The overall goal of this research project is to determine the role of fibronectin (FN) in the attachment of bacteria to the mucosal surface of the oropharynx. To pursue these studies logically, the experiments proposed in this first application are focused on characterizing the interaction of FN with human buccal epithelial cells. Specific efforts will be directed toward: (1) examining buccal cells for content and distribution of FN bound to their surfaces naturally and after experimental treatment, (2) studying the specific characteristics of the binding of exogenous FN to the buccal cells, and (3) determining the major functional domain of plasma FN that mediates binding of the intact molecule to buccal epithelial cells. Pools of buccal epithelial cells will be examined in assays of the amount of labeled anti-FN antibody that binds to the cells, the amount of labeled FN that binds to the cells and the morphological distribution of anti-FN antibodies that bind to the cells. Several different chemical and enzymatic treatments will be used to manipulate FN levels on these cells. Binding studies will be performed to determine the specificity, affinity and number of FN binding sites on buccal epithelial cells. We will pay particular attention to the effects of pH, ionic strength and divalent cation composition and concentration. Well defined FN fragments, and other specific ligands known to interact with FN, will be tested for their ability to inhibit the binding of intact FN to buccal cells. The ability of defined FN fragments to bind to the cells directly will also be tested. Further, defined monoclonal antibodies will be tested for their ability to inhibit the binding of FN to the cells. We believe the proposed experiments on characterization of FN-buccal epithelial cell interactions will be an important step in our efforts to understand the role of FN in bacterial adherence.
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