Sei Kakinuma

Publications (35)211.81 Total impact

• Article: α-fetoprotein levels after interferon therapy and risk of hepatocarcinogenesis in chronic hepatitis C.

  Yasuhiro Asahina, Kaoru Tsuchiya, Takashi Nishimura, Masaru Muraoka, Yuichiro Suzuki, Nobuharu Tamaki, Yutaka Yasui, Takanori Hosokawa, Ken Ueda, Hiroyuki Nakanishi, Jun Itakura, Yuka Takahashi, Masayuki Kurosaki, Nobuyuki Enomoto, Mina Nakagawa, Sei Kakinuma, Mamoru Watanabe, Namiki Izumi
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  ABSTRACT: The effects of interferon (IFN) treatment and the post-IFN treatment α-fetoprotein (AFP) levels on risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C (CHC) are unknown. To determine the relationship between AFP and alanine transaminase (ALT) levels and HCC risk, the cohort consisted of 1818 patients histologically proven to have CHC treated with IFN were studied. Cumulative incidence and HCC risk were analyzed over a mean follow-up period of 6.1 years using the Kaplan-Meier method and Cox proportional hazard analysis. HCC developed in 179 study subjects. According to multivariate analysis, older age, male gender, advanced fibrosis, severe steatosis, lower serum albumin levels, sustained virological response (SVR), and higher post-IFN treatment ALT or AFP levels were identified as independent factors significantly associated with HCC development. Cutoff values for ALT and AFP for prediction of future HCC were determined as 40 IU/L and 6.0 ng/mL, respectively, and negative predictive values of these cutoffs were high at 0.960 in each value. Cumulative incidence of HCC was significantly lower in patients whose post-IFN treatment ALT and AFP levels were suppressed to less than the cutoff values even in non- SVR patients. This suppressive effect was also found in patients whose post-IFN treatment ALT and AFP levels were reduced to less than the cutoff values despite abnormal pre-treatment levels. Conclusion: Post-IFN treatment ALT and AFP levels are significantly associated with the hepatocarcinogenesis. Measurement of these values is useful for predicting future HCC risk after IFN treatment. Suppression of these values after IFN therapy reduces HCC risk even in patients without HCV eradication. (HEPATOLOGY 2013.).
  Hepatology 04/2013; · 11.66 Impact Factor
• Article: Wnt5a signaling mediates biliary differentiation of fetal hepatic stem/progenitor cells.

  Kei Kiyohashi, Sei Kakinuma, Akihide Kamiya, Naoya Sakamoto, Sayuri Nitta, Hideto Yamanaka, Kouhei Yoshino, Junko Fijuki, Miyako Murakawa, Akiko Kusano-Kitazume, Hiromichi Shimizu, Ryuichi Okamoto, Seishin Azuma, Mina Nakagawa, Yasuhiro Asahina, Naoki Tanimizu, Akira Kikuchi, Hiromitsu Nakauchi, Mamoru Watanabe
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  ABSTRACT: The molecular mechanisms regulating differentiation of fetal hepatic stem/progenitor cells, called hepatoblasts, which play pivotal roles in liver development, remain obscure. Wnt signaling pathways regulate the development and differentiation of stem cells in various organs. While a β-catenin-independent non-canonical Wnt pathway is essential for cell adhesion and polarity, the physiological functions of non-canonical Wnt pathways in liver development are unknown. Here we describe a functional role for Wnt5a, a non-canonical Wnt ligand, in the differentiation of mouse hepatoblasts. Wnt5a was expressed in mesenchymal cells and other cells of wild-type mid-gestational fetal liver. We analyzed fetal liver phenotypes in Wnt5a-deficient mice using a combination of histological and molecular techniques. Expression levels of Sox9 and the number of HNF1β-positive HNF4α-negative biliary precursor cells were significantly higher in Wnt5a-deficient liver relative to wild-type liver. In Wnt5a-deficient fetal liver, in vivo formation of primitive bile ductal structures was significantly enhanced relative to wild-type littermates. We also investigated the function of Wnt5a protein and downstream signaling molecules using a three-dimensional culture system that included primary hepatoblasts or a hepatic progenitor cell line. In vitro differentiation assays showed that Wnt5a retarded the formation of bile duct-like structures in hepatoblasts, leading instead to hepatic maturation of such cells. While Wnt5a signaling increased steady-state levels of phosphorylated Calcium/calmodulin-dependent protein kinase II (CaMKII) in fetal liver, inhibition of CaMKII activity resulted in the formation of significantly more and larger-sized bile duct-like structures in vitro compared with those in vehicle-supplemented controls. Conclusions: We demonstrate that Wnt5a-mediated signaling in fetal hepatic stem/progenitor cells suppresses biliary differentiation. We also suggest that activation of CaMKII by Wnt5a signaling suppresses biliary differentiation. (HEPATOLOGY 2013.).
  Hepatology 02/2013; · 11.66 Impact Factor
• Article: Hepatitis C virus NS4B protein targets STING and abrogates RIG-I-mediated type-I interferon-dependent innate immunity.

  Sayuri Nitta, Naoya Sakamoto, Mina Nakagawa, Sei Kakinuma, Kako Mishima, Akiko Kusano-Kitazume, Kei Kiyohashi, Miyako Murakawa, Yuki Nishimura-Sakurai, Seishin Azuma, Megumi Tasaka-Fujita, Yasuhiro Asahina, Mitsutoshi Yoneyama, Takashi Fujita, Mamoru Watanabe
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  ABSTRACT: Hepatitis C virus (HCV) infection blocks cellular interferon (IFN)-mediated antiviral signaling through cleavage of Cardif by HCV-NS3/4A serine protease. Like NS3/4A, NS4B protein strongly blocks IFN-β production signaling mediated by retinoic-acid-inducible protein I (RIG-I) (Tasaka et al. J Gen Virol 2007); however, the underlying molecular mechanisms are not well understood. Recently, the stimulator of interferon genes (STING) was identified as an activator of RIG-I signaling. STING possesses a structural homology domain with flaviviral NS4B, which suggests a direct protein-protein interaction. In the present study, we investigated the molecular mechanisms by which NS4B targets RIG-I induced- and STING-mediated IFN-β production signaling. IFN-β promoter reporter assay showed that IFN-β promoter activation induced by RIG-I or Cardif was significantly suppressed by both NS4B and NS3/4A, while STING-induced IFN-β activation was suppressed by NS4B but not by NS3/4A, suggesting that NS4B had a distinct point of interaction. Immunostaining showed that STING colocalized with NS4B in the endoplasmic reticulum. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays demonstrated that NS4B specifically bound STING. Intriguingly, NS4B expression blocked the protein interaction between STING and Cardif, which is required for robust IFN-β activation. NS4B truncation assays showed that its N-terminus, containing the STING-homology domain, was necessary for the suppression of IFN-β promoter activation. NS4B suppressed residual IFN-β activation by an NS3/4A-cleaved Cardif (Cardif1-508), suggesting that NS3/4A and NS4B may cooperate in the blockade of IFN-β production. CONCLUSION: NS4B suppresses RIG-I-mediated IFN-β production signaling through a direct protein interaction with STING. Disruption of that interaction may restore cellular antiviral responses and may constitute a novel therapeutic strategy for the eradication of HCV. (HEPATOLOGY 2012.).
  Hepatology 08/2012; · 11.66 Impact Factor
• Article: Data mining model using simple and readily available factors could identify patients at high risk for hepatocellular carcinoma in chronic hepatitis C.

  Masayuki Kurosaki, Naoki Hiramatsu, Minoru Sakamoto, Yoshiyuki Suzuki, Manabu Iwasaki, Akihiro Tamori, Kentaro Matsuura, Sei Kakinuma, Fuminaka Sugauchi, Naoya Sakamoto, Mina Nakagawa, Namiki Izumi
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  ABSTRACT: Assessment of the risk of hepatocellular carcinoma (HCC) development is essential for formulating personalized surveillance or antiviral treatment plan for chronic hepatitis C. We aimed to build a simple model for the identification of patients at high risk of developing HCC. Chronic hepatitis C patients followed for at least 5 years (n=1003) were analyzed by data mining to build a predictive model for HCC development. The model was externally validated using a cohort of 1072 patients (472 with sustained virological response (SVR) and 600 with nonSVR to PEG-interferon plus ribavirin therapy). On the basis of factors such as age, platelet, albumin, and aspartate aminotransferase, the HCC risk prediction model identified subgroups with high-, intermediate-, and low-risk of HCC with a 5-year HCC development rate of 20.9%, 6.3-7.3%, and 0-1.5%, respectively. The reproducibility of the model was confirmed through external validation (r(2)=0.981). The 10-year HCC development rate was also significantly higher in the high-and intermediate-risk group than in the low-risk group (24.5% vs. 4.8%; p<0.0001). In the high-and intermediate-risk group, the incidence of HCC development was significantly reduced in patients with SVR compared to those with nonSVR (5-year rate, 9.5% vs. 4.5%; p=0.040). The HCC risk prediction model uses simple and readily available factors and identifies patients at a high risk of HCC development. The model allows physicians to identify patients requiring HCC surveillance and those who benefit from IFN therapy to prevent HCC.
  Journal of Hepatology 03/2012; 56(3):602-8. · 9.26 Impact Factor
• Article: Age and total ribavirin dose are independent predictors of relapse after interferon therapy in chronic hepatitis C revealed by data mining analysis.

  Masayuki Kurosaki, Naoki Hiramatsu, Minoru Sakamoto, Yoshiyuki Suzuki, Manabu Iwasaki, Akihiro Tamori, Kentaro Matsuura, Sei Kakinuma, Fuminaka Sugauchi, Naoya Sakamoto, Mina Nakagawa, Hiroshi Yatsuhashi, Namiki Izumi
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  ABSTRACT: This study aimed to define factors associated with relapse among responders to pegylated interferon (PEG-IFN) plus ribavirin (RBV) therapy in chronic hepatitis C. A cohort of genotype 1b chronic hepatitis C patients treated with PEG-IFN plus RBV and who had an undetectable HCV RNA by week 12 (n=951) were randomly assigned to model derivation (n=636) or internal validation (n=315) groups. An independent cohort (n=598) were used for an external validation. A decision tree model for relapse was explored using data mining analysis. The data mining analysis defined five subgroups of patients with variable rates of relapse ranging from 13% to 52%. The reproducibility of the model was confirmed by internal and external validations (r(2)=0.79 and 0.83, respectively). Patients with undetectable HCV RNA at week 4 had the lowest risk of relapse (13%), followed by patients <60 years with undetectable HCV RNA at week 5-12 who received ≥3.0 g/kg of body weight of RBV (16%). Older patients with a total RBV dose <3.0 g/kg had the highest risk of relapse (52%). Higher RBV dose beyond 3.0 g/kg was associated with further decrease of relapse rate among patients <60 years (up to 11%) but not among older patients whose relapse rate remained stable around 30%. Data mining analysis revealed that time to HCV RNA negativity, age and total RBV dose was associated with relapse. To prevent relapse, ≥3.0 g/kg of RBV should be administered. Higher dose of RBV may be beneficial in patients <60 years.
  Antiviral therapy 01/2012; 17(1):35-43. · 3.16 Impact Factor
• Article: Identification of novel N-(morpholine-4-carbonyloxy) amidine compounds as potent inhibitors against hepatitis C virus replication.

  Akiko Kusano-Kitazume, Naoya Sakamoto, Yukiko Okuno, Yuko Sekine-Osajima, Mina Nakagawa, Sei Kakinuma, Kei Kiyohashi, Sayuri Nitta, Miyako Murakawa, Seishin Azuma, Yuki Nishimura-Sakurai, Masatoshi Hagiwara, Mamoru Watanabe
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  ABSTRACT: To identify novel compounds that possess antiviral activity against hepatitis C virus (HCV), we screened a library of small molecules with various amounts of structural diversity using an HCV replicon-expressing cell line and performed additional validations using the HCV-JFH1 infectious-virus cell culture. Of 4,004 chemical compounds, we identified 4 novel compounds that suppressed HCV replication with 50% effective concentrations of ranging from 0.36 to 4.81 μM. N'-(Morpholine-4-carbonyloxy)-2-(naphthalen-1-yl) acetimidamide (MCNA) was the most potent and also produced a small synergistic effect when used in combination with alpha interferon. Structure-activity relationship (SAR) analyses revealed 4 derivative compounds with antiviral activity. Further SAR analyses revealed that the N-(morpholine-4-carbonyloxy) amidine moiety was a key structural element for antiviral activity. Treatment of cells with MCNA activated nuclear factor κB and downstream gene expression. In conclusion, N-(morpholine-4-carbonyloxy) amidine and other related morpholine compounds specifically suppressed HCV replication and may have potential as novel chemotherapeutic agents.
  Antimicrobial Agents and Chemotherapy 12/2011; 56(3):1315-23. · 4.84 Impact Factor
• Article: Analysis of interferon signaling by infectious hepatitis C virus clones with substitutions of core amino acids 70 and 91.

  Yusuke Funaoka, Naoya Sakamoto, Goki Suda, Yasuhiro Itsui, Mina Nakagawa, Sei Kakinuma, Takako Watanabe, Kako Mishima, Mayumi Ueyama, Izumi Onozuka, Sayuri Nitta, Akiko Kitazume, Kei Kiyohashi, Miyako Murakawa, Seishin Azuma, Kiichiro Tsuchiya, Mamoru Watanabe
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  ABSTRACT: Substitution of amino acids 70 and 91 in the hepatitis C virus (HCV) core region is a significant predictor of poor responses to peginterferon-plus-ribavirin therapy, while their molecular mechanisms remain unclear. Here we investigated these differences in the response to alpha interferon (IFN) by using HCV cell culture with R70Q, R70H, and L91M substitutions. IFN treatment of cells transfected or infected with the wild type or the mutant HCV clones showed that the R70Q, R70H, and L91M core mutants were significantly more resistant than the wild type. Among HCV-transfected cells, intracellular HCV RNA levels were significantly higher for the core mutants than for the wild type, while HCV RNA in culture supernatant was significantly lower for these mutants than for the wild type. IFN-induced phosphorylation of STAT1 and STAT2 and expression of the interferon-inducible genes were significantly lower for the core mutants than for the wild type, suggesting cellular unresponsiveness to IFN. The expression level of an interferon signal attenuator, SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type. Interleukin 6 (IL-6), which upregulates SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type, suggesting interferon resistance, possibly through IL-6-induced, SOCS3-mediated suppression of interferon signaling. Expression levels of endoplasmic reticulum (ER) stress proteins were significantly higher in cells transfected with a core mutant than in those transfected with the wild type. In conclusion, HCV R70 and L91 core mutants were resistant to interferon in vitro, and the resistance may be induced by IL-6-induced upregulation of SOCS3. Those mechanisms may explain clinical interferon resistance of HCV core mutants.
  Journal of Virology 06/2011; 85(12):5986-94. · 5.40 Impact Factor
• Article: Studies on virus kinetics using infectious fluorescence-tagged hepatitis C virus cell culture.

  Machi Yamamoto, Naoya Sakamoto, Tetsuya Nakamura, Yasuhiro Itsui, Mina Nakagawa, Yuki Nishimura-Sakurai, Sei Kakinuma, Seishin Azuma, Kiichiro Tsuchiya, Takanobu Kato, Takaji Wakita, Mamoru Watanabe
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  ABSTRACT: Aim:  Studies of the complete hepatitis C virus (HCV) life cycle have become possible with the development of a HCV-JFH1 cell culture system. Methods:  In this study, we constructed two fluorescence protein-tagged recombinant JFH1 virus clones, JFH1-EYFP and JFH1-AsRed, as well as two corresponding clones with adaptive mutations, JFH1-EYFP mutant and JFH1-AsRed mutant, that and were as effective as JFH1 in producing infectious virus particles, and investigated their viral infection life cycles. Results:  After infection of the fluorescence-tagged mutant viruses, infected cells increased exponentially. In cells, EYFP or AsRed and NS5A were expressed as a fusion protein and co-localized in core proteins. The rate of the cell-cell spread was dependent on the cell densities with a maximum of 10(2.5) /day. Treatment of cells with interferon or a protease inhibitor suppressed expansion of virus-positive cells. Conclusion:  Taken together, these results indicate that fluorescence-tagged HCV is a useful tool to study virus infection life cycles and to assist in the search for novel antiviral compounds.
  Hepatology Research 03/2011; 41(3):258-69. · 2.20 Impact Factor
• Article: Inhibitory effect of a triterpenoid compound, with or without alpha interferon, on hepatitis C virus infection.

  Takako Watanabe, Naoya Sakamoto, Mina Nakagawa, Sei Kakinuma, Yasuhiro Itsui, Yuki Nishimura-Sakurai, Mayumi Ueyama, Yusuke Funaoka, Akiko Kitazume, Sayuri Nitta, Kei Kiyohashi, Miyako Murakawa, Seishin Azuma, Kiichiro Tsuchiya, Shinya Oooka, Mamoru Watanabe
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  ABSTRACT: A lack of patient response to alpha interferon (α-IFN) plus ribavirin (RBV) treatment is a major problem in eliminating hepatitis C virus (HCV). We screened chemical libraries for compounds that enhanced cellular responses to α-IFN and identified a triterpenoid, toosendanin (TSN). Here, we studied the effects and mechanisms of action of TSN on HCV replication and its effect on α-IFN signaling. We treated HCV genotype 1b replicon-expressing cells and HCV-J6/JFH-infected cells with TSN, with or without α-IFN, and the level of HCV replication was quantified. To study the effects of TSN on α-IFN signaling, we detected components of the interferon-stimulated gene factor 3 (ISGF3), phosphorylated signal transducer and activator of transcription 1 (STAT1), and STAT2 by Western blotting analysis; expression levels of mRNA of interferon regulatory factor 9 using real-time reverse transcription-PCR (RT-PCR); and interferon-stimulated response element reporter activity and measured the expression levels of interferon-inducible genes for 2',5'-oligoadenylate synthetase, MxA, protein kinase R, and p56 using real-time RT-PCR. TSN alone specifically inhibited expression of the HCV replicon (50% effective concentration = 20.6 nM, 50% cytotoxic concentration > 3 μM, selectivity index > 146). Pretreatment with TSN prior to α-IFN treatment was more effective in suppressing HCV replication than treatment with either drug alone. Although TSN alone did not activate the α-IFN pathway, it significantly enhanced the α-IFN-induced increase of phosphorylated STATs, interferon-stimulated response element activation, and interferon-stimulated gene expression. TSN significantly increased baseline expression of interferon regulatory factor 9, a component of interferon-stimulated gene factor 3. Antiviral effects of treatment with α-IFN can be enhanced by pretreatment with TSN. Its mechanisms of action could potentially be important to identify novel molecular targets to treat HCV infection.
  Antimicrobial Agents and Chemotherapy 03/2011; 55(6):2537-45. · 4.84 Impact Factor
• Article: Association of IL28B variants with response to pegylated-interferon alpha plus ribavirin combination therapy reveals intersubgenotypic differences between genotypes 2a and 2b.

  Naoya Sakamoto, Mina Nakagawa, Yasuhito Tanaka, Yuko Sekine-Osajima, Mayumi Ueyama, Masayuki Kurosaki, Nao Nishida, Akihiro Tamori, Nishimura-Sakurai Yuki, Yasuhiro Itsui, Seishin Azuma, Sei Kakinuma, Shuhei Hige, Yoshito Itoh, Eiji Tanaka, Yoichi Hiasa, Namiki Izumi, Katsushi Tokunaga, Masashi Mizokami, Mamoru Watanabe
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  ABSTRACT: Genetic polymorphisms of the interleukin 28B (IL28B) locus are associated closely with outcomes of pegylated-interferon (PEG-IFN) plus ribavirin (RBV) combination therapy. The aim of this study was to investigate the relationship between IL28B polymorphism and responses to therapy in patients infected with genotype 2. One hundred twenty-nine chronic hepatitis C patients infected with genotype 2, 77 patients with genotype 2a and 52 patients with genotype 2b, were analyzed. Clinical and laboratory parameters, including genetic variation near the IL28B gene (rs8099917), were assessed. Drug adherence was monitored in each patient. Univariate and multivariate statistical analyses of these parameters and clinical responses were carried out. Univariate analyses showed that a sustained virological response was correlated significantly with IL28B polymorphism, as well as age, white blood cell and neutrophil counts, adherence to RBV, and rapid virological response. Subgroup analysis revealed that patients infected with genotype 2b achieved significantly lower rapid virological response rates than those with genotype 2a. Patients with the IL28B-major allele showed higher virus clearance rates at each time point than those with the IL28B-minor allele, and the differences were more profound in patients infected with genotype 2b than those with genotype 2a. Furthermore, both rapid and sustained virological responses were associated significantly with IL28B alleles in patients with genotype 2b. IL28B polymorphism was predictive of PEG-IFN plus RBV combination treatment outcomes in patients infected with genotype 2 and, especially, with genotype 2b. In conclusion, IL-28B polymorphism affects responses to PEG-IFN-based treatment in difficult-to-treat HCV patients.
  Journal of Medical Virology 02/2011; 83(5):871-8. · 2.82 Impact Factor
• Article: Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice.

  Izumi Onozuka, Sei Kakinuma, Akihide Kamiya, Masato Miyoshi, Naoya Sakamoto, Kei Kiyohashi, Takako Watanabe, Yusuke Funaoka, Mayumi Ueyama, Mina Nakagawa, Naohiko Koshikawa, Motoharu Seiki, Hiromitsu Nakauchi, Mamoru Watanabe
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  ABSTRACT: Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.
  Biochemical and Biophysical Research Communications 02/2011; 406(1):134-40. · 2.48 Impact Factor
• Article: Serum interleukin-6 levels correlate with resistance to treatment of chronic hepatitis C infection with pegylated-interferon-α2b plus ribavirin.

  Mayumi Ueyama, Mina Nakagawa, Naoya Sakamoto, Izumi Onozuka, Yusuke Funaoka, Takako Watanabe, Sayuri Nitta, Kei Kiyohashi, Akiko Kitazume, Miyako Murakawa, Yuki Nishimura-Sakurai, Yuko Sekine-Osajima, Yasuhiro Itsui, Seishin Azuma, Sei Kakinuma, Mamoru Watanabe
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  ABSTRACT: Interleukin (IL)-6, a pleiotropic cytokine, is increased in various types of chronic liver disease, including chronic hepatitis C (CHC). It was reported recently that IL-6 is associated with insulin resistance, iron metabolism and interferon resistance, which may affect the outcome of antiviral treatment. In this study, we investigated the association of serum IL-6 levels with outcomes of pegylated interferon (PEG-IFN) plus ribavirin (RBV) combination therapy. We included 149 CHC patients and measured serum IL-6 levels at baseline and at 4, 8 and 12 weeks, and the end of treatment in 49 patients. We performed univariate and multivariate regression analyses for the association of IL-6 levels and clinical and laboratory parameters and treatment responses. Serum IL-6 levels were significantly higher in CHC patients than healthy subjects. Pretreatment IL-6 levels of male patients were inversely correlated with sustained virological response (SVR) in univariate analysis (P=0.012). In male patients with SVR, serum IL-6 levels decreased significantly at 4 weeks of treatment (P=0.029) and remained significantly lower than those of non-SVR patients after 4, 8 and 12 weeks of PEG-IFN plus RBV therapy. Our results suggest that baseline levels of IL-6, as well as their decrease during treatment, are correlated to outcomes of PEG-IFN plus RBV therapy in male patients. Further analyses of IL-6 may provide new strategies for difficult-to-treat CHC patients and prevention of hepatocarcinogenesis.
  Antiviral therapy 01/2011; 16(7):1081-91. · 3.16 Impact Factor
• Article: IL-6-mediated intersubgenotypic variation of interferon sensitivity in hepatitis C virus genotype 2a/2b chimeric clones.

  Goki Suda, Naoya Sakamoto, Yasuhiro Itsui, Mina Nakagawa, Megumi Tasaka-Fujita, Yusuke Funaoka, Takako Watanabe, Sayuri Nitta, Kei Kiyohashi, Seishin Azuma, Sei Kakinuma, Kiichiro Tsuchiya, Michio Imamura, Nobuhiko Hiraga, Kazuaki Chayama, Mamoru Watanabe
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  ABSTRACT: Mechanisms of difference in interferon sensitivity between hepatitis C virus (HCV) strains have yet to be clarified. Here, we constructed an infectious genotype2b clone and analyzed differences in interferon-alpha sensitivity between HCV-2b and 2a-JFH1 clones using intergenotypic homologous recombination. The HCV-2b/JFH1 chimeric virus able to infect Huh7.5.1 cells and was significantly more sensitive to IFN than JFH1. IFN-induced expression of MxA and 25-OAS was significantly lower in JFH1 than in 2b/JFH1-infected cells. In JFH1-infected cells, expression of SOCS3 and its inducer, IL-6, was significantly higher than in 2b/JFH1-infected cells. The IFN-resistance of JFH1 cells was negated by siRNA-knock down of SOCS3 expression and by pretreatment with anti-IL6 antibody. In conclusion, intergenotypic differences of IFN sensitivity of HCV may be attributable to the sequences of HCV structural proteins and can be determined by SOCS3 and IL-6 expression levels.
  Virology 11/2010; 407(1):80-90. · 3.35 Impact Factor
• Article: ITPA gene variant protects against anemia induced by pegylated interferon‐α and ribavirin therapy for Japanese patients with chronic hepatitis C

  Naoya Sakamoto, Yasuhito Tanaka, Mina Nakagawa, Hiroshi Yatsuhashi, Shuhei Nishiguchi, Nobuyuki Enomoto, Seishin Azuma, Yuki Nishimura-Sakurai, Sei Kakinuma, Nao Nishida, Katsushi Tokunaga, Masao Honda, Kiyoaki Ito, Masashi Mizokami, Mamoru Watanabe
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  ABSTRACT: Aim: Host genetic variants leading to inosine triphosphatase (ITPA) deficiency, a condition not thought to be clinically important, protect against hemolytic anemia in chronic hepatitis C patients receiving ribavirin. In this study, we evaluated the clinical significance of ITPA variants in Japanese hepatitis C patients who were treated with pegylated interferon plus ribavirin.Methods: In this multicenter retrospective cross-sectional study, 474 hepatitis C patients were enrolled who were treated with pegylated interferon plus ribavirin in four geographically different hospitals in Japan. Patients were grouped according to hemoglobin decline of more than 3 g/dL at week 4. Two single nucleotide polymorphisms (SNP) within or adjacent to the ITPA gene (rs6051702, rs1127354) were genotyped.Results: A functional SNP, rs1127354, within the ITPA exon was strongly associated with protection against anemia with only one (0.8%) in 129 patients with the ITPA minor variant A developing severe anemia (P = 5.9 × 10−20). For rs6051702, which had significant association in European-Americans, significant but weak association with severe hemoglobin reduction was found in Japanese (P = 0.009). In patients excluding genotype 1b and high viral load, those with the ITPA minor variant A achieved significantly higher sustained viral response rate than those with the major variant (CC) (96% vs 70%, respectively, P = 0.0066).Conclusion: ITPA SNP, rs1127354, is confirmed to be a useful predictor of ribavirin-induced anemia in Japanese patients. Patients with the ITPA minor variant A (∼27%) have an advantage in pegylated interferon plus ribavirin-based therapies, due to expected adherence of ribavirin doses, resulting in a higher viral clearance rate.
  Hepatology Research 10/2010; 40(11):1063 - 1071. · 2.20 Impact Factor
• Article: Cell culture and in vivo analyses of cytopathic hepatitis C virus mutants.

  Kako Mishima, Naoya Sakamoto, Yuko Sekine-Osajima, Mina Nakagawa, Yasuhiro Itsui, Seishin Azuma, Sei Kakinuma, Kei Kiyohashi, Akiko Kitazume, Kiichiro Tsuchiya, Michio Imamura, Nobuhiko Hiraga, Kazuaki Chayama, Takaji Wakita, Mamoru Watanabe
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  ABSTRACT: HCV-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 8 weeks 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.
  Virology 09/2010; 405(2):361-9. · 3.35 Impact Factor
• Article: Mutations in the interferon sensitivity determining region and virological response to combination therapy with pegylated-interferon alpha 2b plus ribavirin in patients with chronic hepatitis C-1b infection.

  Mina Nakagawa, Naoya Sakamoto, Mayumi Ueyama, Kaoru Mogushi, Satoshi Nagaie, Yasuhiro Itsui, Seishin Azuma, Sei Kakinuma, Hiroshi Tanaka, Nobuyuki Enomoto, Mamoru Watanabe
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  ABSTRACT: Pegylated-interferon-alpha 2b (PEG-IFN) plus ribavirin (RBV) therapy is currently the de-facto standard treatment for hepatitis C virus (HCV) infection. The aims of this study were to analyze the clinical and virological factors associated with a higher rate of response in patients with HCV genotype 1b infection treated with combination therapy. We analyzed, retrospectively, 239 patients with chronic hepatitis C-1b infection who received 48 weeks of combination therapy. We assessed clinical and laboratory parameters, including age, gender, pretreatment hemoglobin, platelet counts, HCV RNA titer, liver histology, the number of interferon sensitivity determining region (ISDR) mutations and substitutions of the core amino acids 70 and 91. Drug adherence was monitored in each patient. We carried out univariate and multivariate statistical analyses of these parameters and clinical responses. On an intention-to-treat (ITT) analysis, 98 of the 239 patients (41%) had sustained virological responses (SVRs). Patients with more than two mutations in the ISDR had significantly higher SVR rates (P<0.01). Univariate analyses showed that stage of fibrosis, hemoglobin, platelet counts, ISDR mutations, serum HCV RNA level, and adherence to PEG-IFN plus RBV were significantly correlated with SVR rates. Multivariate analysis in subjects with good drug adherence extracted the number of ISDR mutations (two or more: odds ratio [OR] 5.181). The number of mutations in the ISDR sequence of HCV-1b (>or=2) is the most effective parameter predicting a favorable clinical outcome of 48-week PEG-IFN plus RBV therapy in patients with HCV genotype 1b infection.
  Journal of Gastroenterology 06/2010; 45(6):656-65. · 4.16 Impact Factor
• Article: Inhibition of hepatitis C virus replication by a specific inhibitor of serine-arginine-rich protein kinase.

  Yuko Karakama, Naoya Sakamoto, Yasuhiro Itsui, Mina Nakagawa, Megumi Tasaka-Fujita, Yuki Nishimura-Sakurai, Sei Kakinuma, Masaya Oooka, Seishin Azuma, Kiichiro Tsuchiya, Hiroshi Onogi, Masatoshi Hagiwara, Mamoru Watanabe
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  ABSTRACT: Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection.
  Antimicrobial Agents and Chemotherapy 05/2010; 54(8):3179-86. · 4.84 Impact Factor
• Article: Comparison of HCV-associated gene expression and cell signaling pathways in cells with or without HCV replicon and in replicon-cured cells.

  Yuki Nishimura-Sakurai, Naoya Sakamoto, Kaoru Mogushi, Satoshi Nagaie, Mina Nakagawa, Yasuhiro Itsui, Megumi Tasaka-Fujita, Yuko Onuki-Karakama, Goki Suda, Kako Mishima, [......], Mayumi Ueyama, Yusuke Funaoka, Takako Watanabe, Seishin Azuma, Yuko Sekine-Osajima, Sei Kakinuma, Kiichiro Tsuchiya, Nobuyuki Enomoto, Hiroshi Tanaka, Mamoru Watanabe
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  ABSTRACT: Hepatitis C virus (HCV) replication is affected by several host factors. Here, we screened host genes and molecular pathways that are involved in HCV replication by comprehensive analyses using two genotypes of HCV replicon-expressing cells, their cured cells and naïve Huh7 cells. Huh7 cell lines that stably expressed HCV genotype 1b or 2a replicon were used. The cured cells were established by treating HCV replicon cells with interferon-alpha. Expression of 54,675 cellular genes was analyzed by GeneChip DNA microarray. The data were analyzed by using the KEGG Pathway database. Hierarchical clustering analysis showed that the gene-expression profiles of each cell group constituted clear clusters of naïve, HCV replicon-expressed, and cured cell lines. The pathway process analysis between the replicon-expressing and the cured cell lines identified significantly altered pathways, including MAPK, steroid biosynthesis and TGF-beta signaling pathways, suggesting that these pathways were affected directly by HCV replication. Comparison of cured and naïve Huh7 cells identified pathways, including steroid biosynthesis and sphingolipid metabolism, suggesting that these pathways were required for efficient HCV replication. Cytoplasmic lipid droplets were obviously increased in replicon-expressing and cured cells as compared to naïve cells. HCV replication was significantly suppressed by peroxisome proliferator-activated receptor (PPAR)-alpha agonists but augmented by PPAR-gamma agonists. Comprehensive gene expression and pathway analyses show that lipid biosynthesis pathways are crucial to support proficient virus replication. These metabolic pathways could constitute novel antiviral targets against HCV.
  Journal of Gastroenterology 12/2009; 45(5):523-36. · 4.16 Impact Factor
• Article: Antiviral effects of the interferon-induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein.

  Yasuhiro Itsui, Naoya Sakamoto, Sei Kakinuma, Mina Nakagawa, Yuko Sekine-Osajima, Megumi Tasaka-Fujita, Yuki Nishimura-Sakurai, Gouki Suda, Yuko Karakama, Kako Mishima, Machi Yamamoto, Takako Watanabe, Mayumi Ueyama, Yusuke Funaoka, Seishin Azuma, Mamoru Watanabe
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  ABSTRACT: Interferons (IFNs) and the interferon-stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We have reported previously that ISGs, including guanylate binding protein 1 (GBP-1), interferon alpha inducible protein (IFI)-6-16, and IFI-27, inhibit HCV subgenomic replication. In this study we investigated the effects of these ISGs against HCV in cell culture and their direct molecular interaction with viral proteins. HCV replication and virus production were suppressed significantly by overexpression of GBP-1, IFI-6-16, or IFI-27. Knockdown of the individual ISGs enhanced HCV RNA replication markedly. A two-hybrid panel of molecular interaction of the ISGs with HCV proteins showed that GBP-1 bound HCV-NS5B directly. A protein truncation assay showed that the guanine binding domain of GBP-1 and the finger domain of NS5B were involved in the interaction. Binding of NS5B with GBP-1 inhibited its guanosine triphosphatase GTPase activity, which is essential for its antiviral effect. Taken together, interferon-induced GBP-1 showed antiviral activity against HCV replication. CONCLUSION: Binding of the HCV-NS5B protein to GBP-1 countered the antiviral effect by inhibition of its GTPase activity. These mechanisms may contribute to resistance to innate, IFN-mediated antiviral defense and to the clinical persistence of HCV infection.
  Hepatology 08/2009; 50(6):1727-37. · 11.66 Impact Factor
• Article: Enrichment and clonal culture of progenitor cells during mouse postnatal liver development in mice.

  Akihide Kamiya, Sei Kakinuma, Yuji Yamazaki, Hiromitsu Nakauchi
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  ABSTRACT: Stem and progenitor cells exist in normal postnatal livers. However, it has not been possible to clonally isolate or analyze postnatal liver stem/progenitor-like cells (PLSCs) derived from noninjured livers because of a lack of specific surface markers. This study aimed to establish a primary culture system for clone-sorted PLSCs. To investigate proliferation and differentiation of PLSCs, subpopulations of nonparenchymal cells derived from noninjured livers were purified and cultured using a single-cell culture system. Cells were grown in fetal liver cell-derived conditioned medium in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632. We identified CD13 and CD133 as markers expressed on the PLSC-containing population in noninjured livers and established an efficient single-cell culture system to clonally analyze PLSCs. Culture of PLSCs is difficult, even using conditioned medium, but the addition of Y-27632 increased PLSC cell proliferation. The proportion of progenitor cells among nonparenchymal cells decreased during postnatal liver development; however, a PLSC population was still preserved in 3-month-old mice. Long-term cultivated cells derived from clone-sorted cells in normal livers were established and were called normal-liver-derived stem-like cells (NLS cells). NLS cells could differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions and underwent self-renewal-like activity in serial reclone-sorted culture. CD13 and CD133 were expressed on progenitor cells derived from fetal and postnatal liver, whereas CD49f (integrin alpha6 subunit) was strongly expressed only on PLSCs. These results demonstrate the presence of progenitor cells in the CD13(+)CD49f(+)CD133(+) subpopulation of nonhematopoietic cells derived from noninjured postnatal livers.
  Gastroenterology 07/2009; 137(3):1114-26, 1126.e1-14. · 11.68 Impact Factor