Fusobacterium nucleatum (FN) cells walls are potent stimulants of human polyclonal B lymphocyte activation (PBA). There is increasing evidence that PBA induced by FN and other gram negative plaque bacteria perpetuates chronic inflammation and is responsible for the unusually large number of activated B cells in periodontitis lesions. As part of our continuing study of nonspecific B cell activation in periodontal diseases, we intend to examine isolated macromolecules from FN cell walls and characterize B cell activation by these components. This proposal has two specific objectives. The first objective is to fractionate cell walls of FN by standard biochemical techniques into the following six fractions: cytoplasmic membranes, peptidoglycan, proteins/lipoproteins, lipopolysaccharide, lipid A, and polysaccharide. These fractions in other gram negative bacteria are potent PBA stimulants and, therefore, are likely candidates of FN-induced PBA. Each fraction will be tested for the capacity to induce PBA in human lymphocyte cultures. Active fractions will characterized and used in subsequent studies. The second objective is to investigate at the cellular level polyclonal B cell activation induced by the purified FN cell wall components. Lymphocytes for these studies will be obtained from peripheral blood of healthy donors. Using monoclonal antibodies, fluorescence activated cell sorting (FACS), and standard rosetting procedures, B and T lymphocytes will be separated and thorougly depleted of contaminating cell types. Initial studies will identify which subset(s) of B cells is activated and the percentage of these cells in peripheral blood. Next, multiple assays of B cell growth and differentiation will be utilized to characterize the events which occur during or following stimulation of B cells. Finally, regulation of PBA by resting or activated T cells will be studied by culturing stimulated B cells with graded doses of unseparated T cells, T cell subsets, or T cell-derived lymphokines.
