Keisuke Hamasaki

Short interfering RNA‐directed inhibition of hepatitis B virus replication

RNA interference (RNAi) is the process by which double‐stranded RNA directs sequence‐specific degradation of mRNA. In mammalian cells, RNAi can be triggered by 21‐nucleotide duplexes of short interfering RNA (siRNA). We examined effects of siRNA on hepatitis B virus (HBV) replication. Human hepatoma cells were transfected with HBV DNA and siRNA against HBV‐pregenome RNA. Transfection experiments demonstrated that the siRNA reduced the amount of HBV‐pregenome RNA and resulted in reduction of the levels of replicative intermediates and viral protein. Our results indicate that siRNA‐mediated gene silencing inhibits HBV replication through suppression of viral RNA, which may be useful as a potential therapeutic modality.

Influence of interleukin-10 gene promoter polymorphisms on disease progression in patients chronically infected with hepatitis B virus

OBJECTIVES:The role of host genetic factors in chronic hepatitis B virus (HBV) infection is not fully understood. We studied the influence of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) gene promoter polymorphisms on disease progression in HBV carriers.METHODS:The sample population included 213 Japanese HBV carriers and 52 healthy volunteers. Of 213 HBV carriers, 66 were considered to be asymptomatic carriers based on the sustained normalization of serum ALT together with seropositivity for the antibody to hepatitis B e antigen (anti-HBe), and 147 were found to have chronic progressive liver disease including cirrhosis. Five biallelic polymorphisms in the TNF-α gene promoter and three biallelic polymorphisms in the IL-10 gene promoter were analyzed by polymerase chain reaction in combination with direct sequencing or restriction fragment length polymorphism assay.RESULTS:Allelic distributions of both gene promoters were not significantly different between HBV carriers and healthy volunteers. In HBV carriers, the TNF-α gene promoter polymorphisms were not linked to disease progression. In contrast, allelic frequencies of T and A at positions −819 and −592, respectively, in the IL-10 gene promoter, as well as the frequencies of ATA haplotype at positions −1082/− 819/− 592 (which is characterized with low capacity for IL-10 production), were significantly higher in asymptomatic carriers than in patients with chronic progressive liver disease. Even after adjusting for individuals positive for anti-HBe, such a relationship could be found between the two groups.CONCLUSION:In chronic HBV infection, inheritance of the IL-10 gene promoter polymorphisms is involved in a host genetic factor that is relevant to disease progression.

Interferon-|[alpha]| sensitizes human hepatoma cells to TRAIL-induced apoptosis through DR5 upregulation and NF-|[kappa]|B inactivation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis in a variety of cancer cells with little or no effect on normal cells. Human hepatoma cells, however, are resistant to TRAIL-induced apoptosis. Since interferon-α (IFN-α) is capable of enhancing TNF-α-induced apoptosis in certain cancer cells, we evaluated the effect of IFN-α on TRAIL-induced apoptosis of human hepatoma cells. IFN-α pretreatment enhanced TRAIL-induced apoptosis of HuH-7 and Hep3B cells, in which IFN-α upregulated the expression of DR5, a death receptor of TRAIL, and downregulated the expression of survivin, which has an antiapoptotic function. In contrast, IFN-α did not enhance TRAIL-induced apoptosis of HepG2 cells, in which expression of DR5 and survivin was not affected by IFN-α. On the other hand, TRAIL activated NF-κB composed of RelA-p50 heterodimer, a key transcription factor regulating cell survival, in HuH-7 and HepG2 cells. However, IFN-α pretreatment repressed the TRAIL-mediated activation of NF-κB and decreased its transcriptional activity in HuH-7 but not in HepG2 cells. Moreover, IFN-α pretreatment clearly augmented TRAIL-mediated caspase-8 activation in HuH-7 cells. Our results suggest that IFN-α could sensitize certain human hepatoma cells to TRAIL-induced apoptosis by stimulating its death signaling and by repressing the survival function in these cells.

Antiangiogenic property of pigment epithelium‐derived factor in hepatocellular carcinoma

Pigment epithelium‐derived factor (PEDF) is one of the most powerful endogenous antiangiogenic reagents discovered to date. Its antiangiogenic potential in neoplastic disease remains unclear. In this study, we investigated antiangiogenic property of PEDF in hepatocellular carcinoma (HCC), a typical hypervascular tumor. In HCC cell lines, constitutive messenger RNA and protein expression of PEDF varied. Genomic DNA encoding the PEDF gene was the same in the cell lines examined by Southern blotting. In chemically induced hypoxic conditions, secreted PEDF protein was suppressed in contrast to elevation of vascular endothelial growth factor protein. When PEDF was overexpressed by gene transfer, proliferation and migration of endothelial cells were inhibited in conditioned media derived from all HCC cell lines. However, the serum concentration of PEDF, as measured by enzyme‐linked immunosorbent assay, was decreased in patients with cirrhosis or HCC complicated by cirrhosis compared to healthy volunteers and patients with chronic hepatitis. According to the endothelial cell proliferation assay, the serum PEDF of patients with HCC had antiangiogenic activity. Moreover, intratumoral injection of a PEDF‐expressing plasmid in athymic mouse models caused significant inhibition of preestablished tumor growth. In conclusion, PEDF plays a role in the angiogenic properties of HCC. Reduction of serum PEDF concentration associated with the development of chronic liver diseases may contribute to the progression of HCC. In addition, gene therapy using PEDF may provide an efficient treatment for HCC. (HEPATOLOGY 2004;40:252–259.)

High viral load is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis B virus infection

Background and Aims:  Hepatitis B virus (HBV) is considered a major risk factor for the progression to liver cirrhosis and hepatocellular carcinoma (HCC). The serum level of HBV‐DNA is correlated with progression of the disease. The aim of the present study was to determine the relationship between the level of HBV‐DNA and hepatocarcinogenesis in patients with chronic HBV infection.

Association Between Liver Fibrosis and Insulin Sensitivity in Chronic Hepatitis C Patients

BACKGROUND:Several clinical studies have suggested a possible link between chronic hepatitis caused by hepatitis C virus (HCV) and the development of diabetes mellitus. We investigated the association between liver fibrosis and glucose intolerance in HCV-infected patients by measuring insulin sensitivity and β-cell function.METHOD:A total of 83 chronic HCV-infected patients were recruited into this study. We evaluated insulin sensitivity and β-cell function of all patients in a fasting state (homeostasis model assessment of insulin resistance [HOMA-R] and homeostasis model assessment of β-cell function [HOMA-β]) and after an oral load of 75 g glucose (whole-body insulin sensitivity index [WBISI] and Δ-insulin/Δ-glucose 30).RESULTS:In a multivariate analysis, severe fibrosis was the only independent factor associated with insulin resistance. There were significant differences in both HOMA-R (P = 0.0063) and WBISI (P = 0.0159) between patients with mild fibrosis (N = 34) and those with severe fibrosis (N = 49). Although HOMA-β was increased significantly in the subjects with severe fibrosis compared with those with mild fibrosis (P = 0.0169), Δ-insulin/Δ-glucose 30 showed no significant difference in stage of liver fibrosis, suggesting an uncertain association between liver fibrosis and β-cell function.CONCLUSION:Our findings suggest that the development of liver fibrosis is associated with insulin resistance in HCV-infected patients.

Association between nonalcoholic fatty liver, markers of obesity, and serum leptin level in young adults.

OBJECTIVES:The aim of the present study was to clarify the risk factors for nonalcoholic fatty liver in young adults.METHODS:One thousand two hundred two students, aged 18–21 yr, received matriculation health examinations, including measurements of body mass index and percent body fat and determination of serum levels of ALT at Nagasaki University in 1998. One hundred twenty-nine were found to have borderline or elevated levels of serum ALT, and 105 of the 129 students (75 men and 30 women) were subjected to further analysis for the presence of fatty liver using ultrasonography, by which both the degree of steatosis and the abdominal wall fat index (AFI) corresponding to the ratio of visceral to s.c. adipose tissue (V/S ratio) were evaluated, in addition to determination of the serum level of leptin.RESULTS:Of 105 students, 74 (70%) had fatty liver. The incidence of moderately to severely fatty liver was significantly higher in men than in women. In parameters related to obesity, the close correlation between body mass index and percent body fat was observed in both sexes. The serum level of leptin correlated well with percent body fat and AFI (V/S ratio) in women, whereas it did not correlate with AFI (V/S ratio) in men. Multiple logistic regression analysis indicated that AFI (V/S ratio) was the only independent risk factor for fatty liver in both sexes.CONCLUSIONS:These results suggest that visceral fat distribution is a key risk factor for nonalcoholic fatty liver in young adults.

Non-alcoholic steatohepatitis and hepatic steatosis in patients with adult onset growth hormone deficiency.

Adult onset growth hormone (GH) deficiency closely resembles syndrome X.1 Patients with syndrome X often suffer from obesity, dyslipidaemia, insulin resistance, and hypertension, and hepatic steatosis and non-alcoholic steatohepatitis (NASH) have also been characterised as symptoms of syndrome X.2,3 However, the relationship of GH deficiency to hepatic steatosis and NASH remains unclear. We looked for the presence of hepatic steatosis using computer tomography (CT) in a study of 18 patients with adult …

Treatment of hepatocellular carcinoma with transcatheter arterial embolization. Analysis of prognostic factors

Background. Transcatheter arterial embolization (TAE) is a useful treatment modality for hepatocellular carcinoma (HCC). To study the prognostic factors, survival time of patients with HCC after TAE was analyzed retrospectively using clinical manifestations of coexisting liver cirrhosis and clinical features of HCC.

Exacerbation of Thyroid Autoimmunity by Interferon α Treatment in Patients with Chronic Viral Hepatitis

In the present studies, the long term effects of IFN alpha on thyroid function and thyroid autoantibodies were evaluated in 42 patients with chronic viral hepatitis type C treated with IFN alpha for at least 4 months. Before IFN treatment, 41 patients tested were all euthyroid. Five (12%) out of 24 patients tested had positive tests for thyroid autoantibodies. MCHA/TPOAb was detected in all 5 and TGHA/TGAb in 3 out of these 5 patients. Six to 10 x 10(6) units (U) of recombinant or natural IFN alpha were given intramuscularly daily for the first 2 to 4 weeks, followed by 3 to 10 x 10(6) U thrice weekly for the subsequent 14 to 22 weeks. Thyroid dysfunction and/or rises in titers of thyroid autoantibodies were observed in 6 patients during IFN alpha treatment; clinically overt thyroid dysfunctions, destructive thyroiditis and thyrotoxicosis of unidentified etiology, developed in 2 patients 4 to 5 months after start of IFN treatment, subclinical hypothyroidism with a slight increase in serum TSH concentrations but no serum thyroid hormone alternations was observed in 2 patients, and increases in titers of thyroid autoantibodies without thyroid dysfunction were found in 2 patients. Thus, IFN alpha exacerbated thyroid autoimmunity exclusively in all patients with positive tests for thyroid autoantibodies prior to treatment, but did not induce thyroid autoimmunity in thyroid autoantibody-negative patients. These data suggest that the prolonged IFN alpha therapy can lead to exacerbation of thyroid autoimmunity in susceptible (thyroid autoantibody-positive) patients.