A Study On The Relationship Between Specificity Of The Autoantibodies To Epitopes On Myeloperoxidase In Neutrophil Cytoplasm And Clinical Damages Of MPO-ANCA Associated Vasculitis

Background myeloperoxidase-specific antineutrophil cytoplasmic autoantibody associated vasculitis, MPO-AAV,mainly including Microscopic polyangiitis, MPA, Granulomatosis with polyangiitis, GPA, Eosinophilic granulomatosis with polyangiitis,EGPA, was not rare in our country. Its clinical manifestation was complex as well as various with rapid progression, and The patients woud die due to delayed diagnosis and treatment. Reporters from clinical,,animal and in vitro experimental studies showed that MPO-ANCA had pathogenicity and theresearches on its epitope identification showed that MPO-ANCA identifying different epitopes on MPO might play different roles in different diseases of AAV.However, by now, there was still no report about systemic research on the relationships between the roles of MPO-ANCA against different epitopes on MPOand the multiplex clinical impairments of MPO-AAV.Objective To investigate the epitope identification specificities of MPO-ANCA in the patients with MPO-AAV and their correlations withtheclinical damagesMethodsBy molecular biological methods involving plasmid recombinant, prokaryotic expression and protein purification, 7 peptide segments from MPO were obtained,which included the light chain(aa 165-272) and 6 peptide segments on the heavy chains: segment H4(aa279-409), segment H5(aa341-474),segment H6(aa410-537),segment H7(aa512-598), segment H8(aa538-745) and segment H9(aa651-745).Their antigenicities were also identified.173 active patients, composed of 165 patientswith primary MPO-AAV and 8 patients with PTU-induced MPO-AAV and all diagnosed for the firsttime,were involved in this study. Their serums and clinical documents were also collected. Clinical impairments of MPO-AAV patients were analyzed. Enzyme linked immunosorbent assay(ELISA) with the 7 recombinant segments as solid phase ligands, was employed to detect MPO-ANCA identification in 173 sera from the selected patients with MPO-AAV and 30 sera from health controls. The specificities of MPO-ANCA to different peptide segmentson MPOwere identified, and their relations with clinical manifestations were analyzed and discussed.Results1. c DNA clones of human MPO protein anditsseven peptide segments were obtainedandthe proteins and polypeptids from them remained theirantigenicities.2. The MPO-ANCA insera of healthy persons could indentify epitopes on H5, H6, H8 and H9.3. Among thesera from 8 PTU-induced MPO-AAV patients, 4 identified peptide segment H8 and 1 identified segment H7.4. Positive rates of MPO-ANCA identifyingall segmentsfrom MPO-AAV patients were higher than those from PTU-secondary patients.5. Among MPO-AAV patients, positive rate of MPO-ACNA identifying light-chain peptide segments and its ELISA-estimated OD values by ELISAfrom the patients with lung damage was significanthigher than thosefrom the patients without lung damage;6. Thedifferences ofpositive rates of MPO-ANCAs againist segment H4 and H5between patients with and without kidney damage were statistically significant and the rates from kidney-involved patients were higher.7. Both the positive rate and OD value for MPO-ACNA against H9 in the patients with renal insufficiency were higher than those from the patients with positivehematuria/positive urine protein only. MPO-ANCA positive rates for the othersegments between themwere not statistically significant.8. Among patients with renal insufficiency, The differences of OD values for the MPO-ANCAs to segment H4, H5, H8 and H9 between acute progressive glomerulonephritis(APGN)and chronicrenal insufficiency(CRI) was statistically significant, OD values in the later group was higher than that in those of the former.9. Comparison of MPO-ANCApositive rates for segment H4 and H5 between APGN and CRI, the positive rates of the later were significanthigher than that of the former.10. For patients with APGN(30 cases), there was a marked positive correlation between the OD value by ELISA for light-chain segment and renal BVAS.11. Between the patients with and withoutjoint injury, The OD values for segment H7 and H8were significantly higherin the formerbut the positive rates of MPO-ANCAs against all the 7 segments had not statistically significantdifference.Conclusion1. 7 peptide segments of MPO proteinwith theirantigenicity were obtained for further research relating to MPO-AAV.2. There were a little ANCA in the sera of few health populations. The epitopes they identified might diffusely distributed on MPO protein(segment H5, H6, H8 and H9,from aa341 to aa754).3. ANCAs within the MPO-AAV patients mainly indentified epitopes on the light and the ends of heavy of MPO protein:light chain, H4, H5, H8 and H9(aa165-272,aa279-474,aa538-745).4. Compared with that fromprimary MPO-AAV patients, MPO epitopes identified by ANCAs from PTU-secondary MPO-AAV patients weremorelimited, andmainly located on the segment Hf(aa538-598). The patients with the second MPO-AAV were younger and had milder disease severity.5. ANCAs identifying the MPO light chain(aa165-272)mightassociate with lung damage of MPO-AAV.6. ANCAs identifying MPO peptide segment H4 and H5(aa279-474)might associatewith kidney damage of MPO-AAV patients. For patients with renal involvement,ANCAs identifying segment H9( aa538-745) might have relationship with their impaired renal function. Amone the patients with renal insufficiency,the patients with ANCA identifying Ha、Hb、Hc(aa279-474)、H8(aa538-745)might be prone to chronic renal insufficiency, in whom, the patients with ANCA identifying the light chian(aa165-272) might be in more severe condition.7. Theantibody level of ANCAs identifying MPO peptide segment H7,H8(aa512-745)might relate to joint injury of MPO-AAV patients.