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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 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. CTLA‐4 blockade without ADCC activity augments the proliferation and CTLA‐4‐independent immunosuppressive functions of Treg cells by enhancing CD28 costimulatory signaling pathways in Treg cells, leading to insufficient tumor regression. This figure was created with BioRender.com.

Hydrogen peroxide (H 2 O 2 ) induces oxidative stress and cytotoxicity, and can be used for treating cancers in combination with radiotherapy. A product comprising H 2 O 2 and sodium hyaluronate has been developed as a radiosensitizer. However, the effects of H 2 O 2 on antitumor immunity remain unclear. To investigate the effects of H 2 O 2 , 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. H 2 O 2 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 H 2 O 2 /RT combination significantly reduced tumor growth at the noninjected/nonirradiated site in the back lesion, whereas H 2 O 2 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 H 2 O 2 /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 H 2 O 2 /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 H 2 O 2 /RT group. Intratumoral injection of H 2 O 2 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 H 2 O 2 /RT therapy combined with cancer immunotherapies, even for advanced cancers.

Recently, lipid droplets have been found to be involved in an important cytoplasmic organelle for hepatitis C virus (HCV) production. However, the mechanisms of HCV assembly, budding, and release remain poorly understood. Retroviruses and some other enveloped viruses require an endosomal sorting complex required for transport (ESCRT) components and their associated proteins for their budding process. To determine whether or not the ESCRT system is needed for HCV production, we examined the infectivity of HCV or the Core levels in culture supernatants as well as HCV RNA levels in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, expressing short hairpin RNA or siRNA targeted to tumor susceptibility gene 101 (TSG101), apoptosis-linked gene 2 interacting protein X (Alix), Vps4B, charged multivesicular body protein 4b (CHMP4b), or Brox, all of which are components of the ESCRT system. We found that the infectivity of HCV in the supernatants was significantly suppressed in these knockdown cells. Consequently, the release of the HCV Core into the culture supernatants was significantly suppressed in these knockdown cells after HCV-JFH1 infection, while the intracellular infectivity and the RNA replication of HCV-JFH1 were not significantly affected. Furthermore, the HCV Core mostly colocalized with CHMP4b, a component of ESCRT-III. In this context, HCV Core could bind to CHMP4b. Nevertheless, we failed to find the conserved viral late domain motif, which is required for interaction with the ESCRT component, in the HCV-JFH1 Core, suggesting that HCV Core has a novel motif required for HCV production. These results suggest that the ESCRT system is required for infectious HCV production.

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.

Persistent infections with hepatitis C virus (HCV) may result in life-threatening liver disease, including cirrhosis and cancer, and impose an important burden on human health. Understanding how the virus is capable of achieving persistence in the majority of those infected is thus an important goal. Although HCV has evolved multiple mechanisms to disrupt and block cellular signaling pathways involved in the induction of interferon (IFN) responses, IFN-stimulated gene (ISG) expression is typically prominent in the HCV-infected liver. Here, we show that Toll-like receptor 3 (TLR3) expressed within uninfected hepatocytes is capable of sensing infection in adjacent cells, initiating a local antiviral response that partially restricts HCV replication. We demonstrate that this is dependent upon the expression of class A scavenger receptor type 1 (MSR1). MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. RNAi-mediated knockdown of MSR1 expression blocks TLR3 sensing of HCV in infected hepatocyte cultures, leading to increased cellular permissiveness to virus infection. Exogenous expression of Myc-MSR1 restores TLR3 signaling in MSR1-depleted cells with subsequent induction of an antiviral state. A series of conserved basic residues within the carboxy-terminus of the collagen superfamily domain of MSR1 are required for binding and transport of dsRNA, and likely facilitate acidification-dependent release of dsRNA at the site of TLR3 expression in the endosome. Our findings reveal MSR1 to be a critical component of a TLR3-mediated pattern recognition receptor response that exerts an antiviral state in both infected and uninfected hepatocytes, thereby limiting the impact of HCV proteins that disrupt IFN signaling in infected cells and restricting the spread of HCV within the liver.

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. By repetitive RBV (100 μM) 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 μM). 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. These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.

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).

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. 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 time-dependent manner, and the mutation rates in the 5'-terminus-NS2 and NS3-NS5B regions were 4.0-9.0×10(-3) and 2.7-4.0×10(-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. Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.

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. Cell‐surface expression of the NK group 2 member D ligand ULBP1 was increased by hepatitis C virus (HCV) in immortalized human hepatocytes and human hepatoma cells. Natural killer (NK) cell mediated‐cytotoxicity and interferon‐γ mRNA expression was enhanced towards HCV‐infected cells and subsequently inhibited viral replication. Our results suggest that NK cells trigger the host innate immune response through the recognition of ULBP1 on HCV‐infected hepatocytes.

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. 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-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect. 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.