Porphyromonas gingivalis is an important pathogen associated with destructive periodontal disease and is able to invade the epithelial cell barrier. Its cysteine proteases are recognized as major virulence factors, and in this study, we examined the interaction of the arginine-specific protease with epithelial cells in culture. Three cell lines (KB, HeLa, and SCC4) were incubated with strain W50 culture supernatant; stained with monoclonal antibody 1A1, which recognizes an epitope on the adhesin (␤) component of the cysteine protease-adhesin (␣/␤) heterodimer; and viewed using immunofluorescence microscopy. Within 1 h, the protease traversed the plasma membrane and was localized around the nucleus before becoming concentrated in the cytoplasm after 24 to 48 h. In contrast, the purified arginine-specific heterodimeric protease (HRgpA) rapidly entered the nucleus within 15 to 30 min. This nuclear targeting (i) was seen with active and N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK)-inactivated HRgpA, indicating it was independent of the proteolytic activity; (ii) occurred at both 4 and 37°C; and (iii) failed to occur with the monomeric protease (RgpA cat ), indicating the importance of the adhesin chain of the HRgpA protease to this process. Rapid cell entry was also observed with recombinant catalytic (␣) and adhesin (␤) chains, with the latter again targeting the nuclear area. After 48 h of incubation with HRgpA, significant dose-dependent stimulation of metabolic activity was observed (measured by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), and a doubling of mitotic activity combined with the presence of apoptotic cells indicated that HRgpA may interfere with cell cycle control mechanisms. These effects were seen with both active and TLCK-inactivated protease, confirming that they were not dependent on proteolytic activity, and thus provide new insights into the functioning of this P. gingivalis protease.Periodontal diseases are chronic inflammatory conditions resulting from the host response to microbial challenge and are characterized by the formation of deepening periodontal pockets and the destruction of both hard and soft tissues supporting the teeth. An essential step in initiating any infection involves adherence to, and/or invasion of, host cells by pathogens or their products. As epithelial cells form the first barrier to invasion by bacteria which colonize the gingival crevice, the nature of their interaction with potential pathogens is likely to have a major influence on the progression of disease.Porphyromonas gingivalis is an anaerobic gram-negative bacterium important in destructive periodontal disease whose main ecological niche is the subgingival crevice adjacent to the tooth surface and the epithelial attachment apparatus of the soft tissues. The organism can invade gingival tissue in advanced periodontitis (18) and can be taken up by gingival and pocket epithelial cells, oral epithelial cell lines, and endothelial cells in vitro (13,23,33,40). The nature of the ...

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Despite extensive investigation of cell kinetic parameters in a wide range of normal and abnormal tissues and their potential value in understanding the pathogenesis of many disease states, as well as in prognosis and treatment planning, their application to oral epithelia remains limited. In part A the methods most commonly applied to the study of epithelial cell kinetics, i.e., isotope labelling, mitotic arrest and flow cytophotorimetry are reviewed with special emphasis on their technical limitations, sources of variation and inapplicability to humans in vivo. The ability of in vitro methods to reliably reproduce in vivo cell kinetic parameters is also considered. The application of cell kinetic studies to normal and abnormal oral tissues will be discussed in part B.

Candida albicans has been associated with epithelial hyperplasia in some diseases of oral mucosa and skin but its etiologic role in these lesions is poorly understood. To test its ability to induce epithelial proliferation, the invasive hyphal form was cultured for 5 h and 23 h in chemically defined medium and yeast-free culture supernatants were injected below the buccal epithelium of young adult Sprague Dawley rats. The mitotic activity was assessed using the metaphase arrest technique 11 h and 31 h after supernatant injection. There was a significant increase in epithelial mitotic activity 31 h after injection with 5 h culture supernatants compared to control media indicating that the supernatants have an effect on epithelial cells, possibly by direct action on them.