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Mechanisms of antibody mediated attenuation of bone loss

Project IV. of Humoral Immunity and Periodontal Disease

PI: Richard P. Darveau, Ph.D., Research Associate Professor, Department of Periodontics, School of Dentistry, University of Washington.

The major thrust of Project IV is to examine the role of antibody in the attenuation in bone loss observed in vaccinated Macaca fascicularis in the ligature induced periodontitis model. Immunized monkeys still harbored millions of P. gingivalis, motivating us to examine non-opsonic mechanisms of antibody mediated bone loss. We have reported previously that rabbit antibodies to P. gingivalis and sera from our Experiment 1 immunized monkeys have the capacity to block LPS-induced activation of monocytes and production of PGE2. We now propose to evaluate the immune sera for activity that blocks cysteine protease-mediated P. Gingivalis adherence and degradation of biologically important proteins such as antibody, complement, and inflammatory mediators such as IL-8.

1. Degradation of IL-8 Assay

IL-8 is a key inflammatory mediator that has been shown to be present in healthy and diseased clinical tissue. In healthy tissue, it functions as a chemokine directing, by a concentration gradient, the orchestrated movement of neutrophils from the vascular compartment to the site of bacterial colonization. In diseased tissue the concentration gradient is missing being replaced by discrete "islands" of IL-8 expression. One possible explanation for this disruption in IL-8 status is localized degradation by cysteine protease. Consistent with this, IL-8 is a substrate for cysteine protease. We have therefore chosen IL-8 as a substrate in our assay to examine anti-cysteine protease immune sera obtained from immunized monkeys. Although we suspect that degradation of IL-8 may be clinically relevant, it also serves as a marker of the catalytic function of cysteine protease regardless of its clinical significance. An assay was developed that measured the ability of whole P. gingivalis to degrade IL-8 after co-incubation for 18 hours . P. gingivalis at concentrations of 1 X 106 and greater completely destroyed IL-8 detection by ELISA. Prior heat inactivation of the P. gingivalis resulted in no loss of IL-8 ELISA reactivity suggesting that the catalytic property of the bacteria was necessary for activity. Additional experiments to confirm degradation by immunoblot analysis are being performed.

2. Cysteine protease mediated adherence assay

An adherence assay was developed that detected the specific adherence of either purified KGP or RGP to human endothelial or primary gingival epithelial cell extracts. We reasoned that the adherence function of KGP and RGP could be detected by direct examination of KGP or RGP binding to extracts of host defense cells. The adherence assay employed monoclonal antibodies directed against KGP and RGP to detect the enzyme binding. Monoclonal antibodies were made by standard procedures employing P. gingivalis cell wall as an immunizing antigen and screening against purified KGP and RGP. A variety of different antibodies was obtained. Initial experiments with whole endothelial and epithelial cells indicated specific binding occurred, yet due to poor sensitivity concentrated cell extracts were employed. A monoclonal antibody, designated 5C4, which specifically bound KGP, and did not cross react with RGP, was employed to detect binding of KGP to endothelial cell extracts. Binding was KGP dose dependent, saturable, and no binding was observed when heat denatured endothelial cell preparations were employed (KGP control). Although not shown, an antibody designated 5G11, that specifically reacted with RGP (did not cross react with KGP) has been employed to demonstrate saturable binding of RGP. In addition, since these antibodies did not cross react, experiments were performed which demonstrated that KGP and RGP competed for the same binding site, demonstrating specificity in binding. In addition, although not shown, similar binding characteristics were observed when primary gingival epithelial cell extracts were employed. . Binding to either non-treated (KGP) or heat denatured extracts (KGP control) was determined by the addition of monoclonal antibody 5C4 in an ELISA format. Antibody was detected by alkaline phosphatase conjugated anti-mouse Ig.

The IL-8 degradation assay will now be used to determine if the monkey immune sera will block the degradation of IL-8 by P. Gingivalis. These experiments serve as an example of others we propose to perform involving cysteine protease degradation of other biologically important molecules. The adherence assay will be used to determine if the monkey immune sera will block the interaction of KGP and RGP to host defense cells

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