To understand how microbial colonization results in tissue destruction, a study is proposed of the molecular and cellular mechanisms of normal and microbially-induced collagen breakdown by resident human gingival cells (fibroblasts, endothelial cells, epithelial cells) and by migrant cells of the immune system (PMN leukocytes, macrophages). 1. The component proteases of the fibroblast collagenolytic cascade, including procollagenase activator, collagenase, gelatinase, and, to a limited extent, plasminogen activators will be isolated from culture media by conventional and HPLC methods and used to produce murine monoclonal and rabbit polyclonal antibodies of unequivocal specificity. The homology of collagenolytic cascades in diverse cell types and relationship of isozymes of individual cascade components will be examined by determination of immunologic cross reactivity. Rate limiting plasmin-dependent and independent cascade pathways for procollagenase activation will be examined and the role of individual cascade components in extracellular collagent breakdown determined. 2. The localization and codistribution of collagenolytic proteases in single cells, colonies of cells and tissue specimens will be studied by fluorescent and ultrastructural immunohistochemical methods, and the role of cellular activation in expression of degradative functions will be examined. In addition, the role of collagenolytic cascade proteases in phagocytic collagen breakdown, and the relationship of extracellular and phagocytic collagen breakdown mechanisms will be studied. 3. The ability of suspected periodontopathogens (prototype strains and wild type isolates) and of unknown cultivatable microorganisms from periodontal disease sites to elicit cytostatic or cytocidal responses in host cells will be investigated. Moreover, the ability of selected prototype, wildtype and unknown strains to generate mitogenic and general activated responses in host cells resulting in induction of collagen degradation will be identified. The responsible microbial mediators will be isolated and characterized and the in site of action in the cascade investigated.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE005817-05
Application #
3219629
Study Section
Oral Biology and Medicine Study Section (OBM)
Project Start
1981-09-01
Project End
1987-08-31
Budget Start
1985-09-01
Budget End
1986-08-31
Support Year
5
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Type
Schools of Dentistry/Oral Hygn
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Birkedal-Hansen, B; Moore, W G; Taylor, R E et al. (1988) Monoclonal antibodies to human fibroblast procollagenase. Inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH2-terminal end of the activated enzyme. Biochemistry 27:6751-8
Birkedal-Hansen, H; Taylor, R E; Zambon, J J et al. (1988) Characterization of collagenolytic activity from strains of Bacteroides gingivalis. J Periodontal Res 23:258-64
Villela, B; Cogen, R B; Bartolucci, A A et al. (1987) Crevicular fluid collagenase activity in healthy, gingivitis, chronic adult periodontitis and localized juvenile periodontitis patients. J Periodontal Res 22:209-11
Birkedal-Hansen, H (1987) Catabolism and turnover of collagens: collagenases. Methods Enzymol 144:140-71
Lin, H Y; Wells, B R; Taylor, R E et al. (1987) Degradation of type I collagen by rat mucosal keratinocytes. Evidence for secretion of a specific epithelial collagenase. J Biol Chem 262:6823-31
Etherington, D J; Birkedahl-Hansen, H (1987) The influence of dissolved calcium salts on the degradation of hard-tissue collagens by lysosomal cathepsins. Coll Relat Res 7:185-99
Birkedal-Hansen, H; Taylor, R E; Bhown, A S et al. (1985) Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form. J Biol Chem 260:16411-7
Wells, B R; Birkedal-Hansen, H (1985) Retinoic acid stimulates degradation of interstitial collagen fibrils by rat mucosal keratinocytes in vitro. J Dent Res 64:1186-90