The objectives of my laboratory are to apply the fundamental concepts and broad technologies of cell/molecular biology to understand hepatic epithelial cell function and dysfunction. Our focus is principally on cholangiocytes (epithelial cells that line intrahepatic bile ducts) because of their biologic and clinical importance, the new hypotheses and techniques we have developed for their study, and the role we have played in advancing cholangiocyte pathobiology, and under-served area of research. Currently, my lab has two major parallel programs focused on cholangiocyte pathobiology each supported by an RO1 grant from NIH.

First, we investigate cholangiocyte water and solute transport by testing the central hypothesis that cholangiocyte bile production is the net result of solute-driven, bidirectional passive movement of water molecules through water selective channels (aquaporins) constituitively expressed on or recycled among cholangiocyte cellular compartments. We utilize new models including: aquaporin knock-out mice; cultured rat cholangiocytes transfected with AQP-green fluorescent fusion constructs; immunoisolation of AQP-1 containing transport vesicles; intact closed or perfused rat bile duct units; subpopulations of rat cholangiocytes isolated from different bile duct segments; and computer-generated three-dimensional reconstruction of the rat biliary tree.

Second, we study the interactions of cholangiocytes with cryptosporidium parvum (C. parvum), an emerging pathogen important in the development of AIDS cholangiopathy. The work tests the central hypothesis that C. parvum induces biliary tract disease in patients with AIDS by two interdependent and complimentary mechanisms: 1) C. parvum inhibits apoptosis in infected cholangiocytes by activation of the NF-kB survival pathway, allowing the organism to propagate in the infected cell; and 2) C. parvum promotes apoptosis in uninfected cholangiocytes by activation of the Fas/FasL proapopoptotic pathway and synergistic effects of HIV dependent soluble factors such as TAT. Our experiments utilize a novel in vitro model of biliary cryptosporidiosis employing cultured human cholangiocytes and live C. parvum sporozoytes.

Our long-term goal is to provide a theoretical framework for development of novel therapeutic strategies for the cholangiopathies, a group of cholestatic genetic and acquired hepatobiliary diseases in which the cholangiocyte is the principle target of diverse destructive processes.

Technologies Used

• The isolation and culture of individual cells derived from rodent and human liver
• Liver organ perfusion
• Experimental rodent models of cholestasis, malignant disease, and immunological cholangitis
• Cell microinjection and transfection
• A range of morphologic techniques including traditional histologic and immunohistologic techniques, ultra-structural approaches including transmission and scanning electron microscopy, and fluorescence imaging techniques, including quantitative fluorescence microscopy, and laser confocal microscopy organelle isolation
• Traditional biochemical techniques (one and two dimensional gel electrophoresis, protein isolation, antibody production and purification, quantitative immunoblotting) and molecular techniques (Northern and Southern blotting, gene cloning, sequencing, and PCR)