Nicholas A. Shackel

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I.

Real‐time quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real‐time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA‐binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real‐time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>107 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (≥1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low‐level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.

Insights into the Pathobiology of Hepatitis C Virus-Associated Cirrhosis: Analysis of Intrahepatic Differential Gene Expression

The pathogenesis of hepatitis C virus (HCV)-associated liver injury involves many genes from multiple pathogenic pathways. cDNA array analysis, which examines the expression of many genes simultaneously, was used to achieve new insights into HCV liver injury. Membrane-based cDNA arrays of 874 genes compared HCV-associated cirrhosis with autoimmune hepatitis-associated cirrhosis as an inflammatory and cirrhotic control, and with nondiseased liver tissue. Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signaling, apoptosis, and oxidative stress. Genes up-regulated in HCV-associated cirrhosis were predominantly associated with a Th1 immune response, fibrosis, cellular proliferation, and apoptosis. Novel observations of differential gene expression included increased expression of secreted apoptosis-related protein 3, a Wnt pathway gene possibly involved in cellular apoptosis. EMMPRIN (CD147) and discoidin domain receptor 1 (CD167) were also shown to be increased and are likely to play a role in liver fibrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction confirmed the increased expression of 15 genes. The comparison of HCV cirrhosis with autoimmune hepatitis cirrhosis showed a marked difference in the apoptosis-associated gene profile with HCV cirrhosis characterized by increased proapoptotic gene expression whereas autoimmune hepatitis was characterized by increased expression of both antiapoptotic and proapoptotic genes. Furthermore, expression of beta-catenin and the fibrosis-associated protein EMMPRIN were localized by immunohistochemistry to the plasma membranes of hepatocytes and biliary epithelium. In conclusion, HCV-associated cirrhosis was characterized by a proinflammatory, profibrotic, and proapoptotic gene expression profile.

Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.

BACKGROUND Primary biliary cirrhosis (PBC) is an autoimmune disease in which the pathogenesis of progressive liver injury is poorly understood. AIM To provide novel insights into the pathogenesis of PBC related liver injury using cDNA array analysis, which simultaneously examines expression of many genes. METHODS Utilising cDNA arrays of 874 genes, PBC was compared with primary sclerosing cholangitis (PSC) associated cirrhosis and non-diseased liver. Differential expression of 10 genes was confirmed by real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signalling, apoptosis, and oxidative stress. PBC was associated with increased expression of both Th1 and Th2 type molecules of the immune response. Fibrosis related gene expression featured upregulation of connective tissue growth factor and transforming growth factor beta3. Many more apoptosis associated molecules exhibited increased expression, consistent with apoptosis being a more active and regulated process, in PSC associated cirrhosis than in PBC. Increased expression of many genes of the Wnt and notch pathways implicated these highly conserved and linked pathways in PBC pathogenesis. The observed increases in expression of c-jun, c-myc, and c-fos related antigen 1 are consistent with increased Wnt pathway activity in PBC. Differential expression of four components of the Wnt pathway, Wnt-5a, Wnt-13, FRITZ, and beta-catenin, was confirmed by quantitative RT-PCR. CONCLUSION Many genes implicated in intrahepatic inflammation, fibrosis, and regeneration were upregulated in PBC cirrhosis. In particular, increased expression of a number of Drosophila homologues was seen in PBC.

Hepatitis C virus drug resistance and immune-driven adaptations: Relevance to new antiviral therapy†

The efficacy of specifically targeted anti‐viral therapy for hepatitis C virus (HCV) (STAT‐C), including HCV protease and polymerase inhibitors, is limited by the presence of drug‐specific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)‐restricted immune responses, which may therefore influence STAT‐C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT‐C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment‐naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT‐C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT‐C drug and HCV genotype/subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA‐driven pressure and therapy selection and identified six HLA‐associated polymorphisms (P ≤ 0.05) at known drug resistance sites. Conclusion: Drug and host immune responses are likely to provide powerful selection forces that shape HCV genetic diversity and replication dynamics. Consideration of HCV viral adaptation in terms of drug resistance as well as host “immune resistance” in the STAT‐C treatment era could provide important information toward an optimized and individualized therapy for chronic hepatitis C. (HEPATOLOGY 2009.)

Molecular pathogenesis of liver disease: an approach to hepatic inflammation, cirrhosis and liver transplant tolerance

Acknowledgments:

Divergent adaptation of hepatitis C virus genotypes 1 and 3 to human leukocyte antigen–restricted immune pressure

Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host–viral interaction. Evidence of HCV adaptation to HLA‐restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA‐associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA‐associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA‐associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. Conclusion: There is little overlap in HLA‐associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design. (HEPATOLOGY 2009.)

Grade of deceased donor liver macrovesicular steatosis impacts graft and recipient outcomes more than the Donor Risk Index.

Background and Aim:  Donor liver steatosis can impact on liver allograft outcomes. The aim of the present study was to comprehensively report on the impact of type and grade of donor steatosis, as well as donor and recipient factors, including the reported Donor Risk Index (DRI), on liver allograft outcomes.

Gene array analysis and the liver

Functional genomics methods promise a previously unparalleled high‐throughput examination of intrahepatic gene expression. Profiling transcriptomes as well as examining the coordinate expression of many genes in diverse pathobiologic pathways is now pssible with techniques such as gene array analysis. However, the nature of the hepatic transcriptome, limitations of the functional genomics methokologies used, and analysis of the data generated are often poorly understood. Further, the use of replicates during analysis and the downstream confirmation of results using a supplemental methokology are important but frequently neglected aspects of experimenta design. This review presents an overview of functional genomics methodoogies in the organ‐specific context of studying intrahepatic gene expression. The focus of the review is the most widely used functional genomic methodology, gene array analysis. The nature of the hepatic transcriptome is discussed, together with a summary of gene array analysis methodologies. Analysis of data generated by gene array analysis, determination of intrahepatic cellular origin of transcript expression, and studies of intrahepatic gene expression using gene array analysis are also reviewed. Finally, the importance of a coordinate experimental approach examining both liver protein and mRNA expression is presented. (HEPATOLOGY 2002;36: 1313‐1325).

Early high peak hepatitis C viral load levels independently predict hepatitis C–related liver failure post–liver transplantation

The aim of this study was to examine the importance of the serum hepatitis C viral load within the first year post–liver transplant in determining posttransplant survival. A retrospective analysis of 118 consecutive hepatitis C virus–positive liver transplant recipients who received an allograft from January 1997 to September 2005 was undertaken with a median duration of follow‐up of 32.4 months. Univariate and multivariate analyses were used to examine the effects of recipient, donor, surgical, and viral factors on posttransplant outcomes. A total of 620 viral load estimations were undertaken in the first 12 months following transplantation. Patient and graft survival rates at 1, 3, and 5 years were 87.8%, 79.9%, and 70.1% and 87.0%, 79.2%, and 68.2%, respectively. According to multivariate analysis, a peak viral load ≥ 107 IU/mL (P = 0.004; hazard ratio, 8.68; 95% confidence interval, 2.04–37.02) and exposure to antirejection therapy (P = 0.05; hazard ratio, 2.26; 95% confidence interval, 1.01–5.38) were both independent predictors of diminished patient and graft survival and hepatitis C–related allograft failure. The only other independent predictor of hepatitis C virus–related outcome after transplant was azathioprine use, which was associated with improved outcomes (P = 0.04; hazard ratio, 0.25; 95% confidence interval, 0.07–0.91). A peak viral load in the first year after transplant of >108, 107 to 108, and <107 IU/mL was associated with a mean survival of 11.8, 70.6, and 89.1 months respectively (P ≤ 0.03). The results emphasize the importance of high viral loads in the early posttransplant period as an independent predictor of recipient outcomes. Liver Transpl 15:709–718, 2009.

The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury

BACKGROUND & AIMS In vertebrates, canonical Hedgehog (Hh) pathway activation requires Smoothened (SMO) translocation to the primary cilium (Pc), followed by a GLI-mediated transcriptional response. In addition, a similar gene regulation occurs in response to growth factors/cytokines, although independently of SMO signalling. The Hh pathway plays a critical role in liver fibrosis/regeneration, however, the mechanism of activation in chronic liver injury is poorly understood. This study aimed to characterise Hh pathway activation upon thioacetamide (TAA)-induced chronic liver injury in vivo by defining Hh-responsive cells, namely cells harbouring Pc and Pc-localised SMO. METHODS C57BL/6 mice (wild-type or Ptc1(+/-)) were TAA-treated. Liver injury and Hh ligand/pathway mRNA and protein expression were assessed in vivo. SMO/GLI manipulation and SMO-dependent/independent activation of GLI-mediated transcriptional response in Pc-positive (Pc(+)) cells were studied in vitro. RESULTS In vivo, Hh activation was progressively induced following TAA. At the epithelial-mesenchymal interface, injured hepatocytes produced Hh ligands. Progenitors, myofibroblasts, leukocytes and hepatocytes were GLI2(+). Pc(+) cells increased following TAA, but only EpCAM(+)/GLI2(+) progenitors were Pc(+)/SMO(+). In vitro, SMO knockdown/hGli3-R overexpression reduced proliferation/viability in Pc(+) progenitors, whilst increased proliferation occurred with hGli1 overexpression. HGF induced GLI transcriptional activity independently of Pc/SMO. Ptc1(+/-) mice exhibited increased progenitor, myofibroblast and fibrosis responses. CONCLUSIONS In chronic liver injury, Pc(+) progenitors receive Hh ligand signals and process it through Pc/SMO-dependent activation of GLI-mediated transcriptional response. Pc/SMO-independent GLI activation likely occurs in Pc(-)/GLI2(+) cells. Increased fibrosis in Hh gain-of-function mice likely occurs by primary progenitor expansion/proliferation and secondary fibrotic myofibroblast expansion, in close contact with progenitors.