Martin Levine - US grants

Studies in humans by the principal investigator have shown that precipitating serum antibodies to oral bacteria are associated with significant differences in dental caries severity and accumulation of bacterial plaque. One antibody precipitates with an epitope found on macromolecules from Actinomyces spp. and the other with the D-alanyl ester moeity of glycerol teichoic acids from Streptococci and Lactobacilli. Non-precipitating serum antibodies to the polyglycerol phosphate moeity of the latter antigen are not associated with caries severity. The D-alanyl ester epitope is stable at pH 5, but hydrolyses rapidly at physiologic pH. The major specific aim of this study is to quantitate the serum and salivary antibody response to each epitope by a sensitive enzymoimmunoassay in order to examine the relationships between epitope-specific local and systemic antibody responses, age, caries severity and plaque accumulation. A secondary aim is to elucidate a role for genetic constitution in response development. Serum IgG and secretory IgA originate from different modes of antigen exposure and respectively indicate systemic or local immune responses. The respective antigens, in culture filtrates or cell extracts of S. mutans GS-4 and A. viscosus ATCC 19246, will be coated on wells of microplates and the antibody content of serum or salivary samples determined. The amount of antibody bound in the absence or presence of appropriate haptens will be measured with alkaline phosphatase covalently conjugated to goat IgG specific for either human IgG or the secretory chain of human IgA. The difference in binding before and after inhibition will reflect epitope-specific antibody binding and will be quantified by comparison with an appropriate, strongly reactive serum. The relationship of the epitope-specific antibody binding contents to precipitin responses will be examined by multiple discriminant analysis. The interrelationship of the respective antibody contents to each other, age, caries severity, length of fluoride exposure, plaque accumulation and gingivitis will be examined by multiple regression analysis over the whole population and within various serum and saliva antibody response groups. Differences in caries severity or expression of HLA-A, B, C and DR specificities respectively by antibody response group will be examined by multiple discriminant analysis. The findings should identify new factors associated with caries protection and susceptibility.

DESCRIPTION: (Adapted from investigator's Abstract) Some patients cannot control tooth attachment loss (periodontitis) after intensive oral hygiene therapy. Recently, successful therapy was found to depend on an increased prevalence of "beneficial" relative to "pathogenic" bacteria in gingival sulci. A simple procedure based on this finding is proposed: an enzyme immunoassay to measure the concentration of specific for Actinomyces spp. (A-Ab), a major component of the beneficial flora. The specific aim of this Phase 1 proposal is to compare the A-Ab concentration in patients who are either refractory or responsive to therapy. High and low A-Ab concentrations will be separated by a cutoff value derived from antigen/antibody precipitation and enzyme immunoassay. Near this value, the determination of high/low A-Ab response requires measuring a control antibody, to unstable D- alanyl esters of lipoteichoic acid from streptococci (S-Ab). The Phase 2 aims are to determine: (1) whether therapy alters A-Ab concentration in longitudinal studies; (2) whether A-Ab concentration affects treatment needs; and (3) whether S-Ab content can be measured with stable surrogate antigens. The proposed assay should identify refractory patients faster and more accurately than clinical skills, or assays for pathogenic bacteria or host-produced destructive enzymes at gingival sulci. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE

DESCRIPTION (provided by applicant): In chronic (formerly adult) periodontitis, bacteria destroy the dento-gingival junction, especially its Dentally attached (DAT) cells, keratinocytes that are activated by persistent trauma from mastication and oral hygiene. These rapidly dividing cells form the internal basement lamina of junctional epithelium (JE) and maintain the epithelial attachment. The DAT cell coronal extremity grows on interstitial fluid that transudes through the JE to the base of a gingival sulcus, near the site of infecting bacteria. We posit that lysine decarboxylase (LDC), a bacterial enzyme in the sulcus, depletes the transudate of lysine, starving the DAT cells. A small amount of LDC causes a cascade of events predisposing to loss of Dental attachment and colonization of the sulci by well-known periodontopathogens. Oral hygiene controls this colonization, but some adults are refractory and discriminated by increased Capnocytophaga spp. and other LDC producers. The activity of LDC from the bacteria in gingival sulci may be critical for determining whether chronic periodontitis can be controlled by current (oral hygiene-based) therapy. The Aims of this study are to: 1) develop new assays for measuring the amount of active LDC in the gingival microbiota (plaque); and 2) use these assays for measuring active enzyme in the sulci from refractory and successfully treated patients. LDC activity will be determined by two methods. The first will determine enzyme activity (cadaverine synthesis) in the presence of a saturating amount of substrate (lysine) in extracts of whole-mouth plaque from refractory and successfully treated patients. The second will use H-a-difluoromethyl DL-lysine (DFML), a suicide inhibitor that forms an adduct at the catalytic center of LDC. DFML is not commercially available and it will be synthesized unlabeled and as a radioactive derivative for this project. Radiolabeled adduct formation should be inhibited by an excess of unlabeled L-DFML or lysine and the amount of radioactivity on blots will indicate the amount of active enzyme after incubation with plaque extracts. The application predicts that there will be more enzyme activity in refractory than in successfully treated patient plaque. The results will indicate the relationship of LDC activity in the gingival sulcular microbiota to therapeutic outcome, and whether LDC inhibitors such as DFML might have utility as a new, alternative pharmacotherapeutic for preventing and controlling chronic periodontitis.