application): This project is intended to characterize the tissue and cellular localization of the Coxsackie and adenovirus receptor (CAR) using a monoclonal antibody (RmcB) that readily detects the protein in human tissue sections and cultured cells. We will determine whether CAR is present in the apical membranes of polarized airway (and other epithelial) cells known to be refractory to adenovirus gene transfer. We will co-localize CAR with the binding sites for recombinant adenovirus fiber in human tissue sections. Basic information of this sort is important from the standpoint of adenoviral tropism, gene therapy targeting, and understanding the refractoriness of differentiated epithelium to adenovirus. However, studies of this sort have not previously been reported. We will also perform subcellular localization studies of CAR in polarized epithelial cells in vitro and compare CAR distribution to the binding sites for recombinant fiber and to the localization of the integrins. These experiments will test the hypothesis that 1) refractoriness of airway and other polarized epithelium to adenovirus gene transfer in vitro and in vivo is due to a failure of fiber binding or CAR expression, 2) in polarized epithelium (as opposed to non-polar cells) refractoriness to adenovirus gene transfer occurs because CAR is confined to the apical membrane compartment and does not have access to aV0 integrins situated in the basolateral membrane, and 3) interventions that safely disrupt tight junctions will permit access of CAR-bound adenoviral particles to the basolateral surface, and will facilitate adenovirus entry and transgene expression.
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