Our goal is to understand the mechanisms of allergic inflammation in the eye. Specifically, in acute ocular allergic inflammation, FcERI-mediated mast cell activation and release of specific cytokines activate conjunctival epithelium resulting in recruitment, adherence, and degranulation of neutrophils and eosinophils. In chronic ocular allergic inflammation (such as Atopic Keratoconjunctivitis [AKC]), colonization with Staphylococcus aureus (a bacteria thought to play a role in AKC) in the presence of mast cell activation results in polyclonal proliferation of T cells and changes in the cytokine environment allowing sustained mast cell activation (i.e., IgG-mediated via FcgammaRI) in the absence of allergen. Mechanisms to be examined, in vitro, involve in the specific cascade of events resulting from mast cell activation (via cytokines, FcEpsilonR1, FcgammaR1) including: mast cell cytokine release (TNFalpha IL-1beta, IFNgamma IL-12) and surface receptor expression (Kit, FcER1, ICAM-1, HLA-DR, DP, DQ, Toll-like receptor 2) activation of epithelial cells (release of IL-8, RANTES, soluble TNF receptor 1; upregulation of ICAM-1, HLA-DR, DP, DQ, TNF receptor 1), adhesion and degranulation of eosinophils and neutrophils, and direct effects of activated mast cells on T cells and monocytes (from peripheral blood) in the presence Staphylococcus aureus proteins (proliferation and release of IL-12, IFNgamma, IL-4, IL-5). These in vitro events will be connected to in vivo comparisons of mediators in tears (IL1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNFalpha, IFNgamma, histamine, soluble TNF receptor 1) and expression of receptors on the ocular surface (ICAM-1, HLA-DR, DP, DQ, CD23) in various ocular allergic diseases. In vitro techniques utilized will include: isolation and purification of conjunctival mast cells and epithelial cells from cadaveric tissues, cell culture (including cell-to-cell interactions through stimulation with activated mast cell supernates and co-culture) adhesion and degranulation assays, flow cytometry (surface and intracellular), ELISA, blocking antibody techniques (to determine specific mechanisms), and proliferation assays. In vivo experiments will include: ocular allergen provocation, collection of ocular surface cells via impression cytology followed by flow cytometry for surface receptors, and tear analysis for cytokines utilizing Becton Dickinson's Cytometric Bead Array flow cytometry.
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