Effect of maintenance therapy with low-dose peginterferon for recurrent hepatitis C after living donor liver transplantation.

Approximately 30% of patients who have recurrent hepatitis C after liver transplantation achieve sustained virological response (SVR) by taking a combination therapy of pegylated interferon and ribavirin. For the remaining non-SVR patients, an effective management treatment has not yet been established. In this study, efficacy of long-term peginterferon maintenance therapy for non-SVR patients was evaluated. Forty patients who had previously received the combination therapy for hepatitis C after living donor liver transplantation were classified into one of the following three groups: the SVR group (n = 11); the non-SVR-IFN group (n =17), which received low-dose peginterferon maintenance therapy for non-SVR patients; and the non-SVR-Withdrawal group (n = 12), which discontinued the interferon treatment. We then compared histological changes among these three groups after 2 or more years follow-up. Activity grade of liver histology improved or remained stable in patients in the SVR and non-SVR-IFN groups, but deteriorated in half of the patients in the non-SVR-Withdrawal group. Fibrosis improved or remained stable in 10 of 11 SVR patients and in 13 of 17 non-SVR-IFN patients, but deteriorated in all non-SVR-Withdrawal patients. Mean changes in fibrosis stage between pretreatment and final liver biopsy were -0.18, +0.06 and +2.2 in the SVR, non-SVR-IFN and non-SVR-Withdrawal groups, respectively. Fibrosis stage deteriorated to F3 or F4 significantly more rapidly in the non-SVR-Withdrawal group than in the other two groups. In conclusion, continuing long-term maintenance therapy with peginterferon prevented histological progression of hepatitis C in patients who had undergone living donor liver transplantation.

Targeting activation-induced cytidine deaminase prevents colon cancer development despite persistent colonic inflammation.

Inflammatory bowel disease (IBD) is an important etiologic factor in the development of colorectal cancer. However, the mechanism underlying carcinogenesis through chronic inflammation is still unknown. Activation-induced cytidine deaminase (AID) is induced by the inflammation and involved in various human carcinogenesis via its mutagenic activity. In the current study, we investigated whether the inflammation/AID axis plays an integral role in the development of colitis-associated cancers. Inflammation in the cecum was more severe than that in other colonic regions, and endogenous AID expression was enhanced most prominently in the inflamed cecal mucosa of interleukin (IL)-10(-/-) mice. Blockade of tumor necrosis factor (TNF)-α and IL-12 significantly suppressed AID expression. Although proinflammatory cytokine expression was comparable between IL-10(-/-)AID(+/+) and IL-10(-/-)AID(-/-) mice, sequencing analyses revealed a significantly lower incidence of somatic mutations in Trp53 gene in the colonic mucosa of IL-10(-/-)AID(-/-) than IL-10(-/-)AID(+/+) mice. Colon cancers spontaneously developed in the cecum in 6 of 22 (27.2%) IL-10(-/-)AID(+/+) mice. In contrast, none of the IL-10(-/-)AID(-/-) mice developed cancers except only one case of neoplasia in the distal colon. These findings suggest that the proinflammatory cytokine-induced aberrant production of AID links colonic inflammation to an enhanced genetic susceptibility to oncogenic mutagenesis. Targeting AID could be a novel strategy to prevent colitis-associated colon carcinogenesis irrespective of ongoing colonic inflammation.

Clinical features of biochemical cholestasis in patients with recurrent hepatitis C after living-donor liver transplantation.

Recurrent hepatitis C after liver transplantation (HepC-LT) progresses faster than hepatitis C in non-transplant settings. Cholestasis has been suggested to be one characteristic of HepC-LT related to the rapid progression. We investigated the clinical features of biochemical cholestasis, which we defined as high serum concentrations of alkaline phosphatase and gamma-glutamyl transpeptidase, in patients with recurrent hepatitis C after living-donor liver transplantation. Eighty patients were diagnosed with post-transplant recurrent hepatitis C after exclusion of other aetiologies of cholestasis by liver biopsy and imaging. The clinical features of biochemical cholestasis in the patients with HepC-LT, including histological changes, the efficacy of interferon therapy and helper T-cell (Th) subsets in the peripheral blood, were analysed. Fifty-five of the 80 patients with HepC-LT (69%) had evidence of biochemical cholestasis. Progression of liver fibrosis to stage F3 or F4 was significantly accelerated in patients with biochemical cholestasis compared with patients without cholestasis. The biochemical cholestasis in patients with HepC-LT improved after interferon therapy in 22 of 39 patients (56%) who showed a virological response to the therapy, suggesting that hepatitis C virus (HCV) caused the biochemical cholestasis in these patients. Patients with biochemical cholestasis who had a biochemical response to interferon therapy showed an increased Th1 responses in peripheral blood. In conclusion, biochemical cholestasis is the characteristic feature of HepC-LT and is related to progression of liver fibrosis. An increased Th1 response is associated with cholestasis caused by HCV after liver transplantation.

Association of genetic polymorphisms with interferon-induced haematologic adverse effects in chronic hepatitis C patients.

Interferon (IFN)-based combination therapy with ribavirin has become the gold standard for the treatment of chronic hepatitis C virus infection. Haematologic toxicities, such as neutropenia, thrombocytopenia, and anaemia, however, frequently cause poor treatment tolerance, resulting in poor therapeutic efficacy. The aim of this study was to identify host genetic polymorphisms associated with the efficacy or haematologic toxicity of IFN-based combination therapy in chronic hepatitis C patients. We performed comprehensive single nucleotide polymorphism detection in all exonic regions of the 12 genes involved in the IFN signalling pathway in 32 healthy Japanese volunteers. Of 167 identified polymorphisms, 35 were genotyped and tested for an association with the efficacy or toxicity of IFN plus ribavirin therapy in 240 chronic hepatitis C patients. Multiple logistic regression analysis revealed that low viral load, viral genotypes 2 and 3, and a lower degree of liver fibrosis, but none of the genetic polymorphisms, were significantly associated with a sustained virologic response. In contrast to efficacy, multiple linear regression analyses demonstrated that two polymorphisms (IFNAR1 10848-A/G and STAT2 4757-G/T) were significantly associated with IFN-induced neutropenia (P = 0.013 and P = 0.011, respectively). Thrombocytopenia was associated with the IRF7 789-G/A (P = 0.031). In conclusion, genetic polymorphisms in IFN signalling pathway-related genes were associated with IFN-induced neutropenia and thrombocytopenia in chronic hepatitis C patients. In contrast to toxicity, the efficacy of IFN-based therapy was largely dependent on viral factors and degree of liver fibrosis.

A novel mouse model of hepatocarcinogenesis triggered by AID causing deleterious p53 mutations.

Activation-induced cytidine deaminase (AID), the only enzyme that is known to be able to induce mutations in the human genome, is required for somatic hypermutation and class-switch recombination in B lymphocytes. Recently, we showed that AID is implicated in the pathogenesis of human cancers including hepatitis C virus (HCV)-induced human hepatocellular carcinoma (HCC). In this study, we established a new AID transgenic mouse model (TNAP-AID) in which AID is expressed in cells producing tissue-nonspecific alkaline phosphatase (TNAP), which is a marker of primordial germ cells and immature stem cells, including ES cells. High expression of TNAP was found in the liver of the embryos and adults of TNAP-AID mice. HCC developed in 27% of these mice at the age of approximately 90 weeks. The HCC that developed in TNAP-AID mice expressed alpha-fetoprotein and had deleterious mutations in the tumour suppressor gene Trp53, some of which corresponded to those found in human cancer. In conclusion, TNAP-AID is a mouse model that spontaneously develops HCC, sharing genetic and phenotypic features with human HCC, which develops in the inflamed liver as a result of the accumulation of genetic changes.

Parkin as a tumor suppressor gene for hepatocellular carcinoma.

The parkin was first identified as a gene implicated in autosomal recessive juvenile Parkinsonism. Deregulation of the parkin gene, however, has been observed in various human cancers, suggesting that the parkin gene may be important in tumorigenesis. To gain insight into the physiologic role of parkin, we generated parkin-/- mice lacking exon 3 of the parkin gene. We demonstrated here that parkin-/- mice had enhanced hepatocyte proliferation and developed macroscopic hepatic tumors with the characteristics of hepatocellular carcinoma. Microarray analyses revealed that parkin deficiency caused the alteration of gene expression profiles in the liver. Among them, endogenous follistatin is commonly upregulated in both nontumorous and tumorous liver tissues of parkin-deficient mice. Parkin deficiency resulted in suppression of caspase activation and rendered hepatocytes resistant to apoptosis in a follistatin-dependent manner. These results suggested that parkin deficiency caused enhanced hepatocyte proliferation and resistance to apoptosis, resulting in hepatic tumor development, partially through the upregulation of endogenous follistatin. The finding that parkin-deficient mice are susceptible to hepatocarcinogenesis provided the first evidence showing that parkin is indeed a tumor suppressor gene.

Expression of activation-induced cytidine deaminase in human hepatocytes via NF-kappaB signaling.

Activation-induced cytidine deaminase (AID) is involved in somatic DNA alterations of the immunoglobulin gene for amplification of immune diversity. The fact that constitutive expression of AID in mice causes tumors in various organs, including lymphoid tissues and lungs, suggests the important role of the aberrant editing activity of AID on various tumor-related genes for carcinogenesis. AID expression, however, is restricted to activated B cells under physiological conditions. We demonstrate here that ectopic AID expression is induced in response to tumor necrosis factor-alpha stimulation in cultured human hepatocytes. The proinflammatory cytokine-mediated expression of AID is achieved by IkappaB kinase-dependent nuclear factor (NF)-kappaB signaling pathways. Hepatitis C virus, one of the leading causes of hepatocellular carcinoma (HCC), enhanced AID expression via NF-kappaB activation through expression of viral core protein. The aberrant expression of AID in hepatoma-derived cells resulted in accumulation of genetic alterations in the c-myc and pim1 genes, suggesting that inappropriate expression of AID acts as a DNA mutator that enhances the genetic susceptibility to mutagenesis in human hepatocytes. Our current findings indicate that the inappropriate expression of AID is induced by proinflammatory cytokine stimulation and may provide the link between hepatic inflammation and the development of HCC.

Beneficial effects of short-term lamivudine treatment for de novo hepatitis B virus reactivation after liver transplantation.

Clearance of hepatitis B surface antigen (HBsAg) by lamivudine is achieved in only a small proportion of patients with chronic hepatitis B virus (HBV) infection. We investigated the effect of lamivudine on de novo HBV reactivation after living-donor liver transplantation when the number of HBV was expected to be very small. Thirty-eight HBV-naive recipients who received liver grafts from antibodies to core antigen-positive donors receiving hepatitis B immunoglobulin (HBIG) were studied. HBsAg appeared in nine cases (23.7 %) despite receiving HBIG for 12-71 months (mean: 35.1 months) after transplantation. Lamivudine treatment was started in six recipients during the acute phase of HBV reactivation. Five of the six recipients achieved complete clearance of HBsAg in sera at a median of 4.6 months (ranging from 21 to 330 days) after lamivudine administration. Although lamivudine was stopped in four cases, all remained negative for HBsAg. Our findings suggested that short-term lamivudine treatment during acute phase of HBV reactivation could achieve complete clearance of HBsAg in a significant number of liver transplant recipients.

Identification of novel defective HCV clones in liver transplant recipients with recurrent HCV infection.

Patients with recurrent hepatitis C after liver transplantation usually have a high viral load and are generally resistant to interferon (IFN)-alpha2b plus ribavirin (RBV) therapy. However, it remains unclear whether pretreatment viral titre determines the effectiveness of combination therapy, especially in patients with a high viral load. The aim of this study was to identify the viral factors associated with a sustained virological response (SVR) to antiviral therapy in patients with recurrent hepatitis C after living-donor liver transplantation. Twenty-three patients with recurrent hepatitis C received combination therapy of IFN-alpha2b plus RBV. SVR was achieved in 7 of the 23 patients (30.4%). Predictive factors for SVR included a 2 log10 decline in Hepatitis C virus (HCV) RNA at 2 weeks after the start of therapy and disappearance of HCV RNA at 4 or 24 weeks after the start of therapy. As the pretreatment high viral load showed no association with SVR, we asked whether other viral factor was associated with the response to the combination therapy in transplant recipients. We found the several novel defective HCV clones in 4 of 12 recipients' sera. All defective HCV clones had deletions in the envelope region. Interestingly, no patients with defective clones showed a prompt decrease in HCV RNA after the start of IFN-alpha2b plus RBV therapy. Thus, early decline in serum HCV RNA after treatment was closely associated with SVR. The circulating defective HCV clones are present and might be associated with the response to the combination therapy in patients with recurrent hepatitis after liver transplantation.

Prevention of hepatitis B virus recurrence after living donor liver transplantation.

The emergence of lamivudine-resistant hepatitis B mutations is a major complication during pretransplantation treatment. The proper time to begin Lamivudine before transplantation is not yet known. Twenty-six patients received preoperative lamivudine treatment followed by combined lamivudine and hepatitis B immunoglobulin after transplantation up to December 2002. The length of preoperative lamivudine treatment ranged from 13 to 200 days (mean, 52 +/- 37 days). Hepatitis B virus-DNA was positive in 22 of 26 (84.6%) patients before preoperative lamivudine prophylaxis and persistently positive among only 4 of 22 patients (18%) who at transplantation did not show a viral mutation. In all patients, hepatitis B virus-DNA became negative immediately after transplantation. At a median follow-up of 34 months, neither a hepatitis B recurrence nor a mutation had occurred in any patient. The ability to schedule the proper time for preoperative lamivudine prophylaxis is an advantage of living donor liver transplantation.

Reactivation of latently infected hepatitis B virus in a leukemia patient with antibodies to hepatitis B core antigen.

A 66-year-old man with chronic B-cell leukemia who had antibody to hepatitis B core antigen (anti-HBc) but not hepatitis B surface antigen (HBsAg) suffered from lethal hepatitis caused by hepatitis B virus (HBV) reactivation. He initially lacked circulating viral genomes in his sera and did not have a past history of liver dysfunction. In this patient, the immunosuppressive condition introduced by disease progression of leukemia induced reactivation of dormant HBV, and the withdrawal of chemotherapy resulted in fatal liver failure. Mutation-specific assay based on competitive polymerase chain reaction (PCR) and sequencing analyses revealed the predominant reactivation of an HBV strain with missence mutation (point mutation G-to-A at nucleotide 1896) in the precore regions, as well as point mutations in the core promoter regions. Therefore, it is important to note the risk of HBV reactivation, with resulting lethal hepatic failure, in anti-HBc-positive healthy individuals, even when they lack HBsAg. who receive immunomodulating therapy.

Regulation of Fas-mediated apoptosis by NF-kappaB activity in human hepatocyte derived cell lines.

Nuclear Factor-kappaB (NF-kappaB) is known to be one of the key regulators of genes involved in survival signaling. The purpose of this study is to investigate the role of NF-kappaB activity in signaling events related to cell survival in hepatocytes, which has been supposed to be one of the most sensitive organs against Fas-induced cytotoxicity. The functions of NF-kappaB activity on Fas-mediated apoptosis in different human cell lines were investigated by a magnetic concentration system for cells with exogenous protein production. We demonstrated that the activation of NF-kappaB was triggered by anti-Fas treatment in hepatocyte derived cell lines, HepG2 and Huh-7 cells. Overexpression of kinase-inactive NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK) inhibited the activation of NF-kappaB introduced by anti-Fas treatment in these cells. Notably, inactivation of NF-kappaB by the production of IkappaB-alpha protein made these cells more susceptible to apoptosis induced by Fas stimulation. In contrast, transient expression of NIK showed a suppressive effect on Fas-mediated apoptosis in the same cell lines. These findings suggest the involvement of NF-kappaB in suppression of Fas-mediated apoptosis in human hepatocyte derived cell lines, in which concomitant activation of this transcriptional factor was observed through the stimulation of Fas signaling.

Cytoplasmic localization is important for transcription factor nuclear factor-kappa B activation by hepatitis C virus core protein through its amino terminal region.

We previously reported that hepatitis C virus core protein (core) activates the transcription factor nuclear factor-kappa B (NF-kappa B) when expressed transiently. In the present study, we investigated the relationship between the NF-kappa B activation capacity and subcellular localization of the core. By changing the subcellular localization of the C-terminally truncated core from the nucleus to the cytoplasm, NF-kappa B was activated. In addition, NF-kappa B activity was augmented by forcing the mutated core to move to the endoplasmic reticulum. It was also suggested that the region from aa 21 to 80 of the core is involved in the activation of NF-kappa B.

TUCAN, an antiapoptotic caspase-associated recruitment domain family protein overexpressed in cancer.

Caspase-associated recruitment domains (CARDs) are protein interaction domains that participate in activation or suppression of CARD-carrying members of the caspase family of apoptosis-inducing proteases. A novel CARD-containing protein was identified that is overexpressed in some types of cancer and that binds and suppresses activation of procaspase-9, which we term TUCAN (tumor-up-regulated CARD-containing antagonist of caspase nine). The CARD domain of TUCAN selectively binds itself and procaspase-9. TUCAN interferes with binding of Apaf1 to procaspase-9 and suppresses caspase activation induced by the Apaf1 activator, cytochrome c. Overexpression of TUCAN in cells by stable or transient transfection inhibits apoptosis and caspase activation induced by Apaf1/caspase-9-dependent stimuli, including Bax, VP16, and staurosporine, but not by Apaf1/caspase-9-independent stimuli, Fas and granzyme B. High levels of endogenous TUCAN protein were detected in several tumor cell lines and in colon cancer specimens, correlating with shorter patient survival. Thus, TUCAN represents a new member of the CARD family that selectively suppresses apoptosis induced via the mitochondrial pathway for caspase activation.