Recent studies suggest that specific periodontal diseases are a result of specific bacterial infections. We hypothesize that these infections are a result of the failure of specific defense effectory mechanisms. It is now known that polymorphonuclear leukocytes (PMN) are critical in the defense of periodontal tissues against the invasive infection by serum-resistant, periodontopathic bacteria. The objective of this study is to identify the bactericidal mechanisms which PMN normally utilize to control specific periodontal pathogens and to examine factors which control the spectra of antimicrobial activity exerted by these bactericidal mechanisms. Bacterial species to be studied include Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus segnis, Haemophilus paraphrophilus, Eikenella corrodens, Capnocytophaga sputigena, Capnocytophaga gingivalis, Capnocytophaga ochracea, Bacteroides intermedius, Bacteroides gingivalis, Wolinella recta, Selenomonas sputigena, Fusobacterium nucleatum, Eubacterium brachy, Eubacterium nodatum, and Eubacterium timidum. Normal human PMN will be examined for their ability to kill these organisms under aerobic and anaerobic conditions and in the presence or absence of heme-enzyme inhibitors in order to classify which type of mechanism is involved in killing by intact cells. Kinetic analysis will be used to discern among the mechanisms involved. PMN bactericidal proteins, peptides, and enzymes will be purified by fast protein liquid chromatography (FPLC) or high pressure liquid chromatography (HPLC). Purity will be confirmed by FPLC, HPLC, and SDS- PAGE. Monoclonal and polyclonal antibodies will be developed to detect bound ligand in binding analyses to inhibit ligand binding and bactericidal activity in studies designed to verify the role of binding in the killing process. Binding will be analyzed by enzymatic, immunoenzyme, or radioimmuno procedures. Bound ligand will be separated from free ligand using microfuge sedimentation. The effect of bacterial surface lipopolysaccharide (LPS) structure will be examined using a series of bacteria with characterized differences in LPS patterns in SDS-PAGE, and using sublethal quantities of a diazaborine-derivative LPS inhibitor. The effect of bacterial exopolysaccharides on binding will be examined. Binding will be characterized by equilibrium binding analysis and Scatchard plots where appropriate. Physical aspects of the binding of bactericidal substances to target organisms will be explored by varying parameters such as pH, ionic strength, counterionic strength, and counterionic valence, and by examining the effects of detergents and hydrogen-bond disrupting agents (such as cyclohexyl-isocyanide) on binding. Binding topography will be determined by electron microscopy and heterobifunctional photoaffinity probes such as radioiodinated sulfosuccinimidyl-2 (P-azidosalicyl - amido)-ethyl - 1, 3'-dithiopropionate. Interactions among the bactericidal substances will be determined by microdilution assay and isobolgram analysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE008161-03
Application #
3221937
Study Section
Oral Biology and Medicine Study Section (OBM)
Project Start
1987-09-01
Project End
1991-12-31
Budget Start
1989-09-01
Budget End
1991-12-31
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Dentistry/Oral Hygn
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Tanaka, D; Miyasaki, K T; Lehrer, R I (2000) Sensitivity of Actinobacillus actinomycetemcomitans and Capnocytophaga spp. to the bactericidal action of LL-37: a cathelicidin found in human leukocytes and epithelium. Oral Microbiol Immunol 15:226-31
Miyasaki, K T; Lehrer, R I (1998) Beta-sheet antibiotic peptides as potential dental therapeutics. Int J Antimicrob Agents 9:269-80
Miyasaki, K T; Iofel, R; Oren, A et al. (1998) Killing of Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia by protegrins. J Periodontal Res 33:91-8
Miyasaki, K T; Iofel, R; Lehrer, R I (1997) Sensitivity of periodontal pathogens to the bactericidal activity of synthetic protegrins, antibiotic peptides derived from porcine leukocytes. J Dent Res 76:1453-9
Miyasaki, K T; Nemirovskiy, E (1997) Myeloperoxidase isoform activities released by human neutrophils in response to dental and periodontal bacteria. Oral Microbiol Immunol 12:27-32
Miyasaki, K T; Qu, X D; Harwig, S S et al. (1995) Identification of CG-1, a natural peptide antibiotic derived from human neutrophil cathepsin G. Adv Dent Res 9:63-6
Flemmig, T F; Miyasaki, K T (1994) Neutrophil lysosomal nonoxidative microbicidal proteins in early-onset periodontitis. Oral Microbiol Immunol 9:272-7
Miyasaki, K T; Bodeau, A L; Pohl, J et al. (1993) Bactericidal activities of synthetic human leukocyte cathepsin G-derived antibiotic peptides and congeners against Actinobacillus actinomycetemcomitans and Capnocytophaga sputigena. Antimicrob Agents Chemother 37:2710-5
Miyasaki, K T; Bodeau, A L; Murthy, A R et al. (1993) In vitro antimicrobial activity of the human neutrophil cytosolic S-100 protein complex, calprotectin, against Capnocytophaga sputigena. J Dent Res 72:517-23
Murthy, A R; Lehrer, R I; Harwig, S S et al. (1993) In vitro candidastatic properties of the human neutrophil calprotectin complex. J Immunol 151:6291-301

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