Dr. D. Montgomery Bissell: The principal focus of research is wound repair in the liver and its clinical manifestation, fibrosis (or cirrhosis). The group originated the concept of stellate cell activation as a central event in the response to injury. Stellate cells are hepatic pericytes, also known as Ito cells, fat-storing cells or lipocytes. In injury they acquire features of myofibroblasts, becoming fibrogenic, migratory and contractile. Both soluble factors (i.e. cytokines) and insoluble constituents of the extracellular milieu (ECM) contribute to stellate cell activation. A major line of investigation is defining the roles of TGF§ and fibronectin, respectively, in this process. Dr. Bissell's group have examined the cellular expression of both of these factors in experimental liver injury in the rat. In the case of fibronectin, injury gives rise to a specific splice variant in which a module termed EIIIA is retained in the mature RNA (termed EDA fibronectin in humans). In liver injury, this form derives largely from sinusoidal endothelial cells and elicits activation of normal stellate cells, implying the presence on stellate cells of receptors for this Fn segment. In vivo, EIIIA-Fn increases significantly in the plasma of rats in parallel with RNA expression after injury. Studies are under way to assess the utility of circulating EDA-Fn as a marker of hepatic fibrogenesis.

Dr. Jacquelyn J. Maher: Dr. Maher is exploring the relationship between inflammation and fibrosis in liver. For these experiments she has utilized 2 genetically engineered strains of mice, one with deficient neutrophil function (IL-8 TG) and another with impaired neutrophil chemotaxis (CXCR2 -/-). The model of liver injury uses bile duct ligation, which induces fibrosis. Prior work from the Maher laboratory showed that neutrophils are prominent in the portal tracts of animals with mechanical cholestasis. Mice with minor deficits in neutrophil function (IL-8 TG) are not protected from hepatic fibrosis after bile duct ligation. Experiments with CXCR2 -/- mice are in progress. The group is also investigating the possibility that CXC chemokines have beneficial effects on hepatocytes. They have observed that mice with high levels of circulating CXC chemokines (IL-8 TG mice) develop less injury in response to galactosamine-endotoxin than wild-type mice. This appears to involve more than simple neutrophil dysfunction, with a suggestion of a direct positive effect on hepatocytes. They are currently exploring the mechanism by which CXC chemokines can promote hepatocyte cytoprotection.

Liver Immunology

Dr. Marion Peters: Local immune responses are thought to be a major factor in autoimmune and viral hepatitis. Dr. Peter's group has developed a transgenic mouse using the albumin promoter to drive liver-specific expression of a membrane form of ovalbumin (OVA-HEP). Because the T cells that develop in these OVA-HEP mice are tolerant, OVA specific T cells were isolated from the spleen of OT-1 or OT-II transgenic mice, restricted by Class I (CD8+ cytoxic T cells) and Class II (CD4+ helper T cells), respectively, with well defined peptide epitopes. The cells were infused into OVA-HEP mice, and cellular responses were evaluated by serum ALT and liver histology. At multiple time points after T cell injection, there was no pathology in lung, kidney, colon or brain. However, the liver displayed portal inflammatory infiltrates of mononuclear cells with apoptotic hepatocytes, lobular inflammation and ballooning degeneration. Serum ALT was maximum 3 days post injection but remained elevated for more than 40 days after a single injection of T cells reactive against OVA and more than 100 days after 3 injections of T cells. The effect of the infused OT-I T cells was proportional to the number infused. Serum ALT changed in parallel with apoptosis. This represents, therefore, a murine model of chronic inflammation in response to expression of "self" antigen on the surface of hepatocytes. The role of T cell subsets and cytokines in the expansion and enhancement of immune injury is being studied in this model, with characterization of the immune cells involved. Dr. Peters has also generated transgenic mice expressing OVA on biliary epithelial cells, towards a model of bile duct-based autoimmunity.

Dr. Jay Ryan: Dr. Ryan's focus is the function of hepatic lymphocytes known as NK (natural killer) cells and their receptors, which have been implicated in the recognition and lysis of neoplastic, foreign and virally infected cells. His work involves the molecular characterization of receptor-ligand interactions and activation pathways as well as gene discovery through eukaryotic expression cloning and eukaryotic complementation.

Gastrointestinal Cancer

Dr. Jonathan Terdiman: In collaboration with investigators at the UCSF Cancer Center, Dr. Terdiman studies germline mutations in families with a high incidence of colon cancer and the genetic alterations of cancer occurring in the setting of inflammatory bowel disease. He is also studying the effect of cyclooxygenase-2 inhibitors on pre-cancerous colonic lesions.

Dr. Michael Korn: Dr. Korn studies cell-cycle regulation with the goal of devising new agents for cancer of the colon, esophagus and liver. A major question has been the way in which tumor cells with altered cell cycle regulation may be uniquely susceptible to infection and lysis by specifically modified adenoviruses. Initially, the focus was on tumors with altered p53, which was thought to be essential for permissive replication of an E2A-deleted adenovirus (Onyx-015). After it was found that some cells with wild-type p53 supported replication of the virus, Dr. Korn showed that mutation of an upstream factor, p14ARF, can mimic the effect of a altered p53. This line of investigation has been extended recently to studying the receptor that binds adenovirus and mediates its transfer into the cell. He is currently characterizing expression of this receptor in intestinal and hepatic tumors, investigating its molecular regulation.

Genetics of Liver Disease: Inherited Cholestasis

Dr. Laura Bull: The main focus is the genetics of liver disease and encompasses several projects. FIC1 and BSEP: FIC1 is mutated in two inherited liver diseases, progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis (BRIC). Dr. Bull's lab is characterizing FIC1 and BSEP mutations in BRIC and PFIC patients, using denaturing high performance liquid chromatography and DNA sequencing. They have so far identified more than 20 different FIC1 disease mutations, and are currently screening 120 additional patients. The mutation screening results will be used to study the relationship between genotype and phenotype, with the goals of enabling more accurate clinical diagnosis of PFIC, and gaining a better understanding of the biology of these disorders. They will determine whether PFIC1 patients are more likely than PFIC2 patients to have manifestations of extrahepatic disease. They will also examine the outcome of treatments, including biliary diversion and liver transplant, in these forms of genetic liver disease. Mouse model of FIC1-associated disease. Dr. Bull's group has generated a mouse carrying one of the principal mutations in Fic1 that underlies PFIC1 in humans. They are currently characterizing the phenotype of this mouse and anticipate that the experiments will provide a better understanding of the biological role of FIC1. Identification of the gene mutated in lymphedema-cholestasis syndrome (LCS). LCS is an autosomal recessive form of hereditary cholestasis that shares some features with PFIC and BRIC. It is most common in Norway, and most patients are descendants of a common ancestor born 400 years ago. Using genetic mapping techniques, Dr. Bull's group has mapped the LCS gene to a region of less than 3 cM on human chromosome 15. Based on this result, the LCS gene appears to be one not previously associated with hereditary cholestasis or lymphedema. Fine mapping is proceeding with additional samples and genetic markers, and sequencing in the LCS region. Intrahepatic cholestasis of pregnancy (ICP), and other forms of secondary liver disease. ICP is relatively common in Chile. In collaboration with Humberto Reyes, Dr. Bull's group is collecting DNA from Chilean ICP families, and evaluating them for evidence of linkage to genes mutated in hereditary cholestasis (FIC1, BSEP, and MDR3), as well as for the presence of mutation in FIC1, and through collaborations, also in BSEP and MDR3. They are also collecting DNA from individuals with other forms of 'secondary' cholestasis, including cholestasis induced by oral contraceptives or other steroids, surgery, sepsis, total parenteral nutrition, or non-steroidal drugs. They will examine whether susceptibility to these conditions is associated with sequence variants in genes involved in hereditary cholestasis (e.g. FIC1, BSEP, and MDR3).

Heme, Heme-Proteins and Bile Pigments

Dr. Almira Correia: The focus is the elucidation of the structure and function relationships of hepatic cytochromes P450 and tryptophan 2,3-dioxygenase (TO), the mechanisms of P450 suicide inactivation by several clinically relevant agents and the heme regulation of TO. Dr. Correia's group has specifically examined heme modification of the proteins of human liver P450 3A4 after their CuOOH-mediated inactivation, a process that brands them for proteolytic removal. Heme-modified peptides have been isolated by HPLC-peptide mapping and analyzed by mass spectrometry. The peptides modified are also in the putative SRS. The proteolytic degradation of these CuOOH-inactivated P450s was found to entail phosphorylation before their ubiquitination and degradation by the 26S proteasome. The role of such phosphorylation and the identity of the kinases involved is being currently explored. P450 2C11 has been expressed in E. coli and the suicide inactivation and proteolytic degradation of the purified enzyme examined. The group has also carried out mechanistic dissection of the specific proteins involved in the ubiquitination and the 26S proteasomal degradation of native, unmodified P450s 2C11 and 3A4 as models of integral ER-proteins in S. cerevisiae. Such findings indicate that while CYP3A4 recruits the Ub-dependent 26S proteasomal pathway, CYP2C11 is degraded by vacuolar (lysosomal) proteases. In parallel, Dr. Correia has examined the role of hepatic heme in the regulation of hepatic TO turnover (synthesis and degradation) and its structure-function relationships after expression of this enzyme, in high yields and a functionally active form in E. coli.

Dr. Antony McDonagh: Dr. McDonagh's group is studying the influence of chemical structure and intramolecular hydrogen bonding on the hepatic uptake, glucuronidation by UGT1.1, and biliary excretion of a series of synthetic dipyrrolic and tetrapyrrolic organic anions related to bilirubin. These metabolic studies are being done in normal rats, using homozygous Gunn rats lacking UGT1.1 activity and homozygous TR- rats deficient in canalicular multidrug resistance protein 2 (MRP2) for comparative studies. They have synthesized the first thiarubin and the first oxo-rubin, analogs of bilirubin with an S and a C=O group, respectively, replacing the CH2 at the C10 position. The thiarubin is considerably more lipophilic than bilirubin and has a tighter-pitched ridge-tile structure. It is metabolized almost identically to bilirubin, being excreted rapidly as mono and di-glucuronides in bile. In contrast, the oxo-rubin has weaker intramolecular hydrogen bonding and is more polar than its CH2 counterpart. While having little apparent effect on hepatic uptake this has a marked effect on hepatic metabolism and canalicular excretion. It is excreted unchanged in bile in Gunn rats without undergoing conjugation and in normal rats is excreted both unchanged and as a mixture of mono and diglucuronides. MRP2 is required for efficient excretion of C10 oxo rubins and their glucuronides since the biliary excretion of both the parent compound and its glucuronides is greatly impaired in TR- rats. In related work they have synthesized two new lipophilic bilirubins with n-butyl substituents on the lactam rings and studied their metabolism in rats. Their results show that glucuronidation is very sensitive to the specific position, endo or exo, of the n-butyl substituent. Substitution in the endo position has only a weak inhibitory effect on glucuronidation, whereas exo substitution interferes markedly with glucuronidation and biliary excretion, probably by blocking access of the lactam carbonyl hydrogen bonding region to a crucial site on the UGT1.1 glucuronosyl transferase enzyme.

Gene Therapy

Dr. Timothy Davern: The goal is to develop novel and effective methods to express transgenes in the liver, both to better understand important biological processes (e.g., hepatic fibrosis and regeneration) in animal models and ultimately to prevent or treat human liver disease Immunomodulation to prolong adenoviral vector mediated gene expression: The duration of adenoviral vector-mediated gene expression in vivo is severely limited by the cellular immune response against vector-transduced cells. The working hypothesis is that specific cell surface interactions between T cells and antigen presenting cells are crucial in developing the destructive immune response directed against cells transduced with adenoviral vectors. They are testing this hypothesis by precisely interrupting these crucial interactions using a recombinant rabbit fusion protein, rCTLA4Ig, that they have previously cloned and characterized. They have demonstrated in preliminary experiments that this protein does in fact prolong Ad vector gene expression in vivo and are now undertaking studies in a well-characterized (Watanabe Heritable Hyperlipidemic) rabbit model of hepatic gene delivery. If successful, this work will have a wide range of clinical and research applications. Adenoviral vector gene delivery to non-parenchmal cells of the liver: Although it has been widely assumed that the hepatocyte is the primary type of liver cell transduced by Ad vectors, Dr. Davern's group has recently demonstrated that Ad vectors also efficiently transduce liver nonparencyhmal cells of rat liver. In particular, hepatic stellate cells, the main source of abnormal collagen matrix in liver disease, appear to be transduced with high efficiency. They plan to build on this finding by testing whether transduction of hepatic stellate cells with an adenovirus expressing a dominant negative (truncated) transforming growth factor beta receptor type 2 (TGF-b RII) prevents fibrogenesis following repetitive CCl4 dosing. They have constructed and characterized an Ad vector expressing a truncated TGF-b RII and are poised to begin experiments aimed at deciphering the in vivo role of TGF-b in hepatic fibrosis, regeneration, and carcinogenesis.

Hepatic Lipid Metabolism

Dr. Richard A. Weisiger: Cellular metabolism depends critically on the intracellular transport of amphipathic molecules, including important molecules involved in energy metabolism (e.g., long chain fatty acids), signal transduction (thyroid and steroid hormones) and intermediary metabolism (bile acids, heme, cholesterol and retinoids). Dr. Weisiger has shown that a cytosolic protein known as fatty acid binding protein (FABP) acts as a true carrier to catalyze transport of these molecules through cytoplasm of liver (L-FABP), heart (H-FABP) and intestine (I-FABP). These mobile carriers may regulate the clearance and metabolism of fatty acids in their respective tissues. Preliminary data indicate that L-FABP is essential for hepatic fatty acid metabolism, and that the rate of hepatic fatty acid utilization increases linearly with rising cytosolic L-FABP concentrations. Current studies focus on measurement of fatty acid gradients within cultured liver cells to determine if intracellular transport mediated by FABP regulates fatty acid metabolism in the liver. The overall goal is to define the fundamental mechanisms that regulate fatty acid metabolism in human cells, a necessary step to understanding how failure of these mechanisms contribute to human disease states to which dysfunction of FABP has been linked, including diabetes, obesity, fatty liver, and blood lipid abnormalities.