Tomoe Shimazaki

Infection of B cells with hepatitis C virus for the development of lymphoproliferative disorders in patients with chronic hepatitis C.

Infection with hepatitis C virus (HCV) is associated with lymphoproliferative disorders, represented by essential mixed cryoglobulinemia and B‐cell non‐Hodgkins lymphoma, but the pathogenic mechanism remains obscure. HCV may infect B cells or interact with their cell surface receptors, and induce lymphoproliferation. The influence of HCV infection of B cells on the development of lymphoproliferative disorders was evaluated in 75 patients with persistent HCV infection. HCV infection was more prevalent (63% vs. 16%, 14%, or 17% P < 0.05 for each), and HCV RNA levels were higher (3.35 ± 3.85 vs. 1.75 ± 2.52, 2.15 ± 2.94 or 2.10 ± 2.90 log copies/100 ng, P < 0.01 for each) in B cells than CD4+, CD8+ T cells or other cells. Negative‐strand HCV RNA, as a marker of viral replication, was detected in B cells from four of the 75 (5%) patients. Markers for lymphoproliferative disorders were more frequent in the 50 patients with chronic hepatitis C than the 32 with chronic hepatitis B, including cryoglobulinemia (26% vs. 0%, P < 0.001), low CH50 levels (48% vs. 3%, P = 0.012), and the clonality of B cells (12% vs. 0%, P < 0.01). By multivariate analysis, HCV RNA in B cells was an independent factor associated with the presence of at least one marker for lymphoproliferation (odds ratio: 1.98 [95% confidence interval: 1.36–7.24], P = 0.027). Based on the results obtained, the infection of B cells with HCV would play an important role in the development of lymphoproliferative disorders. J. Med. Virol. 81:619–627, 2009

Lymphotropic hepatitis C virus has an interferon-resistant phenotype

Summary.  Hepatitis C virus (HCV) infects and associates with B cells, leading to abnormal B‐cell activation and development of lymphoproliferative and autoimmune disorders. This immune perturbation may in turn be associated with the resistance of HCV against the host immune system. The objective of this study was to analyse the effects of HCV infection of B cells on the efficacy of interferon (IFN)‐based therapy. The study enrolled 102 patients with chronic hepatitis C who were treated with pegylated IFN plus ribavirin. HCV RNA titres in B cells were compared in patients with rapid viral responder (RVR) vs non‐RVR, sustained viral responder (SVR) vs non‐SVR and null viral responder (NVR) vs VR. The levels of HCV RNA in B cells were significantly higher in non‐RVR, non‐SVR and NVR groups. Association between the therapy outcome and the positive B‐cell HCV RNA was also investigated in relation to other known viral and host factors. Multivariable analyses showed that the positive B‐cell HCV RNA and the minor single‐nucleotide polymorphism near the IL28B gene (rs8099917) were independent factors associated with NVR in patients infected with HCV genotype 1. When these two factors were combined, the sensitivity, specificity, positive and negative predictive values for NVR were 92.3%, 98.2%, 92.3% and 98.2%, respectively. Genotype 1 and the presence of one or no mutations in the IFN‐sensitivity determining region were associated with higher levels of B‐cell HCV RNA. B‐cell‐tropic HCV appears to have an IFN‐resistant phenotype. B‐cell HCV RNA positivity is a predictive factor for resistance to IFN‐based therapy.

Fibroblast growth factor-2–mediated FGFR/Erk signaling supports maintenance of cancer stem-like cells in esophageal squamous cell carcinoma

In esophageal squamous cell carcinoma (ESCC), a subset of cells defined by high expression of CD44 and low expression of CD24 has been reported to possess characteristics of cancer stem-like cells (CSCs). Novel therapies directly targeting CSCs have the potential to improve prognosis of ESCC patients. Although fibroblast growth factor-2 (FGF-2) expression correlates with recurrence and poor survival in ESCC patients, the role of FGF-2 in regulation of ESCC CSCs has yet to be elucidated. We report that FGF-2 is significantly upregulated in CSCs and significantly increases CSC content in ESCC cell lines by inducing epithelial-mesenchymal transition (EMT). Conversely, the FGFR inhibitor, AZD4547, sharply diminishes CSCs via induction of mesenchymal-epithelial transition. Further experiments revealed that MAPK/Erk kinase (Mek)/extracellular signal-regulated kinases (Erk) pathway is crucial for FGF-2-mediated CSC regulation. Pharmacological inhibition of FGF receptor (FGFR)-mediated signaling via AZD4547 did not affect CSCs in Ras mutated cells, implying that Mek/Erk pathway, downstream of FGFR signaling, might be an important regulator of CSCs. Indeed, the Mek inhibitor, trametinib, efficiently suppressed ESCC CSCs even in the context of Ras mutation. Consistent with these findings in vitro, xenotransplantation studies demonstrated that inhibition of FGF-2-mediated FGFR/Erk signaling significantly delayed tumor growth. Taken together, these findings indicate that FGF-2 is an essential factor regulating CSCs via Mek/Erk signaling in ESCC. Additionally, inhibition of FGFR and/or Mek signaling represents a potential novel therapeutic option for targeting CSCs in ESCC.

Magnitude of CD8+ T‐cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection

Aim:  We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)‐specific CD8+ T‐cell responses and the clinical course of acute HCV infection.

Anti-adipogenic and antiviral effects of l-carnitine on hepatitis C virus infection.

Hepatitis C virus (HCV) has been reported to hijack fatty acid metabolism in infected hepatocytes, taking advantage of lipid droplets for virus assembly. In this study, we analyzed the anti‐HCV activity of l‐carnitine, a substance involved in the transport of fatty acids into mitochondria. JFH‐1 or HCV replicon‐transfected Huh7.5.1 cells were treated with or without l‐carnitine to examine its anti‐HCV effects. The effects of l‐carnitine on HCV entry, HCV‐induced adipogenesis and lipid droplet formation, and HCV‐induced oxidative stress were examined. Treatment of JFH‐1‐infected cells with l‐carnitine inhibited HCV propagation in a concentration‐dependent manner. In contrast, l‐carnitine had no anti‐HCV activity in the HCV replicon system, which is lacking viral assembly. In addition, l‐carnitine did not affect HCV entry. However, l‐carnitine treatment decreased intracellular lipid droplets, which are crucial for HCV assembly in JFH‐1‐infected cells. The expression level of CPT‐1 was decreased in JFH‐1‐infected cells, and l‐carnitine treatment restored this expression. HCV‐infected cells exhibited increased production of reactive oxygen species and glutathione oxidation. l‐carnitine decreased oxidative stress induced by JFH‐1‐infection, as shown by glutathione/glutathione disulfide assays and MitoSOX staining. l‐carnitine exhibited anti‐HCV activity, possibly by inhibiting HCV assembly and through its anti‐adipogenic activity in HCV‐infected cells. Moreover, l‐carnitine has antioxidant properties in HCV‐infected hepatocytes. Overall, these results indicated that l‐carnitine may be an effective adjunctive agent in antiviral therapies to treat chronic hepatitis C. J. Med. Virol. 89:857–866, 2017.

Reactivation of epstein–barr virus in B cells of patients with chronic hepatitis C†

Hepatitis C virus (HCV) infection is associated with lymphoproliferative disorders. HCV infection of B cells is a predictive factor for lymphoproliferative disorders in patients with chronic hepatitis C, although its molecular mechanisms remain unknown. Epstein–Barr virus (EBV) is a B cell‐tropic virus with the potential to cause lymphoproliferative disorders, and its reactivation is induced by several viruses and cytokines. The possibility that HCV infection triggers reactivation of EBV and induces lymphoproliferative disorders were investigated. Expression of EBV mRNAs was analyzed by RT‐PCR in patients infected with HCV and control subjects, and correlations between reactivation of EBV and markers for lymphoproliferative disorders were investigated. BZLF1 mRNA, a starter molecule of reactivation, was detected in peripheral blood mononuclear cells from 12 of 52 (23%), patients infected with HCV and the frequency was higher than in healthy subjects [3 of 43 (9%), P = 0.032]. But the presence of the BZLF1 mRNA was not associated with an abnormality of markers for lymphoproliferative disorders. This study on BZLF1 mRNA expression among lymphoid cell subsets showed that reactivation of EBV was observed specifically in B cells. The BZLF1 mRNA disappeared following anti‐viral therapy and remained negative after eradication of HCV in patients with a sustained viral response, while the EBER1 RNA, a marker for persistence of EBV, was detected throughout the therapy. Infection with HCV induces reactivation of EBV in B cells, but this reactivation was not associated directly with lymphoproliferative disorders triggered by HCV. J. Med. Virol. 82:2064–2072, 2010.

L‐Carnitine Suppresses Loss of Skeletal Muscle Mass in Patients With Liver Cirrhosis

Liver cirrhosis (LC) is a major cause of secondary sarcopenia. Sarcopenia makes the prognosis worse; thus, novel therapeutic options for sarcopenia in patients with LC are urgently required as they are currently limited. In this retrospective study, 158 patients with LC were screened, and 35 of those patients who were treated with L‐carnitine for more than 6 months and for whom skeletal muscle mass changes could be evaluated by computer tomography were enrolled. Of the 158 patients, 79 patients who did not receive L‐carnitine supplementation served as controls. Cases and controls were propensity score matched for age, sex, presence of hepatocellular carcinoma, and branched chain amino acid administration, and changes in skeletal muscle mass and clinical data were compared. The 35 patients who received L‐carnitine supplementation and 35 propensity score‐matched patients who did not receive carnitine supplementation comprised the final enrollment. Compared with control patients, patients who received L‐carnitine had significantly worse liver function, which is associated with rapid progress of skeletal muscle depletion. However, loss of skeletal muscle mass was significantly suppressed in patients receiving L‐carnitine, and a significant effect was observed in patient subgroups stratified by age, sex, presence of hepatocellular carcinoma, and branched chain amino acid administration. The change ratios of most laboratory data, including vitamin D and insulin‐like growth factor 1 levels, were similar in the two groups, but ammonia levels were significantly less in those receiving L‐carnitine. However, even in patients receiving L‐carnitine but not showing an ammonia decrease, loss of skeletal muscle was significantly suppressed. Conclusion: L‐carnitine suppresses loss of skeletal muscle mass and may therefore be a novel therapeutic option for sarcopenia in patients with LC. (Hepatology Communications 2018; 00:000‐000)

Safety and efficacy of elbasvir/grazoprevir for the treatment of chronic hepatitis C: current evidence

Treatments for hepatitis C virus (HCV) have advanced greatly, becoming more efficacious with fewer adverse events, due to the availability of direct-acting antiviral agents, which target specific steps in the HCV life cycle. Recently, a combination regimen consisting of the HCV nonstructural protein 5A inhibitor elbasvir (EBR) and the HCV NS3/4A protease inhibitor grazoprevir (GZR) was approved for the treatment of patients with chronic HCV and genotypes (Gts) 1 and 4 in various countries. In Phase III trials, the combination of EBR/GZR (fixed-dose combination table or single agent) for 12 or 16 weeks of treatment with or without ribavirin resulted in a high sustained virological response at 12 weeks in treatment-naïve and treatment-experienced patients with HCV Gt 1a, 1b, 4, or 6, including special populations, such as individuals with advanced chronic kidney disease, HCV-HIV coinfection, and compensated cirrhosis. In this review, we focus on the mode of action, pharmacokinetics, clinical applications, efficacy, and safety profile of EBR/GZR, including special populations who have been considered refractory from the extensive evidence of clinical trials.

Increased expression of immuno-inhibitory molecules on peripheral blood lymphocytes may suppress disease progression in autoimmune hepatitis.

Accumulation of activated lymphocytes in the liver has been reported in patients with autoimmune liver diseases; the activity of these cells may be related to the disease immunopathogenesis. Although immuno-inhibitory molecules on lymphocytes are thought to play an important role in immunomodulation, there are only a few reports on the status of these molecules in autoimmune liver diseases. We focused on representative immunoinhibitory molecules, such as tumor necrosis factorrelated apoptosis-inducing ligand receptor 2/death receptor 5 (DR-5)/CD262, Fas/APO-1/CD95, programmed cell death-1 (PD-1)/CD279 and cytotoxic T-lymphocyte antigen-4 (CTLA-4)/CD152 on the surface of Tlymphocytes in patients with autoimmune liver disease, and investigated the relationship between inhibitory molecules on lymphocytes and the clinical features of patients with autoimmune liver diseases A limited number of patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and chronic hepatitis C (CHC) (n=10/group) and 10 healthy volunteers (control) were enrolled in this study. All patients and healthy volunteers gave written informed consent according to a protocol approved by the ethics committee of Showa University. Expression of inhibitory molecules DR5, Fas, PD-1 and CTLA-4 on the surface of peripheral CD4 and CD8 cells was assessed by staining withmonoclonal antibodies and by analyzing with flow cytometry. Differences between groups were assessed by ANOVA with Tukey’s honestly significant difference test and were considered statistically significant at P<0.05. Death receptor 5 expression was significantly upregulated on CD4 cells, but not on CD8 cells of patients with AIH compared with the controls (Fig. 1a). We found no differences in the expression of Fas molecules between patients and controls (Fig. 1b). PD-1 expression was more common on CD4 and CD8 cells of patients with AIH than on the control cells (Fig. 1c). The CD4 and CD8 cells of patients with AIH expressed CTLA-4 molecules more frequently than those of the controls, as shown in Figure 1(d). By comparison of the groups, significant difference could not be found between the control group and the disease groups other than AIH. In three AIH patients, the frequency of CTLA-4 positive cells was extremely high (Fig. 1d). The characteristics of these patients are presented in Table 1. They presented high frequencies of DR-5 and PD-1 expression as well. We noticed no signs of hepatitis activation such as elevated serum transaminase levels during the observation period. The other seven AIH patients had been treated with more than 5mg/day of prednisolone to control AIH. High expression of inhibitory molecules on CD4 and CD8 cells may suppress the disease activity of AIH. Upregulation of these immuno-inhibitory molecules may be a strategy for controlling AIH. We have reported that AIH patients have fewer intrahepatic regulatory T cells than do PBC patients. Immuno-inhibitory molecules also play important roles in immune regulation to prevent excessive inflammation. It has been reported that intrahepatic expression of PD-1 may play a crucial role in immunopathogenesis of AIH. Comprehensive study examining expression of inhibitory molecules on the surface of peripheral lymphocytes has not been reported, and it remains unclear whether overexpression of each molecule affects disease progression. Although each characteristic detail of inhibitory molecules has not been defined, several reports have compared CTLA-4 and PD-1. Each molecule inhibits T-cell activation by a different mechanism. PD-1 may be antigen-specific. Elucidation of the role of these molecules in immunerelated diseases would contribute to our understanding of the immunopathogenesis and the development of the treatment strategy. Further investigations including functional assay of inhibitory moleculeoverexpressing cells are needed.

Abstract 5278: Mutation in protein tyrosine phosphatase receptor type K (PTPRK) enhances progression of colon cancer through WNT signaling

Recent comprehensive genome analyses revealed the accumulation of genetic alterations in colorectal cancers (CRCs). However, it is still unknown which genetic alterations are closely associated with certain phenotype of CRCs, such as tumors with highly invasive character regardless of its small size. Here we examined the small invasive CRCs using high-throughput sequencing in order to clarify the common molecular features in those CRCs. Seven small invasive CRCs (types 0-IIa+IIc or 0-Is+IIc, less than 20mm in diameter and with submucosal massive invasion) were examined by exon sequencing. Seven thousands six variants (SNVs and indels) were detected. Among them, PTPRK was frequently mutated (3 cases, 43%; 1 nonsense and 2 missense mutations). We further examined public database (TCGA and COSMIC) and found that 50 of 617 (8.17%) CRCs contain PTPRK mutation. Those cases are frequent in proximal colon, extensively methylated (CIMP-H), microsatellite instability-high (MSI-H) and pathologically mucinous type. Consistent with clinical behavior, invasion assay in CRC cell lines revealed that CRCs with PTPRK domain D1 mutation showed high invasiveness via WNT signaling activity. These data indicate that PTPRK domain D1 mutation contributes to the invasiveness and progression of proximal CRCs, especially CIMP-H and MSI-H cases. Citation Format: Masayuki Tojo, Kazuo Konishi, Keiko Shinjo, Fumiharu Ohka, Keisuke Katushima, Akira Hatanaka, Norihisa Ichimura, Hisako Nozawa, Tomoe Shimazaki, Hitoshi Yoshida, Yutaka Kondo. Mutation in protein tyrosine phosphatase receptor type K (PTPRK) enhances progression of colon cancer through WNT signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5278. doi:10.1158/1538-7445.AM2015-5278