Candida albicans is an opportunistic pathogen which invades the oral mucosa of individuals infected by the human immunodeficiency virus (HIV). In HIV-associated oral mucosal candidiasis, fungal pseudohyphae penetrate into the cytosol of host keratinocytes, becoming inaccessible to host phagocytes and secretory immune defenses. Calprotectin (L1 antigen, MRP-I/MRP-14 complex) is a molecular complex found in the cytosol of nonproliferative host cells such as neutrophils and oral/vaginal upper strata keratinocytes, a distribution suggesting an important role in defense against fungal infection of the mucosae. We have observed that it is fungistatic and that it inhibits hyphal transformation in C. Albicans. We and others have observed that oral keratinocytes increase expression of calprotectin in lesions which are characterized by a chronic inflammatory cell infiltrate in the subjacent connective tissues such as psoriasis and oral lichen planus. We hypothesize that lymphocytes and monocytes stimulate the expression of calprotectin by keratinocytes. This process may be impaired in HIV+ individuals leading to increased invasiveness by intracellular opportunists such as C. Albicans. The goal of this project is to examine the regulation of calprotectin expression in oral keratinocytes and n oral epithelial cell line, HOK-16B. Specifically, we aim to examine the impact of TH1 lymphokines and monokines on the expression of calprotectin mRNA and protein by these keratinocytes. We will test oral mucosal explants and isolated epithelial strips, as well as organotypic cultures formed by purified oral keratinocytes and HOK-16B. Cultures will be exposed to the supernates derived from stimulated monocyes and lymphocytes. As a control and method for assessing the relative importance of various cytokines, we will deplete cytokine activity from these supernates using standard immunoaffinity or neutralization protocols. We will also culture the tissue samples in the presence of TH1 cytokines (IL-2, Il-12, IFN gamma), monokines (IL-1, TNF alpha, and TGF beta), and TH2 cytokines (IL-4, IL-5, and possibly, IL-6). We will quantify calprotectin expression on the mRNA level using Northern blot and RNAse protection assays for both MRP-14 and MRP-8. Alternatively, should we need greater sensitivity, we will use the reverse transcriptase-polymerase chain reaction (RT-PCR) assay. We will quantify calprotectin expression using the avidin-biotin ELISA and Western blots. We believe these studies will lead to knowledge of the regulation of expression of cytosolic antifungal molecules and provide a foundation for new preventive antifungal therapies in the HIV+ individual.
| 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 |
