Only a few autoantibodies that are more or less specific for RA have been described so far. The rheumatoid factor most often tested for is not very specific for RA, while the more specific antiperinuclear factor for several reasons is not routinely used as a serological parameter. Here we show that autoantibodies reactive with synthetic peptides containing the unusual amino acid citrulline, a posttranslationally modified arginine residue, are specifically present in the sera of RA patients. Using several citrulline-containing peptide variants in ELISA, antibodies could be detected in 76% of RA sera with a specificity of 96%. Sera showed a remarkable variety in the reactivity pattern towards different citrulline-containing peptides. Affinity-purified antibodies were shown to be positive in the immunofluorescence-based antiperinuclear factor test, and in the so-called antikeratin antibody test, and were reactive towards filaggrin extracted from human epidermis. The specific nature of these antibodies and the presence of these antibodies early in disease, even before other disease manifestations occur, are indicative for a possible role of citrulline-containing epitopes in the pathogenesis of RA.

Objective. Since modern treatment of rheumatoid arthritis (RA) is shifting toward aggressive antirheumatic therapy in an early phase of the disease, diagnostic tests with high specificity are desirable. A new serologic test (anti-cyclic citrullinated peptide [anti-CCP] enzyme-linked immunosorbent assay [ELISA]) was developed to determine the presence of antibodies directed toward citrullinated peptides, using a synthetic peptide designed for this purpose.Methods. A cyclic peptide variant that contains deiminated arginine (citrulline) was designed and used as antigenic substrate in ELISA. Test parameters and diagnostic characteristics of the test were studied in patients with and without RA, in patients with various infectious diseases, and in a group of patients from an early arthritis clinic (EAC).Results. Using prevalent RA and non-RA sera, the anti-CCP ELISA proved to be extremely specific (98%), with a reasonable sensitivity (68%). Also, in the EAC study group, the anti-CCP ELISA appeared to be highly specific for RA (96%). In comparison with the IgM rheumatoid factor (IgM-RF) ELISA, the anti-CCP ELISA had a significantly higher specificity (96% for CCP versus 91% for IgM-RF; P ‫؍‬ 0.016) at optimal cut-off values. The sensitivity of both tests for RA was moderate: 48% and 54% for the anti-CCP ELISA and the IgM-RF ELISA, respectively (P ‫؍‬ 0.36). Combination of the anti-CCP and the IgM-RF ELISAs resulted in a significantly higher positive predictive value of 91% (P ‫؍‬ 0.013) and a slightly lower negative predictive value of 78% (P ‫؍‬ 0.35) as compared with the use of the IgM-RF ELISA alone. The ability of the 2 tests performed at the first visit to predict erosive disease at 2 years of followup in RA patients was comparable (positive predictive value 91%).Conclusion. The anti-CCP ELISA might be very useful for diagnostic and therapeutic strategies in RA of recent onset.

A cDNA corresponding to a 10-kD protein, designated extracellular protein 2 (EP2), that is secreted by embryogenic cell cultures of carrot was obtained by expression screening. The derived protein sequence and antisera against heterologous plant lipid transfer proteins identified the EP2 protein as a lipid transfer protein. Protein gel blot analysis showed that the EP2 protein is present in cell walls and conditioned medium of cell cultures. RNA gel blot analysis revealed that the EP2 gene is expressed in embryogenic cell cultures, the shoot apex of seedlings, developing flowers, and maturing seeds. In situ hybridization showed expression of the EP2 gene in protoderm cells of somatic and zygotic embryos and transient expression in epidermis cells of leaf primordia and all flower organs. In the shoot apical meristem, expression is found in the tunica and lateral zone. In maturing seeds, the EP2 gene is expressed in the outer epidermis of the integument, the seed coat, and the pericarp epidermis, as well as transiently in between both mericarps. Based on the extracellular location of the EP2 protein and the expression pattern of the encoding gene, we propose a role for plant lipid transfer proteins in the transport of cutin monomers through the extracellular matrix to sites of cutin synthesis.

Objectives-Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) can both present with an erosive arthritis with the small joints of the hands aVected. Therefore a serological marker would be useful to distinguish between these two diseases at onset. In this study anti-RA33 antibodies, which are found in patients with SLE and RA, and anti-citrullinated peptide antibodies (anti-CCP), which have recently been described as highly specific for RA, were assessed. Methods-Two hundred and thirty one patients receiving long term follow up for SLE were evaluated for arthritis and classified as erosive and non-erosive disease. Sixty six patients were tested for anti-RA33 and anti-CCP antibodies. All the patients were tested for rheumatoid factor (RF) and HLA-DR4 status. Results-Ten patients had erosive disease, six of whom were RF positive (60%), and six anti-RA33 positive (60%), whereas only two were anti-CCP positive (20%). Two hundred and twenty one patients had nonerosive disease, 40 of whom were RF positive (18%), 14 were anti-RA33 positive (6%), whereas only one patient was found to be anti-CCP positive (0.5%). Conclusion-The presence of anti-CCP antibodies may be useful in distinguishing RA from erosive SLE. Anti-RA33 antibodies and RF are unhelpful.

Objective To understand the regulation of anti–citrulline‐containing peptide antibody (anti‐CCP) production in rheumatoid arthritis (RA), production of anti‐CCP by B cells derived from peripheral blood (PB), bone marrow (BM), and synovial fluid (SF) was examined. Methods Purified PB and SF B cells were isolated by negative selection and then cultured in the absence or presence of L–CD40 ligand cells and interleukin‐10 or anti‐CD3–activated T cells. Total IgM and IgM–anti‐CCP were detected after 14 days of culture by enzyme‐linked immunosorbent assay. Enzyme‐linked immunospot assays were performed to analyze the frequency of cells that spontaneously produced IgM–anti‐CCP in BM and SF B cells. Results IgM–anti‐CCP autoantibodies were induced in PB B cells from healthy controls and RA patients following coculture with activated T cells or application of the CD40 activation system, whereas no production could be detected when PB B cells were cultured in the absence of a stimulus. SF and BM B cells from anti‐CCP–seropositive RA patients, but not anti‐CCP–seronegative patients, actively produced IgM–anti‐CCP without stimulation. The frequency of spontaneous production of IgM–anti‐CCP among the IgM‐secreting cells ranged from 2.2% to 25%. Conclusion These results indicate the presence of B cell precursors for anti‐CCP autoantibodies that are able to produce antibodies upon stimulation in the PB B cell repertoire of healthy controls and patients with RA. In contrast, B cells that actively secreted anti‐CCP were specifically present in the BM and SF compartment of anti‐CCP–seropositive RA patients. The local presence of anti‐CCP–secreting cells in the inflamed joints provides evidence for an antigen‐driven maturation of CCP‐specific B cells at the site of inflammation in RA.