記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

Abstract

Intracranial metastases are common in nonsmall‐cell lung cancer (NSCLC) patients, whose prognosis is very poor. In addition, intracranial progression is common during systemic treatments due to the inability to penetrate central nervous system (CNS) barriers, whereas the intracranial effects of cancer immunotherapies remain unclear. We analyzed clinical data to evaluate the frequency of intracranial progression in advanced NSCLC patients treated with PD‐1 blockade therapies compared with those treated without PD‐1 blockade therapies, and found that the frequency of intracranial progression in advanced NSCLC patients treated with PD‐1 blockade therapies was significantly lower than that in patients treated with cytotoxic chemotherapies. In murine models, intracranial rechallenged tumors after initial rejection by PD‐1 blockade were suppressed. Accordingly, long‐lived memory precursor effector T cells and antigen‐specific T cells were increased by PD‐1 blockade in intracranial lesions. However, intracranial rechallenged different tumors are not suppressed. Our results indicate that cancer immunotherapies can prevent intracranial progression, maintaining long‐term effects intracranially as well as systemically. If intracranial recurrence occurs during the treatment with PD‐1 blockade therapies, aggressive local therapies could be worthwhile.

DOI： 10.1002/ijc.34700

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hydrogen peroxide (H2 O2 ) induces oxidative stress and cytotoxicity, and can be used for treating cancers in combination with radiotherapy. A product comprising H2 O2 and sodium hyaluronate has been developed as a radiosensitizer. However, the effects of H2 O2 on antitumor immunity remain unclear. To investigate the effects of H2 O2 , especially the abscopal effect when combined with radiotherapy (RT), we implanted murine tumor cells simultaneously in two locations in mouse models: the hind limb and back. H2 O2 mixed with sodium hyaluronate was injected intratumorally, followed by irradiation only at the hind limb lesion. No treatment was administered to the back lesion. The H2 O2 /RT combination significantly reduced tumor growth at the noninjected/nonirradiated site in the back lesion, whereas H2 O2 or RT individually did not reduce tumor growth. Flow cytometric analyses of the tumor-draining lymph nodes in the injected/irradiated areas showed that the number of dendritic cells increased significantly with maturation in the H2 O2 /RT combination group. In addition, analyses of tumor-infiltrating lymphocytes showed that the number of CD8+ (cluster of differentiation 8) T cells and the frequency of IFN-γ+ (interferon gamma) CD8+ T cells were higher in the noninjected/nonirradiated tumors in the H2 O2 /RT group compared to those in the other groups. PD-1 (programmed death receptor 1) blockade further increased the antitumor effect against noninjected/nonirradiated tumors in the H2 O2 /RT group. Intratumoral injection of H2 O2 combined with RT therefore induces an abscopal effect by activating antitumor immunity, which can be further enhanced by PD-1 blockade. These findings promote the development of H2 O2 /RT therapy combined with cancer immunotherapies, even for advanced cancers.

DOI： 10.1111/cas.15911

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Combination therapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death-1 (PD-1) monoclonal antibodies (mAbs) has dramatically improved the prognosis of patients with multiple types of cancer, including renal cell carcinoma (RCC). However, more than half of RCC patients fail to respond to this therapy. Regulatory T cells (Treg cells) are a subset of highly immunosuppressive CD4+ T cells that promote the immune escape of tumors by suppressing effector T cells in the tumor microenvironment (TME) through various mechanisms. CTLA-4 is constitutively expressed in Treg cells and is regarded as a key molecule for Treg cell-mediated immunosuppressive functions, suppressing antigen-presenting cells by binding to CD80/CD86. Reducing Treg cells in the TME with an anti-CTLA-4 mAb with antibody-dependent cellular cytotoxicity (ADCC) activity is considered an essential mechanism to achieve tumor regression. In contrast, we demonstrated that only CTLA-4 blockade without ADCC activity enhanced CD28 costimulatory signaling pathways in Treg cells and promoted Treg-cell proliferation in mouse models. CTLA-4 blockade also augmented CTLA-4-independent immunosuppressive functions, including cytokine production, leading to insufficient antitumor effects. Similar results were also observed in human peripheral blood lymphocytes and tumor-infiltrating lymphocytes from patients with RCC. Our findings highlight the importance of Treg-cell depletion to achieve tumor regression in response to CTLA-4 blockade therapies.

DOI： 10.1111/cas.15756

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

Abstract

Background

Identifying biomarkers to predict immune checkpoint inhibitor (ICI) efficacy is warranted. Considering that somatic mutation-derived neoantigens induce strong immune responses, patients with a high tumour mutational burden reportedly tend to respond to ICIs. However, there are several conflicting data. Therefore, we focused on the original function of neoantigenic mutations and their impact on the tumour microenvironment (TME).

Methods

We evaluated 88 high-frequency microsatellite instability (MSI-H) colorectal cancers and analysed the function of the identified neoantigenic mutations and their influence on programmed cell death 1 (PD-1) blockade efficacy. The results were validated using The Cancer Genome Atlas (TCGA) datasets.

Results

We identified frameshift mutations in RNF43 as a common neoantigenic gene mutation in MSI-H tumours. However, loss-of-function RNF43 mutations induced noninflamed TME by activating the WNT/β-catenin signalling pathway. In addition, loss of RNF43 function induced resistance to PD-1 blockade even in neoantigen-rich tumours. TCGA dataset analyses demonstrated that passenger rather than driver gene mutations were related to the inflamed TME in diverse cancer types.

Conclusions

We propose a novel concept of “paradoxical neoantigenic mutations” that can induce noninflamed TME through their original gene functions, despite deriving neoantigens, suggesting the significance of qualities as well as quantities in neoantigenic mutations.

DOI： 10.1038/s41416-023-02165-6

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その他リンク： https://www.nature.com/articles/s41416-023-02165-6

掲載種別：研究論文（学術雑誌）   出版者・発行元：American Association for Cancer Research (AACR)  

Abstract

Some patients experience mixed response to immunotherapy, whose biological mechanisms and clinical impact have been obscure. We obtained two tumor samples from lymph node (LN) metastatic lesions in a same patient. Whole exome sequencing for the both tumors and single-cell sequencing for the both tumor-infiltrating lymphocytes (TILs) demonstrated a significant difference in tumor clonality and TILs’ characteristics, especially exhausted T cell clonotypes, although a close relationship between the tumor cell and T cell clones were observed as a response of an overlapped exhausted T cell clone to an overlapped neoantigen. To mimic the clinical setting, we generated a mouse model of several clones from a same tumor cell line. Similarly, differential tumor clones harbored distinct TILs, and one responded to PD-1 blockade but the other did not in this model. We further conducted cohort study (n = 503) treated with PD-1 blockade monotherapies to investigate the outcome of mixed response. Patients with mixed responses to PD-1 blockade had a poor prognosis in our cohort. Particularly, there were significant differences in both tumor and T cell clones between the primary and LN lesions in a patient who experienced tumor response to anti-PD-1 mAb followed by disease progression in only LN metastasis. Our results underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome.

DOI： 10.1158/2767-9764.crc-22-0050

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1096/fj.202002678r

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1096/fj.202002678R

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously found that N-89 and its derivative, N-251, which are being developed as antimalarial compounds, showed multiple antiviral activities including hepatitis C virus (HCV). In this study, we focused on the most characterized anti-HCV activity of N-89(N-251) to clarify their antiviral mechanisms. We first prepared cells exhibiting resistance to N-89(N-251) than the parental cells by serial treatment of HCV-RNA-replicating parental cells with N-89(N-251). Then, we newly generated HCV-RNA-replicating cells with the replacement of HCV-RNAs derived from N-89(N-251)-resistant cells and parental cells. Using these cells, we examined the degree of inhibition of HCV-RNA replication by N-89(N-251) and found that the host and viral factors contributed almost equally to the resistance to N-89(N-251). To further examine the contribution of the host factors, we selected several candidate genes by cDNA microarray analysis and found that the upregulated expression of at least RAC2 and CKMT1B genes independently and differently contributed to the acquisition of an N-89(N-251)-resistant phenotype. For the viral factors, we selected several mutation candidates by the genetic comparative analysis of HCV-RNAs and showed that at least one M414I mutation in the HCV NS5B contributed to the resistance to N-89. Moreover, we demonstrated that the combination of host factors (RAC2 and/or CKMT1B) and a viral factor (M414I mutation) additively increased the resistance to N-89. In summary, we identified the host and viral factors contributing to the acquisition of N-89(N-251)-resistance in HCV-RNA replication. These findings will be useful for clarification of the antiviral mechanism of N-89(N-251).

DOI： 10.1096/fba.2020-00107

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) is a rate-limiting enzyme catalyzing triglyceride synthesis. Recently, we demonstrated that the anti-viral drug ribavirin (RBV) reduces GPAM expression by downregulating CCAAT/enhancer-binding protein α (C/EBPα). However, the precise mechanisms of GPAM suppression have remained unclear. Here, we found that RBV suppressed GPAM expression by downregulating not only C/EBPα, but also sterol regulatory element-binding protein-1c (SREBP-1c). We also found that cis-elements regulated by C/EBPα and SREBP-1c functioned as distal and proximal enhancers, respectively, to express hepatocyte- and adipocytes-specific GPAM variants. These results imply that RBV disrupts formation of the enhancer machineries on the GPAM genome by downregulating both transcription factors. Our findings may contribute to the development of treatments for fatty liver diseases caused by aberrant triglyceride synthesis.

DOI： 10.1016/j.bbrc.2020.08.112

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The most characteristic feature of the hepatitis C virus (HCV) genome in patients with chronic hepatitis C is its remarkable variability and diversity. To better understand this feature, we performed genetic analysis of HCV replicons recovered from two human hepatoma HuH-7-derived cell lines after 1, 3, 5, 7, and 9 years in culture: The cell lines 50-1 and sO harbored HCV 1B-1 and O strain-derived HCV replicons established in 2002 and 2003, respectively. The results revealed that genetic variations in both replicons accumulated in a time-dependent manner at a constant rate despite the maintenance of moderate diversity (less than 1.8% difference) between the clones and that the mutation rate in the 50-1 and sO replicons was 2.5 and 2.9 × 10-3 base substitutions/site/year, respectively. We found that the genetic distance of both replicons increased from 7.9% to 10.5% after 9 years in culture. In addition, we observed that the guanine + cytosine (GC) content of both replicon RNAs increased in a time-dependent manner, as observed in our previous studies. Finally, we demonstrated that the high sensitivity of both replicons to direct-acting antivirals was maintained even after 9 years in culture. Our results suggest that long-term cultured HCV replicon-harboring cells are a useful model for understanding the variability and diversity of the HCV genome and the drug sensitivity of HCV in patients with chronic hepatitis C.

DOI： 10.1007/s00705-019-04461-0

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hepatitis B virus (HBV) infection, which increases the risk of cirrhosis and hepatocellular carcinoma and requires lifelong treatment, has become a major global health problem. However, host factors essential to the HBV life cycle are still unclear, and the development of new drugs is needed. Cells derived from the human hepatoma cell line HepG2 and engineered to overexpress sodium taurocholate cotransporting polypeptide (NTCP: a receptor for HBV), termed HepG2/NTCP cells, are widely used as the cell-based HBV infection and replication systems for HBV research. We recently found that human hepatoma cell line Li23-derived cells overexpressing NTCP (A8 cells subcloned from Li23 cells), whose gene expression profile was distinct from that of HepG2/NTCP cells, were also sensitive to HBV infection. However, the HBV susceptibility of A8 cells was around 1/100 that of HepG2/NTCP cells. Since we considered that plural cell assay systems will be needed for the objective evaluation of anti-HBV reagents, as we previously demonstrated in hepatitis C virus research, we here attempted to develop a new Li23 cell-derived assay system equivalent to that using HepG2/NTCP cells. By repeated subcloning of A8 cells, we successfully established a new cell line (A8.15.78.10) exhibiting high HBV susceptibility equal to that of HepG2/NTCP cells. Characterization of A8.15.78.10 cells revealed that the increase of HBV susceptibility was correlated with increases in the protein and glycosylation levels of NTCP, and with decreased expression of STING, a factor contributing to innate immunity. Finally, we performed a comparative evaluation of HBV entry inhibitors (cyclosporin A and rosiglitazone) by an HBV/secNL reporter assay using A8.15.78.10 cells or HepG2/NTCP cells. The results confirmed that cyclosporin A exhibited anti-HBV activity in both cell lines, as previously reported. However, we found that rosiglitazone did not show the anti-HBV activity in A8.15.78.10 cells, although it worked in HepG2/NTCP cells as previously reported. This suggested that the difference in anti-HBV activity between cyclosporin A and rosiglitazone was due to the different types of cells used for the assay. In conclusion, plural assay systems using different types of cells are required for the objective and impartial evaluation of anti-HBV reagents.

DOI： 10.1016/j.bbrc.2019.05.126

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hepatitis B virus (HBV) is a hepatotropic DNA virus causing hepatic diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. To study HBV, human hepatoma HepG2 cells are currently used as an HBV infectious cell culture model worldwide. HepG2 cells exhibit susceptibility to HBV by exogenously expressing sodium taurocholate cotransporting polypeptide (NTCP). We herein demonstrated that human immortalized hepatocyte NKNT-3 cells exhibited susceptibility to HBV by exogenously expressing NTCP (NKNT-3/NTCP cells). By comparing cyclic GMP-AMP synthetase (cGAS)-stimulator of interferon genes (STING) signaling pathway in several NKNT-3/NTCP cell-derived cell clones, we found that STING was highly expressed in cell clones exhibiting resistance but not susceptibility to HBV. High-level expression of STING was implicated in HBV-triggered induction of type III IFN and a pro-inflammatory cytokine, IL-6. In contrast, RNAi-mediated knockdown of STING inhibited type III IFN induction and restored the levels of HBV total transcript in an HBV-infected cell clone exhibiting resistance to HBV. These results suggest that STING regulates susceptibility to HBV by its expression levels. STING may thus be a novel target for anti-HBV strategies.

DOI： 10.1096/fba.1022

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その他リンク： http://orcid.org/0000-0002-2627-8524

掲載種別：研究論文（学術雑誌）  

DOI： 10.1042/BCJ20180680

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrc.2018.08.195

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The chemically synthesized endoperoxide compound N-89 and its derivative N-251 were shown to have potent antimalarial activity. We previously demonstrated that N-89 and N-251 potently inhibited the RNA replication of hepatitis C virus (HCV), which belongs to the Flaviviridae family. Since antimalarial and anti-HCV mechanisms have not been clarified, we were interested whether N-89 and N-251 possessed the activity against viruses other than HCV. In this study, we examined the effects of N-89 and N-251 on other flaviviruses (dengue virus and Japanese encephalitis virus) and hepatitis viruses (hepatitis B virus and hepatitis E virus). Our findings revealed that N-89 and N-251 moderately inhibited the RNA replication of Japanese encephalitis virus and hepatitis E virus, although we could not detect those anti-dengue virus activities. We also observed that N-89 and N-251 moderately inhibited the replication of hepatitis B virus at the step after viral translation. These results suggest the possibility that N-89 and N-251 act on some common host factor(s) that are necessary for viral replications, rather than the possibility that N-89 and N-251 directly act on the viral proteins except for HCV. We describe a new type of antiviral reagents, N-89 and N-251, which are applicable to multiple different viruses.

DOI： 10.1016/j.bbrep.2018.05.007

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley Blackwell  

Natural killer (NK) cells through their NK group 2 member D (NKG2D) receptors recognize NKG2D ligands such as UL16-binding proteins (ULBPs) on virus-infected cells and subsequently trigger the host innate immune response. In the present study, we demonstrated that hepatitis C virus (HCV) induced the cell surface expression of ULBP1 in human immortalized hepatocyte PH5CH8 cells and human hepatoma HuH-7 cell-derived RSc cells. Interestingly, NK cell line NK-92 induced cytotoxicity and interferon-γ mRNA expression and subsequently reduced the levels of HCV RNA replication during co-culture with HCV-infected RSc cells. From these results, we conclude that ULBP1 is a target of the NK cell-mediated innate immune response in HCV-infected human hepatocytes.

DOI： 10.1002/2211-5463.12373

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/hep4.1065

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a major global health problem. Recently developed treatments with direct-acting antivirals (DAAs) have largely improved the sustained virologic response rate of patients with chronic hepatitis C. However, this approach is still hindered by its great expense and the problem of drug resistance. Using our cell-based HCV assay systems, we reported that the preclinical antimalarial drugs N-89 and N-251 exhibited potent anti-HCV activities. In this study we used our assay systems to evaluate the anti-HCV activities of six kinds of DAAs individually or in combination with N-89 or N-251. The results showed that the DAAs had potent anti-HCV activities and N-89 or N-251 contributed additive or synergistic effect. Using DAA-resistant HCV-RNA-replicating cells, which were prepared by continuous treatment with each DAA, we demonstrated that N-89 and N-251 could overcome all of the DAA-resistant HCVs. These preclinical drugs would have been potential as components of a therapeutic regimen that also included combinations of various DAAs. In addition, sequence analysis of the NS3-NS5B regions of the DAA-resistant HCV genomes newly found several amino acid (aa) substitutions that were suggested to contribute to DAA-resistance in addition to the aa substitutions already known to cause DAA-resistance. Among these new aa substitutions, we found that two substitutions in the NS3 region (D79G and S174Y) contributed to simeprevirand/or asunaprevir-resistance.

DOI： 10.1016/j.virusres.2017.03.015

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

During viral replication, the innate immune response is induced through the recognition of viral replication intermediates by host factor(s). One of these host factors, cyclic GMP-AMP synthetase (cGAS), was recently reported to be involved in the recognition of viral DNA derived from DNA viruses. However, it is uncertain whether cGAS is involved in the recognition of hepatitis B virus (HBV), which is a hepatotropic DNA virus. In the present study, we demonstrated that HBV genome-derived double-stranded DNA induced the innate immune response through cGAS and its adaptor protein, stimulator of interferon genes (STING), in human hepatoma Li23 cells expressing high levels of cGAS. In addition, we demonstrated that HBV infection induced ISG56 through the cGAS-STING signaling pathway. This signaling pathway also showed an antiviral response towards HBV through the suppression of viral assembly. From these results, we conclude that the cGAS-STING signaling pathway is required for not only the innate immune response against HBV but also the suppression of HBV assembly. The cGAS-STING signaling pathway may thus be a novel target for anti-HBV strategies.

DOI： 10.1111/febs.13563

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掲載種別：研究論文（学術雑誌）  

DOI： 10.2174/1573408011666151013204142

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その他リンク： http://orcid.org/0000-0002-2627-8524

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Background
Ribavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells.
Methodology/Principal Findings
By repetitive RBV (100 mu M) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 mu M). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor.
Conclusions/Significance
These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.

DOI： 10.1371/journal.pone.0118313

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Upon reacting 3',4'-unsaturated cytosine (8 and 9) and adenine nucleosides (13 and 14) with XeF2/BF3 center dot OEt2, the respective novel 3',4'-difluoro-3'-deoxyribofuranosyl nucleosides (10-12 and 15-18) could be obtained. Formation of anti-adducts (11, 16 and 18) revealed that the fluorination involved oxonium ions as incipient intermediates. TBDMS-protected 3',4'-unsaturated adenosine provided the beta-face adducts as sole stereoisomers whereas a-face-selectivity was observed with the TBDPS-protected adenosine 14. The evaluation of the novel 3'-deoxy-3',4'-difluororibofuranosylcytosine-(19-21) and adenine nucleosides (22-25) against antitumor and antiviral activities revealed that 3',4'-difluorocordycepin (24) was found to possess anti-HCV activity. The SI of 24 was comparable to that of the anti-HCV drug ribavirin. However, sofosbuvir, FDA-approved novel anti-HCV drug, showed better SI value. Our finding revealed that the introduction of the fluoro-substituent into the 4'-position of cordycepin derivatives decreased the cytotoxicity to the host cell with retention of the antiviral activity. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2014.08.024

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Using our recently developed assay system for full-genome-length hepatitis C virus (HCV) RNA replication in human hepatoma-derived Li23 cells (ORL8), we identified 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analog la as a novel HCV inhibitor. Structural modifications of la provided a series of sulfonamides 7 with much more potent HCV RNA replication-inhibitory activity than ribavirin. Compound 7a showed an additive anti-HCV effect in combination with standard anti-HCV therapy (IFN-alpha plus ribavirin). Since 7a generated reactive oxygen species (ROS) in the ORL8 system and its anti-HCV activity was blocked by vitamin E, its anti-HCV activity may be mediated at least in part by ROS. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2014.07.019

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although the sustained virologic response rate in the treatment of genotype 1 using new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has been improved by more than 70%, several severe side effects such as skin rash/ageusia and advanced anemia have become a problem. Under these circumstances, a new type of anti-HCV oral drug with few side effects is needed. Our recently developed HCV drug assay systems, including the HuH-7 cell line-derived OR6 and AH1R, and the Li23 cell line derived ORL8 and ORL11, allow genome-length HCV RNAs (several strains of genotype 1b) encoding renilla luciferase to replicate efficiently. Using these systems as anti-HCV candidates, we have identified numerous existing medicines that can be used against HCV with few side effects, such as statins and teprenon. To obtain additional anti-HCV candidates, we evaluated a number of oral health supplements, and found that the capsule but not the liquid form of Cordyceps militaris (CM) (Ascomycotinanorth, North Chinese caterpillar fungus), which is used as a Chinese herbal medicine, exhibited moderate anti-HCV activity. In combination with interferon-a or ribavirin, CM exhibited an additive inhibitory effect. Among the main components of CM, cordycepin, but not ergosterol, contributed to the anti-HCV activity of CM. In consideration of all these results, we suggest that CM would be useful as an oral anti-HCV agent in combination with interferon-alpha and/or ribavirin. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2014.03.150

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Background: The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells.
Methodology/Principal Findings: Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5'-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a timedependent manner, and the mutation rates in the 5'-terminus-NS2 and NS3-NS5B regions were 4.0-9.0x10(-3) and 2.7-4.0x10(-3) base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3-105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents.
Conclusions/Significance: Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.

DOI： 10.1371/journal.pone.0091156

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

It has been reported that ligand-mediated transcription factor peroxisome proliferator-activated receptor alpha (hPPARα) is involved in hepatitis C virus (HCV) RNA replication, whereas hPPARγ is not, and the effect of hPPARδ is unknown. Here, we show that hPPARδ-selective antagonists effectively inhibit HCV RNA replication. We describe the design, synthesis and pharmacological evaluation of a series of biphenyl-4-carboxylic acid-type hPPARδ antagonists, including previously reported compounds, as candidate anti-HCV agents. A representative compound (4c) dose-dependently inhibited HCV RNA replication (EC50 0.22 μM), while exhibiting relatively weak cytotoxicity to the host cells (CC50 2.5 μM). It also showed an additive and dose-dependent effect on the inhibition of HCV RNA replication by pegylated interferon alpha (Peg-IFNα) alone and by both Peg-IFNα and ribavirin (currently the clinical treatment of choice for HCV infection). Thus, combination of a hPPARδ antagonist with current therapy may improve the efficacy of treatment for HCV infection. © 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2013.07.005

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Background: Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.
Methodology/Principal Findings: Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-alpha demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-alpha-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-alpha and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.
Conclusions/Significance: We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.

DOI： 10.1371/journal.pone.0072519

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

We developed a new cell culture drug assay system (AH1R), in which genome-length hepatitis C virus (HCV) RNA (AH1 strain of genotype 1b derived from a patient with acute hepatitis C) efficiently replicates. By comparing the AH1R system with the OR6 assay system that we developed previously (O strain of genotype 1b derived from an HCV-positive blood donor), we demonstrated that the anti-HCV profiles of reagents including interferon-gamma and cyclosporine A significantly differed between these assay systems. Furthermore, we found unexpectedly that rolipram, an anti-inflammatory drug, showed anti-HCV activity in the AH1R assay but not in the OR6 assay, suggesting that the anti-HCV activity of rolipram differs depending on the HCV strain. Taken together, these results suggest that the AH1R assay system is useful for the objective evaluation of anti-HCV reagents and for the discovery of different classes of anti-HCV reagents.

DOI： 10.1007/s11262-012-0712-2

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. HuH-7 hepatoma-derived cells are widely used as the only cell-based HCV replication system for HCV research, including drug assays. Recently, using different hepatoma Li23-derived cells, we developed an HCV drug assay system (ORL8), in which the genome-length HCV RNA (O strain of genotype 1b) encoding renilla luciferase replicates efficiently. In this study, using the HuH-7-derived OR6 assay system that we developed previously and the ORL8 assay system, we evaluated 26 anti-HCV reagents, which other groups had reported as anti-HCV candidates using HuH-7-derived assay systems other than ORB. The results revealed that more than half of the reagents showed different anti-HCV activities from those in the previous studies, and that anti-HCV activities evaluated by the ORB and ORL8 assays were also frequently different. In further evaluation using the HuH-7-derived AH1R assay system, which was developed using the AH1 strain of genotype 1b, several reagents showed different anti-HCV activities in comparison with those evaluated by the OR6 and ORL8 assays. These results suggest that the different activities of anti-HCV reagents are caused by the differences in cell lines or HCV strains used for the development of assay systems. Therefore, we conclude that plural HCV assay systems developed using different cell lines or HCV strains are required for the objective evaluation of anti-HCV reagents. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.05.061

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記述言語：日本語   出版者・発行元：(一社)日本肝臓学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

The freezing mechanism of water contacted with mesoporous silicas with uniform pore shapes, both cylindrical and cagelike, was studied by thermodynamic and structural analyses with differential scanning calorimetry (DSC) and X-ray diffraction (XRD) together with adsorption measurements. In the DSC data extra exothermic peaks were found at around 230 K for water confined in SBA-15, in addition to that due to the freezing of pore water. These peaks are most likely to be ascribed to the freezing of water present over the micropore and/or mesopore outlets of coronas in SBA-15. Freezing of water confined in SBA-16 was systematically analysed by DSC with changing the pore size. The freezing temperature was found to be around 232 K, close to the homogeneous nucleation temperature of bulk water, independent of the pore size when the pore diameter (d) < 7.0 nm. Water confined in the cagelike pores of SBA-16 is probably surrounded by a water layer (boundary water) at the outlets of channels to interconnect the pores and of fine corona-like pores, which is similar to that present at the outlet of cylindrical pores in MCM-41 and of cylindrical channels in SBA-15. The presence of the boundary water would be a key for water in SBA-16 to freeze at the homogeneous nucleation temperature. This phenomenon is similar to those well known for water droplets in oil and water droplets of clouds in the sky. The XRD data showed that the cubic ice I(c) was formed in SBA-16 as previously found in SBA-15 when d < 8.0 nm.

DOI： 10.1039/c1cp21458f

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開催年月日： 2011年4月

記述言語：日本語  

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