No description available.

The cholestatic liver diseases include Primary Biliary Cirrhosis (PBC) and Primary Sclerosing Cholangitis (PSC). The aetiologies of PBC and PSC are not yet known but it is thought that there are both inherited and environmental factors associated with pathogenesis. In this thesis, I set out to explore the immunologic and virologic basis of cholestatic liver diseases. In recent years a novel human betaretrovirus has been proposed as an antigenic target in PBC. Using separate T cell assays, I analysed responses to both autoantigens and foreign antigens in parallel, backed up with a molecular approach. I have shown that although there is definite evidence of prior encounter with HBRV peptides at the immunologic level the virus is not closely associated with the pathogenesis of PBC. I have shown that there are links between the composition of the cellular infiltrate and the histologic scores as well as the clinical status. In PBC, the accumulation of CD8+ cells in particular correlates with liver fibrosis stage and Foxp3 cells may have an influence on portal and lobular inflammation. In PSC, the frequency of T helper cells and B cells correlate with alkaline phosphatase and liver stage. This may indicate a closer relationship between tissue histochemistry results and fibrosis and clinical markers in PSC than in PBC and may not simply be a result of portal tract inflammation but be associated with disease pathogenesis. The quality of life tool PBC-40 showed similar results to the original Newcastle cohort. Fatigue is the symptom with the greatest apparent impact and there was no association found between symptoms and biological parameters of disease activity and severity. In PSC, there was a negative correlation between the average CD8 count per infiltrate and the emotional and social domain suggesting alterations in CD8 liver cell counts may have an influence on human behaviour or vice versa. Overall these data provide new insight into the pathogenesis of both PBC and PSC, and provide new avenues for immunologic analysis of these diseases in future.

Identification and functional characterization of numerous transport systems at the sinusoidal and canalicular membrane of hepatocytes have significantly expanded our understanding of bilirubin metabolism and contributed to elucidation of molecular basis of hereditary jaundice. Moreover, dysregulation of hepatobiliary transport systems could explain jaundice in many acquired liver disorders. This thesis is focused on the new aspects of bilirubin handling in hepatocytes based on elucidation of the molecular basis of Rotor syndrome. The first study is focused on the antioxidative properties of bilirubin in liver tissue in a model of obstructive cholestasis. In the second part of the thesis we present several novel mutations in ABCC2, the gene associated with Dubin-Johnson syndrome, identified in patients selected for the Rotor locus mapping study. In the key third study concerned with Rotor syndrome we demonstrated that biallelic inactivating mutations causing complete absence of transport proteins OATP1B1 and OATP1B3 result in disruption of hepatic reuptake of bilirubin, which is the molecular basis of Rotor-type jaundice. These results indicate that apart from secretion of conjugated bilirubin into bile, a significant fraction of bilirubin glucuronide is secreted via MRP3 into sinusoidal blood and...

Identification and functional characterization of numerous transport systems at the sinusoidal and canalicular membrane of hepatocytes have significantly expanded our understanding of bilirubin metabolism and contributed to elucidation of molecular basis of hereditary jaundice. Moreover, dysregulation of hepatobiliary transport systems could explain jaundice in many acquired liver disorders. This thesis is focused on the new aspects of bilirubin handling in hepatocytes based on elucidation of the molecular basis of Rotor syndrome. The first study is focused on the antioxidative properties of bilirubin in liver tissue in a model of obstructive cholestasis. In the second part of the thesis we present several novel mutations in ABCC2, the gene associated with Dubin-Johnson syndrome, identified in patients selected for the Rotor locus mapping study. In the key third study concerned with Rotor syndrome we demonstrated that biallelic inactivating mutations causing complete absence of transport proteins OATP1B1 and OATP1B3 result in disruption of hepatic reuptake of bilirubin, which is the molecular basis of Rotor-type jaundice. These results indicate that apart from secretion of conjugated bilirubin into bile, a significant fraction of bilirubin glucuronide is secreted via MRP3 into sinusoidal blood and...

No description available.

No description available.

Intravenous feeding of patients with essential and balanced nutrition is required when enteral feeding is not tolerated, therefore indicating the need for Total Parenteral Nutrition (TPN). This life-saving therapy is also associated with the increase risk of intrahepatic cholestasis. The incidence of TPN-related hepatobiliary complications is common in both adults and infants on TPN. Previous work in in vivo models suggested that one of the potential contributing factors is the aluminum contamination of TPN solutions. The mechanism by which aluminum contributes to the PNAC development, though, was unknown. Aluminum as a risk factor may influence a number of hepatocellular functions to lead to cholestasis but one possible mechanism is the potential for aluminum to cause dysfunction of those transporters responsible in the maintenance of bile flow. To provide some initial information regarding the role of aluminum as a contributing factor to cholestasis and the possible underlying mechanism, cytotoxicity studies were conducted to determine whether aluminum causes direct toxicity of HepG2 cells. Furthermore, the influence of aluminum on the mRNA expression of hepatic biliary transporters (BSEP, MRP2, MATE1, NTCP) and nuclear transcription factor (FXR) in HepG2 cells using real-time RT-PCR analysis was assessed. Since inflammation is a component of cholestasis, these investigations also involved the use of an inflammatory stimulus, lipopolysaccharide (LPS), to determine whether the effects of aluminum were exacerbated by underlying inflammation. My data suggest that for the canalicular hepatic transporters MATE1 and BSEP, aluminum at higher concentration alone as well as with LPS caused increased mRNA expression levels. In addition to this, BSEP mRNA expression was preserved and that of MATE1 was increased on LPS exposure. Given the particular importance of BSEP in the maintenance of bile flow and reported effects of drug-induced inhibition of BSEP to cause hepatic cholestasis, the influence of aluminum on BSEP (and MATE1) protein expression and activity warrant investigation. Further studies may identify that inhibition of BSEP function (and possibly MATE1) by aluminum contamination of total parenteral nutrition formulations may explain, in part, the intrahepatic cholestasis associated with parenteral nutrition.

There are many causes of cholestasis, which results when the flow of bile acids is slowed or stopped. Bile acids are hydrophobic molecules synthesized from cholesterol in the liver, and when present in excess, are cytotoxic to cell membranes. Treatment options for cholestasis are limited, and if left untreated or inadequately treated, many patients will require a liver transplant; thus, underscoring the importance of successfully managing this disease. Activation of nuclear receptors in animal models has been shown to be hepatoprotective during bile acid-induced cholestasis; however, the mechanisms underlying the hepatoprotective effects are poorly understood. Therefore, the over-arching goal of this project is to glean an improved comprehension of the mechanisms of hepatoprotection during bile acid-induced cholestasis. All of the studies involve administration of CAR activators phenobarbital (PB), oltipraz (OPZ), 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene [TCPOBOP (TC)] or corn oil (CO) to C57BL/6 wild type (WT), or WT and CAR knockout (CAR-/-) mice prior to induction of intrahepatic cholestasis using the secondary bile acid lithocholic acid (LCA). Efflux transport proteins such as Mrps 3 and 4 are known to be up-regulated during cholestasis, and this was the first topic of exploration. Unexpectedly, the expression of efflux transporters was not consistently up-regulated in protected mice. However, a decrease in total liver bile acid concentrations was observed. These changes in hepatic bile acids indicated that bile acid biosynthesis may be relevant to hepatoprotection. Indeed decreases in total and individual bile acids correlated with hepatoprotection, and Cyp8b1 expression was also increased which could be suggestive of a shift in the bile acid biosynthesis pathway towards the formation of less toxic bile acid species. CAR may also have a role in cell death via apoptosis by altering Bcl-2 protein expression. Although apoptosis was decreased in hepatoprotected mice, an increase in the expression of Mcl-1 and Bcl-xL was not observed, suggesting hepatoprotection is not a direct result of CAR-induced Mcl-1 expression. These findings add significantly to the body of knowledge surrounding cholestatic liver disease and suggest that studies aimed toward manipulation of nuclear receptors are worthy of further exploration.

Thesis (Ph.D.)--Flinders University, School of Medicine, Dept. of Medical Biochemistry. / Typescript bound. Includes bibliographical references: (leaves 211-230) Also available online.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Veronika Řepová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D., Prof. Ramiro Jover Atienza, Ph.D. Title of diploma thesis: Alterations in gene expression of hepatobiliary transporters as potential mechanisms for drug-induced cholestasis by amoxicillin and clavulanic acid The combination of amoxicillin and clavulanic acid (AMO/CLA) represents one of the most frequent causes of the idiosyncratic type of drug-induced liver injury (DILI) nowadays. Despite difficulties in diagnosis and causality assessment, the clinical features have already been reported and in most of the cases categorized as cholestatic damages. Number of descriptions of the molecular mechanisms of drug-induced cholestasis has been rising recently and the role of hepatobiliary transporters has turned out to be crucial in the pathogenesis. However, the mechanisms of AMO/CLA-induced DILI at the molecular level still remain indistinct. In order to investigate the hepatotoxic effects of AMO/CLA and AMO alone in vitro, HepG2 and human Upcyte hepatocytes were used as hepatocellular models. The mRNA levels of key bile acid (BA) transporters, enzymes and nuclear receptors (NRs) were measured by quantitative real-time polymerase chain...