Cell culture and in vivo analyses of cytopathic hepatitis C virus mutants

AbstractHCV-JFH1 yields subclones that develop cytopathic plaques (Sekine-Osajima Y, et al., Virology 2008; 371:71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection

Complete response to pembrolizumab in advanced hepatocellular carcinoma with microsatellite instability

Hepatocellular carcinoma (HCC) has limited systemic treatment options and a poor prognosis. The immune checkpoint inhibitor pembrolizumab was recently approved for the treatment of solid tumors with microsatellite instability (MSI). However, its clinical utility for the management of HCC remains to be clarified. Here, we present a case of unresectable HCC with MSI that showed an impressive response to pembrolizumab treatment. A 64-year-old man with chronic HCV infection was diagnosed with a large HCC. His severe liver dysfunction and poor performance status prevented any treatment option other than sorafenib. However, sorafenib failed after a few days due to the rapid progression of the tumor. Based on the finding of MSI in a biopsy specimen, immunotherapy using pembrolizumab was initiated. A dramatic improvement in his general condition and a reduction in tumor size were observed after the initiation of pembrolizumab treatment. Among a cohort of 50 consecutive patients with advanced HCC who were refractory to standard systemic therapy, MSI was found only in the present case. Immune checkpoint blockade therapy induced prominent anti-tumor effects in HCC with MSI. Screening for defects in DNA mismatch repair function may be warranted in HCC patients despite the low frequency of MSI

Features of and Mechanisms Underlying Insulitis In aly/aly Male Mice as an Animal Model of Autoimmune Pancreatitis: Activation of CD11c+, CD4+, and Th2 Cells and Predominant Destruction of β-cells

Diabetes mellitus (DM) is observed in patients with autoimmune pancreatitis (AIP). The development of DM in AIP is believed to be due to blood flow obstruction of the endocrine gland that accompanies pancreatitis, as well as injury to the islets caused by inflammation. The latter is called insulitis and the detailed mechanisms underlying its development are not yet clear. The aim of the present study was to elucidate the mechanisms involved in the development of insulitis in AIP using aly mice as an animal model of AIP: results in aly/aly male mice, as the AIP group, were compared with those inaly/+ male mice as a control group. Mice in both groups were killed between 16 and 48 weeks of age, and pancreatitis and insulitis were evaluated histologically. Inflammatory and endocrine cells were evaluated by immunofluorescence staining with anti-CD4, anti-CD8, anti-CD11b, and anti-CD11c antibodies, as well as immunohistochemical analyses using insulin and glucagon antibodies. Plasma levels and the pancreatic content of interferon (IFN)-γ (as a Th1-secreted cytokine) and interleukin (IL)-4 (as a Th2-secreted cytokine) were determined. Pancreatitis was seen in aly/aly mice from 16 weeks of age and it developed gradually thereafter. Insulitis also developed gradually and was seen in mice after 24 weeks of age in association with a decrease in the number of islets. CD11c+ cells and CD4+ T cells were seen to infiltrate into the islets. Although the number of β-cells decreased with time, the number of α-cells was maintained until mice were 48 weeks of age. IFN-γ content peaked in mice at 16 weeks of age and declined rapidly from 20 weeks. There were two peaks in IL-4 content, one at 16 weeks and the other at 32 weeks, suggesting an association between IL-4 content and advanced insulitis after 32 weeks. In conclusion, the results suggest that insulitis in AIP is induced predominantly by the infiltration of CD11c+ cells and CD4+ T cells into the islets, and progression is facilitated by the imbalance of the activation of Th2 rather than Th1. Furthermore, insulitis in AIP predominantly involves β-cells rather than α-cells

Mechanical properties and corrosion resistance of work–hardened titanium on the market

JIS type 2 titanium is cold–worked for work–hardening, and is commercially available (G2W), and this material is used to produce implants. In this study, the mechanical characteristics and corrosion resistance of this work–hardened material (G2W) were compared with those of JIS type 2 titanium (G2), JIS type 4 titanium (G4), and JIS SSH4650–60E (G5). The following results were obtained: ①The tensile strength was highest in G5, and lowest in G2. No marked difference was observed between G2W and G4. ②The proof stress was highest in G5, and lowest in G2. No significant difference was noted between G2W and G4. ③The elongation was highest in G2, and lowest in G5. No significant difference was present between G2W and G4. ④The fracture toughness showed a significant difference between G2 and G2W, whereas no significant differences were observed among G2W, G4, and G5. ⑤The maximum bending load at an inclination angle of 30° was lowest in G2, and no significant differences were noted among G2W, G4, and G5. ⑥The hardness was highest in G5, and lowest in G2. No significant difference was present between G2W and G4. ⑦The released volume of titanium was highest in G5, and lowest in G2W. The released volume was higher in G4 than in G2. Vanadium and aluminum were released from G5. ⑧Work–hardened titanium (G2W) showed mechanical characteristics equivalent to those of G4, and excellent corrosion resistance, and is considered to be useful as an implant material

Assessment of Outcome of Hepatic Arterial Infusion Chemotherapy in Patients with Advanced Hepatocellular Carcinoma by the Combination of RECIST and Tumor Markers

To assess the outcome of stable disease (SD) patients with advanced hepatocellular carcinoma (HCC) by tumor markers after the first course of hepatic arterial infusion chemotherapy (HAIC). The study subjects were 156 HCC patients treated with HAIC and classified as Child Pugh A, with no extrahepatic metastasis, and no history of sorafenib treatment. In the study and validation cohorts, the AFP and DCP ratios of patients who were considered SD to the first course of HAIC were analyzed by AUROC for a prediction of response to the second course of HAIC. The imaging response to the first course of HAIC was classified as partial response (PR), SD and progressive disease (PD) in 29 (18.8%), 80 (51.9%), and 44 (28.6%) patients respectively. For SD patients, the α-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP) ratios of patients who were considered SD to the first course of HAIC were analyzed by the receiver operating characteristic curve for prediction of response to the second course of HAIC in the study cohorts. The area under the curve of AFP ratio was 0.743. The area under the curve of DCP ratio was 0.695. The cut-off values of AFP and DCP ratios were 1.3 and 1.0, respectively. In the validation cohort, the accuracy of the prediction of response in this validation cohort (71.4%) showed no significant difference compared to that in the study cohort (72.4%) (p = 1.0). The results suggested that patients with a high tumor marker ratio could be switched to alternative therapeutic regimens despite the SD response to HAIC

Decreased expression of human heat shock protein 70 in the endometria and pathological lesions of women with adenomyosis and uterine myoma after GnRH agonist therapy

ObjectiveA prominent stress reaction in the pelvis of women with endometriosis and the role of human heat shock protein 70 (HSP70) in inflammation and the growth of endometriosis has been recently demonstrated. We report here expression of HSP70 in tissues derived from GnRH agonist (GnRHa)-untreated and -treated women with adenomyosis and uterine myoma.Study designThis is a case-controlled biological study. Biopsy specimens were collected from pathological lesions and eutopic endometria/autologous myometria of 30 women with adenomyosis, 35 women with uterine myoma and 15 control women during laparoscopy, laparotomy and hysteroscopy. Fourteen women with adenomyosis and 20 women with uterine myoma were treated with GnRHa for a variable period of 3?6 months before surgery. The immunoexpressions of HSP70 and CD68-positive M? in endometria, lesions/myometria were examined by immunohistochemistry. The immunoreactivity of HSP70 in tissues was analyzed by quantitative-histogram (Q-H) scores.ResultsComparing to control women, HSP70 immunoexpression was significantly higher in endometria/myometria and pathological lesions of women with adenomyosis and myoma. A significant positive correlation between Q-H scores of HSP70 and CD68-positive M? numbers was found in the endometria derived from women with adenomyosis (r = 0.388). Treatment with GnRHa significantly decreased Q-H scores of HSP70 in pathological lesions and endometria/myometria of women with adenomyosis and uterine myoma comparing to similar tissues derived from GnRHa-untreated women.ConclusionA variable amount of tissue stress reaction occurred in endometria and pathological lesions of women adenomyosis and uterine myoma that can be effectively suppressed after GnRHa treatment

Hepatitis C Virus Infection Suppresses the Interferon Response in the Liver of the Human Hepatocyte Chimeric Mouse

BACKGROUND AND AIMS: Recent studies indicate that hepatitis C virus (HCV) can modulate the expression of various genes including those involved in interferon signaling, and up-regulation of interferon-stimulated genes by HCV was reported to be strongly associated with treatment outcome. To expand our understanding of the molecular mechanism underlying treatment resistance, we analyzed the direct effects of interferon and/or HCV infection under immunodeficient conditions using cDNA microarray analysis of human hepatocyte chimeric mice. METHODS: Human serum containing HCV genotype 1b was injected into human hepatocyte chimeric mice. IFN-α was administered 8 weeks after inoculation, and 6 hours later human hepatocytes in the mouse livers were collected for microarray analysis. RESULTS: HCV infection induced a more than 3-fold change in the expression of 181 genes, especially genes related to Organismal Injury and Abnormalities, such as fibrosis or injury of the liver (P = 5.90E-16∼3.66E-03). IFN administration induced more than 3-fold up-regulation in the expression of 152 genes. Marked induction was observed in the anti-fibrotic chemokines such as CXCL9, suggesting that IFN treatment might lead not only to HCV eradication but also prevention and repair of liver fibrosis. HCV infection appeared to suppress interferon signaling via significant reduction in interferon-induced gene expression in several genes of the IFN signaling pathway, including Mx1, STAT1, and several members of the CXCL and IFI families (P = 6.0E-12). Genes associated with Antimicrobial Response and Inflammatory Response were also significantly repressed (P = 5.22×10(-10)∼1.95×10(-2)). CONCLUSIONS: These results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy

Hepatitis C virus treatment update — A new era of all-oral HCV treatment

There are estimated to be more than a hundred million hepatitis C virus (HCV) carriers worldwide. About 30% of carriers develop serious liver diseases, such as liver cirrhosis and hepatocellular carcinoma. HCV Genotype 1 is the most common genotype worldwide and the most difficult to treat with interferon-based therapy. Therapy for patients with chronic HCV infection is complicated by poor tolerability and inadequate rates of sustained virological response (SVR). Although the addition of a protease inhibitor in combination with peg-interferon alpha plus ribavirin improved SVR rates and shortened the treatment period, many patients could not tolerate this therapy because of advanced age and clinical conditions such as anemia and low platelet count. Interferon-free therapies that combine two or more direct-acting antiviral (DAA) agents can improve both efficacy and tolerability. Phase III trials of daclatasvir plus asunaprevir, ombitasvir plus parataprevir/r, and sofosbuvir plus ledipasvir all showed high overall SVR rates with few adverse events. However, development of antiviral resistance is a concern with DAA therapies, and it is important to avoid treating patients with existing NS5A Y93H mutations with daclatasvir plus asunaprevir or ombitasvir plus parataprevir/r therapy to prevent viral breakthrough. Fortunately, sofosbuvir plus ledipasvir therapy seems to be less affected by NS5A Y93H variants. An important goal of HCV therapy is to expand treatment to all patients. The current study aims to show the efficacy and safety of these therapies both in clinical trial and real world settings based on our own clinical experiences