Sei Kakinuma

Human Umbilical Cord Blood as a Source of Transplantable Hepatic Progenitor Cells

Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation because of the immaturity of newborn cells compared with adult cells. In contrast to their hematopoietic and mesenchymal potential, it remains unclear whether UCB cells have endodermal competence. Here, with a view to utilize UCB cells for cell transplantation into injured liver, we investigated the hepatic potential of UCB cells both in vitro and in vivo. We determined the most efficient conditions leading UCB cells to produce albumin (ALB). In a novel primary culture system supplemented with a combination of growth/differentiation factors, about 50% of UCB cells in 21‐day cultures expressed ALB, and the ALB+ cells coexpressed hepatocyte lineage markers. The ALB‐expressing cells were able to proliferate in the culture system. Moreover, in the cell‐transplantation model into liver‐injured severe combined immunodeficient mice, inoculated UCB cells developed into functional hepatocytes in the liver, which released human ALB into the sera of the recipient mice. In conclusion, this study demonstrates that human UCB is a source of transplantable hepatic progenitor cells. Our findings may have relevance to clinical application of UCB‐derived cell transplantation as a novel therapeutic option for liver failure.

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

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, a cohort consisting of 1,818 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, non sustained virological response (non‐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. The 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 pretreatment levels. Conclusion: Post‐IFN treatment ALT and AFP levels are significantly associated with 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;58:1253–1262)

Synergistic Inhibition of Intracellular Hepatitis C Virus Replication by Combination of Ribavirin and Interferon-α

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

Antiviral effects of the interferon‐induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein

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 2009.)

Radiation therapy in combination with transcatheter arterial chemoembolization for hepatocellular carcinoma with extensive portal vein involvement

The aim of this study was to examine the effectiveness and toxicity of radiation therapy in combination with transcatheter arterial chemoembolization (TACE) for hepatocellular carcinoma (HCC) with extensive portal vein tumor thrombus (PVTT).

Sall4 Regulates Cell Fate Decision in Fetal Hepatic Stem/Progenitor Cells

BACKGROUND & AIMS Fetal hepatic stem/progenitor cells, called hepatoblasts, differentiate into both hepatocytes and cholangiocytes. The molecular mechanisms regulating this lineage segmentation process remain unknown. Sall4 has been shown to be among the regulators of organogenesis, embryogenesis, maintenance of pluripotency, and early embryonic cell fate decisions in embryonic stem cells. The expression and functional roles of Sall4 during liver development have not been elucidated. We here provide their first description in hepatoblasts. METHODS To investigate functions of Sall4 in fetal liver development, Dlk(+)CD45(-)Ter119(-) hepatoblasts derived from embryonic day 14 mouse livers were purified, and in vitro gain and loss of function analyses and in vivo transplantation analyses were performed using retrovirus- or lentivirus-mediated gene transfer. RESULTS We demonstrated that Sall4 was expressed in fetal hepatoblasts but not adult hepatocytes. The expression level of Sall4 gradually fell during liver development. Overexpression of Sall4 in hepatoblasts significantly inhibited maturation induced by oncostatin M and extracellular matrix in vitro, as evidenced by morphologic changes and suppression of hepatic maturation marker gene expression. When bile duct-like structures were induced by collagen gel-embedded culture, overexpression of Sall4 markedly augmented size and number of cytokeratin19(+)-branching structures. Knockdown of Sall4 inhibited formation of these branching structures. With in vivo transplantation, Sall4 enhanced differentiation of cytokeratin19(+)-bile ducts derived from transplanted hepatoblasts. CONCLUSIONS These results suggest that Sall4 plays a crucial role in controlling the lineage commitment of hepatoblasts not only inhibiting their differentiation into hepatocytes but also driving their differentiation toward cholangiocytes.

Enrichment and clonal culture of progenitor cells during mouse postnatal liver development in mice.

BACKGROUND & AIMS 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. METHODS 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. RESULTS 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. CONCLUSIONS These results demonstrate the presence of progenitor cells in the CD13(+)CD49f(+)CD133(+) subpopulation of nonhematopoietic cells derived from noninjured postnatal livers.

Hepatitis C virus NS4B protein targets STING and abrogates RIG‐I–mediated type I interferon‐dependent innate immunity

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 gene I (RIG‐I); 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, whereas 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 2013)

Lnk regulates integrin αIIbβ3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk-/- mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin alphaIIbbeta3-mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk-/- mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk-/- platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the beta3 integrin subunit, and reduced binding of Fyn to integrin alphaIIbbeta3. These results provide new insight into the mechanism of alphaIIbbeta3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events.

ITPA gene variant protects against anemia induced by pegylated interferon-α and ribavirin therapy for Japanese patients with chronic hepatitis C.

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.