Computational analysis of high-density peptide microarray data with application from systemic sclerosis to multiple sclerosis.

Auto-antibodies are implicated in the pathophysiology of various autoimmune diseases. High-density peptide microarrays incubated with human serum can detect antibody reactivities against thousands of peptides. This enables the identification of new auto-antigens and the determination of the parts of protein antigens (epitopes) that are recognized by antibody paratopes. We discuss the utility of peptide microarrays to investigate epitope-antibody-recognitions (EAR) from systemic sclerosis to multiple sclerosis. The technology can help to establish reliable diagnostic and prognostic biomarkers employing a combination of antigenic peptides. We describe the specifics of peptide microarray data and present bioinformatic methods for their analysis. Quality control, data pre-processing and the filtering of specific peptides are demonstrated on an example data set. Peptide microarrays representing 24 selected proteins by 3235 overlapping 15mer peptides were used to measure antibodies in serum of 10 patients with limited cutaneous systemic sclerosis (SSC) and 10 healthy blood donors. The data showed a sparse and skewed distribution, and we observed strong individual differences since many peptide sequences were bound by antibodies of only one serum sample. In the sera of the SSc patients, but not of the healthy controls, we found antibodies to three peptides MGPRRRSRKPEAPRR, TPTPGPSRRGPSLGA and GPSRRGPSLGASSHQ that share a similar sequence motif (GP-R/S-RR). These peptides map to two known linear epitopes at the N-terminus of centromere protein A (CENPA), demonstrating the utility of peptide microarrays. Presented experimental and bioinformatic approach can be applied in the same manner for multiple sclerosis research.

Hecker M ，Lorenz P ，Steinbeck F ，Hong L ，Riemekasten G ，Li Y ，Zettl UK ，Thiesen HJ ... -  《-》

Naturally occurring and disease-associated auto-antibodies against topoisomerase I: a fine epitope mapping study in systemic sclerosis and systemic lupus erythematosus.

Simon D ，Czömpöly T ，Berki T ，Minier T ，Peti A ，Tóth E ，Czirják L ，Németh P ... -  《-》

The immunodominant epitope of centromere-associated protein A displays homology with the transcription factor forkhead box E3 (FOXE3).

Some patients with systemic sclerosis express autoantibodies to centromere-associated protein A (CENP-A), but the exact CENP-A epitope is unknown and it is possible that another protein primes these antibodies. This study aimed to define the amino acids recognized by these antibodies and discover other proteins that may be targeted by or may prime them. Peptide Ap(17-30), the immunodominant epitope of CENP-A, was used to purify anti-CENP-A Ig from sera of 8 patients. Anti-Ap(17-30) Ig reacted dose-dependently with Ap(17-30). Panning a phage display peptide library with anti-Ap(17-30) IgG identified two overlapping motifs (m), pt1m and pt8m, permitting patients classification into subgroups based on their having antibodies for only pt1m, pt8m, or both. The pt1m matched residues 53-62 of forkhead box E3; this peptide (FOXE3p(53-62)) behaved similarly in binding and inhibition assays with anti-Ap(17-30) IgG and elicited anti-Ap(17-30) antibodies in mice. This study sets the ground for future investigations on a possible relationship between antibody specificity and clinical manifestations of systemic sclerosis, as well as on a possible role of FOXE3 in priming these antibodies.

Perosa F ，Vicenti C ，Racanelli V ，Leone P ，Valentini G ，Dammacco F ... -  《-》

Circulating anticentromere CENP-A and CENP-B antibodies in patients with diffuse and limited systemic sclerosis, systemic lupus erythematosus, and rheumatoid arthritis.

OBJECTIVE:To determine the disease sensitivity and specificity of testing for autoantibodies against 2 of the 3 main human centromere antigenic components, CENP-A and CENP-B (recombinant, expressed in baculovirus). METHODS:ELISA with CENP-A and CENP-B antigens were used to test 45 sera showing a centromere pattern by immunofluorescence (IFA) and sera from 96 patients with systemic sclerosis (SSc), subdivided into diffuse (dSSc) and limited (lSSc) forms. For controls, the same tests were performed on sera from 100 patients with rheumatoid arthritis (RA), 100 with systemic lupus erythematosus (SLE), and 50 random blood donors. Sera from all the patients with SSc were also tested for the presence of anti-Scl70 antibody by ELISA (bovine antigen), and for pattern and titer by IFA (HEp-2 cells). RESULTS:Of the 45 IFA positive sera, 93% were positive for anti-CENP-A and 91% for anti-CENP-B. There was a very good quantitative correlation between the antibody levels against these 2 centromere components (r = 0.597; p<0.001). Anti-CENP-A and B were found in 48% of patients with lSSc, and in 11% and 9%, respectively, of those with dSSc. The difference in the frequency of anti-CENP-A between the 2 patient groups was significant (chi-squared, p<0.001). Similar levels of anticentromere staining pattern by IFA were observed for these 2 groups. Anti-Scl70 was elevated in 8% of lSSc and 25% of dSSc patients; this difference was also significant (chi-squared, p = 0.02). Neither CENP-A nor CENP-B reacted with IgG from SSc patients containing anti-Scl70. The frequency of abnormal levels in patients with SLE and RA was, respectively, 11% and 3% for anti-CENP-A and 4% and 3% for anti-CENP-B. The reaction of IgG from SLE and RA patients with CENP-A was not inhibited by histone H3, i.e., it was not due to recognition of the histone-like domain in CENP-A. Thus, when 96 SSc patients were compared to 200 patients with RA and SLE, the disease specificity of anti-CENP-A and B was 93% and 96.5%, respectively. CONCLUSION:In addition to IFA, ELISA tests for CENP-A and CENP-B yield results with similar sensitivity and specificity for the diagnosis of SSc. CENP-A and CENP-B are primarily associated with lSSc. In SSc the autoantibody response is directed simultaneously and with similar amplitude against these 2 components of the centromere structure, whereas in other autoimmune diseases the response is directed mainly against one of the 2 components.

Russo K ，Hoch S ，Dima C ，Varga J ，Teodorescu M ... -  《JOURNAL OF RHEUMATOLOGY》

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3.

Recke A ，Regensburger AK ，Weigold F ，Müller A ，Heidecke H ，Marschner G ，Hammers CM ，Ludwig RJ ，Riemekasten G ... -  《Frontiers in Immunology》