The basis for my doctoral thesis is the study of the interaction of LPS with human gingival fibroblasts (HGF) with the aim of determining the nature of the initial interaction of LPS with the cells, ie. the prime objective of this study is to determine whether HGF possess functional receptors for LPS. To answer this problem, four specific aims are proposed: I. Determination of the Functional Properties of lipopolysaccharide (LPS) Interaction with Human Gingival Fibroblast. II. Determination of the Presence of LPS Binding Sites on HGF. III. Determination of LPS-Signalling Pathways Involved in Production of PGE2 and lL-1beta by HGF IV. Determine that the HGF """"""""Binding"""""""" Protein(s) Can Act as a """"""""Receptor"""""""" The proposed research strategy will document LPS-induced changes in HGFs. Anti-idiotypic (anti-ld) antibody probes developed to monoclonal antibodies (mAb) raised to the periodontal pathogen Actinobacillus Actinomycetemcomitans (Aa) LPS will then be used to mimic the ability of LPS to stimulate receptor associated intracellular signalling mechanisms. HGFs will be investigated with respect to general alterations of cellular function (cytotoxicity and synesthesis of DNA, RNA, and proteins) in response to incubation with LPS. Thereafter, signalling pathways will be examined to determine the second messengers involved in LPS-induced production of PGE2 and lL-1beta in HGF. Scatchard plot analysis utilizing 125l-labelled LPS will be performed to determine the presence of specific LPS binding sites (their number and binding kinetics) on HGF. Identification of the putative LPS receptor(s) will be determined by crosslinking studies utilizing LPS conjugated to a photoactivatable, heterobifunctional crosslinking agent. Finally, mAb raised to the LPS will be used to immunize a host that does not appear to possess receptors (ie. chickens). Development of internal image, anti-idiotypic antibodies will allow these antibodies to mimic determinants in LPS responsible for binding to the LPS-binding protein. These antibodies will then be analyzed by criteria adopted from signalling pathway and crosslinking studies to determine whether the LPS binding proteins possess functional attributes following interaction with the LPS-like anti-ld antibodies.
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