Actinobacillus actinomycetemcomitans (aa) has been implicated as a possible etiologic agent in juvenile periodontitis. This organism produces several biologically-active products, including a potent leukotoxin which kills isolated human neutrophila (PMNs). It is conceivable that the leukotoxin may be a virulence factor by impairing antibacterial defense systems in the gingival crevice. Our experiments are concerned with the biology of the Aa leukotoxin. We have purified this molecule by molecular sieve and ion exchange chromatography. The isolated toxin is extremely labile and is best stored at -70 C in 10 mM phosphate buffer (pH 6.)) or lyophilized and kept at -20 C. It is inactivated by heat, various proteolytic enzymes, oxidizing and thiol reducing agents. We have been successful in generating monoclonal antibodies against the toxin; these antibodies neutralize the toxin and also serve as useful reagents for identifying toxin-associated peptides in extracts of Aa. We plan to use monoclonal antibodies as an alternate way to purify the leukotoxin (immunoaffinity chromatography) and to determine how the leukotoxin interacts with PMNs. We have also been able to isolate and identify Aa mutants which are defective in the expression of leukotoxic activity. These organisms will be important in studies concerning mechanisms of toxin-PMN interaction, especially in terms of the relationship of toxin subunit structure to biologic activity. Recently, we found that sub-lethal concentrations of Aa leukotoxin were able to alter PMNs so that they responded more vigorously to phagocytic stimulants, such as zymosan. We plan to enlarge upon these observations to ascertain if additional PMN functions as well as the behavior of other cells (e.g., lymphocytes, fibroblasts and endothelial cells) are also affected by low doses of the purified leukotoxin. These studies should provide fundamental information on the pathobiology of Aa in the development of juvenile periodontitis.