Masahiko Kaito

Hepatitis C virus particle detected by immunoelectron microscopic study

To clarify the morphology of hepatitis C virus (HCV), an indirect immunogold electron microscopic study was carried out on two plasma samples with high HCV RNA titres using polyclonal and monoclonal antibodies specific to the putative HCV envelope protein. Spherical virus-like particles, 55 to 65 nm in diameter with spike-like projections, were found in 1.14 to 1.16 g/ml fractions after sucrose density gradient centrifugation. These particles were found only in HCV-infected blood donors and had morphological features similar to those of flaviviruses. Moreover, these particles specifically reacted with the polyclonal and monoclonal antibodies to the putative HCV envelope protein. This is the first known report in which the morphology of the HCV particle is clearly shown.

Influence of hepatitis B virus genotypes on the intra-and extracellular expression of viral DNA and antigens

Various genotypes of the hepatitis B virus (HBV) induce liver disease of distinct severity, but the underlying virological differences are not well defined. Huh7 cells were transfected with plasmids carrying 1.24‐fold the HBV genome of different genotypes/subgenotypes (2 strains each for Aa/A1, Ae/A2, Ba/B2 and D; 3 each for Bj/B1 and C). HBV DNA levels in cell lysates, determined by Southern hybridization, were the highest for C followed by Bj/Ba and D/Ae (P < .01), and the lowest for Aa (P < .01), whereas in culture media, they were the highest for Bj, distantly followed by Ba/C/D and further by Ae/Aa (P < .01). The intracellular expression of core protein was more than 3‐fold lower for Ae/Aa than the others. Hepatitis B e antigen (HBeAg) was excreted in a trend similar to that of HBV DNA with smaller differences. Secretion of hepatitis B surface antigen (HBsAg) was most abundant for Ae followed by Aa, Ba, Bj/C and remotely by D, which was consistent with mRNA levels. Cellular stress determined by the reporter assay for Grp78 promoter was higher for C and Ba than the other genotypes/subgenotypes (P < .01). Severe combined immunodeficiency mice transgenic for urokinase‐type plasminogen activator (uPA/SCID), with the liver replaced for human hepatocytes, were inoculated with virions passed in mouse and recovered from culture supernatants. HBV DNA levels in their sera were higher for C than Ae by 2 logs during 4–7 weeks after inoculation. In conclusion, virological differences among HBV genotypes were demonstrated both in vitro and in vivo. These differences may influence HBV infections with distinct genotypes in clinical and epidemiological settings. (HEPATOLOGY 2006;44:915–924.)

Hepatic iron accumulation is associated with disease progression and resistance to interferon/ribavirin combination therapy in chronic hepatitis C

Background and Aims:  Liver iron accumulation in patients with chronic hepatitis C (CHC) has received increasing attention in recent years. The aim of this study was to determine the prevalence and severity of liver iron deposition in CHC, to assess its relationship with clinical, biochemical and histological characteristics, and to study its influence on the response to interferon (IFN) plus ribavirin combination therapy.

Hepatic oxidative DNA damage is associated with increased risk for hepatocellular carcinoma in chronic hepatitis C

Although the oxidative stress frequently occurs in patients with chronic hepatitis C, its role in future hepatocellular carcinoma (HCC) development is unknown. Hepatic 8-hydroxydeoxyguanosine (8-OHdG) was quantified using liver biopsy samples from 118 naïve patients who underwent liver biopsy from 1995 to 2001. The predictability of 8-OHdG for future HCC development and its relations to epidemiologic, biochemical and histological baseline characteristics were evaluated. During the follow-up period (mean was 6.7±3.3 years), HCC was identified in 36 patients (30.5%). Univariate analysis revealed that 16 variables, including 8-OHdG counts (65.2±20.2 vs 40.0±23.5 cells per 105 μm2, P<0.0001), were significantly different between patients with and without HCC. Cox proportional hazard analysis showed that the hepatic 8-OHdG (P=0.0058) and fibrosis (P=0.0181) were independent predicting factors of HCC. Remarkably, 8-OHdG levels were positively correlated with body and hepatic iron storage markers (vs ferritin, P<0.0001 vs hepatic iron score, P<0.0001). This study showed that oxidative DNA damage is associated with increased risk for HCC and hepatic 8-OHdG levels are useful as markers to identify the extreme high-risk subgroup. The strong correlation between hepatic DNA damage and iron overload suggests that the iron content may be a strong mediator of oxidative stress and iron reduction may reduce HCC incidence in patients with chronic hepatitis C.

Comparison of hepatic oxidative DNA damage in patients with chronic hepatitis B and C

Summary.  8‐Hydroxydeoxyguanosine (8‐OHdG) is a promutagenic DNA lesion produced by hydroxyl radicals and is recognized as a useful marker in estimating DNA damage induced by oxidative stress. The aim of this study was to clarify the clinical significance of hepatic 8‐OHdG levels in patients with chronic viral hepatitis. Hepatic 8‐OHdG accumulation was investigated in patients with chronic hepatitis C (CH‐C) (n = 77) and chronic hepatitis B (CH‐B) (n = 34) by immunohistochemical staining of liver biopsy samples. 8‐OHdG positive hepatocytes were significantly higher in patients with CH‐C compared to CH‐B (median 55.0 vs 18.8 cells/105 μm2, P < 0.0001). The number of positive hepatocytes significantly increased with the elevation of serum aminotransferase levels, especially in CH‐C patients (8‐OHdG vs alanine aminotransferase (ALT)/aspartate aminotrasferase (AST) were r = 0.738/0.720 in CH‐C and 0.506/0.515 in CH‐B). 8‐OHdG reactivity was strongly correlated with body and hepatic iron storage markers in CH‐C (vs serum ferritin, r = 0.615; vs hepatic total iron score, r = 0.520; vs hepatic hepcidin mRNA levels, r = 0.571), although it was related to serum HBV‐DNA titers (r = 0.540) and age of patients (r = –0.559) in CH‐B. These results indicate that hepatic oxidative DNA damage is common in chronic viral hepatitis, in particular chronic HCV‐infected patients, suggesting a possible link between chronic hepatic inflammation and hepatocarcinogenesis. The strong positive correlation between hepatic DNA damage and iron overload suggests that iron content is one of the most likely mediators of hepatic oxidative stress and iron reduction may be beneficial to reduce the incidence of hepatic cancer in CH‐C patients.

Genetic polymorphisms of bilirubin uridine diphosphate‐glucuronosyltransferase gene in Japanese patients with Crigler–Najjar syndrome or Gilbert's syndrome as well as in healthy Japanese subjects

Background and Aim:  Numerous mutations of bilirubin uridine diphosphate‐glucuronosyltransferase gene (UGT1A1) have been reported in patients with familial unconjugated hyperbilirubinemia. The UGT1A1 mutation appears to be considerably different among ethnic groups. To clarify the incidence of this gene mutation in the Japanese population, the presence of UGT1A1 mutation was investigated in a group of Japanese patients with Crigler–Najjar syndrome type 2 (CNS2) and Gilberts syndrome (GS), as well as in healthy anicteric subjects.

Patients with chronic hepatitis C achieving a sustained virological response to peginterferon and ribavirin therapy recover from impaired hepcidin secretion

BACKGROUND/AIMS The aim of this study is to determine the clinical relevance of hepatic producing iron regulatory hormone-hepcidin, on iron overload in patients with chronic hepatitis C (CHC). METHODS Serum hepcidin was measured in 73 CHC patients by surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS), and compared to those of healthy controls and anemia of inflammation patients, and analyzed their relationship to hepatic hepcidin mRNA expression levels and clinical, hematological, and histological findings. The sequential changes of hepcidin were investigated in 27 CHC patients treated with a 48 week-course of pegylated-interferon (PEG-IFN) plus ribavirin therapy. RESULTS Serum hepcidin was positively correlated with hepatic hepcidin mRNA levels, serum ferritin and the degree of hepatic iron deposition in CHC. Serum hepcidin-to-ferritin ratios were significantly lower in HCV positive patients than in HCV negative controls in both hyper- and normal-ferritinemic conditions. This relative impairment of hepcidin production was fully reversible after successful HCV eradication by PEG-IFN plus ribavirin, concomitantly with the improvement of the iron overload condition. CONCLUSIONS The impairment of hepatic hepcidin production occurring with chronic HCV infection may enhance iron toxicity and lead to disease progression, and modulation or supplementation of hepcidin may be beneficial for these conditions in CHC.

Increased lipid peroxidation in patients with non-alcoholic fatty liver disease and chronic hepatitis C as measured by the plasma level of 8-isoprostane.

Background:  Oxidative stress plays an important role in the pathogenesis of chronic liver diseases. The plasma level of 8‐isoprostane, a product of lipid peroxidation, is a marker of oxidative stress in vivo. The aim of the present study was to clarify whether the degree of lipid peroxidation, as measured by the plasma level of 8‐isoprostane, influences the progression of chronic liver diseases and hepatocarcinogenesis.

Increased hepatic and renal expressions of multidrug resistance‐associated protein 3 in Eisai hyperbilirubinuria rats

Background and Aim:  Eisai hyperbilirubinuria rats (EHBR) are animal models of Dubin–Johnson syndrome, which suffer from jaundice due to impaired biliary excretion of bilirubin glucuronides. In EHBR, deficiency of multidrug resistance‐associated protein 2 (mrp2) causes defective biliary excretion of numerous organic anions. However, little is known about the expression of other organic anion transporters in this mrp2‐deficient model. The aim of the present study was to investigate adaptive expressions of mrp1, mrp3, mrp6, organic anion transporting polypeptide 1 (oatp1) and oatp2 in liver and kidney of EHBR.

A significant reduction in serum alanine aminotransferase levels after 3-month iron reduction therapy for chronic hepatitis C: a multicenter, prospective, randomized, controlled trial in Japan

BackgroundIncreasing evidence indicates that iron cytotoxicity plays an important role in the pathogenesis of chronic hepatitis C (CHC). However, the biochemical effects of iron reduction therapy on CHC remain to be confirmed in a controlled study. This study aimed to test whether iron removal by repeated phlebotomy improves serum alanine aminotransferase (ALT) levels in patients with CHC.MethodsPatients were randomly assigned to an iron reduction therapy or control group. The patients in the treatment group received 3-month iron reduction therapy by biweekly phlebotomy, while the patients in the control group were followed up for 3 months with regular blood tests alone.ResultsThirty-three patients completed the 3-month treatment, while 29 patients received the complete follow-up. The serum ALT levels were reduced from 118 ± 79 to 73 ± 39 IU/L in the treatment group, but did not change in the control group (106 ± 45 versus 107 ± 48 IU/L). Posttreatment enzyme activity was decreased significantly from the baseline. Furthermore, it was significantly lower than the 3-month control level. Although 5 patients withdrew from the study, none was affected by any side effects of repeated phlebotomy that required them to discontinue the treatment.ConclusionsThis short-term controlled trial demonstrated the biochemical efficacy and safety of iron reduction therapy for patients with CHC.