Analysis of proteins associated to susceptibility with periodontal desease

Objectives: Polymorphonuclear neutrophils (PMNs) are essential to remove pathogenic bacteria for host defense. PMN's functional abnormalities lead to microbial infection and severe periodontal disease. In this regard, aggressive periodontitis is a destructive form of periodontal disease association with infection by specific Gram-negative bacteria and impaired PMN chemotaxis and phagocytosis. PMN chemotaxis and phagocytosis were influenced by several peptides which expressed on cell surface or intercellular signal molecules. We previously reported that difference of phagocytosis was associated with FcgRIIIb allele. The purpose of present study is to search risk proteins associated with susceptibility periodontal disease in PMNs. Material and Methods: Peripheral blood cells were obtained from healthy volunteers who had undergone the FcgRIIIb genotyping. PMNs were isolated from heparinized peripheral blood cells using a double density gradient purification method. PMNs (2 x 10⁶) were resuspeneded in PBS and stimulated with anti-recombinant 40-kDa outer membrane protein (r40-kDa OMP) of Porphiromonas gingivalis 381 monoclonal antibody for 10 minutes ether at 0° or 37°. For 10 minutes. After twice washing with PBS, PMNs were treated with 2-D lysis buffer for dissolution of proteins. The 2-D lysis buffer consisted of 5M urea, 2M thiourea, 2% CHAPS, 2% SB3-10, and 1% DTT. These cell lysates were removed DNA by centrifuging and then analyzed by gel electrophoresis. The differential spot in 2-D electrophoresis among FcgRIIIb genotypes was detected and extracted peptide. The extracted peptides were analyzed on a Q-TOF mass spectrometer.Results: and conclusion: We have first analyzed an optimal concentration of anti r40-kDa OMP MAb to stimulate PMNs. As a result of having stimulated with anti r40-kDa OMP MAb at 1 ng/ml, differential spots in 2-D electrophoresis were not detected by comparing gels between at 37°and 0°. Further investigation is carried on now.