Host factors influencing disease progression and response to treatment in chronic liver disease

The principal aim of the studies described in this thesis was to examine the influence of steatosis on gene expression and the progression of fibrosis and to examine genetic factors associated with increased oxidative stress in human chronic liver disease. (1) Real-time PCR was used to determine relative mRNA expression levels of apolipoprotein B-100, CD14, collagen I, cytochrome P450 2E1 (CYP 2E1), peroxisome proliferating activated receptor (PPAR)-a and -g, sterol regulatory element binding protein (SREBP)-lc, transforming growth factor-b1, tumour necrosis factor-a (TNF-a), and uncoupling protein-2 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. The results demonstrate that mRNA expression of collagen I, TNF-a and CYP 2E1 was increased in steatotic livers. In addition, mRNA expression of collagen I was increased and SREBP-lc was decreased in more severely fibrotic livers. A strong correlation was observed between TNF-a and collagen I mRNA expression (r=0.598, Pl0.001). Immunohistochemistry locaUsed TNF-a protein to Kupffer cells, bile ducts and portal inflammatory cells. These data provide support that increased expression of TNF-a and CYP 2E1 may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV. (2) The concordance between the use of amplified antisense RNA (aRNA) and total RNA in microarrays was examined in order to assess the fidelity and reliability of the aRNA technique. In addition, regions of least concordance were evaluated to determine which type of hybridisation, either total RNA or aRNA, is most concordant to the expression ratio determined by real-time PCR. Results demonstrate that coefficient of a variation of 16.3% and 17.4% for total RNA and aRNA hybridised microarrays, respectively. A strong linear correlation was observed between total RNA and aRNA expression ratios (r = 0.8530). Moreover, differentially expressed transcripts identified with aRNA based microarray hybridisations were highly concordant (up to 96.7% concordant) to total RNA hybridisations and differences observed in differentially expressed transcripts were related to the inherent variance of the microarray technique and the high gain in signal intensity due to the large increase in targets available for hybridisation following the aRNA amplification procedure. Thus, the use of aRNA for the comprehensive analysis of gene expression in limited quantities of tissue is highly concordant to the use of total RNA. (3) Steatosis is a characteristic feature of non-alcoholic fatty liver disease (NAFLD) and knowledge of factors that are altered in its presence would provide insight into the pathogenesis of this disease. A comprehensive mRNA expression profile of NAFLD was determined in eleven paediatric patients using Ontario Cancer Institute 19,200 element microarrays combined with amplified antisense RNA. A total of 213 transcripts were up-regulated and 64 transcripts were down-regulated in at least seven of the eleven subjects with NAFLD. The results demonstrate several pathways that were differentially expressed in the majority of subjects with NAFLD. These include increased transcription of lipid metabolising enzymes, genes encoding mitochondrial oxidative stress response proteins, activation of retinoid metabolising proteins and altered expression of genes involved in fibrosis and intracellular signalling. (4) The frequency of the H63D mutation in the haemochromatosis gene, HFE, was determined and its influence on total body iron stores was assessed in a well-defined population from Busselton, Australia. Serum transferrin saturation and ferritin levels were correlated with the H63D mutation in 2531 unrelated Caucasian subjects who did not possess the C282Y mutation (from a population of 3011 of whom 480 possessed a C282Y allele). The results demonstrated that 62 subjects (2.1%) were homozygous for the H63D mutation, 711 (23.6%) were heterozygous, and 1758 (58.4%) were wild-type for the H63D mutation. Serum transferrin saturation was significantly increased in male and female H63D homozygotes and heterozygotes compared with wild-types. Serum ferritin levels within each gender were not influenced by H63D genotypes. Elevated transferrin saturation g45 percent was observed in a greater proportion of male H63D carriers than male wild-types. Male H63D homozygotes (9%) and heterozygotes (3%) were more likely to have both elevated transferrin saturation and elevated ferritin g 300 ng/ml than male wild-types (0.7%). Homozygosity for H63D was not associated with the development of clinically significant iron overload. These data demonstrate that the presence of the H63D mutation results in a significant increase in serum transferrin saturation but does not result in significant iron loading. Thus, the effects of the H63D mutation on iron parameters are clinically insignificant. (5) The association between neuropsychiatric side-effects and the inheritance of apolipoprotein E genotypes was examined in 110 patients with chronic hepatitis C treated with interferon-a. A retrospective investigation was conducted by assessing the rates of psychiatric referral and neuropsychiatric symptoms experienced during treatment along with other complaints indicating psychological distress. A highly statistically significant association was observed between apolipoprotein E genotypes and interferon induced neuropsychiatric symptoms. Patients with an 84 allele were more likely to be referred to a psychiatrist (p=0.004) and had more neuropsychiatric symptoms (pl0.05) during antiviral treatment than those without an 84 allele. Additionally, patients with an 84 allele were more likely to experience irritability or anger (p=0.0002) and anxiety or other mood symptoms (p=0.0004). These data provide support that an individual's apolipoprotein E genotype may influence the neuropsychiatric response to antiviral therapy with interferon alpha. Prospective studies evaluating the importance of apolipoprotein E in susceptibility to interferon alpha induced neuropsychiatric complications are needed. Moreover, pathways involving apolipoprotein E should be considered in understanding the pathophysiology of interferon alpha induced neuropsychiatric complications.

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