Kazuyoshi Ohkawa

Hepatitis C virus antibody and hepatitis C virus replication in chronic hepatitis B patients

We assessed hepatitis C virus infection in 156 chronic hepatitis B patients using second-generation hepatitis C virus antibody (anti-HCV). Active virus replication was further investigated in anti-HCV-positive cases by means of polymerase chain reaction assay for the detection of serum hepatitis C virus RNA. Anti-HCV prevalence was higher in patients negative for hepatitis B e antigen (HBeAg) (10/48, 21%) than in HBeAg-positive patients (10/108, 9%) (p < 0.05), and the reactivity (cut-off index) in anti-HCV enzyme-linked immunosorbent assay of the positive cases was significantly higher in HBeAg-negative patients (4.1 +/- 0.1) than in -positive ones (3.6 +/- 0.6) (p < 0.05). The prevalence of hepatitis C virus RNA in anti-HCV-positive cases was also higher in the HBeAg-negative group (9/10, 90%) than in the -positive group (3/10, 30%) (p < 0.01). Viremia was found in association with high reactivity in anti-HCV ELISA (cut-off index > 3.5) in both groups. Nine (90%) of 10 such cases were viremic in the HBeAg-negative group compared with three (43%) of seven in the HBeAg-positive group (p < 0.05). These results suggest that hepatitis C virus replication may be influenced by hepatitis B virus replicative states, indicating possible interference between hepatitis B and C viruses.

Ribavirin dose reduction raises relapse rate dose-dependently in genotype 1 patients with hepatitis C responding to pegylated interferon alpha-2b plus ribavirin

Summary.  The impact of ribavirin exposure on virologic relapse remains controversial in combination therapy with pegylated interferon (Peg‐IFN) and ribavirin for patients with chronic hepatitis C (CH‐C) genotype 1. The present study was conducted to investigate this. Nine hundred and eighty‐four patients with CH‐C genotype 1 were enrolled. The drug exposure of each medication was calculated by averaging the dose actually taken. For the 472 patients who were HCV RNA negative at week 24 and week 48, multivariate logistic regression analysis showed that the degree of fibrosis (P = 0.002), the timing of HCV RNA negativiation (P < 0.001) and the mean doses of ribavirin (P < 0.001) were significantly associated with relapse, but those of Peg‐IFN were not. Stepwise reduction of the ribavirin dose was associated with a stepwise increase in relapse rate from 11% to 60%. For patients with complete early virologic response (c‐EVR) defined as HCV RNA negativity at week 12, only 4% relapse was found in patients given ≥12 mg/kg/day of ribavirin and ribavirin exposure affected the relapse even after treatment week 12, while Peg‐IFN could be reduced to 0.6 μg/kg/week after week 12 without the increase of relapse rate. Ribavirin showed dose‐dependent correlation with the relapse. Maintaining as high a ribavirin dose as possible (≥12 mg/kg/day) during the full treatment period can lead to suppression of the relapse in HCV genotype 1 patients responding to Peg‐IFN alpha‐2b plus ribavirin, especially in c‐EVR patients.

Mcl‐1 and Bcl‐xL cooperatively maintain integrity of hepatocytes in developing and adult murine liver

Anti‐apoptotic members of the Bcl‐2 family, including Bcl‐2, Bcl‐xL, Mcl‐1, Bcl‐w and Bfl‐1, inhibit the mitochondrial pathway of apoptosis. Bcl‐xL and Mcl‐1 are constitutively expressed in the liver. Although previous research established Bcl‐xL as a critical apoptosis antagonist in differentiated hepatocytes, the significance of Mcl‐1 in the liver, especially in conjunction with Bcl‐xL, has not been clear. To examine this question, we generated hepatocyte‐specific Mcl‐1–deficient mice by crossing mcl‐1flox/flox mice and AlbCre mice and further crossed them with bcl‐xflox/flox mice, giving Mcl‐1/Bcl‐xL–deficient mice. The mcl‐1flox/flox AlbCre mice showed spontaneous apoptosis of hepatocytes after birth, as evidenced by elevated levels of serum alanine aminotransferase (ALT) and caspase‐3/7 activity and an increased number of terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate nick‐end labeling (TUNEL)‐positive cells in the liver; these phenotypes were very close to those previously found in hepatocyte‐specific Bcl‐xL–deficient mice. Although mcl‐1flox/+ AlbCre mice did not display apoptosis, their susceptibility to Fas‐mediated liver injury significantly increased. Further crossing of Mcl‐1 mice with Bcl‐xL mice showed that bcl‐xflox/+ mcl‐1flox/+ AlbCre mice also showed spontaneous hepatocyte apoptosis similar to Bcl‐xL–deficient or Mcl‐1–deficient mice. In contrast, bcl‐xflox/flox mcl‐1flox/+ AlbCre, bcl‐xflox/+ mcl‐1flox/flox AlbCre, and bcl‐xflox/flox mcl‐1flox/flox AlbCre mice displayed a decreased number of hepatocytes and a reduced volume of the liver on day 18.5 of embryogenesis and rapidly died within 1 day after birth, developing hepatic failure evidenced by increased levels of blood ammonia and bilirubin. Conclusion: Mcl‐1 is critical for blocking apoptosis in adult liver and, in the absence of Bcl‐xL, is essential for normal liver development. Mcl‐1 and Bcl‐xL are two major anti‐apoptotic Bcl‐2 family proteins expressed in the liver and cooperatively control hepatic integrity during liver development and in adult liver homeostasis in a gene dose‐dependent manner. (HEPATOLOGY 2009.)

Anticancer Chemotherapy Inhibits MHC Class I-Related Chain A Ectodomain Shedding by Downregulating ADAM10 Expression in Hepatocellular Carcinoma

MHC class I-related chain A (MICA) is a ligand for the NKG2D-activating immunoreceptor that mediates activation of natural killer (NK) cells. The ectodomain of MICA is shed from tumor cells, which may be an important means of evading antitumor immunity. We previously reported that patients with hepatocellular carcinoma (HCC) display high levels of soluble MICA in circulation, which could be downregulated by chemotherapy. The present study shows that anti-HCC drugs suppress MICA ectodomain shedding by inhibiting expression of a disintegrin and metalloproteinase 10 (ADAM10). Both ADAM10 and CD44, a typical substrate of the ADAM10 protease, were expressed in human HCC tissues and HCC cells but not in normal liver tissues or cultured hepatocytes. Small interfering RNA-mediated knockdown experiments revealed that ADAM10 is a critical sheddase for both MICA and CD44 in HCC cells. Of interest is the finding that epirubicin clearly downregulated ADAM10 expression and MICA shedding in HCC cells; its suppressive effect on MICA shedding was abolished in ADAM10-depleted cells. Epirubicin treatment also enhanced the NKG2D-mediated NK sensitivity of HCC cells. Patients with HCC had significantly higher levels of serum-soluble CD44, which correlated well with serum-soluble MICA levels, thus suggesting a close link between ADAM10 activity and MICA shedding in these patients. Soluble MICA and CD44 levels were downregulated with a significant correlation in patients treated by transarterial chemoembolization using epirubicin. In conclusion, anticancer drugs can modulate expression of ADAM10, which is critically involved in MICA ectodomain shedding. Epirubicin therapy may have a previously unrecognized effect on antitumor immunity in HCC patients.

Natural killer cell and hepatic cell interaction via NKG2A leads to dendritic cell-mediated induction of CD4+ CD25+ T cells with PD-1-dependent regulatory activities

Summary Natural killer (NK) cells have the ability to control dendritic cell (DC)‐mediated T cell responses. However, the precise mechanisms by which NK receptor‐mediated regulation of NK cells determines the magnitude and direction of DC‐mediated T cell responses remain unclear. In the present study, we applied an in vitro co‐culture system to examine the impact of NK cells cultured with hepatic cells on DC induction of regulatory T cells. We found that interaction of NK cells and non‐transformed hepatocytes (which express HLA‐E) via the NKG2A inhibitory receptor resulted in priming of DCs to induce CD4+ CD25+ T cells with regulatory properties. NKG2A triggering led to characteristic changes of the cytokine milieu of co‐cultured cells; an increase in the transforming growth factor (TGF)‐β involved in the generation of this specific type of DC, and a decrease in the tumour necrosis factor‐α capable of antagonizing the effect of TGF‐β. The regulatory cells induced by NK cell‐primed DCs exert their suppressive actions through a negative costimulator programmed death‐1 (PD‐1) mediated pathway, which differs from freshly isolated CD4+ CD25+ T cells. These findings provide new insight into the role of NK receptor signals in the DC‐mediated induction of regulatory T cells.

Serum levels of soluble major histocompatibility complex (MHC) class I-related chain A in patients with chronic liver diseases and changes during transcatheter arterial embolization for hepatocellular carcinoma

Soluble forms of major histocompatibility complex (MHC) class I‐related chain A and B (MICA/B) are increased in the sera of patients with malignancy and impair the antitumor immune response by downregulating expression of their cognate immunoreceptor natural killer group 2, member D (NKG2D). Recently, soluble MICA/B were reported to appear even in some premalignant diseases, raising questions about the impact of soluble MICA/B produced from tumors on the expression of NKG2D. The present study examined soluble MICA/B in chronic liver disease and hepatocellular carcinoma (HCC) and their involvement in the immune‐cell expression of NKG2D during transcatheter arterial embolization for HCC. The levels of soluble MICA/B were significantly higher in chronic liver disease and HCC patients than in healthy volunteers. The progression of liver disease and that of the tumor were independent determinants for soluble MICA/B levels. Immunohistochemistry revealed that MICA/B were expressed not only in HCC tissue but also on hepatocytes in cirrhotic livers. The transcatheter arterial embolization therapy significantly decreased serum levels of soluble MICA, but not soluble MICB, and increased the NKG2D expression on natural killer cells and CD8‐positive T cells; there was an inverse correlation between changes in soluble MICA levels and in NKG2D expression. In conclusion, although soluble MICA/B are produced from both HCC and premalignant cirrhotic livers, therapeutic intervention for HCC can reduce the levels of soluble MICA and thereby upregulate the expression of NKG2D. Cancer therapy may have a beneficial effect on NKG2D‐mediated antitumor immunity. (Cancer Sci 2008; 99: 1643–1649)

Signal transducer and activator of transcription 3 signaling within hepatocytes attenuates systemic inflammatory response and lethality in septic mice

Sepsis is an infection‐induced syndrome with systemic inflammatory response leading to multiorgan failure and occasionally death. During this process, signal transducer and activator of transcription 3 (STAT3) is activated in the liver, but the significance of this molecule has not been established. We generated hepatocyte‐specific STAT3‐deficient mice (L‐STAT3 KO) and examined the susceptibility of these mice to cecal ligation and puncture–induced peritonitis, a well‐established septic model. L‐STAT3 KO mice showed significantly higher mortality and produced lesser amounts of various acute phase proteins than control littermates. Although blood bacterial infection did not differ between L‐STAT3 KO mice and control mice, the former showed deterioration of the systemic inflammatory response as evidenced by a significant increase in various cytokines such as tumor necrosis factor α, IFN‐γ, IL‐6, IL‐10, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1β. A similar hyperinflammatory response was observed in another septic model caused by lipopolysaccharide (LPS) injection. In vitro analysis revealed that soluble substances derived from hepatocytes and dependent on STAT3 were critical for suppression of cytokine production from LPS‐stimulated macrophage and splenocytes. Conclusion: STAT3 activation in hepatocytes can attenuate a systemic hyperinflammatory response and lethality in sepsis, in part by suppressing immune cell overactivation, implying a critical role of hepatocyte STAT3 signaling in maintaining host homeostasis. (HEPATOLOGY 2007.)

Immunotherapy of murine colon cancer using receptor tyrosine kinase EphA2-derived peptide-pulsed dendritic cell vaccines

Further optimization of dendritic cell (DC)‐based vaccines is required clinically against advanced stage cancer. Given the broad range of expression levels observed in the recently defined tumor antigen EphA2 in a diverse types of cancers, especially in advanced stage or metastatic cancers, the authors evaluated the effectiveness of vaccination using DCs pulsed with EphA2‐derived peptides (Eph‐DCs) in a murine colon cancer model.

Identification of Multiple Transcription Factors, HLF, FTF, and E4BP4, Controlling Hepatitis B Virus Enhancer II

ABSTRACT Hepatitis B virus (HBV) enhancer II (EnII) is a hepatotropiccis element which is responsible for the hepatocyte-specific gene expression of HBV. Multiple transcription factors have been demonstrated to interact with this region. In this study, the region from HBV nucleotides (nt) 1640 to 1663 in EnII was demonstrated to be essential for enhancer activity and to be another target sequence of putative transcription factors. To elucidate the factors which bind to this region, we used a yeast one-hybrid screening system and cloned three transcription factors, HLF, FTF, and E4BP4, from a human adult liver cDNA library. All of these factors had binding affinity to the sequence from nt 1640 to 1663. Investigation of the effects of these factors on transcriptional regulation revealed that HLF and FTF had stimulatory activity on nt 1640 to 1663, whereas E4BP4 had a suppressing effect. FTF coordinately activated both 3.5-kb RNA and 2.4/2.1-kb RNA transcription in a transient transfection assay with an HBV expression vector. HLF, however, activated only 3.5-kb RNA transcription, and in primer extension analysis, HLF strongly stimulated the synthesis of pregenome RNA compared to precore RNA. Thus, FTF stimulated the activity of the second enhancer, while HLF stimulated the activity of the core upstream regulatory sequence, which affects only the core promoter, and had a dominant effect on the pregenome RNA synthesis.

Injection of IL-12 gene-transduced dendritic cells into mouse liver tumor lesions activates both innate and acquired immunity.

Dendritic cell (DC)-based vaccines have been applied clinically in the setting of advanced-stage cancer. To date, the clinical efficacy of these vaccines has been limited, possibly owing to the impairment of transferred DC function in cancer-bearing patients. In this study, we examined the therapeutic efficacy of interleukin-12 (IL-12) gene-transfected DCs isolated from tumor-bearing hosts against liver tumor. The endogenous DCs isolated from subcutaneous (s.c.) CMS4 tumor-bearing mice (CMS4DC) exhibited decreased expression levels of antigen-presenting molecules and low-allostimulatory capacity. CMS4DC produced less IL-12p70 than DCs isolated from normal mice. Adenoviral transfection of IL-12 gene into CMS4DC (AdIL12DC) restored the expression of antigen-presenting molecules and allostimulatory capacity. Intratumoral (i.t.) delivery of AdIL12DC resulted in complete rejection of intrahepatic CMS4 tumors and activation of innate and acquired immune cells. Antibody depletion studies revealed that both CD4+ and CD8+ T cells as well as natural killer cells play critical roles in mediating liver tumor rejection. I.t. treatment of AdIL12DC resulted in long-term protection against s.c. rechallenge with CMS4 tumor cells. These results revealed that IL-12 gene transfer is capable of improving the impaired functions of DC isolated from tumor-bearing hosts, and support the preclinical therapeutic efficacy of intrahepatic injection of AdIL12DC.