IN VIVO STUDIES OF SALIVA ANTIMICROBIAL PROTEINS

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Salivary proteins may influence dental plaque through mechanisms such as cell killing, bacterial aggregation, and adherence to pellicle. In vitro studies have identified saliva proteins involved in each process. However, it has been difficult to show how particular proteins affect plaque composition in vivo. Persons with different saliva protein levels typically are compared. One issue in such comparisons is that saliva proteins form complexes which may function differently than their components. Many proteins also show redundant functions. Those problems make it difficult to isolate the role of salivary proteins in human subjects. This project will employ a novel approach to that problem. Persons who differ greatly for saliva functions will be identified, to test the hypothesis that they differ for saliva and plaque composition. That goal will be addressed through the following specific aims: 1.) Develop microplate methods for quantifying cell- killing, aggregation, and adherence. 2.) Apply those methods to screen unstimulated whole saliva from 170 dental students. Multivariate analysis will be used to group subjects opposite for cell-killing, aggregation, and adherence. 3.) Compare groups by image analysis of native and reducing gels to test the hypothesis that redundancy and complexing will allow salivas with different compositions to exert similar effects. Biotinylated protein overlays will be used to compare proteins and complexes involved in aggregation and adherence. Results from 1-D gels will direct further comparisons using 2-D gels or specific protein assays. 4.) Test hypotheses that saliva proteins exert selective effects in vivo. Plaque will be accumulated on enamel chips placed at sites adjacent to salivary ducts. An arbitrarily- primed PCR method for streptococcal identification will be used to test the hypothesis that indigenous floras differ between groups whose salivas function differently. Saliva coated chips will be treated with anti-idiotype antibodies recognizing an amylase-sIgA complex, to test the hypothesis that redundancy will present inhibition of colonization by cells with a corresponding receptor. Bacteria-coated chips will be treated with the original antibody to the receptor, to test the hypothesis that amylase binding to cells lacking amylase receptors is mediated by amylase-sIgA complexes. Proposed work directly addresses the hypothesis that saliva is an ecological determinant of plaque, and has the potential to validate in vitro studies which suggest that such a relationship exists. By focusing on redundancy and complexing, this project also addresses the question of whether individual proteins exert unique and specific effects in the mouth.