ESTABLISHMENT OF A CLINICAL TREATMENT SYSTEM FOR PERIODONTAL DISEASE

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Periodontal disease is a common inflammatory oral disease characterized by acute progressive lesions of periodontal tissues, excessive leukocyte infiltration, and occurrence of a characteristic microflora. Porphyromonas gingivalis is a Gam-negative anaerobic bacterium that is implicated as a major etiologic agent of some types of periodontitis. This bacterium produces a unique type of cysteine proteinases referred to as gingipains in both cell associated and secretory forms. Gingipains consist of arginine-specific cysteine proteinases (Arg-gingipains, Rgp) and lysine-specific cysteine proteinase (Lys-gingipain, Kgp). The cell-associated gingipains comprise the majority (80%) of Rgp and Kgp activities and thus believed to be responsible for the virulence of the bacterium. Accordingly, the characterization and subsequent control of the cell-associated gingipain complex are thought to be the most important promising therapeutic approaches for periodontitis and related systemic disorders i … More ncluding atherosclerosis. Furthermore, we have shown previously with various P.gingivalis mutants deficient in Rgp- and Kgp-encoding genes that both enzymes play critical roles in most of the virulence of the bacterium and thus indicated that potent inhibitors of gingipains should be useful tools to assess the contribution of their proteolytic activities to the virulence of the bacterium and to facilitate the development if new therapeutic approaches to periodontal diseases. In this research project, thus, we have purified and characterized a major form of cell-associated gingipain complex from the bacterium. The complex comprised the catalytic domains and hemagglutinin domains of both rgpA and kgp gene products. Moreover, lipopolysaccharide (LPS) and phospholipids were associated with the complex. The complex significantly degraded human type I collagen and elastin and strongly disrupted viability of human gingival fibroblasts and umbilical vein endothelial cells with an efficiency which was higher than that of the monomeric gingipains. Importantly, the native complex produced only a small amount of nitrogen dioxide, TNF-□ and IL-6 by macrophages, whereas the heat-denatured complex resulted in increased production, indicating that the functional domains of LPS are structurally masked by the complex proteins. The results indicate the importance of the complex in evasion of host defense mechanisms as well as host tissue breakdown. Furthermore, we designed and synthesized a series of peptides analogues able to inhibit either Rgp or Kgp on the basis of the cleavage site specificity of histatins by each enzyme. Among this series of compounds, we found that KYT-1 and KYT-36 had the most potent and selective inhibitory activities of Rgp and Kgp, respectively. We have also demonstrated that these inhibitors are useful in assessing to what extent the proteolytic activities of Rgp and Kgp contribute to biological activities of P.gingivalis, and indicated that they should facilitate the development of new approaches to periodontal diseases. Less