RESUMO
During the replication of viral genomes, RNA viruses produce double-stranded RNA (dsRNA), through the activity of their RNA-dependent RNA polymerases (RdRps) as viral replication intermediates. Recognition of viral dsRNA by host pattern recognition receptors - such as retinoic acid-induced gene-I (RIG-I)-like receptors and Toll-like receptor 3 - triggers the production of interferon (IFN)-ß via the activation of IFN regulatory factor (IRF)-3. It has been proposed that, during the replication of viral genomes, each of RIG-I and melanoma differentiation-associated gene 5 (MDA5) form homodimers for the efficient activation of a downstream signalling pathway in host cells. We previously reported that, in the non-neoplastic human hepatocyte line PH5CH8, the RdRp NS5B derived from hepatitis C virus (HCV) could induce IFN-ß expression by its RdRp activity without the actual replication of viral genomes. However, the exact mechanism by which HCV NS5B produced IFN-ß remained unknown. In the present study, we first showed that NS5B derived from another Flaviviridae family member, GB virus B (GBV-B), also possessed the ability to induce IFN-ß in PH5CH8 cells. Similarly, HCV NS5B, but not its G317V mutant, which lacks RdRp activity, induced the dimerization of MDA5 and subsequently the activation of IRF-3. Interestingly, immunofluorescence analysis showed that HCV NS5B produced dsRNA. Like HCV NS5B, GBV-B NS5B also triggered the production of dsRNA and subsequently the dimerization of MDA5. Taken together, our results show that HCV NS5B triggers an MDA5-mediated innate immune response by producing dsRNA without the replication of viral genomes in human hepatocytes.
Assuntos
Hepacivirus , Hepatite C , Humanos , Genoma Viral , Hepacivirus/genética , Hepatite C/genética , Imunidade Inata , RNA de Cadeia Dupla , RNA Polimerase Dependente de RNA/genética , Replicação ViralRESUMO
The emergence of hepatitis C virus (HCV) with resistance-associated substitution (RAS), produced by mutations in the HCV genome, is a major problem in direct acting antivirals (DAA) treatment. This study aimed to clarify the mutational spectrum in HCV-RNA and the substitution pattern for the emergence of RASs in patients with chronic HCV infection. HCV-RNA from two HCV replicon cell lines and the serum HCV-RNA of four non-liver transplant and four post-liver transplant patients with unsuccessful DAA treatment were analyzed using high-accuracy single-molecule real-time long-read sequencing. Transition substitutions, especially A>G and U>C, occurred prominently under DAAs in both non-transplant and post-transplant patients, with a mutational bias identical to that occurring in HCV replicon cell lines during 10-year culturing. These mutational biases were reproduced in natural courses after DAA treatment. RASs emerged via both transition and transversion substitutions. NS3-D168 and NS5A-L31 RASs resulted from transversion mutations, while NS5A-Y93 RASs was caused by transition substitutions. The fidelity of the RNA-dependent RNA polymerase, HCV-NS5B, produces mutational bias in the HCV genome, characterized by dominant transition mutations, notably A>G and U>C substitutions. However, RASs are acquired by both transition and transversion substitutions, and the RASs-positive HCV clones are selected and proliferated under DAA treatment pressure.
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Hepatite C Crônica , Hepatite C , Antivirais/farmacologia , Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Genótipo , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/genética , Humanos , Mutação , RNA , Proteínas não Estruturais Virais/genéticaRESUMO
Welding fumes (WFs) can cause occupational pneumonoconiosis and other diseases in workers. WFs have complex chemical composition and morphology depending on the welding conditions. The WF surface is a key factor affecting those diseases. The objective of this study was to establish an analytical method focused on characterizing individual WFs and welding slags (WSs) formed during CO2 arc welding processes for knowledge acquisition of risk assessment. Especially, the characterization was focused on the elemental distributions near the surfaces obtained using fluxing agents and size of the WFs. WFs were collected using personal samplers. After welding, WS was also collected. The fluxing elemental distribution (e.g., Bi) near the surfaces WS and WFs were analyzed through scanning electron microscopy and energy-dispersive X-ray spectroscopy. As a result, some of the micron-sized spherical particles (SPs) grew by incorporating nanosized primary particles composed of other metal species. The fluxing agents formed elemental distribution patterns on the SP surface. Bi were dotted in an agglomerate. Mn amount in WS depends on Mn amount in the WFs. These results obtained through the analysis of both the WS and WF surface as well as the particle sizes will facilitate the establishment of exposure assessment models.
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Poluentes Ocupacionais do Ar , Exposição Ocupacional , Soldagem , Poluentes Ocupacionais do Ar/análise , Humanos , Exposição por Inalação/análise , Exposição Ocupacional/análise , Material Particulado , Aço InoxidávelRESUMO
Sheltering is one of the countermeasures for protection against radiation exposures in nuclear accidents. The effectiveness of sheltering is often expressed by the reduction factor, that is the ratio of the indoor to the outdoor cumulative radioactivity concentrations or doses. The indoor concentration is mainly controlled by the air exchange rate, penetration factor, and indoor deposition rate. The penetration factor and indoor deposition rate depend on the surface and the materials and structure of windows and doors as it is these openings in the building envelope that control penetration. We investigated experimentally these parameters of I2and particles. The experiment was performed in two apartment houses, three single-family houses, and chambers. The obtained penetration factor ranged from 0.3 to 1 for particles of 0.3-1µm and 0.15-0.7 for I2depending on the air exchange rate. The indoor deposition rate for a house room ranged from 0.007 to 0.2 h-1for particles of 0.3-1µm and 0.2-1.5 h-1for I2.
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Poluição do Ar em Ambientes Fechados , Iodo , Exposição à Radiação , Poluição do Ar em Ambientes Fechados/análise , Monitoramento Ambiental , Habitação , JapãoRESUMO
Hepatitis B virus (HBV) is a human hepatotropic pathogen causing hepatocellular carcinoma. We recently obtained HBV-susceptible immortalized human hepatocyte NKNT-3 by exogenously expressing NTCP and its derived cell clones, #28.3.8 and #28.3.25.13 exhibiting different levels of HBV susceptibility. In the present study, we showed that HBV infection activated the ATM-Chk2 signaling pathway in #28.3.25.13 cells but not in #28.3.8 cells. Both the cell culture supernatant and extracellular vesicles (EVs) derived from HBV-infected #28.3.25.13 cells also activated the ATM-Chk2 signaling pathway in naïve #28.3.25.13 cells. Interestingly, EVs derived from HBV-infected #28.3.25.13 cells included higher level of mitochondrial DNA (mtDNA) than those from HBV-infected #28.3.8 cells. Based on our results, we propose the novel model that EVs mediate the activation of ATM-Chk2 signaling pathway by the intercellular transfer of mtDNA in HBV-infected human hepatocyte.
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Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , DNA Mitocondrial/genética , Vesículas Extracelulares/metabolismo , Hepatite B/patologia , Hepatócitos/patologia , Replicação Viral , Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinase do Ponto de Checagem 2/genética , DNA Mitocondrial/metabolismo , Células Hep G2 , Hepatite B/genética , Hepatite B/metabolismo , Hepatite B/microbiologia , Vírus da Hepatite B/fisiologia , Hepatócitos/metabolismo , Hepatócitos/microbiologia , HumanosRESUMO
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).
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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.
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Antivirais/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Ribavirina/farmacologia , Antimetabólitos/farmacologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Linhagem Celular , Glicerol-3-Fosfato O-Aciltransferase , Humanos , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
BACKGROUND AND AIM: There is no report on silent brain infarction (SBI) after minimally invasive cardiac surgery (MICS) with retrograde perfusion. Thus, the current study aimed to investigate the incidence of SBI after MICS using magnetic resonance imaging (MRI). METHODS: This study included 174 patients who underwent MICS with retrograde perfusion between July 2014 and July 2018. Preoperative computed tomography (CT) angiography was routinely performed and vascular pathology was evaluated for patient selection. Postoperative MRI was performed to investigate the occurrence of SBI. RESULTS: Out of the total 174 patients, 26 (14.9%) presented with SBI. A total of 61 SBI lesions were found in the 26 patients; of these, 34 (56%) SBI lesions were in the right hemisphere and 27 (44%) in the left hemisphere. SBIs were primarily observed in the posterior cerebral artery territory. Multivariate analysis revealed aortic stenosis to be the only risk factor of SBI. CONCLUSIONS: Retrograde perfusion via femoral cannulation may not increase the incidence of SBI in selected MICS patients based on preoperative CT findings.
Assuntos
Infarto Encefálico/etiologia , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Procedimentos Cirúrgicos Cardíacos/métodos , Procedimentos Cirúrgicos Minimamente Invasivos/efeitos adversos , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Perfusão/efeitos adversos , Perfusão/métodos , Complicações Pós-Operatórias/etiologia , Idoso , Estenose da Valva Aórtica , Infarto Encefálico/diagnóstico por imagem , Infarto Encefálico/epidemiologia , Angiografia por Tomografia Computadorizada , Feminino , Humanos , Incidência , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico por imagem , Complicações Pós-Operatórias/epidemiologia , Fatores de RiscoRESUMO
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.
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Variação Genética/genética , Hepacivirus/genética , Replicação Viral/genética , Carcinoma Hepatocelular/virologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Genes Reporter/genética , Genoma Viral/genética , Genótipo , Hepatite C Crônica/virologia , Humanos , Neoplasias Hepáticas/virologia , RNA Viral/genética , Replicon/genéticaRESUMO
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 Li23â¯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 A8.15.78.10â¯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 A8.15.78.10â¯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 A8.15.78.10â¯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.
Assuntos
Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Neoplasias Hepáticas/virologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Ciclosporina/farmacologia , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Rosiglitazona/farmacologia , Simportadores/genética , Simportadores/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
Recently, we demonstrated that the anti-viral drug ribavirin (RBV) had the ability to suppress lipogenesis through down-regulation of retinoid X receptor α (RXRα) under the control of the intracellular GTP-level and AMP-activated protein kinase-related kinases, especially microtubule affinity regulating kinase 4 (MARK4). RXRα-overexpression attenuated but did not abolish lipogenesis suppression by RBV, implying that additional factor(s) were involved in this suppressive effect. In the present study, we found that the protein level, but not the mRNA level, of CCAAT/enhancer-binding protein α (C/EBPα) was down-regulated by RBV in hepatic cells. Treatment with proteasome inhibitor attenuated RBV-induced down-regulation of C/EBPα, suggesting that RBV promoted degradation of C/EBPα protein via the ubiquitin-proteasome pathway. Depletion of intracellular GTP through inosine monophosphate dehydrogenase inhibition by RBV led to down-regulation of C/EBPα. In contrast, down-regulation of C/EBPα by RBV was independent of RXRα and MARK4. Knockdown of C/EBPα reduced the intracellular neutral lipid levels and the expression of genes related to the triglyceride (TG) synthesis pathway, especially glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), which encodes the first rate-limiting TG enzyme. Overexpression of C/EBPα yielded the opposite results. We also observed that RBV decreased GPAM expression. Moreover, overexpression of GPAM attenuated RBV-induced reduction in the intracellular neutral lipid levels. These data suggest that down-regulation of C/EBPα by RBV leads to the reduction in GPAM expression, which contributes to the suppression of lipogenesis. Our findings about the mechanism of RBV action in lipogenesis suppression will provide new insights for therapy against the active lipogenesis involved in hepatic steatosis and hepatocellular carcinomas.
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Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação para Baixo/efeitos dos fármacos , Hepatócitos/metabolismo , Lipogênese/efeitos dos fármacos , Ribavirina/farmacologia , Triglicerídeos/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Hepatócitos/citologia , Humanos , Lipogênese/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor X Retinoide alfa , Triglicerídeos/genéticaRESUMO
We examined the effects of various acidic polysaccharides isolated from marine algae on the infection and replication of human immunodeficiency virus type-1 (HIV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-cell leukemia virus type-1 (HTLV-1). It was found that sulfated fucan polysaccharides, ascophyllan, and two fucoidans derived from different sources significantly inhibited the early step of HIV-1 (R9 and JR-FL) infection, while they did not affect the late step. The alginate oligomer consisted of uronic acids and sulfated-galactan porphyran showed no significant inhibitory effects. In addition, ascophyllan and two fucoidans inhibited the early step of HBV infection in a dose-dependent manner. Furthermore, these polysaccharides inhibited the early step of HCV infection but had no inhibitory effects on HTLV-1 replication. To further examine the specificity of these polysaccharides in viral infections, we used vesicular stomatitis virus (VSV)-G-pseudotyped HIV-1 infection. Ascophyllan, the two fucoidans, and alginate oligomer also potently inhibited VSV-G-pseudotyped HIV-1 infection in HeLa cells. Taken together, these results suggest that the acidic polysaccharides used in this study are capable of inhibiting the early step of viral infections depending on the polysaccharides but not in a strict species-specific manner.
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Organismos Aquáticos/química , Polissacarídeos/química , Viroses/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Ácidos/química , Cianobactérias/química , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Hepacivirus/efeitos dos fármacos , Hepacivirus/patogenicidade , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/patogenicidade , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Viroses/virologiaRESUMO
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.
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Hepatitis B virus (HBV) causes hepatic diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. These diseases are closely associated with persistent HBV infection. To prevent the progression of hepatic diseases, it is thus important to suppress persistent HBV infection. Daunorubicin (DNR), a topoisomerase II (Top II) poison, is a clinically used anticancer agent with a wide spectrum of activity against malignancies. DNR was recently reported to cause DNA damage-dependent interferon (IFN)-ß induction through exogenous cyclic GMP-AMP synthetase (cGAS) and subsequently to suppress Ebola virus replication. In the present study, we demonstrated that DNR caused the inhibition of cell proliferation, but not cell death, through the DNA damage response in immortalized human hepatocyte NKNT-3/NTCP cells. Interestingly, DNR triggered the endogenous cGAS-dependent innate immune response and subsequently suppressed viral production of HBV in NKNT-3/NTCP cells. Top II poisons may be anti-HBV drug candidates.
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Daunorrubicina/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Nucleotidiltransferases/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Vírus da Hepatite B/fisiologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Nucleotidiltransferases/genética , Inibidores da Topoisomerase II/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
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.
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BACKGROUND: We previously showed that knockdown of nuclear factor E2-related factor 2 (Nrf2) resulted in suppression of hepatitis C virus (HCV) infection. In this study, whether brusatol, an Nrf2 inhibitor, has dual anti-HCV and anticancer effects was explored. METHODS: The anti-HCV effect of brusatol was investigated by analyzing HCV RNA and proteins in a hepatic cell line persistently-infected with HCV, HPI cells, and by analyzing HCV replication in a replicon-replicating hepatic cell line, OR6 cells. Then, dual anti-HCV and anticancer effects of brusatol and enhancement of the effects by the combination of brusatol with anticancer drugs including sorafenib, which has been reported to have the dual effects, were then investigated. RESULTS: Brusatol suppressed the persistent HCV infection at both the RNA and protein levels in association with a reduction in Nrf2 protein in the HPI cells. Analysis of the OR6 cells treated with brusatol indicated that brusatol inhibited HCV persistence by inhibiting HCV replication. Combination of brusatol with an anticancer drug not only enhanced the anticancer effect but also, in the case of the combination with sorafenib, strongly suppressed HCV infection. CONCLUSIONS: Brusatol has dual anti-HCV and anticancer effects and can enhance the comparable effects of sorafenib. There is therefore the potential for combination therapy of brusatol and sorafenib for HCV-related hepatocellular carcinoma.
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Antineoplásicos/farmacologia , Antivirais/farmacologia , Hepatite C/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Quassinas/farmacologia , Linhagem Celular Tumoral , Humanos , Quassinas/uso terapêutico , RNA Viral/análise , Sorafenibe/farmacologia , Transcriptoma , Replicação Viral/efeitos dos fármacosRESUMO
Although interferon (IFN)based treatment of patients with chronic hepatitis C virus (HCV) infection is widely applied, treatment resistance is often observed in patients with advanced liver fibrosis. Given that the molecular mechanisms of IFN resistance in liver fibrosis remain elusive, the present study investigated the effects of extracellular matrix (ECM) on IFN signaling in hepatic cells. The native HuH7 human hepatoma cell line and HuH7 cells were stably transfected with fulllength HCVRNA fused with Renilla luciferase (OR6 cells) were cultured on ECMcoated dishes or noncoated plastic dishes (NDs), and treated with human IFNα. In Huh7 cells cultured on coated dishes, the IFNstimulated response element (ISRE) luciferase activity was measured following ISRE plasmid transfection and the expression of IFNstimulated genes (ISG) were significantly lower than those in cells cultured on NDs. In addition, after IFNα treatment, the amount of HCVRNA and viral protein produced by OR6 cells cultured on coated dishes was higher than that produced by cells cultured on NDs. When cells were treated with ß1integrinblocking antibody to disrupt the cellmatrix interaction, the ISRE luciferase activity was restored, and the protein expression of ISG was increased, while that of HCV proteins was suppressed. Treatment of cells with integrinlinked kinase (ILK) inhibitor or focal adhesion kinase (FAK) inhibitor restored the ISRE luciferase activity and expression of ISG proteins. These results suggested that ß1integrinmediated signals affected the IFN signaling and promoted HCV replication. Therefore, the accumulation of ECM in liver fibrosis may impair IFN signaling through ß1integrinmediated signaling involving ILK and FAK.
Assuntos
Matriz Extracelular/imunologia , Hepacivirus/fisiologia , Hepatite C/imunologia , Interferon-alfa/imunologia , Transdução de Sinais , Linhagem Celular Tumoral , Matriz Extracelular/virologia , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Proteínas Serina-Treonina Quinases/imunologia , RNA Viral/genética , Replicação ViralRESUMO
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.
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Telomerase-specific replication-competent adenoviruses (Ads), i.e., TRADs, which possess an E1 gene expression cassette driven by the human telomerase reverse transcriptase promoter, are promising agents for cancer treatment. However, even though oncolytic Ads, including TRAD, are intratumorally administered, they are disseminated from the tumor to systemic circulation, causing concern about oncolytic Ad-mediated hepatotoxicity (due mainly to leaky expression of Ad genes in liver). We reported that inhibition of nuclear factor-κB (NF-κB) leads to the suppression of replication-incompetent Ad vector-mediated hepatotoxicity via reduction of the leaky expression of Ad genes in liver. Here, to develop a TRAD with an improved safety profile, we designed a TRAD that carries a liver-specific promoter-driven dominant-negative IκBα (DNIκBα) expression cassette (TRAD-DNIκBα). Compared with a conventional TRAD, TRAD-DNIκBα showed hepatocyte-specific inhibition of NF-κB signaling and significantly reduced Ad gene expression and replication in the normal human hepatocyte cell line. TRAD-induced hepatotoxicity was largely suppressed in mice following intravenous administration of TRAD-DNIκBα. However, the replication profiles and oncolytic activities of TRAD-DNIκBα were comparable with those of the conventional TRAD in human non-hepatic tumor cells. These results indicate that oncolytic Ads containing the liver-specific DNIκBα expression cassette have improved safety profiles without inhibiting oncolytic activities.
RESUMO
OBJECTIVES: The application of multiwall carbon nanotubes (MWCNTs) currently extends to various fields. However, it has been reported that exposure to CNT causes hazardous effects on animals and cells. The purpose of this study was to quantify the exposure to MWCNT in MWCNT/polymer composites for exposure assessment. We focused on catalytic metals included in the MWCNT and the diameter of dust released during the working processes. Although the Co in MWCNTs is not a common catalyst, it was used as a tracer in this study. METHODS: A field survey was conducted in a MWCNT/polymer composite pilot factory. Airborne MWCNTs were monitored using black carbon monitors (BCMs) and optical particle sizers (OPSs) and collected on a filter. The MWCNT powder, all polymer resins used during the working processes, and the filter were analyzed in our lab using inductively coupled plasma mass spectrometry (ICP-MS) and electron microscopic observation. RESULTS: The mean concentration of airborne MWCNT contained in the collected dust was 0.92 µg/m3 a few meters away from the extruder during the working processes (using elemental analysis). The maximum concentration measured using BCMs was shown to be seven times higher than the base concentration during the pelletizing process of polycarbonate (PC) and MWCNT composites. However, free, isolated, and unbound agglomerated MWCNTs were not detected using scanning electron microscopic (SEM) observation. CONCLUSIONS: The result obtained by elemental analysis indicated it was possible to quantify MWCNT in composites. The mean concentration at this factory was lower than the recommended exposure limit. However, additional studies during the pelletizing process are required in the future.