Publications by authors named "Masayuki Sudoh"

29 Publications

  • Page 1 of 1

Epigenetic reprogramming promotes the antiviral action of IFNα in HBV-infected cells.

Cell Death Discov 2021 Jun 2;7(1):130. Epub 2021 Jun 2.

Liver Cancer Prevention Research Unit, RIKEN Cluster for Pioneering Research, Wako, Japan.

Chronic hepatitis B virus (HBV) infections remain a health burden affecting ~250 million people worldwide. Thus far, available interferon-alpha (IFNα)-based therapies have shown unsatisfactory cure rates, and alternative therapeutic molecules are still required. However, their development has been hampered because accessible cell models supporting relevant HBV replication and appropriate antiviral activity are lacking. Strategies that reverse epigenetic alterations offer a unique opportunity for cell reprogramming, which is valuable for restoring altered cellular functions in human cell lines. This work aimed to investigate the feasibility of converting HepG2 cells that stably overexpress the HBV entry receptor (sodium/taurocholate cotransporting polypeptide, NTCP) toward IFNα-responsive cells using epigenetic reprogramming. Herein, we showed that an epigenetic regimen with non-cytotoxic doses of the demethylating compound 5-azacytidine restored the anti-HBV action of IFNα in epigenetically reprogrammed HepG2-NTCP-C4 cells, named REP-HepG2-NTCP cells. Thus, a significant inhibition in HBV DNA levels was measured in REP-HepG2-NTCP cells after IFNα treatment. This inhibitory effect was associated with the enhancement of IFNα-mediated induction of critical interferon-stimulated genes (ISGs), which was limited in non-reprogrammed cells. In particular, our data indicated that re-expression of 2'-5'-oligoadenylate synthetase 1 (OAS1) and interferon regulatory factor 9 (IRF9) was the result of an epigenetically driven unmasking of these genes in reprogrammed cells. At last, we evaluated the therapeutic potential of the IFN analog CDM-3008 in REP-HepG2-NTCP cells and demonstrated the efficiency of this chemical compound in triggering ISG induction and HBV inhibition. In summary, this study shows that epigenetic reprogramming promotes the IFNα response in HBV-infected cells and is potentially attractive for cell-based experimental screening of IFN-like compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41420-021-00515-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8170866PMC
June 2021

An interferon-like small chemical compound CDM-3008 suppresses hepatitis B virus through induction of interferon-stimulated genes.

PLoS One 2019 12;14(6):e0216139. Epub 2019 Jun 12.

Liver Cancer Prevention Research Unit, RIKEN Center for Integrative Medical Sciences, Wako, Saitama, Japan.

Oral administration of nucleotide analogues and injection of interferon-α (IFNα) are used to achieve immediate suppression in replication of hepatitis B virus (HBV). Nucleotide analogs and IFNα inhibit viral polymerase activity and cause long-term eradication of the virus at least in part through removing covalently closed circular DNA (cccDNA) via induction of the APOBEC3 deaminases family of molecules, respectively. This study aimed to explore whether the orally administrable low molecular weight agent CDM-3008 (RO8191), which mimics IFNα through the binding to IFNα/β receptor 2 (IFNAR2) and the activation of the JAK/STAT pathway, can suppress HBV replication and reduce cccDNA levels. In primary cultured human hepatocytes, HBV DNA levels were decreased after CDM-3008-treatment in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) value of 0.1 μM, and this was accompanied by significant reductions in cellular cccDNA levels, both HBeAg and HBsAg levels in the cell culture medium. Using a microarray we comprehensively analyzed and compared changes in gene (mRNA) expression in CDM-3008- and IFNα-treated primary cultured human hepatocytes. As reported previously, CDM-3008 mimicked the induction of genes that participate in the interferon signaling pathway. OAS1 and ISG20 mRNA expression was similarly enhanced by both CDM-3008 and IFNα. Thus, CDM-3008 could suppress pgRNA expression to show anti-HBV activity. APOBEC3F and 3G mRNA expression was also induced by CDM-3008 and IFNα treatments, suggesting that cccDNA could be degraded through induced APOBEC3 family proteins. We identified the genes whose expression was specifically enhanced in CDM-3008-treated cells compared to IFNα-treated cells. The expression of SOCS1, SOCS2, SOCS3, and CISH, which inhibit STAT activation, was enhanced in CDM-3008-treated cells suggesting that a feedback inhibition of the JAK/STAT pathway was enhanced in CDM-3008-treated cells compared to IFNα-treated cells. In addition, CDM-3008 showed an additive effect with a clinically-used nucleoside entecavir on inhibition of HBV replication. In summary, CDM-3008 showed anti-HBV activity through activation of the JAK/STAT pathway, inducing the expression of interferon-stimulated genes (ISGs), with greater feedback inhibition than IFNα.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216139PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561549PMC
February 2020

Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation.

J Biol Chem 2019 04 12;294(15):5759-5773. Epub 2019 Feb 12.

From the Transboundary Animal Diseases Centre, Kagoshima University, Kagoshima-City 890-8580, Japan; Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima-City 890-8580, Japan. Electronic address:

Hepatitis C virus (HCV) establishes a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. The HCV life cycle is closely associated with host factors that promote or restrict viral replication, the characterization of which could help to identify potential therapeutic targets. To this end, here we performed a genome-wide microarray analysis and identified ribonucleotide reductase M2 (RRM2) as a cellular factor essential for HCV replication. We found that RRM2 is up-regulated in response to HCV infection in quiescent hepatocytes from humanized chimeric mouse livers. To elucidate the molecular basis of RRM2 expression in HCV-infected cells, we used HCV-infected hepatocytes from chimeric mice and hepatoma cells infected with the HCV strain JFH1. Both models exhibited increased mRNA and protein expression levels. Moreover, siRNA-mediated silencing of suppressed HCV replication and infection. Of note, RRM2 and RNA polymerase nonstructural protein 5B (NS5B) partially co-localized in cells and co-immunoprecipitated, suggesting that they might interact. knockdown reduced NS5B expression, which depended on the protein degradation pathway, as RNA levels did not decrease and NS5B protein stability correlated with RRM2 protein levels. We also found that silencing decreased levels of hPLIC1 (human homolog 1 of protein linking integrin-associated protein and cytoskeleton), a ubiquitin-like protein that interacts with NS5B and promotes its degradation. This finding suggests that there is a dynamic interplay between RRM2 and the NS5B-hPLIC1 complex that has an important function in HCV replication. Together, these results identify a role of host RRM2 in viral RNA replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.004397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463693PMC
April 2019

Development of an anti-hepatitis B virus (HBV) agent through the structure-activity relationship of the interferon-like small compound CDM-3008.

Bioorg Med Chem 2019 02 5;27(3):470-478. Epub 2018 Dec 5.

Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address:

Hepatitis B, a viral infectious disease caused by hepatitis B virus (HBV), is a life-threatening disease that leads liver cirrhosis and liver cancer. Because the current treatments for HBV, such as an interferon (IFN) formulation or nucleoside/nucleotide analogues, are not sufficient, the development of a more effective agent for HBV is urgent required. CDM-3008 (1, 2-(2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridin-8-yl)-1,3,4-oxadiazole) (RO8191)) is a small molecule with an imidazo[1,2-a][1,8]naphthyridine scaffold that shows anti-HCV activity with an IFN-like effect. Here, we report that 1 was also effective for HBV, although the solubility and metabolic stability were insufficient for clinical use. Through the structure-activity relationship (SAR), we discovered that CDM-3032 (11, N-(piperidine-4-yl)-2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridine-8-carboxamide hydrochloride) was more soluble than 1 (>30 mg/mL for 11 versus 0.92 mg/mL for 1). In addition, the half-life period of 11 was dramatically improved in both mouse and human hepatic microsomes (T1/2, >120 min versus 58.2 min in mouse, and >120 min versus 34.1 min in human, for 11 and 1, respectively).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2018.11.039DOI Listing
February 2019

Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice.

PLoS One 2015 4;10(11):e0142145. Epub 2015 Nov 4.

Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142145PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633119PMC
June 2016

[An approach for anti-fibrotic drug discovery in NASH].

Nihon Yakurigaku Zasshi 2014 Aug;144(2):69-74

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1254/fpj.144.69DOI Listing
August 2014

In vivo therapeutic potential of Dicer-hunting siRNAs targeting infectious hepatitis C virus.

Sci Rep 2014 Apr 23;4:4750. Epub 2014 Apr 23.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.

The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep04750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996463PMC
April 2014

Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

PLoS One 2013 12;8(12):e82094. Epub 2013 Dec 12.

Department of HGF Tissue Repair and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

Background: It has been hypothesized that persistent hepatitis C virus (HCV) infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4), composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation.

Methods: Human C4 was incubated with HCV nonstructural (NS) 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined.

Results: HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease-mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4.

Conclusions: C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082094PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861371PMC
October 2014

A serine palmitoyltransferase inhibitor blocks hepatitis C virus replication in human hepatocytes.

Gastroenterology 2013 Oct 18;145(4):865-73. Epub 2013 Jun 18.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; Research Division, Chugai Pharmaceutical Co., Ltd., Tokyo, Japan.

Background & Aims: Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice.

Methods: We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors.

Results: NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors.

Conclusions: The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2013.06.012DOI Listing
October 2013

An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis.

Int J Exp Pathol 2013 Apr 11;94(2):93-103. Epub 2013 Jan 11.

Fuji-Gotemba Research Laboratories, Chugai Research Institute for Medical Science Inc, Gotemba, Shizuoka, Japan.

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. One of the most common models used in NASH research is a nutritional model where NASH is induced by feeding a diet deficient in both methionine and choline. However, the dietary methionine-/choline-deficient model in mice can cause severe weight loss and liver atrophy, which are not characteristics of NASH seen in human patients. Exclusive, long-term feeding with a high-fat diet (HFD) produced fatty liver and obesity in mice, but the HFD for several months did not affect fibrosis. We aimed to establish a mouse model of NASH with fibrosis by optimizing the methionine content in the HFD. Male mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight. After 1-14 weeks of being fed CDAHFD, the mice were killed. C57BL/6J mice maintained or gained weight when fed CDAHFD, while A/J mice showed a steady decline in body weight (of up to 20% of initial weight). In both strains of mice, plasma levels of alanine aminotransferase increased from week 1, when hepatic steatosis was also observed. By week 6, C57BL/6J mice had developed enlarged fatty liver with fibrosis as assessed by Masson's trichrome staining and by hydroxyproline assay. Therefore, this improved CDAHFD model may be a mouse model of rapidly progressive liver fibrosis and be potentially useful for better understanding human NASH disease and in the development of efficient therapies for this condition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/iep.12008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607137PMC
April 2013

Synthetic lipophilic antioxidant BO-653 suppresses HCV replication.

J Med Virol 2013 Feb 28;85(2):241-9. Epub 2012 Nov 28.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.

The influence of the intracellular redox state on the hepatitis C virus (HCV) life cycle is poorly understood. This study demonstrated the anti-HCV activity of 2,3-dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653), a synthetic lipophilic antioxidant, and examined whether BO-653's antioxidant activity is integral to its anti-HCV activity. The anti-HCV activity of BO-653 was investigated in HuH-7 cells bearing an HCV subgenomic replicon (FLR3-1 cells) and in HuH-7 cells infected persistently with HCV (RMT-tri cells). BO-653 inhibition of HCV replication was also compared with that of several hydrophilic and lipophilic antioxidants. BO-653 suppressed HCV replication in FLR3-1 and RMT-tri cells in a concentration-dependent manner. The lipophilic antioxidants had stronger anti-HCV activities than the hydrophilic antioxidants, and BO-653 displayed the strongest anti-HCV activity of all the antioxidants examined. Therefore, the anti-HCV activity of BO-653 was examined in chimeric mice harboring human hepatocytes infected with HCV. The combination treatment of BO-653 and polyethylene glycol-conjugated interferon-α (PEG-IFN) decreased serum HCV RNA titer more than that seen with PEG-IFN alone. These findings suggest that both the lipophilic property and the antioxidant activity of BO-653 play an important role in the inhibition of HCV replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jmv.23466DOI Listing
February 2013

Enantioselective synthesis of derivatives and structure-activity relationship study in the development of NA255 as a novel host-targeting anti-HCV agent.

Bioorg Med Chem Lett 2013 Jan 30;23(1):336-9. Epub 2012 Oct 30.

Research Division, Chugai Pharmaceutical Co., Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan. Electronic address:

Hepatitis C virus (HCV) infection represents a serious health-care problem. Previously we reported the identification of NA255 from our natural products library using a HCV sub-genomic replicon cell culture system. Herein, we report how the absolute stereochemistry of NA255 was determined and an enantioselective synthetic method for NA255 derivatives was developed. The structure-activity relationship of the NA255 derivatives and rat pharmacokinetic profiles of the representative compounds are disclosed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2012.10.083DOI Listing
January 2013

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

PLoS Pathog 2012 16;8(8):e1002860. Epub 2012 Aug 16.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1002860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420934PMC
December 2012

Raloxifene hydrochloride is an adjuvant antiviral treatment of postmenopausal women with chronic hepatitis C: a randomized trial.

J Hepatol 2012 Dec 10;57(6):1186-92. Epub 2012 Aug 10.

Department of General Internal Medicine, Kyushu University Hospital, Fukuoka, Japan.

Background & Aims: Early menopause in women with chronic hepatitis C virus (HCV) infection is associated with a low likelihood of a sustained virological response (SVR) in conjunction with their antiviral treatment. This is potentially related to their reduced estrogen secretion. The study was done to determine whether selective estrogen receptor modulator administration might improve the efficacy of the current standard of care (SOC) treatment, pegylated interferon (PegIFN) α2a plus ribavirin (RBV), for postmenopausal women.

Methods: One hundred and twenty-three postmenopausal women with genotype 1b chronic hepatitis C were randomly assigned to one of two treatment groups: raloxifene hydrochloride (RLX) (60 mg/day) plus SOC (PegIFNα2a 180 μg/week and RBV 600-1,000 mg/day) (n=62) or SOC only (n=61). Genotyping was performed of the polymorphism in the interleukin-28B (IL28B) gene region (rs8099917) of DNA collected from each patient.

Results: One RLX-treated patient discontinued RLX because of a systemic rash following 2 weeks of treatment. Twenty-four weeks after treatment, the SVR rate was significantly higher for RLX plus SOC patients (61.3%) than for SOC only patients (34.4%) (p=0.0051). Further, the SVR rate was significantly higher for RLX plus SOC patients with IL28B TT (72.5%) than for SOC only patients with IL28B TT (39.2%) (p=0.0014), but no such relationship was observed in patients carrying the minor IL28B allele.

Conclusions: RLX improved the efficacy of SOC in the treatment of postmenopausal women with chronic hepatitis C. RLX shows promise as an adjuvant to the standard antiviral treatment of such patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jhep.2012.08.003DOI Listing
December 2012

An orally available, small-molecule interferon inhibits viral replication.

Sci Rep 2012 10;2:259. Epub 2012 Feb 10.

Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., Kamakura, Kanagawa, Japan.

Most acute hepatitis C virus (HCV) infections become chronic and some progress to liver cirrhosis or hepatocellular carcinoma. Standard therapy involves an interferon (IFN)-α-based regimen, and efficacy of therapy has been significantly improved by the development of protease inhibitors. However, several issues remain concerning the injectable form and the side effects of IFN. Here, we report an orally available, small-molecule type I IFN receptor agonist that directly transduces the IFN signal cascade and stimulates antiviral gene expression. Like type I IFN, the small-molecule compound induces IFN-stimulated gene (ISG) expression for antiviral activity in vitro and in vivo in mice, and the ISG induction mechanism is attributed to a direct interaction between the compound and IFN-α receptor 2, a key molecule of IFN-signaling on the cell surface. Our study highlights the importance of an orally active IFN-like agent, both as a therapy for antiviral infections and as a potential IFN substitute.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep00259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277087PMC
April 2013

Monoclonal antibody 2-152a suppresses hepatitis C virus infection through betaine/GABA transporter-1.

J Infect Dis 2011 Oct;204(8):1172-80

Department of Experimental Phylaxiology, Faculty of Life Sciences, Kumamoto University, Honjo Kumamoto City, Japan.

Background: We recently established a monoclonal antibody (2-152a MAb) that binds to 3β-hydroxysterol-Δ24-reductase (DHCR24) by immunizing mice with cells (RzM6-LC) persistently expressing hepatitis C virus (HCV). Here, we aimed to analyze the activity of 2-152a MAb against HCV replication and explore the molecular mechanism underlying the antiviral activity.

Methods: We characterized the effects of 2-152a MAb on HCV replication and performed a microarray analysis of antibody-treated HCV replicon cells. The molecules showing a significant change after the antibody treatment were screened to examine their relationship with HCV replication.

Results: The antibody had antiviral activity both in vitro and in vivo (chimeric mice). In the microarray analysis, 2-152a MAb significantly suppressed the expression of betaine/GABA transporter-1 (BGT-1) in 2 HCV replicon cell lines but not in HCV-cured cells. Silencing of BGT-1 expression by small interfering RNA (siRNA) revealed significant suppression of HCV replication and infection without cytotoxicity. Further, BGT-1 expression was significantly increased in the presence of HCV (P < .05).

Conclusions: Our results suggest that 2-152a MAb suppresses HCV replication and infection through BGT-1. These findings highlight important roles of BGT-1 in HCV replication and reveal a possible target for anti-HCV therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jir501DOI Listing
October 2011

Host sphingolipid biosynthesis is a promising therapeutic target for the inhibition of hepatitis B virus replication.

J Med Virol 2011 Apr;83(4):587-93

Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

Serine palmitoyltransferase (SPT) catalyzes the first step in the sphingolipid biosynthetic pathway. Myriocin inhibits SPT and was shown to suppress the replication of hepatitis C virus (HCV) in vitro and in vivo. However, its effect on hepatitis B virus (HBV) replication is unknown. In this study, the HBV DNA levels in HuH7 cell culture supernatants were lowered successfully by using myriocin and it was found that the 50% inhibitory concentration of myriocin is approximately 5 µM. Myriocin and/or pegylated interferon (PEG-IFN) were also administered to chimeric mice for 2 weeks and the effects of these compounds on HBV DNA levels were determined. Myriocin alone did not reduce effectively the HBV DNA levels, whereas PEG-IFN alone reduced the DNA levels to 1/10th of the control levels. The combination of myriocin with PEG-IFN reduced the HBV levels to about 1/1,000 th of the control levels and induced a 1.0 log reduction in the levels of the HBV surface antigen and core protein. This latter effect was not observed in the other treatment groups. In conclusion, the combination of myriocin with PEG-IFN represses synergistically HBV replication in vivo without inducing hepatotoxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jmv.21970DOI Listing
April 2011

Establishment of infectious HCV virion-producing cells with newly designed full-genome replicon RNA.

Arch Virol 2011 Feb 19;156(2):295-304. Epub 2011 Jan 19.

Pharmacology Research Laboratories I, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Japan.

Hepatitis C virus (HCV) replicon systems enable in-depth analysis of the life cycle of HCV. However, the previously reported full-genome replicon system is unable to produce authentic virions. On the basis of these results, we constructed newly designed full-genomic replicon RNA, which is composed of the intact 5'-terminal-half RNA extending to the NS2 region flanked by an extra selection marker gene. Huh-7 cells harboring this full-genomic RNA proliferated well under G418 selection and secreted virion-like particles into the supernatant. These particles, which were round and 50 nm in diameter when analyzed by electron microscopy, had a buoyant density of 1.08 g/mL that shifted to 1.19 g/mL after NP-40 treatment; these figures match the putative densities of intact virions and nucleocapsids without envelope. The particles also showed infectivity in a colony-forming assay. This system may offer another option for investigating the life cycle of HCV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00705-010-0859-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032175PMC
February 2011

Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes.

J Hepatol 2011 Sep 22;55(3):512-521. Epub 2010 Dec 22.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.

Background & Aims: We characterized the role of 24-dehydrocholesterol reductase (DHCR24) in hepatitis C virus infection (HCV). DHCR24 is a cholesterol biosynthetic enzyme and cholesterol is a major component of lipid rafts, which is reported to play an important role in HCV replication. Therefore, we examined the potential of DHCR24 as a target for novel HCV therapeutic agents.

Methods: We examined DHCR24 expression in human hepatocytes in both the livers of HCV-infected patients and those of chimeric mice with human hepatocytes. We targeted DHCR24 with siRNA and U18666A which is an inhibitor of both DHCR24 and cholesterol synthesis. We measured the level of HCV replication in these HCV replicon cell lines and HCV infected cells. U18666A was administrated into chimeric mice with humanized liver, and anti-viral effects were assessed.

Results: Expression of DHCR24 was induced by HCV infection in human hepatocytes in vitro, and in human hepatocytes of chimeric mouse liver. Silencing of DHCR24 by siRNA decreased HCV replication in replicon cell lines and HCV JFH-1 strain-infected cells. Treatment with U18666A suppressed HCV replication in the replicon cell lines. Moreover, to evaluate the anti-viral effect of U18666A in vivo, we administrated U18666A with or without pegylated interferon to chimeric mice and observed an inhibitory effect of U18666A on HCV infection and a synergistic effect with interferon.

Conclusions: DHCR24 is an essential host factor which augmented its expression by HCV infection, and plays a significant role in HCV replication. DHCR24 may serve as a novel anti-HCV drug target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jhep.2010.12.011DOI Listing
September 2011

[Suppression of hepatitis C virus (HCV) replication with serine palmitoyltransferase inhibitor].

Yakugaku Zasshi 2010 Feb;130(2):157-61

Department of Microbiology and Cell biology, The Tokyo metropolitan Institute of Medical Science, Tokyo, Japan.

Hepatitis C virus (HCV) persists chronically in most infected patients, eventually causing chronic hepatitis, liver cirrhosis, and in some cases hepatocellular carcinoma. The combination therapy of PEG-IFN and ribavirin improves efficacy in many patients, although it does not lead to sufficient achievements in genotype 1b patients. To aid in invention of new anti-HCV reagents, we focused on host factors that contributed to HCV lifecycle. We identified serine palmitoyltransferase inhibitor as an anti-HCV reagent through high-throughput screening using HCV replicon cells. We investigated the mechanism of anti-HCV effect of SPT inhibitor. It has been reported that sphingolipids and cholesterol compose the lipid raft where replication of HCV occurs. We investigated the influence of SPT inhibitor to lipid rafts by analyzing the detergent-resistant membrane (DRM). The analysis showed that SPT inhibitor moved HCV RNA-dependent RNA polymerase (NS5B) to detergent-soluble fraction from DRM, and Biacore analysis indicated binding of sphingomyelin to NS5B. These results suggest that SPT inhibitor disrupts the interaction between NS5B and sphingomyelin. Moreover, we evaluated the anti-HCV effect of SPT inhibitor in vivo with humanized chimeric mice. SPT inhibitor led to rapid decline in serum HCV-RNA of about 1-2 log within 8 days. Furthermore, combination therapy of SPT inhibitor and PEG-IFN achieved about 3 log reduction in serum HCV-RNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1248/yakushi.130.157DOI Listing
February 2010

Proanthocyanidin from blueberry leaves suppresses expression of subgenomic hepatitis C virus RNA.

J Biol Chem 2009 Aug 16;284(32):21165-76. Epub 2009 Jun 16.

Research Division, Minami Nippon Dairy Co-op Co., Ltd., Miyazaki 885-0073, Japan.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.004945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755839PMC
August 2009

[Suppression of hepatitis C virus with the reagent targetting host factors].

Uirusu 2008 Dec;58(2):207-13

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.

Hepatitis C virus (HCV) develops persistent infection in most infected patients, and eventually cause chronic hepatitis, liver cirrhosis and then hepatocellular carcinoma. The combination therapy of PEG-IFN and ribavirin improves the efficacy in many patients, while it does not lead to sufficient achievements in genotype1b patients. To invent new anti-HCV reagent, we focused on host factors which HCV take advantage of in its life-cycle. We identified serine palmitoyltransferase inhibitor as anti-HCV reagent through high-through put screenig using HCV replicon cells. Moreover, we evaluate the anti-HCV effect of SPT-inhibitor in vivo with humanized chimeric mice. SPT-inhibitor led to rapid decline in serum HCV-RNA of about 1-2log within 8 day, futhermore the combination therapy of SPT-inhibitor and PEG-IFN achieved about 3log reduction in serum HCV-RNA. At last, we investigated the mechanism of anti-HCV effect of SPT-inhibitor. It has been reported that sphingolipids and cholesterol compose the lipid raft, in which the replication of HCV occur. We investigated the influence of SPT-inhibitor to lipid rafts by analysing the detergent resistant membrane (DRM). The analysis proved that SPT inhibitor got HCV RNA dependent RNA polymerase (NS5B) to move to detergent soluble fraction from DRM, and Biacore analysis indicated the binding of sphingomyelin to NS5B. These results suggested SPT inhibitor got NS5B to release from replication complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2222/jsv.58.207DOI Listing
December 2008

Hsp90 inhibitors suppress HCV replication in replicon cells and humanized liver mice.

Biochem Biophys Res Commun 2007 Feb 22;353(4):882-8. Epub 2006 Dec 22.

Department of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.

Persistent infection with hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Here we report that inhibition of heat shock protein 90 (Hsp90) is highly effective in suppressing HCV genome replication. In HCV replicon cells, HCV replication was reduced by Hsp90 inhibitors and by knockdown of endogenous Hsp90 expression mediated by small-interfering RNA (siRNA). The suppression of HCV replication by an Hsp90 inhibitor was prevented by transfection with Hsp90 expression vector. We also tested the anti-HCV effect of Hsp90 inhibition in HCV-infected chimeric mice with humanized liver. Combined administration of an Hsp90 inhibitor and polyethylene glycol-conjugated interferon (PEG-IFN) was more effective in reducing HCV genome RNA levels in serum than was PEG-IFN monotherapy. These results suggest that inhibition of Hsp90 could provide a new therapeutic approach to HCV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2006.12.117DOI Listing
February 2007

Serine palmitoyltransferase inhibitor suppresses HCV replication in a mouse model.

Biochem Biophys Res Commun 2006 Jul 24;346(1):67-73. Epub 2006 May 24.

Department of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.

Serine palmitoyltransferase (SPT) is a first-step enzyme in the sphingolipid biosynthetic pathway. Myriocin is an inhibitor of SPT and suppresses replication of the hepatitis C virus (HCV) replicon. However, it is still unknown whether this SPT inhibitor suppresses HCV replication in vivo. We investigated the anti-HCV effect of myriocin against intact HCV using chimeric mice with humanized liver infected with HCV genotype 1a or 1b. We administered myriocin into HCV infected chimeric mice and succeeded in reducing the HCV RNA levels in serum and liver to 1/10-1/100 of the levels prior to the 8 day treatment. Furthermore, combined treatment with pegylated interferon reduced the HCV RNA levels to less than 1/1000 of the control levels. We strongly suggest that suppression of SPT reduces HCV replication, and therefore that the SPT inhibitor is potentially a novel drug in the treatment of HCV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2006.05.085DOI Listing
July 2006

Host sphingolipid biosynthesis as a target for hepatitis C virus therapy.

Nat Chem Biol 2005 Nov 16;1(6):333-7. Epub 2005 Oct 16.

Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan.

An estimated 170 million individuals worldwide are infected with hepatitis C virus (HCV), a serious cause of chronic liver disease. Current interferon-based therapy for treating HCV infection has an unsatisfactory cure rate, and the development of more efficient drugs is needed. During the early stages of HCV infections, various host genes are differentially regulated, and it is possible that inhibition of host proteins affords a therapeutic strategy for treatment of HCV infection. Using an HCV subgenomic replicon cell culture system, here we have identified, from a secondary fungal metabolite, a lipophilic long-chain base compound, NA255 (1), a previously unknown small-molecule HCV replication inhibitor. NA255 prevents the de novo synthesis of sphingolipids, major lipid raft components, thereby inhibiting serine palmitoyltransferase, and it disrupts the association among HCV nonstructural (NS) viral proteins on the lipid rafts. Furthermore, we found that NS5B protein has a sphingolipid-binding motif in its molecular structure and that the domain was able to directly interact with sphingomyelin. Thus, NA255 is a new anti-HCV replication inhibitor that targets host lipid rafts, suggesting that inhibition of sphingolipid metabolism may provide a new therapeutic strategy for treatment of HCV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio742DOI Listing
November 2005

Transcriptional profiling of the early stages of germination in Candida albicans by real-time RT-PCR.

FEMS Yeast Res 2004 Dec;5(3):287-96

Section of Infection Biology, Department of Functional Bioscience, Fukuoka Dental College, Fukuoka 814-0193, Japan.

By using real-time RT-PCR, we profiled the expression of CGR1, CaMSI3, EFG1, NRG1, and TUP1 in Candida albicans strains JCM9061 and CAI4 under several conditions, including induction of morphological transition, heat shock, and treatment with calcium inhibitors. Expression of CaMSI3 changed under these growth conditions except during heat shock. CGR1 expression increased during the early stages of hyphal growth in JCM9061, while expression was strain-dependent during heat shock. Both EFG1 and NRG1 were similarly expressed under hypha-inducing conditions and heat shock. Expression of TUP1 was slightly different from the expression of EFG1 or NRG1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.femsyr.2004.08.004DOI Listing
December 2004

Isolation and sequencing of the Candida albicans MSI3, a putative novel member of the HSP70 family.

Yeast 2003 Jan;20(2):149-56

Department of Functional Bioscience, Fukuoka Dental College, Fukuoka 814-0193, Japan. chot

We have reported previously that the expression of CGR1 increased at an early stage of the yeast-mycelial transition (morphogenesis) in Candida albicans. We now show that Cgr1p interacts in a yeast two-hybrid system with the C. albicans Msi3p (CaMsi3p), a putative novel member of the heat shock protein 70 (HSP70) family. The DNA sequence of CaMSI3 encodes a predicted protein of 702 amino acids with a molecular mass of 78.6 kDa. The amino acid sequence of CaMsi3p is 63% identical to Msi3p/Sse1p of the HSP70 family of Saccharomyces cerevisiae. Further, CaMSI3 complemented the temperature-sensitive phenotype of the msi3(-) mutant of S. cerevisiae. Other heat shock proteins of C. albicans are required for morphogenesis and are highly antigenic. These observations suggest that CaMSI3 may well provide functions for this organism unrelated to a heat shock function. The DDBJ Accession No. for the sequence reported in this paper is AB061274.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/yea.952DOI Listing
January 2003

The yeast Chs4 protein stimulates the trypsin-sensitive activity of chitin synthase 3 through an apparent protein-protein interaction.

Microbiology (Reading) 2000 Feb;146 ( Pt 2):385-391

Mycology Department, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan1.

Inducible overexpression of the CHS4 gene under the control of the GAL1 promoter increased Chs3p (chitin synthase 3) activity in Saccharomyces cerevisiae several fold. Approximately half of the Chs3p activity in the membranes of cells overexpressing Chs4p was extracted using CHAPS and cholesteryl hemisuccinate. The detergent-extractable Chs3p activity appeared to be non-zymogenic because incubation with trypsin decreased enzyme activity in both the presence and absence of the substrate, UDP-N-acetylglucosamine. Western blotting confirmed that Chs3p was extracted from membranes by CHAPS and cholesteryl hemisuccinate and revealed that Chs4p was also solubilized using these detergents. Yeast two-hybrid analysis with truncated Chs4p demonstrated that the region of Chs4p between amino acids 269 and 563 is indispensable not only for eliciting the non-zymogenic activity of Chs3p but also for binding of Chs4p to Chs3p. Neither the EF-hand motif nor a possible prenylation site in Chs4p was required for these activities. Thus, it was demonstrated that stimulation of non-zymogenic Chs3p activity by Chs4p requires the amino acid region from 269 to 563 of Chs4p, and it seems that Chs4p activates Chs3p through protein-protein interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/00221287-146-2-385DOI Listing
February 2000

The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis.

Microbiology (Reading) 1999 Jul;145 ( Pt 7):1613-1622

Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura-shi, Kanagawa 247-8530, Japan.

The Candida albicans CHS4 gene encoding chitin synthase 4 has been isolated using the Saccharomyces cerevisiae CHS4/SKT5 gene as a probe. The gene contains a 2061 bp open reading frame capable of encoding a protein of 687 amino acids (76053 Da). No intron was observed in the gene. Disruption of CHS4 in C. albicans yielded a Calcofluor-resistant phenotype, indicating that Chs4p contributes to chitin biosynthesis. Consistent with this, overexpression of Chs4p under the regulation of the ScGAL1 promoter enhanced chitin synthase 3 activity in S. cerevisiae 7- to 38-fold. In addition, chs3 and chs4 null mutants were significantly defective in Calcofluor white staining and their chitin content was 10% of that of the parental strain. Chs4p of C. albicans and S. cerevisiae showed 61% identity in the C-terminal half of the proteins and that region of C. albicans Chs4p complemented the Chs4p function of a mutant of S. cerevisiae resistant to Calcofluor white. Therefore, it appears that Chs4p is involved in chitin synthase 3 activity by combining with Chs3p to interact synergistically in chitin biosynthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/13500872-145-7-1613DOI Listing
July 1999
-->