RA synovial fluid samples displayed several distinct patterns of citrullinated proteins. Using mass spectrometry, (fragments of) 192 proteins were identified, including 53 citrullinated proteins, some of which contained multiple citrullinated residues. In addition to previously reported citrullinated proteins in RA synovia (e.g., vimentin and fibrinogen), a series of novel citrullinated proteins, including Apo E, MNDA, ␤-actin, and cyclophilin A, was identified. Immunoprecipitation experiments confirmed the citrullination of Apo E and MNDA. ELISAs demonstrated the presence of autoreactive citrullinated epitopes in Apo E, MNDA, and ␤-actin.Conclusion. Synovial fluid samples from the inflamed joints of patients with RA contain many citrullinated proteins. Citrullinated Apo E, MNDA, and ␤-actin are novel antigens identified in RA synovial fluid, and only a limited number of their citrullinated epitopes are targeted by the immune system in RA.

IntroductionRheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.MethodsEndogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.ResultsOur data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.ConclusionsThe increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.

Antiprothrombin antibodies, measured with phosphatidylserine/prothrombin complex (aPS/PT) ELISA, have been reported to be associated with antiphospholipid syndrome (APS). They are currently being evaluated as a potential classification criterion for this autoimmune disease, characterized by thromboses and obstetric complications. Given the present lack of clinically useful tests for the accurate diagnosis of APS, we aimed to evaluate in-house and commercial assays for determination of aPS/PT as a potential serological marker for APS. We screened 156 patients with systemic autoimmune diseases for antibodies against PS/PT, β 2-glycoprotein I, cardiolipin and for lupus anticoagulant activity. We demonstrated a high degree of concordance between the concentrations of aPS/PT measured with the in-house and commercial assays. Both assays performed comparably relating to the clinical manifestations of APS, such as arterial and venous thromboses and obstetric complications. IgG aPS/PT represented the strongest independent risk factor for the presence of obstetric complications, among all tested aPL. Both IgG and IgM aPS/PT were associated with venous thrombosis, but not with arterial thrombosis. Most importantly, the association between the presence of IgG/IgM aPS/PT and lupus anticoagulant activity was highly significant. Taken together, aPS/PT antibodies detected with the in-house or commercial ELISA represent a promising serological marker for APS and its subsets.

Objective. To determine the prevalence and clinical association of anti-phosphatidylserine/prothrombin antibodies (aPS/PT) in patients with a history of pregnancy complications relevant to antiphospholipid syndrome (APS). Materials and Methods. Two hundred and eleven patients with a history of (a) three or more consecutive miscarriages before 10th week of gestation (WG) (n = 64), (b) death of a morphologically normal fetus beyond 10th WG (n = 72), (c) premature birth of a morphologically normal neonate before 34th WG due to eclampsia, preeclamsia and placental insufficiency (n = 33), and (d) less than three unexplained consecutive miscarriages before 10th WG (n = 42). Subjects sera were analyzed for lupus anticoagulant (LA), anti-cardiolipin (aCL), anti-β 2-glycoprotein I (anti-β 2GPI), and aPS/PT antibodies. Results. 41/169 (24.3%) of patients were positive for at least one measured aPL. The highest prevalence was found for aPS/PT and aCL (13.0% and 12.4%, resp.) followed by LA (7.7%) and anti-β 2GPI (7.1%). 11/169 with APS-related obstetric manifestations had only aPS/PT. 17.8% of patients were positive for LA or aCL and/or anti-β 2GPI; however when adding the aPS/PT results, an additional 7% of patients could be evaluated for APS. Conclusion. aPS/PT are associated with recurrent early or late abortions and with premature delivery irrespective of other aPL.