The general aim of this project is to elucidate mechanisms by which proteins and viruses enter cells by receptor-mediated transport processes and translocate across the lipid bilayer to the cytosol and nuclear compartments. Molecular signals which lead to unique vesicle trafficking are studies. A major new tool is the use of low light level fluorescence digital imaging. Assays are developed which quantitatively define the efficiency of membrane translocation. Macromolecular chemical determinants which affect translocation, and cellular energy sources which drive translocation are determined. Utilizing basic data from such studies, immunotoxins (toxins linked to monoclonal antibodies) are constructed to serve as a new class of pharmacologic reagents to eliminate unwanted cell types such as cancer cells or T-4 lymphocytes and macrophages in AIDS infections, or to manipulate specific cells such as T cell and B cell subsets to correct imbalances which exist in autoimmune diseases that affect the CNS, such as multiple sclerosis and lupus and cause psychosis. In addition, immunotoxins continue to prove useful in diminishing the incidence of graft-versus-host-disease following bone marrow transplantation and thus will also have utility in enzyme replacement therapy and organ transplantation. The relationship between the uptake of exogenous proteins and viruses and the processing of these materials for immunological recognition in association with Class I and Class II MHC molecules is also studies. A clinical application of this program is the development of cells with anti-viral anti-sense oligonucleotides coupled to receptor-mediated protein shuttles via cleavable acid-labile crosslinking reagents.
