Yoshiaki Inui

Diphtheria toxin receptor–mediated conditional and targeted cell ablation in transgenic mice

Specific cell ablation is a useful method for analyzing the in vivo function of cells. We have developed a simple and sensitive method for conditional cell ablation in transgenic mice, called “toxin receptor–mediated cell knockout.” We expressed the diphtheria toxin (DT) receptor in transgenic mice using a hepatocyte-specific promoter and found that injection of DT caused fulminant hepatitis. Three independently established transgenic lines demonstrated a good correlation between the sensitivity of hepatocytes to DT and the expression level of the DT receptors. Moreover, the degree of hepatocyte damage was easily controlled over a wide range of doses of injected DT without any obvious abnormalities in other cells or tissues. This system is useful for generating mouse models of disease and for studying the recovery or regeneration of tissues from cell damage or loss. As DT is a potent inhibitor of protein synthesis in both growing and non-growing cells, the method is applicable to a wide range of cells and tissues in mice or in other DT-insensitive animals.

Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomized controlled trial

Chemotherapy is not effective for hepatocellular carcinoma (HCC). HMG-CoA redutase inhibitors have cytostatic activity for cancer cells, but their clinical usefulness is unknown. To investigate whether pravastatin, a potent HMG-CoA reductase inhibitor, prolongs survival in patients with advanced HCC, this randomized controlled trial was conducted between February 1990 and February 1998 at Osaka University Hospital. 91 consecutive patients <71 years old (mean age 62) with unresectable HCC were enroled in this study. 8 patients were withdrawn because of progressive liver dysfunction; 83 patients were randomized to standard treatment with or without pravastatin. All patients underwent transcatheter arterial embolization (TAE) followed by oral 5-FU 200 mg–1d for 2 months. Patients were then randomly assigned to control (n = 42) and pravastatin (n = 41) groups. Pravastatin was administered at a daily dose of 40 mg. The effect of pravastatin on tumour growth was assessed by ultrasonography. Primary endpoint was death due to progression of HCC. The duration of pravastatin administration was 16.5 ± 9.8 months (mean ± SD). No patients in either group were lost to follow-up. Median survival was 18 months in the pravastatin group versus 9 months in controls (P = 0.006). The Cox proportional hazards model showed that pravastatin was a significant factor contributing to survival. Pravastatin prolonged the survival of patients with advanced HCC, suggesting its value for adjuvant treatment.

Localization of heparin-binding EGF-like growth factor in the smooth muscle cells and macrophages of human atherosclerotic plaques.

Heparin-binding EGF-like growth factor (HB-EGF) is a potent chemoattractant and mitogen for smooth muscle cells (SMC) in culture. To elucidate whether HB-EGF is implicated in the pathogenesis of human atherosclerosis, we examined immunohistochemical localization of HB-EGF in human aortic walls and atherosclerotic plaques. The medial SMC of the aorta in babies and children synthesized HB-EGF protein, while the number of SMC producing HB-EGF was dramatically decreased in young and middle-aged adults. In atherosclerotic plaques, however, marked production of HB-EGF protein was detected in SMC and macrophages of the plaques. Furthermore, EGF receptors, to which HB-EGF is known to bind, were detected in plaque SMC. These data suggest that HB-EGF may be implicated in the migration and proliferation of SMC that occurs in the normal development of arterial walls, and in the formation of atherosclerotic plaques.

Animal model of systemic carnitine deficiency: Analysis in C3H-H-2° strain of mouse associated with juvenile visceral steatosis

Abstract We analyzed carnitine profiles in C3H-H-2° strain of mouse associated with fatty liver, hyperammonemia and hypoglycemia (Koizumi et al., 1988). Carnitine levels in serum, liver and muscle of mouse with fatty liver were markedly decreased in comparison with those of control mouse (littermates without fatty liver). This is a useful animal model to analyze the role of carnitine in lipid, amino acid and carbohydrate metabolism.

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.

Vascular endothelial dysfunction resulting from l-arginine deficiency in a patient with lysinuric protein intolerance

Although L-arginine is the only substrate for nitric oxide (NO) production, no studies have yet been reported on the effect of an L-arginine deficiency on vascular function in humans. Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of dibasic amino acid transport caused by mutations in the SLC7A7 gene, resulting in an L-arginine deficiency. Vascular endothelial function was examined in an LPI patient who was shown to be a compound heterozygote for two mutations in the gene (5.3-kbp Alu-mediated deletion, IVS3+1G-->A). The lumen diameter of the brachial artery was measured in this patient and in healthy controls at rest, during reactive hyperemia (endothelium-dependent vasodilation [EDV]), and after sublingual nitroglycerin administration (endothelium-independent vasodilation [EIV]) using ultrasonography. Both EDV and NO(x) concentrations were markedly reduced in the patient compared with those for the controls. They became normal after an L-arginine infusion. EIV was not significantly different between the patient and controls. Positron emission tomography of the heart and a treadmill test revealed ischemic changes in the patient, which were improved by the L-arginine infusion. Thus, in the LPI patient, L-arginine deficiency caused vascular endothelial dysfunction via a decrease in NO production.

Post-treatment Levels of α-Fetoprotein Predict Incidence of Hepatocellular Carcinoma After Interferon Therapy

BACKGROUND & AIMS In patients with chronic hepatitis C virus (HCV) infection, lack of sustained virologic response (SVR) 24 weeks after the end of interferon therapy is a significant risk factor for hepatocellular carcinoma (HCC). Although many pretreatment factors are known to affect HCC incidence, less is known about post-treatment factors-many change during the course of interferon therapy. METHODS We performed a prospective study, collecting data from 2659 patients with chronic hepatitis C without a history of HCC who had been treated with pegylated interferon (Peg-IFN) plus ribavirin from 2002 through 2008 at hospitals in Japan. Biopsy specimens were collected before treatment; all patients received Peg-IFN plus ribavirin for 48 to 72 weeks (HCV genotype 1) or 24 weeks (HCV genotype 2). Hematologic, biochemical, and virologic data were collected every 4 weeks during treatment and every 6 months after treatment. HCC was diagnosed based on angiography, computed tomography, and/or magnetic resonance imaging findings. RESULTS HCC developed in 104 patients during a mean observation period of 40 months. Older age, male sex, lower platelet counts and higher levels of α-fetoprotein at baseline, and lack of an SVR were significant risk factors for HCC. The cumulative incidence of HCC was significantly lower in patients without SVRs who relapsed than those with no response to treatment. Levels of α-fetoprotein 24 weeks after the end of treatment (AFP24) were significantly lower than levels of α-fetoprotein at baseline in patients with SVRs and those who relapsed, but not in nonresponders. Post-treatment risk factors for HCC among patients with SVRs included higher AFP24 level and older age; among those without SVRs, risk factors included higher AFP24 level, integrated level of alanine aminotransferase, older age, and male sex. AFP24 (≥10 ng/mL, 10-5 ng/mL, and then <5 ng/mL) was a better predictor of HCC incidence than pretreatment level of AFP among patients with and without SVRs. CONCLUSIONS In patients with chronic HCV infection, levels of α-fetoprotein decrease during interferon therapy. High post-treatment levels of α-fetoprotein predict HCC, regardless of whether patients achieve an SVR. University Hospital Medical Information Network Clinical Trials Registry: C000000196, C000000197.

Indications and limitations for aged patients with chronic hepatitis C in pegylated interferon alfa-2b plus ribavirin combination therapy

BACKGROUND & AIMS This study investigated the efficacy and adverse effects of pegylated interferon (Peg-IFN) plus ribavirin therapy in aged patients with chronic hepatitis C (CH-C). METHODS A total of 1040 naïve patients with CH-C (genotype 1, n=759; genotype 2, n=281), of whom 240 (23%) over 65 years old (y.o.), were treated with Peg-IFN alfa-2b plus ribavirin and assessed after being classified into five categories, according to age. RESULTS The discontinuance rate was higher for patients over 70 y.o. (36%), the most common reason being anemia. In the presence of genotype 1, the SVR rate was similar (42-46%) among patients under 65 y.o. and declined (26-29%) among patients over 65 y.o. For patients over 65 y.o., being male (Odds ratio, OR, 3.5, p=0.035) and EVR (OR, 83.3, p<0.001) were significant factors for SVR, in multivariate analysis. The Peg-IFN dose was related to EVR, and when EVR was attained, 76-86% of patients over 65 y.o. achieved SVR. SVR was not achieved (0/35, 0/38, respectively) if a 1-log decrease and a 2-log decrease were not attained at week 4 and week 8, respectively. In the presence of genotype 2, the SVR rate was similar (70-71%) among patients under 70 y.o. and declined among patients over 70 y.o. (43%). CONCLUSIONS Aged patients up to 65 y.o. with genotype 1 and 70 y.o. with genotype 2 can be candidates for Peg-IFN plus ribavirin therapy. The response-guided therapy can be applied for aged patients with genotype 1.

Inhibition of cell growth of human hepatoma cell line (HepG2) by a farnesyl protein transferase inhibitor: A preferential suppression of ras farnesylation

So far, treatment with anti‐cancer agents has failed to achieve satisfactory results in hepatocellular carcinoma. In the process of hepatocarcinogenesis, ras has been shown to play a role, ras requires a farnesyl moiety for activation. It has been found that UCFI‐C (manumycin), an antibiotic, inhibits farnesyl protein transferase, an enzyme that catalyzes farnesylation. Therefore, we investigated the effects of UCFI‐C on cell growth, prenylation of cellular proteins including ras and Rap I, MAP kinase activity, activities of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase, and synthesis of cholesterol in a ras‐activated human hepatoma cell line, Hep G2. Treatment with varying concentrations of UCFI‐C (10–30 μM) for 24 and 72 hr resulted in a time· and dose‐dependent inhibition of cell numbers. 3H‐Thymidine incorporation was also inhibited in a dose‐dependent manner, with 50% inhibition after 44 hr being observed at a concentration of 17 μM. UCFI‐C dose‐dependently inhibited ras farnesylation and MAP kinase activity, but did not decrease Rap I geranylgeranylation or prenylation of 21‐ to 26‐kDa proteins. Neither the activities of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase nor cholesterol synthesis were inhibited. These results suggest that UCFI‐C antagonizes the growth of Hep G2 via the suppression of ras farnesylation and could be a lead for the development of new anti‐cancer agents blocking the function of oncogenic ras associated with human cancer, including hepatocellular carcinoma.

Effect of interferon α-2b plus ribavirin therapy on incidence of hepatocellular carcinoma in patients with chronic hepatitis.

Aim:  The objective of this study was to elucidate the long‐term effects of interferon (IFN)α‐2b plus ribavirin combination therapy and to clarify whether this therapy can reduce the incidence of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C.