chuan2@uky.edu

Genetic finger-printing of oral bacteria: PCR-based genetic finger-printing is carried out to characterize the bacterial ecology of the biofilm in the oral cavity. AP-PCR, REP-PCR, ERIC-PCR, and BOX-PCR are used extensively to characterize the genetic differences among oral bacterial species and strain. Currently, eight major oral bacterial species are the focus of this project. The genomic fingerprinting profiles of 8 oral bacteria species, Porphyromonas gingivalis; Prevotella intermedia; Actinobacillus actinomycetecomitans; Bacteroides forsythus; Treponema denticola; Peptostrepococcus micro; Fusobacterium nucleatum; Selenomonas noxia, and multiple strains of each species are being investigated. Our analysis indicates that these PCR-based genetic fingerprinting is very useful in differentiating species and strain differences. PCR amplification from the genomic DNA of these bacteria produces unique amplicans (DNA fragment). All 8 species and multiple strains can be successfully differentiated at the genetic level. The combined PCR approach can be a valuable tool for clinical research in oral health.  Similar techniques are used to characterize the plaques isolated from gingivitis and periodontal sites. Genetic profiling of the dental plaque can generate valuable information about the complexity of the ecology and bacterial diversity during the disease process.

Interaction between the oral bacteria and HIV-1 infected latent T cells: At the end of 2000 it was estimated that over 36 million people were living with the human immunodeficiency virus. The introduction of HAART regimens (Highly Active Antiretroviral Therapy) has significantly modified the course of HIV disease, with longer survival rates and improvement of life quality in HIV-infected individuals. However, complete eradication of HIV infection cannot be achieved with currently available antiretroviral regimens. This is primarily results from the establishment of a pool of latently infected CD4+ T cells during the very earliest stages of acute HIV infection1 that persists with an extremely long half-life, even with prolonged suppression of plasma viremia to <50 copies/mL2-5. This persistence of HIV-1 within resting CD4+ T cells constitutes a major obstacle in the control of HIV-1 infection. Various exogenous stimuli have been shown to exacerbate HIV-1 infections, including Gram-negative bacteria and LPS, or cytokines/chemokines induced by these stimuli. Evidence of viral recrudescence, as measured by plasma HIV RNA levels (viral load), generally reflects a loss of responsiveness to HAART. Periodontal disease is a chronic, Gram-negative bacterial infection. We propose in this continuation grant that the presence of this chronic infection may serve as a catalyst for the subsequent loss of responsiveness to HAART by exacerbating HIV-1 production from latently infected cells. This research is a continuation of previous research examining microbial characteristics of HIV-infected patients. Based upon these studies, this investigation will focus on an innovative approach to provide a biologic basis for a potential link between periodontal infections and HIV-1 recrudescence. Aims are developed to document the capacity and variation in the ability of oral microorganisms to activate HIV-1, to evaluate the ability of oral microorganisms to stimulate resident host cells releasing mediators that would activate HIV-1, and to determine if the activation requires antigen specific recognition of oral microorganisms. The potential interaction between oral bacterial with the HIV latently infected T cells are studied. This research is to understand if there is such interaction and potential reactivation of these HIV latently infected T cells. The study can be valuable for the care and treatment for HIV-infected patients.


Single-nucleotide genetic polymorphism of inflammatory gene: The predominant population of eastern Kentucky is Caucasian, and recent data from the Division of Neonatology of the University of Kentucky Medical Center reveals that 73% of admitted patients are white, 17% black and 6% Hispanic. During the last 2 decades, the incidence of preterm/lowbirthweight (PTB/LBW) infants has actually increased in the U.S. Public health data from Appalachian counties of Kentucky show rates of PTB/LBW that are 50-100% higher than the national averages. Additional birth data recently compiled by Dr. Arthur Evans (Professor and Director, Maternal Fetal Medicine Division; Department of Obstetrics and Gynecology) indicates that, in the population managed by this "high risk" clinic, 24.7% of births are preterm (<37weeks) and 18.8% are low birth weight (<2500g). Recent comparisons with other rural and metropolitan populations in medical centers with ethnically diverse, socio-economically disadvantaged patients, suggest that the rates of PTB.LBW in the University of Kentucky Medical Center are similar to the high rates seen in ethnic minority groups throughout the country (University of Mississippi Medical Center, low-income African Americans, 26.6% preterm delivery, 20.2% low birth weight; Harlem Hospital upper Manhatten, New York, low socio-economic African American and Latino populations, 19.5% preterm delivery, 19.5% low birthweight; Hennepin County Medical Center, Minneapolis, ethnically diverse with nearly 50% African American, 19.5% preterm delivery, 14.5% low birthweight). Since it appears that a Caucasian, socio-economically disadvantaged population from Central and Eastern Kentucky can have a similar prevalence of PTB/LBW as non-Caucasian minorities, examination of a genetic contribution to potential infectious and/or inflammatory processes in this population would seem appropriate and would add considerably to our understanding of the contributing "risk factors" for preterm and low birth weight in these populations. The study will be to develop the necessary genetic and molecular biology tools to enable investigations of single-nucleotide polymorphisms (SNPs) for inflammatory genes.

Huang C, De Sanctis GT, O'Brien PJ, Mizgerd JP, Friend DS, Drazen JM, Brass LF, and Stevens RL. Evaluation of the Substrate Specificity of Human Mast Cell Tryptase bI and Demonstration of Its Importance in Bacterial Infections of the Lung. 
2001; Journal of Biological Chemistry 276: 26276-26284

Huang C, Morales G, Vagi A, Qiu WT, Chanasyk K, Burklow C, Stevens RL. Formation of enzymatically active, homotypic, and heterotypic tetramers of mouse mast cell tryptases. 
2000; The Journal of Biological Chemistry 275:351-358.

Humphries D, Wong WG, Friend DS, Gurish MF, Qiu WT, Huang C, Sharpe Ah, Stevens RL. Heparin is essential for the storage of specific granule proteases in mast cells. 
1999; Nature 400,769-772.

Huang C, Li L, Krilis SA, Chanasyk K, Tang Y, Stevens RL. Human Tryptase a and b/II are functionally distinct due, in part, to a single amino acid difference in one of the surface loops that forms the substrate-binding cleft. 
1999; The Journal of Biological Chemistry 274:19670-19676.

Huang C, Friends DS, Qiu D, Wong GW, Morales G, Hunt J, Stevens RL. Induction of a selective and persistent extravasation of neutrophils into the peritoneal cavity by the tryptase mouse mast cell protease 6. 
1998; Journal of Immunology 160:1910-1919.