EFFECTS OF LONG TERM USE OF ANTIRETROVIRAL THERAPY ON ORAL INNATE IMMUNITY - DESCRIPTION (provided by applicant): Oral epithelial cells play a critical role in innate mucosal immunity. To maintain tissue integrity, the cells undergo a differentiation resulting in the expression of keratins. Epithelial cells also produced antimicrobial peptides including human a-defensins (hBDs) and secretory leukocyte protease inhibitor (SLPI), and cytokines that play significant roles in the regulation of oral infection. Since prevalence of oral candidiasis is decreased, but oral wart is increased with use of antiretroviral therapy (ART), the immune reconstitution and immune response at mucosal sites may differentially affect epithelial function in defense against infection. It is not known if long-term use of ART can alter epithelial differentiation, structure and function in oral innate immunity. Therefore, the overall objective of this study is to determine changes in expression of keratins, hBD2, and SLPI mRNA and their salivary proteins, as well as oral tissue cytokine profiles and salivary cytokine levels in HIV-infected patients with ART compared with those without ART, and non-HIV healthy subjects. The study is comprised of three aims: 1) To determine changes in epithelial structure and pattern of keratin expression by long term use of ART, 2) to determine if hBD2 and SLPI expression in oral epithelial tissue and saliva are affected by long term use of ART, and 3) to determine changes in pro-inflammatory cytokine profiles in oral epithelial tissues and saliva by long term use of ART. One hundred HIV subjects on ART will be enrolled. One hundred HIV subjects without ART with the same range of CD4 count and viral load, and 50 non-HIV healthy subjects with match age and gender will serve as controls. History taking and oral examination will be performed. Tissue biopsy using an 8-mm diameter punch on oral buccal mucosa will be conducted. One half of the tissue will be stained by hematoxylin and eosin (H&E) and immunohistochemistry method. Pattern of keratin expression will be analyzed and correlated with the use of ART. The other half of the tissue will be used for RNA isolation. The expression of hBD2 and SLPI mRNA will be determined by using a reverse transcriptase polymerase chain reaction (RT-PCR) amplification assay and Quantitative Polymerase Chain Reaction (Real-Time PCR) assay. Whole saliva will be collected, and salivary levels of hBD2 and SLPI proteins will be determined by using enzyme-linked immunosorbent assay (ELISA). Expression of hBD2 and SLPI mRNA in tissue, and their salivary protein levels will be correlated with the advent of ART. Cytokine profiles in tissue from selected representative subjects will be analyzed by microarray. Levels of cytokines of interest in saliva will be determined by ELISA. Salivary cytokine profiles involved in oral innate immunity against Candida and human papilloma virus will be analyzed.
