A senior physician at the Brigham and Women’s Hospital and Professor of Medicine at Harvard Medical School, Dr. Carey was awarded the Beaumont Prize of the AGA in May 2000. His research focuses on several topics involving lipids of the hepatobiliary system important in human health and disease. Dr. Carey’s research on pathophysiology of bile involves physical-chemical and molecular biologic experiments in model systems, ex vivo hepatobiliary tissues, and mouse models. His work on the genetics of cholesterol gallstone formation involves inbred mice of different strains as well as recombinant inbred, congenic, and various “knock-out” and “knock-in” mice to answer specific questions. The overall strategy is to identify and determine the functions of all the major murine cholesterol gallstone (Lith) genes as a framework for human LITH gene discovery. Specific ongoing work concerns: The physical-chemical pathways of cholesterol secretion into bile. The approach involves synthesis of fluorine (F)-labeled cholesterol molecules which exhibit essentially similar membrane occupancy to the native sterol. These fluorosterols will be delivered in recombinant lipoproteins to hepatocyte couplets as well as isolated perfused rat livers and their secretory pathways studied. Because of strategic localization of the F atom (C6, C27), bile salt synthesis from fluorocholesterols will be effectively suppressed. Quantification of fluorocholesterols trafficking through the cell into bile will be carried out by fluorine (19F)NMR spectroscopy as well as by electron spectroscopic imaging. Other studies will be carried out in cells and livers from mice where putative cholesterol transporters are “knocked out,” including Abc1, Mdr2, and SrbI gene disruption. Molecular interactions between bile salts, sphingomyelin, and cholesterol. To understand the roles of sphingomyelin in cholesterol secretion into bile, as well as absorption from the gut, the physical-chemical characteristics of micellar systems, liquid crystalline systems and monolayers containing mixtures of these liquids are being studied by physical-chemical and biophysical techniques. In addition, the chemical activity of cholesterol in equilibrium with bulk micellar and vesicle systems containing sphingomyelin molecules is being quantified by “capture” in polymer beads. This monomeric activity controls tissue uptake into “enterocytes” in culture. This work will elucidate why sphingomyelin on the canalicular membrane may control cholesterol secretion into bile, and explain how sphingomyelin-rich aggregates may control cholesterol absorption from the gut. The biophysical-chemical origin of lipoprotein X. This abnormal plasma lipoprotein, which is vesicular in structure, characterizes all chronic cholestatic syndromes. Its phospholipid molecules are typical of bile, the cholesterol content is unesterified and the bilayers contain traces of bile acids. Its formation depends on an intact lecithin “flippase” (i.e., Mdr2) in hepatocytes since lipoprotein X is absent from plasma in biliary obstructed Mdr2-/- mice. The aims are to track the routes of cholesterol into plasma using fluorocholesterols, and movement of biliary PC by monitoring the subcellular localization of MDR2 by Western blot and Immunohistochemistry. The physical-chemical state of conjugated bilirubins in model and native biles. The Carey laboratory has identified that bilirubin ditaurate is an excellent surrogate for native bilirubin conjugates. It self-associates as well as hetero-associates with bile salts in model systems. Planned studies are to compare by analytical ultracentrifugation and UV-visible, circular dichroic and fluorescent spectroscopy the conjugate’s properties in these complex model systems with those of native human bile. Solubilities of unconjugated bilirubin (UCB) in model biles and pathophysiological correlations with native bile. The fundamental basis of all pigment gallstone formation is precipitation of calcium hydrogen bilirubinate (Ca(HUCB)2) from supersaturated bile. The Carey laboratory has undertaken a systematic study of UCB solubility in model bile systems by potentiometric titration to obtain both metastable and equilibrium solubilities. The work is also relevant to the conditions in the cecal lumen in IBD, in that spilled bile salts from ileal dysfunction solubilize excess UCB. This results in induced enterohepatic circulation of UCB leading to “hyperbilirubinbilia,” a major risk factor for pigment gallstone formation. Mutations in the ileal bile salt transporter as a cause of “black” pigment gallstones. Since an enterohepatic circulation of bilirubin is induced when bile salts spill into the cecum, the focus of this work is to test the hypothesis that in otherwise healthy pigment stone patients there are dysfunctional mutations of the ileal bile acid transporter SLC10A2. In one of three “black” pigment gallstone patients examined in a preliminary study, the gene was found to have a unique mutation in exon 1. Identification of cholesterol gallstone genes in inbred mice. The Carey laboratory, together with the laboratory of Dr. Beverly Paigen at the Jackson Laboratory, Bar Harbor, Maine, is engaged in a program to discover Lith genes by quantitative trait loci (QTL) analysis, testing candidate genes based on pathophysiology, fine mapping of the QTLs and the genes, and then finding differences in their expression, sequence and function in congenic strains. Specific projects are: The molecular pathobiology of canalicular and cholangiocyte function in the pathogenesis of murine cholesterol gallstones. This work will involve isolation of pure preparations of canalicular membranes, as well as cholangiocytes from inbred mouse livers to determine by molecular biological and physical-chemical tools how the major Lith genes, all of which appear to be membrane lipid transporters, induce cholesterol supersaturated bile. The metabolic origin of biliary cholesterol molecules in mice with and without Lith alleles. Drugs that selectively inhibit biosynthetic and transport steps in biliary cholesterol formation and secretion will be utilized to dissect the relative contributions of dietary, recycled biliary, lipoprotein and de novo synthesized cholesterol to biliary cholesterol secretion in mice with mutant and normal Lith alleles. The mucosal immunology of the biliary tree in cholelithogenesis. During cholelithogenesis the entire extrahepatic biliary tree is invaded by chronic inflammatory cells. In the inbred mouse model this occurs as one of the earliest accompaniments of lithogenic bile. However, nothing is known as to their clonal nature, the proportion of acute and chronic (T or B) cells, what immunoglobulin and cytokines they secrete, and what might be the role of inflammatory nucleators, immunoglobulins, adhesion proteins, etc., in cholelithogenesis. It is anticipated that there could be inflammatory and immunological markers in plasma that might be surrogates for a pre-stone diagnosis. Chronic cholestatic liver disease and gallstones in Cystic Fibrosis. CFTR-/- mice will be used to test the hypothesis that bile salt leakage into the large intestine induces enterohepatic circulation of bilirubin, thereby increasing intrahepatic as well as gallbladder levels of bile pigment conjugates. To this is coupled deficient alkalinization of hepatic bile because the nonfunctioning CFTR chloride channel prevents the anionic exchanger isoform 2 (AE2) on cholangiocytes from exchanging chloride for bicarbonate ions. Therefore endogenous b-glucuronidase (pH optimum @4.5) will be highly active, converting bilirubin conjugates to UCB which precipitate as calcium salts in the intrahepatic ductules as well as in the gallbladder.
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