Differential Gene Expression of Liver Tissue and Blood From Individuals With Chronic Viral Hepatitis
Differential Gene Expression of Liver Tissue and Blood From Individuals With Chronic Viral Hepatitis
The purpose of this research is to study body materials like blood proteins as well as white
 blood cell and liver cellular RNA in individuals with liver diseases such as chronic viral
 hepatitis with or without hepatoma and autoimmune liver disease. Presently it is not
 understood how infection with chronic viral hepatitis or autoimmune liver disease damages the
 liver. This research study enroll patients with either chronic viral hepatitis with or
 without hepatoma or autoimmune liver disease.

 The purpose of this study is to find the genes that are expressed in both the circulating
 white blood cells and the liver of patients with varying degrees of liver damage of different
 causes. Genes are biological messengers some of which determine how the body responds to
 injury. We anticipate that results from Differential Gene Expression (DGE) analysis will
 allow us to make predictions about likelihood of disease progression and/or response to
 treatment.

 In addition we will test the blood for markers of injury. The blood collected will be
 prepared differently from the liver tissue. We will use technologies to express pure proteins
 and then we will investigate the functions of these proteins. Nearly all drugs act on
 proteins, not genes, so understanding proteins is the key to really effective new medicines.
 Similarly the first signs of ill health appear in changes to the body's blood proteins,
 making them the most sensitive diagnostic indicators. The studies we plan are called
 proteomics.

 We will later correlate the patterns of gene expression in both circulating white blood cells
 and the liver tissue with clinical outcome and patterns of proteins measured in blood and we
 hope to gain an understanding of how the disease process occurs, which may in turn help us to
 make more precise diagnoses and develop new forms of treatment.

 These techniques that we use are still experimental and so we do not yet know if they will be
 helpful in monitoring changes which may help us to predict the potential severity of your
 liver disease or even if they can be used to indicate who will best respond to treatment.
The objective of this study is to identify genes that are specifically up or down regulated
 in chronic viral hepatitis and autoimmune liver disease and then to examine how these
 expression patterns relate (if at all) to clinical outcome. The expression pattern of
 thousands of genes should provide extremely powerful statistical tools to distinguish between
 different pathophysiological states. It is well recognized that neither hepatitis B or
 hepatitis C is directly cytotoxic, their effect appears to be mediated via an immune
 response. Similarly in individuals with autoimmune liver disease e.g. primary biliary
 cirrhosis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH) immune
 mediated mechanisms appear to be the cause of their liver disease although the eliciting
 antigen(s) remain unknown. The pattern of gene expression can give clues as to both the cause
 and the pathogenesis of disease. For instance, if a disease is caused by an infection, a
 specific cytokine response is observed which will be different if this infection is viral or
 bacterial or parasitic. It is also possible that a certain pattern of response would be
 elicited if the disease is caused as a response to xenobiotic. In patients with autoimmune
 liver disease, it is possible that an endogenous and/or an exogenous antigen initiates the
 disease with a subsequent autoimmune response. Finally, it is also possible that a completely
 unexpected series of cellular events is responsible for pathogenesis, and the microarray
 experiments will enable us to discover these processes.

 HCV - The molecular genetics of hepatitis C viral infection HCV molecular biology: HCV is a
 positive-stranded RNA Flaviviridae virus with a 9.6kB genome. The genome encodes a
 polyprotein of approximately 3000 amino acids which is subsequently cleaved by host and
 virus-specific proteases to yield 4 structural and 6 nonstructural polypeptides. The 4
 functional proteins include a metalloproteinase (NS5A), serine protease (NS3/NS4A), RNA
 helicase (NS3), and a RNA-dependent RNA polymerase (NS5B). Six different genotypes of HCV
 have been described, with genotype 1 being the most prevalent worldwide and genotype 4 being
 the most prevalent in the Middle East.

 The various genotypes interact differently with the host immune system, though the molecular
 basis for this is unclear. Chronic infection by genotypes 1 and 4 is notoriously resistant to
 treatment with IFN, while genotypes 2 and 3 have relatively better responses. This difference
 may be in part due to mutations in the NS5A protein. In cellular models NS5A can interact
 with IFN-induced antiviral enzymes such as 2'5'OAS. Other HCV proteins likely interact with
 cellular proteins to alter responses to immune and IFN antiviral mechanisms: HCV core protein
 dampens lymphocytic Th1 responses and influences IRF, Jak/STAT and iNOS pathways. Although
 these results suggest how the virus may alter known cellular pathways, viral evasion of the
 immune system is multifactorial and clearly complex.

 Research Hypothesis: The processes that lead to persistence of acute HCV infection are
 related, at a molecular level, to those that drive HCV resistance to IFN therapy. Elucidating
 the specifics of the host/viral response in both of these contexts will provide novel targets
 for small-molecule antiviral therapy which can be applied both to acute and chronicHCV.

 Progress Report: CIHR 106800 We have established a large and growing tissue and RNA registry
 for the study of liver disease, and have performed a comprehensive gene array analysis of
 chronic HCV. Some of our most important findings are detailed here.

 HCV is a complex and progressive disease in which the interaction of the host and virus has
 profound effects. The combination of clinical and experimental variability can seriously
 confound the results of a gene expression study and mandates that a large number of patients
 and samples be examined. The fact that liver pathology can be similar among a number of
 different diseases also necessitates that many different patients and diseases are included
 in the analysis. Accordingly, we have analyzed large numbers of patients and controls, and
 have maintained careful databases of clinical details in order to perform multivariate
 analyses of the gene array data. At present we have used a 19K human microarray to determine
 hepatic gene expression in 86 HCV patients and have compared these to 24 normal, 20 HBV and
 14 PBC patients. The array data are of high quality: our rates of confirmation by real-time
 PCR exceed 80%. We have now examined the relative contributions of age, sex, viral genotype
 (1 vs 2/3), degree of fibrosis and disease activity on the genes most upregulated by
 infection with HCV.The most important determinant of consistent gene expression profiles in
 HCV infected livers is viral genotype (genotype 1 vs all others). However, within Genotype 1
 samples there is a clear difference in gene expression.

 This divergent host-virus interaction is reflected in the subsequent response to PegIFN/Rib
 therapy. All of the HCV liver biopsies we have analyzed to date were taken from patients who
 went on to treatment with PegIFN/Rib. At present, treatment has been completed in 49 of these
 patients. In an observation that may have significant clinical utility, we showed that the
 ultimate response to treatment (sustained viral response versus vs nonresponder/relapser) was
 reflected in the original gene expression profiles. We identified 18 genes whose expression
 levels distinguished responders from non-responders/relapsers in with an accuracy of >90%.
 Based on this observation we filed a patent to protect the intellectual property and formed a
 company to commercialize the genetic test. The results have also been submitted for
 publication. Taken together we believe we have described a set of genes, or perhaps a
 biological process, that lies at the heart of the host-HCV interaction.

 Section I. A possible role for the UBP43, ISG15 Pathway in the HCV Interferon While our gene
 expression profiles have distinguished two types of chronic HCV infection - one which
 responds to IFN, and the other that does not - our main objective is to achieve a molecular
 understanding of the roles of specific genes and proteins in the host response to HCV. Though
 an altered gene expression level does not directly implicate the gene product in the
 biological pathway, there are compelling biological reasons to suggest that at least some of
 the genes on our list play an important role in viral-IFN responses. Several of the most
 up-regulated genes are interferon-sensitive (including OAS, Mx1, ISG15, VIPERIN, IFIT, and
 GIP2). Polymorphisms of OAS have been weakly linked to self-limited HCV infection, and
 polymorphisms of Mx1 have been weakly linked to response status. Hepatic mRNA levels for OAS,
 Mx1, and GIP2 are increased in chronic HCV but none, alone, have been linked to treatment
 outcome. The genes that are not directly IFN-responsive may play roles in cellular pathways
 important for IFN responses (PI3AP1, DUSP1), and are involved in inflammatory cell activation
 and maturation (LAP).

 ISG15 and UBP43/USP18, which are components of a newly-recognized IFN regulatory pathway, are
 perhaps the most interesting subset of genes that were found to be differentially expressed
 in our studies. Both genes are expressed more highly in nonresponder (NR) compared with
 responder (R) liver tissue, suggesting that they might interfere with the immune response in
 HCV-infected liver ISG15 is a ubiquitin-like (Ubl) protein which is thought to be important
 to innate immune functions and is covalently linked to proteins following interferon
 activation. How this link is accomplished is controversial and may involve some overlap with
 known E2 Ub enzymes. Interestingly, the E2 Ub enzyme UBCH8 was recently identified as an
 activator of ISG15,34 and is one of the genes upregulated in response to chronic HCV in our
 microarray analysis. The conjugation of ISG15 to its target proteins is reversed by a highly
 specific protease, USP18/UBP43. UBP43 belongs to a family of Ub-specific proteases and is
 induced by IFN, LPS and viral infection; it is degraded by the Skp2 Ub ligase. Loss of USP18
 in mice leads to IFN hypersensitivity. Our data links UBP43 with HCV, and suggests that the
 pathway is important in a divergent host/viral response.

 Research Objective I. To establish the roles of the 18 genes - in particular, of ISG15 and
 UBP43 - associated with a divergent host/ virus interaction, using a HCV replicon model.

 Research Protocols for Section I:

 DNA microarray studies shed light on the transcription state of the cell but do not
 necessarily link specific gene products with the biological problem being addressed.
 Accordingly, it is important to test the roles of individual genes using other approaches. We
 will use in vitro assays of viral replication to study the roles of genes whose expression we
 have found to be altered in HCV infected liver. These are not straightforward experiments,
 since HCV is a difficult virus to study. HCV replicates only in humans and chimpanzees, and
 only poorly in isolated human peripheral blood cells, though these do act as an extrahepatic
 viral pool. Subgenomic RNA replicons can be transfected into certain cell lines and
 maintained, but to date only a few genotypes have been successfully translated into a
 replicon model (1b, 1a, and 2a). Recently, an elegant HCV model was described in Edmonton in
 which human hepatocytes are transplanted into SCID-beige mice. This model supports HCV
 infection and replication; however, since the bulk of HCV effects are due to the immune
 response generated by the virus, this model as it exists currently may not be ideal for
 studying how the virus leads to liver damage and evades antiviral immune responses in humans.

 We have chosen to explore the role of our genes of interest using the full-length HCV
 genotype 1a replicon assay, in collaboration with Dr. Charles Rice (Rockefeller University,
 New York). This replicon is a variant of the full-length HCV H77 strain described recently by
 Dr. Rice, but has higher levels of replication in cell culture than the previous version. The
 replicon model is ideal for rapidly testing the effects of many genes and has been used
 extensively to study HCV mechanisms at a cellular level. This approach will be used for all
 of the genes identified as of potential interest in the studies above, focusing first on the
 ISG15 and UBP43 genes, second on the 16 genes remaining in our response-discriminatory list,
 and finally on novel genes identified in the gene expression studies detailed below.

 Ia. Effect of siRNA knockdown and upregulation (transfection) of individual genes, including
 UBP43 and ISG15, on HCV replicon in Huh7.5 cells.

 We will use RNA interference (RNAi) to "knock down" specific genes in order to evaluate their
 roles on viral replication. Small double-stranded RNA molecules will be used to induce
 sequence-specific degradation of homologous single-stranded RNA. For most of these studies
 stock Huh7.5 cells will be compared to Huh7.5 cells stably expressing the full-length
 Genotype 1a HCV replicon developed in Dr. Charles Rice's laboratory. The methodology detailed
 below is routine in our laboratory and the laboratories of our collaborators.

 Control studies: Baseline gene and protein expression, effect of IFN, effect of replicon:

 The replicon system can be used not only to test the effect of a given gene on viral
 replication, but also for the effects on the IFN response. Previous studies have demonstrated
 that HCV 1b genotype RNA replication is sensitive to IFN-a treatment, even at low doses. In
 fact, the roles of two of the genes on our list of eighteen, MxA and GIP3/IFI6-16, have
 already been examined. The IFN inhibition of HCV 1b replicon RNA replication is independent
 of MxA, and transient transfection of GIP3 does not itself inhibit the 1b replicon but does
 enhance the effect of IFNa. We will now study the roles of the remaining genes in our list
 using the HCV genotype 1a replicon. The studies described below outline our approach for the
 ISG15 and UBP43 genes; however, similar strategies will be employed for the other genes of
 interest.

 Huh7.5 cells and HCV replicon cells will be incubated for 12 hours with increasing doses of
 IFNa2b (0, 1,10 and 100U/ml), IFNb (0,10,100 and 1000 U/ml ), or IFNg (0,10,100 and 1000
 U/ml), and ISG 15, UBP43, and HCV mRNA levels determined by real-time PCR. ISG15 protein
 levels will be determined by western blot (mAb the kind gift of Dr. E.C. Borden, Scripps
 Institute); UBP43 western studies will be performed after we create antibodies or as the
 antibody becomes available (currently it is being developed in the lab of Dr. D.E. Zhang,
 Scripps Institute). RNA isolation will be performed by standard Trizol extraction, and
 protein extraction by lysing the cells in RIPA buffer. These studies will establish mRNA and
 protein levels for ISG15 and UBP43 at baseline, and in response to IFN, replicon and
 replicon/IFN.

 One important issue is confirming that the gene or protein-of-interest is expressed in these
 cells prior to the knock-down experiments. In preliminary studies we have determined that
 there is baseline expression of both ISG15 and UBP43 mRNA in both Huh7.5 and replicon cells
 (real-time PCR) and that IFNa strongly induces protein expression of ISG15 in Huh7.5 cells.
 These results argue that the ISG15 and UBP43 pathway is relevant to this model.

 siRNA studies: We hypothesize that elimination of UBP43 will lead to less HCV RNA replication
 at baseline and will magnify the effect of IFN, and that elimination of ISG15 will increase
 baseline HCV RNA and decrease the effect of IFN. Silencing and control siRNAs will be
 obtained from Ambion. These will be transfected using GenePorter according to the
 manufacturer's instructions; transfection conditions will be optimized using the SV40
 promoter/enhancer luciferase control plasmid, pGL2-control, to ensure that increasing amounts
 of templates results in proportional increases in luciferase activity. Forty-eight hours
 after the transfection, total RNA or protein samples will be prepared as before. Real-time
 PCR will be performed to determine mRNA expression in the presence or absence of siRNA, and
 protein levels will be determined by Western blot. After these control studies have been
 performed, the effect of siRNA inhibition on baseline and IFN-treated HCV RNA replication
 will be tested.

 Transfection studies:

 We hypothesize that upregulation of UBP43 will increase in vitro HCV RNA replication at
 baseline and will decrease the effect of IFN, and that upregulation of ISG15 will decrease
 baseline HCV RNA and increase the effect of IFN. UBP43 expression will be upregulated using a
 UBP43 expression plasmid pcDNA6-UBP43; ISG15 expression plasmid will be obtained by
 subcloning ISG15 gene into pcDNA6 vector. As before, HCV RNA levels will be determined in the
 presence and absence of IFNa using real-time PCR.

 Taken together these studies will define the roles of the genes suggested to play an
 important role in the distinction between clinical PegIFN/Rib treatment responders and
 nonresponders.

 Ib. Mechanism of action of the ISG15/UBP43 pathway:

 If we determine that ISG15 and UBP43 play a role in HCV RNA replication in the genotype 1
 replicon model we will examine how this effect is mediated. We will determine whether the
 effect of UBP43 is dependent on its protease function or on its association with another
 cellular protein. We will also identify which ISG15 protein targets might mediate its effect.

 Role of UBP43 protease activity:

 If elimination of UBP43 is shown to reduce viral replication or magnify the interferon
 response, we hypothesize that inhibition of UBP43 will represent a novel means of treatment
 of HCV. UBP43 would be of particular interest because it has protease activity: proteases are
 known and validated targets for small molecule therapies. Thus, it would be critical to
 establish whether the protease activity of UBP43 is important for its effect on HCV
 replication, and not, for instance, its interaction with another cellular protein. To address
 this question we will over-express UBP43 protease inactive constructs. An inactive form of
 UBP43 will be created by mutation of a critical cysteine residue (Cys61) into serine using
 site-directed mutagenesis of pBK/CMV-UBP43 plasmid as described previously. In order to
 characterize the protease active and inactive forms of UBP43, wild type and mutant UBP43 will
 be expressed as GST fusion proteins in the expression vector pGEX-4T-3 (pGEX-4T-3-UBP43), and
 transformed into E.Coli BL21 (DE3) with a plasmid, pET-ISG15-UBP43-H, that expresses
 ISG15-UBP43 fusion protein.35 ISG15 cleavage from the fusion protein will be shown by Western
 blot. Once we have confirmed that the protease function is eliminated by site-directed
 mutagenesis, wild-type and mutated UBP43 will be overexpressed in Huh7.5 cells with the HCV
 replicon and exposed to increasing doses of IFNa. If the protease function is critical
 overexpression of the inactive (in a dominant negative fashion) will increase responsiveness
 to IFN.

 Protein targets of ISG15:

 Previous work using thymic tissue has identified several targets for ISGylation by ISG15,
 including phospholipase Cg1, Jak1, and ERK1. Whether these are targets in Huh7.5 cells is not
 known, though ERK and JAK have been previously linked to antiviral effects in the HCV
 replicon model. If we find that ISG15 is implicated in HCV replication, we would seek to
 identify substrates for ISG15 modification. We will first perform a series of
 immunoprecipitation studies directed specifically at the proteins previously shown found to
 be modified by ISG15, and explore the differences between infected and uninfected cells. In
 brief, following incubation of Huh7.5 cells +/- replicon with IFNa for 12 hours, cells will
 be lysed in RIPA buffer and immunoprecipitation of ISG15 conjugates performed with the
 anti-ISG15 mAb used above. After resolution on a reducing gel, western blots will be
 performed to determine if the three targets above are conjugated by ISG15, using
 commercially-available mAbs. If any or all of the targets are conjugated, further studies
 will be performed to elucidate the consequences of ISGylation.

 To more fully explore which proteins are modified with ISG15 in cell culture, we will
 undertake a more comprehensive screen using mass-spectroscopy - applying a strategy
 previously used to identify targets of the ubiquitin-like SUMO protein. TAP-tagging
 approaches coupled with MALDI-TOF mass spectrometry will be used. We will transfect cells
 with TAP-tagged or SPA-tagged ISG15 and purify the protein and protein complexes from cells
 treated or untreated with IFN. Using established procedures, we will then purify and identify
 the substrates using gel electrophoresis and mass spectrometry. Gel slices containing the
 proteins will be reductively alkylated and subjected to trypsinolysis. The peptides will be
 purified and analyzed by MALDI-TOF mass spectrometry using a cyano-4-hydroxycin-namic acid
 matrix on a Voyager DE-STR instrument (Applied Biosystems). Identification of the proteins
 using these mass fingerprinting data will be carried out using the ProFound software . Since
 MS cannot be used to quantify protein amounts, we will identify proteins of interest by
 comparing lists of proteins ISGylated under the various conditions and then perform Western
 blot studies to compare protein amounts. In doing these experiments we will use the MS
 equipment and expertise readily available at the Best Institute.

 Conclusions I: Taken together these studies will clearly define the roles of the ISG15 and
 UBP43 pathway in the HCV replicon response to IFN, and will suggest how the effect is
 mediated. Importantly, the methodologies described above, though detailed for ISG15 and
 UBP43, are generic and can be applied to other candidate genes of interest.

 Section II: Gene Expression Profiling of Acute Hepatitis C Viral Infection:

 There are similarities in the pathways that we have identified as important in the chronic
 disease with those that may be important in acute infection. For example, polymorphisms of
 2'5'OAS - a gene found on our list - alter the risk of progressing from acute to chronic
 infection.20 Therefore we hypothesize that this same set of genes is likely to play an
 important role in determining failure to clear acute infection. We will test this hypothesis
 using microarray analysis.

 We plan to examine the role of these and other genes in acute HCV infection in both the
 systemic and local host/virus interaction. Acutely-infected liver tissue is difficult to
 obtain because most acute HCV infections are not identified, and because it is inappropriate
 to perform liver biopsies in these cases. Thus we will use two different approaches. First,
 we will study the gene expression patterns in peripheral blood mononuclear cells (PBMCs)
 collected from Egyptian health care workers with acute HCV (provided by Dr. Sanaa Kamal) and
 compare these to PBMCs from patients with genotype-matched chronic HCV and to healthy
 volunteers. We will develop predictive models to test which gene subsets are most associated
 with clinical outcomes such as the establishment of chronic infection. Second, we will use
 the recurrence of HCV post transplantation as a model of acute hepatitis in vivo. We will
 take biopsies of the donor liver PRIOR to transplantation and then follow gene expression
 levels over time as the virus infects the graft. After transplantation the HCV-naïve liver
 rapidly and universally becomes infected with HCV, though only 70% of patients develop
 histological evidence of recurrence on liver biopsies taken 3 and/or 6 months
 post-transplant. It is these patients who we currently treat for recurrent HCV using
 PegIFN/Rib. As before, we will develop predictive models to associate gene subsets with
 clinical outcomes. We have achieved more than a 60% response rate by encouraging treatment
 adherence despite the even more grueling side effects after transplantation. Thus, as for the
 treatment of HCV prior to transplantation a significant fraction of patients do not respond
 to therapy and are needlessly exposed to morbid side effects: the key clinical outcome we
 will consider is response to therapy with PegIFN/Rib. Another outcome of interest is the
 rapid development of cirrhosis within 5 years in 25-30% of patients, and the development of
 an aggressive fibrosing cholestatic state in 7-9% within 2 years of transplant. Together
 these data will point to the genes important for the immunological response that results in
 disease persistence or elimination (acute HCV in health care workers) and progression of
 liver disease (post-transplantation patients). Highly up- and down-regulated genes will be
 further studied in the HCV replicon model.

 Research Objective 2. To determine the gene expression profiles which are most highly
 associated with clinical outcomes in acute hepatitis, both in circulating immune cells and in
 the liver.

 Research Protocols for Section II:

 We propose three major analyses. First, we will perform gene expression profiling in PBMC
 preparations provided by Dr. Kamal from patients with acute HCV. These PBMC preparations are
 being performed as part of ongoing studies headed by Dr. Kamal. Second, we will use
 microarray analysis to describe the hepatic genes which are altered by HCV infection in the
 post-transplant liver graft in response to infection by HCV. For this we will use portions of
 liver biopsies already being taken as part of routine post-transplant clinical care. Third,
 we will prepare PBMCs from patients with chronic HCV and from these determine gene expression
 profiles. Not only are these a critical comparison to acute HCV, but they will also act as
 the basis for predicting outcomes of treatment in chronic HCV.

 IIa. Patient recruitment, study population and collection of samples and data:

 In all cases, patients will be identified by clinical staff as potential candidates for the
 study and approached by a study nurse. Consent will be obtained for the research use of
 tissue and blood samples, and for the collection of patient clinical data.

 Acute HCV: systemic PBMC response Dr. Kamal follows a population of Egyptian health care
 workers who become infected acutely with HCV, and she routinely isolates PBMCs from them.
 This clinical population is composed primarily of Genotype 1 and Genotype 4 infected
 individuals, and is thoroughly described for patient demographics, RNA titer at the time of
 blood collection, and progression to chronic infection. Given the relative rarity of Genotype
 4 in our local population our initial analysis will focus on Genotype 1 acute HCV, but we
 intend to study genotype 4 as well, since this genotype has great relevance to HCV worldwide.
 Based on our previous work we anticipate needing between 10-20 patients in a given group in
 order to clearly define consistent gene expression changes across a spectrum of clinical
 variability. We expect to collect and study PBMC samples from at least 20 acute genotype 1
 patients per year. Samples from 20 healthy control volunteers will also be obtained over the
 3 year study period (recruited by poster).

 Acute HCV: Hepatic response to HCV recurrence post-transplantation Our center performs 30-40
 liver transplants each year in persons infected with HCV. All clinical data in our liver
 transplant population is entered into an Oracle-based database. Liver biopsies are routinely
 taken from the HCV-naïve graft prior to transplantation and at 12, 24, and 52 weeks
 post-transplantation. Based on previous experience we expect an accrual rate of at least 80%;
 thus, we will be able to collect data and specimens from roughly 24-32 patients per year.
 Biopsies of normal liver are taken as part of an ongoing study in our living donor population
 (PI: I. McGilvray). 30-40 such biopsies are taken every year and represent a unique source of
 control liver tissue.

 Chronic HCV: systemic PBMC response Dr. Heathcote follows a large population of patients with
 chronic HCV. Blood samples will be taken from patients in whom treatment with PegIFN/Rib is
 being considered and PBMCs will be isolated. Based on our experience with the liver biopsy
 studies outlined in the Progress Report above, we expect to obtain PBMC preparations from at
 least 50 patients each year. Roughly 2/3 of these are Genotype 1 patients.

 IIb. Data Analysis:

 Acute HCV: Gene expression changes in response to acute HCV in circulating PBMCs Gene
 expression profiles from PBMCs from patients with acute Genotype 1 HCV will be determined and
 compared to those from normal healthy controls and chronic genotype 1 HCV. Genes will be
 identified by statistical and fold differences between groups (p value of <0.01, fold change
 of at least 1.5). This comparison will define which gene expression alterations are specific
 to acute HCV infection. In order to associate individual genes with the progression to
 chronic disease, subgroup analyses will be done in which gene expression in acutely infected
 individuals is compared between those who went on to chronic infection and those who
 spontaneously eliminated the virus. After real-time PCR confirmation of these genes we will
 use a number of unsupervised (hierarchical clustering, principal components) and supervised
 (nearest neighbours, linear discriminants) analyses to develop predictive gene sets which
 accurately classify patients who go on to develop chronic infection. Taken together these
 results will identify those genes whose expression is altered in the systemic immunologic
 response to acute HCV and how these relate to the establishment of chronic disease. Genes
 which are of interest as possible therapeutic targets will be further studied using the HCV
 replicon model, as described earlier. By the end of the second year of the study we expect to
 have analyzed 40 acute genotype 1 patients, and in the third year we will analyze 20 genotype
 4 patients.

 Acute HCV: local hepatic gene expression in post-transplantation HCV infection Gene
 expression profiles will be determined from liver biopsies at organ retrieval, prior to
 infection by HCV, and at 3, 6 and 12 months after transplantation. Gene changes can therefore
 be followed in the same patient and between patients; genes levels will be compared to normal
 liver tissue and to baseline gene expression in HCV-naïve grafts, using statistical methods
 developed in our lab and at the EBI. Currently 70% of the patients transplanted with HCV in
 our center are treated for recurrent HCV post-transplantation after evidence of recurrent HCV
 on liver biopsy, and in these patients quantitative HCV titers are done at 3 or 6 months
 post-transplantation. Roughly 80% of our patients complete therapy over a 12 month course.
 Thus, by the end of our 3 year study we will know the response to treatment of between 28-36
 patients (we expect a 60% response rate, thus roughly 17-22 responders and 11-14
 nonresponders), and eventually will have data on the 42-54 patients expected to complete
 therapy. With this sample size we can determine which gene subsets are predictive of response
 to treatment (using the methods noted above). Equally we will be able to relate gene
 expression to HCV RNA titer in 50-60 patients. By the end of the three year study we will
 have clinical and genomic data on 72 to 90 patients. Ultimately we will relate gene
 expression to the development of recurrent cirrhosis, and we anticipate that roughly 18-30
 patients will have gone on to this state within 5 years of transplantation. In order to
 control for the effects of the many confounding factors that could influence hepatic gene
 expression post-transplantation (eg. acute rejection, immunosuppression) we will perform
 subgroup analyses using our extensive clinical database. We will compare HCV treatment
 responders to nonresponders, rejection to no rejection, and different immunosuppressive
 regimens, in addition to comparing gene expression profiles from acute hepatic HCV to those
 already determined for chronic HCV. These analyses will allow us to distinguish and describe
 the effects of acute HCV infection on hepatic gene expression.

 Chronic HCV: circulating PBMC response and prediction of treatment outcome:

 Over the first 2 years of our study we will use gene expression profiling to study PBMCs from
 roughly 30 patients with chronic genotype 1 HCV. This data will serve as a crucial control
 for our studies of acute HCV in PBMCs. However, all of these samples are taken from patients
 who will go on to treatment with PegIFN/Rib. Therefore we will use the gene expression data
 from these studies not only for comparison with the acutely infected PBMCs, but also to
 determine which gene subsets are predictive of treatment outcome. These results will form the
 basis for a noninvasive prognostic test.

 Conclusions II: These studies will provide a comprehensive description of the gene expression
 changes that accompany acute HCV infection at both the systemic and the local level. They
 will clearly identify genes which are important to the progression to chronic disease and to
 adverse clinical outcomes, thereby suggesting novel prognostic and therapeutic targets.
Inclusion Criteria: patients who have a liver biopsy as standard of care and are diagnosed
 with either:

 -  patients attending Liver Clinic at Toronto Western Hospital, Toronto, ON, Canada

 -  Hepatitis C

 -  Hepatitis B

 -  Autoimmune Hepatitis

 -  Primary Biliary Cirrhosis

 -  Primary Sclerosing Cholangitis

 Exclusion Criteria: