Akito Nozaki

Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C : A pilot study

Hepatitis C virus (HCV) is associated with the development of cirrhosis and hepatocellular carcinoma. We recently found that bovine lactoferrin, a milk protein belonging to the iron transporter family, effectively prevented HCV infection in cultured human hepatocytes (PH5CH8). We tested the hypothesis that lactoferrin inhibits HCV viremia in patients with chronic hepatitis C. Eleven patients with chronic hepatitis C received an 8‐week course of bovine lactoferrin (1.8 or 3.6 g/day). At the end of lactoferrin treatment, a decrease in serum alanine transaminase and HCV RNA concentrations was apparent in 3 (75%) of 4 patients with low pretreatment serum concentrations of HCV RNA. However, 7 patients with high pretreatment concentrations showed no significant changes in these indices. This pilot study suggests that lactoferrin is one potential candidate as an anti‐HCV reagent that may be effective for the treatment of patients with chronic hepatitis.

Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in vitro.

The hepatitis C virus (HCV) replicon system is a potent tool for understanding the mechanisms of HCV replication and proliferation, and for the development of treatments for patients with HCV. Recently, we established an HCV subgenomic replicon (50-1) using HCV genome RNA obtained from the cultured human T cell line MT-2C infected with HCV (isolate 1B-1) in vitro. In order to further obtain other HCV replicons without difficulty, we generated a replicon RNA library derived from human non-neoplastic hepatocytes infected with HCV (isolate 1B-2) in vitro. Upon transfection of the generated RNA library to cured cells, from which the 50-1 subgenomic replicon was eliminated by prolonged treatment with interferon-alpha, we successfully established a new HCV subgenomic replicon, 1B-2R1. We characterized 1B-2R1 replicon in terms of efficiency of replication, HCV sequence, and sensitivity to interferons. The results revealed that the replication level of the 1B-2R1 replicon was comparable to that of the 50-1 replicon. We also found that the 1B-2R1 replicon possessed an HCV sequence distinct from those of other replicons established to date, and that the 1B-2R1 replicon was sensitive to interferon-alpha, interferon-beta, and interferon-gamma. Taken together, present results indicate that the replicon RNA library generated using an in vitro HCV infection system is useful for the establishment of an HCV subgenomic replicon.

Activation of the Interferon-Inducible 2′-5′-Oligoadenylate Synthetase Gene by Hepatitis C Virus Core Protein

ABSTRACT The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2′-5′-oligoadenylate synthetase (2′-5′-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2′-5′-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2′-5′-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2′-5′-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2′-5′-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2′-5′-OAS gene.

Differential activation of interferon-inducible genes by hepatitis C virus core protein mediated by the interferon stimulated response element.

We previously found that hepatitis C virus (HCV) core protein, which possesses the consensus sequence of genotype 1b, transcriptionally activates the interferon (IFN)-inducible 2-5-oligoadenylate synthetase (2-5-OAS) gene in human hepatocyte cells. To clarify the mechanism of this activation, we further characterized the core protein as an activator of the 2-5-OAS gene. We demonstrated that the activation of the 2-5-OAS gene by the core protein is a general phenomenon, regardless of HCV genotype and strain. We showed that the 20 N-terminal amino acids (aa) of the core protein were important to the activation of the 2-5-OAS gene, although this N-terminal region did not have any effect on the subcellular localization of the core protein. We demonstrated that the core protein was able to activate all promoters possessing the IFN-stimulated response element (ISRE) examined. However, we found that the level of activation of the 2-5-OAS gene promoter possessing a particular variant type of ISRE was significantly higher than that of other IFN-inducible gene promoters. This phenomenon was confirmed using synthetic promoters possessing five repeats of the consensus or a 2-5-OAS-type ISRE. In addition, we showed that gene activation induced by the core protein is mediated by the ISRE. These results imply that the core protein prefers a subclass of IFN-inducible genes, the promoters of which possess the 2-5-OAS-type ISRE. Accordingly, we found that the IFN-inducible double-stranded RNA-specific adenosine deaminase gene promoter, possessing a 2-5-OAS-type ISRE sequence, was also efficiently activated by the core protein. The exact mechanism by which the core protein enhances gene expression was not determined, but we could find no effects of core protein on gene expression and phosphorylation status of the components of the JAK-STAT signaling transduction pathway.

Promotion of Microsatellite Instability by Hepatitis C Virus Core Protein in Human Non-neoplastic Hepatocyte Cells

Hepatitis C virus proteins exert an effect on a variety of cellular functions, including gene expression, signal transduction, and apoptosis, and because they possess oncogenic potentials, they have also been suggested to play an important role in hepatocarcinogenesis. Although the mechanisms of hepatocarcinogenesis remain poorly understood, we hypothesized that the disease may arise because of a disturbance of the DNA repair system by hepatitis C virus proteins. To test this hypothesis, we developed a reproducible microsatellite instability assay system for mismatch-repair using human-cultured cells transducted with pCXpur retrovirus expression vector, in which the puromycin resistance gene was rendered out-of-frame by insertion of a (CA)17 dinucleotide repeat tract immediately following the ATG start codon. Using several human cancer cell lines known to be replication error positive or negative, we demonstrated that this assay system was useful for monitoring the propensity for mismatch-repair in the cells. This assay system was applicable to non-neoplastic human PH5CH8 hepatocytes, which could support hepatitis C virus replication. Using PH5CH8 cells, in which hepatitis C virus proteins were stably expressed by the retrovirus-mediated gene transfer, we found that the core protein promoted microsatellite instability in PH5CH8 cells. Interestingly, such promotion by the core protein only occurred in cells having the core protein belonging to genotype 1b or 2a and did not occur in cells having the core protein belonging to genotype 1a, 2b, or 3a. This is the first report to demonstrate that the core protein may disturb the DNA repair system.

Tandem Repeats of Lactoferrin‐Derived Anti‐Hepatitis C Virus Peptide Enhance Antiviral Activity in Cultured Human Hepatocytes

Previously, we found that bovine and human lactoferrin (LF) specifically inhibited hepatitis C virus (HCV) infection in cultured non‐neoplastic human hepatocyte‐derived PH5CH8 cells, and we identified 33 amino acid residues (termed C‐s3–33; amino acid 600–632) from human LF that were primarily responsible for the binding activity to the HCV E2 envelope protein and for the inhibiting activity against HCV infection. Since the anti‐HCV activity of C‐s3–33 was weaker than that of human LF, we speculated that an increase of E2 protein‐binding activity might contribute to the enhancement of anti‐HCV activity. To test this possibility, we made two repeats [(C‐s3–33)2] and three repeats [(C‐s3–33)3] of C‐s3–33 and characterized them. Far‐Western blot analysis revealed that the E2 protein‐binding activities of (C‐s3–33)2 and (C‐s3–33)3 became stronger than that of the C‐s3–33, and that the binding activity of (C‐s3–33)3 was stronger than that of (C‐s3–33)2. Using an HCV infection system in PH5CH8 cells, we demonstrated that the anti‐HCV activities of (C‐s3–33)2 and (C‐s3–33)3 became stronger than that of the C‐s3–33. Furthermore, using a recently developed infection system with a VSV pseudotype harboring the green fluorescent protein gene and the native E1 and E2 genes, we demonstrated that the antiviral activities of (C‐s3–33)2 and (C‐s3–33)3 were stronger than that of C‐s3–33. These results suggest that tandem repeats of LF‐derived anti‐HCV peptide are useful as anti‐HCV reagents.

Hydroxyurea suppresses HCV replication in humans: a Phase I trial of oral hydroxyurea in chronic hepatitis C patients

BACKGROUNDnHCV is the main causative agent of chronic liver disease, which could progress to liver cirrhosis and hepatocellular carcinoma. By using a recently developed genome-length HCV RNA replication reporter assay system, we found that hydroxyurea (HU), an inhibitor of DNA synthesis, inhibited HCV RNA replication.nnnMETHODSnTo test the hypothesis that HU suppresses HCV replication in humans, we conducted a Phase I trial involving Japanese patients with chronic hepatitis C (CHC) and investigated the safety and effectiveness of a 4-week course of oral HU.nnnRESULTSnA total of nine patients were treated with an HU dose level of 500 mg three times daily. Dose-limiting toxicity was not observed at this dose level. Of the nine patients, eight exhibited a moderate decrease in serum HCV RNA levels during the trial. A decrease in HCV RNA levels to nadir levels was achieved for the eight patients (median -0.27 log(10) IU/ml [range -0.08--0.44]) at various times during the 4 weeks after therapy initiation.nnnCONCLUSIONSnThe results of this Phase I trial suggest that HU has potential as an anti-HCV agent that could be effective for the treatment of CHC patients.

Respiratory motion tracking system of hepatocellular carcinoma treatment using FUS

One of the reasons for the long treatment time of FUS for HCC compared with RFA is that we have to adjust the target lesion which has a respiratory movement. In this study, we evaluated the usefulness of respiratory tracking system for the FUS monitoring images of hepatocellular carcinoma (HCC).

Usefulness of 3D slicer for the planning and monitoring of hepatocellular carcinoma treatment using FUS

FUS is a noninvasive treatment method, as complete coagulation necrosis is achieved without the insertion of any instruments. However, FUS monitor has the poor visualization because of the presence of the multi-reflections, rib shadows and the emergence of the hyperecho after the FUS treatment. 3D Slicer imaging is a diagnostic imaging support system that can provide the same cross-sectional MPR images on the same monitor screen using DICOM volume data from MRI which are not influenced by those artifacts. The purpose of this study was to utilize an interventional navigation system designed for FUS assisted by 3D Slicer was proposed, and a phantom study was carried out to assess the proposed system.