Naoki Hiramatsu

Reduced Numbers and Impaired Ability of Myeloid and Plasmacytoid Dendritic Cells to Polarize T Helper Cells in Chronic Hepatitis C Virus Infection

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries. The mechanism by which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) consist of myeloid and plasmacytoid subsets that play distinct roles in the regulation of antivirus immune responses; however, their roles in the pathogenesis of HCV infection are yet to be determined. We compared the numbers and functions of myeloid and plasmacytoid DCs between 43 patients with chronic hepatitis and 26 age-matched healthy volunteers. Absolute numbers of myeloid DCs, plasmacytoid DCs, and DC progenitors in the periphery were significantly lower in patients with chronic hepatitis than in healthy volunteers. Myeloid and plasmacytoid DCs from the patients had impaired abilities to stimulate allogeneic CD4 T cells and to produce interleukin (IL)-12 p70 and interferon- alpha , compared with those from healthy volunteers. After exposure to naive CD4 T cells, myeloid DCs from the patients were less able to drive the T helper type 1 response, whereas myeloid and plasmacytoid DCs from the patients primed more IL-10-producing cells than did those from healthy volunteers. In conclusion, in chronic HCV infection, both types of blood DCs are reduced and have an impaired ability to polarize T helper cells.

Negative Regulation of NK Cell Activities by Inhibitory Receptor CD94/NKG2A Leads to Altered NK Cell-Induced Modulation of Dendritic Cell Functions in Chronic Hepatitis C Virus Infection

NK cells are potent activators of dendritic cells (DCs), but it remains obscure how third-party cells affect the ability of NK cells to modulate DC functions. We show here that NK cells derived from healthy donors (N-NK), when cocultured with human liver epithelial cells, induced maturation as well as activation of DCs, such as increased migratory capacity as well as T cell stimulatory activity. In contrast, NK cells from chronic hepatitis C virus-infected donors (HCV-NK) were not capable of activating DCs under the same conditions. In comparison to N-NK, HCV-NK showed higher expression of CD94/NKG2A and produced IL-10 and TGFβ when cultured with hepatic cells, most of which express HLA-E, a ligand for CD94/NKG2A. Blockade of NKG2A restored the ability of HCV-NK to activate DCs, which appeared to result from the reduced NK cell production of IL-10 and TGFβ. The blockade also endowed HCV-NK with an ability to drive DCs to generate Th1-polarized CD4+ T cells. These findings show that NK cell modulation of DCs is regulated by third-party cells through NK receptor and its ligand interaction. Aberrant expression of NK receptors may have an impact on the magnitude and direction of DC activation of T cells under pathological conditions, such as chronic viral infection.

Inhibition of autophagy potentiates the antitumor effect of the multikinase inhibitor sorafenib in hepatocellular carcinoma

Multikinase inhibitor sorafenib inhibits proliferation and angiogenesis of tumors by suppressing the Raf/MEK/ERK signaling pathway and VEGF receptor tyrosine kinase. It significantly prolongs median survival of patients with advanced hepatocellular carcinoma (HCC) but the response is disease‐stabilizing and cytostatic rather than one of tumor regression. To examine the mechanisms underlying the relative resistance in HCC, we investigated the role of autophagy, an evolutionarily conserved self‐digestion pathway, in hepatoma cells in vitro and in vivo. Sorafenib treatment led to accumulation of autophagosomes as evidenced by conversion from LC3‐I to LC3‐II observed by immunoblot in Huh7, HLF and PLC/PRF/5 cells. This induction was due to activation of autophagic flux, as there was further increase in LC3‐II expression upon treatment with lysosomal inhibitors, clear decline of the autophagy substrate p62, and an mRFP‐GFP‐LC3 fluorescence change in sorafenib‐treated hepatoma cells. Sorafenib inhibited the mammalian target of rapamycin complex 1 and its inhibition led to accumulation of LC3‐II. Pharmacological inhibition of autophagic flux by chloroquine increased apoptosis and decreased cell viability in hepatoma cells. siRNA‐mediated knockdown of the ATG7 gene also sensitized hepatoma cells to sorafenib. Finally, sorafenib induced autophagy in Huh7 xenograft tumors in nude mice and coadministration with chloroquine significantly suppressed tumor growth compared with sorafenib alone. In conclusion, sorafenib administration induced autophagosome formation and enhanced autophagic activity, which conferred a survival advantage to hepatoma cells. Concomitant inhibition of autophagy may be an attractive strategy for unlocking the antitumor potential of sorafenib in HCC.

Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice

The tumor suppressor p53 has been implicated in the pathogenesis of non-cancer-related conditions such as insulin resistance, cardiac failure, and early aging. In addition, accumulation of p53 has been observed in the hepatocytes of individuals with fibrotic liver diseases, but the significance of this is not known. Herein, we have mechanistically linked p53 activation in hepatocytes to liver fibrosis. Hepatocyte-specific deletion in mice of the gene encoding Mdm2, a protein that promotes p53 degradation, led to hepatocyte synthesis of connective tissue growth factor (CTGF; the hepatic fibrogenic master switch), increased hepatocyte apoptosis, and spontaneous liver fibrosis; concurrent removal of p53 completely abolished this phenotype. Compared with wild-type controls, mice with hepatocyte-specific p53 deletion exhibited similar levels of hepatocyte apoptosis but decreased liver fibrosis and hepatic CTGF expression in two models of liver fibrosis. The clinical significance of these data was highlighted by two observations. First, p53 upregulated CTGF in a human hepatocellular carcinoma cell line by repressing miR-17-92. Second, human liver samples showed a correlation between CTGF and p53-regulated gene expression, which were both increased in fibrotic livers. This study reveals that p53 induces CTGF expression and promotes liver fibrosis, suggesting that the p53/CTGF pathway may be a therapeutic target in the treatment of liver fibrosis.

The Bcl-xL inhibitor, ABT-737, efficiently induces apoptosis and suppresses growth of hepatoma cells in combination with sorafenib†

Tumor cells are characterized by uncontrolled proliferation, often driven by activation of oncogenes, and apoptosis resistance. The oncogenic kinase inhibitor sorafenib can significantly prolong median survival of patients with advanced hepatocellular carcinoma (HCC), although the response is disease‐stabilizing and cytostatic rather than one of tumor regression. Bcl‐xL (B cell lymphoma extra large), an antiapoptotic member of the B cell lymphoma‐2 (Bcl‐2) family, is frequently overexpressed in HCC. Here, we present in vivo evidence that Bcl‐xL overexpression is directly linked to the rapid growth of solid tumors. We also examined whether ABT‐737, a small molecule that specifically inhibits Bcl‐xL but not myeloid cell leukemia‐1 (Mcl‐1), could control HCC progression, especially when used with sorafenib. Administration of ABT‐737, even at an in vivo effective dose, failed to suppress Huh7 xenograft tumors in mice. ABT‐737 caused the levels of Mcl‐1 expression to rapidly increase by protein stabilization. This appeared to be related to resistance to ABT‐737, because decreasing Mcl‐1 expression levels to the baseline by a small interfering RNA–mediated strategy made hepatoma cells sensitive to this agent. Importantly, administration of ABT‐737 to Mcl‐1 knockout mice induced severe liver apoptosis, suggesting that tumor‐specific inhibition of Mcl‐1 is required for therapeutic purposes. Sorafenib transcriptionally down‐regulated Mcl‐1 expression specifically in tumor cells and abolished Mcl‐1 up‐regulation induced by ABT‐737. Sorafenib, not alone but in combination with ABT‐737, efficiently induced apoptosis in hepatoma cells. This combination also led to stronger suppression of xenograft tumors than sorafenib alone. Conclusion: Bcl‐xL inactivation by ABT‐737 in combination with sorafenib was found to be safe and effective for anti‐HCC therapy in preclinical models. Direct activation of the apoptosis machinery seems to unlock the antitumor potential of oncogenic kinase inhibitors and may produce durable clinical responses against HCC. (HEPATOLOGY 2010)

Declining Incidence of Hepatocellular Carcinoma in Osaka, Japan, from 1990 to 2003

Context Hepatitis C virus (HCV) infection in Japan began to spread during the 1920s, increased after World War II with an explosion in parenteral amphetamine use and paid blood donation, and decreased in the 1950s to 1960s with voluntary blood donation and penalties against amphetamine use. Evidence linking the trends in HCV infection to hepatocellular carcinoma rates in Japan is limited. Contribution Data from the Osaka Cancer Registry and 10 Osaka hospitals suggest that hepatocellular carcinoma rates began to decrease in 2000, mainly because of a decrease in HCV-associated cancer. Implication Control of HCV transmission within a population seems to be followed by a decrease in hepatocellular carcinoma. The Editors Primary liver cancer was the fifth most common cancer worldwide by 2000, with approximately 551000 new cases recorded (1). In most countries, hepatocellular carcinoma (HCC) comprises 85% to 90% of primary liver cancer cases. With some exceptions, developed countries, including the United States, have been experiencing an increase in the incidence of primary liver cancer, considered to be due at least in part to increased prevalence of chronic hepatitis C virus (HCV) infection (2). Japan has had one of the highest incidence rates of primary liver cancer among developed countries (age-standardized incidence rate in 1995, 25.5 per 100000 men and 7.7 per 100000 women) (3). Approximately 90% of liver cancer cases are HCC, which, in Japan, is mainly caused by chronic HCV infection rather than chronic hepatitis B virus infection (4). A recent report on the age-standardized incidence of primary liver cancer among Japanese men, which was calculated from 6 population-based cancer registries, showed a sharp increase that started in the mid-1970s but leveled off in the mid-1990s (5). These distinctive trends were thought to be due to the spread of HCV infection, which began in the 1920s and increased after World War II (68). Thus, HCV penetrated Japan earlier than Spain, Egypt, the United States, the former Soviet Union, South Africa, and Hong Kong, as evidenced by molecular clock analysis of the sequences of HCV isolates (8). However, recent temporal trends regarding incidence rates of HCC and the contribution of HCV infection have not been clearly documented in the Japanese population. We analyzed temporal trends for HCC incidence rates between 1981 and 2003 in Osaka Prefecture (population in 2005, 8.8 million) and interpreted these in the context of HCV infection rates. Methods Data Collection on Incident HCC Cases We obtained data on incident HCC cases from the Osaka Cancer Registry, which was established by the Osaka Prefectural Government in 1962. The registry collects reports on patients with newly diagnosed cancer, including demographic and cancer-related information, from all medical institutions in Osaka Prefecture (9). These have been routinely supplemented by death certificates gathered by the Osaka Prefectural Government (9). For patients with cancer who were enrolled in the registry on the basis of their death certificate, we contacted the issuing hospital to obtain information on diagnosis and treatment and to establish the date of HCC incidence, which we determined to be the time of diagnosis at that hospital. We site-coded the data according to the International Classification of Diseases for Oncology, Third Edition (10). We included patients with HCC (codes 8170 through 8180). The protocol was approved by the ethics committee of the Osaka Medical Center for Cancer and Cardiovascular Diseases. From 1981 to 2003, 48166 men and 15696 women with HCC were documented in the Osaka Cancer Registry. We calculated the annual age-standardized incidence rates of HCC (world population as a standard population) by sex between 1981 and 2003. To characterize temporal trends for HCC, we assessed 10-year, age-specific incidence rates of HCC between 1981 and 2003 in individuals age 50 to 79 years. We studied these particular age-specific rates because most HCV-related HCC cases in the Japanese population occur between the ages of 50 and 79 years (4). We used the annual population estimates from 1981 to 2003, which were based on the average population in each sex and age category for the Osaka Prefecture during the particular period, as denominators for calculating incidence rates. The annual population estimates were based on data from the 1980, 1985, 1990, 1995, 2000, and 2005 Japanese population censuses, with linear interpolation for the years in between. Statistical Analysis To identify years when a statistically significant change in the slope of the temporal trend in the incidence occurred, we applied the joinpoint regression model by using the Joinpoint Regression Program, version 3.0 (U.S. National Cancer Institute, Bethesda, Maryland). We assumed constant variance and uncorrelated errors (11) because we could not detect heteroskedasticity by the White test or autocorrelation by the Durbin-Watson test in men or women in any age group. We computed the estimated slopes describing the average annual change of incidence rate per 100000 persons and the corresponding 95% CIs for each trend by fitting a piecewise regression line to the rates, using calendar year as a regression variable. We used the permutation test method to identify years when a statistically significant change had occurred (P< 0.05) and set the number of randomly permuted data sets at 4499. We set the number of joinpoints to a minimum of 0 and a maximum of 3 in the Joinpoint Regression Program. Data Collection on Prevalence of HCV Infection among Patients with HCC The Osaka Cancer Registry does not collect serologic data on HCV infection in the registered patients. Therefore, we used data on HCV seropositivity from patients with HCC that was diagnosed at 10 hospitals in Osaka Prefecture (1 university hospital, 2 cancer centers, and 7 general hospitals) to estimate the prevalence of HCV infection in patients with HCC. We considered the HCC diagnosis confirmed when the patient had positive histologic or positive radiologic results by enhanced computed tomography or hepatic angiography. We collected data on the patients sex, date of birth, date of diagnosis between 1990 and 2003, first Chinese letter of the family name, and presence of hepatitis B surface antigen and antibody to hepatitis C (anti-HCV) as assessed by any commercially available kit. We did not collect the full first and family name for reasons of confidentiality. Because anti-HCV testing first became available in Japan in 1990, we collected data on patients whose HCC diagnosis was between 1990 and 2003. One investigator checked for duplication of the data set, because some patients might have been registered multiple times among the participating hospitals as a result of referrals and recurrence of HCC. We defined HCV-related HCC as occurring in patients who were HCV-seropositive at the time of diagnosis. We calculated the sex-specific, age-specific (50 to 59, 60 to 69, or 70 to 79 years), and period-specific (1990 to 1992, 1993 to 1995, 1996 to 1998, 1999 to 2001, or 2002 to 2003) prevalences of HCV seropositivity for patients with HCC. We then multiplied prevalence rates by the corresponding strata of the HCC incidence rate obtained from the Osaka Cancer Registry data. Thus, we derived the denominators from the general population in Osaka through the denominators of the HCC incidence rate and obtained the numerators by multiplying the prevalence rates by the HCC incidence rate. We calculated the incidence rate of nonHCV-related HCC by subtracting HCV-related HCC from total HCC. Thus, we describe trends for the estimated incidence rates of HCV-related and nonHCV-related HCC between 1990 and 2003 in Osaka Prefecture. We calculated the CI of the estimated rates by multiplying the lower and upper limits of the CI of the prevalence based on SE by the corresponding HCC incidence rate. Role of the Funding Source This study was supported by the Osaka Prefectural Government between 1990 and 2000 and Grants-in-Aid for Hepatitis Research of the Japanese Ministry of Health, Labor, and Welfare. There is no conflict of interest in the study. The funding sources had no role in the collection, management, or analysis of data. Results The age-standardized incidence rate of HCC in men increased between 1981 and 1987 from 29.2 to 41.9 cases per 100000 persons, then fluctuated until 1995. After that, it steadily decreased to 24.0 cases per 100000 persons in 2003 (Figure 1). Among women, the age-standardized incidence rate of HCC increased between 1981 and 1996 from 6.6 to 10.8 cases per 100000 persons, then gradually decreased to 7.3 cases per 100000 persons in 2003 (Figure 1). Figure 1. Trends in age-standardized (world population) incidence of hepatocellular carcinoma in Osaka, Japan, 19812003. Figure 2 shows the trends in the incidence of HCC among men and women age 50 to 59 years, 60 to 69 years, and 70 to 79 years in Osaka between 1981 and 2003. The HCC incidence rate increased from 1981 to 1986 among men age 50 to 59 years, from 1981 to 1995 among men age 60 to 69 years, and from 1981 to 2000 among men age 70 to 79 years (average annual change of the incidence rate [per 100000 persons], 10.0, 10.7, and 6.2, respectively) (Table 1). A striking downward trend occurred after the year of peak incidence in the 3 age groups (7.9 until 1996, 22.3 until 2003, and 12.4 until 2003, respectively). Among men age 50 to 59 years, there was a second joinpoint (a change from rapid to moderate decrease) in 1996, resulting in a slope of 3.1 until 2003. Among women age 50 to 59 years, 60 to 69 years, and 70 to 79 years, the incidence rates of HCC peaked in 1991, 1997, and 2000, respectively (Table 1). The rates in women seemed to increase slightly from 1981 until the year of the joinpoint, with slopes of 0.43, 2.07, and 3.10, respectively. Thereafter, HCC incidence rates in women decreased through 2003 at a

Oral Glucose Tolerance Test Predicts Prognosis of Patients with Liver Cirrhosis

OBJECTIVE:The aim of this study was to evaluate whether oral glucose tolerance test (OGTT) was useful in evaluating the prognosis of patients with liver cirrhosis.METHODS:Fifty-six patients with liver cirrhosis were enrolled in a prospective cohort study. In all cases, glucose tolerance was diagnosed by a 75-g OGTT according to World Health Organization (WHO) criteria. The relationship of clinical variables to the cirrhosis-related prognosis was investigated using univariate and multivariate regression models.RESULTS:Diabetes mellitus (DM) was diagnosed in 21 subjects (38%), impaired glucose tolerance (IGT) in 13 subjects (23%), and normal glucose tolerance (NGT) in 22 subjects (39%) using OGTT. The cumulative survival rates of patients with liver cirrhosis and NGT were 94.7% at 5 yr; liver cirrhosis and IGT, 68.8% at 5 yr; liver cirrhosis and DM, 56.6% at 5 yr. The survival rates of patients with liver cirrhosis and DM significantly differed from those with NGT. Univariate analysis demonstrated that serum albumin, total bilirubin, prothrombin activity, Child-Pugh scores, and glucose intolerance were highly significant prognostic factors. Multiple regression analysis yielded albumin and DM as the most powerful independent negative predictors of survival.CONCLUSIONS:OGTT appears to be useful for evaluating the prognosis of cirrhotic patients.

Alterations in microRNA expression profile in HCV-infected hepatoma cells: involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway.

The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

Detection of Hepatitis C Virus RNA in Chronic Non-A, Non-B Liver Disease

Serum samples were tested for detection of hepatitis C virus (HCV) RNA from 156 patients with chronic non-A, non-B liver disease. HCV RNA was detected in 121 (93.8%) of 129 patients positive for anti-C100-3 but was also found in 15 (55.6%) of 27 patients negative for anti-C100-3. The rate of positivity for HCV RNA was not significantly different among various stages of liver diseases. These results showed that HCV continues to replicate even in advanced liver disease and that it seems to be related to half of the cases of chronic non-A, non-B liver disease negative for anti-C100-3.

Ribavirin dose reduction raises relapse rate dose-dependently in genotype 1 patients with hepatitis C responding to pegylated interferon alpha-2b plus ribavirin

Summary.  The impact of ribavirin exposure on virologic relapse remains controversial in combination therapy with pegylated interferon (Peg‐IFN) and ribavirin for patients with chronic hepatitis C (CH‐C) genotype 1. The present study was conducted to investigate this. Nine hundred and eighty‐four patients with CH‐C genotype 1 were enrolled. The drug exposure of each medication was calculated by averaging the dose actually taken. For the 472 patients who were HCV RNA negative at week 24 and week 48, multivariate logistic regression analysis showed that the degree of fibrosis (P = 0.002), the timing of HCV RNA negativiation (P < 0.001) and the mean doses of ribavirin (P < 0.001) were significantly associated with relapse, but those of Peg‐IFN were not. Stepwise reduction of the ribavirin dose was associated with a stepwise increase in relapse rate from 11% to 60%. For patients with complete early virologic response (c‐EVR) defined as HCV RNA negativity at week 12, only 4% relapse was found in patients given ≥12 mg/kg/day of ribavirin and ribavirin exposure affected the relapse even after treatment week 12, while Peg‐IFN could be reduced to 0.6 μg/kg/week after week 12 without the increase of relapse rate. Ribavirin showed dose‐dependent correlation with the relapse. Maintaining as high a ribavirin dose as possible (≥12 mg/kg/day) during the full treatment period can lead to suppression of the relapse in HCV genotype 1 patients responding to Peg‐IFN alpha‐2b plus ribavirin, especially in c‐EVR patients.