IN VIVO STUDIES OF SALIVA ANTIMICROBIAL PROTEINS

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Attempts to relate saliva levels of lysozyme (Lz), lactoferrin (Lf), salivary peroxidase (Spx), and secretory IgA (sIgA) to oral health and ecology have yielded inconsistent results. It has been difficult to define measures of the action of these antimicrobial proteins in vivo, and it also has been difficult to devise controls for factors which may influence those actions. Proposed research will address those questions in clinical studies of Lz, Lf, Spx, and sIgA binding to Streptococcus sanguis monolayers placed in the mouth. Studies will be designed with controls for variation due to oral clearance of antimicrobial proteins, interaction between antimicrobial proteins, bacterial affinity for antimicrobial proteins, and localization of antimicrobial proteins in different oral sites. Research will proceed in stages defined by these specific aims: (1) Combine electronic recording of swallowing activity with sensitive enzyme-linked immunoassay (ELISA) to develop a protocol for quantitation of Lz, Lf, Spx, and sIgA secreted during single swallowing cycles. (2) Combine that protocol with multivariate statistical methods to screen a population of 200 or 40 subjects with high or low levels of Lz, Lf, Spx, and sIgA to be recalled for in vivo studies. (3) Provide an outcome variable for in vivo studies by developing ELISAs to measure Lz, Lf, Spx, and sIgA bound to S. sanguis monolayers formed on bovine enamel chips. (4) Use chip ELISAs to screen a panel of S. sanguis isolates for two strains which differ greatly in binding of Lz, Lf, Spx, and sIgA. (5) Carry out in vivo studies by placing chips with S. sanguis monolayers on anterior and posterior teeth in upper and lower jaws. The binding of Lz, Lf, Spx, and sIgA to high and low affinity strains will be compared between subjects previously found to secrete high or low levels of those proteins in a swallowing cycle. The hypothesis is to be tested is that saliva levels of Lz, Lf, Spx, and sIgA determine binding of those proteins to oral bacteria in vivo when variation attributable to oral sites, bacterial strains, saliva clearance, and interaction between antimicrobial proteins is controlled. Results may clarify the interpretation of previous clinical studies. Approaches developed also will have broad applications for future studies of antimicrobial protein action in vivo.