MEMBRANE PERTURBATION DURING INFLAMMATION

此提案是为了回应最近的小额拨款计划

This proposal is in response to the small grants program for recently trained, less experienced basic scientists. The specific aims and long-term objectives are to develop the bovine corneal cup as a method for studying changes in membrane during the inflammatory process. This method will allow dissection of the inflammatory cell-corneal endothelial cell interaction from a morphologic and functional viewpoint. Many ramifications of the inflammatory process within the eye can eventually be explored by use of specific cell types, stimulated cells (i.e., activated neutrophils, lymphocytes, macrophages), specific humoral or chemical mediators of inflammation, viral or bacterial disease states, and blocking receptors such as lectins. Several specific questions will be addressed in this initial study: 1) What are the changes in corneal endothelial cell surface morphology and carbohydrate structure during inflammatory cell adhesion and transmigration?; 2) Are there changes in the morphology or function of endothelial intercellular junctions?; and 3) Are these changes dependent on a specific inflammatory cell type? In order to begin answering these questions the bovine corneal cup model will be established with several techniques being applied to the model. Specific peripheral blood leukocytes will be isolated. Changes in endothelial cell surface morphology will be determined by the use of scanning electron microscopy and the freeze-fracture technique with quantitative data accumulated by determination of intramembranous particle size and density distribution. Changes in cell surface carbohydrate structure will be determined with a battery of lectins complexed with hemocyanin which then can be quantitated by use of scanning electron microscopy. Changes in intercellular junctions will be identified by quantitative assessment of junction size and distribution by use of the freeze-fracture technique. Establishment of the corneal cup model will allow a more controlled and detailed examination of inflammatory cell-endothelial cell interaction.