白澤 浩

Purpose: Oncolytic viral therapy for neuroblastoma (NB) cells with Sindbis virus (SINV) is a promising strategy for treating high-risk NB. Here, we evaluated the possibility of using SINV structural proteins as therapeutic agents for NB since UV-inactivated SINV could induce cytopathogenic effects. Methods: The cytotoxicity of UV-inactivated SINV toward human NB cell lines NB69, NGP, GOTO, NLF, SK-N-SH, SH-SY5Y, CHP134, NB-1, IMR32, and RT-BM-1 were analyzed. Apoptosis was confirmed by TUNEL assays. To determine the components of SINV responsible for the cytotoxicity of UV-inactivated SINV, expression vectors encoding the structural proteins, namely capsid, E2, and E1, were transfected in NB cells. Cytotoxicity was evaluated by MTT assays. Results: UV-inactivated SINV elicited more significant cytotoxicity in NB69, NGP, and RT-BM-1 than in normal human fibroblasts. Results of the transfection experiments showed that all NB cell lines susceptible to UV-inactivated SINV were highly susceptible to the E1 protein, whereas fibroblasts transfected with vectors harboring capsid, E1, or E2 were not. Conclusions: We demonstrated that the cytotoxicity of the UV-inactivated SINV is due to apoptosis induced by the E1 structural protein of SINV, which can be used selectively as a therapeutic agent for NB.

PURPOSE: Oncolytic viral therapy for neuroblastoma (NB) cells with Sindbis virus (SINV) is a promising strategy for treating high-risk NB. Here, we evaluated the possibility of using SINV structural proteins as therapeutic agents for NB since UV-inactivated SINV could induce cytopathogenic effects. METHODS: The cytotoxicity of UV-inactivated SINV toward human NB cell lines NB69, NGP, GOTO, NLF, SK-N-SH, SH-SY5Y, CHP134, NB-1, IMR32, and RT-BM-1 were analyzed. Apoptosis was confirmed by TUNEL assays. To determine the components of SINV responsible for the cytotoxicity of UV-inactivated SINV, expression vectors encoding the structural proteins, namely capsid, E2, and E1, were transfected in NB cells. Cytotoxicity was evaluated by MTT assays. RESULTS: UV-inactivated SINV elicited more significant cytotoxicity in NB69, NGP, and RT-BM-1 than in normal human fibroblasts. Results of the transfection experiments showed that all NB cell lines susceptible to UV-inactivated SINV were highly susceptible to the E1 protein, whereas fibroblasts transfected with vectors harboring capsid, E1, or E2 were not. CONCLUSIONS: We demonstrated that the cytotoxicity of the UV-inactivated SINV is due to apoptosis induced by the E1 structural protein of SINV, which can be used selectively as a therapeutic agent for NB.

In Japan, routine immunization for polio using the oral polio vaccine (OPV) was suspended in September 2012 subsequently, an immunization program with inactivated polio vaccines (IPVs), the conventional IPV (cIPV) derived from virulent strains, and IPV derived from Sabin strains (sIPV), was introduced. However, the immunity induced by sIPV is not well characterized. This study assessed and compared neutralizing antibodies produced against poliovirus in cases who received doses of OPV or IPV. Serum samples (n = 1186) were collected yearly between 2013 and 2016 as part of the National Epidemiological Surveillance of Vaccine–Preventable Disease. The neutralizing antibody titers for Sabin strain types 1, 2, and 3 in 224 children, aged between 0 and 90 months, were assessed. Seropositive rates after vaccination with OPV or IPV were more than 90%. Neutralizing antibody titers for Sabin type 1 after vaccination with IPV were lower than those with OPV, while those for Sabin types 2 and 3 after vaccination with IPV were significantly higher than those with OPV. Analyses of antibody titer dynamics revealed that the decay of antibody titers for Sabin types 1, 2, and 3 in cases vaccinated with IPV was steeper than those with OPV. Thus, our study showed that although IPV induced a sufficient level of neutralizing antibody, the immunity induced by IPV was not maintained as long as that by OPV. Our study suggested that a long-term survey should be conducted for polio vaccination using IPV and that it might be necessary to consider booster vaccination for IPVs.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis including acute liver failure. Hepatitis B infection (HBV) occurs worldwide, with the highest rates in Asian and African countries, and there are several reports that HAV infection may have a more severe clinical course in patients with chronic HBV infection. We previously demonstrated that Japanese miso extracts have inhibitory effects on HAV replication. In the present study, we examined the replication of HAV and HBV in a hepatocyte superinfection model and the inhibitory effects of Japanese miso extracts on both viruses. According to the results, HAV infection inhibited HBV replication in superinfected hepatocytes, and Japanese rice-koji miso extracts had inhibitory effects on HAV replication. Our findings provide useful information for clinicians in managing HAV infection in patients with chronic HBV infection.

Hepatitis A virus (HAV) infection is one of the major causes of acute hepatitis and acute liver failure in developing and developed countries. Although effective vaccines for HAV infection are available, outbreaks of HAV infection still cause deaths, even in developed countries. One approach to control HAV infection is prevention through diet, which can inhibit HAV propagation and replication. Glucose-regulated protein 78 (GRP78) is a member of the heat shock protein 70 family of molecular chaperone required for endoplasmic reticulum stress and stress-induced autophagy. We previously showed that the elevation of GRP78 expression inhibits HAV replication. It has been reported that Japanese miso extracts, which was made from rice-koji, enhance GRP78 expression. In the present study, we used human hepatoma Huh7 cells and human hepatocyte PXB cells to examine the efficacy of Japanese miso extracts as antiviral agents against HAV. Japanese miso extracts enhanced GRP78 expression and inhibited HAV replication in human hepatocytes. Together, these results demonstrate that Japanese miso extracts may partly modulate GRP78 expression and additively or synergistically work as antivirals against HAV infection. Japanese miso extracts can be used as effective dietary supplements for severe hepatitis A.

Myanmar is adjacent to India, Bangladesh, Thailand, Laos and China. In Myanmar, the prevalence of hepatitis B virus (HBV) infections is 6.5% and accounts for 60% of hepatocellular carcinoma. HBV has nine genotypes that have been identified by molecular genetic analysis. HBV genotypes are associated with several clinical features. We reviewed the prevalence of HBV genotypes in Myanmar and neighboring countries. We also reviewed HBV genotypes in refugees from Myanmar. HBV subgenotype C1 is predominant in Myanmar. As HBV genotype C is associated with hepatocellular carcinoma (HCC), it is important to screen for cirrhosis and HCC and to prevent their development in HBV-infected individuals of Myanmar.

Infection with hepatitis A virus (HAV) is a major cause of acute hepatitis globally and it is important to identify the mechanisms of HAV replication. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) chaperone and serves a role in unfolded protein response pathways. Previous studies have demonstrated that GRP78 functions as an endogenous antiviral factor. In the present study, two loss-of-function studies using GRP78 were completed to elucidate the role of GRP78 in HAV infection. HAV replication was observed to be enhanced by deficient GRP78 although GRP78-deficiency also led to lower expression of ER stress molecules downstream of GRP78. Therefore, GRP78 appears to be a potential novel defensive molecule against HAV in hepatocytes.

The contributions of splash from vomiting and the dispersion of dried-up virus from a contaminated floor surface to community gastroenteritis outbreaks caused by Norovirus (NoV) were evaluated, using Feline calicivirus (FCV) as an NoV surrogate. There was no difference in the size distribution of FCV-containing particles around 0.75 µm) collected from a virus-sprayed chamber 1 and 12 hr after nebulization. FCV clearly dispersed after hitting a floor surface contaminated with dried virus. These results suggest that NoV can likely form airborne droplet nuclei, and dust may be the main route of infection transmission. J. Med. Virol. 89:931–935, 2017. © 2016 Wiley Periodicals, Inc.

Although the interaction between host and hepatitis A virus (HAV) factors could lead to severe hepatitis A, the exact mechanism of acute liver failure caused by HAV infection is not yet fully understood. The effects of metabolic diseases such as dyslipidemia or diabetes mellitus on HAV replication are still unknown. Here, we examined the effects of free fatty acids or high-concentration glucose on HAV replication and the effects on mitogen-activated protein kinase signaling pathway-related genes in human hepatocytes. We discovered a novel effect of free fatty acids or high-concentration glucose on HAV replication in association with a reduction in the expression of glucose-regulated protein 78 (GRP78). We also observed that thapsigargin induced GRP78 expression and inhibited HAV replication. These findings may provide a new interpretation of the relationship between metabolic diseases and severity of hepatitis A and suggest a new understanding of the mechanism of severe HAV infection.

Background: Genetic variation near the interferon lambda 3 (IFNL3) is known to be associated with response to pegylated interferon (pegIFN) and ribavirin combination therapy in patients with chronic hepatitis C virus (HCV) infection which is often accompanied by hepatic steatosis. Aims: We examined whether this genetic variation is associated with host lipids and treatment response. Methods: A total of 101 Japanese patients who had underwent liver biopsy before treatment with pegIFN and ribavirin for HCV genotype 1b infection were retrospectively analyzed for association between IFNL3 genotypes (rs8099917) and clinical factors including histopathological features of the liver. The presence of >5% steatosis in the liver specimen was defined as hepatic steatosis. Results: Forty patients (40%) had liver steatosis before therapy. Patients with IFNL3 minor genotype (non-TT) showed lower low-density lipoprotein cholesterol level (p=0.0045), higher.-glutamyl transpeptidase level (p=0.0003) and higher prevalence of hepatic steatosis (p=0.0002). Advanced fibrosis [odds ratio (OR) 4.63, p=0.03] and IFNL3 major genotype (OR 0.13, p= 0.001) were 2 independent factors for determining the presence of hepatic steatosis. Among the factors associated with sustained virological response, IFNL3 genotype was the most significant predictor, as per multivariate analysis. Conclusions: Our results confirmed that IFNL3 genotype is associated with hepatic steatosis as well as IFN response.

Determination of hepatitis C virus (HCV) genotypes plays an important role in the direct-acting agent era. Discrepancies between HCV genotyping and serotyping assays are occasionally observed. Eighteen samples with discrepant results between genotyping and serotyping methods were analyzed. HCV serotyping and genotyping were based on the HCV nonstructural 4 (NS4) region and 5'-untranslated region (5'-UTR), respectively. HCV core and NS4 regions were chosen to be sequenced and were compared with the genotyping and serotyping results. Deep sequencing was also performed for the corresponding HCV NS4 regions. Seventeen out of 18 discrepant samples could be sequenced by the Sanger method. Both HCV core and NS4 sequences were concordant with that of genotyping in the 5'-UTR in all 17 samples. In cloning analysis of the HCV NS4 region, there were several amino acid variations, but each sequence was much closer to the peptide with the same genotype. Deep sequencing revealed that minor clones with different subgenotypes existed in two of the 17 samples. Genotyping by genome amplification showed high consistency, while several false reactions were detected by serotyping. The deep sequencing method also provides accurate genotyping results and may be useful for analyzing discrepant cases. HCV genotyping should be correctly determined before antiviral treatment.

Background The induction of apoptosis in hepatic stellate cells (HSCs) is a promising therapeutic strategy against hepatitis B virus (HBV)-related hepatic fibrosis. The underlying mechanisms of apoptosis in HSCs, however, are unknown under consideration of HBV infection. In this study, the effects of HBV on apoptosis and endoplasmic reticulum (ER) stress signaling in HSCs were examined. Methods The effects of conditioned media (CM) from HepG2.2.15 on apoptosis induced by the proteasome inhibitor MG132 in LX-2 and HHSteC were studied in regard to c-Jun. In combination with c-Fos, c-Jun forms the AP-1 early response transcription factor, leading to AP-1 activation, signal transduction, endoplasmic reticulum (ER) stress and apoptosis. Results In LX-2 cells, MG132 treatment was associated with the phosphorylation of c-Jun, activation of AP-1 and apoptosis. However, in the presence of CM from HepG2.2.15, these phenomena were attenuated. In HHSteC cells, similar results were observed. HBV genomic DNA is not involved in the process of HSC apoptosis. It is possible that HBeAg has an inhibitory effect on MG132-induced apoptosis in LX-2. We also observed the upregulation of several ER stress-associated genes, such as cAMP responsive element binding protein 3-like 3, inhibin-beta A and solute carrier family 17-member 2, in the presence of CM from HepG2.2.15, or CM from PXB cells infected with HBV. Conclusions HBV inhibits the activation of c-Jun/AP-1 in HSCs, contributing to the attenuation of apoptosis and resulting in hepatic fibrosis. HBV also up-regulated several ER stress genes associated with cell growth and fibrosis. These mechanistic insights might shed new light on a treatment strategy for HBV-associated hepatic fibrosis.

<p></p><p> 国際化は医学教育の世界にも影響を及ぼし, 千葉大学においても医学留学制度の拡充を図ってきた. 本稿では, 一医学生がどのような形で留学準備, 英語力の向上を実現させたかを実体験を踏まえて述べ, また6年間の留学の軌跡を提示する. 更に医学留学の意義として, 世界の医学界の第一人者に出会うこと, 同世代の世界の医学生との交友を持つ中で, 日本人としての医学生の能力を世界標準と照らし合わせること, 長い医療者人生の中で学生の段階においてのみ, フレキシブルに世界を目にすることの3点を挙げ, 今後のキャリアへの活かし方, 及び, 医学生に対する医学留学への提言を行う.</p>

The search for human cancer viruses triggered by the discovery of animal tumor viruses in the early twentieth century was unsuccessful until the end of the 1970s, although the concept of oncogenes and tumor-suppressor genes developed by the research on tumor viruses yielded substantial advances in understanding the etiology of cancer. Currently, the advent of molecular biology has led to the identification of seven human viruses as cancer viruses. The histories of tumor virology and the discoveries of human cancer viruses are discussed.

Some tumor-specific near-infrared (NIR) fluorescent dyes such as indocyanine green (ICG), IDRye800CW, and 5-aminolevulinic acid have been used clinically for detecting tumor margins or micro-cancer lesions. In this study, we evaluated the physicochemical properties of liposomally formulated phospholipid-conjugated ICG, denoted by LP-iDOPE, as a clinically translatable NIR imaging nanoparticle for brain tumors. We also confirmed its brain-tumor-specific biodistribution and its characteristics as the intra-operative NIR imaging nanoparticles for brain tumor surgery. These properties of LP-iDOPE may enable neurosurgeons to achieve more accurate identification and more complete resection of brain tumor.

With current treatment regimens, high-risk neuroblastoma (NB) remains largely incurable. Oncolytic viral therapy uses replication-competent viruses, like Sindbis virus (SINV), to kill cancers. The SINV AR339 strain is blood borne and relatively non-virulent. We evaluated the feasibility of SINV AR339 for treating human NB. The cytotoxicity and viral growth of SINV AR339 were evaluated for five human NB cell lines, SK-N-SH, IMR-32, LAN-5, GOTO, and RT-BM-1. SINV-induced apoptosis was confirmed by TUNEL assays and PARP-1 cleavage. In vivo effects of SINV on neuroblastoma cell xenografts in nude mice were assessed by intratumoral or intravenous SINV inoculation. In five human NB cell lines, SINV infections induced remarkable cytotoxicity. The mRNA expressions of anti-apoptotic genes, Bcl-2 and Bcl-xL, in LAN-5 and RT-BM-1, which were less sensitive to SINV infection, increased in response to SINV infection, while the other NB cell lines sensitive to SINV infection failed to respond. In nude mice, intratumoral and intravenous SINV inoculations caused significant regression of NB xenograft tumors. Our results suggested that SINV AR339 was significantly oncolytic against human NB. Thus, SINV showed promise as a novel therapy for treating NB.

Epigenetics plays a role in the regulation of gene expression. Epigenetic changes control gene expression at the transcriptional level. Our previous study suggested that the La protein, which is mainly localized in the nucleus, was associated with hepatitis A virus (HAV) internal ribosomal entry site (IRES)-mediated translation and HAV replication. The aim of this study was to investigate whether epigenetic compounds have effects on HAV IRES-mediated translation and HAV replication. Sirtinol, a sirtuin inhibitor, inhibited HAV IRES-mediated translation in COS7-HAV-IRES cells. Treatment with 10 mu M sirtinol resulted in a significant reduction in the intracellular RNA levels of HAV HA11-1299 genotype IIIA in Huh7 cells. Epigenetic treatment with a sirtuin inhibitor may represent a new treatment option for HAV infection. In conclusion, epigenetic control was involved in HAV IRES-dependent translation and HAV replication. Special attention should also be paid to underlying viral diseases in the clinical use of epigenetic treatments for malignancies. (C) 2015 Elsevier Inc. All rights reserved.

Due to the lack of efficient hepatitis B virus (HBV) infection systems, progress in understanding the role of innate immunity in HBV infection has remained challenging. Here we used human hepatocytes from a humanized severe combined immunodeficiency albumin promoter/enhancer driven-urokinase-type plasminogen activator mouse model for HBV infection. HBV DNA levels in culture medium from these human hepatocytes were 4.8-5.7 log IU/mL between day 16 and day 66 post-infection by HBV genotype C inoculum. HBV surface antigen (HBsAg) was also detected by chemiluminescent immunoassay from day 7 to day 66 post-infection. Western blot analysis revealed that major histocompatibility complex class I-related chain A (MICA), which plays a role in the innate immune system, was induced in HBV-infected human hepatocytes 27 days after infection compared with the uninfected control. MICA was reduced at day 62 and undetectable at day 90. Of interest, MICA expression by human hepatocytes increased after HBV infection and decreased before HBsAg loss. Human hepatocytes derived from chimeric mice with hepatocyte-humanized liver could support HBV genome replication. Further studies of the association between HBV replication and MICA induction should be conducted. (C) 2015 Elsevier Inc. All rights reserved.

We examined whether hepatitis C virus (HCV) genotype 1b core- and NS5A-region mutations are associated with response to peginterferon -2b plus ribavirin combination therapy. A total of 103 patients with high HCV genotype 1b viral loads (100 KIU/mL) were treated with the combination therapy. Pretreatment mutations in the core region and interferon sensitivity determining region (ISDR) in the NS5A region were analyzed. In univariate analysis, arginine and leucine at positions 70 and 91 in the core region, defined as double wild (DW)-type, were associated with early virologic response (p = 0.002), sustained virologic response (SVR) (p = 0.004), and non-response (p = 0.005). Non-threonine at position 110 was associated with SVR (p = 0.004). Multivariate analysis showed the following pretreatment predictors of SVR: hemoglobin level 14 g/dL (odds ratio (OR) 6.2, p = 0.04); platelet count 14 x 10(4)/mm(3) (OR 5.2, p = 0.04); aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio &lt; 0.9 (OR 6.17, p = 0.009); DW-type (OR 6.8, p = 0.02); non-threonine at position 110 (OR 14.5, p = 0.03); and 2 mutations in the ISDR (OR 12.3, p = 0.02). Patients with non-DW-type, non-threonine at position 110, and &lt;2 ISDR mutations showed significantly lower SVR rates than others (11/45 (24.4%) vs. 27/37 (73.0%), respectively; p &lt; 0.001). SVR can be predicted through core and NS5A region mutations and host factors like hemoglobin, platelet count, and AST/ALT ratio in HCV genotype 1b-infected patients treated with peginterferon and ribavirin combination therapy.

Background Hepatitis C virus (HCV) infection is one of the major causes of cirrhosis and hepatocellular carcinoma. HCV nonstructural protein 5A (NS5A) is an attractive antiviral target and plays an important role in HCV replication as well as hepatocarcinogenesis. The aim of this study was to assess the effect of HCV NS5A protein in the abrogation of apoptotic cell death induced by the proteasome inhibitor MG132. Methods Apoptotic responses to MG132 and the expression of molecules involved in NF-kappa B signaling pathways in human hepatocytes were investigated with or without the expression of HCV NS5A. Results HCV NS5A protected HepG2 cells against MG132-induced apoptosis, in line with NF-kappa B-nuclear translocation. A similar NF-kappa B-nuclear translocation was observed in Huh7 cells infected with HCV JFH1. In agreement with this, after treatment with MG132, HCV NS5A could elevate the transcription of several NF-kappa B target genes such as BCL2 and BCLXL to inhibit MG132-induced apoptosis in hepatocytes. HCV HCV NS5A also enhanced phosphorylation of I kappa B alpha. Consistent with a conferred prosurvival advantage, HCV NS5A reduced MG132-induced poly(adenosine diphosphate-ribose) polymerase cleavage. Conclusions HCV NS5A expression enhances phosphorylation of I kappa B alpha, liberates NF-kappa B for nuclear translocation and downregulates MG132-induced apoptotic pathways in human hepatocytes. It is possible that the disruption of proteasome-associated apoptosis plays a role in the pathogenesis of HCV infection.

We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G(1) phase preferred to proliferate during S/G(2) phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G(1) phase than in cells infected during S/G(2) phase. (C) 2015 The Authors. Published by Elsevier Inc.

The JAK2 inhibitor AZD1480 has been reported to inhibit La protein expression. We previously demonstrated that the inhibition of La expression could inhibit hepatitis A virus (HAV) internal ribosomal entry-site (IRES)-mediated translation and HAV replication in vitro. In this study, we analyzed the effects of AZD1480 on HAV IRES-mediated translation and replication. HAV IRES-mediated translation in COS7-HAV-IRES cells was inhibited by 0.1-1 mu M AZD1480, a dosage that did not affect cell viability. Results showed a significant reduction in intracellular HAV HA11-1299 genotype IIIA RNA levels in Huh7 cells treated with AZD1480. Furthermore, AZD1480 inhibited the expression of phosphorylated-(Tyr-705)-signal transducer and activator of transcription 3 (STAT3) and La in Huh7 cells. Therefore, we propose that AZD1480 can inhibit HAV IRES activity and HAV replication through the inhibition of the La protein. (C) 2015 Elsevier Inc. All rights reserved.

Patients who are infected with hepatitis C virus (HCV) and also have advanced fibrosis or cirrhosis have been recognized as "difficult-to-treat" patients during an era when peginterferon and ribavirin combination therapy is the standard of care. Recent guidelines have clearly stated that treatment should be prioritized in this population to prevent complications such as decompensation and hepatocellular carcinoma. Recent advances in the treatment of chronic hepatitis C have been achieved through the development of direct-acting antiviral agents (DAAs). Boceprevir and telaprevir are first-generation DAAs that inhibit the HCV NS3/4A protease. Boceprevir or telaprevir, in combination with peginterferon and ribavirin, improved the sustained virological response rates compared with peginterferon and ribavirin alone and were tolerated in patients with HCV genotype 1 infection without cirrhosis or compensated cirrhosis. However, the efficacy is lower especially in prior non-responders with or without cirrhosis. Furthermore, a high incidence of adverse events was observed in patients with advanced liver disease, including cirrhosis, in real-life settings. Current guidelines in the United States and in some European countries no longer recommend these regimens for the treatment of HCV. Next-generation DAAs include second-generation HCV NS3/4A protease inhibitors, HCV NS5A inhibitors and HCV NS5B inhibitors, which have a high efficacy and a lower toxicity. These drugs are used in interferon-free or in interferon-based regimens with or without ribavirin in combination with different classes of DAAs. Interferon-based regimens, such as simeprevir in combination with peginterferon and ribavirin, are well tolerated and are highly effective especially in treatment-naïve patients and in patients who received treatment but who relapsed. The efficacy is less pronounced in null-responders and in patients with cirrhosis. Interferon-free regimens in combination with ribavirin and/or two or more DAAs could be used for treatment-naïve, treatment-experienced and even for interferon-ineligible or interferon-intolerant patients. Some clinical trials have demonstrated promising results, and have shown that the efficacy and safety were not different between patients with and without cirrhosis. There are also promising regimens for genotypes other than genotype 1. Interferon is contraindicated in patients with decompensated cirrhosis, and further studies are needed to establish the optimal treatment regimen for this population. In the future, interferon-free and ribavirin-free regimens with high efficacy and improved safety are expected for HCV-infected patients with advanced liver diseases.

Background: We previously reported that the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) down-regulates TLR4 signaling and lipopolysaccharide-induced apoptosis of hepatocytes. There have been several reports regarding the association between HCV infection and endoplasmic reticulum (ER) stress. Here, we examined the regulation of HCV NS5A on the apoptosis of hepatocytes induced by thapsigargin, an inducer of ER stress. Methods: The apoptotic response to thapsigargin and the expression of molecules involved in human hepatocyte apoptotic pathways were examined in the presence or absence of HCV NS5A expression. Results: HCV JFH1 infection induced ER stress in the Huh7 cell line. HCV NS5A protected HepG2 cells against thapsigargin-induced apoptosis, the effect of which was linked to the enhanced expression of the 78-kDa glucose-regulated protein/immunoglobulin heavy-chain binding protein (GRP78). Consistent with a conferred pro-survival advantage, HCV NS5A reduced poly(adenosine diphosphate-ribose) polymerase cleavage and activation of caspases-3, -7 and -9, and Bax expression, while increasing the expressions of the anti-apoptotic molecules XIAP and c-FLIP. HCV NS5A weakly interacts with GRP78 and enhances GRP78 expression in hepatocytes. Conclusion: HCV NS5A enhances GRP78 expression, resulting in the inhibition of apoptotic properties, and inhibits thapsigargin-induced apoptotic pathways in human hepatocytes, suggesting that disruption of ER stress-mediated apoptosis may have a role in the pathogenesis of HCV infection. Thus, HCV NS5A might engender the survival of HCV-infected hepatocytes contributing to the establishment of persistent infection.

Background: Despite the development and availability of hepatitis A virus (HAV) vaccine, HAV infection is still a major cause of acute hepatitis that occasionally leads to fatal liver disease. HAV internal ribosomal entry-site (IRES) is one of the attractive targets of antiviral agents against HAV. The aim of the present study is to evaluate the impact of La, one of the cellular proteins, on HAV IRES-mediated translation and HAV replication. Methods and Findings: We investigated the therapeutic feasibility of siRNAs specific for cellular cofactors for HAV IRES-mediated translation in cell culture. It was revealed that siRNA against La could inhibit HAV IRES activities as well as HAV subgenomic replication. We also found that the Janus kinase (JAK) inhibitors SD-1029 and AG490, which reduce La expression, could inhibit HAV IRES activities as well as HAV replication. Conclusions: Inhibition of La by siRNAs and chemical agents could lead to the efficient inhibition of HAV IRES-mediated translation and HAV replication in cell culture models. La might play important roles in HAV replication and is being exploited as one of the therapeutic targets of host-targeting antivirals.

Hepatitis A virus (HAV) is a causative agent of acute viral hepatitis for which an effective vaccine has been developed. Here we describe ultra-deep pyrosequences (UDPSs) of HAV 5'-untranslated region (5'UTR) among cases of the same outbreak, which arose from a single source, associated with a revolving sushi bar. We determined the reference sequence from HAV-derived clone from an attendant by the Sanger method. Sixteen UDPSs from this outbreak and one from another sporadic case were compared with this reference. Nucleotide errors yielded a UDPS error rate of &lt; 1%. This study confirmed that nucleotide substitutions of this region are transition mutations in outbreak cases, that insertion was observed only in non-severe cases, and that these nucleotide substitutions were different from those of the sporadic case. Analysis of UDPSs detected low-prevalence HAV variations in 5'UTR, but no specific mutations associated with severity in these outbreak cases. To our surprise, HAV strains in this outbreak conserved HAV IRES sequence even if we performed analysis of UDPSs. UDPS analysis of HAV 5'UTR gave us no association between the disease severity of hepatitis A and HAV 5'UTR substitutions. It might be more interesting to perform ultra-deep sequencing of full length HAV genome in order to reveal possible unknown genomic determinants associated with disease severity. Further studies will be needed.

Group C rotavirus (GCRV), astrovirus (AstV), and adenovirus (subgenus F AdenoV) are etiologic agents of acute nonbacterial gastroenteritis, which often represents community outbreaks. For the efficient detection of GCRV, AstV, and subgenus F AdenoV in stool specimens, a multiplex real-time PCR assay was developed to detect these three viruses simultaneously, with high sensitivity and specificity. In total, 8404 clinical specimens were collected between April 2008 and March 2011 and tested for GCRV, AstV, and subgenus F AdenoV by the multiplex real-time PCR, as well as for norovirus (NoV), sapovirus (Say), and group A rotavirus (GARV) by non-multiplex real-time PCR. Forty-one specimens were positive for GCRV, AstV, or subgenus F AdenoV, including 15 specimens that were also positive for NoV, SaV, or GARV. Multiple viruses were detected simultaneously in 29 out of 4596 (0.63%) specimens infected with at least one virus. The association rates of AstV and subgenus F AdenoV with other viruses were significantly higher than those of NoV, SaV, GARV, or GCRV. (C) 2012 Elsevier B.V. All rights reserved.

We previously reported that hepatitis B virus (HBV) e antigen (HBeAg) inhibits production of interleukin 6 by suppressing NF-kappa B activation. NF-kappa B is known to be activated through receptor-interacting serine/threonine protein kinase 2 (RIPK2), and we examined the mechanisms of interleukin 6 regulation by HBeAg. HBeAg inhibits RIPK2 expression and interacts with RIPK2, which may represent 2 mechanisms through which HBeAg blocks nucleotide-binding oligomerization domain-containing protein 1 ligand-induced NF-kappa B activation in HepG2 cells. Our findings identified novel molecular mechanisms whereby HBeAg modulates intracellular signaling pathways by targeting RIPK2, supporting the concept that HBeAg could impair both innate and adaptive immune responses to promote chronic HBV infection.

Recent studies have implicated nuclear receptors (NRs) in the development of hepatocarcinogenesis. We assumed that hepatitis B virus (HBV) alters the expression of NRs and coregulators, and compared the gene expression profiling for 84 NRs and related genes between HpeG2.2.15, which secretes complete HBV virion, and HepG2 by real-time RT-PCR with SyBr green. Forty (47.6%) genes were upregulated 2-fold or greater, and only 5(5.9%) were down-regulated 2-fold or more, in HepG2.2.15 compared to HepG2. These results suggest that HBV affects NRs and their related signal transduction, and that they play important roles in viral replication and HBV-related hepatocarcinogenesis. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

1)学習成果基盤型教育で順次性のあるカリキュラムを作成するためにHardenらが提唱したラセン型カリキュラムの考え方を参考にした。2)カリキュラムの順次性を医師の育成過程として捉え、Millerの学習ピラミッドを応用して医師へと続くコンピテンスの発達モデルを作成した。3)発達モデルに基づくコンピテンスレベルをユニットの学習目標に反映し、授業とコンピテンスの関係を学習ガイドで学生、教育双方が理解できるように可視化している。(著者抄録)

Background. Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to modulate multiple cellular processes, including apoptosis. The aim of this study was to assess the effects of HCV NS5A on apoptosis induced by Toll- like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Methods. Apoptotic responses to TLR4 ligands and the expression of molecules involved in TLR signaling pathways in human hepatocytes were examined with or without expression of HCV NS5A. Results. HCV NS5A protected HepG2 hepatocytes against LPS-induced apoptosis, an effect linked to reduced TLR4 expression. A similar downregulation of TLR4 expression was observed in Huh-7-expressing genotype 1b and 2a. In agreement with these findings, NS5A inhibited the expression of numerous genes encoding for molecules involved in TLR4 signaling, such as CD14, MD-2, myeloid differentiation primary response gene 88, interferon regulatory factor 3, and nuclear factor-kappa B2. Consistent with a conferred prosurvival advantage, NS5A diminished the poly(adenosine diphosphate-ribose) polymerase cleavage and the activation of caspases 3, 7, 8, and 9 and increased the expression of anti-apoptotic molecules Bcl-2 and c-FLIP. Conclusions. HCV NS5A downregulates TLR4 signaling and LPS-induced apoptotic pathways in human hepatocytes, suggesting that disruption of TLR4-mediated apoptosis may play a role in the pathogenesis of HCV infection.

Background: Cervical cancer is caused primarily by human papillomaviruses (HPV). The polymorphism rs1042522 at codon 72 of the TP53 tumour-suppressor gene has been investigated as a genetic cofactor. More than 80 studies were done between 1998 and 2006, after it was initially reported that women who are homozygous for the arginine allele had a risk for cervical cancer seven times higher than women who were heterozygous for the allele. However, results have been inconsistent. Here we analyse pooled data from 49 studies to determine whether there is an association between TP53 codon 72 polymorphism and cervical cancer. Methods: Individual data on 7946 cases and 7888 controls from 49 different studies worldwide were reanalysed. Odds ratios (OR) were estimated using logistic regression, stratifying by study and ethnic origin. Subgroup analyses were done for infection with HPV, ethnic origin, Hardy-Weinberg equilibrium, study quality, and the material used to determine TP53 genotype. Findings: The pooled estimates (OR) for invasive cervical cancer were 1·22 (95% CI 1·08-1·39) for arginine homozygotes compared with heterozygotes, and 1·13 (0·94-1·35) for arginine homozygotes versus proline homozygotes. Subgroup analyses showed significant excess risks only in studies where controls were not in Hardy-Weinberg equilibrium (1·71 [1·21-2·42] for arginine homozygotes compared with heterozygotes), in non-epidemiological studies (1·35 [1·15-1·58] for arginine homozygotes compared with heterozygotes), and in studies where TP53 genotype was determined from tumour tissue (1·39 [1·13-1·73] for arginine homozygotes compared with heterozygotes). Null results were noted in studies with sound epidemiological design and conduct (1·06 [0·87-1·29] for arginine homozygotes compared with heterozygotes), and studies in which TP53 genotype was determined from white blood cells (1·06 [0·87-1·29] for arginine homozygotes compared with heterozygotes). Interpretation: Subgroup analyses indicated that excess risks were most likely not due to clinical or biological factors, but to errors in study methods. No association was found between cervical cancer and TP53 codon 72 polymorphism when the analysis was restricted to methodologically sound studies. Funding: German Research Foundation (DFG). © 2009 Elsevier Ltd. All rights reserved.

BACKGROUND: Sindbis virus (SIN) infection causes no or only mild symptoms (fever, rash, and arthralgia) in humans. However, SIN has a strong cytopathic effect (CPE) on various cancer cells. This study focuses on the oncolytic activity of SIN AR399 on oral cancer cells compared with reovirus, a well-known oncolytic virus that targets cancer cells. METHODS: We analysed the cytotoxicity and growth of SIN in 13 oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, H-1, Sa-3, KON, KOSC-2, OK-92, HO-1-N1, SCC-4, SAT, SKN-3) and normal human oral keratinocytes (NHOKs). RESULTS: Sindbis virus infection induced CPE in 12 OSCC cell lines at a low multiplicity of infection (MOI) of 0.01, but not in the OSCC cell line, HSC-4 or NHOKs. Sindbis viral growth was not observed in NHOKs, whereas high SIN growth was observed in all OSCC cell lines, including HCS-4. The cytotoxicity and growth of SIN was the same as reovirus at an MOI of 20 in 12 OSCC cell lines. The CPE was shown, by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assays, to be apoptotic cell death. Furthermore, quantitative RT-PCR of mRNA in HSC-3 and HSC-4 cells after SIN infection showed that activation of caspases, cytochrome c, and I kappa B alpha was associated with SIN-induced apoptosis. CONCLUSION: As a replication-competent oncolytic virus, SIN may be a useful therapeutic modality for oral cancers. British Journal of Cancer (2009) 101, 684-690. doi: 10.1038/sj.bjc.6605209 www.bjcancer.com Published online 28 July 2009 (C) 2009 Cancer Research UK