| BackgroundSince nonspecific clinical manifestations, particularly the lack of specific biomarker, systemic vasculitis is a challenge for rheumatologists to diagnose early and has a poor prognosis with a higher morbidity and mortality. Currently, screening of specific and facilitative biomarkers has become a hot field in clinical studies on systemic vasculitis. During the last years, protein fingerprinting technology (also named as SELDI-TOF-MS) has been applied widely in medical field for the detection of diseases, especially for cancers without traditional tumor makers. An increasing number of cancer-related biomarkers for diagnosis, progression and prognosis have been identified successfully using SELDI-TOF-MS. When about systemic vasculitis, only one study on Wegener's granulomatosis has been reported. On the other hand, in a previous study, we found the positive relation between anti-moesin antibody and the damage of vascular and vasculitis. So for the first time, we carry out studies on Behcet's disease(BD), Takayasu arteritis(TA) and ANCA-associated systemic vasculitis (AASV) using SELDI-TOF-MS and detect the positive rate of anti-moesin antibody in patients with systemic vasculitis and evaluate its clinical significance.Objective1. To detect serum proteomic fingerprinting of patients with systemic vasculitis, and screen differentially expressed proteins, and establish classification tree models or serum biomarker pattern for diagnosis and monitoring on disease activity.2. To screen differential proteins and analyze their relationships with the clinical and immunological patterns of systemic vasculitis.3. To identify the aim proteins by the proteomics technique.4. To detect the positive rate of anti-moesin antibody in patients with systemic vasculitis, and analyze their clinical significance. Method1. Record the clinical dates of all subjects and follow up the experimental group with the clinical record in hospitalization and/or out-patient clinic. The experimental group is composed of 138 patients with systemic vasculitis, including 59 patients with BD, 42 with TA, 37 with AASV. 30 patients with lupus nephritis, 15 patients with coronary heart disease, 115 healthy persons were collected as controls. Serum samples of all subjects were collected when they entered the study.2. Detect all serum samples using SELDI-TOF-MS and obtain serum proteomic fingerprinting, and screen differentially expressed proteins using Ciphergen Biomarker Wizard, and establish classification tree models or serum biomarker pattern for diagnosis and monitoring on disease activity.using using Ciphergen Biomarker Wizard.3. Using Ciphergen Biomarker Wizard, screen differentially expressed proteins and analyze their relationships with the clinical and immunological patterns of systemic vasculitis.4. To identify the aim proteins by weak cation exchange interaction magnetic bead(WCX), SDS-PAGE, SELDI-TOF-MS and MALDI-TOF-MS.5. With recombinant moesin as antigen, sera were screened for the presence of anti-moesin antibody in all subjects by Western blotting and discuss its clinical significance.Results1. The diagnosis classification tree model for BD is composed of m/z 7625.7, m/z 3937.5 and m/z 12555.8 ions, while for TA and AASV are composed of m/z 7618.6 and m/z 8337.8 respectively, the sensitivities as follows respectively: 78.9%,91.7%,91.7%, and the specificities are as follows respectively 80.0%,80.0%,100.0%.2. The peak intensity of m/z 7813.2 gave 74.0% sensitivity and 77.8% specificity for active BD versus remission one at the cut-off point 1.20. The combination of m/z 8690.9,m/z 16508.8 and m/z 4346.8 has a sensitivity of 76.9% for active TA and a specificity of 100.0%, while the combination of m/z 11449.2,m/z 3276.6 and m/z 11671.9 has a sensitivity of 96.7% for active AASV and a specificity of 100.0%.3. The peak intensity of m/z 13751.6 has 84.2% sensitivity and 100.0% specificity for BD patients with systemic involvement at the cut-off point 0.64. 4. The peak intensity of m/z 11689.7 has 80.0% sensitivity and 87.5% specificity for BD patients failed to respond to therapy at the cut-off point 3.04.5. With SELDI-TOF-MS, the aim proteins m/z 28.9 and m/z 16.7 was purified and enriched by WCX magnetic bead, SDS-PAGE, and identified with MALDI-TOF-MS as mixture for the band 28.9kDa and immunoglobulin kappa 1 light chain for the band 16.7kDa respectively.6. Taking recombinant moesin as antigen, sera were screened for the presence of anti-moesin antibody in all subjects by Western blotting. The positive rates are follows: 32.6% (45/138) in systemic vasculitis, including 42.4% (25/59) in BD, 33.3% (14/42) in TA and 16.2% (6/37). 20.0% (4/20) in lupus nephritis, 6.7% (1/15) in coronary heart disease and 5.0% (1/20) in healthy persons.7. No relationship has been found between anti-moesin antibody and the clinical features of BD. While higher rate has been found in TA patients with the type of brachiocephalic artery involvememt versus those with the type of general involvememt.8. As a diagnosis method of systemic vasculitis, the sensitivity and specificity are 32.6% and 89.1% respectively.Conclusion1. Protein fingerprinting technology is a potential tool for discovery of novel biomarker in systemic vasculitis.2. The diagnosis classification tree models, including m/z 7625.7, m/z 3937.5 and m/z 12555.8 for BD, m/z 7618.6 for TA and m/z 8337.8 for AASV, have a certain value for diagnosis.3. Serum biomarker patterns, such as single ion m/z 7813.2 in BD, combination of m/z 8690.9,m/z 16508.8 and m/z 4346.8 in TA , and combination of m/z 11449.2,m/z 3276.6 and m/z 11671.9 in AASV, have significantly predictive value for active disease and better than ERS and CRP.4. Ion m/z13751.6 has a strong predictive value for systemic involvement in BD patients.5. Ion m/z11689.7 is a moderate predictive of patients with BD response to therapy.6. All of these differentially expressed proteins could be aimed as objects in the future studies. 7. Combination of WCX magnetic bead, Tricine-SDS-PAGE and SELDI-TOF-MS, would be a powerful arm to studies on serum differential proteome.8. The positive rate of anti-moesin antibody is higher in patients with systemic vasculitis than in those with coronary heart disease and healthy persons, and equal as patients in lupus nephritis. Among systemic vasculitis, patients with BD and TA have higher positive rate than those with AASV. Anti-moesin antibody has no relationship with the clinical features of BD.9. Anti-moesin antibody has moderate value for the diagnosis of systemic vasculitis. |
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