Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in rheumatoid arthritis.

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.

Use of anti-citrullinated peptide and anti-RA33 antibodies in distinguishing erosive arthritis in patients with systemic lupus erythematosus and rheumatoid arthritis.

OBJECTIVES: Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) can both present with an erosive arthritis with the small joints of the hands affected. 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 non-erosive 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.

The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide.

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.

The prognostic value of the antiperinuclear factor, anti-citrullinated peptide antibodies and rheumatoid factor in early rheumatoid arthritis.

OBJECTIVE: To study the prognostic value of the antiperinuclear factor (APF), determined by an indirect immunofluorescence test (IIF) and a recently developed anti-citrullinated cyclic peptide (CCP) ELISA, in combination with rheumatoid factor (RF) status, in early RA (< 1 year). METHODS: A total of 249 participants in a randomized trial of treatment strategies were divided into 4 groups according to their APF (or CCP) and RF status at baseline. Differences in disability, joint involvement and radiological damage over a 3-year period were analysed. RESULTS: APF-IIF results differed from CCP-ELISA in 42 cases (17%); 38 of the 42 had a positive IIF and negative ELISA value. Disability after 3 years did not differ significantly between the RF and APF groups. APF- patients had significantly lower Thompson joint scores compared to APF+ patients (6 vs 24 for CCP-ELISA; 2 vs 24 for IIF). RF+APF+ patients exhibited more radiological damage compared to RF-APF- patients. RF+APF- and RF-APF+ patients had intermediate scores. Within the RF+ and RF- groups, APF+ was associated with more radiological damage and thus yielded prognostic information in addition to RF. In this respect, the results of ELISA and IIF were comparable. Thirty percent of the RF+APF+ patients had a radiological score higher than 45, compared to 13% of the RF+APF-, none of the RF-APF+, and 2% of RF-APF- patients (p < 0.001). In addition, more large joints were affected in APF+ than in APF- patients, while no difference was observed between RF+ and RF- patients. CONCLUSION: APF has prognostic value in addition to RF for joint involvement and radiological damage in early RA. The CCP-ELISA technique for APF assessment may facilitate its use in clinical practice. However, the prognostic value of the two tests lies in their ability to predict mild disease. Reliable identification at baseline of individual patients with progressive disease is still not possible.

Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies.

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.

Kinetic analysis of synthetic analogues of linear-epitope peptides of glycoprotein D of herpes simplex virus type 1 by surface plasmon resonance.

The interaction between mAb A16 and glycoprotein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface-plasmon-resonance biosensor. mAb A16 belongs to group VII antibodies, which recognize residues 11-19 of gD. In a previous study, three critical residues, Asp13, Arg16 and Phe17, of this epitope were identified by screening a phage display library that contained a random 15-amino-acid insert with the antibody. The contribution to binding of these residues in the motif DXXRF was further analyzed by an amino-acid-replacement study of the epitope gD-(9-19)-peptide and of a gD-(9-19)-peptide mimotope, previously obtained by screening the phage display library. Amino acid residues of the motif were replaced by a neutral amino acid residue, an amino acid residue with opposite charge and a corresponding D-amino acid residue. Kinetic parameters of peptide analogues were determined with a surface plasmon-resonance biosensor. The kinetic parameters of the peptide analogues were compared with the kinetic parameters of the interaction between mAb A16 and the epitope gD-(9-19)-peptide. The minimal size of the gD epitope for mAb A16 was also determined in this study. The kinetic constants of the resulting gD-(11-17)-peptide were found to be similar to those of entire gD. The kinetic analysis precisely defined the epitope on gD for mAb A16 to residues 11-17, identified Arg16 as an essential residue and suggested that Asp13 and Phe17 are mainly involved in stabilization of the secondary structure of the peptide.

The use of dedicated peptide libraries permits the discovery of high affinity binding peptides.

The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been obtained from a phage display peptide library (Schellekens et al., 1994). Binding studies with different length variants of this peptide identified a 12mer as a suitable lead compound for our library study. Two incomplete dedicated resin-bound synthetic peptide libraries were generated. Both consisted of 2 x 10(6) 12mers, in which positions were alternately fixed (amino acids identical to the lead sequence) and random. The libraries were screened with mAb A16 and beads with binding peptides were sequenced using Edman degradation. This resulted in a unique peptide binding motif, essentially comprising a 7mer core sequence. Comparison of the sequence of the natural epitope with the binding motif revealed that its sequence was identical to the motif except for one position. Substitution of a methionine in the natural epitope by a tyrosine or a phenylalanine at that position, as dictated by the motif, resulted in a peptide with an affinity for binding to mAb A16 about 50 times higher than that of the natural epitope. Thus, if a lead sequence is available, the use of incomplete, dedicated synthetic peptide libraries provides a fast and powerful tool for the detection of high affinity peptides.

Identification of the core residues of the epitope of a monoclonal antibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a random peptide library.

Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virus type 1 (gD-1). The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD-1 sequence. Synthetic peptides were prepared based on the sequence of the original epitope and the phage-derived epitope. The binding constants (Ka) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology. The RPL-derived peptide and peptide 9-19 of gD-1 had approximately the same affinity for mAb A16. This suggests that those residues within the epitope that are essential for binding were identified. The synthesis of shorter versions of the RPL-derived peptide restricted the binding region to seven amino acid residues. These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution. Immunization of mice with the phage-derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD-1 derived peptide.

Cell-specific expression of the carrot EP2 lipid transfer protein gene.

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.