As an essential first step in understanding the interaction between subgingival species and gingival epithelial cells, is to determine which species are present on or in epithelial cells recovered from the human gingival crevice or periodontal pocket.
The Specific Aims of this project will be as follows: 1) to isolate and identify cultivable bacterial species which are attached to or invade into epithelial cells derived from the gingival crevice or periodontal pocket, 2) to seek uncultivable species which invade into or adhere to epithelial cells, 3) to enumerate predominant bacterial species, using DNA probes, which are on or in epithelial cells derived from subjects of different age and racial/ethnic groups and different states of health or disease and 4) to use light and electron microscopy to determine the species, numbers and location (on and/or in) of bacteria associated with crevicular epithelial cells. Epithelial cells will be isolated from samples taken from the gingival crevice or periodontal pocket of subjects of different ages, gender and racial/ethnic group. Initial cultural microbiology studies will be performed on samples from 32 subjects. Cultivable bacteria recovered from such samples will be used to define a set of DNA probes for subsequent Specific Aims. PCR techniques will be used to seek uncultivable species from harvested crevicular epithelial cells and DNA probes to such species added to the battery defined by cultural studies. The complete set of probes will be used to examine samples from a larger group of 60 Black, 60 Hispanic, 60 Asiatic and 60 White subjects. Each racial/ethnic group will consist of equal numbers of male and female, young and old and periodontally healthy and periodontally diseased subjects. 4 to 8 epithelial call samples will be taken from each subject and assayed for their content of up 50 species using the DNA probes. Finally, epithelial cell samples will be examined by light and electron microscopy for the number and location of bacteria of different morphotypes. Bacterial cells will be identified in such samples by immunocytochemical and/or in situ hybridization techniques.
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