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  1. Hepatic Cells Derived from Induced Pluripotent Stem Cells of Pigtail Macaques Support Hepatitis C Virus infection

    PubMed Central

    Sourisseau, Marion; Goldman, Orit; He, Wenqian; Gori, Jennifer L.; Kiem, Hans-Peter; Gouon-Evans, Valerie; Evans, Matthew J.

    2013-01-01

    The narrow species tropism of hepatitis C virus (HCV) limits animal studies. We found that pigtail macaque (Macaca nemestrina) hepatic cells derived from induced pluripotent stem cells support the entire HCV life cycle, although infection efficiency was limited by defects in the HCV cell entry process. This block was overcome by either increasing occludin expression, complementing the cells with human CD81, or infecting them with a strain of HCV with less-restricted requirements for CD81. Using this system, we can modify viral and host cell genetics to make pigtail macaques a suitable, clinically relevant model for the study of HCV infection. PMID:23891978

  2. Extra-hepatic replication and infection of hepatitis E virus in neuronal-derived cells.

    PubMed

    Drave, S A; Debing, Y; Walter, S; Todt, D; Engelmann, M; Friesland, M; Wedemeyer, H; Neyts, J; Behrendt, P; Steinmann, E

    2016-07-01

    Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. Infection usually leads to acute hepatitis that can become fulminant, particularly among pregnant women and in patients with preexisting liver disease, or may evolve to a chronic state, especially in immunosuppressed individuals. HEV has been shown to produce a range of extra-hepatic manifestations including aplastic anaemia, acute thyroiditis, glomerulonephritis as well as neurological disorders such as Guillain-Barré syndrome, neuralgic amyotrophy and encephalitis. The pathogenesis of these neurological injuries remains largely unknown, and it is also uncertain whether or not HEV can directly infect neuronal cells. In this study, we investigated whether HEV is capable of completing the viral life cycle in human neuronal-derived cell lines such as neuroepithelioma (SK-N-MC), desmoplastic cerebellar medulloblastoma (DAOY), glioblastoma multiforme (DBTRG), glioblastoma astrocytoma (U-373 MG) and oligodendrocytic (M03.13) cells. Following transfection of these cells with HEV Gaussia luciferase reporter virus, all tested cell lines supported HEV RNA replication. Furthermore, extra- and intracellular viral capsid was detected by an HEV antigen ELISA as a marker for virus assembly and release. Permissiveness for HEV cell entry could be demonstrated for the oligodendrocytic cell line M03.13. In conclusion, these results indicate that HEV tropism is not restricted to the liver and HEV can potentially complete the full viral life cycle in neuronal-derived tissues explaining neurologic disorders during HEV infection. PMID:26891712

  3. Early activated hepatic stellate cell-derived molecules reverse acute hepatic injury

    PubMed Central

    Chang, Wen-Ju; Song, Lu-Jun; Yi, Tuo; Shen, Kun-Tang; Wang, Hong-Shan; Gao, Xiao-Dong; Li, Min; Xu, Jian-Min; Niu, Wei-Xin; Qin, Xin-Yu

    2015-01-01

    AIM: To test whether hepatic stellate cells (HSCs) at different activation stages play different roles in acetaminophen (APAP)-induced acute liver injury (ALI). METHODS: HSCs were isolated from mouse liver and cultured in vitro. Morphological changes of initiation HSCs [HSCs (5d)] and perpetuation HSCs [HSCs (p3)] were observed by immunofluorescence and transmission electron microscopy. The protective effects of HSC-derived molecules, cell lysates and HSC-conditioned medium (HSC-CM) were tested in vivo by survival and histopathological analyses. Liver injury was determined by measuring aminotransferase levels in the serum and by histologic examination of tissue sections under a light microscope. Additionally, to determine the molecular mediators of the observed protective effects of initiation HSCs, we examined HSC-CM using a high-density protein array. RESULTS: HSCs (5d) and HSCs (p3) had different morphological and phenotypic traits. HSCs (5d) presented a star-shaped appearance with expressing α-SMA at non-uniform levels between cells. However, HSCs (p3) evolved into myofibroblast-like cells without lipid droplets and expressed a uniform and higher level of α-SMA. HSC-CM (5d), but not HSC-CM (p3), provided a significant survival benefit and showed a dramatic reduction of hepatocellular necrosis and panlobular leukocyte infiltrates in mice exposed to APAP. However, this protective effect was abrogated at higher cell masses, indicating a therapeutic window of effectiveness. Furthermore, the protein array screen revealed that HSC-CM (5d) was composed of many chemokines and growth factors that correlated with inflammatory inhibition and therapeutic activity. When compared with HSC-CM (p3), higher levels of monocyte chemoattractant protein-1, macrophage inflammatory protein-1γ, hepatocyte growth factor, interleukin-10, and matrix metalloproteinase-2, but lower levels of stem cell factor and Fas-Ligand were observed in HSC-CM (5d). CONCLUSION: These data indicated

  4. Emerging roles of myeloid derived suppressor cells in hepatic inflammation and fibrosis

    PubMed Central

    Hammerich, Linda; Tacke, Frank

    2015-01-01

    Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immune cells that are potent suppressors of immune responses. MDSC emerge in various compartments in the body, such as blood, bone marrow or spleen, especially in conditions of cancer, infections or inflammation. MDSC usually express CD11b, CD33, and low levels of human leukocyte antigen-DR in humans or CD11b and Gr1 (Ly6C/G) in mice, and they can be further divided into granulocytic or monocytic MDSC. The liver is an important organ for MDSC induction and accumulation in hepatic as well as extrahepatic diseases. Different hepatic cells, especially hepatic stellate cells, as well as liver-derived soluble factors, including hepatocyte growth factor and acute phase proteins (SAA, KC), can promote the differentiation of MDSC from myeloid cells. Importantly, hepatic myeloid cells like neutrophils, monocytes and macrophages fulfill essential roles in acute and chronic liver diseases. Recent data from patients with liver diseases and animal models linked MDSC to the pathogenesis of hepatic inflammation, fibrosis and hepatocellular carcinoma (HCC). In settings of acute hepatitis, MDSC can limit immunogenic T cell responses and subsequent tissue injury. In patients with chronic hepatitis C, MDSC increase and may favor viral persistence. Animal models of chronic liver injury, however, have not yet conclusively clarified the involvement of MDSC for hepatic fibrosis. In human HCC and mouse models of liver cancer, MDSC are induced in the tumor environment and suppress anti-tumoral immune responses. Thus, the liver is a primary site of MDSC in vivo, and modulating MDSC functionality might represent a promising novel therapeutic target for liver diseases. PMID:26301117

  5. Emerging roles of myeloid derived suppressor cells in hepatic inflammation and fibrosis.

    PubMed

    Hammerich, Linda; Tacke, Frank

    2015-08-15

    Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immune cells that are potent suppressors of immune responses. MDSC emerge in various compartments in the body, such as blood, bone marrow or spleen, especially in conditions of cancer, infections or inflammation. MDSC usually express CD11b, CD33, and low levels of human leukocyte antigen-DR in humans or CD11b and Gr1 (Ly6C/G) in mice, and they can be further divided into granulocytic or monocytic MDSC. The liver is an important organ for MDSC induction and accumulation in hepatic as well as extrahepatic diseases. Different hepatic cells, especially hepatic stellate cells, as well as liver-derived soluble factors, including hepatocyte growth factor and acute phase proteins (SAA, KC), can promote the differentiation of MDSC from myeloid cells. Importantly, hepatic myeloid cells like neutrophils, monocytes and macrophages fulfill essential roles in acute and chronic liver diseases. Recent data from patients with liver diseases and animal models linked MDSC to the pathogenesis of hepatic inflammation, fibrosis and hepatocellular carcinoma (HCC). In settings of acute hepatitis, MDSC can limit immunogenic T cell responses and subsequent tissue injury. In patients with chronic hepatitis C, MDSC increase and may favor viral persistence. Animal models of chronic liver injury, however, have not yet conclusively clarified the involvement of MDSC for hepatic fibrosis. In human HCC and mouse models of liver cancer, MDSC are induced in the tumor environment and suppress anti-tumoral immune responses. Thus, the liver is a primary site of MDSC in vivo, and modulating MDSC functionality might represent a promising novel therapeutic target for liver diseases. PMID:26301117

  6. CD40 dependent exacerbation of immune mediated hepatitis by hepatic CD11b+ Gr-1+ myeloid derived suppressor cells in tumor bearing mice

    PubMed Central

    Kapanadze, Tamar; Medina-Echeverz, José; Gamrekelashvili, Jaba; Weiss, Jonathan M.; Wiltrout, Robert H.; Kapoor, Veena; Hawk, Nga; Terabe, Masaki; Berzofsky, Jay A.; Manns, Michael P.; Wang, Ena; Marincola, Francesco M.; Korangy, Firouzeh; Greten, Tim F.

    2015-01-01

    Immunosuppressive CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) accumulate in the livers of tumor-bearing mice. We studied hepatic MDSC in two murine models of immune mediated hepatitis. Unexpectedly, treatment of tumor bearing mice with Concanavalin A or α-Galactosylceramide resulted in increased ALT and AST serum levels in comparison to tumor free mice. Adoptive transfer of hepatic MDSC into naïve mice exacerbated Concanavalin A induced liver damage. Hepatic CD11b+Gr-1+ cells revealed a polarized pro-inflammatory gene signature after Concanavalin A treatment. An interferon gamma- dependent up-regulation of CD40 on hepatic CD11b+Gr-1+ cells along with an up-regulation of CD80, CD86, and CD1d after Concanavalin A treatment was observed. Concanavalin A treatment resulted in a loss of suppressor function by tumor-induced CD11b+Gr-1+ MDSC as well as enhanced reactive oxygen species-mediated hepatotoxicity. CD40 knockdown in hepatic MDSC led to increased arginase activity upon Concanavalin A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40−/− tumor-induced myeloid cells resulted in exacerbation of hepatitis and increased reactive oxygen species production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSC act as pro-inflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner. PMID:25616156

  7. Ionone Derivatives from the Mycelium of Phellinus linteus and the Inhibitory Effect on Activated Rat Hepatic Stellate Cells

    PubMed Central

    Huang, Shiow-Chyn; Kuo, Ping-Chung; Hung, Hsin-Yi; Pan, Tai-Long; Chen, Fu-An; Wu, Tian-Shung

    2016-01-01

    Three new γ-ionylideneacetic acid derivatives, phellinulins A–C (1–3), were characterized from the mycelium extract of Phellinus linteus. The chemical structures were established based on the spectroscopic analysis. In addition, phellinulin A (1) was subjected to the examination of effects on activated rat hepatic stellate cells and exhibited significant inhibition of hepatic fibrosis. PMID:27164091

  8. Large scale production of a mammalian cell derived quadrivalent hepatitis C virus like particle vaccine.

    PubMed

    Earnest-Silveira, L; Christiansen, D; Herrmann, S; Ralph, S A; Das, S; Gowans, E J; Torresi, J

    2016-10-01

    A method for the large-scale production of a quadrivalent mammalian cell derived hepatitis C virus-like particles (HCV VLPs) is described. The HCV core E1 and E2 coding sequences of genotype 1a, 1b, 2a or 3a were co-expressed in Huh7 cell factories using a recombinant adenoviral expression system. The structural proteins self-assembled into VLPs that were purified from Huh7 cell lysates by iodixanol ultracentrifugation and Stirred cell ultrafiltration. Electron microscopy, revealed VLPs of the different genotypes that are morphologically similar. Our results show that it is possible to produce large quantities of individual HCV genotype VLPs with relative ease thus making this approach an alternative for the manufacture of a quadrivalent mammalian cell derived HCV VLP vaccine. PMID:27373602

  9. Metabolic regulation of hepatitis B immunopathology by myeloid-derived suppressor cells

    PubMed Central

    Pallett, Laura J.; Gill, Upkar S.; Quaglia, Alberto; Sinclair, Linda V.; Jover-Cobos, Maria; Schurich, Anna; Singh, Kasha P.; Thomas, Niclas; Das, Abhishek; Chen, Antony; Fusai, Giuseppe; Bertoletti, Antonio; Cantrell, Doreen A.; Kennedy, Patrick T.; Davies, Nathan A.; Haniffa, Muzlifah; Maini, Mala K.

    2015-01-01

    Infection with hepatitis B virus (HBV) results in disparate degrees of tissue injury: it can replicate without pathological consequences or trigger immune-mediated necroinflammatory liver damage. We investigated the potential for myeloid-derived suppressor cells (MDSC) to suppress T cell-mediated immunopathology in this setting. Granulocytic MDSC (gMDSC) expanded transiently in acute resolving HBV, decreasing before peak hepatic injury. In persistent infection, arginase-expressing gMDSC (and circulating arginase) increased most in phases characterized by HBV replication without immunopathology, whilst L-arginine decreased. gMDSC expressed liver-homing chemokine receptors and accumulated in the liver, their expansion being supported by hepatic stellate cells. We provide in vitro and ex vivo evidence that gMDSC potently inhibited T cells in a partially arginase-dependent manner. L-arginine-deprived T cells upregulated system-L amino acid transporters to increase uptake of essential nutrients and attempt metabolic reprogramming. These data demonstrate the capacity of expanded arginase-expressing gMDSC to regulate liver immunopathology in HBV infection. PMID:25962123

  10. Cytoglobin as a Marker of Hepatic Stellate Cell-derived Myofibroblasts

    PubMed Central

    Kawada, Norifumi

    2015-01-01

    Myofibroblasts play important roles in inflammation, fibrosis and tumorigenesis in chronically inflamed liver. Liver myofibroblasts originate from hepatic stellate cells, portal fibroblasts or mesothelial cells, and they are localized in and around fibrotic septum and portal tracts. Liver myofibroblasts are the source of extracellular matrix materials, including type I collagen and multiple fibrogenic growth factors, such as transforming growth factor-β and vascular endothelial growth factor. Although a detailed characterization of the function of individual myofibroblasts has not been conducted, owing to the lack of appropriate cell markers, recent lineage-tracing technology has revealed the limited contribution of myofibroblasts that are derived from portal fibroblasts to various types of liver fibrosis, as compared with the contribution of hepatic stellate cells. In addition, cytoglobin, which is the fourth globin in mammals and function as a local gas sensor, provides a new perspective on the involvement of stellate cells in fibrosis and carcinogenesis, possibly through its anti-oxidative properties and is a promising new marker that discriminates between myofibroblasts derived from stellate cells and those from portal fibroblasts. PMID:26617531

  11. Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus

    PubMed Central

    Kaneko, Shun; Kakinuma, Sei; Asahina, Yasuhiro; Kamiya, Akihide; Miyoshi, Masato; Tsunoda, Tomoyuki; Nitta, Sayuri; Asano, Yu; Nagata, Hiroko; Otani, Satoshi; Kawai-Kitahata, Fukiko; Murakawa, Miyako; Itsui, Yasuhiro; Nakagawa, Mina; Azuma, Seishin; Nakauchi, Hiromitsu; Nishitsuji, Hironori; Ujino, Saneyuki; Shimotohno, Kunitada; Iwamoto, Masashi; Watashi, Koichi; Wakita, Takaji; Watanabe, Mamoru

    2016-01-01

    Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies. PMID:27386799

  12. Alcohol Increases Liver Progenitor Populations and Induces Disease Phenotypes in Human IPSC-Derived Mature Stage Hepatic Cells

    PubMed Central

    Tian, Lipeng; Deshmukh, Abhijeet; Prasad, Neha; Jang, Yoon-Young

    2016-01-01

    Alcohol consumption has long been a global problem affecting human health, and has been found to influence both fetal and adult liver functions. However, how alcohol affects human liver development and liver progenitor cells remains largely unknown. Here, we used human induced pluripotent stem cells (iPSCs) as a model to examine the effects of alcohol, on multi-stage hepatic cells including hepatic progenitors, early and mature hepatocyte-like cells derived from human iPSCs. While alcohol has little effect on endoderm development from iPSCs, it reduces formation of hepatic progenitor cells during early hepatic specification. The proliferative activities of early and mature hepatocyte-like cells are significantly decreased after alcohol exposure. Importantly, at a mature stage of hepatocyte-like cells, alcohol treatment increases two liver progenitor subsets, causes oxidative mitochondrial injury and results in liver disease phenotypes (i.e., steatosis and hepatocellular carcinoma associated markers) in a dose dependent manner. Some of the phenotypes were significantly improved by antioxidant treatment. This report suggests that fetal alcohol exposure may impair generation of hepatic progenitors at early stage of hepatic specification and decrease proliferation of fetal hepatocytes; meanwhile alcohol injury in post-natal or mature stage human liver may contribute to disease phenotypes. This human iPSC model of alcohol-induced liver injury can be highly valuable for investigating alcoholic injury in the fetus as well as understanding the pathogenesis and ultimately developing effective treatment for alcoholic liver disease in adults. PMID:27570479

  13. Alcohol Increases Liver Progenitor Populations and Induces Disease Phenotypes in Human IPSC-Derived Mature Stage Hepatic Cells.

    PubMed

    Tian, Lipeng; Deshmukh, Abhijeet; Prasad, Neha; Jang, Yoon-Young

    2016-01-01

    Alcohol consumption has long been a global problem affecting human health, and has been found to influence both fetal and adult liver functions. However, how alcohol affects human liver development and liver progenitor cells remains largely unknown. Here, we used human induced pluripotent stem cells (iPSCs) as a model to examine the effects of alcohol, on multi-stage hepatic cells including hepatic progenitors, early and mature hepatocyte-like cells derived from human iPSCs. While alcohol has little effect on endoderm development from iPSCs, it reduces formation of hepatic progenitor cells during early hepatic specification. The proliferative activities of early and mature hepatocyte-like cells are significantly decreased after alcohol exposure. Importantly, at a mature stage of hepatocyte-like cells, alcohol treatment increases two liver progenitor subsets, causes oxidative mitochondrial injury and results in liver disease phenotypes (i.e., steatosis and hepatocellular carcinoma associated markers) in a dose dependent manner. Some of the phenotypes were significantly improved by antioxidant treatment. This report suggests that fetal alcohol exposure may impair generation of hepatic progenitors at early stage of hepatic specification and decrease proliferation of fetal hepatocytes; meanwhile alcohol injury in post-natal or mature stage human liver may contribute to disease phenotypes. This human iPSC model of alcohol-induced liver injury can be highly valuable for investigating alcoholic injury in the fetus as well as understanding the pathogenesis and ultimately developing effective treatment for alcoholic liver disease in adults. PMID:27570479

  14. Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-γ expression.

    PubMed

    Mwangi, Simon Musyoka; Peng, Sophia; Nezami, Behtash Ghazi; Thorn, Natalie; Farris, Alton B; Jain, Sanjay; Laroui, Hamed; Merlin, Didier; Anania, Frank; Srinivasan, Shanthi

    2016-01-15

    Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease. PMID:26564715

  15. Modulating Innate Immunity Improves Hepatitis C Virus Infection and Replication in Stem Cell-Derived Hepatocytes

    PubMed Central

    Zhou, Xiaoling; Sun, Pingnan; Lucendo-Villarin, Baltasar; Angus, Allan G.N.; Szkolnicka, Dagmara; Cameron, Kate; Farnworth, Sarah L.; Patel, Arvind H.; Hay, David C.

    2014-01-01

    Summary In this study, human embryonic stem cell-derived hepatocytes (hESC-Heps) were investigated for their ability to support hepatitis C virus (HCV) infection and replication. hESC-Heps were capable of supporting the full viral life cycle, including the release of infectious virions. Although supportive, hESC-Hep viral infection levels were not as great as those observed in Huh7 cells. We reasoned that innate immune responses in hESC-Heps may lead to the low level of infection and replication. Upon further investigation, we identified a strong type III interferon response in hESC-Heps that was triggered by HCV. Interestingly, specific inhibition of the JAK/STAT signaling pathway led to an increase in HCV infection and replication in hESC-Heps. Of note, the interferon response was not evident in Huh7 cells. In summary, we have established a robust cell-based system that allows the in-depth study of virus-host interactions in vitro. PMID:25068132

  16. Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection.

    PubMed

    Shirasago, Yoshitaka; Shimizu, Yoshimi; Tanida, Isei; Suzuki, Tetsuro; Suzuki, Ryosuke; Sugiyama, Kazuo; Wakita, Takaji; Hanada, Kentaro; Yagi, Kiyohito; Kondoh, Masuo; Fukasawa, Masayoshi

    2016-05-01

    It is well known that occludin (OCLN) is involved in hepatitis C virus (HCV) entry into hepatocytes, but there has been no conclusive evidence that OCLN is essential for HCV infection. In this study, we first established an OCLN-knockout cell line derived from human hepatic Huh7.5.1-8 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, in which two independent targeting plasmids expressing single-guide RNAs were used. One established cell clone, named OKH-4, had the OCLN gene truncated in the N-terminal region, and a complete defect of the OCLN protein was shown using immunoblot analysis. Infection of OKH-4 cells with various genotypes of HCV was abolished, and exogenous expression of the OCLN protein in OKH-4 cells completely reversed permissiveness to HCV infection. In addition, using a co-culture system of HCV-infected Huh7.5.1-8 cells with OKH-4 cells, we showed that OCLN is also critical for cell-to-cell HCV transmission. Thus, we concluded that OCLN is essential for HCV infection of human hepatic cells. Further experiments using HCV genomic RNA-transfected OKH-4 cells or HCV subgenomic replicon-harboring OKH-4 cells suggested that OCLN is mainly involved in the entry step of the HCV life cycle. It was also demonstrated that the second extracellular loop of OCLN, especially the two cysteine residues, is critical for HCV infection of hepatic cells. OKH-4 cells may be a useful tool for understanding not only the entire mechanism of HCV entry, but also the biological functions of OCLN. PMID:26887345

  17. A comparison of the chemical and liver extract-induced hepatic differentiation of adipose derived stem cells.

    PubMed

    Nhung, Truong Hai; Nam, Nguyen Hai; Nguyen, Nguyen Thi Kim; Nghia, Huynh; Van Thanh, Nguyen; Ngoc, Phan Kim; Van Pham, Phuc

    2015-11-01

    Adipose-derived stem cells (ADSCs) have been put forward as promising therapeutics for end-stage liver disease (ESLD). In the present study, we compared the effects of defined chemicals and liver extract on the hepatic differentiation of ADSCs. ADSCs were isolated according to the method described in our previously published study. Subsequently, the differentiation of ADSCs was induced separately by chemicals (including hepatic growth factor (HGF), fibroblast growth factor (FGF), and oncostatin M (OSM)) and liver extract (30 μg/ml) in a total period of 21 d. The efficiency of hepatic differentiation was evaluated by changes in the cell morphology, gene expression, and cellular function. The results showed that the liver extract promoted the hepatic differentiation of ADSCs to a significantly greater extent than the chemicals. In the group of ADSCs treated with liver extract, changes in the cell morphology began sooner, and the expression of alpha-FP and albumin genes was higher than that in the chemically treated group. The ADSCs in both the groups stained positive for anti-alpha trypsin (AAT) and albumin markers. The cells also exhibited glycogen storage capacity. Therefore, we concluded that the liver extract could efficiently induce the differentiation of ADSCs into hepatocyte-like cells. This study reveals the potential of mesenchymal stem cell differentiation in the liver extract, which supports further preclinical and clinical research on the application of ADSCs in ESLD treatment. PMID:26275888

  18. Let-7f microRNA negatively regulates hepatic differentiation of human adipose tissue-derived stem cells.

    PubMed

    Davoodian, Nahid; Lotfi, Abbas S; Soleimani, Masoud; Mola, Seyed Javad; Arjmand, Sare

    2014-09-01

    MicroRNAs (miRNAs) are noncoding RNAs involved in the regulation of the diverse biological processes such as metabolism, proliferation, and cell cycle, in addition to regulation of differentiation. So far, some miRNAs have been recognized to have important role in regulating hepatic functions. Statistically, let-7f has been revealed as a negative regulator of hepatic differentiation. In the present study, we investigated the effect of let-7f on hepatic differentiation of human adipose tissue-derived stem cells (hADSCs). hADSCs were transduced with recombinant lentivirus containing human inhibitor let-7 f. The expression of hepatocyte nuclear factors alpha (HNF4a), albumin (ALB), alpha fetoprotein (AFP), cytokeratin 18 (CK18), and cytokeratin 19 (CK19) was evaluated using quantitative real-time PCR (qRT-PCR). Immunocytochemistry was used to investigate the expression levels of the hepatocyte markers including ALB, AFP, and HNF4a, and biochemical analysis was implemented for hepatic function, glycogen deposition, and urea secretion. qRT-PCR showed significant upregulation in HNF4a, ALB, AFP, CK18, and CK19 expression in cells transduced with let-7f inhibitor lentiviruses. Moreover, positive staining was detected for ALB, AFP, and HNF4a using immunocytochemistry. Urea production and glycogen deposits were also found in the treated cells, the two specific features of the hepatic cells. Therefore, let-7f silencing led to the increased expression of the hepatocyte-specific factors and the accelerated hADSCs hepatic differentiation. Summing all these finding together, our present report has provided evidences that inhibition of let-7f would facilitate induction of hADSCs into hepatocyte-like cells and possibly in regenerative therapy of the liver disease in a wider spectrum. PMID:25077652

  19. Activated hepatic stellate cells promote liver cancer by induction of myeloid-derived suppressor cells through cyclooxygenase-2.

    PubMed

    Xu, Yaping; Zhao, Wenxiu; Xu, Jianfeng; Li, Jie; Hong, Zaifa; Yin, Zhenyu; Wang, Xiaomin

    2016-02-23

    Hepatic stellate cells (HSCs) are critical mediators of immunosuppression and the pathogenesis of hepatocellular carcinoma (HCC). Our previous work indicates that HSCs promote HCC progression by enhancing immunosuppressive cell populations including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). MDSCs are induced by inflammatory cytokines (e.g., prostaglandins) and are important in immune suppression. However, how HSCs mediate expansion of MDSCs is uncertain. Thus, we studied activated HSCs that could induce MDSCs from bone marrow cells and noted that HSC-induced MDSCs up-regulated immunosuppressive activity via iNOS, Arg-1, and IL-4Rα. After treating cells with a COX-2 inhibitor or an EP4 antagonist, we established that HSC-induced MDSC accumulation was mediated by the COX2-PGE2-EP4 signaling. Furthermore, in vivo animal studies confirmed that inhibition of HSC-derived PGE2 could inhibit HSC-induced MDSC accumulation and HCC growth. Thus, our data show that HSCs are required for MDSC accumulation mediated by the COX2-PGE2-EP4 pathway, and these data are the first to link HSC and MDSC subsets in HCC immune microenvironment and provide a rationale for targeting PGE2 signaling for HCC therapy. PMID:26758420

  20. Activated hepatic stellate cells promote liver cancer by induction of myeloid-derived suppressor cells through cyclooxygenase-2

    PubMed Central

    Xu, Jianfeng; Li, Jie; Hong, Zaifa; Yin, Zhenyu; Wang, Xiaomin

    2016-01-01

    Hepatic stellate cells (HSCs) are critical mediators of immunosuppression and the pathogenesis of hepatocellular carcinoma (HCC). Our previous work indicates that HSCs promote HCC progression by enhancing immunosuppressive cell populations including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). MDSCs are induced by inflammatory cytokines (e.g., prostaglandins) and are important in immune suppression. However, how HSCs mediate expansion of MDSCs is uncertain. Thus, we studied activated HSCs that could induce MDSCs from bone marrow cells and noted that HSC-induced MDSCs up-regulated immunosuppressive activity via iNOS, Arg-1, and IL-4Rα. After treating cells with a COX-2 inhibitor or an EP4 antagonist, we established that HSC-induced MDSC accumulation was mediated by the COX2-PGE2-EP4 signaling. Furthermore, in vivo animal studies confirmed that inhibition of HSC-derived PGE2 could inhibit HSC-induced MDSC accumulation and HCC growth. Thus, our data show that HSCs are required for MDSC accumulation mediated by the COX2-PGE2-EP4 pathway, and these data are the first to link HSC and MDSC subsets in HCC immune microenvironment and provide a rationale for targeting PGE2 signaling for HCC therapy. PMID:26758420

  1. Novel Radiolytic Rotenone Derivative, Rotenoisin B with Potent Anti-Carcinogenic Activity in Hepatic Cancer Cells

    PubMed Central

    Badaboina, Srilatha; Bai, Hyoung-Woo; Na, Yun Hee; Park, Chul-Hong; Kim, Tae Hoon; Lee, Tae-Hoon; Chung, Byung Yeoup

    2015-01-01

    Rotenone, isolated from roots of derris plant, has been shown to possess various biological activities, which lead to attempting to develop a potent drug against several diseases. However, recent studies have demonstrated that rotenone has the potential to induce several adverse effects such as a neurodegenerative disease. Radiolytic transformation of the rotenone with gamma-irradiation created a new product, named rotenoisin B. The present work was designed to investigate the anticancer activity of rotenoisin B with low toxicity and its molecular mechanism in hepatic cancer cells compared to a parent compound, rotenone. Our results showed rotenoisin B inhibited hepatic cancer cells’ proliferation in a dose dependent manner and increased in apoptotic cells. Interestingly, rotenoisin B showed low toxic effects on normal cells compared to rotenone. Mitochondrial transmembrane potential has been decreased, which leads to cytochrome c release. Down regulation of anti-apoptotic Bcl-2 levels as well as the up regulation of proapoptotic Bax levels were observed. The cleaved PARP (poly ADP-ribose polymerase) level increased as well. Moreover, phosphorylation of extracellular signal regulated kinase (ERK) and p38 slightly up regulated and intracellular reactive oxygen species (ROS) increased as well as cell cycle arrest predominantly at the G2/M phase observed. These results suggest that rotenoisin B might be a potent anticancer candidate similar to rotenone in hepatic cancer cells with low toxicity to normal cells even at high concentrations compared to rotenone. PMID:26213921

  2. Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

    PubMed Central

    Sharma, Ruchi; Greenhough, Sebastian; Medine, Claire N.; Hay, David C.

    2010-01-01

    The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays. PMID:20169088

  3. Identification of a Bipotential Precursor Cell in Hepatic Cell Lines Derived from Transgenic Mice Expressing Cyto-Met in the Liver

    PubMed Central

    Spagnoli, Francesca M.; Amicone, Laura; Tripodi, Marco; Weiss, Mary C.

    1998-01-01

    Met murine hepatocyte (MMH) lines were established from livers of transgenic mice expressing constitutively active human Met. These lines harbor two cell types: epithelial cells resembling the parental populations and flattened cells with multiple projections and a dispersed growth habit that are designated palmate. Epithelial cells express the liver-enriched transcription factors HNF4 and HNF1α, and proteins associated with epithelial cell differentiation. Treatments that modulate their differentiation state, including acidic FGF, induce hepatic functions. Palmate cells show none of these properties. However, they can differentiate along the hepatic cell lineage, giving rise to: (a) epithelial cells that express hepatic transcription factors and are competent to express hepatic functions; (b) bile duct-like structures in three-dimensional Matrigel cultures. Derivation of epithelial from palmate cells is confirmed by characterization of the progeny of individually fished cells. Furthermore, karyotype analysis confirms the direction of the phenotypic transition: palmate cells are diploid and the epithelial cells are hypotetraploid. The clonal isolation of the palmate cell, an immortalized nontransformed bipotential cell that does not yet express the liver-enriched transcription factors and is a precursor of the epithelial-hepatocyte in MMH lines, provides a new tool for the study of mechanisms controlling liver development. PMID:9817765

  4. Identification of a bipotential precursor cell in hepatic cell lines derived from transgenic mice expressing cyto-Met in the liver.

    PubMed

    Spagnoli, F M; Amicone, L; Tripodi, M; Weiss, M C

    1998-11-16

    Met murine hepatocyte (MMH) lines were established from livers of transgenic mice expressing constitutively active human Met. These lines harbor two cell types: epithelial cells resembling the parental populations and flattened cells with multiple projections and a dispersed growth habit that are designated palmate. Epithelial cells express the liver-enriched transcription factors HNF4 and HNF1alpha, and proteins associated with epithelial cell differentiation. Treatments that modulate their differentiation state, including acidic FGF, induce hepatic functions. Palmate cells show none of these properties. However, they can differentiate along the hepatic cell lineage, giving rise to: (a) epithelial cells that express hepatic transcription factors and are competent to express hepatic functions; (b) bile duct-like structures in three-dimensional Matrigel cultures. Derivation of epithelial from palmate cells is confirmed by characterization of the progeny of individually fished cells. Furthermore, karyotype analysis confirms the direction of the phenotypic transition: palmate cells are diploid and the epithelial cells are hypotetraploid. The clonal isolation of the palmate cell, an immortalized nontransformed bipotential cell that does not yet express the liver-enriched transcription factors and is a precursor of the epithelial-hepatocyte in MMH lines, provides a new tool for the study of mechanisms controlling liver development. PMID:9817765

  5. Injury mechanism dictates contribution of bone marrow-derived cells to murine hepatic vascular regeneration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stem and progenitor cells derived from adult marrow have been shown to regenerate vascular cells in response to injury. However, it is unclear whether the type of injury dictates the contribution of such cells to neovascularization and which subpopulations of cells contribute to vascular regeneratio...

  6. Completion of the Entire Hepatitis C Virus Life Cycle in Vero Cells Derived from Monkey Kidney

    PubMed Central

    Murayama, Asako; Sugiyama, Nao; Wakita, Takaji

    2016-01-01

    ABSTRACT A hepatitis C virus (HCV) cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122), an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI) was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE), a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells. PMID:27302754

  7. Cell Fusion Reprogramming Leads to a Specific Hepatic Expression Pattern during Mouse Bone Marrow Derived Hepatocyte Formation In Vivo

    PubMed Central

    Arza, Elvira; Alvarez-Barrientos, Alberto; Fabregat, Isabel; Garcia-Bravo, Maria; Meza, Nestor W.; Segovia, Jose C.

    2012-01-01

    The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation. PMID:22457803

  8. Evaluation of hepatotoxicity of chemicals using hepatic progenitor and hepatocyte-like cells derived from mouse embryonic stem cells: effect of chemicals on ESC-derived hepatocyte differentiation.

    PubMed

    Kang, Seok-Jin; Jeong, Sang-Hee; Kim, Eun-Joo; Cho, Joon-Hyoung; Park, Young-Il; Park, Sung-Won; Shin, Hyo-Sook; Son, Seong-Wan; Kang, Hwan-Goo

    2013-02-01

    Embryonic stem cell testing is an alternative model system to assess drug and chemical toxicities because of its similar developmental characteristics with in vivo embryogenesis and organogenesis. This study evaluated the toxicity of chemicals at specific developmental stages of mouse embryonic stem cell (ESC)-derived hepatic differentiation; hepatic progenitor cells (HPCs), and hepatocyte-like cells (HCs). The toxic effects of carbon tetrachloride (CCl(4)), 5-fluorouracil (5-FU), and arsanilic acid (Ars) were evaluated by measuring the expressions of Cytokeratin (CK18) and GATA binding protein 4 (GATA-4) and the activities of aspartate transaminase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) during the hepatic differentiation process. Non-toxic doses of three chemicals at a range of 25 to 500 μM for CCl(4), 12.5 to 800 nM for 5-FU and 6.25 to 400 mM for Ars were treated. In the CCl(4)-treated group, significant decreases (P < 0.05) of the marker expression were observed by more than 300 μM from day 10 in CK18 and by more than 400 μM of CCl(4) from day 22 in GATA-4, respectively. However, both markers were decreased (P < 0.01) by treatments of all doses at day 40. In the 5-FU-treated group, the expressions of two proteins were not affected by any of the doses at day 10 and 22, whereas the GATA-4 expression was decreased (P < 0.05) by more than 400 nM of 5-FU at days 28 and 40. In the Ars-treated group, the CK18 expression was inhibited (P < 0.05) by more than 100 mM of Ars at day 22 but showed a tendency to recover. Although the GATA-4 was inhibited by all doses at day 22, the inhibition of GATA-4 recovered at days 28 and 40. ALP activities of three chemicals were significantly increased (P < 0.05) by a dose-dependent manner. The activities of AST and LDH were prone to be increased by more than 300 μM of CCl(4,) but not affected by all doses of 5-FU except for 800 nM of 5-FU in AST activities. In the Ars, the

  9. In vitro assessment of drug-induced liver steatosis based on human dermal stem cell-derived hepatic cells.

    PubMed

    Rodrigues, Robim M; Branson, Steven; De Boe, Veerle; Sachinidis, Agapios; Rogiers, Vera; De Kock, Joery; Vanhaecke, Tamara

    2016-03-01

    Steatosis, also known as fatty liver disease (FLD), is a disorder in which the lipid metabolism of the liver is disturbed, leading to the abnormal retention of lipids in hepatocytes. FLD can be induced by several drugs, and although it is mostly asymptomatic, it can lead to steatohepatitis, which is associated with liver inflammation and damage. Drug-induced liver injury is currently the major cause of postmarketing withdrawal of pharmaceuticals and discontinuation of the development of new chemical entities. Therefore, the potential induction of steatosis must be evaluated during preclinical drug development. However, robust human-relevant in vitro models are lacking. In the present study, we explore the applicability of hepatic cells (hSKP-HPCs) derived from postnatal skin precursors, a stem cell population residing in human dermis, to investigate the steatosis-inducing effects of sodium valproate (Na-VPA). Exposure of hSKP-HPC to sub-cytotoxic concentrations of this reference steatogenic compound showed an increased intracellular accumulation of lipid droplets, and the modulation of key factors involved in lipid metabolism. Using a toxicogenomics approach, we further compared Na-VPA-treated hSKP-HPC and Na-VPA-treated primary human hepatocytes to liver samples from patients suffering from mild and advanced steatosis. Our data show that in hSKP-HPC exposed to Na-VPA and liver samples of patients suffering from mild steatosis, but not in primary human hepatocytes, "liver steatosis" was efficiently identified as a toxicological response. These findings illustrate the potential of hSKP-HPC as a human-relevant in vitro model to identify hepatosteatotic effects of chemical compounds. PMID:25716160

  10. Establishment and characterization of a cell line, EH-GB2, derived from hepatic metastasis of gallbladder cancer.

    PubMed

    Wang, Jing-Han; Li, Lin-Fang; Yu, Yong; Li, Bin; Jin, Hua-Jun; Shen, Dong-Hao; Li, Jiang; Jiang, Xiao-Qing; Qian, Qi-Jun

    2012-03-01

    Gallbladder cancer is a fatal neoplasia with an extremely low survival rate. Liver invasion and metastasis are the most common causes of death; however, the metastatic mechanism is still unclear, and no effective treatment methods are available. To provide comprehensive and profound approaches in investigating the metastatic mechanism and treatment methods, new cell lines derived from liver metastasis are urgently needed. A hepatic metastasis lesion was obtained from a 65-year-old patient, and was treated using a primary culture method to establish a novel gallbladder cancer cell line. Different in vitro/in vivo methods were used to characterize the phenotypes of this cell line. The gallbladder cancer cell line was named EH-GB2, with a roughly 48-h doubling time. The cell line represents stronger colony formation and migration abilities than the control group. The cells showed complicated chromosomal abnormalities. EH-GB2 cells showed epithelial-to-mesenchymal transition (EMT) and the mRNA expression levels of E-cadherin and integrin were decreased, and those of vimentin, Snail, Twist, matrix metalloproteinase-1 (MMP-1) and MMP-2 were increased in comparison with control cells. The in vivo study demonstrated that EH-GB2 cells show significant tumorigenicity in nude mice. The EH-GB2 established gallbladder cancer cell line is useful for future studies of gallbladder cancer development, progression, metastasis and therapy. PMID:22134783

  11. Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

    PubMed

    Earnest-Silveira, L; Chua, B; Chin, R; Christiansen, D; Johnson, D; Herrmann, S; Ralph, S A; Vercauteren, K; Mesalam, A; Meuleman, P; Das, S; Boo, I; Drummer, H; Bock, C-T; Gowans, E J; Jackson, D C; Torresi, Joseph

    2016-08-01

    An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV. PMID:27147296

  12. Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate hepatic-like differentiation.

    PubMed

    Chen, Y K; Huang, Anderson H C; Chan, Anthony W S; Lin, L M

    2016-06-01

    Reviewing the literature, hepatic differentiation of human dental pulp stem cells (hDPSCs) from cryopreserved dental pulp tissues of vital extracted teeth with disease has not been studied. This study is aimed to evaluate the hypothesis that hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease could possess potential hepatic differentiation. Forty vital extracted teeth with disease recruited for hDPSCs isolation, stem cell characterization and hepatic differentiation were randomly and equally divided into group A (liquid nitrogen-stored dental pulp tissues) and group B (freshly derived dental pulp tissues). Samples of hDPSCs isolated from groups A and B but without hepatic growth factors formed negative controls. A well-differentiated hepatocellular carcinoma cell line was employed as a positive control. All the isolated hDPSCs from groups A and B showed hepatic-like differentiation with morphological change from a spindle-shaped to a polygonal shape and normal karyotype. Differentiated hDPSCs and the positive control expressed hepatic metabolic function genes and liver-specific genes. Glycogen storage of differentiated hDPSCs was noted from day 7 of differentiation-medium culture. Positive immunofluorescence staining of low-density lipoprotein and albumin was observed from day 14 of differentiation-medium culture; urea production in the medium was noted from week 6. No hepatic differentiation was observed for any of the samples of the negative controls. We not only demonstrated the feasibility of hepatic-like differentiation of hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease but also indicated that the differentiated cells possessed normal karyotype and were functionally close to normal hepatic-like cells. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23950016

  13. Hepatic Stellate Cell-Derived Microvesicles Prevent Hepatocytes from Injury Induced by APAP/H2O2

    PubMed Central

    Huang, Renwei; Wang, Yan; Liang, Yaolong; Liao, Xiaorong; Li, Mingyi

    2016-01-01

    Hepatic stellate cells (HSCs), previously described for liver-specific mesenchymal stem cells (MSCs), appear to contribute to liver regeneration. Microvesicles (MVs) are nanoscale membrane fragments, which can regulate target cell function by transferring contents from their parent cells. The aim of this study was to investigate the effect of HSC-derived MVs on xenobiotic-induced liver injury. Rat and human hepatocytes, BRL-3A and HL-7702, were used to build hepatocytes injury models by n-acetyl-p-aminophenol n-(APAP) or H2O2 treatment. MVs were prepared from human and rat HSCs, LX-2, and HST-T6 and, respectively, added to injured BRL-3A and HL-7702 hepatocytes. MTT assay was utilized to determine cell proliferation. Cell apoptosis was analyzed by flow cytometry and hoechst33258 staining. Western blot was used for analyzing the expression of activated caspase-3. Liver injury indicators, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in culture medium were also assessed. Results showed that (1) HSC-MVs derived from LX-2 and HST-T6 were positive to CD90 and annexin V surface markers; (2) HSC-MVs dose-dependently improved the viability of hepatocytes in both injury models; (3) HSC-MVs dose-dependently inhibited the APAP/H2O2 induced hepatocytes apoptosis and activated caspase-3 expression and leakage of LDH, ALT, and AST. Our results demonstrate that HSC-derived MVs protect hepatocytes from toxicant-induced injury. PMID:27239205

  14. MicroRNA-27b Enhances the Hepatic Regenerative Properties of Adipose-Derived Mesenchymal Stem Cells

    PubMed Central

    Chen, Kuang-Den; Huang, Kuang-Tzu; Lin, Chih-Che; Weng, Wei-Teng; Hsu, Li-Wen; Goto, Shigeru; Nakano, Toshiaki; Lai, Chia-Yun; Kung, Chao-Pin; Chiu, King-Wah; Wang, Chih-Chi; Cheng, Yu-Fan; Ma, Yen-Ying; Chen, Chao-Long

    2016-01-01

    Adipose-derived mesenchymal stem cells (ASCs) are readily available multipotent mesenchymal progenitor cells and have become an attractive therapeutic tool for regenerative medicine. We herein investigated the mechanistic role of how miR-27b modulated regenerative capacities of ASCs. Intravenous administration of miR-27b-transfected ASCs (ASCs-miR-27b) was conducted after 70% partial hepatectomy (PH). After PH, rats injected with ASCs-miR-27b had decreased inflammatory cytokines and increased hepatocyte growth factor and other related growth factors. We showed that the nature of ASCs-miR-27b to inhibit hepatic stellate cell activation was dependent upon peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) in vitro. Moreover, expression of miR-27b in ASCs induced heme oxygenase-1 (HO-1), resulting in increased production of ATP, protective cytokines/growth factors, and genes involved in mitochondrial biogenesis in a PGC-1α-dependent manner. RNA sequencing (RNA-Seq) analysis revealed drastic transcriptional changes in livers treated with ASCs-miR-27b after PH. The differentially expressed genes classified into “regeneration,” “fibrosis,” and “mitochondrial biogenesis” clusters were mainly mitochondrial. The potential biological context reflecting the effects of PGC-1α by ASCs-miR-27b treatment was also observed by the subnetwork analysis with HO-1 and PGC-1α being the top-ranked regulatory genes. We demonstrate autologous ASCs-miR-27b enhances liver regeneration and, importantly, preserves hepatic function through paracrine actions which offers a viable therapeutic option to facilitate rapid recovery after liver injury. PMID:26836372

  15. Clinical Significance of Myeloid-Derived Suppressor Cells in Human Immunodeficiency Virus-1/ Hepatitis C Virus-coinfected Patients.

    PubMed

    Lei, A H; Yang, Q; Cai, W P; Liu, Y F; Lan, Y; Qin, A P; Hu, F Y; Zhou, J

    2016-06-01

    Myeloid-derived suppressor cells (MDSCs) are known to accumulate during chronic viral infection, including human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) infection, and play a critical role in suppressing immune responses. However, the role of MDSCs in HIV/HCV coinfection is unclear. Here, we observed a dramatic increase in monocytic MDSCs (M-MDSCs) level in the peripheral blood of HIV/HCV-coinfected patients compared to that of healthy controls; the level of M-MDSCs proportion in coinfection was not higher than that in HIV or HCV monoinfection. Interestingly, we found the M-MDSCs level in coinfected patients correlated well with CD4(+) T cell loss (r = -0.5680; P = 0.0058), HIV-1 load (r = 0.6011; P = 0.0031), HCV load (r = 0.6288; P = 0.0017) and activated CD38(+) T cells (r = 0.5139; P = 0.0144). Initiation of highly active antiretroviral therapy considerably reduced both M-MDSCs and CD8(+) CD38(+) -activated T cell proportion in coinfected patients, and they showed a parallel course of decline. Thus, our results suggest that HIV-1 infection and high chronic immune activation may contribute to the expansion of M-MDSCs and accelerate the disease progression in HIV/HCV-coinfected patients. PMID:26972443

  16. Myeloid-derived suppressor cells correlate with patient outcomes in hepatic arterial infusion chemotherapy for hepatocellular carcinoma.

    PubMed

    Mizukoshi, Eishiro; Yamashita, Tatsuya; Arai, Kuniaki; Terashima, Takeshi; Kitahara, Masaaki; Nakagawa, Hidetoshi; Iida, Noriho; Fushimi, Kazumi; Kaneko, Shuichi

    2016-06-01

    Hepatic arterial infusion chemotherapy (HAIC) has been employed as an alternative therapy to sorafenib for the patients with advanced hepatocellular carcinoma (HCC). In this study, we performed a comparative analysis of various immune cell responses including tumor-associated antigen (TAA)-specific T cells, regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in advanced HCC patients treated with HAIC. Thirty-six HCC patients were examined in the study. Interferon gamma enzyme-linked immunospot assays were performed to examine the frequency of TAA-specific T cells. The frequencies of Tregs and MDSCs were examined by multicolor fluorescence-activated cell sorting analysis. The treatment with HAIC using interferon (IFN)/5-fluorouracil (FU) or IFN/FU + cisplatin modulated the frequencies of various immune cells. In 22.2 % of patients, the frequency of TAA-specific T cells increased after HAIC. Although the frequency of Tregs decreased after HAIC, it was not associated with the prognosis of patients. An analysis of prognostic factors for overall survival identified diameter of the tumor (<3.0 cm), absence of major portal vein invasion, absence of distant metastasis, Union Internationale Contre Le Cancer tumor lymph node metastasis stage (I or II), neutrophil lymphocytic ratio (<2.1) and the frequency of MDSCs (<30.5 %) as factors that prolonged overall survival time after HAIC. Even in the group adjusted with progressive levels of tumors, patients with a low frequency of MDSCs had a significantly longer overall survival time. In conclusion, the frequency of MDSCs before the treatment is a prognostic factor in HAIC against HCC. PMID:27083166

  17. Hepatitis C virus-induced myeloid-derived suppressor cells regulate T-cell differentiation and function via the signal transducer and activator of transcription 3 pathway.

    PubMed

    Ren, Jun P; Zhao, Juan; Dai, Jun; Griffin, Jeddidiah W D; Wang, Ling; Wu, Xiao Y; Morrison, Zheng D; Li, Guang Y; El Gazzar, Mohamed; Ning, Shun B; Moorman, Jonathan P; Yao, Zhi Q

    2016-08-01

    T cells play a pivotal role in controlling viral infection; however, the precise mechanisms responsible for regulating T-cell differentiation and function during infections are incompletely understood. In this study, we demonstrated an expansion of myeloid-derived suppressor cells (MDSCs), in particular the monocytic MDSCs (M-MDSCs; CD14(+) CD33(+) CD11b(+) HLA-DR(-/low) ), in patients with chronic hepatitis C virus (HCV) infection. Notably, HCV-induced M-MDSCs express high levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and interleukin-10 (IL-10) compared with healthy subjects. Blocking STAT3 signalling reduced HCV-mediated M-MDSC expansion and decreased IL-10 expression. Importantly, we observed a significant increase in the numbers of CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells following incubation of healthy peripheral blood mononuclear cells (PBMCs) with MDSCs derived from HCV-infected patients or treated with HCV core protein. In addition, depletion of MDSCs from PBMCs led to a significant reduction of Foxp3(+) Treg cells developed during chronic HCV infection. Moreover, depletion of MDSCs from PBMCs significantly increased interferon-γ production by CD4(+) T effector (Teff) cells derived from HCV patients. These results suggest that HCV-induced MDSCs promote Treg cell development and inhibit Teff cell function, suggesting a novel mechanism for T-cell regulation and a new strategy for immunotherapy against human viral diseases. PMID:27149428

  18. Effect of Chromatin-Remodeling Agents in Hepatic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells In Vitro and In Vivo

    PubMed Central

    Ye, Danna; Li, Tong; Heraud, Philip; Parnpai, Rangsun

    2016-01-01

    Epigenetic events, including covalent histone modifications and DNA methylation, play fundamental roles in the determination of lineage-specific gene expression and cell fates. The aim of this study was to determine whether the DNA methyltransferase inhibitor (DNMTi) 5-aza-2′-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) promote the hepatic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) and their therapeutic effect on liver damage. 1 μM TSA and 20 μM 5-aza-dC were added to standard hepatogenic medium especially at differentiation and maturation steps and their potential function on hepatic differentiation in vitro and in vivo was determined. Exposure of rBM-MSCs to 1 μM TSA at both the differentiation and maturation steps considerably improved hepatic differentiation. TSA enhanced the development of the hepatocyte shape, promoted the chronological expression of hepatocyte-specific markers, and improved hepatic functions. In contrast, treatment of rBM-MSCs with 20 μM 5-aza-dC alone or in combination with TSA was ineffective in improving hepatic differentiation in vitro. TSA and/or 5-aza-dC derived hepatocytes-like cells failed to improve the therapeutic potential in liver damage. We conclude that HDACis enhance hepatic differentiation in a time-dependent manner, while DNMTis do not induce the hepatic differentiation of rBM-MSCs in vitro. Their in vivo function needs further investigation. PMID:27242905

  19. Innate immune responses in human hepatocyte-derived cell lines alter genotype 1 hepatitis E virus replication efficiencies

    PubMed Central

    Devhare, Pradip B.; Desai, Swapnil; Lole, Kavita S.

    2016-01-01

    Hepatitis E virus (HEV) is a significant health problem in developing countries causing sporadic and epidemic forms of acute viral hepatitis. Hepatitis E is a self-limiting disease; however, chronic HEV infections are being reported in immunocompromised individuals. The disease severity is more during pregnancy with high mortality (20–25%), especially in third trimester. Early cellular responses after HEV infection are not completely understood. We analyzed innate immune responses associated with genotype-I HEV replication in human hepatoma cell lines (Huh7, Huh7.5 and HepG2/C3A) using HEV replicon system. These cells supported HEV replication with different efficiencies due to the cell type specific innate immune responses. HepG2/C3A cells were less supportive to HEV replication as compared to Huh7.5 and S10-3 cells. Reconstitution of the defective RIG-I and TLR3 signaling in Huh7.5 cells enabled them to induce higher level antiviral responses and restrict HEV replication, suggesting the involvement of both RIG-I and TLR3 in sensing HEV RNA and downstream activation of interferon regulatory factor 3 (IRF3) to generate antiviral responses. Inhibition of IRF3 mediated downstream responses in HepG2/C3A cells by pharmacological inhibitor BX795 significantly improved HEV replication efficiency implying the importance of this study in establishing a better cell culture system for future HEV studies. PMID:27230536

  20. Hepatitis C virus regulates the production of monocytic myeloid-derived suppressor cells from peripheral blood mononuclear cells through PI3K pathway and autocrine signaling.

    PubMed

    Pang, Xiaoli; Song, Hongxiao; Zhang, Qianqian; Tu, Zhengkun; Niu, Junqi

    2016-03-01

    Hepatitis C virus (HCV) infection is a major liver disease that ultimately develops into chronic hepatitis. Consequently, such patients are predisposed to serious complications, such as hepatocellular carcinoma. In HCV-infected patients, impaired T-cell responses are associated with persistent infection. Myeloid-derived suppressor cells (MDSCs) play a pivotal role in suppressing T-cell responses. In this study, we investigated the capacity and mechanism through which HCV transforms CD14+ monocytes into monocytic (Mo)-MDSCs. We showed that HCV core protein promotes CD14+ monocytes to develop a CD14+HLA-DR/low phenotype with upregulated indoleamine 2,3-dioxygenase (IDO) expression and suppressed T-cell proliferation. Importantly, HCV-induced Mo-MDSC production was attributed to the PI3K pathway via induction of IL-10 and TNF-α secretion. This process could be reversed by polyinosinic:polycytidylic acid (polyI:C) treatment. In conclusion, our results suggest that HCV regulates Mo-MDSC production from monocytes through the PI3K pathway and autocrine cytokines. The latter can serve as effective targets for novel HCV therapies. PMID:26821305

  1. In Vitro and In Vivo Infectivity and Pathogenicity of the Lymphoid Cell-Derived Woodchuck Hepatitis Virus

    PubMed Central

    Lew, Yuan-Yee; Michalak, Tomasz I.

    2001-01-01

    Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (∼103 virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses. PMID:11160675

  2. Hepatitis C Virus-Induced Myeloid-Derived Suppressor Cells Suppress NK Cell IFN-γ Production by Altering Cellular Metabolism via Arginase-1.

    PubMed

    Goh, Celeste C; Roggerson, Krystal M; Lee, Hai-Chon; Golden-Mason, Lucy; Rosen, Hugo R; Hahn, Young S

    2016-03-01

    The hepatitis C virus (HCV) infects ∼ 200 million people worldwide. The majority of infected individuals develop persistent infection, resulting in chronic inflammation and liver disease, including cirrhosis and hepatocellular carcinoma. The ability of HCV to establish persistent infection is partly due to its ability to evade the immune response through multiple mechanisms, including suppression of NK cells. NK cells control HCV replication during the early phase of infection and regulate the progression to chronic disease. In particular, IFN-γ produced by NK cells limits viral replication in hepatocytes and is important for the initiation of adaptive immune responses. However, NK cell function is significantly impaired in chronic HCV patients. The cellular and molecular mechanisms responsible for impaired NK cell function in HCV infection are not well defined. In this study, we analyzed the interaction of human NK cells with CD33(+) PBMCs that were exposed to HCV. We found that NK cells cocultured with HCV-conditioned CD33(+) PBMCs produced lower amounts of IFN-γ, with no effect on granzyme B production or cell viability. Importantly, this suppression of NK cell-derived IFN-γ production was mediated by CD33(+)CD11b(lo)HLA-DR(lo) myeloid-derived suppressor cells (MDSCs) via an arginase-1-dependent inhibition of mammalian target of rapamycin activation. Suppression of IFN-γ production was reversed by l-arginine supplementation, consistent with increased MDSC arginase-1 activity. These novel results identify the induction of MDSCs in HCV infection as a potent immune evasion strategy that suppresses antiviral NK cell responses, further indicating that blockade of MDSCs may be a potential therapeutic approach to ameliorate chronic viral infections in the liver. PMID:26826241

  3. In vitro interactions between rat bone marrow-derived endothelial progenitor cells and hepatic stellate cells: interaction between EPCs and HSCs.

    PubMed

    Liu, Feng; Liu, Zhi-da; Wu, Nan; Wang, Jiang-Hua; Zhang, Heng-Hui; Fei, Ran; Cong, Xu; Chen, Hong-song; Wei, Lai

    2013-08-01

    Transplantation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) has been reported to improve liver fibrosis, but there is no direct evidence for the mechanism of improvement. We investigated the mechanism in vitro by coculturing BM-derived EPCs with activated hepatic stellate cells (HSCs) to mimic the hepatic environment. EPCs and HSCs were cultured alone and indirectly cocultured at a 1:1 ratio in a Transwell system. The characteristics of HSCs and EPCs were examined at different time points. An invasion assay showed the time-dependent effect on degradation of the extracellular matrix (ECM) layer in EPCs cultured alone. Real-time PCR and enzyme-linked immunosorbent assay analysis revealed that EPCs served as a source of matrix metalloproteinase-9 (MMP-9), and MMP-9 expression levels significantly increased during the 2 d of coculture. CFSE labeling showed that EPCs inhibited proliferation of HSCs. Annexin-V/PI staining, erminal deoxynucleotidyl transferase X-dUTP nick end labeling analysis, and (cleaved) caspase-3 activity revealed that EPCs promoted HSC apoptosis. However, the proliferation and apoptosis of EPCs were unaffected by cocultured HSCs. Coculturing increased the expression of inducible nitric oxide synthase, vascular endothelial growth factor, and hepatocyte growth factor (HGF) in EPCs, promoted differentiation of EPCs, and reduced the expression of types I and III collagens and transforming growth factor beta 1. Knockdown of HGF expression attenuated EPC-induced activation of HSC apoptosis and profibrotic ability. These findings demonstrated that BM-derived EPCs could degrade ECM, promoting activated HSC apoptosis, suppressing proliferation and profibrotic ability of activated HSCs. HGF secretion by EPCs plays a key role in inducing activated HSC apoptosis and HSC profibrotic ability. PMID:23722413

  4. Enhanced expression of hepatocyte-specific microRNAs in valproic acid mediated hepatic trans-differentiation of human umbilical cord derived mesenchymal stem cells.

    PubMed

    Raut, Akshata; Khanna, Aparna

    2016-05-01

    MicroRNAs (miRNAs) play an important role in the control of cell fate determination during differentiation. In this study, we analyzed the expression pattern of microRNAs (miRNAs) during hepatic trans-differentiation. The protocol employed the use of histone deacetylase inhibitor (HDACI), valproic acid (VPA) to induce hepatic trans-differentiation of human umbilical cord Wharton's jelly derived mesenchymal stem cells (hUC-MSCs). The differentiated hepatocyte like cells (HLCs) from hUC-MSCs shared typical characteristics with mature hepatocytes, including morphology, expression of hepatocyte -specific genes at the molecular and cellular level. Moreover, the functionality of HLCs was confirmed through various liver function tests such as periodic acid-Schiff (PAS) stain for glycogen accumulation, enzyme-linked immunosorbent assay (ELISA) for synthesis of albumin and release of urea. The aim of the present work was to examine the effect of VPA treatment on miRNA expression during hepatic trans-differentiation. The analysis at miRNA level showed that there was a significant increase in expression of miRNAs involved in hepatic differentiation, due to VPA pre-treatment during differentiation. The study, thus demonstrated that improved expression of hepatocyte-specific miRNAs, miR-23b cluster (miR-27b-3p, miR-24-1-5p and miR-23b-3p), miR-30a-5p, miR-26a-5p, miR-148a-3p, miR-192-5p, miR-122-5p due to VPA pre-treatment contributed to a more efficient hepatic trans-differentiation from hUC-MSCs. The putative targets of these upregulated miRNAs were predicted using Bioinformatics analysis. Finally, miR-122-5p, highly upregulated miRNA during hepatic differentiation, was selected for target verification studies. Thus, this study also provides the basis for the function of miR-122-5p during hepatic differentiation of hUC-MSCs. PMID:27001466

  5. A Gaussia Luciferase Cell-Based System to Assess the Infection of Cell Culture- and Serum-Derived Hepatitis C Virus

    PubMed Central

    Koutsoudakis, George; Pérez-del-Pulgar, Sofía; González, Patricia; Crespo, Gonzalo; Navasa, Miquel; Forns, Xavier

    2012-01-01

    Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted Gaussia luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the Gaussia was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ź-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their in vitro infection and replication potential. Approximately 12% of the sera contained in vitro replication-competent viruses, as deduced by the Gaussia signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of in vitro replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds. PMID:23300900

  6. Generalized Liver- and Blood-Derived CD8+ T-Cell Impairment in Response to Cytokines in Chronic Hepatitis C Virus Infection

    PubMed Central

    Burke Schinkel, Stephanie C.; Carrasco-Medina, Lorna; Cooper, Curtis L.; Crawley, Angela M.

    2016-01-01

    Generalized CD8+ T-cell impairment in chronic hepatitis C virus (HCV) infection and the contribution of liver-infiltrating CD8+ T-cells to the immunopathogenesis of this infection remain poorly understood. It is hypothesized that this impairment is partially due to reduced CD8+ T-cell activity in response to cytokines such as IL-7, particularly within the liver. To investigate this, the phenotype and cytokine responsiveness of blood- and liver-derived CD8+ T-cells from healthy controls and individuals with HCV infection were compared. In blood, IL-7 receptor α (CD127) expression on bulk CD8+ T-cells in HCV infection was no different than controls yet was lower on central memory T-cells, and there were fewer naïve cells. IL-7-induced signalling through phosphorylated STAT5 was lower in HCV infection than in controls, and differed between CD8+ T-cell subsets. Production of Bcl-2 following IL-7 stimulation was also lower in HCV infection and inversely related to the degree of liver fibrosis. In liver-derived CD8+ T-cells, STAT5 activation could not be increased with cytokine stimulation and basal Bcl-2 levels of liver-derived CD8+ T-cells were lower than blood-derived counterparts in HCV infection. Therefore, generalized CD8+ T-cell impairment in HCV infection is characterized, in part, by impaired IL-7-mediated signalling and survival, independent of CD127 expression. This impairment is more pronounced in the liver and may be associated with an increased potential for apoptosis. This generalized CD8+ T-cell impairment represents an important immune dysfunction in chronic HCV infection that may alter patient health. PMID:27315061

  7. Enhanced hepatic differentiation of rat bone marrow-derived mesenchymal stem cells in spheroidal aggregate culture on a decellularized liver scaffold

    PubMed Central

    Bao, Ji; Wu, Qiong; Wang, Yujia; Li, Yi; Li, Li; Chen, Fei; Wu, Xiujuan; Xie, Mingjun; Bu, Hong

    2016-01-01

    In the present study, we aimed to determine whether the combination of aggregate culture and decellularized liver scaffolds (DLSs) promoted the hepatic differentiation of murine bone marrow-derived mesenchymal stem cells (BM-MSCs) into high yields of mature hepatocytes in vitro. Four culturing methods for differentiation [single cell (2D), spheroids (3D), 2D + DLS and 3D + DLS] were studied. To determine the differentiation stages of the MSCs, RT-qPCR of the hepatocyte genes, immunostaining of hepatocyte markers, and functional analyses were all performed. Compared with the other groups, hepatocyte-like cells which differentiated from BM-MSC spheroids on extracellular matrix (ECM) exhibited more intensive staining of stored glycogen, an elevated level of urea biosynthesis and albumin secretion as well as the higher expression of hepatocyte-specific genes. Our results indicated that DLSs combined with spheroidal aggregate culture may be used as an effective method to facilitate the hepatic maturation of BM-MSCs and may have future applications in stem cell-based liver regenerative medicine. PMID:27314916

  8. Enhanced hepatic differentiation of rat bone marrow-derived mesenchymal stem cells in spheroidal aggregate culture on a decellularized liver scaffold.

    PubMed

    Bao, Ji; Wu, Qiong; Wang, Yujia; Li, Yi; Li, Li; Chen, Fei; Wu, Xiujuan; Xie, Mingjun; Bu, Hong

    2016-08-01

    In the present study, we aimed to determine whether the combination of aggregate culture and decellularized liver scaffolds (DLSs) promoted the hepatic differentiation of murine bone marrow-derived mesenchymal stem cells (BM-MSCs) into high yields of mature hepatocytes in vitro. Four culturing methods for differentiation [single cell (2D), spheroids (3D), 2D + DLS and 3D + DLS] were studied. To determine the differentiation stages of the MSCs, RT-qPCR of the hepatocyte genes, immunostaining of hepatocyte markers, and functional analyses were all performed. Compared with the other groups, hepatocyte-like cells which differentiated from BM‑MSC spheroids on extracellular matrix (ECM) exhibited more intensive staining of stored glycogen, an elevated level of urea biosynthesis and albumin secretion as well as the higher expression of hepatocyte-specific genes. Our results indicated that DLSs combined with spheroidal aggregate culture may be used as an effective method to facilitate the hepatic maturation of BM-MSCs and may have future applications in stem cell-based liver regenerative medicine. PMID:27314916

  9. Duck hepatitis B virus integrations in LMH chicken hepatoma cells: identification and characterization of new episomally derived integrations.

    PubMed Central

    Gong, S S; Jensen, A D; Wang, H; Rogler, C E

    1995-01-01

    While the cytoplasmic phase of the hepadnavirus replication cycle is well understood, very little is known about the nuclear phase. In contrast to retroviruses, proviral integration is not required for hepadnavirus replication; however, some of the viral DNAs in the nucleus are diverted into an integration pathway. Under certain conditions these integrations function as carcinogenic agents. In order to study the integration process, we have utilized LMH-D2 cells, which replicate wild-type duck hepatitis B virus (DHBV), to develop the first protocol to detect and characterize integrations of DHBV originating from episomal viral DNAs. Contrary to expectations, our results showed that stable new integrations are readily detectable in subclones of LMH-D2 cells. Complete characterization of one integration revealed a single-genome-length integrant with the structure of double-stranded linear (DSL) DHBV DNAs which are produced by in situ priming during viral replication. The integration contained a terminal redundancy of 6 bp from the r region of the virus DNA minus strand as well as a direct repeat of 70 bp of cellular DNA. On the basis of the structure of the integrant and the cellular DNA target site, we propose a molecular model for the integration mechanism that has some similarities to that of retroviruses. Identification of DSL hepadnavirus DNA integration suggests the possibility that modified DSL viral DNAs may be the precursors to a class of simple, unrearranged hepadnavirus integrations. PMID:7494330

  10. Antiproliferative effects of triterpenoidal derivatives, obtained from the marine sponge Siphonochalina sp., on human hepatic and colorectal cancer cells.

    PubMed

    Abdel-Lateff, Ahmed; Al-Abd, Ahmed M; Alahdal, Abdulrahman M; Alarif, Walied M; Ayyad, Seif-Eldin N; Al-Lihaibi, Sultan S; Hegazy, Mohamed E; Al Mohammadi, Ameen; Abdelghany, Tamer M; Abdel-Naim, Ashraf B; Moustafa, Mohamed A A; Banjer, Zainy M; Azhar, Ahmad S

    2016-01-01

    Three triterpenoidal derivatives [Sipholenol A (1), sipholenol L (2) and sipholenone A (3)] were isolated from the Red Sea sponge Siphonochalina sp. The structures were determined based on spectroscopic measurements (NMR, UV, IR and MS). The isolated compounds were evaluated for their cytotoxic activity against three cancer cell lines; HepG2, Caco-2 and HT-29. Moreover, the effects of these metabolites on cell cycle progression as well as cell cycle regulating proteins were assessed. Compounds 1, 2 and 3 showed moderate activity against HepG2 cells with IC(50) values of 17.18 ± 1.18, 24.01 ± 0.59 and 35.06 ± 1.10 μM, respectively. Compounds 1 and 2 exerted a considerable antiproliferative effect with IC(50) values of 4.80 ± 0.18 and 26.64 ± 0.30 μM, respectively, against Caco-2 cells. Finally, 1 and 2 exhibited antiproliferative activity against colorectal cancer cells (HT-29) with IC(50) values of 24.65 ± 0.80 and 4.48 ± 0.1 μM, respectively. Cell cycle analysis indicated that these compounds induced cell cycle arrest particularly in G0/G1 and S phases. Furthermore, the triterpenoids increased the expression of cyclin-B1, cyclin-D1 and cleaved caspase-3, as determined by immunofluorescence, indicating an important role of apoptosis in cell death induced by these compounds. PMID:26845717

  11. Hepatic Differentiation from Human Ips Cells Using M15 Cells.

    PubMed

    Umeda, Kahoko; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    Here, we describe a procedure of human iPS cells differentiation into the definitive endoderm, further into albumin-expressing and albumin-secreting hepatocyte, using M15, a mesonephros- derived cell line. Approximately 90 % of human iPS cells differentiated into SOX17-positive definitive endoderm then approximately 50 % of cells became albumin-positive cells, and secreted ALB protein. This M15 feeder system for endoderm and hepatic differentiation is a simple and efficient method, and useful for elucidating molecular mechanisms for hepatic fate decision, and could represent an attractive approach for a surrogate cell source for pharmaceutical studies. PMID:25417065

  12. A Novel Matrine Derivative WM130 Inhibits Activation of Hepatic Stellate Cells and Attenuates Dimethylnitrosamine-Induced Liver Fibrosis in Rats

    PubMed Central

    Xu, Yang; Peng, Zhangxiao; Ji, Weidan; Li, Xiang; Lin, Xuejing; Qian, Liqiang; Li, Xiaoya; Chai, Xiaoyun; Wu, Qiuye; Gao, Quangen; Su, Changqing

    2015-01-01

    Activation of hepatic stellate cells (HSCs) is a critical event in process of hepatic fibrogenesis and cirrhosis. Matrine, the active ingredient of Sophora, had been used for clinical treatment of acute/chronic liver disease. However, its potency was low. We prepared a high potency and low toxicity matrine derivate, WM130 (C30N4H40SO5F), which exhibited better pharmacological activities on antihepatic fibrosis. This study demonstrated that WM130 results in a decreased proliferative activity of HSC-T6 cells, with the half inhibitory concentration (IC50) of 68 μM. WM130 can inhibit the migration and induce apoptosis in HSC-T6 cells at both concentrations of 68 μM (IC50) and 34 μM (half IC50). The expression of α-SMA, Collagen I, Collagen III, and TGF-β1 could be downregulated, and the protein phosphorylation levels of EGFR, AKT, ERK, Smad, and Raf (p-EGFR, p-AKT, p-ERK, p-Smad, and p-Raf) were also decreased by WM130. On the DMN-induced rat liver fibrosis model, WM130 can effectively reduce the TGF-β1, AKT, α-SMA, and p-ERK levels, decrease the extracellular matrix (ECM) formation, and inhibit rat liver fibrosis progression. In conclusion, this study demonstrated that WM130 can significantly inhibit the activation of HSC-T6 cells and block the rat liver fibrosis progression by inducing apoptosis, suppressing the deposition of ECM, and inhibiting TGF-β/Smad and Ras/ERK pathways. PMID:26167476

  13. A Novel Matrine Derivative WM130 Inhibits Activation of Hepatic Stellate Cells and Attenuates Dimethylnitrosamine-Induced Liver Fibrosis in Rats.

    PubMed

    Xu, Yang; Peng, Zhangxiao; Ji, Weidan; Li, Xiang; Lin, Xuejing; Qian, Liqiang; Li, Xiaoya; Chai, Xiaoyun; Wu, Qiuye; Gao, Quangen; Su, Changqing

    2015-01-01

    Activation of hepatic stellate cells (HSCs) is a critical event in process of hepatic fibrogenesis and cirrhosis. Matrine, the active ingredient of Sophora, had been used for clinical treatment of acute/chronic liver disease. However, its potency was low. We prepared a high potency and low toxicity matrine derivate, WM130 (C30N4H40SO5F), which exhibited better pharmacological activities on antihepatic fibrosis. This study demonstrated that WM130 results in a decreased proliferative activity of HSC-T6 cells, with the half inhibitory concentration (IC50) of 68 μM. WM130 can inhibit the migration and induce apoptosis in HSC-T6 cells at both concentrations of 68 μM (IC50) and 34 μM (half IC50). The expression of α-SMA, Collagen I, Collagen III, and TGF-β1 could be downregulated, and the protein phosphorylation levels of EGFR, AKT, ERK, Smad, and Raf (p-EGFR, p-AKT, p-ERK, p-Smad, and p-Raf) were also decreased by WM130. On the DMN-induced rat liver fibrosis model, WM130 can effectively reduce the TGF-β1, AKT, α-SMA, and p-ERK levels, decrease the extracellular matrix (ECM) formation, and inhibit rat liver fibrosis progression. In conclusion, this study demonstrated that WM130 can significantly inhibit the activation of HSC-T6 cells and block the rat liver fibrosis progression by inducing apoptosis, suppressing the deposition of ECM, and inhibiting TGF-β/Smad and Ras/ERK pathways. PMID:26167476

  14. A telomerase-derived peptide regulates reactive oxygen species and hepatitis C virus RNA replication in HCV-infected cells via heat shock protein 90.

    PubMed

    Lee, Seoung-Ae; Kim, Jinhee; Sim, Jihyun; Kim, Sang-Gyune; Kook, Yoon-Hoh; Park, Chung-Gyu; Kim, Hang-Rae; Kim, Bum-Joon

    2016-02-26

    GV1001, a synthetic peptide derived from human telomerase, has a range of diverse biological activities, including an antioxidant function. Here, we investigated the role of GV1001 in hepatitis C virus (HCV)-infected Huh7.5 (JFH-1) cells. We showed that GV1001 inhibited the production of ROS with decreased MAP kinase signaling. Interestingly, GV1001 lost its antioxidant activity as ROS levels decreased, resulting in a reduction in extracellular heat shock protein 90 (eHSP90) as low-density lipoprotein receptor-related protein 1 (LRP1) was blocked or knocked-down. GV1001 binds to eHSP90 and is delivered into the cell by endocytosis via LRP1. Endocytosed GV1001 finally suppressed ROS generation, presumably by hindering the interaction between eHSP90 and NADPH oxidase (NOX). Importantly, GV1001 suppressed HCV RNA replication in JFH-1 cells by inhibiting the binding of HSP90 to FKBP8, a member of the FK506-binding protein family. We also found that HSP90 expression was high in HCV-infected hepatocytes. Therefore, our data suggest that GV1001 may be a good therapeutic agent by controlling HCV RNA replication, as well as by preferentially targeting cells under conditions of oxidative stress. PMID:26828270

  15. Gene regulation in hepatic stellate cell.

    PubMed

    Lang, A; Brenner, D A

    1999-03-01

    Hepatic stellate cells are now recognized as the major source of extracellular matrix in hepatic fibrosis. Following liver injury the hepatic stellate cell changes from a quiescent to an activated cell. The activation process includes an increased proliferation rate, a phenotypic change to a myofibroblast-like cell, loss of vitamin A stores, increased extra-cellular matrix protein synthesis and contractility. Furthermore, hepatic stellate cells have been implicated in hepatic inflammation through their ability to secrete cytokines and chemokines. Here, we review the literature on the molecular pathogenesis of hepatic stellate cells activation with emphasis on the most recent findings. The reviewed topics include transcriptional and post-transcriptional regulation of the genes encoding type I collagen in hepatic stellate cells; the role of the transcription factor nuclear factor Kappa B in the hepatic stellate cell activation; focal adhesion kinase and integrin-mediated signal transduction in hepatic stellate cell, and apoptosis in hepatic stellate cells. New insight into hepatic stellate cell activation and death may lead to the development of novel therapies for hepatic fibrosis. PMID:10363203

  16. Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factor-β receptor-mediated ERK and p38 pathways

    SciTech Connect

    Zhang, Feng; Ni, Chunyan; Kong, Desong; Zhang, Xiaoping; Zhu, Xiaojing; Chen, Li; Lu, Yin; Zheng, Shizhong

    2012-11-15

    Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with accumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fibrosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSC activation stimulated by hydrogen peroxide (H{sub 2}O{sub 2}), an in vitro model mimicking the oxidative stress in liver fibrogenesis, and to elucidate the possible mechanisms. Our results demonstrated that H{sub 2}O{sub 2} at 5 μM significantly stimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazine dose-dependently suppressed proliferation and induced apoptosis in H{sub 2}O{sub 2}-activated HSCs, and attenuated expression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factor-β receptor (PDGF-βR) expression and blocked the phosphorylation of extracellular regulated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-βR. Further molecular evidence suggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-βR and might be involved in ligustrazine reduction of fibrotic marker gene expression under H{sub 2}O{sub 2} stimulation. Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H{sub 2}O{sub 2}-stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by oxidative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis. Highlights: ► Ligustrazine inhibits oxidative stress-induced HSC activation.

  17. Cytotoxic T Lymphocytes Derived from Patients with Chronic Hepatitis C Virus Infection Kill Bystander Cells via Fas-FasL Interaction

    PubMed Central

    Gremion, Christel; Grabscheid, Benno; Wölk, Benno; Moradpour, Darius; Reichen, Jürg; Pichler, Werner; Cerny, Andreas

    2004-01-01

    The role of Fas-mediated lysis of hepatocytes in hepatitis C virus (HCV)-induced injury is frequently discussed. We therefore analyzed the effect of the number of HCV antigen-expressing cells, the mode of antigen presentation, and the number of cytotoxic T lymphocytes in a coculture system mimicking cellular components of the liver. Here, we show that endogenously processed HCV proteins are capable of inducing bystander killing. We further demonstrate that 0.8 to 1.5% of cells presenting HCV antigens suffice to induce lysis of 10 to 29% of bystander cells, suggesting that the mechanism may be operative at low fractions of infected versus uninfected hepatocytes in vivo. Our data underscore the role of the Fas pathway in HCV-related liver injury and support the exploration of Fas-based treatment strategies for patients with chronic hepatitis C virus infection. PMID:14747581

  18. The induction of human myeloid derived suppressor cells through hepatic stellate cells is dose-dependently inhibited by the tyrosine kinase inhibitors nilotinib, dasatinib and sorafenib, but not sunitinib.

    PubMed

    Heine, Annkristin; Schilling, Judith; Grünwald, Barbara; Krüger, Achim; Gevensleben, Heidrun; Held, Stefanie Andrea Erika; Garbi, Natalio; Kurts, Christian; Brossart, Peter; Knolle, Percy; Diehl, Linda; Höchst, Bastian

    2016-03-01

    Increased numbers of immunosuppressive myeloid derived suppressor cells (MDSCs) correlate with a poor prognosis in cancer patients. Tyrosine kinase inhibitors (TKIs) are used as standard therapy for the treatment of several neoplastic diseases. However, TKIs not only exert effects on the malignant cell clone itself but also affect immune cells. Here, we investigate the effect of TKIs on the induction of MDSCs that differentiate from mature human monocytes using a new in vitro model of MDSC induction through activated hepatic stellate cells (HSCs). We show that frequencies of monocytic CD14(+)HLA-DR(-/low) MDSCs derived from mature monocytes were significantly and dose-dependently reduced in the presence of dasatinib, nilotinib and sorafenib, whereas sunitinib had no effect. These regulatory effects were only observed when TKIs were present during the early induction phase of MDSCs through activated HSCs, whereas already differentiated MDSCs were not further influenced by TKIs. Neither the MAPK nor the NFκB pathway was modulated in MDSCs when any of the TKIs was applied. When functional analyses were performed, we found that myeloid cells treated with sorafenib, nilotinib or dasatinib, but not sunitinib, displayed decreased suppressive capacity with regard to CD8+ T cell proliferation. Our results indicate that sorafenib, nilotinib and dasatinib, but not sunitinib, decrease the HSC-mediated differentiation of monocytes into functional MDSCs. Therefore, treatment of cancer patients with these TKIs may in addition to having a direct effect on cancer cells also prevent the differentiation of monocytes into MDSCs and thereby differentially modulate the success of immunotherapeutic or other anti-cancer approaches. PMID:26786874

  19. [Study of plasma and hepatic hydrolysis of nitroxinil derivatives].

    PubMed

    Baziard-Mouysset, G; Alvinerie, M; Galtier, P; Ane-Margail, M; Floch, R; Payard, M

    1996-01-01

    Seven esters derivatives of Nitroxinil were prepared and their structures were assigned by IR and 1H-NMR spectroscopy. The rate of plasma and hepatic hydrolysis were evaluated in vitro in sheep and rabbit. In view of this profile of activity, pivaloyl derivative merits evaluation, in vivo. PMID:8953797

  20. Pharmacological Intervention in Hepatic Stellate Cell Activation and Hepatic Fibrosis

    PubMed Central

    Schon, Hans-Theo; Bartneck, Matthias; Borkham-Kamphorst, Erawan; Nattermann, Jacob; Lammers, Twan; Tacke, Frank; Weiskirchen, Ralf

    2016-01-01

    The activation and transdifferentiation of hepatic stellate cells (HSCs) into contractile, matrix-producing myofibroblasts (MFBs) are central events in hepatic fibrogenesis. These processes are driven by autocrine- and paracrine-acting soluble factors (i.e., cytokines and chemokines). Proof-of-concept studies of the last decades have shown that both the deactivation and removal of hepatic MFBs as well as antagonizing profibrogenic factors are in principle suitable to attenuate ongoing hepatic fibrosis. Although several drugs show potent antifibrotic activities in experimental models of hepatic fibrosis, there is presently no effective pharmaceutical intervention specifically approved for the treatment of liver fibrosis. Pharmaceutical interventions are generally hampered by insufficient supply of drugs to the diseased liver tissue and/or by adverse effects as a result of affecting non-target cells. Therefore, targeted delivery systems that bind specifically to receptors solely expressed on activated HSCs or transdifferentiated MFBs and delivery systems that can improve drug distribution to the liver in general are urgently needed. In this review, we summarize current strategies for targeted delivery of drugs to the liver and in particular to pro-fibrogenic liver cells. The applicability and efficacy of sequestering molecules, selective protein carriers, lipid-based drug vehicles, viral vectors, transcriptional targeting approaches, therapeutic liver- and HSC-specific nanoparticles, and miRNA-based strategies are discussed. Some of these delivery systems that had already been successfully tested in experimental animal models of ongoing hepatic fibrogenesis are expected to translate into clinically useful therapeutics specifically targeting HSCs. PMID:26941644

  1. Angiotensin II enhances epithelial-to-mesenchymal transition through the interaction between activated hepatic stellate cells and the stromal cell-derived factor-1/CXCR4 axis in intrahepatic cholangiocarcinoma.

    PubMed

    Okamoto, Koichi; Tajima, Hidehiro; Nakanuma, Shinichi; Sakai, Seisho; Makino, Isamu; Kinoshita, Jun; Hayashi, Hironori; Nakamura, Keishi; Oyama, Katsunobu; Nakagawara, Hisatoshi; Fujita, Hideto; Takamura, Hiroyuki; Ninomiya, Itasu; Kitagawa, Hirohisa; Fushida, Sachio; Fujimura, Takashi; Harada, Shinichi; Wakayama, Tomohiko; Iseki, Shoichi; Ohta, Tetsuo

    2012-08-01

    We previously reported that hepatic stellate cells (HSCs) activated by angiotensin II (AngII) facilitate stromal fibrosis and tumor progression in intrahepatic cholangiocarcinoma (ICC). AngII has been known as a growth factor which can promote epithelial-to-mesenchymal transition (EMT) in renal epithelial cells, alveolar epithelial cells and peritoneal mesothelial cells. However, in the past, the relationship between AngII and stromal cell-derived factor-1 (SDF-1) in the microenvironment around cancer and the role of AngII on EMT of cancer cells has not been reported in detail. SDF-1 and its specific receptor, CXCR4, are now receiving attention as a mechanism of cell progression and metastasis. In this study, we examined whether activated HSCs promote tumor fibrogenesis, tumor progression and distant metastasis by mediating EMT via the AngII/AngII type 1 receptor (AT-1) and the SDF-1/CXCR4 axis. Two human ICC cell lines and a human HSC line, LI-90, express CXCR4. Significantly higher concentration of SDF-1α was released into the supernatant of LI-90 cells to which AngII had been added. SDF-1α increased the proliferative activity of HSCs and enhanced the activation of HSCs as a growth factor. Furthermore, addition of SDF-1α and AngII enhanced the increase of the migratory capability and vimentin expression, reduced E-cadherin expression, and translocated the expression of β-catenin into the nucleus and cytoplasm in ICC cells. Co-culture with HSCs also enhanced the migratory capability of ICC cells. These findings suggest that SDF-1α, released from activated HSCs and AngII, play important roles in cancer progression, tumor fibrogenesis, and migration in autocrine and paracrine fashion by mediating EMT. Our mechanistic findings may provide pivotal insights into the molecular mechanism of the AngII and SDF-1α-initiated signaling pathway that regulates fibrogenesis in cancerous stroma, tumor progression and meta-stasis of tumor cells expressing AT-1 and CXCR4

  2. Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons

    PubMed Central

    Anggakusuma; Frentzen, Anne; Gürlevik, Engin; Yuan, Qinggong; Steinmann, Eike; Ott, Michael; Staeheli, Peter; Schmid-Burgk, Jonathan; Schmidt, Tobias; Hornung, Veit; Kuehnel, Florian

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) efficiently infects only humans and chimpanzees. Although the detailed mechanisms responsible for this narrow species tropism remain elusive, recent evidence has shown that murine innate immune responses efficiently suppress HCV replication. Therefore, poor adaptation of HCV to evade and/or counteract innate immune responses may prevent HCV replication in mice. The HCV NS3-4A protease cleaves human MAVS, a key cellular adaptor protein required for RIG-I-like receptor (RLR)-dependent innate immune signaling. However, it is unclear if HCV interferes with mouse MAVS function equally well. Moreover, MAVS-dependent signaling events that restrict HCV replication in mouse cells were incompletely defined. Thus, we quantified the ability of HCV NS3-4A to counteract mouse and human MAVS. HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signaling in human and mouse cells. Moreover, replicon-encoded protease cleaved a similar fraction of both MAVS variants. Finally, FLAG-tagged MAVS proteins repressed HCV replication to similar degrees. Depending on MAVS expression, HCV replication in mouse liver cells triggered not only type I but also type III IFNs, which cooperatively repressed HCV replication. Mouse liver cells lacking both type I and III IFN receptors were refractory to MAVS-dependent antiviral effects, indicating that the HCV-induced MAVS-dependent antiviral state depends on both type I and III IFN receptor signaling. IMPORTANCE In this study, we found that HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signaling in human and mouse cells. Therefore, it is unlikely that ineffective cleavage of mouse MAVS per se precludes HCV propagation in immunocompetent mouse liver cells. Hence, approaches to reinforce HCV replication in mouse liver cells (e.g., by expression of essential human replication cofactors) should not be thwarted by the poor ability of HCV to counteract MAVS-dependent antiviral signaling

  3. Synthesis of hemslecin A derivatives: a new class of hepatitis B virus inhibitors.

    PubMed

    Guo, Rui-Hua; Geng, Chang-An; Huang, Xiao-Yan; Ma, Yun-Bao; Zhang, Quan; Wang, Li-Jun; Zhang, Xue-Mei; Zhang, Rong-Ping; Chen, Ji-Jun

    2013-03-01

    A series of hemslecin A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities, namely, inhibiting the secretion of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV DNA replication on HepG 2.2.15 cells. Most of the derivatives showed enhanced anti-HBV activities, of which compounds A1-A7, B5, C and E exhibited significant activities inhibiting HBV DNA replication with IC(50) values of 2.8-11.6 μM, comparable to that of the positive control, tenofovir. Compounds A1-A3, A5, B5, and C displayed low cytotoxicities, which resulted in high SI values of 89.7, 55.6, 77.8, >83.4, >55.8, and >150.5, respectively. PMID:23385212

  4. Natural Killer Cells in Viral Hepatitis

    PubMed Central

    Rehermann, Barbara

    2015-01-01

    Natural killer (NK) cells are traditionally regarded as first-line effectors of the innate immune response, but they also have a distinct role in chronic infection. Here, we review the role of NK cells against hepatitis C virus (HCV) and hepatitis B virus (HBV), two agents that cause acute and chronic hepatitis in humans. Interest in NK cells was initially sparked by genetic studies that demonstrated an association between NK cell–related genes and the outcome of HCV infection. Viral hepatitis also provides a model to study the NK cell response to both endogenous and exogenous type I interferon (IFN). Levels of IFN-stimulated genes increase in both acute and chronic HCV infection and pegylated IFNα has been the mainstay of HCV and HBV treatment for decades. In chronic viral hepatitis, NK cells display decreased production of antiviral cytokines. This phenotype is found in both HCV and HBV infection but is induced by different mechanisms. Potent antivirals now provide the opportunity to study the reversibility of the suppressed cytokine production of NK cells in comparison with the antigen-induced defect in IFNγ and tumor necrosis factor-α production of virus-specific T cells. This has implications for immune reconstitution in other conditions of chronic inflammation and immune exhaustion, such as human immunodeficiency virus infection and cancer. PMID:26682281

  5. Matrix metalloproteinase-14 mediates formation of bile ducts and hepatic maturation of fetal hepatic progenitor cells.

    PubMed

    Otani, Satoshi; Kakinuma, Sei; Kamiya, Akihide; Goto, Fumio; Kaneko, Shun; Miyoshi, Masato; Tsunoda, Tomoyuki; Asano, Yu; Kawai-Kitahata, Fukiko; Nitta, Sayuri; Nakata, Toru; Okamoto, Ryuichi; Itsui, Yasuhiro; Nakagawa, Mina; Azuma, Seishin; Asahina, Yasuhiro; Yamaguchi, Tomoyuki; Koshikawa, Naohiko; Seiki, Motoharu; Nakauchi, Hiromitsu; Watanabe, Mamoru

    2016-01-22

    Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver development; however, the molecular mechanisms regulating the phenotype of these cells have not been completely elucidated. Matrix metalloproteinase (MMP)-14 is a type I transmembrane proteinase regulating pericellular proteolysis of the extracellular matrix and is essential for the activation of several MMPs and cytokines. However, the physiological functions of MMP-14 in liver development are unknown. Here we describe a functional role for MMP-14 in hepatic and biliary differentiation of mouse hepatoblasts. MMP-14 was upregulated in cells around the portal vein in perinatal stage liver. Formation of bile duct-like structures in MMP-14-deficient livers was significantly delayed compared with wild-type livers in vivo. In vitro biliary differentiation assays showed that formation of cholangiocytic cysts derived from MMP-14-deficient hepatoblasts was completely impaired, and that overexpression of MMP-14 in hepatoblasts promoted the formation of bile duct-like cysts. In contrast, the expression of molecules associated with metabolic functions in hepatocytes, including hepatic nuclear factor 4α and tryptophan 2,3-dioxygenase, were significantly increased in MMP-14-deficient livers. Expression of the epidermal growth factor receptor and phosphorylation of mitogen-activated protein kinases were significantly upregulated in MMP-14-deficient livers. We demonstrate that MMP-14-mediated signaling in fetal hepatic progenitor cells promotes biliary luminal formation around the portal vein and negatively controls the maturation of hepatocytes. PMID:26724533

  6. Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

    PubMed Central

    Dong, Xue-Jun; Zhang, Guo-Rong; Zhou, Qing-Jun; Pan, Ruo-Lang; Chen, Ye; Xiang, Li-Xin; Shao, Jian-Zhong

    2009-01-01

    AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells

  7. (+)-Catechin attenuates activation of hepatic stellate cells.

    PubMed

    Bragança de Moraes, Cristina Machado; Bitencourt, Shanna; de Mesquita, Fernanda Cristina; Mello, Denizar; de Oliveira, Leticia Paranhos; da Silva, Gabriela Viegas; Lorini, Vinicius; Caberlon, Eduardo; de Souza Basso, Bruno; Schmid, Julia; Ferreira, Gabriela Acevedo; de Oliveira, Jarbas Rodrigues

    2014-04-01

    (+)-Catechin is a type of catechin present in large amounts in açaí fruits and cocoa seeds. Besides its antioxidant and anti-inflammatory activities, little is known about its effects in the liver, especially during hepatic fibrosis. We report here the effects of (+)-catechin on hepatic stellate cells. (+)-Catechin induced quiescent phenotype in GRX cells, along with an increase in lipid droplets. Proliferator-activated receptor γ mRNA expression was upregulated, whereas type I collagen mRNA expression was downregulated. Pro-inflammatory cytokines were not influenced by (+)-catechin, whereas the levels of interleukin 10 were significantly increased. The data provide evidence that (+)-catechin can reduce hepatic stellate cell activation. PMID:24353036

  8. Effects of T cell immunoglobulin and mucin domain-containing molecule-3 signaling molecule on human monocyte-derived dendritic cells with hepatitis B virus surface antigen stimulation in vitro.

    PubMed

    Yu, Zhenjun; Jiang, Ting; Zhu, Min; Pan, Kechuan; Yan, Fei; Zhu, Jiansheng

    2016-03-01

    The aim of the present study was to investigate the in vitro effects of hepatitis B virus surface antigen (HBsAg) on the immune function of human monocyte-derived dendritic cells (MD‑DCs), and the moderating role of T cell immunoglobulin and mucin domain‑containing molecule‑3 (Tim‑3) signaling molecule. The monocytes, obtained from healthy adult peripheral blood, were incubated with recombinant human granulocyte‑macrophage colony‑stimulating factor and interleukin (IL)‑4 to induce DCs. DC‑associated cell markers were detected using flow cytometry. MD‑DCs were treated with HBsAg (5 µg/ml) in vitro for 48 h and subsequently, cell markers, lymphocyte stimulatory capacity, signaling protein and downstream cytokines were assessed. In addition, a Tim‑3 monoclonal antibody was used to inhibit the Tim‑3 signaling pathway, and subsequently the immune responses of MD‑DCs to HBsAg stimulation were determined using the aforementioned method. The cell phenotype expressions of MD‑DCs were all significantly increased with cluster of differentiation (CD)11c at 70.09±0.57%, human leukocyte antigen‑DR at 79.83±2.12%, CD80 at 48.33±7.34% and CD86 at 44.21±5.35%. The treatment of MD‑DCs with HBsAg resulted in a CD80 and CD86 enhanced expression, enhanced lymphocyte stimulatory capacity, upregulated expression of Tim‑3 and nuclear factor‑κB (NF‑κB), as well as enhanced cytokine secretion of IL‑6, IL‑10 and interferon (IFN)‑γ. However, a reduced immune response of MD‑DCs in response to HBsAg stimulation was observed when the Tim‑3 signaling pathway was inhibited prior to stimulation. The expression of NF‑κB was decreased and the cytokine secretion level of IL‑6, IL‑10 and IFN‑γ were downregulated. The treatment with HBsAg in vitro resulted in an enhanced immune response of MD‑DCs, which may be positively regulated by the Tim-3 signaling molecule. PMID:26820685

  9. Hepatitis C virus infection of cholangiocarcinoma cell lines.

    PubMed

    Fletcher, Nicola F; Humphreys, Elizabeth; Jennings, Elliott; Osburn, William; Lissauer, Samantha; Wilson, Garrick K; van IJzendoorn, Sven C D; Baumert, Thomas F; Balfe, Peter; Afford, Simon; McKeating, Jane A

    2015-06-01

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo. PMID:25701818

  10. Hepatitis C virus infection of cholangiocarcinoma cell lines

    PubMed Central

    Fletcher, Nicola F.; Humphreys, Elizabeth; Jennings, Elliott; Osburn, William; Lissauer, Samantha; Wilson, Garrick K.; van IJzendoorn, Sven C. D.; Baumert, Thomas F.; Balfe, Peter; Afford, Simon

    2015-01-01

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo. PMID:25701818

  11. The development of hepatic stellate cells in normal and abnormal human fetuses – an immunohistochemical study

    PubMed Central

    Loo, Christine K C; Pereira, Tamara N; Pozniak, Katarzyna N; Ramsing, Mette; Vogel, Ida; Ramm, Grant A

    2015-01-01

    The precise embryological origin and development of hepatic stellate cells is not established. Animal studies and observations on human fetuses suggest that they derive from posterior mesodermal cells that migrate via the septum transversum and developing diaphragm to form submesothelial cells beneath the liver capsule, which give rise to mesenchymal cells including hepatic stellate cells. However, it is unclear if these are similar to hepatic stellate cells in adults or if this is the only source of stellate cells. We have studied hepatic stellate cells by immunohistochemistry, in developing human liver from autopsies of fetuses with and without malformations and growth restriction, using cellular Retinol Binding Protein-1 (cRBP-1), Glial Fibrillary Acidic Protein (GFAP), and α-Smooth Muscle Actin (αSMA) antibodies, to identify factors that influence their development. We found that hepatic stellate cells expressing cRBP-1 are present from the end of the first trimester of gestation and reduce in density throughout gestation. They appear abnormally formed and variably reduced in number in fetuses with abnormal mesothelial Wilms Tumor 1 (WT1) function, diaphragmatic hernia and in ectopic liver nodules without mesothelium. Stellate cells showed similarities to intravascular cells and their presence in a fetus with diaphragm agenesis suggests they may be derived from circulating stem cells. Our observations suggest circulating stem cells as well as mesothelium can give rise to hepatic stellate cells, and that they require normal mesothelial function for their development. PMID:26265759

  12. Myeloid derived suppressor cells

    PubMed Central

    Waldron, Todd J.; Quatromoni, Jon G.; Karakasheva, Tatiana A.; Singhal, Sunil; Rustgi, Anil K.

    2013-01-01

    The goal of achieving measurable response with cancer immunotherapy requires counteracting the immunosuppressive characteristics of tumors. One of the mechanisms that tumors utilize to escape immunosurveillance is the activation of myeloid derived suppressor cells (MDSCs). Upon activation by tumor-derived signals, MDSCs inhibit the ability of the host to mount an anti-tumor immune response via their capacity to suppress both the innate and adaptive immune systems. Despite their relatively recent discovery and characterization, anti-MDSC agents have been identified, which may improve immunotherapy efficacy. PMID:23734336

  13. Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systems.

    PubMed

    Rodrigues, Robim M; Heymans, Anja; De Boe, Veerle; Sachinidis, Agapios; Chaudhari, Umesh; Govaere, Olivier; Roskams, Tania; Vanhaecke, Tamara; Rogiers, Vera; De Kock, Joery

    2016-01-01

    Primary human hepatocytes (hHEP), human HepaRG and HepG2 cell lines are the most used human liver-based in vitro models for hepatotoxicity testing, including screening of drug-induced liver injury (DILI)-inducing compounds. hHEP are the reference hepatic in vitro system, but their availability is limited and the cells available for toxicology studies are often of poor quality. Hepatic cell lines on the other hand are highly proliferative and represent an inexhaustible hepatic cell source. However, these hepatoma-derived cells do not represent the population diversity and display reduced hepatic metabolism. Alternatively, stem cell-derived hepatic cells, which can be produced in high numbers and can differentiate into multiple cell lineages, are also being evaluated as a cell source for in vitro hepatotoxicity studies. Human skin-derived precursors (hSKP) are post-natal stem cells that, after conversion towards hepatic cells (hSKP-HPC), respond to hepatotoxic compounds in a comparable way as hHEP. In the current study, four different human hepatic cell systems (hSKP-HPC, hHEP, HepaRG and HepG2) are evaluated for their capacity to predict hepatic toxicity. Their hepatotoxic response to acetaminophen (APAP) exposure is compared to data obtained from patients suffering from APAP-induced acute liver failure (ALF). The results indicate that hHEP, HepaRG and hSKP-HPC identify comparable APAP-induced hepatotoxic functions and that HepG2 cells show the slightest hepatotoxic response. Pathway analyses further points out that HepaRG cells show the highest predicted activation of the functional genes related to 'damage of liver', followed by hSKP-HPC and hHEP cells that generated similar results. HepG2 did not show any activation of this function. PMID:26497421

  14. Regenerative cells for transplantation in hepatic failure.

    PubMed

    Ishikawa, Tetsuya; Banas, Agnieszka; Teratani, Takumi; Iwaguro, Hideki; Ochiya, Takahiro

    2012-01-01

    Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have an enormous potential; however, their potential clinical application is being arrested due to various limitations such as teratoma formation followed by tumorigenesis, emergent usage, and the quality control of cells, as well as safety issues regarding long-term culture are also delaying their clinical application. In addition, human ES cells have two crucial issues: immunogenicity and ethical issues associated with their clinical application. The efficient generation of human iPS cells requires gene transfer, yet the mechanism underlying pluripotent stem cell induction has not yet been fully elucidated. Otherwise, although human adult regenerative cells including mesenchymal stem cells have a limited capacity for differentiation, they are nevertheless promising candidates for tissue regeneration in a clinical setting. This review highlights the use of regenerative cells for transplantation in hepatic failure. PMID:22793046

  15. Generation and In Vitro Expansion of Hepatic Progenitor Cells from Human iPS Cells.

    PubMed

    Yanagida, Ayaka; Nakauchi, Hiromitsu; Kamiya, Akihide

    2016-01-01

    Stem cells have the unique properties of self-renewal and multipotency (producing progeny belonging to two or more lineages). Induced pluripotent stem (iPS) cells can be generated from somatic cells by simultaneous expression of pluripotent factors (Oct3/4, Klf4, Sox2, and c-Myc). They share the same properties as embryonic stem (ES) cells and can differentiate into several tissue cells, i.e., neurons, hematopoietic cells, and liver cells. Therefore, iPS cells are suitable candidate cells for regenerative medicine and analyses of disease mechanisms.The liver is the major organ that regulates a multitude of metabolic functions. Hepatocytes are the major cell type populating the liver parenchyma and express several metabolic enzymes that are necessary for liver functions. Although hepatocytes are essential for maintaining homeostasis, it is difficult to alter artificial and transplanted cells because of their multifunctionality, donor shortage, and immunorejection risk. During liver development, hepatic progenitor cells in the fetal liver differentiate into both mature hepatocytes and cholangiocytes. As hepatic progenitor cells have bipotency and high proliferation ability, they could present a potential source for generating transplantable cells or as a liver study model. Here we describe the induction and purification of hepatic progenitor cells derived from human iPS cells. These cells can proliferate for a long term under suitable culture conditions. PMID:25697415

  16. Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.

    PubMed

    Nakatani, Kazuki; Seki, Shuichi; Kawada, Norifumi; Kitada, Takuya; Yamada, Takao; Sakaguchi, Hiroki; Kadoya, Hirokazu; Ikeda, Kazuo; Kaneda, Kenji

    2002-11-01

    Secreted protein, acidic and rich in cysteine (SPARC), which functions in tissue remodeling, has been reported to be expressed by myofibroblasts in liver cirrhosis and hepatocellular carcinoma. This study aimed to reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARC was expressed by nerve fibers and hepatic stellate cells (HSCs) in the liver parenchyma and myofibroblasts in the fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope. Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smooth muscle actin were co-expressed by HSCs. Quantitative analysis demonstrated that, while SPARC-positive HSCs were sparse in control livers, they significantly increased in number in the livers with chronic hepatitis. There were, however, no significant differences in number among the grades of activity, the stages of fibrosis, or etiology (virus-infected or autoimmune, hepatitis B virus or hepatitis C virus). In liver cirrhosis, however, they significantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs in chronic hepatitis, suggesting the involvement of SPARC in hepatic fibrogenesis after chronic injuries. PMID:12447677

  17. The improving effects on hepatic fibrosis of interferon-γ liposomes targeted to hepatic stellate cells

    NASA Astrophysics Data System (ADS)

    Li, Qinghua; Yan, Zhiqiang; Li, Feng; Lu, Weiyue; Wang, Jiyao; Guo, Chuanyong

    2012-07-01

    No satisfactory anti-fibrotic therapies have yet been applied clinically. One of the main reasons is the inability to specifically target the responsible cells to produce an available drug concentration and the side-effects. Exploiting the key role of the activated hepatic stellate cells (HSCs) in both hepatic fibrogenesis and over-expression of platelet-derived growth factor receptor-β (PDGFR-β), we constructed targeted sterically stable liposomes (SSLs) modified by a cyclic peptide (pPB) with affinity for the PDGFR-β to deliver interferon (IFN)-γ to HSCs. The pPB-SSL-IFN-γ showed satisfactory size distribution. In vitro pPB-SSL could be taken up by activated HSCs. The study of tissue distribution via living-body animal imaging showed that the pPB-SSL-IFN-γ mostly accumulated in the liver until 24 h. Furthermore, the pPB-SSL-IFN-γ showed more significant remission of hepatic fibrosis. In vivo the histological Ishak stage, the semiquantitative score for collagen in fibrotic liver and the serum levels of collagen type IV-C in fibrotic rats treated with pPB-SSL-IFN-γ were less than those treated with SSL-IFN-γ, IFN-γ and the control group. In vitro pPB-SSL-IFN-γ was also more effective in suppressing activated HSC proliferation and inducing apoptosis of activated HSCs. Thus the data suggest that pPB-SSL-IFN-γ might be a more effective anti-fibrotic agent and a new opportunity for clinical therapy of hepatic fibrosis.

  18. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting.

    PubMed

    Ma, Xuanyi; Qu, Xin; Zhu, Wei; Li, Yi-Shuan; Yuan, Suli; Zhang, Hong; Liu, Justin; Wang, Pengrui; Lai, Cheuk Sun Edwin; Zanella, Fabian; Feng, Gen-Sheng; Sheikh, Farah; Chien, Shu; Chen, Shaochen

    2016-02-23

    The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling. PMID:26858399

  19. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

    PubMed Central

    Ma, Xuanyi; Qu, Xin; Zhu, Wei; Li, Yi-Shuan; Yuan, Suli; Zhang, Hong; Liu, Justin; Wang, Pengrui; Lai, Cheuk Sun Edwin; Zanella, Fabian; Feng, Gen-Sheng; Sheikh, Farah; Chien, Shu; Chen, Shaochen

    2016-01-01

    The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling. PMID:26858399

  20. Natural killer cells in hepatitis C: Current progress

    PubMed Central

    Yoon, Joo Chun; Yang, Chang Mo; Song, Youkyong; Lee, Jae Myun

    2016-01-01

    Patients infected with the hepatitis C virus (HCV) are characterized by a high incidence of chronic infection, which results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The functional impairment of HCV-specific T cells is associated with the evolution of an acute infection to chronic hepatitis. While T cells are the important effector cells in adaptive immunity, natural killer (NK) cells are the critical effector cells in innate immunity to virus infections. The findings of recent studies on NK cells in hepatitis C suggest that NK cell responses are indeed important in each phase of HCV infection. In the early phase, NK cells are involved in protective immunity to HCV. The immune evasion strategies used by HCV may target NK cells and might contribute to the progression to chronic hepatitis C. NK cells may control HCV replication and modulate hepatic fibrosis in the chronic phase. Further investigations are, however, needed, because a considerable number of studies observed functional impairment of NK cells in chronic HCV infection. Interestingly, the enhanced NK cell responses during interferon-α-based therapy of chronic hepatitis C indicate successful treatment. In spite of the advances in research on NK cells in hepatitis C, establishment of more physiological HCV infection model systems is needed to settle unsolved controversies over the role and functional status of NK cells in HCV infection. PMID:26819513

  1. Cell entry of hepatitis C virus

    SciTech Connect

    Bartosch, Birke . E-mail: Birke.Bartosch@ens-lyon.fr; Cosset, Francois-Loic . E-mail: Francois-Loic.Cosset@ens-lyon.fr

    2006-04-25

    Hepatitis C virus (HCV), an important human pathogen, is an enveloped, positive-stranded RNA virus classified in the hepacivirus genus of the Flaviviridae family. Cell attachment of flaviviruses generally leads to endocytosis of bound virions. Systems that support HCV replication and particle formation in vitro are emerging only now, 16 years after the discovery of the virus. Albeit this limitation, the route of HCV cell entry as well as 'capture' molecules involved in low-affinity interactions for the initial contact of HCV with target cells and potential high-affinity receptor candidates that may mediate HCV trafficking and fusion has been described. The objective of this review is to summarize the contribution of different HCV model systems to our current knowledge about structure of the HCV GPs E1 and E2 and their roles in cell entry comprising cell attachment, interactions with cellular receptors, endocytosis, and fusion.

  2. CD4+/CD8+ ratio of liver-derived lymphocytes is related to viraemia and not to hepatitis C virus genotypes in chronic hepatitis C.

    PubMed Central

    Pham, B N; Martinot-Peignoux, M; Mosnier, J F; Njapoum, C; Marcellin, P; Bougy, F; Degott, C; Erlinger, S; Cohen, J H; Degos, F

    1995-01-01

    The pathogenic mechanisms that lead to chronic hepatitis C are unknown. As hepatitis C virus (HCV) has been shown to induce T cell response, we assessed whether a particular T lymphocyte subset could be preferentially detected in the liver of patients with chronic hepatitis C in relation to viraemia or HCV genotypes. The immunophenotypes of liver-derived lymphocytes were analysed in 26 patients by flow cytometry and immunohistochemistry. Viraemia was quantified by branched DNA assay. Using this assay, HCV RNA was not detectable in six patients. HCV RNA was detected in 20 patients, and titres ranged from 8 to 137 x 10(6) Eq/ml. Genotyping was performed using a line probe assay. Type 1a, 1b, 2a, 3a and 4a were found to infect 2, 10, 2, 7 and 3 patients, respectively. The CD4+/CD8+ ratio of liver-derived lymphocytes was significantly higher (P < 0.01) in patients with detectable viraemia than in patients without detectable viraemia. In contrast, neither the percentage of gamma/delta T lymphocytes nor that of CD2+CD57+ cells was different in the groups. When comparing the CD4+/CD8+ ratio, the percentage of gamma/delta T lymphocytes or CD2+CD57+ cells according to genotype, the differences were not significant. These results suggest that the CD4+/CD8+ ratio of liver-derived lymphocytes is related to viraemia but not to HCV genotypes in patients with chronic hepatitis C, and that T lymphocytes may be involved in the pathogenesis of liver lesions in chronic hepatitis C. PMID:7586685

  3. Laminin-511 and laminin-521-based matrices for efficient hepatic specification of human pluripotent stem cells.

    PubMed

    Kanninen, Liisa K; Harjumäki, Riina; Peltoniemi, Pasi; Bogacheva, Mariia S; Salmi, Tuuli; Porola, Pauliina; Niklander, Johanna; Smutný, Tomáš; Urtti, Arto; Yliperttula, Marjo L; Lou, Yan-Ru

    2016-10-01

    Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied, only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511, laminin-521, and fibronectin, found in human liver progenitor cells, as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Altogether, we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells. PMID:27372423

  4. Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

    PubMed

    Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

    2005-04-01

    After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro. PMID:15793284

  5. Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

    NASA Astrophysics Data System (ADS)

    Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

    2016-04-01

    Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

  6. Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

    PubMed Central

    Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

    2016-01-01

    Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue. PMID:27063397

  7. Transcriptional regulation of hepatic stellate cells.

    PubMed

    Mann, Jelena; Mann, Derek A

    2009-07-01

    Hepatic stellate cell (HSC) activation is a process of cellular transdifferentiation in which, upon liver injury, the quiescent vitamin A storing perisinusoidal HSC is converted into a wound-healing myofibroblast and acquires potent pro-inflammatory and pro-fibrogenic activities. This remarkable phenotypic transformation is underpinned by changes in the expression of a vast number of genes. In this review we survey current knowledge of the transcription factors that either control HSC activation or which regulate specific fibrogenic functions of the activated HSC such as collagen expression, proliferation and resistance to apoptosis. PMID:19393271

  8. Hepatic Stellate Cells: Protean, Multifunctional, and Enigmatic Cells of the Liver

    PubMed Central

    Friedman, Scott L.

    2010-01-01

    The hepatic stellate cell has surprised and engaged physiologists, pathologists, and hepatologists for over 130 years, yet clear evidence of its role in hepatic injury and fibrosis only emerged following the refinement of methods for its isolation and characterization. The paradigm in liver injury of activation of quiescent vitamin A-rich stellate cells into proliferative, contractile, and fibrogenic myofibroblasts has launched an era of astonishing progress in understanding the mechanistic basis of hepatic fibrosis progression and regression. But this simple paradigm has now yielded to a remarkably broad appreciation of the cell's functions not only in liver injury, but also in hepatic development, regeneration, xenobiotic responses, intermediary metabolism, and immunoregulation. Among the most exciting prospects is that stellate cells are essential for hepatic progenitor cell amplification and differentiation. Equally intriguing is the remarkable plasticity of stellate cells, not only in their variable intermediate filament phenotype, but also in their functions. Stellate cells can be viewed as the nexus in a complex sinusoidal milieu that requires tightly regulated autocrine and paracrine cross-talk, rapid responses to evolving extracellular matrix content, and exquisite responsiveness to the metabolic needs imposed by liver growth and repair. Moreover, roles vital to systemic homeostasis include their storage and mobilization of retinoids, their emerging capacity for antigen presentation and induction of tolerance, as well as their emerging relationship to bone marrow-derived cells. As interest in this cell type intensifies, more surprises and mysteries are sure to unfold that will ultimately benefit our understanding of liver physiology and the diagnosis and treatment of liver disease. PMID:18195085

  9. [Hepatic and intestinal toxicity of furazan and furoxan derivatives].

    PubMed

    Fundarò, A; Cassone, M C

    1980-11-30

    Histological examination revealed that 4-methylfurazan-3-carboxylic acid hydrazide (I); 3-methylfuroxan-4-carboxylic acid hydrazide (II); 4-methylfurazan-3-carboxylic acid amide(III); 4-methylfuroxan-3-carboxylic acid amide (IV), caused hepatic and intestinal lesion in the mouse. At the higher doses a destruction of the intestinal mucosa and a vasodilatation of the hepatic sinusoid was observed. At lower doses hepatic steatosis was observed. PMID:7225247

  10. Bile acids induce hepatic differentiation of mesenchymal stem cells

    PubMed Central

    Sawitza, Iris; Kordes, Claus; Götze, Silke; Herebian, Diran; Häussinger, Dieter

    2015-01-01

    Mesenchymal stem cells (MSC) have the potential to differentiate into multiple cell lineages and their therapeutic potential has become obvious. In the liver, MSC are represented by stellate cells which have the potential to differentiate into hepatocytes after stimulation with growth factors. Since bile acids can promote liver regeneration, their influence on liver-resident and bone marrow-derived MSC was investigated. Physiological concentrations of bile acids such as tauroursodeoxycholic acid were able to initiate hepatic differentiation of MSC via the farnesoid X receptor and transmembrane G-protein-coupled bile acid receptor 5 as investigated with knockout mice. Notch, hedgehog, transforming growth factor-β/bone morphogenic protein family and non-canonical Wnt signalling were also essential for bile acid-mediated differentiation, whereas β-catenin-dependent Wnt signalling was able to attenuate this process. Our findings reveal bile acid-mediated signalling as an alternative way to induce hepatic differentiaion of stem cells and highlight bile acids as important signalling molecules during liver regeneration. PMID:26304833

  11. Hepatitis

    MedlinePlus

    ... Got Homework? Here's Help White House Lunch Recipes Hepatitis KidsHealth > For Kids > Hepatitis Print A A A ... an important digestive liquid called bile . What Is Hepatitis? Hepatitis is an inflammation (say: in-fluh-MAY- ...

  12. Development and characterization of a new human hepatic cell line.

    PubMed

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  13. Development and characterization of a new human hepatic cell line

    PubMed Central

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  14. New Pyrazolobenzothiazine Derivatives as Hepatitis C Virus NS5B Polymerase Palm Site I Inhibitors

    PubMed Central

    2015-01-01

    We have previously identified the pyrazolobenzothiazine scaffold as a promising chemotype against hepatitis C virus (HCV) NS5B polymerase, a validated and promising anti-HCV target. Herein we describe the design, synthesis, enzymatic, and cellular characterization of new pyrazolobenzothiazines as anti-HCV inhibitors. The binding site for a representative derivative was mapped to NS5B palm site I employing a mutant counterscreen assay, thus validating our previous in silico predictions. Derivative 2b proved to be the best selective anti-HCV derivative within the new series, exhibiting a IC50 of 7.9 μM against NS5B polymerase and antiviral effect (EC50 = 8.1 μM; EC90 = 23.3 μM) coupled with the absence of any antimetabolic effect (CC50 > 224 μM; SI > 28) in a cell based HCV replicon system assay. Significantly, microscopic analysis showed that, unlike the parent compounds, derivative 2b did not show any significant cell morphological alterations. Furthermore, since most of the pyrazolobenzothiazines tested altered cell morphology, this undesired aspect was further investigated by exploring possible perturbation of lipid metabolism during compound treatment. PMID:24654886

  15. Surface expression and cytolytic function of natural killer cell receptors is altered in chronic hepatitis C

    PubMed Central

    Nattermann, J; Feldmann, G; Ahlenstiel, G; Langhans, B; Sauerbruch, T; Spengler, U

    2006-01-01

    Introduction Impaired activity of natural killer (NK) cells has been proposed as a mechanism contributing to viral persistence in hepatitis C virus (HCV) infection. As the function of NK cells is primarily regulated by NK cell receptors (NKR), we analysed whether decreased NK cell function in hepatitis C may be related to dysregulated NKR expression. Patients and methods Expression of NK cell was analysed by flow cytometry on lymphocytes from HCV(+) subjects (n = 30), patients who became HCV(−) after antiviral therapy (n = 10), healthy individuals (n = 10), and hepatitis B virus (HBV) infected patients (n = 9). Cytolytic function of lymphocytes was studied in a redirected lysis assay and in a standard 51chromium release cytotoxicity assay, respectively. Results In patients with chronic hepatitis C, we found a significantly reduced proportion of NKp46 and NKp30 expressing NK cells compared with healthy and HBV infected subjects. Low expression of natural cytotoxicity receptor (NCR) was also confirmed in in vitro activated NK cell populations derived from HCV patients compared with uninfected donors. In contrast, patients who cleared HCV under antiviral therapy showed normal expression of NKp44, NKp30, and NKp46. Reduced NCR expression in chronic hepatitis C was associated with a parallel decrease in NCR mediated target cell killing. Furthermore, we found a significantly increased proportion of NKG2A expressing NK cells and CD8+ T cells in HCV positive patients, resulting in a reduced cytolytic activity against cells incubated with the HLA‐E stabilising peptide HCV core35–44. Conclusion The present study indicates that defective expression of NKR represents a novel mechanism contributing to impaired function of NK cells and CD8+ T cells in chronic hepatitis C. PMID:16322112

  16. Viral hepatitis: A new HCV cell culture model for the next clinical challenges.

    PubMed

    Colpitts, Che C; Baumert, Thomas F

    2015-11-01

    Despite advances in hepatitis C treatment, substantial clinical hurdles remain to achieve universal cure and global control of infection. Saeed et al. identified SEC14L2 as a host factor permitting replication of clinical HCV isolates in cell culture, providing a novel system to model infection of patient-derived viruses. PMID:26441247

  17. A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

    SciTech Connect

    Shikanai, Mima; Asahina, Kinji; Iseki, Sachiko Teramoto, Kenichi; Nishida, Tomohiro; Shimizu-Saito, Keiko; Ota, Masato; Eto, Kazuhiro; Teraoka, Hirobumi

    2009-04-03

    Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or {alpha}-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

  18. Targeted TFO delivery to hepatic stellate cells.

    PubMed

    Yang, Ningning; Singh, Saurabh; Mahato, Ram I

    2011-10-30

    Triplex-forming oligonucleotides (TFOs) represent an antigene approach for gene regulation through direct interaction with genomic DNA. While this strategy holds great promise owing to the fact that only two alleles need silencing to impact gene regulation, delivering TFOs to target cells in vivo is still a challenge. Our recent efforts have focused on conjugating TFOs to carrier molecules like cholesterol to enhance their cellular uptake and mannose-6-phosphate-bovine serum albumin (M6P-BSA) to target TFO delivery to hepatic stellate cells (HSCs) for treating liver fibrosis. These approaches however are rendered less effective owing to a lack of targeted delivery, as seen with lipid-conjugates, and the potential immune reactions due to repeated dosing with high molecular weight BSA conjugated TFO. In this review, we discuss our latest efforts to enhance the effectiveness of TFO for treating liver fibrosis. We have shown that conjugation of TFOs to M6P-HPMA can enhance TFO delivery to HSCs and has the potential to treat liver fibrosis by inhibiting collagen synthesis. This TFO conjugate shows negligible immunogenicity owing to the use of HPMA, one of the least immunogenic copolymers, thereby making it a suitable and more effective candidate for antifibrotic therapy. PMID:21763370

  19. Targeted TFO Delivery to Hepatic Stellate Cells

    PubMed Central

    Yang, Ningning; Singh, Saurabh; Mahato, Ram I

    2011-01-01

    Triplex-forming oligonucleotides (TFOs) represent an antigene approach for gene regulation through direct interaction with genomic DNA. While this strategy holds great promise owing to the fact that only two alleles need silencing to impact gene regulation, delivering TFOs to target cells in vivo is still a challenge. Our recent efforts have focused on conjugating TFOs to carrier molecules like cholesterol to enhance their cellular uptake and mannose-6-phosphate-bovine serum albumin (M6P-BSA) to target TFO delivery to hepatic stellate cells (HSCs) for treating liver fibrosis. These approaches however are rendered less effective owing to a lack of targeted delivery, as seen with lipid-conjugates, and the potential immune reactions due to repeated dosing with high molecular weight BSA conjugated TFO. In this review, we discuss our latest efforts to enhance the effectiveness of TFO for treating liver fibrosis. We have shown that conjugation of TFOs to M6P-HPMA can enhance TFO delivery to HSCs and has the potential to treat liver fibrosis by inhibiting collagen synthesis. This TFO conjugate shows negligible immunogenicity owing to the use of HPMA, one of the least immunogenic copolymers, thereby making it a suitable and more effective candidate for antifibrotic therapy. PMID:21763370

  20. Replacement of Diseased Mouse Liver by Hepatic Cell Transplantation

    NASA Astrophysics Data System (ADS)

    Rhim, Jonathan A.; Sandgren, Eric P.; Degen, Jay L.; Palmiter, Richard D.; Brinster, Ralph L.

    1994-02-01

    Adult liver has the unusual ability to fully regenerate after injury. Although regeneration is accomplished by the division of mature hepatocytes, the replicative potential of these cells is unknown. Here, the replicative capacity of adult liver cells and their medical usefulness as donor cells for transplantation were investigated by transfer of adult mouse liver cells into transgenic mice that display an endogenous defect in hepatic growth potential and function. The transplanted liver cell populations replaced up to 80 percent of the diseased recipient liver. These findings demonstrate the enormous growth potential of adult hepatocytes, indicating the feasibility of liver cell transplantation as a method to replace lost or diseased hepatic parenchyma.

  1. Hepatic cancer stem cells may arise from adult ductal progenitors

    PubMed Central

    Nikolaou, Kostas C; Talianidis, Iannis

    2016-01-01

    Cancer stem cells (CSCs) are defined as cells within tumors that can self-renew and differentiate into heterogeneous lineages of cancerous cells. The origin of CSCs is not well understood. Recent evidence suggests that CSCs in hepatocellular carcinoma could be generated via oncogenic transformation and partial differentiation of adult hepatic ductal progenitor cells.

  2. Primary Hepatic Small-Cell Carcinoma Developed during Antiviral Treatment for Chronic Hepatitis B

    PubMed Central

    Kim, Suk Bae

    2015-01-01

    Previously reported cases of primary hepatic small-cell carcinoma were all detected at progressed state with associated symptoms. Therefore, the natural course of primary hepatic small-cell carcinoma remains unknown. This case shows the natural course of primary hepatic small-cell carcinoma. We detected a 1.2 cm hypodense nodule 6 months ago in a patient with cirrhosis who had been taking entecavir. It was suspected to be a regenerating or degenerating nodule. Three months later, liver computed tomography (CT) revealed that the mass was increased to 2.1 cm with the same characteristics. The patient wanted to do a follow-up CT scan after 3 months instead of a biopsy. Another 3 months later, the mass was markedly increased, involving the whole left lobe and was confirmed as small-cell carcinoma on biopsy. Here, we report the first case of primary hepatic small-cell carcinoma developed during treatment for chronic hepatitis B with cirrhosis. PMID:26951743

  3. HIV Through the Looking Glass: Insights Derived from Hepatitis B

    PubMed Central

    Rivera, Maria M.; Soza, Alejandro; Jazwinski, Alison; Mi, Lijun; Kleiner, David E.; Zhao, Xiongce; Zuber, Charma; Brust, Douglas; Hsu, Emory; Simpson, Jennifer; Hoofnagle, Jay H.; Heller, Theo

    2014-01-01

    Background While higher levels of hepatitis B virus (HBV) replication in HIV-HBV co-infection may relate to liver disease progression, this has not been completely elucidated. We utilized expression of hepatitis B core antigen (HBcAg) in liver biopsies from HIV-HBV co-infected and HBV mono-infected patients as a marker for HBV replication, and related these findings to clinical and histological parameters. Methods Data from 244 HBV patients were compared to 34 HIV-HBV patients. Liver biopsies were scored for inflammation, fibrosis, HBcAg, and hepatitis B surface antigen (HBsAg). Univariate and multivariate analyses were performed. Results HBcAg, but not HBsAg, staining was stronger in HIV co-infected than in HBV mono-infected. Co-infected and HBV mono-infected had similar ALT, inflammatory and fibrosis scores, and hepatitis B e Antigen (HBeAg) status. HBcAg staining correlated with HIV after correcting for HBV DNA and HBeAg. CD4 counts and HIV RNA level did not correlate with intensity of HBcAg staining. HBV DNA levels were higher in HIV co-infected and correlated with HBcAg staining. Conclusions By looking at HBcAg as a reflection of HBV replication in HIV-HBV co-infected with controlled HIV, our findings suggest that these patients may have subtle immune function defects, which could lead to adverse liver disease outcomes. PMID:25590267

  4. Discovery, Optimization, and Characterization of Novel Chlorcyclizine Derivatives for the Treatment of Hepatitis C Virus Infection

    PubMed Central

    2015-01-01

    Recently, we reported that chlorcyclizine (CCZ, Rac-2), an over-the-counter antihistamine piperazine drug, possesses in vitro and in vivo activity against hepatitis C virus. Here, we describe structure–activity relationship (SAR) efforts that resulted in the optimization of novel chlorcyclizine derivatives as anti-HCV agents. Several compounds exhibited EC50 values below 10 nM against HCV infection, cytotoxicity selectivity indices above 2000, and showed improved in vivo pharmacokinetic properties. The optimized molecules can serve as lead preclinical candidates for the treatment of hepatitis C virus infection and as probes to study hepatitis C virus pathogenesis and host–virus interaction. PMID:26599718

  5. Epigenetic Changes during Hepatic Stellate Cell Activation

    PubMed Central

    Götze, Silke; Schumacher, Eva C.; Kordes, Claus; Häussinger, Dieter

    2015-01-01

    Background and Aims Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC. Methods and Results The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism. Conclusions In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism. PMID:26065684

  6. Hepatitis B viral infection of hepatic progenitor cells. Resolving unresolved questions?

    PubMed

    Minuk, G Y; Baruch, Y

    2016-06-01

    Accumulated data to date do not entirely explain the; propensity of the hepatitis B virus (HBV) to cause chronic infections in newborns; failure of antiviral agents to resolve infections or precise mechanism whereby HBV causes hepatocellular carcinoma (HCC). Based on the increased numbers of hepatic stem/progenitor cells (HPCs) present within the neonatal liver, the refractoriness of these cells to the effects of interferons and xenobiotics and their ability to undergo malignant transformation, we hypothesize that HBV infection of HPCs could explain these and perhaps other clinical features of chronic HBV. PMID:27142136

  7. CD133(+) human umbilical cord blood stem cells enhance angiogenesis in experimental chronic hepatic fibrosis.

    PubMed

    Elkhafif, Nagwa; El Baz, Hanan; Hammam, Olfat; Hassan, Salwa; Salah, Faten; Mansour, Wafaa; Mansy, Soheir; Yehia, Hoda; Zaki, Ahmed; Magdy, Ranya

    2011-01-01

    The in vivo angiogenic potential of transplanted human umbilical cord blood (UCB) CD133(+) stem cells in experimental chronic hepatic fibrosis induced by murine schistosomiasis was studied. Enriched cord blood-derived CD133(+) cells were cultured in primary medium for 3 weeks. Twenty-two weeks post-Schistosomiasis infection in mice, after reaching the chronic hepatic fibrotic stage, transplantation of stem cells was performed and mice were sacrificed 3 weeks later. Histopathology and electron microscopy showed an increase in newly formed blood vessels and a decrease in the fibrosis known for this stage of the disease. By immunohistochemical analysis the newly formed blood vessels showed positive expression of the human-specific angiogenic markers CD31, CD34 and von Willebrand factor. Few hepatocyte-like polygonal cells showed positive expression of human vascular endothelial growth factor and inducible nitric oxide synthase. The transplanted CD133(+) human stem cells primarily enhanced hepatic angiogenesis and neovascularization and contributed to repair in a paracrine manner by creating a permissive environment that enabled proliferation and survival of damaged cells rather than by direct differentiation to hepatocytes. A dual advantage of CD133(+) cell therapy in hepatic disease is suggested based on its capability of hematopoietic and endothelial differentiation. PMID:21143528

  8. Idiopathic neonatal giant cell hepatitis presenting with acute hepatic failure on postnatal day one.

    PubMed

    Correa, Kimberley K; Nanjundiah, Prathiba; Wirtschafter, David D; Alshak, Najeeb S

    2002-01-01

    We report a term male infant presenting on postnatal day 1 with fulminant hepatic failure. Described congenital infection, metabolic disorders, and cardiovascular etiologies of acute neonatal liver failure were assessed and eliminated. A liver biopsy on postnatal day 10 showed neonatal giant cell hepatitis (NGCH) with an unusual degree of fibrosis for this early postnatal age. NGCH is a clinical diagnosis of cholestatic disorders of unknown etiology in the newborn, and, to our knowledge, has not been previously associated with immediate neonatal hepatic failure. The giant cell transformation is a common response to a variety of insults and only rarely occurs beyond the neonatal period. Most cases present with cholestatic jaundice and varying degrees of coagulopathy, and, many, as in this case, show progressive resolution. PMID:11948391

  9. Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex.

    PubMed

    Grzyb, Katarzyna; Czarnota, Anna; Brzozowska, Agnieszka; Cieślik, Anna; Rąbalski, Łukasz; Tyborowska, Jolanta; Bieńkowska-Szewczyk, Krystyna

    2016-01-01

    Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate. PMID:27481352

  10. Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex

    PubMed Central

    Grzyb, Katarzyna; Czarnota, Anna; Brzozowska, Agnieszka; Cieślik, Anna; Rąbalski, Łukasz; Tyborowska, Jolanta; Bieńkowska-Szewczyk, Krystyna

    2016-01-01

    Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate. PMID:27481352

  11. Overactivation of the nuclear factor (erythroid-derived 2)-like 2-antioxidant response element pathway in hepatocytes decreases hepatic ischemia/reperfusion injury in mice.

    PubMed

    Lee, Lung-Yi; Harberg, Calvin; Matkowskyj, Kristina A; Cook, Shelly; Roenneburg, Drew; Werner, Sabine; Johnson, Jeffrey; Foley, David P

    2016-01-01

    Hepatic ischemia/reperfusion injury (IRI) is a critical component of hepatic surgery. Oxidative stress has long been implicated as a key player in IRI. In this study, we examine the cell-specific role of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-antioxidant response element pathway in warm hepatic IRI. Nrf2 knockout (KO) and wild-type (WT) animals and novel transgenic mice expressing a constitutively active nuclear factor (erythroid-derived 2)-like 2 (caNrf2) mutant in hepatocytes (AlbCre+/caNrf2+) and their littermate controls underwent partial hepatic ischemia or sham surgery. The animals were killed 6 hours after reperfusion, and their serum and tissue were collected for analysis. As compared to WT animals after ischemia/reperfusion (IR), Nrf2 KO mice had increased hepatocellular injury with increased serum alanine aminotransferase and aspartate aminotransferase, Suzuki score, apoptosis, an increased inflammatory infiltrate, and enhanced inflammatory cytokine expression. On the other hand, AlbCre+/caNrf2+ that underwent IR had significantly reduced serum transaminases, less necrosis on histology, and a less pronounced inflammatory infiltrate and inflammatory cytokine expression as compared to the littermate controls. However, there were no differences in apoptosis. Taken together, Nrf2 plays a critical role in our murine model of warm hepatic IRI, with Nrf2 deficiency exacerbating hepatic IRI and hepatocyte-specific Nrf2 overactivation providing protection against warm hepatic IRI. PMID:26285140

  12. Mitochondrial uncouplers inhibit hepatic stellate cell activation

    PubMed Central

    2012-01-01

    Background Mitochondrial dysfunction participates in the progression of several pathologies. Although there is increasing evidence for a mitochondrial role in liver disease, little is known about its contribution to hepatic stellate cell (HSC) activation. In this study we investigated the role of mitochondrial activity through mild uncoupling during in vitro activation of HSCs. Methods Cultured primary human and mouse HSCs were treated with the chemical uncouplers FCCP and Valinomycin. ATP levels were measured by luciferase assay and production of reactive oxygen species was determined using the fluorescent probe DCFH-DA. Possible cytotoxicity by uncoupler treatment was evaluated by caspase 3/7 activity and cytoplasmic protease leakage. Activation of HSCs and their response to the pro-fibrogenic cytokine TGF-β was evaluated by gene expression of activation markers and signal mediators using RT-qPCR. Proliferation was measured by incorporation of EdU and protein expression of α-smooth muscle actin was analyzed by immunocytochemistry and western blot. Results FCCP and Valinomycin treatment mildly decreased ATP and reactive oxygen species levels. Both uncouplers increased the expression of mitochondrial genes such as Tfam and COXIV while inducing morphological features of quiescent mouse HSCs and abrogating TGF-β signal transduction. Mild uncoupling reduced HSC proliferation and expression of pro-fibrogenic markers of mouse and human HSCs. Conclusions Mild mitochondrial uncoupling inhibits culture-induced HSC activation and their response to pro-fibrogenic cytokines like TGF-β. These results therefore suggest mitochondrial uncoupling of HSCs as a strategy to reduce progression of liver fibrosis. PMID:22686625

  13. Hepatitis

    MedlinePlus

    ... has been associated with drinking contaminated water. Hepatitis Viruses Type Transmission Prognosis A Fecal-oral (stool to ... risk for severe disease. Others A variety of viruses can affect the liver Signs and Symptoms Hepatitis ...

  14. Efficient Generation of Hepatic Cells from Multipotent Adult Mouse Germ-Line Stem Cells Using an OP9 Co-Culture System

    PubMed Central

    Streckfuss-Bömeke, Katrin; Jende, Jörg; Cheng, I-Fen; Hasenfuss, Gerd

    2014-01-01

    Abstract On the basis of their self-renewal capacity and their ability to differentiate into derivatives of all three germ layers, germ line–derived multipotent adult stem cells (maGSCs) from mouse testis might serve as one of preferable sources for pluripotent stem cells in regenerative medicine. In our study, we aimed for an efficient hepatic differentiation protocol that is applicable for both maGSCs and embryonic stem cells (ESCs). We attempted to accomplish this goal by using a new established co-culture system with OP9 stroma cells for direct differentiation of maGSCs and ESCs into hepatic cells. We found that the hepatic differentiation of maGSCs was induced by the OP9 co-culture system in comparison to the gelatin culture. Furthermore, we showed that the combination of OP9 co-culture with activin A resulted in the increased expression of endodermal and early hepatic markers Gata4, Sox17, Foxa2, Hnf4, Afp, and Ttr compared to differentiated cells on gelatin or on OP9 alone. Moreover, the hepatic progenitors were capable of differentiating further into mature hepatic cells, demonstrated by the expression of liver-specific markers Aat, Alb, Tdo2, Krt18, Krt8, Krt19, Cps1, Sek, Cyp7a1, Otc, and Pah. A high percentage of maGSC-derived hepatic progenitors (51% AFP- and 61% DLK1-positive) and mature hepatic-like cells (26% ALB-positive) were achieved using this OP9 co-culture system. These generated hepatic cells successfully demonstrated in vitro functions associated with mature hepatocytes, including albumin and urea secretion, glycogen storage, and uptake of low-density lipoprotein. The established co-culture system for maGSCs into functional hepatic cells might serve as a suitable model to delineate the differentiation process for the generation of high numbers of mature hepatocytes in humans without genetic manipulations and make germ line–derived stem cells a potential autologous and alternative cell source for hepatic transplants in metabolic liver

  15. Combinatorial insulin secretion dynamics of recombinant hepatic and enteroendocrine cells.

    PubMed

    Durvasula, Kiranmai; Thulé, Peter M; Sambanis, Athanassios

    2012-04-01

    One of the most promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-β cells that secrete insulin in response to physiologic stimuli. A normal pancreatic β cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal β cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with 1 of 3 adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards preclinical studies. PMID:22094821

  16. Combinatorial Insulin Secretion Dynamics of Recombinant Hepatic and Enteroendocrine Cells

    PubMed Central

    Durvasula, Kiranmai; Thulé, Peter M.; Sambanis, Athanassios

    2012-01-01

    One of the more promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-β cells that secrete insulin in response to physiologic stimuli. A normal pancreatic β cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal β cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with one of three adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards pre-clinical studies. PMID:22094821

  17. Two populations of Thy1-positive mesenchymal cells regulate in vitro maturation of hepatic progenitor cells.

    PubMed

    Kamo, Naoko; Yasuchika, Kentaro; Fujii, Hideaki; Hoppo, Toshitaka; Machimoto, Takafumi; Ishii, Takamichi; Fujita, Naoya; Tsuruo, Takashi; Yamashita, Jun K; Kubo, Hajime; Ikai, Iwao

    2007-02-01

    We previously reported that the in vitro maturation of CD49f(+)Thy1(-)CD45(-) (CD49f positive) fetal hepatic progenitor cells (HPCs) is supported by Thy1-positive mesenchymal cells derived from the fetal liver. These mesenchymal cell preparations contain two populations, one of a cuboidal shape and the other spindle shaped in morphology. In this study, we determined that the mucin-type transmembrane glycoprotein gp38 could distinguish cuboidal cells from spindle cells by immunocytochemistry. RT-PCR analysis revealed differences between isolated CD49f(+/-)Thy1(+)gp38(+)CD45(-) (gp38 positive) cells and CD49f(+/-)Thy1(+)gp38(-)CD45(-) (gp38 negative) cells, whereas both cells expressed mesenchymal cell markers. The coculture with gp38-positive cells promoted the maturation of CD49f-positive HPCs, which was estimated by positivity for periodic acid-Schiff (PAS) staining, whereas the coculture with gp38-negative cells maintained CD49f-positive HPCs negative for PAS staining. The expression of mature hepatocyte markers, such as tyrosine aminotransferase, tryptophan-2,3-dioxygenase, and glucose-6-phosphatase, were upregulated on HPCs by coculture with gp38-positive cells. Furthermore, transmission electron microscopy revealed the acquisition of mature hepatocyte features by HPCs cocultured with gp38-positive cells. This effect on maturation of HPCs was inhibited by the addition of conditioned medium derived from gp38-negative cells. By contrast, the upregulation of bromodeoxyuridine incorporation by HPCs demonstrated the proliferative effect of coculture with gp38-negative cells. In conclusion, these results suggest that in vitro maturation of HPCs promoted by gp38-positive cells may be opposed by an inhibitory effect of gp38-negative cells, which likely maintain the immature, proliferative state of HPCs. PMID:16990447

  18. Development and molecular composition of the hepatic progenitor cell niche.

    PubMed

    Vestentoft, Peter Siig

    2013-05-01

    End-stage liver diseases represent major health problems that are currently treated by liver transplantation. However, given the world-wide shortage of donor livers novel strategies are needed for therapeutic treatment. Adult stem cells have the ability to self-renew and differentiate into the more specialized cell types of a given organ and are found in tissues throughout the body. These cells, whose progeny are termed progenitor cells in human liver and oval cells in rodents, have the potential to treat patients through the generation of hepatic parenchymal cells, even from the patient's own tissue. Little is known regarding the nature of the hepatic progenitor cells. Though they are suggested to reside in the most distal part of the biliary tree, the canal of Hering, the lack of unique surface markers for these cells has hindered their isolation and characterization. Upon activation, they proliferate and form ductular structures, termed "ductular reactions", which radiate into the hepatic parenchyma. The ductular reactions contain activated progenitor cells that not only acquire a phenotype resembling that observed in developing liver but also display markers of differentiation shared with the cholangiocytic or hepatocytic lineages, the two parenchymal hepatic cell types. Interactions between the putative progenitor cells, the surrounding support cells and the extracellular matrix scaffold, all constituting the progenitor cell niche, are likely to be important for regulating progenitor cell activity and differentiation. Therefore, identifying novel progenitor cell markers and deciphering their microenvironment could facilitate clinical use. The aims of the present PhD thesis were to expand knowledge of the hepatic progenitor cell niche and characterize it both during development and in disease. Several animal models of hepatic injury are known to induce activation of the progenitor cells. In order to identify possible progenitor cell markers and niche components

  19. Synthesis of some benzimidazole derivatives endowed with 1,2,3-triazole as potential inhibitors of hepatitis C virus.

    PubMed

    Youssif, Bahaa G M; Mohamed, Yaseen A M; Salim, Mohammed T A; Inagaki, Fuyuhiko; Mukai, Chisato; Abdu-Allah, Hajjaj H M

    2016-06-01

    New derivatives of 2-thiobenzimidazole incorporating triazole moiety were synthesized, characterized and tested in vitro for antiviral activity against hepatitis C virus (HCV) and hepatitis B virus (HBV). Their cytotoxicity was determined by the reduction in the number of viable cell. All of the synthesized compounds are inactive against HBV and some showed activity against HCV. In particular, two compounds showed significant activity, 2-{4-[(1-benzoylbenzimidazol-2-ylthio)methyl]-1H-1,2,3-triazol-1-yl}-N-(p-nitro-phenyl)-acetamide (13) and 2-(4-{[1-(p-chlorobenzoyl)-benzimidazol-2-ylthio)methyl]-1H-1,2,3-triazol-1-yl}-N-(p-nitrophenyl)-acetamide (17). The results give an insight into the importance of the substituent at position 2 of benzimidazole for the inhibition of HCV. PMID:27279065

  20. A Novel Pyridazinone Derivative Inhibits Hepatitis B Virus Replication by Inducing Genome-Free Capsid Formation

    PubMed Central

    Wang, Ya-Juan; Lu, Dong; Xu, Yi-Bin; Xing, Wei-Qiang; Tong, Xian-Kun; Wang, Gui-Feng; Feng, Chun-Lan; He, Pei-Lan; Zuo, Jian-Ping

    2015-01-01

    Here we first identified a novel pyridazinone derivative, compound 3711, as a nonnucleosidic hepatitis B virus (HBV) inhibitor in a cell model system. 3711 decreased extracellular HBV DNA levels by 50% (50% inhibitory concentration [IC50]) at 1.5 ± 0.2 μM and intracellular DNA levels at 1.9 ± 0.1 μM, which demonstrated antiviral activity at levels far below those associated with toxicity. Both the 3TC/ETV dually resistant L180M/M204I mutant and the adefovir (ADV)-resistant A181T/N236T mutant were as susceptible to 3711 as wild-type HBV. 3711 treatment induced the formation of genome-free capsids, a portion of which migrated faster on 1.8% native agarose gel. The induced genome-free capsids sedimented more slowly in isopycnic CsCl gradient centrifugation without significant morphological changes. 3711 treatment decreased levels of HBV DNA contained in both secreted enveloped virion and naked virus particles in supernatant. 3711 could interfere with capsid formation of the core protein (Cp) assembly domain. A Cp V124W mutant, which strengthens capsid interdimer interactions, recapitulated the effect of 3711 on capsid assembly. Pyridazinone derivative 3711, a novel chemical entity and HBV inhibitor, may provide a new opportunity to combat chronic HBV infection. PMID:26349829

  1. Host cell kinases and the hepatitis C virus life cycle.

    PubMed

    Colpitts, Che C; Lupberger, Joachim; Doerig, Christian; Baumert, Thomas F

    2015-10-01

    Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. PMID:25896387

  2. Human mesenchymal stem cell-engineered hepatic cell sheets accelerate liver regeneration in mice

    PubMed Central

    Itaba, Noriko; Matsumi, Yoshiaki; Okinaka, Kaori; Ashla, An Afida; Kono, Yohei; Osaki, Mitsuhiko; Morimoto, Minoru; Sugiyama, Naoyuki; Ohashi, Kazuo; Okano, Teruo; Shiota, Goshi

    2015-01-01

    Mesenchymal stem cells (MSCs) are an attractive cell source for cell therapy. Based on our hypothesis that suppression of Wnt/β-catenin signal enhances hepatic differentiation of human MSCs, we developed human mesenchymal stem cell-engineered hepatic cell sheets by a small molecule compound. Screening of 10 small molecule compounds was performed by WST assay, TCF reporter assay, and albumin mRNA expression. Consequently, hexachlorophene suppressed TCF reporter activity in time- and concentration-dependent manner. Hexachlorophene rapidly induced hepatic differentiation of human MSCs judging from expression of liver-specific genes and proteins, PAS staining, and urea production. The effect of orthotopic transplantation of human mesenchymal stem cell-engineered hepatic cell sheets against acute liver injury was examined in one-layered to three-layered cell sheets system. Transplantation of human mesenchymal stem cell-engineered hepatic cell sheets enhanced liver regeneration and suppressed liver injury. The survival rates of the mice were significantly improved. High expression of complement C3 and its downstream signals including C5a, NF-κB, and IL-6/STAT-3 pathway was observed in hepatic cell sheets-grafted tissues. Expression of phosphorylated EGFR and thioredoxin is enhanced, resulting in reduction of oxidative stress. These findings suggest that orthotopic transplantation of hepatic cell sheets manufactured from MSCs accelerates liver regeneration through complement C3, EGFR and thioredoxin. PMID:26553591

  3. Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes

    PubMed Central

    Takayama, Kazuo; Morisaki, Yuta; Kuno, Shuichi; Nagamoto, Yasuhito; Harada, Kazuo; Furukawa, Norihisa; Ohtaka, Manami; Nishimura, Ken; Imagawa, Kazuo; Sakurai, Fuminori; Tachibana, Masashi; Sumazaki, Ryo; Noguchi, Emiko; Nakanishi, Mahito; Hirata, Kazumasa; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2014-01-01

    Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPS-HLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drug-metabolizing capacity and drug responsiveness. PMID:25385620

  4. RMP Plays Distinct Roles in the Proliferation of Hepatocellular Carcinoma Cells and Normal Hepatic Cells

    PubMed Central

    Yang, Sijun; Wang, Hongmin; Guo, Yunlan; Chen, Shaomu; Zhang, Mei-Yin; Shen, Jian; Yu, Huijun; Miao, Jingcheng; Wang, Hui-Yun; Wei, Wenxiang

    2013-01-01

    RMP has been shown to function in the transcription regulation through association with RNA polymerase (RNAP) II subunit RPB5. It also has been shown to be required for the proliferation of hepatocellular carcinoma (HCC) cells with an antiapoptotic property. In this article, we further demonstrate that RMP displays distinct features in HCC cells compared with normal hepatic cells. RMP expression is remarkably increased in various cancer cell lines including HCC cells when compared with normal cells. Depletion of RMP could inhibit the proliferation of HCC cells, but not the normal hepatic cells. RMP significantly prevented apoptosis of HCC cells in SMMC-7721 and HepG2, but had little effect on apoptosis in the normal hepatic cells. The mechanisms of RMP's distinct features rely on different responsive expressions of apoptosis factors induced by RMP in HCC and hepatic cells. Either overexpression or depletion of RMP significantly affected the expression of apoptosis factors in HCC cells. However, normal hepatic cells showed a tendency to resist RMP for the regulation of apoptosis. In the clinical samples, the increased expression of RMP in HCCs was also observed when compared with the matched non-tumor tissues from 30 HCC patients. The different expression levels of and distinct responses to RMP between HCC and hepatic cells suggest that RMP might serve as not only a biomarker for the diagnosis of HCC, but also a potential target for the HCC therapy. PMID:23847445

  5. Identification of cells responsible for synthesis of sulphated glycosaminoglycans in schistosome-induced hepatic granulomas.

    PubMed Central

    Silva, L. C.; Borojevic, R.; Mourão, P. A.

    1990-01-01

    Sulphated glycosaminoglycans were isolated from schistosome-induced hepatic granuloma and from the pericellular, intracellular and extracellular compartments of two murine cell lines derived from granulomas: the primary cell line GR, and the permanent cell line GRX, established spontaneously from GR. The glycosaminoglycans composition in the whole granuloma was similar to that observed in the intracellular and extracellular compartments of GR cells. This result suggests that GR cells may be the major cell population involved in the synthesis and accumulation of glycosaminoglycans in the granulomas, and play an important role in the process of hepatic fibrosis. The conversion of the primary cell line GR into the established GRX cells did not modify the ratios that prevail among different glycosaminoglycans of the cell surface. However, it decreased the synthesis and secretion of glycosaminoglycans, reduced the proportion of iduronic acid units in the chondroitin sulphate, and increased the proportion of heparan sulphate in intracellular and extracellular pools. These characteristics of the GRX cells are similar to those observed in long-term cultures of smooth-muscle cells. In agreement with the general phenomenon of progressive de-differentiation during in-vitro culture of primary cell lines, these data indicate that the connective tissue cells of liver may belong to the myofibroblastic cell lineage. Images Fig. 1 PMID:2126187

  6. Alcoholic hepatitis: The pivotal role of Kupffer cells.

    PubMed

    Suraweera, Duminda B; Weeratunga, Ashley N; Hu, Robert W; Pandol, Stephen J; Hu, Richard

    2015-11-15

    Kupffer cells play a central role in the pathogenesis of alcoholic hepatitis (AH). It is believed that alcohol increases the gut permeability that results in raised levels of serum endotoxins containing lipopolysaccharides (LPS). LPS binds to LPS-binding proteins and presents it to a membrane glycoprotein called CD14, which then activates Kupffer cells via a receptor called toll-like receptor 4. This endotoxin mediated activation of Kupffer cells plays an important role in the inflammatory process resulting in alcoholic hepatitis. There is no effective treatment for AH, although notable progress has been made over the last decade in understanding the underlying mechanism of alcoholic hepatitis. We specifically review the current research on the role of Kupffer cells in the pathogenesis of AH and the treatment strategies. We suggest that the imbalance between the pro-inflammatory and the anti-inflammatory process as well as the increased production of reactive oxygen species eventually lead to hepatocyte injury, the final event of alcoholic hepatitis. PMID:26600966

  7. Alcoholic hepatitis: The pivotal role of Kupffer cells

    PubMed Central

    Suraweera, Duminda B; Weeratunga, Ashley N; Hu, Robert W; Pandol, Stephen J; Hu, Richard

    2015-01-01

    Kupffer cells play a central role in the pathogenesis of alcoholic hepatitis (AH). It is believed that alcohol increases the gut permeability that results in raised levels of serum endotoxins containing lipopolysaccharides (LPS). LPS binds to LPS-binding proteins and presents it to a membrane glycoprotein called CD14, which then activates Kupffer cells via a receptor called toll-like receptor 4. This endotoxin mediated activation of Kupffer cells plays an important role in the inflammatory process resulting in alcoholic hepatitis. There is no effective treatment for AH, although notable progress has been made over the last decade in understanding the underlying mechanism of alcoholic hepatitis. We specifically review the current research on the role of Kupffer cells in the pathogenesis of AH and the treatment strategies. We suggest that the imbalance between the pro-inflammatory and the anti-inflammatory process as well as the increased production of reactive oxygen species eventually lead to hepatocyte injury, the final event of alcoholic hepatitis. PMID:26600966

  8. 5-azacytidine promotes terminal differentiation of hepatic progenitor cells.

    PubMed

    He, Yun; Cui, Jiejie; He, Tongchuan; Bi, Yang

    2015-08-01

    5-azacytidine (5-azaC) is known to induce cardiomyocyte differentiation. However, its function in hepatocyte differentiation is unclear. The present study investigated the in vitro capability of 5-azaC to promote maturation and differentiation of mouse embryonic hepatic progenitor cells, with the aim of developing an approach for improving hepatic differentiation. Mouse embryonic hepatic progenitor cells (HP14.5 cells) were treated with 5-azaC at concentrations from 0 to 20 μmol/l, in addition to hepatocyte induction culture medium. Hepatocyte induction medium induces HP14.5 cell differentiation. 5-azaC may enhance the albumin promotor-driven Gaussia luciferase (ALB-GLuc) activity in induced HP14.5 cells. In the present study 2 μmol/l was found to be the optimum concentration with which to achieve this. The expression of hepatocyte-associated factors was not significantly different between the group treated with 5-azaC alone and the control group. The mRNA levels of ALB; cytokeratin 18 (CK18); tyrosine aminotransferase (TAT); and cytochrome p450, family 1, member A1 (CYP1A1); in addition to the protein levels of ALB, CK18 and uridine diphosphate glucuronyltransferase 1A (UGT1A) in the induced group with 5-azaC, were higher than those in the induced group without 5-azaC, although no significant differences were detected in expression of the hepatic stem cell markers, DLK and α-fetoprotein, between the two groups. Treatment with 5-azaC alone did not affect glycogen synthesis or indocyanine green (ICG) metabolic function in HP14.5 cells, although it significantly increased ICG uptake and periodic acid-Schiff-positive cell numbers amongst HP14.5 cells. Therefore, the present study demonstrated that treatment with 5-azaC alone exerted no effects on the maturation and differentiation of HP14.5 cells. However, 5-azaC exhibited a synergistic effect on the terminal differentiation of induced hepatic progenitor cells in association with a hepatic induction medium. PMID

  9. Hepatitis B virus e antigen induces activation of rat hepatic stellate cells

    SciTech Connect

    Zan, Yanlu; Zhang, Yuxia; Tien, Po

    2013-06-07

    Highlights: •HBeAg expression in HSCs induced production of ECM protein and liver fibrotic markers. •The activation and proliferation of HSCs were mediated by TGF-β. •HBeAg protein purified from cell medium directly activated HSCs. -- Abstract: Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-β1 (TGF-β), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-β secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-β antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-β, and HBeAg protein purified from cell medium can directly activate HSCs.

  10. Hepatitis

    MedlinePlus

    ... be serious. Some can lead to scarring, called cirrhosis, or to liver cancer. Sometimes hepatitis goes away by itself. If it does not, it can be treated with drugs. Sometimes hepatitis lasts a lifetime. Vaccines can help prevent some viral forms.

  11. Forced expression of Hnf1b/Foxa3 promotes hepatic fate of embryonic stem cells.

    PubMed

    Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Hakhamaneshi, Mohammad Saeed; Ebadifar, Asghar; Fathi, Fardin; Baharvand, Hossein

    2016-05-20

    Embryonic stem (ES) cell-derived hepatocytes have the potential to be used for basic research, regenerative medicine, and drug discovery. Recent reports demonstrated that in addition to conventional differentiation inducers such as chemical compounds and cytokines, overexpression of lineage-specific transcription factors could induce ES cells to differentiate to a hepatic fate. Here, we hypothesized that lentivirus-mediated inducible expression of hepatic lineage transcription factors could enhance mouse ES cells to hepatocyte-like cells. We screened the effects of candidate transcription factors Hnf1b, Hnf1a, Hnf4a, Foxa1, Foxa3 and Hex, and determined that the combination of Hnf1b/Foxa3 promoted expression of several hepatic lineage-specific markers and proteins, in addition to glycogen storage, ICG uptake, and secretion of albumin and urea. The differentiated cells were engraftable and expressed albumin when transplanted into a carbon tetrachloride-injured mouse model. These results demonstrated the crucial role of Hnf1b and Foxa3 in hepatogenesis in vitro and provided a valuable tool for the efficient differentiation of HLCs from ES cells. PMID:27107701

  12. Summary of clinical findings on Engerix-B, a genetically engineered yeast derived hepatitis B vaccine.

    PubMed

    André, F E; Safary, A

    1987-01-01

    Between February 1984 and August 1986 results have been obtained in 58 completed or ongoing clinical studies by 33 investigators in 19 countries on a yeast-derived recombinant DNA hepatitis B vaccine (Engerix-B). Among the 6100 subjects enrolled in these studies, 5664 subjects (150 normal neonates, 178 neonates of hepatitis B carrier mothers, 330 children aged 3-10 years, 3697 young healthy adults, 438 homosexual males, 110 older healthy adults, 139 drug addicts, 262 institutionalized mentally retarded patients, 59 thalassaemics, 25 sicklaemics, 270 patients on chronic haemodialysis and 6 haemophiliacs) received one or more (up to 4) injections of different doses of the yeast-derived vaccine according to either a 0, 1, 2, and 12 month or a 0, 1, and 6 month vaccination schedule. In randomized comparative studies 436 subjects received either one of two commercially available plasma-derived vaccines. The results reviewed in the present summary have shown that Engerix-B is safe, clinically well tolerated, gives an anti-HBs response which is qualitatively and quantitatively similar to that obtained with plasma-derived vaccines and confers protection against infection and disease. Engerix-B can be considered as a valid alternative to existing hepatitis B vaccines. PMID:3317357

  13. Expression of Stromal Cell-Derived Factor-1 and of Its Receptor CXCR4 in Liver Regeneration from Oval Cells in Rat

    PubMed Central

    Mavier, Philippe; Martin, Nadine; Couchie, Dominique; Préaux, Anne-Marie; Laperche, Yannick; Zafrani, Elie Serge

    2004-01-01

    Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway. PMID:15579440

  14. Inhibition of concanavalin A-induced mice hepatitis by coumarin derivatives.

    PubMed

    Okamoto, T; Yoshida, S; Kobayashi, T; Okabe, S

    2001-01-01

    The effects of coumarin derivatives, osthole, imperatorin, Pd-Ia, Pd-II and Pd-III, on mice concanavalin A (Con A) (0.2 mg/mouse, i.v.)-induced hepatitis were studied. At the dose of 200 mg/kg (i.p.), these coumarins inhibited more than 90% of the Con A-induced elevation of plasma alanine aminotransferase activity, but glycyrrhizin (200 mg/kg, i.p.) caused only 45% inhibition. At the dose of 100 mg/kg (i.p.), osthole produced the strongest inhibition among these coumarins. The inhibitory activity of osthole is lost when its 7-methoxy group is replaced by a 7-hydroxy group to form osthenol. The present results showed that coumarin derivatives inhibited Con A-induced hepatitis, with osthole being the most inhibitory. PMID:11243581

  15. Apicobasal polarity controls lymphocyte adhesion to hepatic epithelial cells.

    PubMed

    Reglero-Real, Natalia; Alvarez-Varela, Adrián; Cernuda-Morollón, Eva; Feito, Jorge; Marcos-Ramiro, Beatriz; Fernández-Martín, Laura; Gómez-Lechón, Maria José; Muntané, Jordi; Sandoval, Pilar; Majano, Pedro L; Correas, Isabel; Alonso, Miguel A; Millán, Jaime

    2014-09-25

    Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1) adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α). We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery. PMID:25242329

  16. Involvement of LSECtin in the hepatic natural killer cell response.

    PubMed

    Yang, Juntao; Wang, He; Wang, Min; Liu, Biao; Xu, Hui; Xu, Feng; Zhao, Dianyuan; Hu, Bin; Zhao, Na; Wang, Junyi; Liu, Di; Tang, Li; He, Fuchu

    2016-07-15

    Accumulating evidence has indicated that natural killer cells (NK cells) play an important role in immune responses generated in the liver. However, the underlying molecular basis for local immune regulation is poorly understood. Mice were intraperitoneally injected with polyinosinic-polycytidylic acid (PolyI:C) at a dose of 20 mg/kg body wt. The percentage and absolute number of NK cells in the liver were analysed with flow cytometry. LSECtin knockout mice and LSECtin cDNA plasmids were used for analyze the role of LSECtin in hepatic NK cell regulation in vivo. Here, we show that the C-type lectin LSECtin, a member of the DC-SIGN family, is a novel liver regulator for NK cells. LSECtin could bind to NK cells in a carbohydrate-dependent manner and could regulate the number of hepatic NK cells. In the NK cell-mediated acute liver injury model induced with PolyI:C, the exogenous expression of LSECtin accelerated NK cell-induced liver injury, whereas the absence of LSECtin ameliorated this condition. Our results reveal that LSECtin is a novel, liver-specific NK cell regulator that may be a target for the treatment of inflammatory diseases in the liver. PMID:27184407

  17. An epoxysuccinic acid derivative(loxistatin)-induced hepatic injury in rats and hamsters

    SciTech Connect

    Fukushima, K.; Arai, M.; Kohno, Y.; Suwa, T.; Satoh, T. )

    1990-08-01

    Loxistatin is a possible therapeutic agent of muscular dystrophy. A single oral administration of loxistatin to male rats caused focal necrosis of the liver with inflammatory cell infiltration. The severity of the lesions was dose-dependent up to 200 mg/kg and also manifest by an increase in serum alanine aminotransferase and aspartate aminotransferase activities. Hepatic glutathione (GSH) levels decreased with a maximum 20% depletion within 5 hr after the oral administration of loxistatin. Pretreatment with diethyl maleate did not potentiate the loxistatin-induced hepatic injury. On the other hand, the hepatoprotective effect of cysteamine was observed when cysteamine was administered 24 hr before loxistatin dosing, but the effect was not observed when the antidote was administered concomitantly with loxistatin. Pretreatment of rats with phenobarbital or trans-stilbene oxide provided partial protection against the hepatotoxic effect of loxistatin. Pretreatment with SKF-525A resulted in increased hepatic injury, while pretreatment with piperonyl butoxide, cimetidine, or 3-methylcholanthrene had no effect on hepatic damage by loxistatin. Five hours after (14C)loxistatin administration to rats, the covalent binding of the radioactivity to proteins was greatest in the liver, followed by the kidney, then muscle and blood to a lesser extent. (14C)Loxistatin acid, the pharmacologically active form of loxistatin, irreversibly bound to rat liver microsomal proteins; more binding occurred when the NADPH-generating system was omitted and when the microsomes were boiled first. GSH did not alter the extent of irreversible binding, whereas N-ethylmaleimide decreased the binding of (14C)loxistatin acid to rat liver microsomal proteins by 75%. Unlike the rat, administration of loxistatin to hamsters caused neither hepatic injury nor hepatic GSH depletion.

  18. Successful Interferon Therapy Reverses Enhanced Hepatic Progenitor Cell Activation in Patients with Chronic Hepatitis C.

    PubMed

    Noritake, Hidenao; Kobayashi, Yoshimasa; Ooba, Yukimasa; Matsunaga, Erika; Ohta, Kazuyoshi; Shimoyama, Shin; Yamazaki, Satoru; Chida, Takeshi; Kawata, Kazuhito; Sakaguchi, Takanori; Suda, Takafumi

    2015-12-01

    The enhanced accumulation of hepatic progenitor cells (HPCs) is related to the risk of progression to hepatocellular carcinoma (HCC). Interferon (IFN) treatment reduces HCC risk in patients with chronic hepatitis C virus (HCV) infection. However, the underlying mechanisms remain unclear. The aim of this study was to examine the effects of IFN treatment on HPC activation in HCV patients. Immunohistochemical detection and computer-assisted quantitative image analyses of cytokeratin 7 (CK7) were performed to evaluate HPC activation in paired pre- and post-treatment liver biopsies from 18 HCV patients with sustained virological response (SVR) to IFN-based therapy and from 23 patients without SVR, as well as normal liver tissues obtained from surgical resection specimens of 10 patients. Pretreatment HCV livers showed increased CK7 immunoreactivity, compared with normal livers (HCV: median, 1.38%; normal: median, 0.69%, P=0.006). IFN treatment reduced hepatic CK7 immunoreactivity (median, 1.57% pre-IFN vs. 0.69% post-IFN, P=0.006) in SVR patients, but not in non-SVR patients. The development of HCC following IFN treatment was encountered in 3 non-SVR patients who showed high post-IFN treatment CK7 immunoreactivity (>4%). Successful IFN therapy can reverse enhanced HPC activation in HCV patients, which may contribute to the reduced risk of HCC development in these patients. PMID:26308703

  19. Inhibition of cytokine-induced microvascular arrest of tumor cells by recombinant endostatin prevents experimental hepatic melanoma metastasis.

    PubMed

    Mendoza, Lorea; Valcárcel, María; Carrascal, Teresa; Egilegor, Eider; Salado, Clarisa; Sim, B Kim Lee; Vidal-Vanaclocha, Fernando

    2004-01-01

    We investigated effects of endostatin (ES) in the prometastatic microenvironment of inflammation occurring during the microvascular phase of cancer cell infiltration in the liver. We used a model of intrasplenic injection of B16 melanoma (B16M) cells leading to hepatic metastasis through vascular cell adhesion molecule-(VCAM-1)-mediated capillary arrest of cancer cells via interleukin-18 (IL-18)-dependent mechanism. We show that administration of 50 mg/kg recombinant human (rh) ES 30 min before B16M, plus repetition of same dose for 3 additional days decreased metastasis number by 60%. A single dose of rhES before B16M injection reduced hepatic microvascular retention of luciferase-transfected B16M by 40% and inhibited hepatic production of tumor necrosis factor alpha (TNF-alpha) and IL-18 and VCAM-1 expression by hepatic sinusoidal endothelia (HSE). Consistent with these data, rhES inhibited VCAM-1-dependent B16M cell adhesion to primary cultured HSE receiving B16M conditioned medium, and it abolished the HSE cell production of TNF-alpha and IL-18 induced by tumor-derived vascular endothelial cell growth factor (VEGF). rhES abrogated recombinant murine VEGF-induced tyrosine phosphorylation of KDR/flk-1 receptor in HSE cells, preventing the proinflammatory action of tumor-derived VEGF on HSE. rhES also abolished hepatic production of TNF-alpha, microvascular retention of luciferase-transfected B16M, and adhesion of B16M cells to isolated HSE cells, all of them induced in mice given 5 micro g/kg recombinant murine VEGF for 18 h. This capillary inflammation-deactivating capability constitutes a nonantiangiogenic antitumoral action of endostatin that decreases cancer cell arrest within liver microvasculature and prevents metastases promoted by proinflammatory cytokines induced by VEGF. PMID:14729638

  20. The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation.

    PubMed

    Wang, Qiwei; Wang, Hai; Sun, Yu; Li, Shi-Wu; Donelan, William; Chang, Lung-Ji; Jin, Shouguang; Terada, Naohiro; Cheng, Henrique; Reeves, Westley H; Yang, Li-Jun

    2013-08-15

    Induced pluripotent stem cells (iPSCs) hold great promise for cell therapy. However, their low efficiency of lineage-specific differentiation and tumorigenesis severely hinder clinical translation. We hypothesized that reprogramming of somatic cells into lineage-specific progenitor cells might allow for large-scale expansion, avoiding the tumorigenesis inherent with iPSCs and simultaneously facilitating lineage-specific differentiation. Here we aimed at reprogramming rat hepatic WB cells, using four Yamanaka factors, into pancreatic progenitor cells (PPCs) or intermediate (IM) cells that have characteristics of PPCs. IM clones were selected based on their specific morphology and alkaline phosphatase activity and stably passaged under defined culture conditions. IM cells did not have iPSC properties, could be stably expanded in large quantity, and expressed all 14 genes that are used to define the PPC developmental stage. Directed differentiation of IM and WB cells by Pdx1-Ngn3-MafA (PNM) into pancreatic beta-like cells revealed that the IM cells are more susceptible to directed beta cell differentiation because of their open chromatin configuration, as demonstrated by expression of key pancreatic beta cell genes, secretion of insulin in response to glucose stimulation, and easy access to exogenous PNM proteins at the rat insulin 1 and Pdx1 promoters. This notion that IM cells are superior to their parental cells is further supported by the epigenetic demonstration of accessibility of Pdx1 and insulin 1 promoters. In conclusion, we have developed a strategy to derive and expand PPC cells from hepatic WB cells using conventional cell reprogramming. This proof-of-principal study may offer a novel, safe and effective way to generate autologous pancreatic beta cells for cell therapy of diabetes. PMID:23750005

  1. Hepatic effector CD8+ T-cell dynamics

    PubMed Central

    Iannacone, Matteo

    2015-01-01

    CD8+ T cells play a critical role in hepatitis B virus (HBV) pathogenesis. During acute, self-limited infections, these cells are instrumental to viral clearance; in chronic settings, they sustain repetitive cycles of hepatocellular necrosis that promote hepatocellular carcinoma development. Both CD8+ T-cell defensive and destructive functions are mediated by antigen-experienced effector cells and depend on the ability of these cells to migrate to the liver, recognize hepatocellular antigens and perform effector functions. Understanding the signals that modulate the spatiotemporal dynamics of CD8+ T cells in the liver, particularly in the context of antigen recognition, is therefore critical to gaining insight into the pathogenesis of acute and chronic HBV infection. Here, we highlight recent data on how effector CD8+ T cells traffic within the liver, and we discuss the potential for novel imaging techniques to shed light on this important aspect of HBV pathogenesis. PMID:25242274

  2. Xanthohumol, a chalcon derived from hops, inhibits hepatic inflammation and fibrosis.

    PubMed

    Dorn, Christoph; Kraus, Birgit; Motyl, Magdalena; Weiss, Thomas S; Gehrig, Manfred; Schölmerich, Jürgen; Heilmann, Jörg; Hellerbrand, Claus

    2010-07-01

    Xanthohumol (XN) is a major prenylated chalcone found in hops, which is used to add bitterness and flavor to beer. In this study, we first investigated the effects of XN on hepatocytes and hepatic stellate cells (HSC), the central mediators of liver fibrogenesis. XN inhibited the activation of primary human HSC and induced apoptosis in activated HSC in vitro in a dose dependent manner (0-20 microM). In contrast, XN doses as high as 50 microM did not impair viability of primary human hepatocytes. However, in both cell types XN inhibited activation of the transcription factor NFkappaB and expression of NFkappaB dependent proinflammatory genes. In vivo, feeding of XN reduced hepatic inflammation and expression of profibrogenic genes in a murine model of non-alcoholic steatohepatitis. These data indicate that XN has the potential as functional nutrient for the prevention or treatment of non-alcoholic steatohepatitis or other chronic liver disease. PMID:20087858

  3. Patient-Derived Antibody Targets Tumor Cells

    Cancer.gov

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  4. HLA class I-restricted human cytotoxic T cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen.

    PubMed Central

    Bertoletti, A; Ferrari, C; Fiaccadori, F; Penna, A; Margolskee, R; Schlicht, H J; Fowler, P; Guilhot, S; Chisari, F V

    1991-01-01

    Knowledge of the immune effector mechanisms responsible for clearance of hepatitis B virus (HBV)-infected cells has been severely limited by the absence of reproducible systems to selectively expand and to characterize HBV-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of patients with viral hepatitis. By using a strategy involving sequential stimulation with HBV nucleocapsid synthetic peptides followed by autologous, or HLA class I-matched, HBV nucleocapsid transfectants, we now report the existence of CTLs able to lyse target cells that express endogenously synthesized HBV nucleocapsid antigen in the peripheral blood of patients with acute viral hepatitis B. The CTL response is HLA-A2 restricted, mediated by CD8-positive T cells, and specific for a single epitope, located between amino acid residues 11 and 27 of HBV core protein; these residues are shared with the secretable precore-derived hepatitis B e antigen. Equivalent lysis of target cells that express each of these proteins suggests that their intracellular trafficking pathways may intersect. The current report provides definitive evidence that HLA class I-restricted, CD8-positive CTLs that recognize endogenously synthesized HBV nucleocapsid antigen are induced during acute HBV infection in humans and establishes a strategy that should permit a detailed analysis of the role played by HBV-specific CTLs in the immunopathogenesis of viral hepatitis. PMID:1660137

  5. Peripheral blood mononuclear cells and regulatory T cells in acute viral hepatitis.

    PubMed Central

    Barnaba, V; Tamburrini, E; Laghi, V; Cauda, R; Levrero, M; Ruocco, G; Ortona, L; Balsano, F

    1985-01-01

    During acute viral hepatitis, we observed a significant decrease in OKT4/OKT8 ratio with a significant increase in the OKT8 positive subset in acute type B and non-A-non-B hepatitis. This altered ratio persisted in type B for a long time until HBsAg antibody became detectable, while it soon returned to normal in type A and non-A-non-B hepatitis. In the majority of acute hepatitis the altered ratio is because of an increase and not to a decrease in the whole T cell population, as described in chronic HBV infection. The number of HNK-1 positive cells remained raised during the recovery phase of type B and non-A-non-B hepatitis, a finding consistent with the hypothesis that NK cells play a role in the host defence against B and non-A-non-B virus infections. Serum beta 2-microglobulin concentrations were increased only in acute hepatitis B and non-A-non-B where immunological mechanisms are suspected to be involved, and showed a good correlation with the population of activated OKIa positive cells. PMID:2862096

  6. Human hepatic stem cells from fetal and postnatal donors

    PubMed Central

    Schmelzer, Eva; Zhang, Lili; Bruce, Andrew; Wauthier, Eliane; Ludlow, John; Yao, Hsin-lei; Moss, Nicholas; Melhem, Alaa; McClelland, Randall; Turner, William; Kulik, Michael; Sherwood, Sonya; Tallheden, Tommi; Cheng, Nancy; Furth, Mark E.; Reid, Lola M.

    2007-01-01

    Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies. PMID:17664288

  7. NK Cells Help Induce Anti-Hepatitis B Virus CD8+ T Cell Immunity in Mice.

    PubMed

    Zheng, Meijuan; Sun, Rui; Wei, Haiming; Tian, Zhigang

    2016-05-15

    Although recent clinical studies demonstrate that NK cell function is impaired in hepatitis B virus (HBV)-persistent patients, whether or how NK cells play a role in anti-HBV adaptive immunity remains to be explored. Using a mouse model mimicking acute HBV infection by hydrodynamic injection of an HBV plasmid, we observed that although serum hepatitis B surface Ag and hepatitis B envelope Ag were eliminated within 3 to 4 wk, HBV might persist for >8 wk in CD8(-/-) mice and that adoptive transfer of anti-HBV CD8(+) T cells restored the ability to clear HBV in HBV-carrier Rag1(-/-) mice. These results indicate that CD8(+) T cells are critical in HBV elimination. Furthermore, NK cells increased IFN-γ production after HBV plasmid injection, and NK cell depletion led to significantly increased HBV persistence along with reduced frequency of hepatitis B core Ag-specific CD8(+) T cells. Adoptive transfer of IFN-γ-sufficient NK cells restored donor CD8(+) T cell function, indicating that NK cells positively regulated CD8(+) T cells via secreting IFN-γ. We also observed that NK cell depletion correlated with decreased effector memory CD8(+) T cell frequencies. Importantly, adoptive transfer experiments showed that NK cells were involved in anti-HBV CD8(+) T cell recall responses. Moreover, DX5(+)CD49a(-) conventional, but not DX5(-)CD49a(+) liver-resident, NK cells were involved in improving CD8(+) T cell responses against HBV. Overall, the current study reveals that NK cells, especially DX5(+)CD49a(-) conventional NK cells, promote the antiviral activity of CD8(+) T cell responses via secreting IFN-γ in a mouse model mimicking acute HBV infection. PMID:27183639

  8. Discriminating dengue-infected hepatic cells (WRL-68) using dielectrophoresis.

    PubMed

    Yafouz, Bashar; Kadri, Nahrizul Adib; Rothan, Hussin A; Yusof, Rohana; Ibrahim, Fatimah

    2016-02-01

    Dielectrophoresis (DEP), the induced movement of dielectric particles placed in a nonuniform electric field, has been used as a potential technique for manipulation and separation of many biological samples without destructive consequences to the cell. Cells of the same genotype in different physiological and pathological states have unique morphological and structural features, therefore, it is possible to differentiate between them using their DEP responses. This paper reports the experimental discrimination of normal and dengue-infected human hepatic fetal epithelial cells (WRL-68 cells) based on their DEP crossover frequency, at which no resultant movement occurs in the cells in response to the DEP force. A microarray dot electrode was used to conduct the DEP experiments. The DEP forces applied to the cells were quantified by analyzing the light intensity shift within the electrode's dot region based on the Cumulative Modal Intensity Shift image analysis technique. The differences in dielectric properties between infected and uninfected cells were exploited by plotting a unique DEP spectrum for each set of cells. We observed that the crossover frequency decreased from 220 kHz for the normal WRL-68 cells to 140 kHz after infection with the dengue virus in a medium conductivity of 100 μS/cm. We conclude that the change in the DEP crossover frequency between dengue-infected cells and their healthy counterparts should allow direct characterization of these cell types by exploiting their electrophysiological properties. PMID:26530354

  9. Regulation of the survival and differentiation of hepatic stem/progenitor cells by acyclic retinoid.

    PubMed

    Kamiya, Akihide

    2015-01-01

    During embryonic liver development, hepatic stem/progenitor cells (HpSCs) have a high proliferative ability and bipotency to differentiate into hepatocytes and cholangiocytes. Retinoic acid is a derivative of vitamin A and is involved in the proliferation and differentiation of stem/progenitor cells in several tissues. However, whether retinoic acid regulates the characteristics of HpSCs in the normal liver is still unknown. A recent study has shown that acyclic retinoid regulates the survival and proliferation of HpSCs derived from mouse foetal liver. Acyclic retinoid suppressed the expansion of CD29(+)CD49f(+) HpSCs through the induction of hepatocytic differentiation and progression of apoptosis. PMID:26021438

  10. Evaluation of hepatocyteprotective and anti-hepatitis B virus properties of Cichoric acid from Cichorium intybus leaves in cell culture.

    PubMed

    Zhang, Hong-Li; Dai, Ling-Hao; Wu, Yi-Hang; Yu, Xiao-Ping; Zhang, Yong-Yong; Guan, Rong-Fa; Liu, Tao; Zhao, Jun

    2014-01-01

    Hepatitis B is the most common serious liver infection in the world. To date, there is still no complete cure for chronic hepatitis B. Natural caffeic acid analogues possess prominent antiviral activity, especially anti-hepatitis B virus (HBV) and anti-human immunodeficiency virus effects. Cichoric acid is a caffeic acid derivative from Cichorium intybus. In the study, the anti-hepatitis B property of cichoric acid was evaluated by the D-galactosamine (D-GalN)-induced normal human HL-7702 hepatocyte injury model, the duck hepatitis B virus (DHBV)-infected duck fetal hepatocytes and the HBV-transfected cell line HepG2.2.15 cells, respectively. The results showed that cichoric acid attenuated significantly D-GalN-induced HL-7702 hepatocyte injury at 10-100 µg/mL and produced a maximum protection rate of 56.26%. Moreover, cichoric acid at 1-100 µg/mL inhibited markedly DHBV DNA replication in infected duck fetal hepatocytes. Also, cichoric acid at 10-100 µg/mL reduced significantly the hepatitis B surface and envelope antigen levels in HepG2.2.15 cells and produced the maximum inhibition rates of 79.94% and 76.41%, respectively. Meanwhile, test compound at 50-100 µg/mL inhibited markedly HBV DNA replication. In conclusion, this study verifies the anti-hepatitis B effect of cichoric acid from Cichorium intybus leaves. In addition, cichoric acid could be used to design the antiviral agents. PMID:24759764

  11. Pan-genotypic Hepatitis C Virus Inhibition by Natural Products Derived from the Wild Egyptian Artichoke

    PubMed Central

    Elsebai, Mahmoud Fahmi; Koutsoudakis, George; Saludes, Verónica; Pérez-Vilaró, Gemma; Turpeinen, Ari; Mattila, Sampo; Pirttilä, Anna Maria; Fontaine-Vive, Fabien; Mehiri, Mohamed; Meyerhans, Andreas

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases. Water extracts of the leaves of the wild Egyptian artichoke (WEA) [Cynara cardunculus L. var. sylvestris (Lam.) Fiori] have been used for centuries in the Sinai Peninsula to treat hepatitis symptoms. Here we isolated and characterized six compounds from the water extracts of WEA and evaluated their HCV inhibition capacities in vitro. Importantly, two of these compounds, grosheimol and cynaropicrin, inhibited HCV with half-maximal effective concentrations (EC50s) in the low micromolar range. They inhibited HCV entry into target cells and were active against both cell-free infection as well as cell-cell transmission. Furthermore, the antiviral activity of both compounds was pan-genotypic as HCV genotypes 1a, 1b, 2b, 3a, 4a, 5a, 6a, and 7a were inhibited. Thus, grosheimol and cynaropicrin are promising candidates for the development of new pan-genotypic entry inhibitors of HCV infection. IMPORTANCE Because there is no preventive HCV vaccine available today, the discovery of novel anti-HCV cell entry inhibitors could help develop preventive measures against infection. The present study describes two compounds isolated from the wild Egyptian artichoke (WEA) with respect to their structural elucidation, absolute configuration, and quantitative determination. Importantly, both compounds inhibited HCV infection in vitro. The first compound was an unknown molecule, and it was designated “grosheimol,” while the second compound is the known molecule cynaropicrin. Both compounds belong to the group of sesquiterpene lactones. The mode of action of these compounds occurred during the early steps of the HCV life cycle, including cell-free and cell-cell infection inhibition. These natural compounds present promising candidates for further development into anti-HCV therapeutics. PMID:26656684

  12. Coinfection of hepatic cell lines with human immunodeficiency virus and hepatitis B virus leads to an increase in intracellular hepatitis B surface antigen.

    PubMed

    Iser, David M; Warner, Nadia; Revill, Peter A; Solomon, Ajantha; Wightman, Fiona; Saleh, Suha; Crane, Megan; Cameron, Paul U; Bowden, Scott; Nguyen, Tin; Pereira, Cândida F; Desmond, Paul V; Locarnini, Stephen A; Lewin, Sharon R

    2010-06-01

    Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals. PMID:20357083

  13. Inhibitory effect of oestradiol on activation of rat hepatic stellate cells in vivo and in vitro

    PubMed Central

    Shimizu, I; Mizobuchi, Y; Yasuda, M; Shiba, M; Ma, Y; Horie, T; Liu, F; Ito, S

    1999-01-01

    Background—Hepatic stellate cells play a key role in the pathogenesis of hepatic fibrosis. 
Aims—To examine the inhibitory effect of oestradiol on stellate cell activation. 
Methods—In vivo, hepatic fibrosis was induced in rats by dimethylnitrosamine or pig serum. In vitro, rat stellate cells were activated by contact with plastic dishes resulting in their transformation into myofibroblast-like cells. 
Results—In the dimethylnitrosamine and pig serum models, treatment with oestradiol at gestation related doses resulted in a dose dependent suppression of hepatic fibrosis with restored content of hepatic retinyl palmitate, reduced collagen content, lower areas of stellate cells which express α smooth muscle actin (α-SMA) and desmin, and lower procollagen type I and III mRNA levels in the liver. In cultured stellate cells, oestradiol inhibited type I collagen production, α-SMA expression, and cell proliferation. These findings suggest that oestradiol is a potent inhibitor of stellate cell transformation. 
Conclusion—The antifibrogenic role of oestradiol in the liver may contribute to the sex associated differences in the progression from hepatic fibrosis to cirrhosis. 

 Keywords: hepatic stellate cells; hepatic fibrosis; oestradiol; α smooth muscle actin; retinyl palmitate PMID:9862839

  14. Hepatitis B Virus Replication in CD34+ Hematopoietic Stem Cells From Umbilical Cord Blood.

    PubMed

    Huang, Yanxin; Yan, Qin; Fan, Rongshan; Song, Shupeng; Ren, Hong; Li, Yongguo; Lan, Yinghua

    2016-01-01

    BACKGROUND Hepatitis B virus (HBV) is a hepatotropic virus that can infect extrahepatic tissue. Whether hematopoietic stem cells (HSCs) can be infected by HBV and serve as a potential virus reservoir is still unknown. In this study, the susceptibility of CD34+ HSCs to HBV was investigated. MATERIAL AND METHODS Cord blood-derived CD34+ HSCs were exposed to HBV in vitro, and immunocytochemistry, transmission electron microscopy, and RT-PCR were used to identify viral-related proteins and specific viral genomic sequences. Then, CD34+ HSCs were challenged by different titers of HBV, and intracellular and supernatant HBV DNA, and hepatitis B surface antigen (HBsAg) levels, were examined. In addition, CD34+ peripheral blood stem cells (PBSCs) from chronic HBV carriers were isolated and cultured, and HBV DNA levels were measured. RESULTS HBV-infected CD34+ cells showed positive signals for HBsAg by DAB staining and TRITC staining, and HBV particles were identified. RT-PCR results showed that the 403 bp PCR products corresponding to the amplified hepatitis B S gene fragment were observed in CD34+ HSCs infected by HBV. In addition, supernatant and intracellular HBV DNA increased with the proliferation of CD34+ HSCs. Similar results were obtained from intracellular HBsAg quantification tests. In addition, HBV DNA levels both in cells and in supernatants of CD34+ PBSCs increased proportionally, and the increments of HBV DNA in the supernatants paralleled those found in cells. CONCLUSIONS HBV can replicate in CD34+ HSCs in cord blood or peripheral blood of chronic HBV carriers. PMID:27188537

  15. Hepatitis B Virus Replication in CD34+ Hematopoietic Stem Cells From Umbilical Cord Blood

    PubMed Central

    Huang, Yanxin; Yan, Qin; Fan, Rongshan; Song, Shupeng; Ren, Hong; Li, Yongguo; Lan, Yinghua

    2016-01-01

    Background Hepatitis B virus (HBV) is a hepatotropic virus that can infect extrahepatic tissue. Whether hematopoietic stem cells (HSCs) can be infected by HBV and serve as a potential virus reservoir is still unknown. In this study, the susceptibility of CD34+ HSCs to HBV was investigated. Material/Methods Cord blood–derived CD34+ HSCs were exposed to HBV in vitro, and immunocytochemistry, transmission electron microscopy, and RT-PCR were used to identify viral-related proteins and specific viral genomic sequences. Then, CD34+ HSCs were challenged by different titers of HBV, and intracellular and supernatant HBV DNA, and hepatitis B surface antigen (HBsAg) levels, were examined. In addition, CD34+ peripheral blood stem cells (PBSCs) from chronic HBV carriers were isolated and cultured, and HBV DNA levels were measured. Results HBV-infected CD34+ cells showed positive signals for HBsAg by DAB staining and TRITC staining, and HBV particles were identified. RT-PCR results showed that the 403 bp PCR products corresponding to the amplified hepatitis B S gene fragment were observed in CD34+ HSCs infected by HBV. In addition, supernatant and intracellular HBV DNA increased with the proliferation of CD34+ HSCs. Similar results were obtained from intracellular HBsAg quantification tests. In addition, HBV DNA levels both in cells and in supernatants of CD34+ PBSCs increased proportionally, and the increments of HBV DNA in the supernatants paralleled those found in cells. Conclusions HBV can replicate in CD34+ HSCs in cord blood or peripheral blood of chronic HBV carriers. PMID:27188537

  16. Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C

    PubMed Central

    Zhou, Ping; Cai, Qing; Chen, You-Chun; Zhang, Mu-Sen; Guan, Jian; Li, Xiao-Juan

    1997-01-01

    AIM: To investigate the existence and clinical significance of hepatitis C virus (HCV) RNA in the serum and peripheral blood mononuclear cells (PBMC) of patients with hepatitis C. METHODS: HCV RNA was detected by nested polymerase chain reaction (Nested PCR) in serum and in PBMC of 46 patients with acute hepatitis C (AHC) and in 42 patients with chronic hepatitis C (CHC). RESULTS: The positive rate of HCV RNA in PBMC of patients with CHC was markedly higher than that of patients with AHC (P < 0.01). The positive rates of HCV RNA in serum of patients with AHC and CHC and in PBMC of patients with CHC were significantly higher than those of anti-HCV positive patients with normal alanine aminotransferase (ALT) levels (P < 0.01). HCV RNA was negative in the serum of two patients, but could be detected in PBMC. In 12 patients, anti HCV was negative while HCV RNA was positive in serum. CONCLUSION: (1) detection of serum HCV RNA by nested PCR might be helpful in the early diagnosis of anti-HCV negative hepatitis C; (2) liver damage in patients with hepatitis C might be correlated with HCV-viremia; (3) infection of PBMC by HCV might play an important role in chronic liver damage in patients with HCV and in the chronicity of its clinical course; and (4) PBMC might be considered as a “reservoir” for HCV. PMID:27041960

  17. Entry of hepatitis C virus into the cell: a therapeutic target.

    PubMed

    Del Campo, José Antonio; Rojas, Ángela; Romero-Gómez, Manuel

    2012-09-01

    Several receptors have been identified as implicated on viral entry into the hepatocyte; and, this interaction between the virus and potential receptors could modulate infection, spontaneous viral clearance, persistence of the infection and the widespread of the virus as outbreak. Nevertheless, the playing role of each of them remains controversial. The Niemann-Pick type C1 like 1 gene (NPC1L1) receptor has been recently implicated on hepatitis C virus (HCV) entry into the cell and ezetimibe, an anti-cholesterol drug seems to block that, emerging the idea to control hepatitis C outbreak modulating lipid-related receptors. Hepatitis C infection seems to modulate lipid metabolism according to host genetic background. Indeed, it circulates like a lipoviroparticle. The main aim of this field of vision would be to discuss the role of hepatocyte receptors implicated on virus entry, especially NPC1L1 and the therapeutic options derived from the better knowledge about HCV-lipids- receptors interaction. PMID:22969220

  18. Production of infectious duck hepatitis B virus in a human hepatoma cell line.

    PubMed Central

    Galle, P R; Schlicht, H J; Fischer, M; Schaller, H

    1988-01-01

    The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks. Images PMID:2833623

  19. Research Advancements in Porcine Derived Mesenchymal Stem Cells.

    PubMed

    Bharti, Dinesh; Shivakumar, Sharath Belame; Subbarao, Raghavendra Baregundi; Rho, Gyu-Jin

    2016-01-01

    In the present era of stem cell biology, various animals such as Mouse, Bovine, Rabbit and Porcine have been tested for the efficiency of their mesenchymal stem cells (MSCs before their actual use for stem cell based application in humans. Among them pigs have many similarities to humans in the form of organ size, physiology and their functioning, therefore they have been considered as a valuable model system for in vitro studies and preclinical assessments. Easy assessability, few ethical issues, successful MSC isolation from different origins like bone marrow, skin, umbilical cord blood, Wharton's jelly, endometrium, amniotic fluid and peripheral blood make porcine a good model for stem cell therapy. Porcine derived MSCs (pMSCs have shown greater in vitro differentiation and transdifferention potential towards mesenchymal lineages and specialized lineages such as cardiomyocytes, neurons, hepatocytes and pancreatic beta cells. Immunomodulatory and low immunogenic profiles as shown by autologous and heterologous MSCs proves them safe and appropriate models for xenotransplantation purposes. Furthermore, tissue engineered stem cell constructs can be of immense importance in relation to various osteochondral defects which are difficult to treat otherwise. Using pMSCs successful treatment of various disorders like Parkinson's disease, cardiac ischemia, hepatic failure, has been reported by many studies. Here, in this review we highlight current research findings in the area of porcine mesenchymal stem cells dealing with their isolation methods, differentiation ability, transplantation applications and their therapeutic potential towards various diseases. PMID:26201864

  20. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology

    NASA Astrophysics Data System (ADS)

    Mederacke, Ingmar; Hsu, Christine C.; Troeger, Juliane S.; Huebener, Peter; Mu, Xueru; Dapito, Dianne H.; Pradere, Jean-Philippe; Schwabe, Robert F.

    2013-11-01

    Although organ fibrosis causes significant morbidity and mortality in chronic diseases, the lack of detailed knowledge about specific cellular contributors mediating fibrogenesis hampers the design of effective antifibrotic therapies. Different cellular sources, including tissue-resident and bone marrow-derived fibroblasts, pericytes and epithelial cells, have been suggested to give rise to myofibroblasts, but their relative contributions remain controversial, with profound differences between organs and different diseases. Here we employ a novel Cre-transgenic mouse that marks 99% of hepatic stellate cells (HSCs), a liver-specific pericyte population, to demonstrate that HSCs give rise to 82-96% of myofibroblasts in models of toxic, cholestatic and fatty liver disease. Moreover, we exclude that HSCs function as facultative epithelial progenitor cells in the injured liver. On the basis these findings, HSCs should be considered the primary cellular target for antifibrotic therapies across all types of liver disease.

  1. Anti-fibrotic effect of thymoquinone on hepatic stellate cells.

    PubMed

    Ghazwani, Mohammed; Zhang, Yifei; Gao, Xiang; Fan, Jie; Li, Jiang; Li, Song

    2014-02-15

    Hepatic stellate cells (HSCs) are the major cell type involved in the production of extracellular matrix in liver. After liver injury, HSCs undergo transdifferentiation process from quiescent state to activated state, which plays an important role in liver fibrosis. Previous studies have shown that thymoquinone (TQ) might have protective effect against liver fibrosis in animal models; however, the underlying mechanism of action is not fully understood. The aim of this study is to examine whether TQ has any direct effect on HSCs. Our results showed that pretreatment of mice with TQ has protective effect against CCl4-induced liver injury compared to control group (untreated), which is consistent with previous studies. Moreover, our in vivo study showed that COL1A1 and α-SMA mRNA levels were significantly downregulated by TQ treatment. Similarly, in vitro study confirmed that TQ downregulated COL1A1, COL3A1 and α-SMA mRNA levels in activated rat HSCs and LX2 cells, an immortalized human hepatic stellate cell line. Pretreatment with TQ also inhibited the LPS-induced proinflammatory response in LX2 cells as demonstrated by reduced mRNA expression of IL-6 and MCP-1. Mechanistically, inactivation of NF-κB pathway is likely to play a role in the TQ-mediated inhibition of proinflammatory response in HSCs. Finally, we have shown that TQ inhibited the culture-triggered transdifferentiation of freshly isolated rat HSCs as shown by significant downregulation of mRNA expression of several fibrosis-related genes. In conclusion, our study suggests that TQ has a direct effect on HSCs, which may contribute to its overall antifibrotic effect. PMID:24182989

  2. Hepatitis C virus-specific T-cell response correlates with hepatitis activity and donor IL28B genotype early after liver transplantation.

    PubMed

    Tsuzaki, Ryuichiro; Takaki, Akinobu; Yagi, Takahito; Ikeda, Fusao; Koike, Kazuko; Iwasaki, Yoshiaki; Shiraha, Hidenori; Miyake, Yasuhiro; Sadamori, Hiroshi; Shinoura, Susumu; Umeda, Yuzo; Yoshida, Ryuichi; Nobuoka, Daisuke; Utsumi, Masashi; Nakayama, Eiichi; Fujiwara, Toshiyoshi; Yamamoto, Kazuhide

    2014-01-01

    It is not known how the immune system targets hepatitis C virus (HCV)-infected HLA-mismatched hepatocytes under immune-suppressed conditions after orthotopic liver transplantation (OLT). In addition, the relationship between the HCV-specific immune response and IL28B variants as predictors of HCV clearance has not been well-characterized. We determined the IL28B polymorphisms for 57 post-OLT HCV carriers, and we assessed the HCV-specific immune responses by measuring the peripheral blood mononuclear cell-derived HCV-specific interferon-gamma (IFN-γ) response using an enzyme-linked immunospot assay. At 1-3 years after OLT, patients with no active hepatitis showed higher total spots on the immunospot assay. At>3 years after OLT, patients with resolved HCV showed higher levels of core, NS3, NS5A, and total spots compared to the chronic hepatitis patients. The IL28B major genotype in the donors correlated with higher spot counts for NS5A and NS5B proteins at 1-3 years after OLT. In the post-OLT setting, the HCV-specific immune response could be strongly induced in patients with no active hepatitis with an IL28B major donor or sustained virological response. Strong immune responses in the patients with no active hepatitis could only be maintained for 3 years and diminished later. It may be beneficial to administer IFN treatment starting 3 years after OLT, to induce the maximum immunological effect. PMID:25338486

  3. Hepatitis B virus antigens impair NK cell function.

    PubMed

    Yang, Yinli; Han, Qiuju; Zhang, Cai; Xiao, Min; Zhang, Jian

    2016-09-01

    An inadequate immune response of the host is thought to be a critical factor causing chronic hepatitis B virus (CHB) infection. Natural killer (NK) cells, as one of the key players in the eradication and control of viral infections, were functionally impaired in CHB patients, which might contribute to viral persistence. Here, we reported that HBV antigens HBsAg and HBeAg directly inhibited NK cell function. HBsAg and/or HBeAg blocked NK cell activation, cytokine production and cytotoxic granule release in human NK cell-line NK-92 cells, which might be related to the downregulation of activating receptors and upregulation of inhibitory receptor. Furthermore, the underlying mechanisms likely involved the suppression of STAT1, NF-κB and p38 MAPK pathways. These findings implicated that HBV antigen-mediated inhibition of NK cells might be an efficient strategy for HBV evasion, targeting the early antiviral responses mediated by NK cells and resulting in the establishment of chronic virus infection. Therefore, this study revealed the relationship between viral antigens and human immune function, especially a potential important interaction between HBV and innate immune responses. PMID:27341035

  4. Adult langerhans cell histiocytosis with hepatic and pulmonary involvement.

    PubMed

    Araujo, Bruno; Costa, Francisco; Lopes, Joanne; Castro, Ricardo

    2015-01-01

    Langerhans cell histiocytosis (LCH) is a rare proliferative disorder of Langerhans cells of unknown etiology. It can involve multiple organ systems with different clinical presentation, which complicates the diagnosis. It can range from isolated to multisystem disease with different prognosis. Although common among children, liver involvement is relatively rare in adults and frequently overlooked. Natural history of liver LCH fits into two stages: an early stage with infiltration by histiocytes and a late stage with sclerosis of the biliary tree. Pulmonary findings are more common and include multiple nodules in different stages of cavitation, predominantly in the upper lobes. We present a case of adult LCH with pulmonary and biopsy proven liver involvement with resolution of the hepatic findings after treatment. PMID:25977828

  5. Tissue-Derived Stem and Progenitor Cells

    PubMed Central

    Tesche, Leora J.; Gerber, David A.

    2010-01-01

    The characterization and isolation of various stem cell populations, from embryonic through tissue-derived stem cells, have led a rapid growth in the field of stem cell research. These research efforts have often been interrelated as to the markers that identify a select cell population are frequently analyzed to determine their expression in cells of distinct organs/tissues. In this review, we will expand the current state of research involving select tissue-derived stem cell populations including the liver, central nervous system, and cardiac tissues as examples of the success and challenges in this field of research. Lastly, the challenges of clinical therapies will be discussed as it applies to these unique cell populations. PMID:21048854

  6. Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

    PubMed Central

    Fagoonee, Sharmila; Famulari, Elvira Smeralda; Silengo, Lorenzo; Tolosano, Emanuela; Altruda, Fiorella

    2015-01-01

    One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine. PMID:26323094

  7. Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells.

    PubMed

    Niemietz, Christoph J; Sauer, Vanessa; Stella, Jacqueline; Fleischhauer, Lutz; Chandhok, Gursimran; Guttmann, Sarah; Avsar, Yesim; Guo, Shuling; Ackermann, Elizabeth J; Gollob, Jared; Monia, Brett P; Zibert, Andree; Schmidt, Hartmut H-J

    2016-01-01

    Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene, predominantly expressed in the liver. Two compounds that knockdown TTR, comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx), are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background, this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown, siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (>80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds. PMID:27584576

  8. Oxidative stress modulation in hepatitis C virus infected cells

    PubMed Central

    Lozano-Sepulveda, Sonia A; Bryan-Marrugo, Owen L; Cordova-Fletes, Carlos; Gutierrez-Ruiz, Maria C; Rivas-Estilla, Ana M

    2015-01-01

    Hepatitis C virus (HCV) replication is associated with the endoplasmic reticulum, where the virus can induce cellular stress. Oxidative cell damage plays an important role in HCV physiopathology. Oxidative stress is triggered when the concentration of oxygen species in the extracellular or intracellular environment exceeds antioxidant defenses. Cells are protected and modulate oxidative stress through the interplay of intracellular antioxidant agents, mainly glutathione system (GSH) and thioredoxin; and antioxidant enzyme systems such as superoxide dismutase, catalase, GSH peroxidase, and heme oxygenase-1. Also, the use of natural and synthetic antioxidants (vitamin C and E, N-acetylcysteine, glycyrrhizin, polyenylphosphatidyl choline, mitoquinone, quercetin, S-adenosylmethionine and silymarin) has already shown promising results as co-adjuvants in HCV therapy. Despite all the available information, it is not known how different agents with antiviral activity can interfere with the modulation of the cell redox state induced by HCV and decrease viral replication. This review describes an evidence-based consensus on molecular mechanisms involved in HCV replication and their relationship with cell damage induced by oxidative stress generated by the virus itself and cell antiviral machinery. It also describes some molecules that modify the levels of oxidative stress in HCV-infected cells. PMID:26692473

  9. Hhex Is Necessary for the Hepatic Differentiation of Mouse ES Cells and Acts via Vegf Signaling

    PubMed Central

    Arterbery, Adam S.; Bogue, Clifford W.

    2016-01-01

    Elucidating the molecular mechanisms involved in the differentiation of stem cells to hepatic cells is critical for both understanding normal developmental processes as well as for optimizing the generation of functional hepatic cells for therapy. We performed in vitro differentiation of mouse embryonic stem cells (mESCs) with a null mutation in the homeobox gene Hhex and show that Hhex-/- mESCs fail to differentiate from definitive endoderm (Sox17+/Foxa2+) to hepatic endoderm (Alb+/Dlk+). In addition, hepatic culture elicited a >7-fold increase in Vegfa mRNA expression in Hhex-/- cells compared to Hhex+/+ cells. Furthermore, we identified VEGFR2+/ALB+/CD34- in early Hhex+/+ hepatic cultures. These cells were absent in Hhex-/- cultures. Finally, through manipulation of Hhex and Vegfa expression, gain and loss of expression experiments revealed that Hhex shares an inverse relationship with the activity of the Vegf signaling pathway in supporting hepatic differentiation. In summary, our results suggest that Hhex represses Vegf signaling during hepatic differentiation of mouse ESCs allowing for cell-type autonomous regulation of Vegfr2 activity independent of endothelial cells. PMID:26784346

  10. Hhex Is Necessary for the Hepatic Differentiation of Mouse ES Cells and Acts via Vegf Signaling.

    PubMed

    Arterbery, Adam S; Bogue, Clifford W

    2016-01-01

    Elucidating the molecular mechanisms involved in the differentiation of stem cells to hepatic cells is critical for both understanding normal developmental processes as well as for optimizing the generation of functional hepatic cells for therapy. We performed in vitro differentiation of mouse embryonic stem cells (mESCs) with a null mutation in the homeobox gene Hhex and show that Hhex(-/-) mESCs fail to differentiate from definitive endoderm (Sox17(+/)Foxa2(+)) to hepatic endoderm (Alb(+)/Dlk(+)). In addition, hepatic culture elicited a >7-fold increase in Vegfa mRNA expression in Hhex(-/-) cells compared to Hhex(+/+) cells. Furthermore, we identified VEGFR2(+)/ALB(+/)CD34(-) in early Hhex(+/+) hepatic cultures. These cells were absent in Hhex(-/-) cultures. Finally, through manipulation of Hhex and Vegfa expression, gain and loss of expression experiments revealed that Hhex shares an inverse relationship with the activity of the Vegf signaling pathway in supporting hepatic differentiation. In summary, our results suggest that Hhex represses Vegf signaling during hepatic differentiation of mouse ESCs allowing for cell-type autonomous regulation of Vegfr2 activity independent of endothelial cells. PMID:26784346

  11. Human Skin-Derived Stem Cells as a Novel Cell Source for In Vitro Hepatotoxicity Screening of Pharmaceuticals

    PubMed Central

    De Kock, Joery; Branson, Steven; Vinken, Mathieu; Meganathan, Kesavan; Chaudhari, Umesh; Sachinidis, Agapios; Govaere, Olivier; Roskams, Tania; De Boe, Veerle; Vanhaecke, Tamara; Rogiers, Vera

    2014-01-01

    Human skin-derived precursors (hSKP) are postnatal stem cells with neural crest properties that reside in the dermis of human skin. These cells can be easily isolated from small (fore) skin segments and have the capacity to differentiate into multiple cell types. In this study, we show that upon exposure to hepatogenic growth factors and cytokines, hSKP acquire sufficient hepatic features that could make these cells suitable in vitro tools for hepatotoxicity screening of new chemical entities and already existing pharmaceutical compounds. Indeed, hepatic differentiated hSKP [hSKP-derived hepatic progenitor cells (hSKP-HPC)] express hepatic progenitor cell markers (EPCAM, NCAM2, PROM1) and adult hepatocyte markers (ALB), as well as key biotransformation enzymes (CYP1B1, FMO1, GSTA4, GSTM3) and influx and efflux drug transporters (ABCC4, ABCA1, SLC2A5). Using a toxicogenomics approach, we could demonstrate that hSKP-HPC respond to acetaminophen exposure in a comparable way to primary human hepatocytes in culture. The toxicological responses “liver damage”, “liver proliferation”, “liver necrosis” and “liver steatosis” were found to be significantly enriched in both in vitro models. Also genes associated with either cytotoxic responses or induction of apoptosis (BCL2L11, FOS, HMOX1, TIMP3, and AHR) were commonly upregulated and might represent future molecular biomarkers for hepatotoxicity. In conclusion, our data gives a first indication that hSKP-HPC might represent a suitable preclinical model for in vitro screening of hepatotoxicity. To the best of our knowledge, this is the first report in which human postnatal stem cells derived from skin are described as a potentially relevant cell source for in vitro hepatotoxicity testing of pharmaceutical compounds. PMID:23952781

  12. [Human papillomavirus, neonatal giant cell hepatitis and biliary duct atresia].

    PubMed

    Drut, R; Gómez, M A; Drut, R M; Cueto, R E; Lojo, M

    1998-01-01

    We previously recognized the presence of HPV-DNA in cases of idiopathic neonatal giant cell hepatitis (INGCH) and extrahepatic biliary duct atresia (EBDA) in archivated tissue using the PCR technique. In order to investigate a possible vertical transmission we looked for the presence of HPV-DNA in cervical swabs in the mothers along with formalin-fixed paraffin-embedded hepatic tissue from 3 infants with INGCH and 4 patients with EBDA by nested-PCR. Cervical smears showed koilocytosis consistent with HPV infection in 2 cases. Delivery was vaginal except for one that was by cesarean section. All infants were males. Amplification of HPV-DNA was demonstrated in all cases, the types being concordant in infants and mothers. Although this is a small group, the findings appear in line with previous data. The presence of the same type of HPV-DNA in the infants' livers and their mothers' cervical swabs is another argument supporting the possibility of vertical transmission of the virus. PMID:9607071

  13. Peroxisome Proliferator-activated Receptor α Positively Regulates Complement C3 Expression but Inhibits Tumor Necrosis Factor α-mediated Activation of C3 Gene in Mammalian Hepatic-derived Cells*

    PubMed Central

    Mogilenko, Denis A.; Kudriavtsev, Igor V.; Shavva, Vladimir S.; Dizhe, Ella B.; Vilenskaya, Ekaterina G.; Efremov, Alexander M.; Perevozchikov, Andrej P.; Orlov, Sergey V.

    2013-01-01

    Complement C3 is a pivotal component of three cascades of complement activation. The liver is the main source of C3 in circulation and expression and secretion of C3 by hepatocytes is increased during acute inflammation. However, the mechanism of the regulation of the C3 gene in hepatocytes is not well elucidated. We showed that the C3 gene is the direct target for peroxisome proliferator-activated receptor α (PPARα) in human hepatoma HepG2 cells and mouse liver. Using PPARα siRNA and synthetic PPARα agonist WY-14643 and antagonist MK886 we showed that activation of PPARα results in up-regulation of C3 gene expression and protein secretion by HepG2 cells. The PPAR response element (PPRE), which is able to bind PPARα in vitro and in vivo, was found in the human C3 promoter. PPRE is conserved between human and mouse, and WY-14643 stimulates mouse C3 expression in the liver. TNFα increases C3 gene via NF-κB and, to a lesser extent, MEK1/2 signaling pathways, whereas TNFα-mediated stimulation of C3 protein secretion depends on activation of MEK1/2, p38, and JNK in HepG2 cells. Activation of PPARα abolishes TNFα-mediated up-regulation of C3 gene expression and protein secretion due to interference with NF-κB via PPRE-dependent mechanism in HepG2 cells. TNFα decreases PPARα protein content via NF-κB and MEK1/2 signaling pathways and inhibits PPARα binding with the human C3 promoter in HepG2 cells. These results suggest novel mechanism controlling C3 expression in hepatocytes during acute phase inflammation and demonstrate a crosstalk between PPARα and TNFα in the regulation of complement system. PMID:23168409

  14. In vitro differentiation of unrestricted somatic stem cells into functional hepatic-like cells displaying a hepatocyte-like glucose metabolism.

    PubMed

    Waclawczyk, Simon; Buchheiser, Anja; Flögel, Ulrich; Radke, Teja F; Kögler, Gesine

    2010-11-01

    The hepatic-like phenotype resulting from in vitro differentiation of unrestricted somatic stem cells (USSC) derived from human umbilical cord blood (CB) was analyzed with regard to functional and metabolic aspects. USSC can be differentiated into cells of all three germ layers in vitro and in vivo and, although they share many features with mesenchymal stroma cells (MSC), can be distinguished from these by their expression of DLK1 as well as a restricted adipogenic differentiation potential. For the differentiation procedure described herein, a novel three-stage differentiation protocol resembling embryonic developmental processes of hepatic endoderm was applied. Hepatic pre-induction was performed by activinA and FGF4 resulting in enhanced SOX17 and FOXA2 expression. Further differentiation was achieved sequentially by retinoic acid, FGF4, HGF, EGF, and OSM resulting in a hepatic endodermal identity, characterized by the expression of AFP and HNF1alpha. Thereafter, expression of G6PC, ARG1, FBP1, and HNF4alpha was observed, thus indicating progressive differentiation. Functional studies concerning albumin secretion, urea formation, and cytochrome-p450-3A4 (CYP3A4) enzyme activity confirmed the hepatic-like phenotype. In order to characterize the differentiated cells at a metabolic level, USSC were incubated with [1-(13)C]glucose. By tracing the fate of the molecule's label via isotopomer analysis using (13)C NMR spectroscopy, formation of both glycogen and some gluconeogenetic activity could be observed providing evidence of a hepatocyte-like glucose metabolism in differentiated USSC. In conclusion, the results of the present study indicate that USSC represent a stem cell source with a substantial hepatic differentiation capacity which hold the potential for clinical applications. PMID:20458755

  15. Accumulated myeloid-derived suppressor cells demonstrate distinct phenotypes and functions in two non-alcoholic steatohepatitis mouse models

    PubMed Central

    Tsunashima, Hiromichi; Tsuneyama, Koichi; Moritoki, Yuki; Hara, Masumi

    2015-01-01

    Background To examine the steady state of hepatic myeloid-derived suppressor cells (MDSCs) and the lipid accumulation and inflammation-related changes in these cells, we analyzed the presence and functions of hepatic MDSCs in the following two non-alcoholic steatohepatitis (NASH) mouse models. Methods Monosodium glutamate (MSG) model; MSG was subcutaneously injected into neonatal male C57BL/6J mice that were fed with normal diet up to 18 weeks of age. Methionine/choline-deficient diet (MCD) model; 16-week-old male C57BL/6J mice were fed with an MCD for 2 weeks. Those hepatic MDSCs were evaluated by flow cytometry and immunohistochemically. Results Both MSG and MCD mice exhibited greater numbers of hepatic lipid droplets than 18-week-old male control mice. Hepatocellular ballooning was obvious in MSG, whereas inflammatory cell infiltration were apparent in MCD mice. CD11b, CD115, and Gr-1-positive hepatic MDSCs were increased in both models but higher in MCD mice, and demonstrated higher expression of an M2 macrophage marker CD206 mean fluorescence intensity (MFI) in MSG compared to MCD mice. Degree of reactive oxygen species production was evaluated using the DCFDA MFI values, which were significantly elevated in hepatic MDSCs from MCD mice. MSG mouse livers demonstrated Gr-1 positive cell accumulation around lipid droplets, mimicking crown-like structures in adipose tissues. In contrast, hepatic Gr-1 positive cells were primarily located in inflammatory cell aggregates in MCD mice. Conclusions These results suggest that hepatic fatty changes promote MDSC accumulation, and inflammatory changes induce phenotypic and functional alteration in hepatic MDSCs in NASH mouse models. PMID:26605278

  16. Possible Involvement of Hepatitis B Virus Infection of Hepatocytes in the Attenuation of Apoptosis in Hepatic Stellate Cells

    PubMed Central

    Sasaki, Reina; Kanda, Tatsuo; Nakamura, Masato; Nakamoto, Shingo; Haga, Yuki; Wu, Shuang; Shirasawa, Hiroshi; Yokosuka, Osamu

    2016-01-01

    Background The induction of apoptosis in hepatic stellate cells (HSCs) is a promising therapeutic strategy against hepatitis B virus (HBV)-related hepatic fibrosis. The underlying mechanisms of apoptosis in HSCs, however, are unknown under consideration of HBV infection. In this study, the effects of HBV on apoptosis and endoplasmic reticulum (ER) stress signaling in HSCs were examined. Methods The effects of conditioned media (CM) from HepG2.2.15 on apoptosis induced by the proteasome inhibitor MG132 in LX-2 and HHSteC were studied in regard to c-Jun. In combination with c-Fos, c-Jun forms the AP-1 early response transcription factor, leading to AP-1 activation, signal transduction, endoplasmic reticulum (ER) stress and apoptosis. Results In LX-2 cells, MG132 treatment was associated with the phosphorylation of c-Jun, activation of AP-1 and apoptosis. However, in the presence of CM from HepG2.2.15, these phenomena were attenuated. In HHSteC cells, similar results were observed. HBV genomic DNA is not involved in the process of HSC apoptosis. It is possible that HBeAg has an inhibitory effect on MG132-induced apoptosis in LX-2. We also observed the upregulation of several ER stress-associated genes, such as cAMP responsive element binding protein 3-like 3, inhibin-beta A and solute carrier family 17-member 2, in the presence of CM from HepG2.2.15, or CM from PXB cells infected with HBV. Conclusions HBV inhibits the activation of c-Jun/AP-1 in HSCs, contributing to the attenuation of apoptosis and resulting in hepatic fibrosis. HBV also up-regulated several ER stress genes associated with cell growth and fibrosis. These mechanistic insights might shed new light on a treatment strategy for HBV-associated hepatic fibrosis. PMID:26731332

  17. Gastrointestinal and hepatic complications of hematopoietic stem cell transplantation

    PubMed Central

    Tuncer, Hande H; Rana, Naveed; Milani, Cannon; Darko, Angela; Al-Homsi, Samer A

    2012-01-01

    Recognition and management of gastrointestinal and hepatic complications of hematopoietic stem cell transplantation has gained increasing importance as indications and techniques of transplantation have expanded in the last few years. The transplant recipient is at risk for several complications including conditioning chemotherapy related toxicities, infections, bleeding, sinusoidal obstruction syndrome, acute and chronic graft-versus-host disease (GVHD) as well as other long-term problems. The severity and the incidence of many complications have improved in the past several years as the intensity of conditioning regimens has diminished and better supportive care and GVHD prevention strategies have been implemented. Transplant clinicians, however, continue to be challenged with problems arising from human leukocyte antigen-mismatched and unrelated donor transplants, expanding transplant indications and age-limit. This review describes the most commonly seen transplant related complications, focusing on their pathogenesis, differential diagnosis and management. PMID:22563164

  18. Converting Skin Fibroblasts into Hepatic-like Cells by Transient Programming.

    PubMed

    Zhu, Xiang-Qing; Pan, Xing-Hua; Yao, Ling; Li, Wei; Cui, Jiuwei; Wang, Guanjun; Mrsny, Randall J; Hoffman, Andrew R; Hu, Ji-Fan

    2016-03-01

    Transplantation of hepatocytes is a promising therapy for end-stage liver disease, but the availability of functional cells currently precludes its clinical application. We now report a simple transient reprogramming approach to convert fibroblasts into hepatic-like cells. Human skin fibroblasts were treated with fish egg extracts to become the transiently remodeled cells (TRCs). After infected with retroviral EGFP, they were directly injected into the fetal monkey liver, where they underwent in situ differentiation in the hepatic niche. The hepatic-like cells were functional as shown by the synthesis of hepatic markers in vivo, including albumin, cytokeratin-18, and hepatic serum antigen. Similarly, when implanted in the mouse liver, the TRCs were differentiated into hepatic-like cells that synthesize albumin and CK18 and became completely integrated into the liver parenchyma. The potency of TRCs was mechanistically related to the activation of several signal pathways, which reactivate endogenous genes related to cell potency. This study demonstrates the feasibility of a simple and inexpensive epigenetic remodeling approach to convert human fibroblasts into therapeutic hepatic-like cells for the treatment of end-stage liver disease. PMID:26312781

  19. Interferon-γ facilitates hepatic antiviral T cell retention for the maintenance of liver-induced systemic tolerance.

    PubMed

    Zeng, Zhutian; Li, Lu; Chen, Yongyan; Wei, Haiming; Sun, Rui; Tian, Zhigang

    2016-05-30

    Persistent exposure to liver pathogens leads to systemic antigen-specific tolerance, a major cause of chronicity during hepatotropic infection. The mechanism regarding how this systemic tolerance is maintained remains poorly elucidated. In a well established mouse model of hepatitis B virus (HBV) persistence-induced systemic tolerance, we observed that interferon-γ (IFN-γ) deficiency led to complete loss of tolerance, resulting in robust anti-HBV responses upon peripheral vaccination. The recovery of vaccine-induced anti-HBV responses was mainly caused by the retained antigen-specific CD4(+) T cells rather than decreased functional inhibitory cells in the periphery. Mechanistically, HBV persistence induced sustained hepatic CD4(+) T cell-derived IFN-γ production. IFN-γ was found to promote CXCL9 secretion from liver-resident macrophages. This T cell chemokine facilitated the retention of antiviral CD4(+) T cells in the liver in a CXCR3-dependent manner. Hepatic sequestrated antiviral CD4(+) T cells subsequently underwent local apoptotic elimination partially via cytotoxic T lymphocyte-associated protein 4 ligation. These findings reveal an unexpected tolerogenic role for IFN-γ during viral persistence in the liver, providing new mechanistic insights regarding the maintenance of systemic antigen-specific tolerance during HBV persistence. PMID:27139489

  20. Trophoblast lineage cells derived from human induced pluripotent stem cells

    SciTech Connect

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  1. Coupling to the surface of liposomes alters the immunogenicity of hepatitis C virus-derived peptides and confers sterile immunity.

    PubMed

    Takagi, Akira; Kobayashi, Nobuharu; Taneichi, Maiko; Uchida, Tetsuya; Akatsuka, Toshitaka

    2013-01-01

    We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen presenting cells to cytotoxic T lymphocytes (CTLs). Liposomal form of immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus exhibited highly efficient antiviral CTL responses in immunized mice. In this study, we coupled 15 highly conserved immunodominant CTL epitope peptides derived from hepatitis C virus (HCV) to the surface of liposomes. We also emulsified the peptides in incomplete Freund's adjuvant, and compared the immune responses of the two methods of presenting the peptides by cytotoxicity induction and interferon-gamma (IFN-γ) production by CD8(+) T cells of the immunized mice. We noticed significant variations of the immunogenicity of each peptide between the two antigen delivery systems. In addition, the immunogenicity profiles of the peptides were also different from those observed in the mice infected with recombinant adenoviruses expressing HCV proteins as previously reported. Induction of anti-viral immunity by liposomal peptides was tested by the challenge experiments using recombinant vaccinia viruses expressing corresponding HCV epitopes. One D(b)-restricted and three HLA-A(*)0201-restricted HCV CTL epitope peptides on the surface of liposomes were found to confer complete protection to immunized mice with establishment of long-term memory. Interestingly, their protective efficacy seemed to correlate with the induction of IFN-γ producing cells rather than the cytotoxicity induction suggesting that the immunized mice were protected through non-cytolytic mechanisms. Thus, these liposomal peptides might be useful as HCV vaccines not only for prevention but also for therapeutic use. PMID:23159619

  2. Hepatic cell lines for drug hepatotoxicity testing: limitations and strategies to upgrade their metabolic competence by gene engineering.

    PubMed

    Donato, M Teresa; Jover, Ramiro; Gómez-Lechón, M José

    2013-11-01

    One key issue in the pharmaceutical development of new compounds is knowledge on metabolism, the enzymes involved and the potential hepatotoxicity of a drug. Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, human hepatocytes show phenotypic instability and have restricted accessibility and high batch-to-batch functional variability, which seriously complicates their use in routine testing. Therefore, several liver-derived cell models have been developed for drug metabolism and hepatotoxicity screening to circumvent these drawbacks. Hepatoma cell lines offer important advantages, availability, an unlimited life span and a stable phenotype, thus rendering them suitable models for such studies. However, currently available human hepatoma cell lines are not a good alternative to cultured hepatocytes as they show very limited expression for most drug-metabolising enzymes. Other approaches have been developed to generate immortalised hepatic cells with metabolic competence (use of plasmids encoding immortalising genes to transform human hepatocytes, cell lines obtained from transgenic animals, hepatocytomes or hydrid cells). Recombinant models heterologously expressing cytochrome P450 enzymes in hepatoma cells have also been generated, and are widely used in drug metabolism and toxicity evaluations. In recent years, new approaches to up-regulate the expression of drug-biotransformation enzymes in human cell lines (i.e., transfection with the expression vectors encoding key hepatic transcription factors) have also been investigated. This paper reviews the features of liver-derived cell lines, their suitability for drug metabolism and hepatotoxicity studies, and the state-of-the-art strategies pursued to generate metabolically competent hepatic cell lines. PMID:24160292

  3. Tannic Acid Inhibits Hepatitis C Virus Entry into Huh7.5 Cells

    PubMed Central

    Hagedorn, Curt H.

    2015-01-01

    Chronic infection with the hepatitis C virus (HCV) is a cause of cirrhosis and hepatocellular carcinoma worldwide. Although antiviral therapy has dramatically improved recently, a number of patients remain untreated and some do not clear infection with treatment. Viral entry is an essential step in initiating and maintaining chronic HCV infections. One dramatic example of this is the nearly 100% infection of newly transplanted livers in patients with chronic hepatitis C. HCV entry inhibitors could play a critical role in preventing HCV infection of newly transplanted livers. Tannic acid, a polymer of gallic acid and glucose molecules, is a plant-derived polyphenol that defends some plants from insects and microbial infections. It has been shown to have a variety of biological effects, including antiviral activity, and is used as a flavoring agent in foods and beverages. In this study, we demonstrate that tannic acid is a potent inhibitor of HCV entry into Huh7.5 cells at low concentrations (IC50 5.8 μM). It also blocks cell-to-cell spread in infectious HCV cell cultures, but does not inhibit HCV replication following infection. Moreover, experimental results indicate that tannic acid inhibits an early step of viral entry, such as the docking of HCV at the cell surface. Gallic acid, tannic acid’s structural component, did not show any anti-HCV activity including inhibition of HCV entry or replication at concentrations up to 25 μM. It is possible the tannin structure is related on the effect on HCV inhibition. Tannic acid, which is widely distributed in plants and foods, has HCV antiviral activity in cell culture at low micromolar concentrations, may provide a relative inexpensive adjuvant to direct-acting HCV antivirals and warrants future investigation. PMID:26186636

  4. Hepatitis C virus - associated B cell non-Hodgkin's lymphoma.

    PubMed

    Mihăilă, Romeo-Gabriel

    2016-07-21

    The hepatitis C virus (HCV) infected patients are prone to develop bone marrow or various tissue infiltrates with monoclonal B cells, monoclonal B lymphocytosis or different types of B cell non-Hodgkin's lymphoma (BCNHL), of which the most common are splenic marginal zone BCNHL, diffuse large BCNHL and follicular lymphoma. The association between chronic HCV infection and non Hodgkin's lymphoma has been observed especially in areas with high prevalence of this viral infection. Outside the limitations of some studies that have been conducted, there are also geographic, environmental, and genetic factors that contribute to the epidemiological differences. Various microenvironmental signals, such as cytokines, viral antigenic external stimulation of lymphocyte receptors by HCV antigens, and intercellular interactions contribute to B cell proliferation. HCV lymphotropism and chronic antigenic stimulation are involved in B-lymphocyte expansion, as mixted cryoglobulinemia or monoclonal gammopathy of undetermined significance, which can progress to BCNHL. HCV replication in B lymphocytes has oncogenic effect mediated by intracellular HCV proteins. It is also involved in an important induction of reactive oxygen species that can lead to permanent B lymphocyte damage, as DNA mutations, after binding to surface B-cell receptors. Post-transplant lymphoproliferative disorder could appear and it has a multiclonal potentiality that may develop into different types of lymphomas. The hematopoietic stem cell transplant made for lymphoma in HCV-infected patients can increase the risk of earlier progression to liver fibrosis and cirrhosis. HCV infected patients with indolent BCNHL who receive antiviral therapy can be potentially cured. Viral clearance was related to lymphoma response, fact that highlights the probable involvement of HCV in lymphomagenesis. Direct acting antiviral drugs could be a solution for the patients who did not tolerate or respond to interferon, as they seem to

  5. Hepatitis C virus - associated B cell non-Hodgkin's lymphoma

    PubMed Central

    Mihăilă, Romeo-Gabriel

    2016-01-01

    The hepatitis C virus (HCV) infected patients are prone to develop bone marrow or various tissue infiltrates with monoclonal B cells, monoclonal B lymphocytosis or different types of B cell non-Hodgkin’s lymphoma (BCNHL), of which the most common are splenic marginal zone BCNHL, diffuse large BCNHL and follicular lymphoma. The association between chronic HCV infection and non Hodgkin’s lymphoma has been observed especially in areas with high prevalence of this viral infection. Outside the limitations of some studies that have been conducted, there are also geographic, environmental, and genetic factors that contribute to the epidemiological differences. Various microenvironmental signals, such as cytokines, viral antigenic external stimulation of lymphocyte receptors by HCV antigens, and intercellular interactions contribute to B cell proliferation. HCV lymphotropism and chronic antigenic stimulation are involved in B-lymphocyte expansion, as mixted cryoglobulinemia or monoclonal gammopathy of undetermined significance, which can progress to BCNHL. HCV replication in B lymphocytes has oncogenic effect mediated by intracellular HCV proteins. It is also involved in an important induction of reactive oxygen species that can lead to permanent B lymphocyte damage, as DNA mutations, after binding to surface B-cell receptors. Post-transplant lymphoproliferative disorder could appear and it has a multiclonal potentiality that may develop into different types of lymphomas. The hematopoietic stem cell transplant made for lymphoma in HCV-infected patients can increase the risk of earlier progression to liver fibrosis and cirrhosis. HCV infected patients with indolent BCNHL who receive antiviral therapy can be potentially cured. Viral clearance was related to lymphoma response, fact that highlights the probable involvement of HCV in lymphomagenesis. Direct acting antiviral drugs could be a solution for the patients who did not tolerate or respond to interferon, as they

  6. Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.

    PubMed

    Stecklum, Maria; Wulf-Goldenberg, Annika; Purfürst, Bettina; Siegert, Antje; Keil, Marlen; Eckert, Klaus; Fichtner, Iduna

    2015-02-01

    In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction. PMID:25270685

  7. Elevated Levels of Endocannabinoids in Chronic Hepatitis C May Modulate Cellular Immune Response and Hepatic Stellate Cell Activation

    PubMed Central

    Patsenker, Eleonora; Sachse, Philip; Chicca, Andrea; Gachet, María Salomé; Schneider, Vreni; Mattsson, Johan; Lanz, Christian; Worni, Mathias; de Gottardi, Andrea; Semmo, Mariam; Hampe, Jochen; Schafmayer, Clemens; Brenneisen, Rudolf; Gertsch, Jürg; Stickel, Felix; Semmo, Nasser

    2015-01-01

    The endocannabinoid (EC) system is implicated in many chronic liver diseases, including hepatitis C viral (HCV) infection. Cannabis consumption is associated with fibrosis progression in patients with chronic hepatitis C (CHC), however, the role of ECs in the development of CHC has never been explored. To study this question, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were quantified in samples of HCV patients and healthy controls by gas and liquid chromatography mass spectrometry. Fatty acid amide hydrolase (FAAH) and monoaclyglycerol lipase (MAGL) activity was assessed by [3H]AEA and [3H]2-AG hydrolysis, respectively. Gene expression and cytokine release were assayed by TaqMan PCR and ELISpot, respectively. AEA and 2-AG levels were increased in plasma of HCV patients, but not in liver tissues. Hepatic FAAH and MAGL activity was not changed. In peripheral blood mononuclear cells (PBMC), ECs inhibited IFN-γ, TNF-α, and IL-2 secretion. Inhibition of IL-2 by endogenous AEA was stronger in PBMC from HCV patients. In hepatocytes, 2-AG induced the expression of IL-6, -17A, -32 and COX-2, and enhanced activation of hepatic stellate cells (HSC) co-cultivated with PBMC from subjects with CHC. In conclusion, ECs are increased in plasma of patients with CHC and might reveal immunosuppressive and profibrogenic effects. PMID:25826533

  8. Indole derivatives inhibit hepatitis C virus replication through induction of pro-inflammatory cytokines.

    PubMed

    Lee, S; Jin, G; Kim, D; Son, S; Lee, K; Lee, C

    2015-03-01

    Previously, we discovered a series of indole derivatives as a new class of hepatitis C virus (HCV) replication inhibitors by using a target-free chemical genetic strategy. Through a structure-activity relationship study, the compound 12e was identified as the most potent inhibitor of this class (EC50 = 1.1 μmol/l) with minimal cytotoxicity (CC50 = 61.8 μmol/l). In order to gain insight into its detailed antiviral mechanism of action, we performed PCR array analyses and found that 12e was able to activate transcription of a number of pro-inflammatory as well as antiviral cytokine genes including CXCL-8, IL-1α, TNF-α, IL-3, IRAK-1, and DDX58. Their induction by 12e was verified by individual RT-PCR analyses. In addition, 12e was found to stimulate secretion of soluble factors with anti-HCV replication activity. Among the 12e-induced pro-inflammatory cytokines, CXCL-8 showed a strong positive correlation between its transcriptional activation and antiviral potency. Interestingly, a recombinant CXCL-8 protein also reduced HCV replication, though only moderately. In conclusion, we found a novel mode of action of indole derivatives in inhibiting HCV replication, particularly the induction of pro-inflammatory cytokines. PMID:25790053

  9. Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury.

    PubMed

    Ryu, Kyung-Ha; Kim, So-Yeon; Kim, Ye-Ryung; Woo, So-Youn; Sung, Sun Hee; Kim, Han Su; Jung, Sung-Chul; Jo, Inho; Park, Joo-Won

    2014-08-01

    Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease. PMID:24954408

  10. Effect of adipocyte-secreted factors on EpCAM+/CD133+ hepatic stem cell population.

    PubMed

    Firtina Karagonlar, Zeynep; Koç, Doğukan; Şahin, Eren; Avci, Sanem Tercan; Yilmaz, Mustafa; Atabey, Neşe; Erdal, Esra

    2016-06-01

    Recent epidemiological studies have associated obesity with a variety of cancer types including HCC. However, the tumor initiating role of obesity in hepatocarcinogenesis is still unknown. The objective of this paper is to investigate the effect of adipocyte-secreted factors on EpCAM+/CD133+ cancer stem cells and to identify which factors play a role in modulating hepatic cancer stem cell behavior. Our results demonstrated that adipocyte-secreted factors affect motility and drug resistance of EpCAM+/CD133+ cells. When incubated with adipocyte conditioned media, EpCAM+/CD133+ cells exhibited augmented motility and reduced sorafenib-induced apoptosis. Using array-based system, we identified secretion of several cytokines such as IL6, IL8 and MCP1 by cultured adipocytes and activation of c-Met, STAT3 and ERK1/2 signaling pathways in EpCAM+/CD133+ cells incubated with adipocyte conditioned media. Treating EpCAM+/CD133+ cancer stem cells with IL6 receptor blocking antibody or c-Met inhibitor SU11274 both reduced the increase in motility; however SU11274 had greater effect on relieving protection from sorafenib-induced apoptosis. These results indicate that adipocyte-secreted factors might regulate cancer stem cell behavior through several signaling molecules including c-Met, STAT3 and ERK1/2 and inhibition of these signaling pathways offer novel strategies in targeting the effect of adipose-derived cytokines in cancer. PMID:27131739

  11. Hepatic differentiation of human embryonic stem cells as microscaled multilayered colonies leading to enhanced homogeneity and maturation.

    PubMed

    Yao, Rui; Wang, Jingyu; Li, Xiaokang; Jung Jung, Da; Qi, Hao; Kee, Keh Kooi; Du, Yanan

    2014-11-12

    Directed differentiation of human embryonic stem cells (hESCs) towards hepatocyte-like cells on planar tissue culture plates has been extensively investigated with great promise to provide alternative cell sources for drug metabolism/toxicity testing. Recently, hepatic differentiation of hESCs in 3D configuration with better mimicry of embryonic liver development represents incremental efforts to improve the differentiation efficiency and cellular maturation. However, most of the present 3D differentiation configurations involved interruptive operations during the multi-staged differentiation process, which might impose unwanted influence on cellular differentiation. Most of the current researches resulted in generation of hepatocytes with high expression of AFP, which is minimally expressed in primary hepatocytes. Here, off-the-shelf micro-stencil arrays are developed to generate adherent multilayered colonies composed of hESCs-derived cells. Uninterrupted cellular differentiation and proliferation is achieved to recapitulate the continuous and multi-stage liver development. Compared with conventional 2D format, the micro-scaled multilayered colonies with uniform and defined sizes constrained within the microwells are composed of more homogenous and mature hepatocyte-like cells with significantly lowered AFP expression and elevated hepatic functions. The multilayered colonies as novel 3D configuration for hepatic differentiation of hESCs represent a significant step toward efficient generation of functional hepatocytes for regenerative medicine and drug discovery. PMID:25059765

  12. Natural killer cells contribute to hepatic injury and help in viral persistence during progression of hepatitis B e-antigen-negative chronic hepatitis B virus infection.

    PubMed

    Ghosh, S; Nandi, M; Pal, S; Mukhopadhyay, D; Chakraborty, B C; Khatun, M; Bhowmick, D; Mondal, R K; Das, S; Das, K; Ghosh, R; Banerjee, S; Santra, A; Chatterjee, M; Chowdhury, A; Datta, S

    2016-08-01

    Hepatitis B e-antigen negative (e(-)) chronic HBV infection (CHI) encompasses a heterogeneous clinical spectrum ranging from inactive carrier (IC) state to e(-) chronic hepatitis B (CHB), cirrhosis and hepatic decompensation. In the backdrop of dysfunctional virus-specific T cells, natural killer (NK) cells are emerging as innate effectors in CHI. We characterized CD3(-) CD56(+) NK cells in clinically well-defined, treatment-naive e(-) patients in IC, e(-)CHB or decompensated liver cirrhosis (LC) phase to appraise their role in disease progression. The NK cell frequencies increased progressively with disease severity (IC 8.2%, e(-)CHB 13.2% and LC 14.4%). Higher proportion of NK cells from LC/e(-)CHB expressed CD69, NKp46, NKp44, TRAIL and perforin, the last two being prominent features of CD56(bright) and CD56(dim) NK subsets, respectively. The frequencies of CD3(-) CD56(+) NK cells together with TRAIL(+) CD56(bright) and Perforin(+) CD56(dim) NK cells correlated positively with serum alanine transaminase levels in e(-)CHB/LC. K562 cell-stimulated NK cells from e(-)CHB/LC exhibited significantly greater degranulation but diminished interferon-γ production than IC. Further, Perforin(+) NK cell frequency inversely correlated with autologous CD4(+) T-cell count in e(-) patients and ligands of NK receptors were over-expressed in CD4(+) T cells from e(-)CHB/LC relative to IC. Co-culture of sorted CD56(dim) NK cells and CD4(+) T cells from e(-)CHB showed enhanced CD4(+) T-cell apoptosis, which was reduced by perforin inhibitor, concanamycin A, suggesting a possible perforin-dependent NK cell-mediated CD4(+) T-cell depletion. Moreover, greater incidence of perforin-expressing NK cells and decline in CD4(+) T cells were noticed intrahepatically in e(-)CHB than IC. Collectively, NK cells contribute to the progression of e(-)CHI by enhanced TRAIL- and perforin-dependent cytolytic activity and by restraining anti-viral immunity through reduced interferon-γ secretion and

  13. Endothelial Cells Derived From Nuclear Reprogramming

    PubMed Central

    Wong, Wing Tak; Huang, Ngan F.; Botham, Crystal M.; Sayed, Nazish; Cooke, John P.

    2012-01-01

    The endothelium plays a pivotal role in vascular homeostasis, regulating the tone of the vascular wall, and its interaction with circulating blood elements. Alterations in endothelial functions facilitate the infiltration of inflammatory cells and permit vascular smooth muscle proliferation and platelet aggregation. Therefore, endothelial dysfunction is an early event in disease processes including atherosclerosis, and because of its critical role in vascular health the endothelium is worthy of the intense focus it has received. However, there are limitations to studying human endothelial function in vivo, or human vascular segments ex vivo. Thus, methods for endothelial cell culture have been developed and refined. More recently, methods to derive endothelial cells from pluripotent cells have extended the scientific range of human endothelial cell studies. Pluripotent stem cells may be generated, expanded and then differentiated into endothelial cells for in vitro studies. Constructs for molecular imaging can also be employed to facilitate tracking these cells in vivo. Furthermore, one can generate patient-specific endothelial cells to study the effects of genetic or epigenetic alterations on endothelial behavior. Finally, there is the opportunity to apply these cells for vascular therapy. This review focuses on the generation of endothelial cells from stem cells; their characterization by genetic, histological and functional studies; and their translational applications. PMID:23104878

  14. Myeloid-derived suppressor cells in B cell malignancies.

    PubMed

    Yazdani, Yaghoub; Mohammadnia-Afrouzi, Mousa; Yousefi, Mehdi; Anvari, Enayat; Ghalamfarsa, Ghasem; Hasannia, Hadi; Sadreddini, Sanam; Jadidi-Niaragh, Farhad

    2015-09-01

    Tumor cells use several mechanisms such as soluble immune modulators or suppressive immune cells to evade from anti-tumor responses. Immunomodulatory cytokines, such as transforming growth factor-β, interleukin (IL)-10, and IL-35, soluble factors, such as adenosine, immunosuppressive cells, such as regulatory T cells, NKT cells and myeloid-derived suppressor cells (MDSCs), are the main orchestra leaders involved in immune suppression in cancer by which tumor cells can freely expand without immune cell-mediated interference. Among them, MDSCs have attracted much attention as they represent a heterogenous population derived from myeloid progenitors that are expanded in tumor condition and can also shift toward other myeloid cells, such as macrophages and dendritic cells, after tumor clearing. MDSCs exert their immunosuppressive effects through various immune and non-immune mechanisms which make them as potent tumor-promoting cells. Although, there are several studies regarding the immunobiology of MDSCs in different solid tumors, little is known about the precise characteristics of these cells in hematological malignancies, particularly B cell malignancies. In this review, we tried to clarify the precise role of MDSCs in B cell-derived malignancies. PMID:26330296

  15. Hepatitis C Virus Infection in Phenotypically Distinct Huh7 Cell Lines

    PubMed Central

    Sainz, Bruno; Barretto, Naina; Uprichard, Susan L.

    2009-01-01

    In 2005, the first robust hepatitis C virus (HCV) infectious cell culture system was developed based on the HCV genotype 2a JFH-1 molecular clone and the human-derived hepatoma cell line Huh7. Although much effort has been made to dissect and expand the repertoire of JFH-1-derived clones, less attention has been given to the host cell despite the intriguing facts that thus far only Huh7 cells have been found to be highly permissive for HCV infection and furthermore only a limited number of Huh7 cell lines/stocks appear to be fully permissive. As such, we compiled a panel of Huh7 lines from disparate sources and evaluated their permissiveness for HCV infection. We found that although Huh7 lines from different laboratories do vary in morphology and cell growth, the majority (8 out of 9) were highly permissive for infection, as demonstrated by robust HCV RNA and de novo infectious virion production following infection. While HCV RNA levels achieved in the 8 permissive cell lines were relatively equivalent, three Huh7 lines demonstrated higher infectious virion production suggesting these cell lines more efficiently support post-replication event(s) in the viral life cycle. Consistent with previous studies, the single Huh7 line found to be relatively resistant to infection demonstrated a block in HCV entry. These studies not only suggest that the majority of Huh7 cell lines in different laboratories are in fact highly permissive for HCV infection, but also identify phenotypically distinct Huh7 lines, which may facilitate studies investigating the cellular determinants of HCV infection. PMID:19668344

  16. Myeloid derived suppressor cells and autoimmunity.

    PubMed

    Boros, Peter; Ochando, Jordi; Zeher, Margit

    2016-08-01

    Myeloid-derived suppressor cells are a heterogeneous group of immature myeloid cells with immunoregulatory function. When activated and expanded, these cells can suppress T cell functions via cell-to cell interactions as well as soluble mediators. Recent studies investigated the involvement of MDSC in autoimmune diseases. Some papers have described beneficial effect of MDSC during the course of autoimmune diseases, and suggest a potential role as a treatment option, while others failed to detect these effects. Their contributions to autoimmune diseases are not fully understood, and many questions and some controversies remain as to the expansion, activation, and inhibitory functions of MDSC. This review aims to summarize current knowledge of MDSC in autoimmune disorders. PMID:27240453

  17. Hepatitis C virus cell-cell transmission and resistance to direct-acting antiviral agents.

    PubMed

    Xiao, Fei; Fofana, Isabel; Heydmann, Laura; Barth, Heidi; Soulier, Eric; Habersetzer, François; Doffoël, Michel; Bukh, Jens; Patel, Arvind H; Zeisel, Mirjam B; Baumert, Thomas F

    2014-05-01

    Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs. PMID:24830295

  18. Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents

    PubMed Central

    Heydmann, Laura; Barth, Heidi; Soulier, Eric; Habersetzer, François; Doffoël, Michel; Bukh, Jens; Patel, Arvind H.; Zeisel, Mirjam B.; Baumert, Thomas F.

    2014-01-01

    Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs. PMID:24830295

  19. Modelling the Impact of Cell-To-Cell Transmission in Hepatitis B Virus

    PubMed Central

    2016-01-01

    Cell-free virus is a well-recognized and efficient mechanism for the spread of hepatitis B virus (HBV) infection in the liver. Cell-to-cell transmission (CCT) can be a more efficient means of virus propagation. Despite experimental evidence implying CCT occurs in HBV, its relative impact is uncertain. We develop a 3-D agent-based model where each hepatocyte changes its viral state according to a dynamical process driven by cell-free virus infection, CCT and intracellular replication. We determine the relative importance of CCT in the development and resolution of acute HBV infection in the presence of cytolytic (CTL) and non-CTL mechanisms. T cell clearance number is defined as the minimum number of infected cells needed to be killed by each T cell at peak infection that results in infection clearance within 12 weeks with hepatocyte turnover (HT, number of equivalent livers) ≤3. We find that CCT has very little impact on the establishment of infection as the mean cccDNA copies/cell remains between 15 to 20 at the peak of the infection regardless of CCT strength. In contrast, CCT inhibit immune-mediated clearance of acute HBV infection as higher CCT strength requires higher T cell clearance number and increases the probability of T cell exhaustion. An effective non-CTL inhibition can counter these negative effects of higher strengths of CCT by supporting rapid, efficient viral clearance and with little liver destruction. This is evident as the T cell clearance number drops by approximately 50% when non-CTL inhibition is increased from 10% to 80%. Higher CCT strength also increases the probability of the incidence of fulminant hepatitis with this phenomenon being unlikely to arise for no CCT. In conclusion, we report the possibility of CCT impacting HBV clearance and its contribution to fulminant hepatitis. PMID:27560827

  20. Modelling the Impact of Cell-To-Cell Transmission in Hepatitis B Virus.

    PubMed

    Goyal, Ashish; Murray, John M

    2016-01-01

    Cell-free virus is a well-recognized and efficient mechanism for the spread of hepatitis B virus (HBV) infection in the liver. Cell-to-cell transmission (CCT) can be a more efficient means of virus propagation. Despite experimental evidence implying CCT occurs in HBV, its relative impact is uncertain. We develop a 3-D agent-based model where each hepatocyte changes its viral state according to a dynamical process driven by cell-free virus infection, CCT and intracellular replication. We determine the relative importance of CCT in the development and resolution of acute HBV infection in the presence of cytolytic (CTL) and non-CTL mechanisms. T cell clearance number is defined as the minimum number of infected cells needed to be killed by each T cell at peak infection that results in infection clearance within 12 weeks with hepatocyte turnover (HT, number of equivalent livers) ≤3. We find that CCT has very little impact on the establishment of infection as the mean cccDNA copies/cell remains between 15 to 20 at the peak of the infection regardless of CCT strength. In contrast, CCT inhibit immune-mediated clearance of acute HBV infection as higher CCT strength requires higher T cell clearance number and increases the probability of T cell exhaustion. An effective non-CTL inhibition can counter these negative effects of higher strengths of CCT by supporting rapid, efficient viral clearance and with little liver destruction. This is evident as the T cell clearance number drops by approximately 50% when non-CTL inhibition is increased from 10% to 80%. Higher CCT strength also increases the probability of the incidence of fulminant hepatitis with this phenomenon being unlikely to arise for no CCT. In conclusion, we report the possibility of CCT impacting HBV clearance and its contribution to fulminant hepatitis. PMID:27560827

  1. Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells

    SciTech Connect

    Sakai, Hiroshi; Tagawa, Yoh-ichi; Tamai, Miho; Motoyama, Hiroaki; Ogawa, Shinichiro; Soeda, Junpei; Nakata, Takenari; Miyagawa, Shinichi

    2010-12-17

    Research highlights: {yields} Hepatic progenitor cells were isolated from the portal branch-ligated liver of mice. {yields} Portal branch ligation-stimulated hepatic progenitor cells (PBLHCs) express Hmga2. {yields} PBLHCs have bidirectional differentiation capability in vitro. -- Abstract: Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

  2. Determining the Cellular Diversity of Hepatitis C Virus Quasispecies by Single-Cell Viral Sequencing

    PubMed Central

    McLauchlan, John

    2013-01-01

    Single-cell genomics is emerging as an important tool in cellular biology. We describe for the first time a system to investigate RNA virus quasispecies diversity at the cellular level utilizing hepatitis C virus (HCV) replicons. A high-fidelity nested reverse transcription (RT)-PCR assay was developed, and validation using control transcripts of known copy number indicated a detection limit of 3 copies of viral RNA/reaction. This system was used to determine the cellular diversity of subgenomic JFH-1 HCV replicons constitutively expressed in Huh7 cells. Each cell contained a unique quasispecies that was much less diverse than the quasispecies of the bulk cell population from which the single cells were derived, suggesting the occurrence of independent evolution at the cellular level. An assessment of the replicative fitness of the predominant single-cell quasispecies variants indicated a modest reduction in fitness compared to the wild type. Real-time RT-PCR methods capable of determining single-cell viral loads were developed and indicated an average of 113 copies of replicon RNA per cell, correlating with calculated RNA copy numbers in the bulk cell population. This study introduces a single-cell RNA viral-sequencing method with numerous potential applications to explore host-virus interactions during infection. HCV quasispecies diversity varied greatly between cells in vitro, suggesting different within-cell evolutionary pathways. Such divergent trajectories in vivo could have implications for the evolution and establishment of antiviral-resistant variants and host immune escape mutants. PMID:24049174

  3. Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide

    PubMed Central

    Okuyama-Dobashi, Kaori; Kasai, Hirotake; Tanaka, Tomohisa; Yamashita, Atsuya; Yasumoto, Jun; Chen, Wenjia; Okamoto, Toru; Maekawa, Shinya; Watashi, Koichi; Wakita, Takaji; Ryo, Akihide; Suzuki, Tetsuro; Matsuura, Yoshiharu; Enomoto, Nobuyuki; Moriishi, Kohji

    2015-01-01

    Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection. PMID:26592202

  4. Perinatal hepatitis B virus infection caused by antihepatitis Be positive maternal mononuclear cells.

    PubMed

    Shimizu, H; Mitsuda, T; Fujita, S; Yokota, S

    1991-06-01

    To investigate the infectivity of hepatitis B virus (HBV) from mothers to their newborn offspring, HBV-DNA in plasma and peripheral mononuclear cells from 28 antihepatitis Be positive, hepatitis B surface antigen positive carrier mothers was examined by a highly sensitive polymerase chain reaction/Southern hybridisation technique. HBV specific DNA was detected in three maternal mononuclear cell samples, but was absent in plasma. Two of four infants born to the three mothers with HBV-DNA positive mononuclear cells developed acute or fulminant hepatitis within three months after birth. Two infants were effectively prevented from infection with HBV by combined hepatitis B immunoglobulin/HBV vaccine administration. The 25 infants born to the HBV-DNA negative mothers were free of HBV infection within the next seven months to 3.5 years. These results suggest that latent infection with HBV in maternal mononuclear cells is responsible for perinatal HBV infection. PMID:2053794

  5. Giant cell hepatitis associated with systemic lupus erythematosus.

    PubMed Central

    Cairns, A; McMahon, R F

    1996-01-01

    Giant hepatocytes are commonly found in several neonatal and infantile liver diseases, but are rarely found in adult liver disease. A 42 year old white woman presented with a five month history of paraesthesia and numbness of both the upper and lower limbs and with vague abdominal pain. Abnormal liver function was noted on routine screening. Ultrasound scan of the abdomen showed gallstones; barium enema, ERCP and computed tomography scan were all normal. IgG antibodies to double stranded DNA were present at a titre of 40 units. Anti-cardiolipin antibodies, anti-mitochondrial antibodies and rheumatoid factor were not detected. Serology for hepatitis A, B, C, and paramyxoviruses was negative, as was the Paul Bunnell test. A clinical diagnosis of systemic lupus erythematosus (SLE) with an axonal sensory polyneuropathy was made, the latter confirmed on biopsy of the sural nerve. Giant cells were noted on liver biopsy. The patient was treated with corticosteroids; liver function had improved after two years of follow up. When extensive giant cell transformation is noted on liver biopsy, particularly when neuropathy is also a feature, the possibility of an association with SLE should be considered. Images PMID:8655694

  6. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

    NASA Astrophysics Data System (ADS)

    Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

    1995-02-01

    Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

  7. Serum Amyloid A Induces Inflammation, Proliferation and Cell Death in Activated Hepatic Stellate Cells

    PubMed Central

    Siegmund, Sören V.; Schlosser, Monika; Schildberg, Frank A.; Seki, Ekihiro; De Minicis, Samuele; Uchinami, Hiroshi; Kuntzen, Christian; Knolle, Percy A.; Strassburg, Christian P.; Schwabe, Robert F.

    2016-01-01

    Serum amyloid A (SAA) is an evolutionary highly conserved acute phase protein that is predominantly secreted by hepatocytes. However, its role in liver injury and fibrogenesis has not been elucidated so far. In this study, we determined the effects of SAA on hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Serum amyloid A potently activated IκB kinase, c-Jun N-terminal kinase (JNK), Erk and Akt and enhanced NF-κB-dependent luciferase activity in primary human and rat HSCs. Serum amyloid A induced the transcription of MCP-1, RANTES and MMP9 in an NF-κB- and JNK-dependent manner. Blockade of NF-κB revealed cytotoxic effects of SAA in primary HSCs with signs of apoptosis such as caspase 3 and PARP cleavage and Annexin V staining. Serum amyloid A induced HSC proliferation, which depended on JNK, Erk and Akt activity. In primary hepatocytes, SAA also activated MAP kinases, but did not induce relevant cell death after NF-κB inhibition. In two models of hepatic fibrogenesis, CCl4 treatment and bile duct ligation, hepatic mRNA levels of SAA1 and SAA3 were strongly increased. In conclusion, SAA may modulate fibrogenic responses in the liver in a positive and negative fashion by inducing inflammation, proliferation and cell death in HSCs. PMID:26937641

  8. Serum Amyloid A Induces Inflammation, Proliferation and Cell Death in Activated Hepatic Stellate Cells.

    PubMed

    Siegmund, Sören V; Schlosser, Monika; Schildberg, Frank A; Seki, Ekihiro; De Minicis, Samuele; Uchinami, Hiroshi; Kuntzen, Christian; Knolle, Percy A; Strassburg, Christian P; Schwabe, Robert F

    2016-01-01

    Serum amyloid A (SAA) is an evolutionary highly conserved acute phase protein that is predominantly secreted by hepatocytes. However, its role in liver injury and fibrogenesis has not been elucidated so far. In this study, we determined the effects of SAA on hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Serum amyloid A potently activated IκB kinase, c-Jun N-terminal kinase (JNK), Erk and Akt and enhanced NF-κB-dependent luciferase activity in primary human and rat HSCs. Serum amyloid A induced the transcription of MCP-1, RANTES and MMP9 in an NF-κB- and JNK-dependent manner. Blockade of NF-κB revealed cytotoxic effects of SAA in primary HSCs with signs of apoptosis such as caspase 3 and PARP cleavage and Annexin V staining. Serum amyloid A induced HSC proliferation, which depended on JNK, Erk and Akt activity. In primary hepatocytes, SAA also activated MAP kinases, but did not induce relevant cell death after NF-κB inhibition. In two models of hepatic fibrogenesis, CCl4 treatment and bile duct ligation, hepatic mRNA levels of SAA1 and SAA3 were strongly increased. In conclusion, SAA may modulate fibrogenic responses in the liver in a positive and negative fashion by inducing inflammation, proliferation and cell death in HSCs. PMID:26937641

  9. Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines.

    PubMed

    Ghosheh, Nidal; Olsson, Björn; Edsbagge, Josefina; Küppers-Munther, Barbara; Van Giezen, Mariska; Asplund, Annika; Andersson, Tommy B; Björquist, Petter; Carén, Helena; Simonsson, Stina; Sartipy, Peter; Synnergren, Jane

    2016-01-01

    Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models. PMID:26949401

  10. Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines

    PubMed Central

    Ghosheh, Nidal; Olsson, Björn; Edsbagge, Josefina; Küppers-Munther, Barbara; Van Giezen, Mariska; Asplund, Annika; Andersson, Tommy B.; Björquist, Petter; Carén, Helena; Simonsson, Stina; Sartipy, Peter; Synnergren, Jane

    2016-01-01

    Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models. PMID:26949401

  11. The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets.

    PubMed

    Nagamoto, Yasuhito; Tashiro, Katsuhisa; Takayama, Kazuo; Ohashi, Kazuo; Kawabata, Kenji; Sakurai, Fuminori; Tachibana, Masashi; Hayakawa, Takao; Furue, Miho Kusuda; Mizuguchi, Hiroyuki

    2012-06-01

    Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1α. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening. PMID:22445253

  12. Evaluation of a new standardized enzymatic isolation protocol for human umbilical cord-derived stem cells.

    PubMed

    Buyl, Karolien; Vanhaecke, Tamara; Desmae, Terry; Lagneaux, Laurence; Rogiers, Vera; Najar, Mehdi; De Kock, Joery

    2015-09-01

    The umbilical cord (UC) represents an important source of mesenchymal stem cells (MSC). These human UC-derived MSC (UC-MSC) have already been isolated using a protocol based on the migratory and plastic adhesive properties of MSC (UC-MSC-Mig). The UC-MSC-Mig isolation method, however, is difficult to standardize. Therefore, we developed an enzymatic isolation protocol (UC-MSC-Enz) to overcome the above mentioned disadvantages. First, we investigated the UC-MSC-Enz for their MSC properties. We found that UC-MSC-Enz express the MSC markers CD73, CD90 and CD105 and are able to differentiate into osteoblasts, adipocytes and chondroblasts fulfilling the MSC criteria of the International Society for Cellular Therapy. Previously we found that UC-MSC-Mig are unique among MSCs due to their significant expression of several hepatic (progenitor) markers. Therefore, we also investigated the expression of hepatic transcription factors and other hepatic markers in UC-MSC-Enz at both the mRNA and protein level. We found that the expression of hepatic transcription factors (GATA4, GATA6, SOX9 and SOX17) and hepatic markers (AFP, DPP4, CX43, DKK1, DSG2, KRT18 and KRT19) in UC-MSC-Enz was not significantly different from those of UC-MSC-Mig. Consequently, this optimized enzyme-based method represents a fast, robust and standardized way to isolate UC-MSC for a broad range of applications. PMID:25541070

  13. Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

    PubMed Central

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L.; Wolf, Don; Mitalipov, Shoukhrat

    2013-01-01

    SUMMARY Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state. PMID:23683578

  14. CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.

    PubMed Central

    Saile, B.; Knittel, T.; Matthes, N.; Schott, P.; Ramadori, G.

    1997-01-01

    During liver tissue repair, hepatic stellate cells (HSC), a pericyte-like mesenchymal liver cell population, transform from a "quiescent" status ("resting" HSC) into myofibroblast-like cells ("activated" HSC) with the latter representing the principle matrix synthesizing cell of the liver. Presently, the mechanisms that terminate HSC cell proliferation when tissue repair is concluded are poorly understood. Controlled cell death known as apoptosis could be a mechanism underlying this phenomenon. Therefore, apoptosis and its regulation were studied in HSC using an in vitro and in vivo approach. Spontaneous apoptosis became detectable in parallel with HSC activation because resting cells (2 days after isolation) displayed no sign of apoptosis, whereas apoptosis was present in 8% (+/- 5%) of "transitional" cells (day 4) and in 18% (+/- 8%) of fully activated cells (day 7). Both CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) became increasingly expressed during the course of activation. Apoptosis could be fully blocked by CD95-blocking antibodies in normal cells and HSC already entering the apoptotic cycle. Using CD95-activating antibodies, transition of more than 95% cells into apoptosis was evident at each activation step. The apoptosis-regulating proteins Bcl-2 and p53 could not be detected in resting cells but were found in increasing amounts at days 4 and 7 of cultivation. Whereas p53 expression was induced by the CD95-activating antibody, no change was inducible in Bcl-2 expression. The Bcl-2-related protein bax could be found at days 2 and 4 in similar expression, was considerably up-regulated at day 7, but was not regulated by CD95-agonistic antibodies. In vivo, acute tissue damage was first accompanied by activation and proliferation of HSC displaying no sign of apoptosis. In the recovery phase, apoptotic HSC were detectable in parallel to a reduction in the total number of HSC present in the liver tissue. The data demonstrate that apoptosis becomes detectable

  15. Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

    PubMed Central

    Takahara, Yoshiyuki; Takahashi, Mitsuo; Wagatsuma, Hiroki; Yokoya, Fumihiko; Zhang, Qing-Wei; Yamaguchi, Mutsuyo; Aburatani, Hiroyuki; Kawada, Norifumi

    2006-01-01

    AIM: To determine the gene expression profile data for the whole liver during development of dimethylni-trosamine (DMN)-induced hepatic fibrosis. METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells), and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells. RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSC-specific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis, suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocyte-specific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis. CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis. PMID:17072980

  16. Bile acids are toxic for isolated cardiac mitochondria: a possible cause for hepatic-derived cardiomyopathies?

    PubMed

    Ferreira, Manuela; Coxito, Pedro M; Sardão, Vilma A; Palmeira, Carlos M; Oliveira, Paulo J

    2005-01-01

    Cholestasis and other liver diseases may affect the heart through the toxic effects of the retained bile acids on cardiac mitochondria, which could explain the origin of hepatic-derived cardiomyopathies. The objective of this work was to test the hypothesis that bile acids are toxic to heart mitochondria for concentrations that are relevant for cholestasis. Heart mitochondria were isolated from rat and subjected to incubation with selected bile acids (litocholic acid [LCA], deoxycholic acid [DCA], chenodeoxycholic acid [CDCA], glycochenodeoxycholic acid [GCDC], taurodeoxycholic acid [TDCA], and glycoursodeoxycholic acid [GUDC]). We observed that the most toxic bile acids were also the most lipophilic ones (LCA, DCA, and CDCA), inducing a decrease on state 3 respiration, respiratory control ratio, and membrane potential and causing the induction of the mitochondrial permeability transition. GUDC was the bile acid with lower indexes of toxicity on isolated heart mitochondria. The results of this research indicate that at toxicologically relevant concentrations, most bile acids (mainly the most lipophilic) alter mitochondrial bioenergetics. The impairment of cardiac mitochondrial function may be an important cause for the observed cardiac alterations during cholestasis. PMID:15738586

  17. Cadmium and mercury toxicity in a human fetal hepatic cell line (WRL-68 cells).

    PubMed

    Bucio, L; Souza, V; Albores, A; Sierra, A; Chávez, E; Cárabez, A; Gutiérrez-Ruiz, M C

    1995-09-18

    The toxic effects of cadmium (Cd) and mercury (Hg), as chloride salts, were studied using an hepatic human fetal cell line (WRL-68 cells). From viability curves and the proliferative capacity of the cell in the presence of the metal, three different cell treatments were chosen, (1) 0.5 microM of the metal chloride for 24 h (acute low dose treatment), (2) 0.5 microM of the metal chloride for 7 days (chronic treatment), and (3) 5 microM of the metal chloride for 24 h (acute high dose treatment). WRL-68 cells grown in the presence of Cd exhibited the same proliferative curve as control cells, whereas in the case of Hg, the cells increased their proliferative capacity. Both metals produced ultrastructural alterations in different degrees, mainly observed as mitochondrial and RER structural changes, depending of the treatment and concentration of the metal used. Cytotoxicity was assessed by measuring the release of lactate dehydrogenase from the cells. Acutely high dose-treated cells showed the highest value for this parameter, and Cd-treated cells presented higher lactate dehydrogenase release than the Hg-treated ones. Cell damage was also measured by alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities. Acute high dose Cd treatment caused the highest value of enzymatic release. Lipid peroxidation was significantly different with respect to control cells in chronic and acute high dose treatments with both metals. Metallothionein (MT) induction in response to Hg treatment was not detected. However, a dramatic induction of this protein occurred in Cd-treated cells. WRL-68 cells differentially respond to Cd and Hg making this hepatic fetal human cell line a useful tool in investigating the mechanism of toxicity of these heavy metals. PMID:7482568

  18. Cell-Free Replication of the Hepatitis C Virus Subgenomic Replicon

    PubMed Central

    Ali, Naushad; Tardif, Keith D.; Siddiqui, Aleem

    2002-01-01

    The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5′ noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3′ NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [α32P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents. PMID:12414942

  19. Hepatic Stellate Cells and microRNAs in Pathogenesis of Liver Fibrosis

    PubMed Central

    Kitano, Mio; Bloomston, P. Mark

    2016-01-01

    microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by either blocking translation or inducing degradation of target mRNA. miRNAs play essential roles in diverse biological and pathological processes, including development of hepatic fibrosis. Hepatic stellate cells (HSCs) play a central role in development of hepatic fibrosis and there are intricate regulatory effects of miRNAs on their activation, proliferation, collagen production, migration, and apoptosis. There are multiple differentially expressed miRNAs in activated HSCs, and in this review we aim to summarize current data on miRNAs that participate in the development of hepatic fibrosis. Based on this review, miRNAs may serve as biomarkers for diagnosis of liver disease, as well as markers of disease progression. Most importantly, dysregulated miRNAs may potentially be targeted by novel therapies to treat and reverse progression of hepatic fibrosis. PMID:26999230

  20. Reconstruction of hepatic stellate cell-incorporated liver capillary structures in small hepatocyte tri-culture using microporous membranes.

    PubMed

    Kasuya, Junichi; Sudo, Ryo; Masuda, Genta; Mitaka, Toshihiro; Ikeda, Mariko; Tanishita, Kazuo

    2015-03-01

    In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC-incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri-culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell-cell interactions, which are essential to form HSC-EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d, l-lactide-co-glycolide) (PLGA) microporous membranes in SH-HSC-EC tri-culture to reconstruct the HSC-incorporated liver capillary structures in vitro. First, the formation of EC capillary-like structures was induced on Matrigel-coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary-like structures. This process was mediated in part by platelet-derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary-like structures with their long cytoplasmic processes. In the HSC-incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro. PMID:23086892

  1. An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

    PubMed Central

    Qian, Hui; Deng, Xing; Huang, Zhao-Wei; Wei, Ji; Ding, Chen-Hong; Feng, Ren-Xin; Zeng, Xin; Chen, Yue-Xiang; Ding, Jin; Qiu, Lei; Hu, Zhen-Lin; Zhang, Xin; Wang, Hong-Yang; Zhang, Jun-Ping; Xie, Wei-Fen

    2015-01-01

    Hepatocytes are critical for the maintenance of liver homeostasis, but its involvement in hepatic fibrogenesis remains elusive. Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor that plays a key role in hepatocyte function. Our previous study revealed a significant inhibitory effect of HNF1α on hepatocellular carcinoma. In this study, we report that the expression of HNF1α is significantly repressed in both human and rat fibrotic liver. Knockdown of HNF1α in the liver significantly aggravates hepatic fibrogenesis in either dimethylnitrosamine (DMN) or bile duct ligation (BDL) model in rats. In contrast, forced expression of HNF1α markedly alleviates hepatic fibrosis. HNF1α regulates the transcriptional expression of SH2 domain-containing phosphatase-1 (SHP-1) via directly binding to SHP-1 promoter in hepatocytes. Inhibition of SHP-1 expression abrogates the anti-fibrotic effect of HNF1α in DMN-treated rats. Moreover, HNF1α repression in primary hepatocytes leads to the activation of NF-κB and JAK/STAT pathways and initiates an inflammatory feedback circuit consisting of HNF1α, SHP-1, STAT3, p65, miR-21 and miR-146a, which sustains the deregulation of HNF1α in hepatocytes. More interestingly, a coordinated crosstalk between hepatocytes and hepatic stellate cells (HSCs) participates in this positive feedback circuit and facilitates the progression of hepatocellular damage. Our findings demonstrate that impaired hepatocytes play an active role in hepatic fibrogenesis. Early intervention of HNF1α-regulated inflammatory feedback loop in hepatocytes may have beneficial effects in the treatment of chronic liver diseases. PMID:26169608

  2. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    PubMed

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes. PMID:25751085

  3. Endothelial cells derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  4. Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis

    PubMed Central

    Blaya, Delia; Morales-Ibanez, Oriol; Coll, Mar; Millán, Cristina; Altamirano, José; Arroyo, Vicente; Caballería, Joan; Bataller, Ramón; Ginès, Pere; Sancho-Bru, Pau

    2015-01-01

    Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis. PMID:26691857

  5. Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

    PubMed Central

    Sautto, Giuseppe A; Wisskirchen, Karin; Clementi, Nicola; Castelli, Matteo; Diotti, Roberta A; Graf, Julia; Clementi, Massimo; Burioni, Roberto; Protzer, Ulrike; Mancini, Nicasio

    2016-01-01

    Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool. PMID:25661083

  6. Follicular cell-derived thyroid cancer.

    PubMed

    Dralle, Henning; Machens, Andreas; Basa, Johanna; Fatourechi, Vahab; Franceschi, Silvia; Hay, Ian D; Nikiforov, Yuri E; Pacini, Furio; Pasieka, Janice L; Sherman, Steven I

    2015-01-01

    Follicular cell-derived thyroid cancers are derived from the follicular cells in the thyroid gland, which secrete the iodine-containing thyroid hormones. Follicular cell-derived thyroid cancers can be classified into papillary thyroid cancer (80-85%), follicular thyroid cancer (10-15%), poorly differentiated thyroid cancer (<2%) and undifferentiated (anaplastic) thyroid cancer (<2%), and these have an excellent prognosis with the exception of undifferentiated thyroid cancer. The advent and expansion of advanced diagnostic techniques has driven and continues to drive the epidemic of occult papillary thyroid cancer, owing to overdiagnosis of clinically irrelevant nodules. This transformation of the thyroid cancer landscape at molecular and clinical levels calls for the modification of management strategies towards personalized medicine based on individual risk assessment to deliver the most effective but least aggressive treatment. In thyroid cancer surgery, for instance, injuries to structures outside the thyroid gland, such as the recurrent laryngeal nerve in 2-5% of surgeries or the parathyroid glands in 5-10% of surgeries, negatively affect quality of life more than loss of the expendable thyroid gland. Furthermore, the risks associated with radioiodine ablation may outweigh the risks of persistent or recurrent disease and disease-specific mortality. Improvement in the health-related quality of life of survivors of follicular cell-derived thyroid cancer, which is decreased despite the generally favourable outcome, hinges on early tumour detection and minimization of treatment-related sequelae. Future opportunities include more widespread adoption of molecular and clinical risk stratification and identification of actionable targets for individualized therapies. PMID:27188261

  7. Risk factors and prognosis of hepatic acute GvHD after allogeneic hematopoietic cell transplantation.

    PubMed

    Arai, Y; Kanda, J; Nakasone, H; Kondo, T; Uchida, N; Fukuda, T; Ohashi, K; Kaida, K; Iwato, K; Eto, T; Kanda, Y; Nakamae, H; Nagamura-Inoue, T; Morishima, Y; Hirokawa, M; Atsuta, Y; Murata, M

    2016-01-01

    Hepatic acute GvHD (aGvHD) is associated with high mortality owing to poor response to immunosuppressive therapy. The pathogenesis of hepatic aGvHD differs from that of other lesions, and specific risk factors related to pre-transplant liver conditions should be determined. We conducted a cohort study by using a Japanese transplant registry database (N=8378). Of these subjects, 1.5% had hepatitis C virus Ab (HCV-Ab) and 9.4% had liver dysfunction (elevated transaminase or bilirubin levels) before hematopoietic cell transplantation (HCT). After HCT, the cumulative incidence of hepatic aGvHD was 6.7%. On multivariate analyses, HCV-Ab positivity (hazard ratio (HR), 1.93; P=0.02) and pre-transplant liver dysfunction (HR, 1.85; P<0.01), as well as advanced HCT risk, unrelated donors, HLA mismatch and cyclosporine as GvHD prophylaxis, were significant risk factors for hepatic aGvHD, whereas hepatitis B virus surface Ag was not. Hepatic aGvHD was a significant risk factor for low overall survival and high transplant-related mortality in all aGvHD grades (P<0.01). This study is the first to show the relationship between pre-transplant liver conditions and hepatic aGvHD. A prospective study is awaited to validate the results of this study and establish a new strategy especially for high-risk patients. PMID:26367230

  8. Cadmium uptake by a human hepatic cell line (WRL-68 cells).

    PubMed

    Souza, V; Bucio, L; Gutiérrez-Ruiz, M C

    1997-07-11

    A hepatic human cell line (WRL-68 cells) was employed to investigate the uptake of the toxic heavy metal cadmium. Cd accumulation in WRL-68 cells is a time-, temperature- and concentration-dependent process. A rapid initial phase of uptake was followed by a second slower phase. The transport does not require energy and 55% of Cd transport occurs by temperature-insensitive processes, possibly by diffusion. The rest of Cd transport (45%) occurs by temperature-sensitive processes, probably ion channels and carriers, that involve interaction with sulfhydryl groups. The calcium channel blockers nifedipine and verapamil inhibit the uptake of cadmium, with an inhibition of 35% after 30 min incubation with 100 microM verapamil and 10 microM Cd. These data suggest that about one third of the Cd enters WRL-68 cells through the calcium channels. The toxic metals appear to use the transport pathways that exist for biologically essential metals. Our results in human hepatic cells are very similar to those reported in cultured rat hepatocytes. It appears that transport pathways available for Cd uptake are similar and independent of the species of hepatocyte origin. Moreover, the WRL-68 cell line seems to be an excellent in vitro model to study the mechanism of cell damage due to Cd. PMID:9217308

  9. Hepatic Tumor Metastases Cause Enhanced PEGylated Liposome Uptake by Kupffer Cells.

    PubMed

    Ukawa, Masami; Fujiwara, Yukako; Ando, Hidenori; Shimizu, Taro; Ishida, Tatsuhiro

    2016-01-01

    Kupffer cells in livers bearing tumor metastases were found to have promoted tumor invasion and exacerbated the metastasis. This implies that the function of Kupffer cells might differ between animals bearing hepatic metastases and those that are healthy. Kupffer cells are considered responsible for the accumulation of liposomes in the liver. In this study, we hypothesized that the alteration in the function of Kupffer cells by hepatic metastasis would also affect the biodistribution of liposomes following intravenous administration. The hepatic accumulation and the blood concentration of PEGylated liposomes were compared between healthy mice and tumor-bearing mice. We noted that hepatic accumulation and elimination from the blood were significantly accelerated in tumor-bearing mice, indicating that our hypothesis was correct. In the tumor-bearing mice, the proportion of Kupffer cells taking up liposomes was significantly increased. Intravenous injection of oxaliplatin (l-OHP) containing PEGylated liposomes decreased the fraction of Kupffer cells, but this administration caused no injury to the hepatocytes. These results suggest that PEGylated liposomes containing l-OHP may have the potential to treat metastatic hepatic cancer-not only via the direct killing of the cancer cells but also via a reduction in tumor-supportive Kupffer cells. PMID:26830481

  10. Differential effect of p7 inhibitors on hepatitis C virus cell-to-cell transmission☆

    PubMed Central

    Meredith, L.W.; Zitzmann, N.; McKeating, J.A.

    2013-01-01

    Inhibitors targeting the hepatitis C virus (HCV) encoded viroporin, p7 prevent virus release in vitro. HCV can transmit by cell-free particle infection of new target cells and via cell-to-cell dependent contact with limited exposure to the extracellular environment. The role of assembly inhibitors in preventing HCV transmission via these pathways has not been studied. We compared the efficacy of three published p7 inhibitors to inhibit cell-free and cell-to-cell transmission of two chimeric HCV strains encoding genotype 2 (GT2) or 5 (GT5) p7 using a recently developed single cycle co-culture assay. The inhibitors reduced the infectivity of extracellular GT2 and GT5 virus by 80–90% and GT2 virus cell-to-cell transmission by 50%. However, all of the p7 inhibitors had minimal effect on GT5 cell contact dependent transmission. Screening a wider panel of diverse viral genotypes demonstrated that p7 viroporin inhibitors were significantly more effective at blocking cell-free virus than cell-to-cell transmission. These results suggest an altered assembly or trafficking of cell-to-cell transmitted compared to secreted virus. These observations have important implications for the validation, therapeutic design and testing of HCV assembly inhibitors. PMID:24157306

  11. Three Peptides from Soy Glycinin Modulate Glucose Metabolism in Human Hepatic HepG2 Cells.

    PubMed

    Lammi, Carmen; Zanoni, Chiara; Arnoldi, Anna

    2015-01-01

    Ile-Ala-Val-Pro-Gly-Glu-Val-Ala (IAVPGEVA), Ile-Ala-Val-Pro-Thr-Gly-Val-Ala (IAVPTGVA) and Leu-Pro-Tyr-Pro (LPYP), three peptides deriving from soy glycinin hydrolysis, are known to regulate cholesterol metabolism in human hepatic HepG2 cells. We have recently demonstrated that the mechanism of action involves the activation of adenosine monophosphate-activated protein kinase (AMPK). This fact suggested a potential activity of the same peptides on glucose metabolism that prompted us to also investigate this aspect in the same cells. After treatment with IAVPGEVA, IAVPTGVA and LPYP, HepG2 cells were analyzed using a combination of molecular techniques, including western blot analysis, glucose uptake experiments and fluorescence microscopy evaluation. The results showed that these peptides are indeed able to enhance the capacity of HepG2 cells to uptake glucose, via glucose transporter 1 GLUT1 and glucose transporter 4 GLUT4 activation, through the stimulation of protein kinase B Akt and adenosine monophosphate-activated protein kinase AMPK pathways, both involved in glucose metabolism. PMID:26580610

  12. Three Peptides from Soy Glycinin Modulate Glucose Metabolism in Human Hepatic HepG2 Cells

    PubMed Central

    Lammi, Carmen; Zanoni, Chiara; Arnoldi, Anna

    2015-01-01

    Ile-Ala-Val-Pro-Gly-Glu-Val-Ala (IAVPGEVA), Ile-Ala-Val-Pro-Thr-Gly-Val-Ala (IAVPTGVA) and Leu-Pro-Tyr-Pro (LPYP), three peptides deriving from soy glycinin hydrolysis, are known to regulate cholesterol metabolism in human hepatic HepG2 cells. We have recently demonstrated that the mechanism of action involves the activation of adenosine monophosphate-activated protein kinase (AMPK). This fact suggested a potential activity of the same peptides on glucose metabolism that prompted us to also investigate this aspect in the same cells. After treatment with IAVPGEVA, IAVPTGVA and LPYP, HepG2 cells were analyzed using a combination of molecular techniques, including western blot analysis, glucose uptake experiments and fluorescence microscopy evaluation. The results showed that these peptides are indeed able to enhance the capacity of HepG2 cells to uptake glucose, via glucose transporter 1 GLUT1 and glucose transporter 4 GLUT4 activation, through the stimulation of protein kinase B Akt and adenosine monophosphate-activated protein kinase AMPK pathways, both involved in glucose metabolism. PMID:26580610

  13. Immunogenicity of umbilical cord tissue derived cells.

    PubMed

    Cho, Patricia S; Messina, Darin J; Hirsh, Erica L; Chi, Nina; Goldman, Stephanie N; Lo, Diana P; Harris, Ian R; Popma, Sicco H; Sachs, David H; Huang, Christene A

    2008-01-01

    Umbilical cord tissue provides a unique source of cells with potential for tissue repair. Umbilical cord tissue-derived cells (UTCs) are MHC class I (MHCI) dull and negative for MHC class II (MHCII), but can be activated to increase MHCI and to express MHCII with IFN-gamma stimulation. Mesenchymal stem cells with similar characteristics have been inferred to be nonimmunogenic; however, in most cases, immunogenicity was not directly assessed. Using UTC from Massachusetts General Hospital MHC-defined miniature swine, we assessed immunogenicity across a full MHC barrier. Immunogenicity was assessed by in vitro assays including mixed lymphocyte reaction (MLR) and flow cytometry to detect serum alloantibody. A single injection of MHC-mismatched unactivated UTCs did not induce a detectable immune response. When injected in an inflamed region, injected repeatedly in the same region or stimulated with IFN-gamma prior to injection, UTCs were immunogenic. As clinical cellular repair strategies may involve injection of allogeneic cells into inflamed regions of damaged tissue or repeated doses of cells to achieve the desired benefit, our results on the immunogenicity of these cells in these circumstances may have important implications for optimal success and functional improvement for this cellular treatment strategy for diseased tissues. PMID:17909081

  14. Immortalized human hepatic cell lines for in vitro testing and research purposes

    PubMed Central

    Ramboer, Eva; Vanhaecke, Tamara; Rogiers, Vera; Vinken, Mathieu

    2015-01-01

    Summary The ubiquitous shortage of primary human hepatocytes has urged the scientific community to search for alternative cell sources, such as immortalized hepatic cell lines. Over the years, several human hepatic cell lines have been produced, whether or not using a combination of viral oncogenes and human telomerase reverse transcriptase protein. Conditional approaches for hepatocyte immortalization have also been established and allow generation of growth-controlled cell lines. A variety of immortalized human hepatocytes have already proven useful as tools for liver-based in vitro testing and fundamental research purposes. The present chapter describes currently applied immortalization strategies and provides an overview of the actually available immortalized human hepatic cell lines and their in vitro applications. PMID:26272134

  15. Retrospective study on the impact of hepatitis B and hepatitis C virus infection on hematopoietic stem cell transplantation in Japan.

    PubMed

    Hamaguchi, Motohiro; Yamada, Hironori; Gondo, Hisashi; Takemoto, Yoshinobu; Morishima, Yasuo; Kodera, Yoshihisa

    2002-04-01

    We performed a retrospective survey in 62 hematopoietic cell transplantation (HCT) centers in Japan in which all HCTs performed between 1986 and 1998 were reviewed, and those involving hepatitis B virus surface antigen (HBsAg)-positive donors were identified. One hundred and thirty-five patients who underwent allogeneic HCT (alloHCT) were studied for complications related to hepatitis B virus (HBV) or hepatitis C virus (HCV). The median follow-up period was 24 months. Positivity for HBsAg was observed in 32 patients (24%) throughout the study. Twenty-six of the 32 patients were HBsAg carriers before alloHCT, whereas the remaining 6 became HBsAg(+) after alloHCT. Forty-two recipients were anti-HBs antibody (HBsAb)-positive, and 58 recipients (43%) were HCV Ab(+). Eleven of 26 (42%) HBsAg(+) recipients survived between >4 and >119 months. Six of 26 cases received transplants from HBsAg(+) donors, and, although they had not developed acute graft-versus-host disease, 4 of 6 died of hepatic and renal failure within 10 months after HCT. After transplantation, 5 patients showed serologic evidence of HBV reactivation, whereas 4 patients showed evidence of an immune response to HBV. Viral reactivation occurred during the tapering of the immunosuppressive agent. However, 3 of 5 were alive at the time of this report, suggesting that reactivation is not directly correlated with severe liver dysfunction. Seventeen patients (13%) of 135 recipients developed hepatic failure. Eight (47%) of 17 were diagnosed with fulminant hepatitis and 5 (29%) with veno-occlusive disease (VOD). VOD was observed in 12% of both HBsAg(+) and HCVAb(+) patients. In this study, the relatively high incidence of HBV events occurred after alloHCT, and, therefore, we should consider a protocol for active immunization of donors and recipients against HBV. Moreover, although the presence of HBV or HCV is not a contraindication for alloHCT, we recommend a careful follow-up of recipients after transplantation

  16. Development of Murine Hepatic NK Cells during Ontogeny: Comparison with Spleen NK Cells

    PubMed Central

    Wu, Xian; Chen, Yongyan; Wei, Haiming; Sun, Rui; Tian, Zhigang

    2012-01-01

    The phenotype of developing liver NK cells (CD3−NK1.1+) was investigated during mouse ontogeny comparing with spleen NK cells. The highest percentage of hepatic CD27−CD11b− NK cells occurred at the fetal stage. After birth, the percentage of CD27−CD11b−NK cells in both the liver and spleen gradually decreased to their lowest level at 6 weeks. More CD27+CD11b−NK cells were detected in the liver than that in spleen from week 1 to 6. Expression of NKG2A on liver NK cells was decreased but still much higher than that of spleen NK cells after 1 week. The NKG2D expression on liver NK cells increased to its highest level and was significantly higher than on spleen NK cells till 4 weeks. During mouse ontogeny, weaker expression of NKp46 and CD2 and stronger expression of CD69, CD11c, 2B4, and CD73 were observed on liver NK cells. Furthermore, neonatal liver NK cells express higher IFN-γ and perforin than adult .These results suggest that the maturation process of NK cells is unique in the livers, and liver microenvironments might play critical roles to keep NK cells in an immature status. PMID:22203859

  17. Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening

    PubMed Central

    Baxter, Melissa A.; Rowe, Cliff; Alder, Jane; Harrison, Sean; Hanley, Karen Piper; Park, B. Kevin; Kitteringham, Neil R.; Goldring, Chris E.; Hanley, Neil A.

    2012-01-01

    Hepatotoxicity is an enormous and increasing problem for the pharmaceutical industry. Early detection of problems during the drug discovery pathway is advantageous to minimize costs and improve patient safety. However, current cellular models are sub-optimal. This review addresses the potential use of pluripotent stem cells in the generation of hepatic cell lineages. It begins by highlighting the scale of the problem faced by the pharmaceutical industry, the precise nature of drug-induced liver injury and where in the drug discovery pathway the need for additional cell models arises. Current research is discussed, mainly for generating hepatocyte-like cells rather than other liver cell-types. In addition, an effort is made to identify where some of the major barriers remain in translating what is currently hypothesis-driven laboratory research into meaningful platform technologies for the pharmaceutical industry. PMID:20483202

  18. Matrix Metalloproteinase-3 in Odontoblastic Cells Derived from Ips Cells: Unique Proliferation Response as Odontoblastic Cells Derived from ES Cells

    PubMed Central

    Hiyama, Taiki; Ozeki, Nobuaki; Mogi, Makio; Yamaguchi, Hideyuki; Kawai, Rie; Nakata, Kazuhiko; Kondo, Ayami; Nakamura, Hiroshi

    2013-01-01

    We previously reported that matrix metalloproteinase (MMP)-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL)-1β and interferon-γ) induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES) cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS) and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (P<0.05). Exogenous MMP-3 was found to induce cell proliferation in odontoblast-like cells derived from iPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P<0.05). Taken

  19. Hepatitis E virus derived from different sources exhibits different behaviour in virus inactivation and/or removal studies with plasma derivatives.

    PubMed

    Yunoki, Mikihiro; Tanaka, Hiroyuki; Takahashi, Kadue; Urayama, Takeru; Hattori, Shinji; Ideno, Shoji; Furuki, Rie; Sakai, Kaoru; Hagiwara, Katsuro; Ikuta, Kazuyoshi

    2016-09-01

    Hepatitis E virus (HEV) causes viral hepatitis, and is considered a risk factor for blood products. Although some HEV inactivation/removal studies have been reported, detailed investigations of different manufacturing steps as heat treatment, partitioning during cold ethanol fractionation, low pH treatment, and virus filtration have yet to be reported for plasma-derived medicinal products. In this study, human serum- and swine faeces-derived HEVs, with and without detergent treatment, were used. The kinetic patterns of inactivation, log reduction value, or partitioning during the process were evaluated. In addition, the mouse encephalomyocarditis virus (EMCV) and canine and porcine parvoviruses (CPV/PPV) were also evaluated as model viruses for HEV. Small pore size (19 or 15 nm) virus filtration demonstrated effective removal of HEV. Middle pore size (35 nm) virus filtration and 60 °C liquid heating demonstrated moderate inactivation/removal. Ethanol fractionation steps demonstrated limited removal of HEV. Unpurified HEV exhibited different properties than the detergent-treated HEV, and both forms displayed differences when compared with EMCV, CPV, and PPV. Limited or no inactivation of HEV was observed during low pH treatment. Untreated plasma-derived HEV from humans showed different properties compared to that of HEV treated with detergent or derived from swine faeces. Therefore, HEV spike preparation requires more attention. PMID:27461242

  20. Cell-derived microparticles and the lung.

    PubMed

    Nieri, Dario; Neri, Tommaso; Petrini, Silvia; Vagaggini, Barbara; Paggiaro, Pierluigi; Celi, Alessandro

    2016-09-01

    Cell-derived microparticles are small (0.1-1 μm) vesicles shed by most eukaryotic cells upon activation or during apoptosis. Microparticles carry on their surface, and enclose within their cytoplasm, molecules derived from the parental cell, including proteins, DNA, RNA, microRNA and phospholipids. Microparticles are now considered functional units that represent a disseminated storage pool of bioactive effectors and participate both in the maintenance of homeostasis and in the pathogenesis of diseases. The mechanisms involved in microparticle generation include intracellular calcium mobilisation, cytoskeleton rearrangement, kinase phosphorylation and activation of the nuclear factor-κB. The role of microparticles in blood coagulation and inflammation, including airway inflammation, is well established in in vitro and animal models. The role of microparticles in human pulmonary diseases, both as pathogenic determinants and biomarkers, is being actively investigated. Microparticles of endothelial origin, suggestive of apoptosis, have been demonstrated in the peripheral blood of patients with emphysema, lending support to the hypothesis that endothelial dysfunction and apoptosis are involved in the pathogenesis of the disease and represent a link with cardiovascular comorbidities. Microparticles also have potential roles in patients with asthma, diffuse parenchymal lung disease, thromboembolism, lung cancer and pulmonary arterial hypertension. PMID:27581826

  1. Alginate Hydrogel Protects Encapsulated Hepatic HuH-7 Cells against Hepatitis C Virus and Other Viral Infections

    PubMed Central

    Tran, Nhu-Mai; Dufresne, Murielle; Helle, François; Hoffmann, Thomas Walter; François, Catherine; Brochot, Etienne; Paullier, Patrick; Legallais, Cécile; Duverlie, Gilles; Castelain, Sandrine

    2014-01-01

    Cell microencapsulation in alginate hydrogel has shown interesting applications in regenerative medicine and the biomedical field through implantation of encapsulated tissue or for bioartificial organ development. Although alginate solution is known to have low antiviral activity, the same property regarding alginate gel has not yet been studied. The aim of this work is to investigate the potential protective effect of alginate encapsulation against hepatitis C virus (HCV) infection for a hepatic cell line (HuH-7) normally permissive to the virus. Our results showed that alginate hydrogel protects HuH-7 cells against HCV when the supernatant was loaded with HCV. In addition, alginate hydrogel blocked HCV particle release out of the beads when the HuH-7 cells were previously infected and encapsulated. There was evidence of interaction between the molecules of alginate hydrogel and HCV, which was dose- and incubation time-dependent. The protective efficiency of alginate hydrogel towards HCV infection was confirmed against a variety of viruses, whether or not they were enveloped. This promising interaction between an alginate matrix and viruses, whose chemical mechanisms are discussed, is of great interest for further medical therapeutic applications based on tissue engineering. PMID:25310111

  2. New naphthoquinone derivatives against glioma cells.

    PubMed

    Redaelli, Marco; Mucignat-Caretta, Carla; Isse, Abdirisak Ahmed; Gennaro, Armando; Pezzani, Raffaele; Pasquale, Riccardo; Pavan, Valeria; Crisma, Marco; Ribaudo, Giovanni; Zagotto, Giuseppe

    2015-01-01

    This work was aimed to the development of a set of new naphtoquinone derivatives that can act against glioma. The compounds were tested in order to find out their ability to inhibit the growth of glioma cells, and the results of these assays were correlated with electrochemical analysis and NMR-based reoxidation kinetic studies, suggesting that a redox mechanism underlies and may explain the observed biological behavior. In addition to a full description of the synthetic pathways, electrochemistry, NMR and single crystal X-ray diffraction data are provided. PMID:25916907

  3. Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line.

    PubMed

    Miyamae, Yusaku; Nishito, Yukina; Nakai, Naomi; Nagumo, Yoko; Usui, Takeo; Masuda, Seiji; Kambe, Taiho; Nagao, Masaya

    2016-08-12

    Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. PMID:27270032

  4. Back to the drawing board: Understanding the complexity of hepatic innate lymphoid cells.

    PubMed

    Marotel, Marie; Hasan, Uzma; Viel, Sébastien; Marçais, Antoine; Walzer, Thierry

    2016-09-01

    Recent studies of immune populations in nonlymphoid organs have highlighted the great diversity of the innate lymphoid system. It has also become apparent that mouse and human innate lymphoid cells (ILCs) have distinct phenotypes and properties. In this issue of the European Journal of Immunology, Harmon et al. [Eur. J. Immunol. 2016. 46: 2111-2120] characterized human hepatic NK-cell subsets. The authors report that hepatic CD56(bright) NK cells resemble mouse liver ILC1s in that they express CXCR6 and have an immature phenotype. However, unlike mouse ILC1s, they express high levels of Eomes and low levels of T-bet, and upon stimulation with tumor cells, secrete low amounts of cytokines. These unexpected findings further support the differences between human and mouse immune populations and prompt the study of the role of hepatic ILC subsets in immune responses. PMID:27600673

  5. Stromal cell-derived factor-1 (SDF-1) as a target in liver diseases.

    PubMed

    Liepelt, Anke; Tacke, Frank

    2016-08-01

    The chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is constitutively expressed in healthy liver. However, its expression increases following acute or chronic liver injury. Liver sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC), and malignant hepatocytes are important sources of SDF-1/CXCL12 in liver diseases. CXCL12 is able to activate two chemokine receptors with different downstream signaling pathways, CXCR4 and CXCR7. CXCR7 expression is relevant on LSEC, while HSC, mesenchymal stem cells, and tumor cells mainly respond via CXCR4. Here, we summarize recent developments in the field of liver diseases involving this chemokine and its receptors. SDF-1-dependent signaling contributes to modulating acute liver injury and subsequent tissue regeneration. By activating HSC and recruiting mesenchymal cells from bone marrow, CXCL12 can promote liver fibrosis progression, while CXCL12-CXCR7 interactions endorse proregenerative responses in chronic injury. Moreover, the SDF-1 pathway is linked to development of hepatocellular carcinoma (HCC) by promoting tumor growth, angiogenesis, and HCC metastasis. High hepatic CXCR4 expression has been suggested as a biomarker indicating poor prognosis of HCC patients. Tumor-infiltrating myeloid-derived suppressor cells (MDSC) also express CXCR4 and migrate toward CXCL12. Thus CXCL12 inhibition might not only directly block HCC growth but also modulate the tumor microenvironment (angiogenesis, MDSC), thereby sensitizing HCC patients to conventional or emerging novel cancer therapies (e.g., sorafenib, regorafenib, nivolumab, pembrolizumab). We herein summarize the current knowledge on the complex interplay between CXCL12 and CXCR4/CXCR7 in liver diseases and discuss approaches on the therapeutic targeting of these axes in hepatitis, fibrosis, and liver cancer. PMID:27313175

  6. All-trans and 9-cis retinoic acid alter rat hepatic stellate cell phenotype differentially

    PubMed Central

    Hellemans, K; Grinko, I; Rombouts, K; Schuppan, D; Geerts, A

    1999-01-01

    BACKGROUND—Hepatic stellate cells exert specific functions in the liver: storage of large amounts of retinyl esters, synthesis and breakdown of hepatic extracellular matrix, secretion of a variety of cytokines, and control of the diameter of the sinusoids.
AIMS—To examine the influence of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9RA) on extracellular matrix production and proliferation of activated hepatic stellate cells.
METHODS—Cells were isolated using collagenase/pronase, purified by centrifugation in nycodenz, and cultured for two weeks. At this time point the cells exhibited the activated phenotype. Cells were exposed to various concentrations of ATRA and 9RA. The expression of procollagens I, III, and IV, of fibronectin and of laminin were analysed by immunoprecipitation and northern hybridisation.
RESULTS—ATRA exerted a significant inhibitory effect on the synthesis of procollagens type I, III, and IV, fibronectin, and laminin, but did not influence stellate cell proliferation, whereas 9RA showed a clear but late effect on proliferation. 9RA increased procollagen I mRNA 1.9-fold, but did not affect the expression of other matrix proteins.
CONCLUSION—Results showed that ATRA and 9RA exert different, often contrary effects on activated stellate cells. These observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or in animals subjected to fibrogenic stimuli.


Keywords: hepatic stellate cells; retinoic acid; extracellular matrix proteins; proliferation PMID:10369717

  7. Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells.

    PubMed

    Gentilini, A; Feliers, D; Pinzani, M; Woodruff, K; Abboud, S

    1998-02-01

    Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin-like growth factor-I (IGF-I) and IGF binding protein (IGFBP) activity. IGF-I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF-I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP-1 through IGFBP-6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF-I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP-2 through IGFBP-6 mRNAs and release detectable levels of IGFBP-2 through IGFBP-5. Because IGF-I, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-beta (TGF-beta) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF-I induced IGFBP-3 and IGFBP-5 proteins in a time-dependent manner without an increase in the corresponding mRNAs. IGFBP-4 protein levels decreased in response to IGF-I. TGF-beta stimulated IGFBP-3 mRNA and protein but decreased IGFBP-5 mRNA and protein. In contrast, PDGF-BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP-3 (rhIGFBP-3) was then tested for its effect on IGF-I-induced mitogenesis in HSCs. rhIGFBP-3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner, with a peak effect observed at 25 nM IGFBP-3. Because TGF-beta is highly expressed in cirrhotic liver tissue, we determined whether IGFBP-3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral-induced chronic active hepatitis. In the majority of these samples, IGFBP-3 mRNA was increased compared with normal

  8. Maternal-Derived Hepatitis B Virus e Antigen Alters Macrophage Function in Offspring to Drive Viral Persistence after Vertical Transmission.

    PubMed

    Tian, Yongjun; Kuo, Cheng-Fu; Akbari, Omid; Ou, Jing-Hsiung James

    2016-05-17

    In contrast to horizontal transmission of hepatitis B virus (HBV) between adults, which often leads to self-limited acute infection, vertical transmission of HBV from mother to child often leads to chronic infection. However, the mechanisms linking vertical transmission with chronic infection are not known. We developed a mouse model to study the effect of maternal HBV infection on HBV persistence in offspring and found that HBV carried by the mother impaired CD8(+) T cell responses to HBV in her offspring, resulting in HBV persistence. This impairment of CD8(+) T cell responses was mediated by hepatic macrophages, which were predisposed by maternal HBV e antigen (HBeAg) to support HBV persistence by upregulation of inhibitory ligand PD-L1 and altered polarization upon restimulation with HBeAg. Depletion of hepatic macrophages led to CD8(+) T cell activation and HBV clearance in the offspring, raising the possibility of targeting macrophages to treat chronic HBV patients. PMID:27156385

  9. Generation of a human hepatoma cell line supporting efficient replication of a lamivudine resistant hepatitis B virus.

    PubMed

    Zhang, Yijun; Zhang, Yongmei; Kang, Yaoyue; Wang, Jinyu; Liu, Hongyan; Zhu, Haoxiang; Qin, Yanli; Mao, Richeng; Lin, Xu; Lu, Mengji; Zhang, Jiming

    2014-06-01

    Emergence of lamivudine (LAM) resistance causes treatment failure in patients with chronic hepatitis B and compromise the efficacy of subsequent salvage therapies with other nucleot(s)ide analogs (NAs). Establishment of cell-based assays supporting LAM-resistant hepatitis B virus (HBV) replication will not only provide tools for investigating the replication property, but also screening for antiviral agents efficiently inhibiting the replication of LAM-resistant HBV variants. Accordingly, a human hepatoma (HepG2)-derived cell line was established by stable transfection of a plasmid containing a 1.2 unit length of HBV genome harboring rtL180M and rtM204V mutations that confer LAM resistance. In addition to support efficient viral genome replication, the cell line also produces high levels of HBV virions and subviral particles. As expected, HBV DNA replication in this cell line is completely resistant to lamivudine, but sensitive to adefovir (ADV), entecavir (ETV) and tenofovir (TDF). The cell line is suitable for screening for antiviral agents that inhibit LAM-resistant HBV replication and inhibitors of HBsAg biosynthesis and secretion, which may reduce HBsAg antigenemia and ultimately help to restore host antiviral immune response against HBV and cure chronic HBV infection. PMID:24583110

  10. Mechanisms of liver fibrosis associated with experimental Fasciola hepatica infection: roles of Fas2 proteinase and hepatic stellate cell activation.

    PubMed

    Marcos, Luis A; Terashima, Angélica; Yi, Pedro; Andrade, Roy; Cubero, Francisco J; Albanis, Efsevia; Gotuzzo, Eduardo; Espinoza, Jose R; Friedman, Scott L

    2011-02-01

    We have evaluated the possible mechanisms of liver fibrosis caused by Fasciola hepatica in an animal model and in culture using immortalized human stellate cells. Liver biopsies of F. hepatica-infected rats were performed at wk 8 and 16. Serum-starved LX-2 cells, a human stellate cell line, were exposed to increasing concentrations of Fas2 antigen. The expression of key fibrosis-related genes was evaluated by qRT-PCR. There was a significant correlation between fibrogenic gene expression and both intensity and duration of infection. LX-2 cells exposed to Fas2 showed progressively increased expression of mRNAs for Collagen I, alpha-smooth muscle-actin, platelet-derived growth factor beta receptor, and tissue inhibitor of metalloproteinase II; inhibition of Fas2 cysteine proteinase activity by E-64 abrogated these increases, suggesting that the protease activity of Fas2 is involved in fibrogenic stimulation. In summary, F. hepatica infection is associated with up-regulation of mRNAs associated with hepatic fibrogenesis in vivo and in activated hepatic stellate cells. PMID:21348611

  11. Efficient Virus Assembly, but Not Infectivity, Determines the Magnitude of Hepatitis C Virus-Induced Interferon Alpha Responses of Plasmacytoid Dendritic Cells

    PubMed Central

    Grabski, Elena; Wappler, Ilka; Pfaender, Stephanie; Steinmann, Eike; Haid, Sibylle; Dzionek, Andrzej

    2014-01-01

    ABSTRACT Worldwide, approximately 160 million people are chronically infected with hepatitis C virus (HCV), seven distinct genotypes of which are discriminated. The hallmarks of HCV are its genetic variability and the divergent courses of hepatitis C progression in patients. We assessed whether intragenotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental Japanese fulminant hepatitis C virus (JFH1) did not induce interferon alpha (IFN-α), whereas the intragenotypic chimera Jc1 triggered massive IFN-α responses. Purified Jc1 retained full infectivity but no longer induced IFN-α. Coculture of pDC with HCV-infected hepatoma cells retrieved the capacity to induce IFN-α, whereas Jc1-infected cells triggered stronger responses than JFH1-infected cells. Since the infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different stages of particle assembly. These experiments revealed that efficient assembly and core protein envelopment were critically needed to trigger IFN-α. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced the IFN-α responses of pDC. These data showed that viral determinants shaped host innate IFN-α responses to HCV. IMPORTANCE Although pegylated IFN-α plus ribavirin currently is the standard of care for the treatment of chronic hepatitis C virus infection, not much is known about the relevance of early interferon responses in the pathogenesis of hepatitis C virus infection. Here, we addressed whether intragenotypic variations of hepatitis C virus would account for differential induction of type I interferon responses mounted by primary blood-derived plasmacytoid dendritic cells. Surprisingly, a chimeric genotype 2a virus carrying the

  12. Hepatitis A in the US Army: epidemiology and vaccine development.

    PubMed

    Hoke, C H; Binn, L N; Egan, J E; DeFraites, R F; MacArthy, P O; Innis, B L; Eckels, K H; Dubois, D; D'Hondt, E; Sjogren, M H

    1992-01-01

    Control of hepatitis A has been an important concern for US military forces in war and peace. Immune serum globulin, although effective, is exceedingly cumbersome to use. The prevalence of antibody against hepatitis A is decreasing in young American soldiers, putting them at risk of hepatitis A during deployment. The US Army has been an active participant in development of hepatitis A vaccine. The first successful cell-culture-derived, formalin-inactivated hepatitis A vaccine was developed at the Walter Reed Army Institute of Research. This prototype vaccine was shown, in 1986, to be safe and immunogenic for humans. Since then we have evaluated the following issues related to the use of inactivated hepatitis A vaccines in military populations. Immunogenicity of vaccine derived from the CLF and HM175 strains; immunogenicity of hepatitis A vaccine given by jet injector; immunogenicity of hepatitis A vaccine when given with hepatitis B vaccine; immunogenicity when given in shortened schedules; safety and immunogenicity in Thai children; and efficacy under field conditions in the tropics. The hepatitis A vaccines which we tested are safe and highly immunogenic. Immunization by jet gun confers immunity equivalent to immunization by needle. Hepatitis A vaccine is equally potent when given with hepatitis B vaccine. Data on rapid immunization schedules and efficacy are under evaluation. We conclude that hepatitis A vaccine is a major improvement in our ability to prevent hepatitis A in soldiers. PMID:1335665

  13. Regenerative medicine: Hepatic progenitor cells up their game in the therapeutic stakes.

    PubMed

    Alison, Malcolm R; Lin, Wey-Ran

    2015-11-01

    Bipotential hepatic progenitor cells (HPCs) are recognized as making modest contributions to hepatocyte regeneration, though never credited with major liver repopulation. A new study in mice demonstrates HPCs can make a massive contribution to hepatocyte replacement, suggesting HPCs have the potential to be an effective cell therapy for liver failure. PMID:26441248

  14. Sofosbuvir and Simeprevir Treatment of a Stem Cell Transplanted Teenager With Chronic Hepatitis C Infection.

    PubMed

    Fischler, Björn; Priftakis, Peter; Sundin, Mikael

    2016-06-01

    There have been no previous reports on the use of interferon-free combinations in pediatric patients with chronic hepatitis C infection. An infected adolescent with severe sickle cell disease underwent stem cell transplantation and subsequent treatment with sofosbuvir and simeprevir during ongoing immunosuppression. Despite the emergence of peripheral edema as a side effect, treatment was continued with sustained antiviral response. PMID:26928522

  15. Enriched retinal ganglion cells derived from human embryonic stem cells

    PubMed Central

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  16. Enriched retinal ganglion cells derived from human embryonic stem cells.

    PubMed

    Gill, Katherine P; Hung, Sandy S C; Sharov, Alexei; Lo, Camden Y; Needham, Karina; Lidgerwood, Grace E; Jackson, Stacey; Crombie, Duncan E; Nayagam, Bryony A; Cook, Anthony L; Hewitt, Alex W; Pébay, Alice; Wong, Raymond C B

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  17. Attenuation of Experimental Autoimmune Hepatitis by Exogenous and Endogenous Cannabinoids: Involvement of Regulatory T Cells

    PubMed Central

    Hegde, Venkatesh L.; Hegde, Shweta; Cravatt, Benjamin F.; Hofseth, Lorne J.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2009-01-01

    Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. Natural cannabinoids such as Δ9-tetrahydrocannabinol (THC) effectively modulate immune cell function, and they have shown therapeutic potential in treating inflammatory diseases. We investigated the effects of THC in a murine model of concanavalin A (ConA)-induced hepatitis. Intraperitoneal administration of THC after ConA challenge inhibited hepatitis as shown by significant decrease in liver enzymes and reduced liver tissue injury. Furthermore, THC treatment resulted in significant suppression of crucial inflammatory cytokines in ConA-induced hepatitis. It is noteworthy that THC treatment in ConA-injected mice led to significant increase in absolute number of Forkhead helix transcription factor p3+ T regulatory cells in liver. We were surprised to find that select cannabinoid receptor (CB1 or CB2) agonists were not able to block hepatitis either independently or in combination. However, CB1/CB2 mixed agonists were able to efficiently attenuate hepatitis similar to THC. The modulatory effect of THC in ConA-induced hepatitis was reversed by both CB1 and CB2 antagonists. We also observed that endogenous cannabinoid anandamide was able to reduce hepatitis by suppressing cytokine levels. In addition, deficiency or inhibition of endocannabinoid hydrolyzing enzyme fatty acid amide hydrolase (FAAH), which leads to increased levels of endogenous cannabinoids, resulted in decreased liver injury upon ConA challenge. Our data demonstrate that targeting cannabinoid receptors using exogenous or endogenous cannabinoids and use of FAAH inhibitors may constitute novel therapeutic modalities to treat immune-mediated liver inflammation. PMID:18388242

  18. FGF2 mediates hepatic progenitor cell formation during human pluripotent stem cell differentiation by inducing the WNT antagonist NKD1.

    PubMed

    Twaroski, Kirk; Mallanna, Sunil K; Jing, Ran; DiFurio, Francesca; Urick, Amanda; Duncan, Stephen A

    2015-12-01

    Fibroblast growth factors (FGFs) are required to specify hepatic fate within the definitive endoderm through activation of the FGF receptors (FGFRs). While the signaling pathways involved in hepatic specification are well understood, the mechanisms through which FGFs induce hepatic character within the endoderm are ill defined. Here we report the identification of genes whose expression is directly regulated by FGFR activity during the transition from endoderm to hepatic progenitor cell. The FGFR immediate early genes that were identified include those encoding transcription factors, growth factors, and signaling molecules. One of these immediate early genes encodes naked cuticle homolog 1 (NKD1), which is a repressor of canonical WNT (wingless-type MMTV integration site) signaling. We show that loss of NKD1 suppresses the formation of hepatic progenitor cells from human induced pluripotent stem cells and that this phenotype can be rescued by using a pharmacological antagonist of canonical WNT signaling. We conclude that FGF specifies hepatic fate at least in large part by inducing expression of NKD1 to transiently suppress the canonical WNT pathway. PMID:26637527

  19. FGF2 mediates hepatic progenitor cell formation during human pluripotent stem cell differentiation by inducing the WNT antagonist NKD1

    PubMed Central

    Twaroski, Kirk; Mallanna, Sunil K.; Jing, Ran; DiFurio, Francesca; Urick, Amanda; Duncan, Stephen A.

    2015-01-01

    Fibroblast growth factors (FGFs) are required to specify hepatic fate within the definitive endoderm through activation of the FGF receptors (FGFRs). While the signaling pathways involved in hepatic specification are well understood, the mechanisms through which FGFs induce hepatic character within the endoderm are ill defined. Here we report the identification of genes whose expression is directly regulated by FGFR activity during the transition from endoderm to hepatic progenitor cell. The FGFR immediate early genes that were identified include those encoding transcription factors, growth factors, and signaling molecules. One of these immediate early genes encodes naked cuticle homolog 1 (NKD1), which is a repressor of canonical WNT (wingless-type MMTV integration site) signaling. We show that loss of NKD1 suppresses the formation of hepatic progenitor cells from human induced pluripotent stem cells and that this phenotype can be rescued by using a pharmacological antagonist of canonical WNT signaling. We conclude that FGF specifies hepatic fate at least in large part by inducing expression of NKD1 to transiently suppress the canonical WNT pathway. PMID:26637527

  20. Permanently Blocked Stem Cells Derived from Breast Cancer Cell Lines

    PubMed Central

    Sajithlal, Gangadharan B.; Rothermund, Kristi; Zhang, Fang; Dabbs, David J.; Latimer, Jean J.; Grant, Stephen G.; Prochownik, Edward V.

    2016-01-01

    Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. An unanticipated result, however, was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immuno-compromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs. PMID:20506227

  1. Murine viral hepatitis involves NK cell depletion associated with virus-induced apoptosis

    PubMed Central

    LEHOUX, M; JACQUES, A; LUSIGNAN, S; LAMONTAGNE, L

    2004-01-01

    Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent animal model for the study of immunological disorders related to acute and chronic hepatitis. In this study, we have verified if the fulminant hepatitis induced by MHV3 could be related to an impairment of innate immunity. Groups of three C57BL/6 mice were infected with the pathogenic L2-MHV3 or attenuated YAC-MHV3 viruses, and the natural killer (NK) cell populations from liver, spleen and bone marrow were analysed. The percentage of intrahepatic NK1·1+T cell receptor (TCR)− cells did not increase while NK1·1+TCRinter cells decreased in both L2-MHV3- and YAC-MHV3-infected mice. Concurrently, splenic and myeloid NK1·1+ cells decreased in L2-MHV3-infected mice. However, the cytotoxic activity of NK cells increased in liver and decreased in bone marrow from pathogenic L2-MHV3-infected mice while no modification was detected in YAC-MHV3-infected mice. Flow cytometric analysis revealed that both normal and larger splenic or myeloid NK cells decreased more in pathogenic L2-MHV3-infected mice than in attenuated YAC-MHV3-infected mice. In vitro viral infections of interleukin (IL)-15-stimulated lymphoid cells from liver and bone marrow revealed that L2-MHV3 induced higher decreases in cell viability of NK1·1+ cells than the YAC-MHV3 variant. The NK cell decreases were due to the viral permissivity leading to cytopathic effects characterized by cell rounding, syncytia formation and apoptosis. Larger NK+ syncytia were observed in L2-MHV3-infected cells than in YAC-MHV3-infected cells. These results suggest that NK cell production is impaired by viral infection favouring fulminant hepatitis. PMID:15196242

  2. Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

    NASA Astrophysics Data System (ADS)

    Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

    1995-02-01

    The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

  3. Lipid-lowering agents inhibit hepatic steatosis in a non-alcoholic steatohepatitis-derived hepatocellular carcinoma mouse model.

    PubMed

    Orime, Kazuki; Shirakawa, Jun; Togashi, Yu; Tajima, Kazuki; Inoue, Hideaki; Nagashima, Yoji; Terauchi, Yasuo

    2016-02-01

    Non-alcoholic fatty liver disease (NAFLD) is associated with various metabolic disorders, and the therapeutic strategies for treating NAFLD and non-alcoholic steatohepatitis (NASH) have not been fully established. In the present study, we examined whether lipid-lowering agents inhibited the progression of NAFLD and tumorigenesis in a non-alcoholic steatohepatitis-derived hepatocellular carcinoma model mouse (STAM mice) generated by streptozotocin injection and a high-fat diet. Seven-week-old STAM mice were divided into groups fed a high-fat diet (Ctl) or a high-fat diet supplemented with ezetimibe (Ez), fenofibrate (Ff), rosuvastatin (Rs), ezetimibe plus fenofibrate (EF), or ezetimibe plus rosuvastatin (ER) for 4 weeks. At the end of the experiments, an oral glucose tolerance test, an insulin tolerance test, biochemical analyses using serum and liver, and a histological analysis of liver were performed in 11-week-old STAM mice. The lipid-lowering agents did not affect the body weight or the casual blood glucose levels in any of the groups. The serum triglyceride level was significantly decreased by Ff, Rs, and EF. Glucose tolerance was improved by Ez and Ff, but none of these agents improved insulin sensitivity. A histochemical analysis revealed that the lipid-lowering agents, with the exception of Rs, significantly inhibited the progression of hepatic steatosis. Nonetheless, no significant changes in the incidence of hepatic tumors were observed in any of the groups. Lipid-lowering agents inhibited the progression of hepatic steatosis without suppressing tumorigenesis in STAM mice. Our data has implications for the mechanism underlying steatosis-independent hepatic tumorigenesis in mice. PMID:26724391

  4. Sustainable inflammation transforms hepatic cells by causing oxidative stress injury and potential epithelial-mesenchymal transition.

    PubMed

    Lu, Kun; Liu, Guoyan; Yang, Ling; Liu, Fan; Gao, Libin; Shi, Jingxian; Deng, Xiaoling; Li, Qifu; Xu, Donghui; Shi, Songlin

    2016-09-01

    The inflammatory microenvironment promotes tumorigenesis. However, the mechanism through which inflammation transforms hepatic cells in precancerous lesions remains unclear. Hepatic cells undergo significant changes in metabolism before carcinogenesis, but the specific alterations in gene expression and cellular functions in response to precancerous inflammation have not been elucidated. In this study, a hepatitis-hepatoma mouse model was successfully established. Label-free quantitative (LFQ) proteomics coupled with bioinformatics analysis was then performed to identify differentially expressed proteins and their functions in hepatic cells with precancerous inflammation. We found that different chemical treatments induced several common changes in the model. Hepatic cells underwent serious oxidative stress injury. Canonical pathway analysis using IPA revealed the activation of signaling pathways, such as integrin signaling, signaling by Rho family GTPases, IL-8 signaling, and ILK signaling, as well as the inhibition of RhoGDI signaling. Analysis of the KEGG pathway indicated alteration in the pathways for focal adhesion and regulation of actin cytoskeleton. Results from western blot analysis demonstrated the upregulation of proteins, including p-STAT3, TWIST, SNAIL, Vimentin, and MMP-9, which are involved in epithelial-mesenchymal transition (EMT). These results indicated that hepatic cells were likely to undergo EMT. Interestingly, the expression of E-cadherin was upregulated, but this observation must be further investigated. In conclusion, the results revealed that notable functional and pathway changes occurred during the precancerous inflammation stage in the liver. Our study contributes to understanding of the roles of inflammation in tumorigenesis and provides a molecular basis for further studies on the tumorigenesis of hepatocellular carcinoma. PMID:27315196

  5. Universal immunization of infants with low doses of a low-cost, plasma-derived hepatitis B vaccine in South Africa.

    PubMed Central

    Schoub, B. D.; Matai, U.; Singh, B.; Blackburn, N. K.; Levin, J. B.

    2002-01-01

    OBJECTIVE: To evaluate the effectiveness of universal vaccination against viral hepatitis B in South Africa among 18-month-old rural children. METHODS: Children were immunized with a course of low-dose (1.5 microg), plasma-derived hepatitis B vaccine at 6, 10 and 14 weeks of age, and blood samples from the children were tested for three hepatitis B markers: hepatitis B surface antigen (HBsAg), anti-HBs and anti-HBc. FINDINGS: One year after vaccination, a protective anti-HBs antibody titre of at least 10 IU/l was present in 669/769 (87.0%) of blood serum samples tested. Only 3/756 children (0.4%) were HBsAg positive and a fourth child was anti-HBc positive (HBsAg negative). This is a marked decrease compared to the hepatitis B prevalences reported in previous studies. Among rural migrant mine-workers, for example, HBsAg prevalence was 9.9%, and was 10.1% among children 0-6 years of age in the Eastern Cape Province. CONCLUSION: The low-dose, plasma-derived hepatitis B vaccine, which is affordable to most developing countries, was very successful in controlling endemic hepatitis B infection, where the virus is predominantly spread by horizontal transmission among infants and young children. PMID:12075363

  6. Dioxin TECs in lake trout derived with liver cell lines from rainbow trout and rat

    SciTech Connect

    Whyte, J.J.; Clemons, J.H.; Heuvel, M.R. van den; Dixon, D.G.; Bols, N.C.

    1995-12-31

    The RTL-WL rainbow trout liver and H4IIE rat hepatoma cell lines were exposed to organic solvent extracts from livers of lake trout collected from two Lake Superior sites (Jackfish Bay -- a pulp mill site and Black Bay -- a reference site) and one Lake Ontario site (Eastern Basin -- a PCB-contaminated site). These two bioassays measure the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalent concentrations (TECs) of the residues as indicated by their relative ability to induce 7-ethoxyresorufin-o-deethylase (EROD) activity. This estimates the burden of compounds that could act through the Ah receptor and are potentially toxic, Subsamples of liver were also analyzed for hepatic EROD activity. For Jackfish Bay extracts, the H4IIE mean TEC was found to be significantly higher than that derived using RTL-WL, indicating a more potent contaminant mixture to this mammalian model. No such difference was seen between mean TECs derived for Lake Ontario extracts. The reference site, Black Bay, had TECs significantly lower than those from the other two sites. A significant positive correlation between TECs derived with H4IIE vs. RTL-WL was seen for Jackfish Bay, but not Lake Ontario. Lake Ontario extracts had a significantly higher mean TEC than the mean TEC from Jackfish Bay regardless of the cell line used. Hepatic EROD activities of liver subsamples from Jackfish Bay were found to be significantly higher than subsamples from Black Bay, while Lake Ontario subsamples were variable. No significant correlation was found between hepatic EROD activity and TEC values. The RTL-WL cell line bioassay was found to have excellent utility for rapid TEC determination and should be generally suitable for estimating the toxic potency of environmental samples.

  7. Hepatic bilirubin UDP-glucuronyltransferase in patients with sickle cell anemia.

    PubMed

    Maddrey, W C; Cukier, J O; Maglalang, A C; Boitnott, J K; Odell, G B

    1978-02-01

    In sickle cell anemia the shortened survival of red blood cells presents the liver with an augmented load of bilirubin for hepatic clearance. To determine the effects of this excessive bilirubin load on the microsomal conjugating enzyme, hepatic bilirubin UDP-glucuronyltransferase, levels of this enzyme were measured in liver biopsies from patients with sickle cell anemia and several comparison groups. UDP-glucuronyltransferase activity in 14 patients with sickle cell anemia was 2-fold greater (P less than 0.005) than in 14 nonjaundiced comparison patients without liver disease. The elevated UDP-glucuronyltransferase activity in sickle cell anemia was similar to that found in 10 patients who chronically ingested drugs (barbiturates or estrogens) known to increase UDP-glucuronyltransferase activity. These observations suggest enhanced conjugation of bilirubin in patients with sickle cell anemia may result from substrate (bilirubin) induction of UDP-glycuronyltransferase. PMID:413760

  8. Comparison of human adipose-derived stem cells and bone marrow-derived stem cells in a myocardial infarction model.

    PubMed

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Holst-Hansen, Claus; Kastrup, Jens; Baandrup, Ulrik; Zachar, Vladimir; Fink, Trine; Simonsen, Ulf

    2014-02-01

    Treatment of myocardial infarction (MI) with bone marrow-derived mesenchymal stem cells and recently also adipose-derived stem cells has shown promising results. In contrast to clinical trials and their use of autologous bone marrow-derived cells from the ischemic patient, the animal MI models are often using young donors and young, often immune-compromised, recipient animals. Our objective was to compare bone marrow-derived mesenchymal stem cells with adipose-derived stem cells from an elderly ischemic patient in the treatment of MI using a fully grown non-immune-compromised rat model. Mesenchymal stem cells were isolated from adipose tissue and bone marrow and compared with respect to surface markers and proliferative capability. To compare the regenerative potential of the two stem cell populations, male Sprague-Dawley rats were randomized to receive intramyocardial injections of adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, or phosphate-buffered saline 1 week following induction of MI. After 4 weeks, left ventricular ejection fraction (LVEF) was improved in the adipose-derived stem cell group, and scar wall thickness was greater compared with the saline group. Adipose-derived as well as bone marrow-derived mesenchymal stem cells prevented left ventricular end diastolic dilation. Neither of the cell groups displayed increased angiogenesis in the myocardium compared with the saline group. Adipose-derived stem cells from a human ischemic patient preserved cardiac function following MI, whereas this could not be demonstrated for bone marrow-derived mesenchymal stem cells, with only adipose-derived stem cells leading to an improvement in LVEF. Neither of the stem cell types induced myocardial angiogenesis, raising the question whether donor age and health have an effect on the efficacy of stem cells used in the treatment of MI. PMID:23211469

  9. Viral Load Analysis of Hepatitis C Virus in Huh7.5 Cell Culture System

    PubMed Central

    Teimourpour, Roghayeh; Meshkat, Zahra; Gholoubi, Aida; Nomani, Hosein; Rostami, Sina

    2015-01-01

    Background: Previous studies using cell culture systems for the replication of hepatitis C virus have opened new research dimensions, and paved the ways for further and detailed studies of the virus in vitro. Objectives: The purpose of the present study was to cultivate hepatitis C virus in a cell culture system and evaluate viral amplification. Materials and Methods: In order to propagate hepatitis C virus, cloned whole genome of virus, JFH-1, was used. JFH-1 cDNA was introduced into strain JM109 of Escherichia coli and plasmid, containing the viral genome was purified from transformed bacteria. After XbaI digestion, RNA synthesis was induced using T7 RNA polymerase enzyme. Next, eukaryotic cell line Huh 7.5 was transfected by the purified RNA. Finally, Huh-7.5 cell line was infected with replicated virus and viral load was determined using real-time PCR (Polymerase Chain Reaction). Results: The amount of viral load, which was measured using real-time PCR was 17592 IU/mL. Conclusions: In the present study, using cell culture, a high titer (in acceptable range) of infectious hepatitis C virus was produced. This method could be used in future studies. PMID:26290686

  10. Stem/Progenitor Cell Niches Involved in Hepatic and Biliary Regeneration

    PubMed Central

    Carpino, Guido; Renzi, Anastasia; Franchitto, Antonio; Cardinale, Vincenzo; Onori, Paolo; Reid, Lola; Alvaro, Domenico; Gaudio, Eugenio

    2016-01-01

    Niches containing stem/progenitor cells are present in different anatomical locations along the human biliary tree and within liver acini. The most primitive stem/progenitors, biliary tree stem/progenitor cells (BTSCs), reside within peribiliary glands located throughout large extrahepatic and intrahepatic bile ducts. BTSCs are multipotent and can differentiate towards hepatic and pancreatic cell fates. These niches' matrix chemistry and other characteristics are undefined. Canals of Hering (bile ductules) are found periportally and contain hepatic stem/progenitor cells (HpSCs), participating in the renewal of small intrahepatic bile ducts and being precursors to hepatocytes and cholangiocytes. The niches also contain precursors to hepatic stellate cells and endothelia, macrophages, and have a matrix chemistry rich in hyaluronans, minimally sulfated proteoglycans, fetal collagens, and laminin. The microenvironment furnishes key signals driving HpSC activation and differentiation. Newly discovered third niches are pericentral within hepatic acini, contain Axin2+ unipotent hepatocytic progenitors linked on their lateral borders to endothelia forming the central vein, and contribute to normal turnover of mature hepatocytes. Their relationship to the other stem/progenitors is undefined. Stem/progenitor niches have important implications in regenerative medicine for the liver and biliary tree and in pathogenic processes leading to diseases of these tissues. PMID:26880956

  11. Turning Hepatic Cancer Stem Cells Inside Out – A Deeper Understanding through Multiple Perspectives

    PubMed Central

    Chan, Lok-Hei; Luk, Steve T.; Ma, Stephanie

    2015-01-01

    Hepatocellular carcinoma (HCC), a highly malignant disease and the third leading cause of all cancer mortalities worldwide, often responses poorly to current treatments and results in dismal outcomes due to frequent chemoresistance and tumor relapse. The heterogeneity of HCC is an important attribute of the disease. It is the outcome of many factors, including the cross-talk between tumor cells within the tumor microenvironment and the acquisition and accumulation of genetic and epigenetic alterations in tumor cells. In addition, there is accumulating evidence in recent years to show that the malignancy of HCC can be attributed partly to the presence of cancer stem cell (CSC). CSCs are capable to self-renew, differentiate and initiate tumor formation. The regulation of the stem cell-like properties by several important signaling pathways have been found to endow the tumor cells with an increased level of tumorigenicity, chemoresistance, and metastatic ability. In this review, we will discuss the recent findings on hepatic CSCs, with special emphasis on their putative origins, relationship with hepatitis viruses, regulatory signaling networks, tumor microenvironment, and how these factors control the stemness of hepatic CSCs. We will also discuss some novel therapeutic strategies targeted at hepatic CSCs for combating HCC and perspectives of future investigation. PMID:25666349

  12. Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System

    PubMed Central

    Taniguchi, Miki; Tasaka-Fujita, Megumi; Nakagawa, Mina; Watanabe, Takako; Kawai-Kitahata, Fukiko; Otani, Satoshi; Goto, Fumio; Nagata, Hiroko; Kaneko, Shun; Nitta, Sayuri; Murakawa, Miyako; Nishimura-Sakurai, Yuki; Azuma, Seishin; Itsui, Yasuhiro; Mori, Kenichi; Yagi, Shintaro; Kakinuma, Sei; Asahina, Yasuhiro; Watanabe, Mamoru

    2016-01-01

    Background and Aims: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. Methods: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. Results were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. Results: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). Conclusions: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment. PMID:27047766

  13. Natural killer cells in highly exposed hepatitis C-seronegative injecting drug users.

    PubMed

    Mina, M M; Cameron, B; Luciani, F; Vollmer-Conna, U; Lloyd, A R

    2016-06-01

    Injecting drug use remains the major risk factor for hepatitis C (HCV) transmission. A minority of long-term injecting drug users remain seronegative and aviraemic, despite prolonged exposure to HCV - termed highly exposed seronegative subjects. Natural killer (NK) cells have been implicated in this apparent protection. A longitudinal nested, three group case-control series of subjects was selected from a prospective cohort of seronegative injecting drug users who became incident cases (n = 11), remained seronegative (n = 11) or reported transient high-risk behaviour and remained uninfected (n = 11). The groups were matched by age, sex and initial risk behaviour characteristics. Stored peripheral blood mononuclear cells were assayed in multicolour flow cytometry to enumerate natural killer cell subpopulations and to assess functional activity using Toll-like receptor ligands before measurement of activation, cytokine production and natural cytotoxicity receptor expression. Principal components were derived to describe the detailed phenotypic characteristics of the major NK subpopulations (based on CD56 and CD16 co-expression), before logistic regression analysis to identify associations with exposed, seronegative individuals. The CD56(dim) CD16(+) (P = 0.05, OR 6.92) and CD56(dim) CD16(-) (P = 0.05, OR 6.07) principal components differed between exposed, seronegative individuals and pre-infection samples of the other two groups. These included CD56(dim) CD16(+) and CD56(dim) CD16(-) subsets with CD56(dim) CD16(+) IFN-γ and TNF-α on unstimulated cells, and CD56(dim) CD16(-) CD69(+) , CD107a(+) , IFN-γ and TNF-α following TLR stimulation. The cytotoxic CD56(dim) NK subset thus distinguished highly exposed, seronegative subjects, suggesting NK cytotoxicity may contribute to protection from HCV acquisition. Further investigation of the determinants of this association and prospective assessment of protection against HCV infection are warranted. PMID:26833632

  14. The discovery of indole derivatives as novel hepatitis C virus inhibitors.

    PubMed

    Han, Zhiqiang; Liang, Xiao; Wang, Yaxin; Qing, Jie; Cao, Lin; Shang, Luqing; Yin, Zheng

    2016-06-30

    In this study, a library of in-house small molecule was screened using a HCV cell-based assay and a compound (1) containing an N-protected indole scaffold (NINS) was identified as a novel anti-HCV inhibitor. Through structure activity relationship (SAR) study, it was observed that the racemic inhibitor (10m) displayed good anti-HCV activity (EC50 = 1.02 ± 0.10 μM) with the excellent selectivity index (SI = 45.56). Interestingly, R-enantiomer ((R)-10m) showed better anti-HCV activity and lower cytotoxicity than S-enantiomer ((S)-10m). (R)-10m gave the best anti-HCV potency (EC50 = 0.72 ± 0.09 μM) with the highest selectivity index (SI > 69.44). In addition, the mechanism of action study of NINS derivatives demonstrated that NINS derivatives interfere with the early step (viral entry) of the HCV life cycle. PMID:27061978

  15. Kupffer Cells Support Hepatitis B Virus-Mediated CD8+ T Cell Exhaustion via Hepatitis B Core Antigen-TLR2 Interactions in Mice.

    PubMed

    Li, Min; Sun, Rui; Xu, Long; Yin, Wenwei; Chen, Yongyan; Zheng, Xiaodong; Lian, Zhexiong; Wei, Haiming; Tian, Zhigang

    2015-10-01

    Hepatitis B virus (HBV) persistence is a fundamental process in chronic HBV infection and a key factor in all related liver diseases; however, the mechanisms have yet to be elucidated. We studied the role of TLR2 in HBV persistence using a well-established HBV-carrier mouse model generated by hydrodynamically injecting a phospho-adeno-associated virus/HBV1.2 plasmid into mice. We found that a genetic deficiency in TLR2 improves HBV elimination, whereas activating TLR2 led to more stable HBV persistence, suggesting that TLR2 activation is critical in HBV persistence. Furthermore, we noted that TLR2 activation could inhibit CD8(+) T cell function, causing the exhaustion phenotype in HBV-carrier mice, because TLR2 deficiency might rescue CD8(+) T cell function in a cellular adoptive experiment. TLR2 expression on Kupffer cells (KCs) was upregulated in HBV-carrier mice, which accounts for HBV persistence, because the difference in anti-HBV immunity between HBV-carrier wild-type and Tlr2(-/-) mice did not exist after KC depletion. In addition, similar to TLR2 deficiency, after KC depletion, CD8(+) T cells were more efficiently activated in HBV-carrier mice, leading to rapid HBV elimination. KCs produced more IL-10 upon TLR2 activation in response to direct hepatitis B core Ag stimulation, and the elevated IL-10 inhibited CD8(+) T cell function in HBV-carrier mice, because IL-10 deficiency or anti-IL-10R treatment resulted in CD8(+) T cells with stronger antiviral function. In conclusion, KCs support liver tolerance by inducing anti-HBV CD8(+) T cell exhaustion via IL-10 production after TLR2 activation by hepatitis B core Ag stimulation. PMID:26304988

  16. Fibrosing cholestatic hepatitis C after hematopoietic cell transplantation: report of 3 fatal cases.

    PubMed

    Evans, Ashley T; Loeb, Keith R; Shulman, Howard M; Hassan, Sajida; Qiu, Wan Chong; Hockenbery, David M; Ioannou, George N; Chauncey, Thomas R; Gretch, David R; McDonald, George B

    2015-02-01

    Development of liver disease after hematopoietic cell transplantation is common and the causes diverse. Infection by hepatitis C virus (HCV) can be seen in patients who are chronically infected before transplant or from passage of virus from an infected donor; the normal 10-year course of hepatitis C after transplant is one of waxing and waning of serum aminotransferase enzymes, with little morbidity. In the series of 3 patients reported here, the course of hepatitis C was rapidly fatal, with the onset of jaundice at day 60 to 80 after transplant and liver histology typical of fibrosing cholestatic hepatitis (marked bile ductular proliferation, ballooned hepatocytes, and associated collagenous fibrosis centered around ductules). The bile ductular reaction pattern varied from elongated structures without a recognizable lumen to a pattern of cuboidal cells with a clear lumen. There was significant cholestasis with bile within hepatocytes and canalicular bile plugs. In situ HCV RNA hybridization studies from 1 patient showed a robust infection with high levels of HCV-infected hepatocytes and active viral replication. All 3 patients were on immunosuppressive drugs after transplant, including mycophenolate mofetil (MMF), which irreversibly inhibits inosine monophosphate dehydrogenase, on which T and B lymphocytes are dependent. We speculate that fatal fibrosing cholestatic hepatitis C in these cases was related to the immunosuppressive effects of MMF, as we had not recognized this presentation of HCV infection before the introduction of MMF. PMID:25517948

  17. Thymoquinone attenuates liver fibrosis via PI3K and TLR4 signaling pathways in activated hepatic stellate cells.

    PubMed

    Bai, Ting; Lian, Li-Hua; Wu, Yan-Ling; Wan, Ying; Nan, Ji-Xing

    2013-02-01

    Thymoquinone (TQ) is the major active compound derived from the medicinal Nigella sativa. In the present study, we investigated the anti-fibrotic mechanism of TQ in lipopolysaccharide (LPS)-activated rat hepatic stellate cells line, T-HSC/Cl-6. T-HSC/Cl-6 cells were treated with TQ (3.125, 6.25 and 12.5μM) prior to LPS (1μg/ml). Our data demonstrated that TQ effectively decreased activated T-HSC/Cl-6 cell viability. TQ significantly attenuated the expression of CD14 and Toll-like receptor 4 (TLR4). TQ also significantly inhibited phosphatidylinositol 3-kinase (PI3K) and serine/threonine kinase-protein kinase B (Akt) phosphorylation. The expression of α-SMA and collagen-I were significantly decreased by TQ. Furthermore, TQ decreased X linked inhibitor of apoptosis (XIAP) and cellular FLIP (c-FLIPL) expression, which are related with the regulation of apoptosis. Furthermore, TQ significantly increased the survival against LPS challenge in d-galactosamine (d-GlaN)-sensitized mice, and decreased the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were in line with in vitro results. Our data demonstrated that TQ attenuates liver fibrosis partially via blocking TLR4 expression and PI3K phosphorylation on the activated HSCs. Therefore, TQ may be a potential candidate for the therapy of hepatic fibrosis. PMID:23318601

  18. 27-Hydroxycholesterol modulation of low density lipoprotein metabolism in cultured human hepatic and extrahepatic cells.

    PubMed

    Bellosta, S; Corsini, A; Bernini, F; Granata, A; Didoni, G; Mazzotti, M; Fumagalli, R

    1993-10-11

    27-Hydroxycholesterol**, 25-hydroxycholesterol and cholesterol suppressed LDL uptake and degradation in human extrahepatic and hepatic cell lines in a concentration-dependent manner. Cholesterol was the least potent, and the inhibitory effect of oxysterols was more pronounced in skin fibroblasts and in endothelial cell line EAhy 926 than in hepatoma HepG2 cells. Shorter incubations were required for oxysterols to achieve 50% inhibition of LDL uptake and degradation in all cultured cells. The inhibition of LDL catabolism in extrahepatic cells by 27-hydroxycholesterol occurred at concentrations close to those observed in human plasma (0.2-0.6 microM). The results support a possible role of 27-hydroxycholesterol, a physiological oxysterol, in the regulation of cellular cholesterol homeostasis in non-hepatic tissues. PMID:8405424

  19. Role of antigen presenting cell invariant chain in the development of hepatic steatosis in mouse model.

    PubMed

    Mishra, Alaknanda; Iyer, Srikanth; Kesarwani, Ashwani; Baligar, Prakash; Arya, Satya Pal; Arindkar, Shailendra; Kumar, M J Mahesh; Upadhyay, Pramod; Majumdar, Subeer S; Nagarajan, Perumal

    2016-08-15

    The role of Invariant chain (CD74 or Ii) in antigen presentation via Antigen Presenting Cells (APC), macrophage recruitment as well as survival, T cell activation and B cell differentiation has been well recognized. However, the aspect of CD74 which is involved in the development of hepatic steatosis and the pathways through which it acts remain to be studied. In this study, we investigated the role of CD74 in the inflammatory pathway and its contribution to development of hepatic steatosis. For this, wild type C57BL/6J and CD74 deficient mice (Ii(-/-) mice) were fed with high fat high fructose (HFHF) diet for 12 weeks. Chronic consumption of this feed did not develop hepatic steatosis, glucose intolerance or change in the level of immune cells in Ii(-/-) mice. Moreover, there was relatively delayed expression of genes involved in development of non alcoholic fatty liver disease (NAFLD) in HFHF fed Ii(-/-) mice as compared to that of C57BL/6J phenotype. Taken together, the data suggest that HFHF diet fed Ii(-/-) mice fail to develop hepatic steatosis, suggesting that Ii mediated pathways play a vital role in the initiation and propagation of liver inflammation. PMID:27371158

  20. Specific CD8+ T cell response immunotherapy for hepatocellular carcinoma and viral hepatitis

    PubMed Central

    Moreno-Cubero, Elia; Larrubia, Juan-Ramón

    2016-01-01

    Hepatocellular carcinoma (HCC), chronic hepatitis B (CHB) and chronic hepatitis C (CHC) are characterized by exhaustion of the specific CD8+ T cell response. This process involves enhancement of negative co-stimulatory molecules, such as programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), 2B4, Tim-3, CD160 and LAG-3, which is linked to intrahepatic overexpression of some of the cognate ligands, such as PD-L1, on antigen presenting cells and thereby favouring a tolerogenic environment. Therapies that disrupt these negative signalling mechanisms represent promising therapeutic tools with the potential to restore reactivity of the specific CD8+ T cell response. In this review we discuss the impressive in vitro and in vivo results that have been recently achieved in HCC, CHB and CHC by blocking these negative receptors with monoclonal antibodies against these immune checkpoint modulators. The article mainly focuses on the role of CTLA-4 and PD-1 blocking monoclonal antibodies, the first ones to have reached clinical practice. The humanized monoclonal antibodies against CTLA-4 (tremelimumab and ipilimumab) and PD-1 (nivolumab and pembrolizumab) have yielded good results in testing of HCC and chronic viral hepatitis patients. Trelimumab, in particular, has shown a significant increase in the time to progression in HCC, while nivolumab has shown a remarkable effect on hepatitis C viral load reduction. The research on the role of ipilimumab, nivolumab and pembrolizumab on HCC is currently underway.

  1. Specific CD8(+) T cell response immunotherapy for hepatocellular carcinoma and viral hepatitis.

    PubMed

    Moreno-Cubero, Elia; Larrubia, Juan-Ramón

    2016-07-28

    Hepatocellular carcinoma (HCC), chronic hepatitis B (CHB) and chronic hepatitis C (CHC) are characterized by exhaustion of the specific CD8(+) T cell response. This process involves enhancement of negative co-stimulatory molecules, such as programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), 2B4, Tim-3, CD160 and LAG-3, which is linked to intrahepatic overexpression of some of the cognate ligands, such as PD-L1, on antigen presenting cells and thereby favouring a tolerogenic environment. Therapies that disrupt these negative signalling mechanisms represent promising therapeutic tools with the potential to restore reactivity of the specific CD8(+) T cell response. In this review we discuss the impressive in vitro and in vivo results that have been recently achieved in HCC, CHB and CHC by blocking these negative receptors with monoclonal antibodies against these immune checkpoint modulators. The article mainly focuses on the role of CTLA-4 and PD-1 blocking monoclonal antibodies, the first ones to have reached clinical practice. The humanized monoclonal antibodies against CTLA-4 (tremelimumab and ipilimumab) and PD-1 (nivolumab and pembrolizumab) have yielded good results in testing of HCC and chronic viral hepatitis patients. Trelimumab, in particular, has shown a significant increase in the time to progression in HCC, while nivolumab has shown a remarkable effect on hepatitis C viral load reduction. The research on the role of ipilimumab, nivolumab and pembrolizumab on HCC is currently underway. PMID:27605882

  2. Localization of Xenobiotic Transporter OCTN1/SLC22A4 in Hepatic Stellate Cells and Its Protective Role in Liver Fibrosis.

    PubMed

    Tang, Yaliang; Masuo, Yusuke; Sakai, Yoshio; Wakayama, Tomohiko; Sugiura, Tomoko; Harada, Ryuichi; Futatsugi, Azusa; Komura, Takuya; Nakamichi, Noritaka; Sekiguchi, Hirotaka; Sutoh, Keita; Usumi, Koji; Iseki, Shoichi; Kaneko, Shuichi; Kato, Yukio

    2016-05-01

    Xenobiotic transporters play key roles in disposition of certain therapeutic agents, although limited information is available on their roles other than pharmacokinetic issues. Here, suppressive effect of multispecific organic cation transporter OCTN1/SLC22A4 on liver fibrosis was proposed in liver injury models. After injection of hepatotoxins such as dimethylnitrosamine (DMN) or concanavalin A, hepatic fibrosis, and oxidative stress, evaluated in terms of Sirius red and 4-hydroxy-2-nonenal staining, respectively, were more severe in liver of octn1/slc22a4 gene knockout (octn1(-/-)) mice than that in wild-type mice. DMN treatment markedly increased α-smooth muscle actin and F4/80, markers of activated stellate and Kupffer cells, respectively, in liver of octn1(-/-), but had less effect in wild-type mice. Thus, octn1/slc22a4 gene deletion results in more severe hepatic fibrosis, oxidative stress, and inflammation. DMN-treated wild-type mice showed increased Octn1 staining and hepatic concentration of its food-derived antioxidant ergothioneine (ERGO). The upregulated Octn1 was co-localized with α-smooth muscle actin. Functional expression of Octn1 was demonstrated in activated human hepatic stellate cell lines, LI90 and LX-2. Provision of ERGO-rich feed ameliorated DMN-induced liver fibrosis and oxidative stress. Overall, Octn1 is upregulated in activated stellate cells, resulting in increased delivery of its substrate antioxidant ERGO and a protective effect against liver fibrosis. PMID:27020986

  3. Caveolin-1 protects against hepatic ischemia/reperfusion injury through ameliorating peroxynitrite-mediated cell death.

    PubMed

    Gao, Lei; Chen, Xingmiao; Peng, Tao; Yang, Dan; Wang, Qi; Lv, Zhiping; Shen, Jiangang

    2016-06-01

    Nitrative stress is considered as an important pathological process of hepatic ischemia and reperfusion injury but its regulating mechanisms are largely unknown. In this study, we tested the hypothesis that caveolin-1 (Cav-1), a plasma membrane scaffolding protein, could be an important cellular signaling against hepatic I/R injury through inhibiting peroxynitrite (ONOO(-))-induced cellular damage. Male wild-type mice and Cav-1 knockout (Cav-1(-/-)) were subjected to 1h hepatic ischemia following 1, 6 and 12h of reperfusion by clipping and releasing portal vessels respectively. Immortalized human hepatocyte cell line (L02) was subjected to 1h hypoxia and 6h reoxygenation and treated with Cav-1 scaffolding domain peptide. The major discoveries included: (1) the expression of Cav-1 in serum and liver tissues of wild-type mice was time-dependently elevated during hepatic ischemia-reperfusion injury. (2) Cav-1 scaffolding domain peptide treatment inhibited cleaved caspase-3 expression in the hypoxia-reoxygenated L02 cells; (3) Cav-1 knockout (Cav-1(-/-)) mice had significantly higher levels of serum transaminases (ALT&AST) and TNF-α, and higher rates of apoptotic cell death in liver tissues than wild-type mice after subjected to 1h hepatic ischemia and 6hour reperfusion; (4) Cav-1(-/-) mice revealed higher expression levels of iNOS, ONOO(-) and 3-nitrotyrosine (3-NT) in the liver than wild-type mice, and Fe-TMPyP, a representative peroxynitrite decomposition catalyst (PDC), remarkably reduced level of ONOO(-) and 3-NT and ameliorated the serum ALT, AST and TNF-α levels in both wild-type and Cav-1(-/-) mice. Taken together, we conclude that Cav-1 could play a critical role in preventing nitrative stress-induced liver damage during hepatic ischemia-reperfusion injury. PMID:27021966

  4. Hepatic non-parenchymal cells: Master regulators of alcoholic liver disease?

    PubMed Central

    Seo, Wonhyo; Jeong, Won-Il

    2016-01-01

    Chronic alcohol consumption is one of the most common causes of the progression of alcoholic liver disease (ALD). In the past, alcohol-mediated hepatocyte injury was assumed to be a significantly major cause of ALD. However, a huge number of recent and brilliant studies have demonstrated that hepatic non-parenchymal cells including Kupffer cells, hepatic stellate cells, liver sinusoidal endothelial cells and diverse types of lymphocytes play crucial roles in the pathogenesis of ALD by producing inflammatory mediators such as cytokines, oxidative stress, microRNA, and lipid-originated metabolites (retinoic acid and endocannabinoids) or by directly interacting with parenchymal cells (hepatocytes). Therefore, understanding the comprehensive roles of hepatic non-parenchymal cells during the development of ALD will provide new integrative directions for the treatment of ALD. This review will address the roles of non-parenchymal cells in alcoholic steatosis, inflammation, and liver fibrosis and might help us to discover possible therapeutic targets and treatments involving modulating the non-parenchymal cells in ALD. PMID:26819504

  5. Hepatic non-parenchymal cells: Master regulators of alcoholic liver disease?

    PubMed

    Seo, Wonhyo; Jeong, Won-Il

    2016-01-28

    Chronic alcohol consumption is one of the most common causes of the progression of alcoholic liver disease (ALD). In the past, alcohol-mediated hepatocyte injury was assumed to be a significantly major cause of ALD. However, a huge number of recent and brilliant studies have demonstrated that hepatic non-parenchymal cells including Kupffer cells, hepatic stellate cells, liver sinusoidal endothelial cells and diverse types of lymphocytes play crucial roles in the pathogenesis of ALD by producing inflammatory mediators such as cytokines, oxidative stress, microRNA, and lipid-originated metabolites (retinoic acid and endocannabinoids) or by directly interacting with parenchymal cells (hepatocytes). Therefore, understanding the comprehensive roles of hepatic non-parenchymal cells during the development of ALD will provide new integrative directions for the treatment of ALD. This review will address the roles of non-parenchymal cells in alcoholic steatosis, inflammation, and liver fibrosis and might help us to discover possible therapeutic targets and treatments involving modulating the non-parenchymal cells in ALD. PMID:26819504

  6. Enhanced Metabolizing Activity of Human ES Cell-Derived Hepatocytes Using a 3D Culture System with Repeated Exposures to Xenobiotics.

    PubMed

    Kim, Jong Hyun; Jang, Yu Jin; An, Su Yeon; Son, Jeongsang; Lee, Jaehun; Lee, Gyunggyu; Park, Ji Young; Park, Han-Jin; Hwang, Dong-Youn; Kim, Jong-Hoon; Han, Jiyou

    2015-09-01

    Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the cell-based therapy of liver disease and the development of effective in vitro toxicity screening tools. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized hepatic differentiation protocol, which produces >90% (BGO1 and CHA15) albumin-positive HLCs with no purification process from human embryonic stem cell lines. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating 3D spheroidal aggregate of HLCs, compared with 2D HLCs. The 3D differentiation method increased expression of nuclear receptors (NRs) that regulate the proper expression of key hepatic enzymes. Furthermore, significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids, compared with 2D HLCs. HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated further functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic metabolizing ability of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models. PMID:26089346

  7. Quantification of Hepatitis C Virus Cell-to-Cell Spread Using a Stochastic Modeling Approach

    PubMed Central

    Martin, Danyelle N.; Perelson, Alan S.; Dahari, Harel

    2015-01-01

    ABSTRACT It has been proposed that viral cell-to-cell transmission plays a role in establishing and maintaining chronic infections. Thus, understanding the mechanisms and kinetics of cell-to-cell spread is fundamental to elucidating the dynamics of infection and may provide insight into factors that determine chronicity. Because hepatitis C virus (HCV) spreads from cell to cell and has a chronicity rate of up to 80% in exposed individuals, we examined the dynamics of HCV cell-to-cell spread in vitro and quantified the effect of inhibiting individual host factors. Using a multidisciplinary approach, we performed HCV spread assays and assessed the appropriateness of different stochastic models for describing HCV focus expansion. To evaluate the effect of blocking specific host cell factors on HCV cell-to-cell transmission, assays were performed in the presence of blocking antibodies and/or small-molecule inhibitors targeting different cellular HCV entry factors. In all experiments, HCV-positive cells were identified by immunohistochemical staining and the number of HCV-positive cells per focus was assessed to determine focus size. We found that HCV focus expansion can best be explained by mathematical models assuming focus size-dependent growth. Consistent with previous reports suggesting that some factors impact HCV cell-to-cell spread to different extents, modeling results estimate a hierarchy of efficacies for blocking HCV cell-to-cell spread when targeting different host factors (e.g., CLDN1 > NPC1L1 > TfR1). This approach can be adapted to describe focus expansion dynamics under a variety of experimental conditions as a means to quantify cell-to-cell transmission and assess the impact of cellular factors, viral factors, and antivirals. IMPORTANCE The ability of viruses to efficiently spread by direct cell-to-cell transmission is thought to play an important role in the establishment and maintenance of viral persistence. As such, elucidating the dynamics of cell-to-cell

  8. Characterisation of the hepatic progenitor cell compartment in normal liver and in hepatitis: an immunohistochemical comparison between dog and man.

    PubMed

    Ijzer, J; Schotanus, B A; Vander Borght, S; Roskams, T A D; Kisjes, R; Penning, L C; Rothuizen, J; van den Ingh, T S G A M

    2010-06-01

    The liver progenitor cell compartment in the normal canine liver and in spontaneous canine acute (AH) and chronic hepatitis (CH) was morphologically characterised and compared to its human equivalents. Immunohistochemistry was performed for cytokeratin-7 (CK7), human hepatocyte marker (Hep Par 1), multidrug resistance-associated protein-2 (MRP2), and breast cancer resistance protein (BCRP) on paraffin and frozen sections from canine and human tissues. Normal liver showed similar morphology and immunohistochemical reaction of the progenitor cell compartment/canal of Hering in man and dog. In addition, a ductular reaction, comparable in terms of severity, location and immunohistochemical characteristics, was observed in canine and human AH and CH. CK7 was a good marker for canine progenitor cells, including intermediate cells, which were positively identified in cases of AH and CH. In both species, BCRP was expressed in both hepatocytes and bile ducts of the normal liver, and in ductular reaction in AH and CH. MRP2 detected bile canalicular membranes in man and dog. These findings underline the similarities between canine and human liver reaction patterns and may offer mutual advantage for comparative research in human and canine spontaneous liver diseases. PMID:19369099

  9. The isolation and in vitro expansion of hepatic Sca-1 progenitor cells

    SciTech Connect

    Clayton, Elizabeth

    2009-04-17

    The intra-hepatic population of liver progenitor cells expands during liver injury when hepatocyte proliferation is inhibited. These cells can be purified by density gradient centrifugation and cultured. Separated by size only this population contains small cells of hematopoietic, epithelial and endothelial lineages and is thought to contain liver stem cells. The identity of liver stem cells remains unknown although there is some evidence that tissue Sca1{sup +} CD45{sup -} cells display progenitor cell characteristics. We identified both intra-hepatic and gall bladder Sca1{sup +} cells following liver injury and expanded ex vivo Sca1 cells as part of heterogenous cell culture or as a purified population. We found significant difference between the proliferation of Sca-1 cells when plated on laminin or collagen I while proliferation of heterogenous population was not affected by the extracellular matrix indicating the necessity for culture of Sca1{sup +} cells with laminin matrix or laminin producing cells in long term liver progenitor cell cultures.

  10. N-Acetylglucosaminyltransferase V regulates TGF-β response in hepatic stellate cells and the progression of steatohepatitis.

    PubMed

    Kamada, Yoshihiro; Mori, Kanako; Matsumoto, Hitoshi; Kiso, Shinichi; Yoshida, Yuichi; Shinzaki, Shinichiro; Hiramatsu, Naoki; Ishii, Mayuko; Moriwaki, Kenta; Kawada, Norifumi; Takehara, Tetsuo; Miyoshi, Eiji

    2012-06-01

    N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in tumor metastasis and carcinogenesis. Although the expression of GnT-V is induced in chronic liver diseases, the biological meaning of GnT-V in the diseases remains unknown. The aim of this study was to investigate the effects of GnT-V on the progression of chronic hepatitis, using GnT-V transgenic (Tg) mice fed a high fat and high cholesterol (HFHC) diet, an experimental model of murine steatohepatitis. Although enhanced hepatic lymphocytes infiltration and fibrosis were observed in wild-type (WT) mice fed the HFHC diet, they were dramatically prevented in Tg mice. In addition, the gene expression of inflammatory Th1 cytokines in the liver was significantly decreased in Tg mice than WT mice. Inhibition of liver fibrosis was due to the dysfunction of hepatic stellate cells (HSCs), which play pivotal roles in liver fibrosis through the production of transforming growth factor (TGF)-β1. Although TGF-β1 signaling was enhanced in Tg mouse-derived HSCs (Tg-HSCs) compared with WT mouse-derived HSCs (WT-HSCs), collagen expression was significantly reduced in Tg-HSCs. As a result from DNA microarray, cyclooxygenase-2 (COX2) expression, known as a negative feedback signal for TGF-β1, was significantly elevated in Tg-HSCs compared with WT-HSCs. Prostaglandin E2 (PGE2), the product of COX2, production was also significantly elevated in Tg-HSCs. COX2 inhibition by celecoxib decreased PGE2 and increased collagen expression in Tg-HSCs. In conclusion, GnT-V prevented steatohepatitis progression through modulating lymphocyte and HSC functions. PMID:22294551

  11. Blockade of Retinol Metabolism Protects T Cell-Induced Hepatitis by Increasing Migration of Regulatory T Cells.

    PubMed

    Lee, Young-Sun; Yi, Hyon-Seung; Suh, Yang-Gun; Byun, Jin-Seok; Eun, Hyuk Soo; Kim, So Yeon; Seo, Wonhyo; Jeong, Jong-Min; Choi, Won-Mook; Kim, Myung-Ho; Kim, Ji Hoon; Park, Keun-Gyu; Jeong, Won-Il

    2015-11-01

    Retinols are metabolized into retinoic acids by alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (Raldh). However, their roles have yet to be clarified in hepatitis despite enriched retinols in hepatic stellate cells (HSCs). Therefore, we investigated the effects of retinols on Concanavalin A (Con A)-mediated hepatitis. Con A was injected into wild type (WT), Raldh1 knock-out (Raldh1(-/-)), CCL2(-/-) and CCR2(-/-) mice. For migration study of regulatory T cells (Tregs), we used in vivo and ex vivo adoptive transfer systems. Blockade of retinol metabolism in mice given 4-methylpyrazole, an inhibitor of ADH, and ablated Raldh1 gene manifested increased migration of Tregs, eventually protected against Con A-mediated hepatitis by decreasing interferon-γ in T cells. Moreover, interferon-γ treatment increased the expression of ADH3 and Raldh1, but it suppressed that of CCL2 and IL-6 in HSCs. However, the expression of CCL2 and IL-6 was inversely increased upon the pharmacologic or genetic ablation of ADH3 and Raldh1 in HSCs. Indeed, IL-6 treatment increased CCR2 expression of Tregs. In migration assay, ablated CCR2 in Tregs showed reduced migration to HSCs. In adoptive transfer of Tregs in vivo and ex vivo, Raldh1-deficient mice showed more increased migration of Tregs than WT mice. Furthermore, inhibited retinol metabolism increased survival rate (75%) compared with that of the controls (25%) in Con A-induced hepatitis. These results suggest that blockade of retinol metabolism protects against acute liver injury by increased Treg migration, and it may represent a novel therapeutic strategy to control T cell-mediated acute hepatitis. PMID:26537191

  12. Hepatic veno-occlusive disease after hematopoietic stem cell transplantation: Prophylaxis and treatment controversies

    PubMed Central

    Cheuk, Daniel KL

    2012-01-01

    Hepatic veno-occlusive disease (VOD), also known as sinusoidal obstruction syndrome, is a major complication of hematopoietic stem cell transplantation and it carries a high mortality. Prophylaxis for hepatic VOD is commonly given to transplant recipients from the start of conditioning through the early weeks of transplant. However, high quality evidence from randomized controlled trials is scarce with small sample sizes and the trials yielded conflicting results. Although various treatment options for hepatic VOD are available, most have not undergone stringent evaluation with randomized controlled trial and therefore it remains uncertain which treatment offers real benefit. It remains controversial whether VOD prophylaxis should be given, which prophylactic therapy should be given, who should receive prophylaxis, and what treatment should be offered once VOD is established. PMID:24175193

  13. Bmi1 Is Required for Hepatic Progenitor Cell Expansion and Liver Tumor Development

    PubMed Central

    Wang, Chunmei; Tao, Junyan; Ho, Coral; Jiang, Lijie; Gui, Bing; Huang, Shiang; Evert, Matthias; Calvisi, Diego F.; Chen, Xin

    2012-01-01

    Bmi1 is a polycomb group transcriptional repressor and it has been implicated in regulating self-renewal and proliferation of many types of stem or progenitor cells. In addition, Bmi1 has been shown to function as an oncogene in multiple tumor types. In this study, we investigated the functional significance of Bmi1 in regulating hepatic oval cells, the major type of bipotential progenitor cells in adult liver, as well as the role of Bmi1 during hepatocarcinogenesis using Bmi1 knockout mice. We found that loss of Bmi1 significantly restricted chemically induced oval cell expansion in the mouse liver. Concomitant deletion of Ink4a/Arf in Bmi1 deficient mice completely rescued the oval cell expansion phenotype. Furthermore, ablation of Bmi1 delayed hepatocarcinogenesis induced by AKT and Ras co-expression. This antineoplastic effect was accompanied by the loss of hepatic oval cell marker expression in the liver tumor samples. In summary, our data demonstrated that Bmi1 is required for hepatic oval cell expansion via deregulating the Ink4a/Arf locus in mice. Our study also provides the evidence, for the first time, that Bmi1 expression is required for liver cancer development in vivo, thus representing a promising target for innovative treatments against human liver cancer. PMID:23029524

  14. Pluripotent Stem Cells Derived From Mouse and Human White Mature Adipocytes

    PubMed Central

    Abdmaulen, Raushan; Ly, Albert; Cubberly, Mark R.; Shahmirian, Laurine J.; Heydarkhan-Hagvall, Sepideh; Dumesic, Daniel A.; Yao, Yucheng

    2014-01-01

    White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5–7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies. PMID:24396033

  15. The canine hepatic progenitor cell niche: molecular characterisation in health and disease.

    PubMed

    Kruitwagen, H S; Spee, B; Viebahn, C S; Venema, H B; Penning, L C; Grinwis, G C M; Favier, R P; van den Ingh, T S G A M; Rothuizen, J; Schotanus, B A

    2014-09-01

    Hepatic progenitor cells (HPCs) are an adult stem cell compartment in the liver that contributes to liver regeneration when replication of mature hepatocytes is insufficient. In this study, laser microdissection was used to isolate HPC niches from the livers of healthy dogs and dogs with lobular dissecting hepatitis (LDH), in which HPCs are massively activated. Gene expression of HPC, hepatocyte and biliary markers was determined by quantitative reverse transcriptase PCR. Expression and localisation of selected markers were further studied at the protein level by immunohistochemistry and immunofluorescent double staining in samples of normal liver and liver from dogs with LDH, acute and chronic hepatitis, and extrahepatic cholestasis. Activated HPC niches had higher gene expression of the hepatic progenitor markers OPN, FN14, CD29, CD44, CD133, LIF, LIFR and BMI1 compared to HPCs from normal liver. There was lower expression of albumin, but activated HPC niches were positive for the biliary markers SOX9, HNF1β and keratin 19 by immunohistochemistry and immunofluorescence. Laminin, activated stellate cells and macrophages are abundant extracellular matrix and cellular components of the canine HPC niche. This study demonstrates that the molecular and cellular characteristics of canine HPCs are similar to rodent and human HPCs, and that canine HPCs are distinctively activated in different types of liver disease. PMID:24923752

  16. Cyclooxygenase-2 contributes to the selective induction of cell death by the endocannabinoid 2-arachidonoyl glycerol in hepatic stellate cells.

    PubMed

    Siegmund, S V; Wojtalla, A; Schlosser, M; Schildberg, F A; Knolle, P A; Nüsing, R M; Zimmer, A; Strassburg, C P; Singer, M V

    2016-02-12

    The endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is an anti-fibrotic lipid mediator that induces apoptosis in hepatic stellate cells (HSCs), but not in hepatocytes. However, the exact molecular mechanisms of this selective induction of HSC death are still unresolved. Interestingly, the inducible isoform of cyclooxygenase, COX-2, can metabolize 2-AG to pro-apoptotic prostaglandin glycerol esters (PG-GEs). We analyzed the roles of COX-2 and endocannabinoid-derived PG-GEs in the differential susceptibility of primary activated HSCs and hepatocytes toward 2-AG-induced cell death. HSCs displayed significant COX-2 expression in contrast to hepatocytes. Similar to 2-AG, treatment of HSCs with PGD2-GE dose-dependently induced cell death independently from cannabinoid receptors that was accompanied by PARP- and caspase 3-cleavage. In contrast to 2-AG, PGD2-GE failed to induce significant ROS formation in HSCs, and depletion of membrane cholesterol did not rescue HSCs from PGD2-GE-induced apoptosis. These findings indicate differential engagement of initial intracellular signaling pathways by 2-AG and its COX-2-derived metabolite PGD2-GE, but similar final cell death pathways. Other PG-GEs, such as PGE2-or PGF2α-GE did not induce apoptosis in HSCs. Primary rat hepatocytes were mainly resistant against 2-AG- and PGD2-GE-induced apoptosis. HSCs, but not hepatocytes were able to metabolize 2-AG to PGD2-GE. As a proof of principle, HSCs from COX-2(-/-) mice lacked PDG2-GE production after 2-AG treatment. Accordingly, COX-2(-/-) HSCs were resistant against 2-AG-induced apoptosis. In conclusion, the divergent expression of COX-2 in HSCs and hepatocytes contributes to the different susceptibility of these cell types towards 2-AG-induced cell death due to the generation of pro-apoptotic PGD2-GE by COX-2 in HSCs. Modulation of COX-2-driven metabolization of 2-AG may provide a novel physiological concept allowing the specific targeting of HSCs in liver fibrosis. PMID

  17. Hepatitis virus vaccines: present status.

    PubMed Central

    Krugman, S.

    1982-01-01

    During the past decade there has been extraordinary progress toward the development of vaccines for the prevention of type A and type B hepatitis. The successful propagation of hepatitis A virus in cell culture in 1979 was followed by the preparation of experimental live attenuated hepatitis A vaccines that have been shown to induce antibody in marmosets and chimpanzees and protect immunized marmosets against challenge with hepatitis A virus. The first human immunization trials will begin in mid-1982. An inactivated hepatitis B vaccine that was licensed in the United States in November 1981 has been shown to be safe, immunogenic, and effective. When this vaccine becomes available for use in July 1982, it will be recommended for persons who are considered to be at increased risk of contracting hepatitis B infection. Future generations of hepatitis B vaccines may be prepared from hepatitis B surface antigen derived from DNA recombinant technology or by in vitro synthesis of HBs Ag determinants by chemical means. PMID:6295013

  18. Antiproliferative and cytotoxic effects of purple pitanga (Eugenia uniflora L.) extract on activated hepatic stellate cells.

    PubMed

    Denardin, Cristiane C; Parisi, Mariana M; Martins, Leo A M; Terra, Silvia R; Borojevic, Radovan; Vizzotto, Márcia; Perry, Marcos L S; Emanuelli, Tatiana; Guma, Fátima T C R

    2014-01-01

    The presence of phenolic compounds in fruit- and vegetable-rich diets has attracted researchers' attention due to their health-promoting effects. The objective of this study was to evaluate the effects of purple pitanga (Eugenia uniflora L.) extract on cell proliferation, viability, mitochondrial membrane potential, cell death and cell cycle in murine activated hepatic stellate cells (GRX). Cell viability by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was significantly decreased on cells treated with 50 and 100 µg ml(-1) of purple pitanga extract for 48 and 72 h, and the percentage of dead cell stained with 7-amino-actinomycin D was significantly higher in treated cells. The reduction of cell proliferation was dose dependent, and we also observed alterations on cell cycle progression. At all times studied, GRX cells treated with 50 and 100 µg ml(-1) of purple pitanga showed a significant reduction in cellular mitochondrial content as well as a decrease in mitochondrial membrane potential. Furthermore, our results indicated that purple pitanga extract induces early and late apoptosis/necrosis and necrotic death in GRX cells. This is the first report describing the antiproliferative, cytotoxic and apoptotic activity for E. uniflora fruits in hepatic stellate cells. The present study provides a foundation for the prevention and treatment of liver fibrosis, and more studies will be carried to elucidate this effect. PMID:23475531

  19. Amnion-derived stem cells: in quest of clinical applications

    PubMed Central

    2011-01-01

    In the promising field of regenerative medicine, human perinatal stem cells are of great interest as potential stem cells with clinical applications. Perinatal stem cells could be isolated from normally discarded human placentae, which are an ideal cell source in terms of availability, the fewer number of ethical concerns, less DNA damage, and so on. Numerous studies have demonstrated that some of the placenta-derived cells possess stem cell characteristics like pluripotent differentiation ability, particularly in amniotic epithelial (AE) cells. Term human amniotic epithelium contains a relatively large number of stem cell marker-positive cells as an adult stem cell source. In this review, we introduce a model theory of why so many AE cells possess stem cell characteristics. We also describe previous work concerning the therapeutic applications and discuss the pluripotency of the AE cells and potential pitfalls for amnion-derived stem cell research. PMID:21596003

  20. Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering

    PubMed Central

    Jiang, Wei-Cheng; Cheng, Yu-Hao; Yen, Meng-Hua; Chang, Yin; Yang, Vincent W.; Lee, Oscar K.

    2015-01-01

    Liver transplantation is the ultimate treatment for severe hepatic failure to date. However, the limited supply of donor organs has severely hampered this treatment. So far, great potentials of using mesenchymal stem cells (MSCs) to replenish the hepatic cell population have been shown; nevertheless, there still is a lack of an optimal three-dimensional scaffold for generation of well-transplantable hepatic tissues. In this study, we utilized a cryo-chemical decellularization method which combines physical and chemical approach to generate acellular liver scaffolds (ALS) from the whole liver. The produced ALS provides a biomimetic three-dimensional environment to support hepatic differentiation of MSCs, evidenced by expression of hepatic-associated genes and marker protein, glycogen storage, albumin secretion, and urea production. It is also found that hepatic differentiation of MSCs within the ALS is much more efficient than two-dimensional culture in vitro. Importantly, the hepatic-like tissues (HLT) generated by repopulating ALS with MSCs are able to act as functional grafts and rescue lethal hepatic failure after transplantation in vivo. In summary, the cryo-chemical method used in this study is suitable for decellularization of liver and create acellular scaffolds that can support hepatic differentiation of MSCs and be used to fabricate functional tissue-engineered liver constructs. PMID:24462361

  1. Changes in the protein expression profiles of the Hepa-T1 cell line when exposed to Cu2+.

    PubMed

    Chen, Dong-Shi; Chan, King Ming

    2009-09-14

    Copper is an essential element in a variety of biological processes, but it can be toxic when present in excessive amounts. The central regulators of cellular copper metabolism include copper-binding proteins, copper transporters, metal membrane active transporters and copper-dependent enzymes. However, the way in which cupric ions (Cu(2+)) cause cellular changes in proteins and lead to toxic effects is less well-known. The aim of this study is to identify the proteins related to Cu(2+) toxicity or detoxification mechanisms in tilapia (Oreochromis niloticus) using a proteomic approach. A cell line derived from the liver of tilapia, Hepa-T1, was used as a model and exposed to two sub-lethal concentrations of waterborne copper for 96 h. The proteins expressed in Hepa-T1 were investigated by differential protein profiling using two-dimensional gel electrophoresis (2DE). It was found that Cu(2+) (120 and 300 microM) caused the differential expression of 93 different proteins, 18 of which were further verified by real-time quantitative polymerase chain reaction (PCR) analysis. Following analysis with ingenuity pathway software, several proteins were found to be involved in lipid metabolism, tissue connective development and cell cycle control, thus indicating that copper toxicity affects these cellular functions. PMID:19616320

  2. TLR4-Dependent Secretion by Hepatic Stellate Cells of the Neutrophil-Chemoattractant CXCL1 Mediates Liver Response to Gut Microbiota

    PubMed Central

    Ebrahimkhani, Mohammad R.; Shimizu-Albergine, Masami; Campbell, Jean S.; Crispe, Ian N.

    2016-01-01

    Background & Aims The gut microbiota significantly influences hepatic immunity. Little is known on the precise mechanism by which liver cells mediate recognition of gut microbes at steady state. Here we tested the hypothesis that a specific liver cell population was the sensor and we aimed at deciphering the mechanism by which the activation of TLR4 pathway would mediate liver response to gut microbiota. Methods Using microarrays, we compared total liver gene expression in WT versus TLR4 deficient mice. We performed in situ localization of the major candidate protein, CXCL1. With an innovative technique based on cell sorting, we harvested enriched fractions of KCs, LSECs and HSCs from the same liver. The cytokine secretion profile was quantified in response to low levels of LPS (1ng/mL). Chemotactic activity of stellate cell-derived CXCL1 was assayed in vitro on neutrophils upon TLR4 activation. Results TLR4 deficient liver had reduced levels of one unique chemokine, CXCL1 and subsequent decreased of neutrophil counts. Depletion of gut microbiota mimicked TLR4 deficient phenotype, i.e., decreased neutrophils counts in the liver. All liver cells were responsive to low levels of LPS, but hepatic stellate cells were the major source of chemotactic levels of CXCL1. Neutrophil migration towards secretory hepatic stellate cells required the TLR4 dependent secretion of CXCL1. Conclusions Showing the specific activation of TLR4 and the secretion of one major functional chemokine—CXCL1, the homolog of human IL-8-, we elucidate a new mechanism in which Hepatic Stellate Cells play a central role in the recognition of gut microbes by the liver at steady state. PMID:27002851

  3. Kupffer cell inactivation by carbon monoxide bound to red blood cells preserves hepatic cytochrome P450 via anti-oxidant and anti-inflammatory effects exerted through the HMGB1/TLR-4 pathway during resuscitation from hemorrhagic shock.

    PubMed

    Ogaki, Shigeru; Taguchi, Kazuaki; Maeda, Hitoshi; Watanabe, Hiroshi; Ishima, Yu; Otagiri, Masaki; Maruyama, Toru

    2015-10-01

    Red blood cell (RBC) transfusions for controlling hemorrhaging induce systemic ischemia reperfusion, resulting in a decrease in hepatic cytochrome P450 (CYP) levels. Carbon monoxide (CO), when bound to red blood cells (CO-RBC) has the potential to protect the hepatic CYP protein to produce a resuscitative effect in a hemorrhagic shock rat model. The aim of this study was to investigate the mechanism by which CO-RBC resuscitation from a massive hemorrhage protects against a decrease in hepatic CYP. In the early phase (∼1h) after a hemorrhage and RBC resuscitation, hepatic CYP protein levels were significantly decreased with increasing hepatic free heme levels, but were maintained by a pre-treatment of gadolinium chloride (GdCl3), a Kupffer cell inhibitor, and Trolox, an anti-oxidant agent, as well as CO-RBC resuscitation. Under these conditions, the production of reactive oxygen species (ROS) derived from activated Kupffer cells was increased, but this increase was suppressed by CO-RBC resuscitation. At a late phase (6∼24h), CYP mRNA levels decreased after hemorrhage and RBC resuscitation, but not in the case of CO-RBC resuscitation. The increases in plasma IL-6 and TNF-α levels were decreased by CO-RBC resuscitation via the suppression of the toll-like receptor-4 (TLR-4) and the expression of the high mobility group box-1 (HMGB-1). Hepatic CYP protection after a hemorrhage and CO-RBC resuscitation can be attributed to the inactivation of Kupffer cells, resulting in the suppression of ROS production in the early phase and the suppression of inflammatory cytokine production via the TLR-4/HMGB-1signal pathway in the late phase. PMID:26232728

  4. Graphene and its derivatives for cell biotechnology.

    PubMed

    Yang, Mei; Yao, Jun; Duan, Yixiang

    2013-01-01

    Every few years, a novel material with salient and often unique properties emerges and attracts both academic and industrial interest from the scientific community. The latest blockbuster is graphene, an increasingly important nanomaterial with atomically thin sheets of carbon, which has become a shining star and has shown great promise in the field of material science and nanotechnology. In recent years, it has changed from being the exclusive domain of physicists to the new passion of chemists and biologists. Graphene and its derivatives are now at the forefront of nearly every rapidly developing field of science and engineering, including biochemistry, biomedicine and certain cutting-edge interdisciplines that have intense popularity. The aim of this review is, firstly, to provide readers with a comprehensive, systematic and in-depth prospective of graphene's band structure and properties, and secondly, to concentrate on the recent progress in producing graphene-based nanomaterials, including mechanical exfoliation, chemical vapor deposition, plasma enhanced chemical vapor deposition, chemical reduction of graphene oxide, total organic synthesis, electrochemical synthesis and other fabrication strategies widely accepted by research scientists. At the same time, important definitions related to graphene are also introduced. The focus of this Tutorial Review is to emphasize the current situation and significance of using this new kind of two-dimensional material in the hot and emerging fields that are closely related to human life quality, for instance, cell biochemistry, bioimaging along with other frontier areas. Finally, the latest developments and possible impact that affect the heart of the whole scientific community have been discussed. In addition, the future trends along with potential challenges of this rapidly rising layered carbon have been pointed out in this paper. PMID:23115773

  5. Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

    SciTech Connect

    Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J.

    2013-04-15

    Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional program encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation. - Highlights: ► Circadian-dependent physiological variation impacts therapeutic efficacy. ► Hepatic leukemia factor inhibits cell death and is a candidate circadian factor. ► Hepatic leukemia factor anti-death program is conserved in murine and human cells. ► Transcriptomics indicates the anti-death program results from a systems response.

  6. Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

    SciTech Connect

    Corcelle, V.; Stieger, B.; Gjinovci, A.; Wollheim, C.B.; Gauthier, B.R. . E-mail: Benoit.Gauthier@medecine.unige.ch

    2006-09-10

    Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl{sub 4}. Livers were removed 9 to 13 days post-CCl{sub 4} treatment. Immunohistochemistry was performed using anti-c-kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c-kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b{sup +} cells fail to propagate while c-kit {sup +}-HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c-kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c-kit {sup +}-HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion.

  7. Degradable hydrogels derived from PEG-diacrylamide for hepatic tissue engineering.

    PubMed

    Stevens, Kelly R; Miller, Jordan S; Blakely, Brandon L; Chen, Christopher S; Bhatia, Sangeeta N

    2015-10-01

    Engineered tissue constructs have the potential to augment or replace whole organ transplantation for the treatment of liver failure. Poly(ethylene glycol) (PEG)-based systems are particularly promising for the construction of engineered liver tissue due to their biocompatibility and amenability to modular addition of bioactive factors. To date, primary hepatocytes have been successfully encapsulated in non-degradable hydrogels based on PEG-diacrylate (PEGDA). In this study, we describe a hydrogel system based on PEG-diacrylamide (PEGDAAm) containing matrix-metalloproteinase sensitive (MMP-sensitive) peptide in the hydrogel backbone that is suitable for hepatocyte culture both in vitro and after implantation. By replacing hydrolytically unstable esters in PEGDA with amides in PEGDAAm, resultant hydrogels resisted non-specific hydrolysis, while still allowing for MMP-mediated hydrogel degradation. Optimization of polymerization conditions, hepatocellular density, and multicellular tissue composition modulated both the magnitude and longevity of hepatic function in vitro. Importantly, hepatic PEGDAAm-based tissues survived and functioned for over 3 weeks after implantation ectopically in the intraperitoneal (IP) space of nude mice. Together, these studies suggest that MMP-sensitive PEGDAAm-based hydrogels may be a useful material system for applications in tissue engineering and regenerative medicine. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3331-3338, 2015. PMID:25851120

  8. Degradable hydrogels derived from PEG-diacrylamide for hepatic tissue engineering

    PubMed Central

    Stevens, Kelly R.; Miller, Jordan S.; Blakely, Brandon L.; Chen, Christopher S.; Bhatia, Sangeeta N.

    2016-01-01

    Engineered tissue constructs have the potential to augment or replace whole organ transplantation for the treatment of liver failure. Poly(ethylene glycol) (PEG)-based systems are particularly promising for the construction of engineered liver tissue due to their biocompatibility and amenability to modular addition of bioactive factors. To date, primary hepatocytes have been successfully encapsulated in non-degradable hydrogels based on PEG-diacrylate (PEGDA). In this study, we describe a hydrogel system based on PEG-diacrylamide (PEGDAAm) containing matrix-metalloproteinase sensitive (MMP-sensitive) peptide in the hydrogel backbone that is suitable for hepatocyte culture both in vitro and after implantation. By replacing hydrolytically unstable esters in PEGDA with amides in PEGDAAm, resultant hydrogels resisted non-specific hydrolysis, while still allowing for MMP-mediated hydrogel degradation. Optimization of polymerization conditions, hepatocellular density, and multicellular tissue composition modulated both the magnitude and longevity of hepatic function in vitro. Importantly, hepatic PEGDAAm-based tissues survived and functioned for over three weeks after implantation ectopically in the intraperitoneal (IP) space of nude mice. Together, these studies suggest that MMP-sensitive PEGDAAm-based hydrogels may be a useful material system for applications in tissue engineering and regenerative medicine. PMID:25851120

  9. Naturally derived anti-hepatitis B virus agents and their mechanism of action.

    PubMed

    Wu, Yi-Hang

    2016-01-01

    Despite that some approved drugs and genetically engineered vaccines against hepatitis B virus (HBV) are available for HBV patients, HBV infection is still a severe public health problem in the world. All the approved therapeutic drugs (including interferon-alpha and nucleoside analogues) have their limitations. No drugs or therapeutic methods can cure hepatitis B so far. Therefore, it is urgently needed to discover and develop new anti-HBV drugs, especially non-nucleoside agents. Naturally originated compounds with enormous molecular complexity and diversity offer a great opportunity to find novel anti-HBV lead compounds with specific antiviral mechanisms. In this review, the natural products against HBV are discussed according to their chemical classes such as terpenes, lignans, phenolic acids, polyphenols, lactones, alkaloids and flavonoids. Furthermore, novel mode of action or new targets of some representative anti-HBV natural products are also discussed. The aim of this review is to report new discoveries and updates pertaining to anti-HBV natural products in the last 20 years, especially novel skeletons and mode of action. Although many natural products with various skeletons have been reported to exhibit potent anti-HBV effects to date, scarcely any of them are found in the list of conventional anti-HBV drugs worldwide. Additionly, in anti-HBV mechanism of action, only a few references reported new targets or novel mode of action of anti-HBV natural products. PMID:26755870

  10. Naturally derived anti-hepatitis B virus agents and their mechanism of action

    PubMed Central

    Wu, Yi-Hang

    2016-01-01

    Despite that some approved drugs and genetically engineered vaccines against hepatitis B virus (HBV) are available for HBV patients, HBV infection is still a severe public health problem in the world. All the approved therapeutic drugs (including interferon-alpha and nucleoside analogues) have their limitations. No drugs or therapeutic methods can cure hepatitis B so far. Therefore, it is urgently needed to discover and develop new anti-HBV drugs, especially non-nucleoside agents. Naturally originated compounds with enormous molecular complexity and diversity offer a great opportunity to find novel anti-HBV lead compounds with specific antiviral mechanisms. In this review, the natural products against HBV are discussed according to their chemical classes such as terpenes, lignans, phenolic acids, polyphenols, lactones, alkaloids and flavonoids. Furthermore, novel mode of action or new targets of some representative anti-HBV natural products are also discussed. The aim of this review is to report new discoveries and updates pertaining to anti-HBV natural products in the last 20 years, especially novel skeletons and mode of action. Although many natural products with various skeletons have been reported to exhibit potent anti-HBV effects to date, scarcely any of them are found in the list of conventional anti-HBV drugs worldwide. Additionly, in anti-HBV mechanism of action, only a few references reported new targets or novel mode of action of anti-HBV natural products. PMID:26755870

  11. TCR gene-modified T cells can efficiently treat established hepatitis C-associated hepatocellular carcinoma tumors.

    PubMed

    Spear, Timothy T; Callender, Glenda G; Roszkowski, Jeffrey J; Moxley, Kelly M; Simms, Patricia E; Foley, Kendra C; Murray, David C; Scurti, Gina M; Li, Mingli; Thomas, Justin T; Langerman, Alexander; Garrett-Mayer, Elizabeth; Zhang, Yi; Nishimura, Michael I

    2016-03-01

    The success in recent clinical trials using T cell receptor (TCR)-genetically engineered T cells to treat melanoma has encouraged the use of this approach toward other malignancies and viral infections. Although hepatitis C virus (HCV) infection is being treated with a new set of successful direct anti-viral agents, potential for virologic breakthrough or relapse by immune escape variants remains. Additionally, many HCV(+) patients have HCV-associated disease, including hepatocellular carcinoma (HCC), which does not respond to these novel drugs. Further exploration of other approaches to address HCV infection and its associated disease are highly warranted. Here, we demonstrate the therapeutic potential of PBL-derived T cells genetically engineered with a high-affinity, HLA-A2-restricted, HCV NS3:1406-1415-reactive TCR. HCV1406 TCR-transduced T cells can recognize naturally processed antigen and elicit CD8-independent recognition of both peptide-loaded targets and HCV(+) human HCC cell lines. Furthermore, these cells can mediate regression of established HCV(+) HCC in vivo. Our results suggest that HCV TCR-engineered antigen-reactive T cells may be a plausible immunotherapy option to treat HCV-associated malignancies, such as HCC. PMID:26842125

  12. Hepatic stem cells: A viable approach for the treatment of liver cirrhosis

    PubMed Central

    Habeeb, Md Aejaz; Vishwakarma, Sandeep Kumar; Bardia, Avinash; Khan, Aleem Ahmed

    2015-01-01

    Liver cirrhosis is characterized by distortion of liver architecture, necrosis of hepatocytes and regenerative nodules formation leading to cirrhosis. Various types of cell sources have been used for the management and treatment of decompensated liver cirrhosis. Knowledge of stem cells has offered a new dimension for regenerative therapy and has been considered as one of the potential adjuvant treatment modality in patients with end stage liver diseases (ESLD). Human fetal hepatic progenitor cells are less immunogenic than adult ones. They are highly propagative and challenging to cryopreservation. In our earlier studies we have demonstrated that fetuses at 10-18 wk of gestation age contain a large number of actively dividing hepatic stem and progenitor cells which possess bi-potent nature having potential to differentiate into bile duct cells and mature hepatocytes. Hepatic stem cell therapy for the treatment of ESLD is in their early stage of the translation. The emerging technology of decellularization and recellularization might offer a significant platform for developing bioengineered personalized livers to come over the scarcity of desired number of donor organs for the treatment of ESLD. Despite these significant advancements long-term tracking of stem cells in human is the most important subject nowadays in order to answer several unsettles issues regarding the route of delivery, the choice of stem cell type(s), the cell number and the time-point of cell delivery for the treatment in a chronic setting. Answering to these questions will further contribute to the development of safer, noninvasive, and repeatable imaging modalities that could discover better cell therapeutic approaches from bench to bed-side. Combinatorial approach of decellularization and nanotechnology could pave a way towards the better understanding in determination of cell fate post-transplantation. PMID:26131316

  13. Hepatic stem cells: A viable approach for the treatment of liver cirrhosis.

    PubMed

    Habeeb, Md Aejaz; Vishwakarma, Sandeep Kumar; Bardia, Avinash; Khan, Aleem Ahmed

    2015-06-26

    Liver cirrhosis is characterized by distortion of liver architecture, necrosis of hepatocytes and regenerative nodules formation leading to cirrhosis. Various types of cell sources have been used for the management and treatment of decompensated liver cirrhosis. Knowledge of stem cells has offered a new dimension for regenerative therapy and has been considered as one of the potential adjuvant treatment modality in patients with end stage liver diseases (ESLD). Human fetal hepatic progenitor cells are less immunogenic than adult ones. They are highly propagative and challenging to cryopreservation. In our earlier studies we have demonstrated that fetuses at 10-18 wk of gestation age contain a large number of actively dividing hepatic stem and progenitor cells which possess bi-potent nature having potential to differentiate into bile duct cells and mature hepatocytes. Hepatic stem cell therapy for the treatment of ESLD is in their early stage of the translation. The emerging technology of decellularization and recellularization might offer a significant platform for developing bioengineered personalized livers to come over the scarcity of desired number of donor organs for the treatment of ESLD. Despite these significant advancements long-term tracking of stem cells in human is the most important subject nowadays in order to answer several unsettles issues regarding the route of delivery, the choice of stem cell type(s), the cell number and the time-point of cell delivery for the treatment in a chronic setting. Answering to these questions will further contribute to the development of safer, noninvasive, and repeatable imaging modalities that could discover better cell therapeutic approaches from bench to bed-side. Combinatorial approach of decellularization and nanotechnology could pave a way towards the better understanding in determination of cell fate post-transplantation. PMID:26131316

  14. Discovery of cytoglobin and its roles in physiology and pathology of hepatic stellate cells

    PubMed Central

    YOSHIZATO, Katsutoshi; THUY, Le Thi Thanh; SHIOTA, Goshi; KAWADA, Norifumi

    2016-01-01

    Cytoglobin (CYGB), a new member of the globin family, was discovered in 2001 as a protein associated with stellate cell activation (stellate cell activation-associated protein [STAP]). Knowledge of CYGB, including its crystal, gene, and protein structures as well as its physiological and pathological importance, has increased progressively. We investigated the roles of oxygen (O2)-binding CYGB as STAP in hepatic stellate cells (HSCs) to understand the part played by this protein in their pathophysiological activities. Studies involving CYGB-gene-deleted mice have led us to suppose that CYGB functions as a regulator of O2 homeostasis; when O2 homeostasis is disrupted, HSCs are activated and play a key role(s) in hepatic fibrogenesis. In this review, we discuss the rationale for this hypothesis. PMID:26972599

  15. Discovery of cytoglobin and its roles in physiology and pathology of hepatic stellate cells.

    PubMed

    Yoshizato, Katsutoshi; Thuy, Le Thi Thanh; Shiota, Goshi; Kawada, Norifumi

    2016-01-01

    Cytoglobin (CYGB), a new member of the globin family, was discovered in 2001 as a protein associated with stellate cell activation (stellate cell activation-associated protein [STAP]). Knowledge of CYGB, including its crystal, gene, and protein structures as well as its physiological and pathological importance, has increased progressively. We investigated the roles of oxygen (O2)-binding CYGB as STAP in hepatic stellate cells (HSCs) to understand the part played by this protein in their pathophysiological activities. Studies involving CYGB-gene-deleted mice have led us to suppose that CYGB functions as a regulator of O2 homeostasis; when O2 homeostasis is disrupted, HSCs are activated and play a key role(s) in hepatic fibrogenesis. In this review, we discuss the rationale for this hypothesis. PMID:26972599

  16. Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells.

    PubMed Central

    Sakamuro, D; Furukawa, T; Takegami, T

    1995-01-01

    Clinical evidence suggests that hepatitis C virus (HCV) is etiologically involved in hepatic cancer and liver cirrhosis. To investigate whether the HCV nonstructural protein NS3 has oncogenic activity, NIH 3T3 cells were transfected with an expression vector containing cDNA for the 5'- or 3'-half sequence of the HCV genome segment encoding NS3. Only cells transfected with the 5'-half cDNA rapidly proliferated, lost contact inhibition, grew anchorage independently in soft agar, and formed tumors in nude mice. PCR analysis confirmed the presence of the 5'-half DNA in the transfectants. These results suggest that the 5' region of the HCV genome segment encoding NS3 is involved in cell transformation. PMID:7745741

  17. Antifibrotic activity of coumarins from Cnidium monnieri fruits in HSC-T6 hepatic stellate cells.

    PubMed

    Shin, Eunjin; Lee, Chul; Sung, Sang Hyun; Kim, Young Choong; Hwang, Bang Yeon; Lee, Mi Kyeong

    2011-04-01

    The CHCl(3) fraction of Cnidium monnieri fruits significantly inhibited the proliferation of hepatic stellate cells in an in-vitro assay system employing HSC-T6 hepatic stellate cell lines. Activity-guided fractionation of the CHCl(3) fraction of C. monnieri led to the isolation of ten coumarins: osthol (1), meranzin (2), auraptenol (3), meranzin hydrate (4), 7-hydroxy-8-methoxy coumarin (5), imperatorin (6), xanthotoxol (7), xanthotoxin (8), bergapten (9) and isopimpinellin (10). Of these, compounds 1 and 6 significantly inhibited proliferation of HSCs in a time- and concentration-dependent manner. In addition, compounds 1 and 6 significantly reduced collagen content in HSC-T6 cells. PMID:21082271

  18. Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells[S

    PubMed Central

    Röhrl, Clemens; Eigner, Karin; Winter, Katharina; Korbelius, Melanie; Obrowsky, Sascha; Kratky, Dagmar; Kovacs, Werner J.; Stangl, Herbert

    2014-01-01

    Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function. PMID:24179149

  19. Bone marrow-derived cell regulation of skeletal muscle regeneration.

    PubMed

    Sun, Dongxu; Martinez, Carlo O; Ochoa, Oscar; Ruiz-Willhite, Lourdes; Bonilla, Jose R; Centonze, Victoria E; Waite, Lindsay L; Michalek, Joel E; McManus, Linda M; Shireman, Paula K

    2009-02-01

    Limb regeneration requires the coordination of multiple stem cell populations to recapitulate the process of tissue formation. Therefore, bone marrow (BM) -derived cell regulation of skeletal muscle regeneration was examined in mice lacking the CC chemokine receptor 2 (CCR2). Myofiber size, numbers of myogenic progenitor cells (MPCs), and recruitment of BM-derived cells and macrophages were assessed after cardiotoxin-induced injury of chimeric mice produced by transplanting BM from wild-type (WT) or CCR2(-/-) mice into irradiated WT or CCR2(-/-) host mice. Regardless of the host genotype, muscle regeneration and recruitment of BM-derived cells and macrophages were similar in mice replenished with WT BM, whereas BM-derived cells and macrophage accumulation were decreased and muscle regeneration was impaired in all animals receiving CCR2(-/-) BM. Furthermore, numbers of MPCs (CD34(+)/Sca-1(-)/CD45(-) cells) were significantly increased in mice receiving CCR2(-/-) BM despite the decreased size of regenerated myofibers. Thus, the expression of CCR2 on BM-derived cells regulated macrophage recruitment into injured muscle, numbers of MPC, and the extent of regenerated myofiber size, all of which were independent of CCR2 expression on host-derived cells. Future studies in regenerative medicine must include consideration of the role of BM-derived cells, possibly macrophages, in CCR2-dependent events that regulate effective skeletal muscle regeneration. PMID:18827026

  20. Human Amnion-Derived Mesenchymal Stem Cell Transplantation Ameliorates Liver Fibrosis in Rats

    PubMed Central

    Kubo, Kimitoshi; Ohnishi, Shunsuke; Hosono, Hidetaka; Fukai, Moto; Kameya, Ayano; Higashi, Ryosuke; Yamada, Takahiro; Onishi, Reizo; Yamahara, Kenichi; Takeda, Hiroshi; Sakamoto, Naoya

    2015-01-01

    Background Mesenchymal stem cells (MSCs) are a valuable cell source in regenerative medicine. Recently, several studies have shown that MSCs can be easily isolated from human amnion. In this study, we investigated the therapeutic effect of transplantation of human amnion-derived MSCs (hAMSCs) in rats with liver fibrosis. Methods Liver fibrosis was induced by an intraperitoneal injection of 2 mL/kg of 50% carbon tetrachloride twice a week for 6 weeks. At 3 weeks, hAMSCs (1 × 106 cells) were transplanted intravenously. Rats were sacrificed at 7 weeks, and histological analyses and quantitative reverse-transcription polymerase chain reaction were performed. In vitro experiments were conducted to investigate the effect of hAMSCs on the activation of Kupffer cells. Results Transplantation of hAMSCs significantly reduced the fibrotic area, deposition of type-I collagen, the number of α-smooth muscle actin–positive hepatic stellate cells, and CD68-positive Kupffer cells in the livers. messenger RNA expression of α-smooth muscle actin and tissue inhibitor of metalloproteinase-1 was significantly decreased and the expression of matrix metalloproteinase-9 and hepatocyte growth factor was significantly increased in the liver of hAMSC-treated rats. Transplantation of hAMSCs at 3 weeks plus 5 weeks did not have an additive effect. In vitro experiments demonstrated that Kupffer cell activation induced by lipopolysaccharide was significantly decreased by culturing with conditioned medium obtained from hAMSCs. Conclusions Transplantation of hAMSCs provided significant improvement in a rat model of liver fibrosis, possibly through the inhibition of Kupffer cell and hepatic stellate cell activation. hAMSCs may be a potential new treatment for liver fibrosis.

  1. Synthesis and Quantitative Structure-activity Relationships Study for Arylpropenamide Derivatives as Inhibitors of Hepatitis B Virus Replication.

    PubMed

    Min, Ma; Xingjun, Jiang; Xueding, Wang; Hao, Zou; Weiqing, Yang; Yuanyuan, Zhang; Changrong, Peng; Zicheng, Li; Jing, Yang; Quan, Du; Menglin, Ma

    2016-09-01

    A series of new arylpropenamide derivatives containing different aryl groups were synthesized, characterized, and evaluated for their anti-hepatitis B virus (HBV) activities. A new high accuracy QSAR model of arylpropenamide was constructed based on a more completely activities data and calculation parameter. The 2D-QSAR equations, by using DFT and multiple linear regression analysis methods, revealed that higher value of thermal energy (TE) and lower entropy (S(ө) ) increase the anti-HBV activities of the arylpropenamide molecules. Predictive 3D-QSAR models were established by SYBYL multifit molecular alignment rule. The optimum models were all statistically significant with cross-validated and conventional coefficients, indicating that they were reliable enough for activity prediction. PMID:27085815

  2. Biomarkers distinguish apoptotic and necrotic cell death during hepatic ischemia/reperfusion injury in mice.

    PubMed

    Yang, Min; Antoine, Daniel J; Weemhoff, James L; Jenkins, Rosalind E; Farhood, Anwar; Park, B Kevin; Jaeschke, Hartmut

    2014-11-01

    Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery (Pringle maneuver) and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. To address this important issue with a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA 122, full-length cytokeratin 18 (FK18), and high-mobility group box 1 (HMGB1) protein and plasma biomarkers of apoptosis such as plasma caspase-3 activity and caspase-cleaved fragment of cytokeratin 18 (CK18) coupled with markers of inflammation (hyperacetylated HMGB1) were compared by histological features in hematoxylin and eosin-stained and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-stained liver sections. After 45 minutes of hepatic ischemia and 1 to 24 hours of reperfusion, all necrosis markers increased dramatically in plasma by 40- to >10,000-fold over the baseline with a time course similar to that of alanine aminotransferase. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL positive; initially (≤3 hours of reperfusion), the staining was restricted to nuclei, but it later spread to the cytosol, and this is characteristic of karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except for a minor increase at 3 hours of reperfusion) in plasma. A quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be the elucidation of necrotic signaling mechanisms to

  3. BIOMARKERS DISTINGUISH APOPTOTIC AND NECROTIC CELL DEATH DURING HEPATIC ISCHEMIA-REPERFUSION INJURY IN MICE

    PubMed Central

    Yang, Min; Antoine, Daniel J.; Weemhoff, James L.; Jenkins, Rosalind E.; Farhood, Anwar; Park, B. Kevin; Jaeschke, Hartmut

    2014-01-01

    Hepatic ischemia-reperfusion (IRP) injury is a significant clinical problem during tumor resection surgery (Pringle maneuver), and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. In order to address this important issue in a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA-122, full-length cytokeratin-18 (FK18) and high mobility group box-1 (HMGB1) protein, and apoptosis including plasma caspase-3 activity and caspase-cleaved cytokeratin-18 (CK18), coupled with markers of inflammation (hyper-acetylated HMGB1) were compared with histological features in H&E- and TUNEL-stained liver sections. After 45 min of hepatic ischemia and 1–24h of reperfusion, all necrosis markers increased dramatically in plasma by 40-to->10,000-fold over baseline with a time course similar to ALT. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL-positive; initially (≤ 3h of RP) the staining was restricted to nuclei but later spread to the cytosol characteristic for karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except a minor increase at 3h RP) in plasma. The quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated the dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be on the elucidation of necrotic signaling mechanisms to identify relevant targets, which may be used to attenuate hepatic IRP injury. PMID:25046819

  4. Exosomes mediate intercellular transfer of pro-fibrogenic connective tissue growth factor (CCN2) between hepatic stellate cells, the principal fibrotic cells in the liver

    PubMed Central

    Charrier, Alyssa; Chen, Ruju; Chen, Li; Kemper, Sherri; Hattori, Takako; Takigawa, Masaharu; Brigstock, David R.

    2014-01-01

    Background Fibrogenic pathways in the liver are principally regulated by hepatic stellate cells (HSC) which produce and respond to fibrotic mediators such as connective tissue growth factor (CCN2). The aim of this study was to determine whether CCN2 is shuttled between HSC in membraneous nanovesicles, or “exosomes”. Methods Exosomes were incubated with HSC after isolation from conditioned medium of control or CCN2-GFP-transfected primary mouse HSC or human LX-2 HSC. Some exosomes were flourescently stained with PKH26. HSC co-culture experiments were performed in the presence of GW4869 exosome inhibitor. CCN2 or CCN2-GFP were evaluated by qRT-PCR or Western blot. Results HSC-derived exosomes contained CCN2 or CCN2 mRNA, each of which increased in concentration during HSC activation or after transfection of HSC with CCN2-GFP. Exosomes, stained with either PKH26 or purified from CCN2-GFP-transfected cells, were taken up by activated or quiescent HSC resulting in CCN2-GFP delivery, as shown by their direct addition to recipient cells or by the GW4869-dependency of donor HSC. Conclusions CCN2 is packaged into secreted nano-sized exosomes which mediate its intercellular transfer between HSC. Exosomal CCN2 may amplify or fine-tune fibrogenic signaling and may, in conjunction with other exosome constituents, have utility as a noninvasive biomarker to assess hepatic fibrosis. PMID:24882759

  5. MIF, secreted by human hepatic sinusoidal endothelial cells, promotes chemotaxis and outgrowth of colorectal cancer in liver prometastasis.

    PubMed

    Hu, Chun-Ting; Guo, Li-Li; Feng, Na; Zhang, Lei; Zhou, Na; Ma, Li-Li; Shen, Lan; Tong, Gui-Hui; Yan, Qian-Wen; Zhu, Shi-Jie; Bian, Xiu-Wu; Lai, Mao D; Deng, Yong-Jian; Ding, Yan-Qing

    2015-09-01

    Growth and invasion of metastatic colorectal cancer (CRC) cells in the liver depend on microenvironment. Here, we showed that human hepatic sinusoidal endothelial cells (HHSECs) induce chemotaxis and outgrowth of CRC cells. Macrophage migration inhibitory factor (MIF), released by HHSECs, stimulated chemotaxis of CRC cells. MIF secreted by HHSECs, but not by CRC cells themselves, promoted migration and epithelial-mesenchymal transition (EMT) and facilitated proliferation and apoptotic resistance of CRC cells. In orthotopic implantation models in nude mice, exogenous MIF stimulated growth of CRC cells and metastasis. Furthermore, MIF accelerated mobility of CRC cells by suppressing F-actin depolymerization and phosphorylating cofilin. Noteworthy, MIF levels were correlated with the size of hepatic metastases. We suggest that HHSECs and paracrine MIF promote initial migration and proliferation of CRC cells in the hepatic sinusoids to generate liver metastases. PMID:26087187

  6. Thy-1 Is Expressed in Hepatic Myofibroblasts and Not Oval Cells in Stem Cell-Mediated Liver Regeneration

    PubMed Central

    Dezső, Katalin; Jelnes, Peter; László, Viktória; Baghy, Kornélia; Bödör, Csaba; Paku, Sándor; Tygstrup, Niels; Bisgaard, Hanne Cathrine; Nagy, Peter

    2007-01-01

    Thy-1, a marker of hematopoietic stem cells, has been reported to be expressed by oval cells proliferating during stem cell-mediated regeneration in rat liver, suggesting a relationship between the two cell populations. Consequently, Thy-1 has become an accepted cell surface marker to sort hepatic oval cells. In the present study we used the well-characterized 2-acetylaminfluorene/partial hepatectomy model to induce transit-amplification of hepatic oval cells in the regenerating liver and characterized Thy-1 expression using Northern hybridization, quantitative reverse transcriptase-polymerase chain reaction analysis, immunofluorescence confocal microscopy, and immunoelectronmicroscopy. We found that Thy-1 expression was induced during transit-amplification of the oval cell population, but Thy-1 mRNA was not present in the α-fetoprotein-expressing oval cells. Thy-1 protein was consistently present outside the basement membrane surrounding the oval cells. It overlapped frequently with smooth muscle actin staining. A similar cellular localization of the Thy-1 protein was found on human liver specimens with ductular reactions obtained from patients with fulminant liver failure. Furthermore, Thy-1 was expressed by myofibroblasts in experimental liver fibrosis models without oval cell proliferation. We conclude that Thy-1 is not a marker of oval cells but is present on a subpopulation of myofibroblasts/stellate cells. PMID:17884967

  7. History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.

    PubMed

    Geerts, A

    2001-08-01

    In 1876, von Kupffer described liver Sternzellen (star-shaped cells). The functions of these cells remained enigmatic for 75 years until Ito observed lipid-containing perisinusoidal cells in human liver. In 1971, Wake demonstrated that the Sternzellen of von Kupffer and the fat-storing cells described by Ito were identical. Wake also established that these cells were important sites of vitamin A storage. Soon thereafter, Kent and Popper demonstrated that the stellate cells were intimately linked to the pathogenesis of hepatic fibrosis. Since then, these cells have been studied in detail. Quiescent stellate cells represent 5-8% of the total number of liver cells. They play a cardinal role in storage and controlled release of retinoids. They control extracellular matrix (ECM) turnover in the space of Disse by secreting the correct amounts of a limited number of ECM molecules, and by releasing matrix metalloproteinases and their inhibitors. By virtue of their long cytoplasmic processes, quiescent stellate cells presumably contribute to the control of blood flow through the sinusoidal capillaries. They are important sources of paracrine, autocrine, juxtacrine, and chemoattractant factors that maintain homeostasis in the microenvironment of the hepatic sinusoid. PMID:11586463

  8. HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells

    PubMed Central

    DeLaForest, Ann; Nagaoka, Masato; Si-Tayeb, Karim; Noto, Fallon K.; Konopka, Genevieve; Battle, Michele A.; Duncan, Stephen A.

    2011-01-01

    The availability of pluripotent stem cells offers the possibility of using such cells to model hepatic disease and development. With this in mind, we previously established a protocol that facilitates the differentiation of both human embryonic stem cells and induced pluripotent stem cells into cells that share many characteristics with hepatocytes. The use of highly defined culture conditions and the avoidance of feeder cells or embryoid bodies allowed synchronous and reproducible differentiation to occur. The differentiation towards a hepatocyte-like fate appeared to recapitulate many of the developmental stages normally associated with the formation of hepatocytes in vivo. In the current study, we addressed the feasibility of using human pluripotent stem cells to probe the molecular mechanisms underlying human hepatocyte differentiation. We demonstrate (1) that human embryonic stem cells express a number of mRNAs that characterize each stage in the differentiation process, (2) that gene expression can be efficiently depleted throughout the differentiation time course using shRNAs expressed from lentiviruses and (3) that the nuclear hormone receptor HNF4A is essential for specification of human hepatic progenitor cells by establishing the expression of the network of transcription factors that controls the onset of hepatocyte cell fate. PMID:21852396

  9. Impact of natural killer cells on chronic hepatitis C and hepatocellular carcinoma.

    PubMed

    Tatsumi, Tomohide; Takehara, Tetsuo

    2016-03-01

    Natural killer (NK) cells are involved in the pathogenesis of hepatitis C viral (HCV) infection and hepatocellular carcinoma (HCC). Recent immunological progresses have revealed the molecular mechanisms of activation or inhibition of NK cells. In patients infected with HCV, the percentages of NK cells are decreased and the NK receptor expression and function of NK cells including cytotoxicity and cytokine production are altered. These alterations in NK cells are associated with persistent infection with HCV, liver injury, liver fibrosis and liver carcinogenesis. In HCV treatment, NK cells play a role in the eradication of HCV in both interferon (IFN)-based therapy and IFN-free therapy using direct-acting antivirals (DAA). In HCC patients, the exhaustion of NK cells that represents lower cytotoxicity and impaired cytokine production may contribute to the progression of HCC. Several immunotherapies targeting NK cells have been reported. NK cell transfer and NK-activating gene therapy have been demonstrated to be effective in mouse liver cancer models and several clinical trials are ongoing. Recently, the role of major histocompatibility complex class I-related chain A (MICA), a human ligand of NKG2D, has attracted attention in the development of HCC. The expression of MICA could be controlled by anti-HCC drugs including sorafenib. A new chemo-immunotherapy may be expected in the treatment of HCC. In this review, we summarize the impact of NK cells on chronic hepatitis C and HCC. PMID:26574168

  10. Can engineered "designer" T cells outsmart chronic hepatitis B?

    PubMed

    Protzer, U; Abken, H

    2010-01-01

    More than 350 million people worldwide are persistently infected with human heptatitis B virus (HBV) and at risk to develop liver cirrhosis and hepatocellular carcinoma making long-term treatment necessary. While a vaccine is available and new antiviral drugs are being developed, elimination of persistently infected cells is still a major issue. Recent efforts in adoptive cell therapy are experimentally exploring immunotherapeutic elimination of HBV-infected cells by means of a biological attack with genetically engineered "designer" T cells. PMID:21188203

  11. Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope.

    PubMed

    Qi, Ying; Zhang, Feng; Zhang, Li; Harrison, Tim J; Huang, Weijin; Zhao, Chenyan; Kong, Wei; Jiang, Chunlai; Wang, Youchun

    2015-01-01

    The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15 g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06 g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope. PMID:26161670

  12. Apoptosis induced by tumor necrosis factor-alpha in rat hepatocyte cell lines expressing hepatitis B virus.

    PubMed Central

    Guilhot, S.; Miller, T.; Cornman, G.; Isom, H. C.

    1996-01-01

    Three well differentiated SV40-immortalized rat hepatocyte cell lines, CWSV1, CWSV2, and CWSV14, and Hepatitis B Virus (HBV)-producing cell lines derived from them were examined for sensitivity to tumor necrosis factor (TNF)-alpha. CWSV1, CWSV2, and CWSV14 cells were co-transfected with a DNA construct containing a dimer of the HBV genome and the neo gene and selected in G418 to generate stable cell lines. Characterization of these cell lines indicated that they contain integrated HBV DNA, contain low molecular weight HBV DNA compatible with the presence of HBV replication intermediates, express HBV transcripts, and produce HBV proteins. The viability of CWSV1, CWSV2, and CWSV2 cells was not significantly altered when they were treated with TNF-alpha at concentrations as high as 20,000 U/ml. The HBV-expressing CWSV1 cell line, SV1di36, and the HBV-expressing CWSV14 cell line, SV14di208, were also not killed when treated with TNF-alpha. However, the HBV-expressing CWSV2 cell line, SV2di366, was extensively killed when treated with TNF-alpha at concentrations ranging from 200 to 20,000 U/ml. Analysis of several different HBV-producing CWSV2 cell lines indicated that TNF-alpha killing depended upon the level of HBV expression. The TNF-alpha-induced cell killing in high HBV-producing CWSV2 cell lines was accompanied by the presence of an oligonucleosomal DNA ladder characteristic of apoptosis. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 9 Figure 10 Figure 11 PMID:8774135

  13. Midgut of the diplopod Urostreptus atrobrunneus: structure, function, and redefinition of hepatic cells.

    PubMed

    Moreira-de-Sousa, C; Iamonte, M; Fontanetti, C S

    2016-07-11

    Diplopods are considered important macroarthropods the soil as part of its maintenance and balance. These animals usually do not occur in high densities, but population explosions caused by environmental disturbances, climate changes, and use of pesticides that eliminate possible competitors, have been reported. The millipede Urostreptus atrobrunneus Pierozzi and Fontanetti, 2006 have become a nuisance to humans in infestation sites in urban centers of the state of Sao Paulo, Brazil. As a contribution to the understanding of this potential pest, this study describes the histology, histochemistry, and ultrastructure of the U. atrobrunneus midgut, and presents the redefinition of hepatic cells somewhat controversial in the literature. The region of the midgut is characterized by the absence of a cuticular intima, and composed of a pseudostratified epithelium on a thick basal membrane, followed by a muscle layer, a layer of hepatic cells, lined by an external membrane. The morphology observed in U. atrobrunneus is similar to that reported for other species of diplopods. The hepatic cells have been previously described as randomly without forming a layer, however, the present results clearly demonstrate that these cells form a continuous layer over the whole midgut. PMID:27409229

  14. Hepatitis B transmission by cell and tissue allografts: How safe is safe enough?

    PubMed Central

    Solves, Pilar; Mirabet, Vicente; Alvarez, Manuel

    2014-01-01

    More than 2 million human tissue transplants (bone, tendon, cartilage, skin, cornea, amniotic membrane, stem cells, heart valve, blood vessel, etc.), are performed worldwide every year. Cells and tissues are shared between countries which have different regulations and laboratory equipment and represent a risk of hepatitis B virus (HBV) transmission that has become a global safety concern. While the risk of transfusion-transmitted HBV infection from blood donations has been estimated, the rate of HBV transmission from donors to recipients of allografts is unknown and varies between different tissues. There are various important ways of reducing the transmission risk, but donor screening and donor testing are still the main factors for preventing HBV transmission. HBV detection is included in the routine screening tests for cell and tissue donors. The standard test for preventing transplant-transmitted hepatitis B is the hepatitis B surface antigen. The implementation of methods involving nucleic acid amplification and the new generation of reactives to detect viral antibodies or antigens with an immunoassay, has increased the sensitivity and the specificity of the screening tests. The objective of our research was to review the literature and critically analyse the different steps for avoiding HBV transmission in cell and tissue donors, focusing on the screening tests performed. PMID:24966613

  15. Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor.

    PubMed

    Kimura, Kumi; Tanida, Mamoru; Nagata, Naoto; Inaba, Yuka; Watanabe, Hitoshi; Nagashimada, Mayumi; Ota, Tsuguhito; Asahara, Shun-ichiro; Kido, Yoshiaki; Matsumoto, Michihiro; Toshinai, Koji; Nakazato, Masamitsu; Shibamoto, Toshishige; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2016-03-15

    Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity. PMID:26947072

  16. Multiple Effects of Berberine Derivatives on Colon Cancer Cells

    PubMed Central

    Guamán Ortiz, Luis Miguel; Dutto, Ilaria; Arcamone, Andrea G.; Buzzetti, Franco

    2014-01-01

    The pharmacological use of the plant alkaloid berberine is based on its antibacterial and anti-inflammatory properties; recently, anticancer activity has been attributed to this compound. To exploit this interesting feature, we synthesized three berberine derivatives, namely, NAX012, NAX014, and NAX018, and we tested their effects on two human colon carcinoma cell lines, that is, HCT116 and SW613-B3, which are characterized by wt and mutated p53, respectively. We observed that cell proliferation is more affected by cell treatment with the derivatives than with the lead compound; moreover, the derivatives proved to induce cell cycle arrest and cell death through apoptosis, thus suggesting that they could be promising anticancer drugs. Finally, we detected typical signs of autophagy in cells treated with berberine derivatives. PMID:25045712

  17. Cell culture-derived HCV cannot infect synovial fibroblasts

    PubMed Central

    Nadeem, Abd-Elshafy D.; Thomas, Pietschmann; Ulf, Müller-Ladner; Elena, Neumann; Anggakusuma, A; Mohamed, Bahgat M.; Frank, Pessler; Patrick, Behrendt

    2015-01-01

    Worldwide 170 million individuals are infected with hepatitis C virus (HCV), up to 45 million of whom are affected by arthropathy. It is unclear whether this is due to viral infection of synovial cells or immune-mediated mechanisms. We tested the capacity of primary synovial fibroblasts to support HCV propagation. Out of the four critical HCV receptors, only CD81 was expressed to any significant extent in OASF and RASF. Consistent with this, pseudotyped HCV particles were unable to infect these cells. Permissiveness for HCV replication was investigated by transfecting cells with a subgenomic replicon of HCV encoding a luciferase reporter. OASF and RASF did not support replication of HCV, possibly due to low expression levels of miR-122. In conclusion, primary human synovial fibroblasts are unable to support propagation of HCV in vitro. HCV-related arthropathy is unlikely due to direct infection of these cells. PMID:26643193

  18. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    SciTech Connect

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  19. An in vitro model for hepatocyte-like cell differentiation from Wharton’s jelly derived-mesenchymal stem cells by cell-base aggregates

    PubMed Central

    Talaei-Khozani, Tahereh; Borhani-Haghighi, Maryam; Ayatollahi, Maryam; Vojdani, Zahra

    2015-01-01

    Aim: The present study investigated the differentiation potential of human Umbilical Cord Mesenchymal Stem Cells (UCMSCs) into hepatic lineage through embryonic body-like aggregate formation in the presence of IGF-1. Background: Cells derived from Wharton’s jelly have been reported to display a wide multilineage differentiation potential, showing some similarities to both embryonic (ESC) and mesenchymal stem cells (MSCs). Patients and methods: Human MSCs isolated from the umbilical cord were plated in 20 μL micro drops. A two-step differentiation protocol was used and the cell aggregates were exposed to the media supplemented with IGF, HGF, oncostatin M, and dexamethasone for 21 days. Immunoperoxidase and immuno-fluorescence were performed for cyrokeratins 18, 19 and albumin. Functional assays were done by periodic acid Schiff (PAS) and indocyanine green. Results: The expression of cytokeratin 19 was shown to be higher in the cells derived from 3D spheroids compared to those cultured in conventional protocol. They showed a polygonal shape after being exposed to hepatogenic media. Immunostaining demonstrated the expression of cytokeratin-18, 19 and albumin by the differentiated cells. Besides, PAS staining revealed glycogen storage in differentiated cells. Also, a greater number of large size differentiated cells were found at the periphery of the expanded cell aggregates. Conclusion: We established a protocol for UCMSC differentiation into hepatocytes and these cells were morphologically and functionally similar to hepatocytes. Thus, hepatocyte differentiation may be facilitated by the UCMSCs aggregate formation before administration of the differentiation protocols. PMID:26328041

  20. Novel management of acute or secondary biliary liver conditions using hepatically differentiated human dental pulp cells.

    PubMed

    Ishkitiev, Nikolay; Yaegaki, Ken; Imai, Toshio; Tanaka, Tomoko; Fushimi, Naho; Mitev, Vanyo; Okada, Mio; Tominaga, Noriko; Ono, Sachie; Ishikawa, Hiroshi

    2015-02-01

    The current definitive treatment for acute or chronic liver condition, that is, cirrhosis, is liver transplantation from a limited number of donors, which might cause complications after donation. Hence, bone marrow stem cell transplantation has been developed, but the risk of carcinogenesis remains. We have recently developed a protocol for hepatic differentiation of CD117(+) stem cells from human exfoliated deciduous teeth (SHED). In the present study, we examine whether SHED hepatically differentiated (hd) in vitro could be used to treat acute liver injury (ALI) and secondary biliary cirrhosis. The CD117(+) cell fraction was magnetically separated from SHED and then differentiated into hepatocyte-like cells in vitro. The cells were transplanted into rats with either ALI or induced secondary biliary cirrhosis. Engraftment of human liver cells was determined immunohistochemically and by in situ hybridization. Recovery of liver function was examined by means of histochemical and serological tests. Livers of transplanted animals were strongly positive for human immunohistochemical factors, and in situ hybridization confirmed engraftment of human hepatocytes. The tests for recovery of liver function confirmed the presence of human hepatic markers in the animals' blood serum and lack of fibrosis and functional integration of transplanted human cells into livers. No evidence of malignancy was found. We show that in vitro hdSHED engraft morphologically and functionally into the livers of rats having acute injury or secondary biliary cirrhosis. SHED are readily accessible adult stem cells, capable of proliferating in large numbers before differentiating in vitro. This makes SHED an appropriate and safe stem cell source for regenerative medicine. PMID:25234861

  1. Homing in on the hepatic scar: recent advances in cell-specific targeting of liver fibrosis

    PubMed Central

    Dobie, Ross; Henderson, Neil C.

    2016-01-01

    Despite the high prevalence of liver disease globally, there are currently no approved anti-fibrotic therapies to treat patients with liver fibrosis. A major goal in anti-fibrotic therapy is the development of drug delivery systems that allow direct targeting of the major pro-scarring cell populations within the liver (hepatic myofibroblasts) whilst not perturbing the homeostatic functions of other mesenchymal cell types present within both the liver and other organ systems. In this review we will outline some of the recent advances in our understanding of myofibroblast biology, discussing both the origin of myofibroblasts and possible myofibroblast fates during hepatic fibrosis progression and resolution. We will then discuss the various strategies currently being employed to increase the precision with which we deliver potential anti-fibrotic therapies to patients with liver fibrosis. PMID:27508067

  2. Endogenous Hepatitis C Virus Homolog Fragments in European Rabbit and Hare Genomes Replicate in Cell Culture

    PubMed Central

    Silva, Eliane; Marques, Sara; Osório, Hugo; Carvalheira, Júlio; Thompson, Gertrude

    2012-01-01

    Endogenous retroviruses, non-retroviral RNA viruses and DNA viruses have been found in the mammalian genomes. The origin of Hepatitis C virus (HCV), the major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans, remains unclear since its discovery. Here we show that fragments homologous to HCV structural and non-structural (NS) proteins present in the European rabbit (Oryctolagus cuniculus) and hare (Lepus europaeus) genomes replicate in bovine cell cultures. The HCV genomic homolog fragments were demonstrated by RT-PCR, PCR, mass spectrometry, and replication in bovine cell cultures by immunofluorescence assay (IFA) and immunogold electron microscopy (IEM) using specific MAbs for HCV NS3, NS4A, and NS5 proteins. These findings may lead to novel research approaches on the HCV origin, genesis, evolution and diversity. PMID:23185448

  3. T helper cell dysregulation with hepatitis B and rebalance with glucocorticoids

    PubMed Central

    Lu, Zhong-Hua; Huang, Xiao-Ping; Sun, Wei; Zhu, Yi-Ling; Cui, Juan-Juan; Chen, Wei; Huang, Li-Hua; Kuai, Shou-Gang; Du, He-Juan; Ju, Zhao-Xia; Gan, Jian-He

    2014-01-01

    AIM: To investigate T helper 17/regulatory T cell alterations in early severe hepatitis B and the effect of glucocorticoids. METHODS: The study included 20 patients in the early stage of severe hepatitis B (SHB) and 11 healthy controls. All patients had elevated T helper 17 (Th17) levels, decreased regulatory T (Treg) cell levels, and significant Th17/Treg ratios. RESULTS: After glucocorticoid treatment, 16 patients showed improvement with significant decreases in Th17 levels, increases in Treg, and rebalanced Th17/Treg ratios. The four patients who showed no improvement had increases in both Th17 and Treg levels and an even higher Th17/Treg ratio than before. CONCLUSION: Glucocorticoid treatment can rectify Th17/Treg dysregulation in patients with SHB. PMID:25561802

  4. Epigenetic Alterations of IL-6/STAT3 Signaling by Placental Stem Cells Promote Hepatic Regeneration in a Rat Model with CCl4-induced Liver Injury

    PubMed Central

    Jung, Jieun; Moon, Ji Wook; Choi, Jong-Ho; Lee, Yong Woo; Park, Sun-Hwa; Kim, Gi Jin

    2015-01-01

    Background Human chorionic plate-derived mesenchymal stem cells (CP-MSCs) isolated from the placenta have been reported to demonstrate therapeutic effects in animal models of liver injury; however, the underlying epigenetic mechanism of this effect has not been elucidated. Thus, we investigated whether CP-MSCs influence epigenetic processes during regeneration of the injured liver. Methods CP-MSCs were engrafted into a carbon tetrachloride (CCl4)-injured rat model through direct transplantation into the liver (DTX), intrasplenic transplantation (STX), and intravenous transplantation via the tail vein (TTX). Non-transplanted (NTX) rats were maintained as sham controls. Liver tissues were analyzed after transplantation using immunohistochemistry, western blot analysis, and quantitative methylation-specific polymerase chain reaction. Proliferation and human interleukin-6 (hIL-6) enzyme-linked immunosorbent assays were performed using CCl4-treated hepatic cells that were co-cultured with CP-MSCs. Results The Ki67 labeling index, cell cyclins, albumin, IL-6, and gp130 levels were elevated in the CP-MSC transplantation groups. The concentration of hIL-6 in supernatants and the proliferation of CCl4-treated rat hepatic cells were enhanced by co-culturing with CP-MSCs (p<0.05), while the methylation of IL-6/IL-6R and STAT3 by CP-MSC transplantation decreased. Conclusion These results suggest that administration of CP-MSCs promotes IL-6/STAT3 signaling by decreasing the methylation of the IL-6/SATA3 promoters and thus inducing the proliferation of hepatic cells in a CCl4-injured liver rat model. These data advance our understanding of the therapeutic mechanisms in injured livers, and can facilitate the development of cell-based therapies using placenta-derived stem cells. PMID:26019757

  5. Oleoylethanolamide, an endogenous PPAR-α ligand, attenuates liver fibrosis targeting hepatic stellate cells

    PubMed Central

    Chen, Junde; Li, Lei; Zheng, Zihan; Ren, Jie; Qiu, Yan

    2015-01-01

    Oleoylethanolamide (OEA), an endocannabinoid-like molecule, was revealed to modulate lipid metabolism through a peroxisome proliferator-activated receptor-α (PPAR-α) mediated mechanism. In present study, we further investigated the activities and mechanisms of OEA in ameliorating hepatic fibrosis in Sv/129 mice induced by a methionine choline-deficient (MCD) diet or thioacetamide (TAA) treatment. Liver fibrosis development was assessed by Hematoxylin-eosin and Sirius red staining. Treatment with OEA (5 mg/kg/day, intraperitoneal injection, i.p.) significantly attenuated the progress of liver fibrosis in both two experimental animal models by blocking the activation of hepatic stellate cells (HSCs). Gene expression analysis of hepatic tissues indicated that OEA inhibited the expression of α-smooth muscle action (α-SMA) and collagen matrix, fibrosis markers, and genes involved in inflammation and extracellular matrix remodeling. In vitro studies showed that OEA inhibited transforming growth factor β1-stimulated HSCs activation through suppressing Smad2/3 phosphorylation, α-SMA expression and myofibroblast transformation. These improvements could not be observed in PPAR-α knockout mice models with OEA administration, which suggested all the anti-fibrotic effects of OEA in vivo and in vitro were mediated by PPAR-α activation. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating hepatic fibrosis development through the inhibition of HSCs activation in liver and therefore may be a potential therapeutic agent for liver fibrosis. PMID:26729705

  6. Geniposide Suppresses Hepatic Glucose Production via AMPK in HepG2 Cells.

    PubMed

    Guo, Lixia; Zheng, Xuxu; Liu, Jianhui; Yin, Zhongyi

    2016-01-01

    Geniposide is one of the main compounds in Gardenia jasminoides ELLIS and has many pharmacological activities, but its anti-hyperglycemic activity has not yet been fully explored. This study was designed to determine, for the first time, how geniposide from G. jasminoides regulates hepatic glucose production, and the underlying mechanisms. During in vitro study, we found the inhibitory effect of geniposide on the hepatic glucose production is partly through AMP-activated protein kinase (AMPK) activation in HepG2 cells. Geniposide significantly inhibited hepatic glucose production in a dose-dependent manner. AMPK, acetyl coenzyme A synthetase (ACC) and forkhead box class O1 (FoxO1) phosphorylation were stimulated by different concentrations of geniposide. In addition, the enzyme activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were all significantly suppressed. What is important is that these effects were partly reversed by (1) inhibition of AMPK activity by compound C, a selective AMPK inhibitor, and by (2) suppression of AMPKα expression by small interfering RNA (siRNA). In summary, geniposide potentially ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis by modulation of the AMPK-FoxO1 signaling pathway. Geniposide or geniposide-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes on gluconeogenesis. PMID:26830672

  7. Oleoylethanolamide, an endogenous PPAR-α ligand, attenuates liver fibrosis targeting hepatic stellate cells.

    PubMed

    Chen, Ling; Li, Long; Chen, Junde; Li, Lei; Zheng, Zihan; Ren, Jie; Qiu, Yan

    2015-12-15

    Oleoylethanolamide (OEA), an endocannabinoid-like molecule, was revealed to modulate lipid metabolism through a peroxisome proliferator-activated receptor-α (PPAR-α) mediated mechanism. In present study, we further investigated the activities and mechanisms of OEA in ameliorating hepatic fibrosis in Sv/129 mice induced by a methionine choline-deficient (MCD) diet or thioacetamide (TAA) treatment. Liver fibrosis development was assessed by Hematoxylin-eosin and Sirius red staining. Treatment with OEA (5 mg/kg/day, intraperitoneal injection, i.p.) significantly attenuated the progress of liver fibrosis in both two experimental animal models by blocking the activation of hepatic stellate cells (HSCs). Gene expression analysis of hepatic tissues indicated that OEA inhibited the expression of α-smooth muscle action (α-SMA) and collagen matrix, fibrosis markers, and genes involved in inflammation and extracellular matrix remodeling. In vitro studies showed that OEA inhibited transforming growth factor β1-stimulated HSCs activation through suppressing Smad2/3 phosphorylation, α-SMA expression and myofibroblast transformation. These improvements could not be observed in PPAR-α knockout mice models with OEA administration, which suggested all the anti-fibrotic effects of OEA in vivo and in vitro were mediated by PPAR-α activation. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating hepatic fibrosis development through the inhibition of HSCs activation in liver and therefore may be a potential therapeutic agent for liver fibrosis. PMID:26729705

  8. Novel fullerene derivatives as dual inhibitors of Hepatitis C virus NS5B polymerase and NS3/4A protease.

    PubMed

    Kataoka, Hiroki; Ohe, Tomoyuki; Takahashi, Kyoko; Nakamura, Shigeo; Mashino, Tadahiko

    2016-10-01

    We evaluated the Hepatitis C virus (HCV) NS5B polymerase and HCV NS3/4A protease inhibition activities of a new set of proline-type fullerene derivatives. All of the compounds had the potential to inhibit both the enzymes, indicating that the fullerene derivatives may be dual inhibitors against NS5B and NS3/4A and could be novel lead compounds for the treatment of HCV infections. PMID:27597249

  9. Granulin-Epithelin Precursor Is an Oncofetal Protein Defining Hepatic Cancer Stem Cells

    PubMed Central

    Cheung, Phyllis Fung Yi; Cheng, Christine Kei Chin; Wong, Nicholas Chun Lim; Ho, Jenny Chung Yee; Yip, Chi Wai; Lui, Vincent Chi Hang; Cheung, Annie Nga Yin; Fan, Sheung Tat; Cheung, Siu Tim

    2011-01-01

    Background and Aims Increasing evidence has suggested that hepatocellular carcinoma (HCC) might originate from a distinct subpopulation called cancer stem cells (CSCs), which are responsible for the limited efficacy of conventional therapies. We have previously demonstrated that granulin-epithelin precursor (GEP), a pluripotent growth factor, is upregulated in HCC but not in the adjacent non-tumor, and that GEP is a potential therapeutic target for HCC. Here, we characterized its expression pattern and stem cell properties in fetal and cancerous livers. Methods Protein expression of GEP in fetal and adult livers was examined in human and mouse models by immunohistochemical staining and flow cytometry. Liver cancer cell lines, isolated based on their GEP and/or ATP-dependent binding cassette (ABC) drug transporter ABCB5 expression, were evaluated for hepatic CSC properties in terms of colony formation, chemoresistance and tumorigenicity. Results We demonstrated that GEP was a hepatic oncofetal protein that expressed in the fetal livers, but not in the normal adult livers. Importantly, GEP+ fetal liver cells co-expressed the embryonic stem (ES) cell-related signaling molecules including β-catenin, Oct4, Nanog, Sox2 and DLK1, and also hepatic CSC-markers CD133, EpCAM and ABCB5. Phenotypic characterization in HCC clinical specimens and cell lines revealed that GEP+ cancer cells co-expressed these stem cell markers similarly as the GEP+ fetal liver cells. Furthermore, GEP was shown to regulate the expression of ES cell-related signaling molecules β-catenin, Oct4, Nanog, and Sox2. Isolated GEPhigh cancer cells showed enhanced colony formation ability and chemoresistance when compared with the GEPlow counterparts. Co-expression of GEP and ABCB5 better defined the CSC populations with enhanced tumorigenic ability in immunocompromised mice. Conclusions Our findings demonstrate that GEP is a hepatic oncofetal protein regulating ES cell-related signaling molecules. Co

  10. Effects of cellular origin on differentiation of human induced pluripotent stem cell–derived endothelial cells

    PubMed Central

    Hu, Shijun; Zhao, Ming-Tao; Jahanbani, Fereshteh; Shao, Ning-Yi; Lee, Won Hee; Chen, Haodong; Snyder, Michael P.; Wu, Joseph C.

    2016-01-01

    Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. PMID:27398408

  11. Derivation of three new human embryonic stem cell lines.

    PubMed

    Bradley, Cara K; Chami, Omar; Peura, Teija T; Bosman, Alexis; Dumevska, Biljana; Schmidt, Uli; Stojanov, Tomas

    2010-04-01

    Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines. PMID:20198447

  12. Hepatitis C virus-specific cytotoxic T cell response restoration after treatment-induced hepatitis C virus control

    PubMed Central

    Larrubia, Juan-Ramón; Moreno-Cubero, Elia; Miquel, Joaquín; Sanz-de-Villalobos, Eduardo

    2015-01-01

    Hepatitis C virus (HCV)-specific cytotoxic T cell (CTL) response plays a major role in viral control during spontaneous infection resolution. These cells develop an exhausted and pro-apoptotic status during chronic onset, being unable to get rid of HCV. The role of this response in contributing to sustained viral response (SVR) after anti-HCV is controversial. Recent studies show that after successful interferon-based anti-HCV treatment, HCV traces are still detectable and this correlates with a peak of HCV-specific CTL response activation, probably responsible for maintaining SVR by subsequent complete HCV clearing. Moreover, SVR patients’ serum is still able to induce HCV infection in naïve chimpanzees, suggesting that the infection could be under the control of the immune system after a successful treatment, being transmissible in absence of this adaptive response. At least theoretically, treatment-induced viral load decrease could allow an effective HCV-specific CTL response reestablishment. This effect has been recently described with anti-HCV interferon-free regimes, based on direct-acting antivirals. Nevertheless, this is to some extent controversial with interferon-based therapies, due to the detrimental immunoregulatory α-interferon effect on T cells. Moreover, HCV-specific CTL response features during anti-HCV treatment could be a predictive factor of SVR that could have clinical implications in patient management. In this review, the recent knowledge about the role of HCV-specific CTL response in the development of SVR after anti-HCV treatment is discussed. PMID:25834312

  13. Design, synthesis, assessment, and molecular docking of novel pyrrolopyrimidine (7-deazapurine) derivatives as non-nucleoside hepatitis C virus NS5B polymerase inhibitors.

    PubMed

    Mohamed, Mosaad S; Sayed, Amira I; Khedr, Mohammed A; Soror, Sameh H

    2016-05-01

    Hepatitis C virus (HCV) infection is highly persistent and presents an unmet medical need requiring more effective treatment options. This has spurred intensive efforts to discover novel anti-HCV agents. The RNA-dependent RNA polymerase (RdRp), NS5B of HCV, constitutes a selective target for drug discovery due to its absence in human cells; also, it is the centerpiece for viral replication. Here, we synthesized novel pyrrole, pyrrolo[2,3-d]pyrimidine and pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives. The non-toxic doses of these compounds on Huh 7.5 cell line were determined and their antiviral activity against HCVcc genotype 4a was examined. Compounds 7j, 7f, 5c, 12i and 12f showed significant anti HCV activity. The percent of reduction for the non-toxic doses of 7j, 7f, 5c, 12i and 12f were 90%, 76.7±5.8%, 73.3±5.8%, 70% and 63.3±5.8%, respectively. The activity of these compounds was interpreted by molecular docking against HCV NS5B polymerase enzyme. PMID:27052365

  14. Altered effector functions of NK cells in chronic hepatitis C are associated with IFNL3 polymorphism.

    PubMed

    Rogalska-Taranta, Magdalena; Markova, Antoaneta A; Taranta, Andrzej; Lunemann, Sebastian; Schlaphoff, Verena; Flisiak, Robert; Manns, Michael P; Cornberg, Markus; Kraft, Anke R M; Wedemeyer, Heiner

    2015-08-01

    Interferon α-mediated effector functions of NK cells may contribute to the control of HCV replication and the pathogenesis of liver disease. The single-nucleotide polymorphism rs12979860 near IFNL3 (previously known as IL28B) is important in response to IFN-α treatment and in spontaneous resolution of acute hepatitis C. The role of the IFNL3 polymorphism in NK cell function is unclear. Thus, we investigated the role of IFNL3 polymorphism in type I IFN-dependent regulation of NK cell functions in patients with cHC and healthy control subjects. We demonstrated a marked polarization of NK cells toward cytotoxicity in response to IFN-α stimulation in patients with hepatitis C. That TRAIL up-regulation was present, particularly in patients with the IFNL3-TT allele, was supported by a shift in the pSTAT-1:pSTAT-4 ratios toward pSTAT-1. In patients bearing the IFNL3-TT allele, NK cell effector function correlated with liver disease activity. In contrast, higher cytokine production of NK cells was observed in healthy individuals with the IFNL3-CC genotype, which may support spontaneous HCV clearance in acute infection. Overall, these findings show that the role of NK cells may differ in chronic infection vs. early antiviral defense and that the IFNL3 genotype differentially influences NK cell function. PMID:26034208

  15. Reversal of Hepatic Fibrosis by Human CD34(+) Stem/Progenitor Cell Transplantation in Rats.

    PubMed

    Abdel Aziz, M T; El Asmar, Mf; Mostafa, S; Salama, H; Atta, H M; Mahfouz, S; Roshdy, N K; Rashed, L A; Sabry, D; Hasan, N; Mahmoud, M; Elderwy, D

    2010-05-01

    Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation. Here, we transplant UCB cells into injured liver fibrosis, investigated the hepatic potential of UCB cells both in vitro and in vivo. a CCl4 rat model with liver fibrosis was prepared. Human (UCB) CD34(+) stem cell was separated with MACS (magnetic cell sorting). Cells were cultured with and without hepatic differentiation medium. Rats were divided into 3 groups; group (1): control healthy, group (2): CCl4 injected rats and group 3: CCl4/CD34(+)injected rats with human differentiated and undifferentiated cells through intrahepatic (IH) and intravenous (IV) routes. A significant elevation was detected in serum albumin in CCl4/CD34(+) compared to the CCl4 group (p<0.001). Serum ALT, had a significant decrease of its level after administration of stem cells compared to the CCl4 group (p<0.001). However, it was still significantly higher than control (p<0.001) with no significant difference between the groups that received stem cells. Histopathological examination of liver tissue showed that stem cells have a significant antifibrotic effect. Concerning gene expression, the collagen gene (rat) was highly expressed in the CCl4 group whereas its expression was significantly decreased after administration of stem cells. Human albumin and matrix metalloproteinase (MMP2) genes were expressed in liver tissues in the groups that received stem cells. Highest expression was in the group that received un-differentiated cells I.V. human UCB CD34(+) stem cells can ameliorate liver fibrosis in rats. PMID:24855554

  16. Hepatic stellate cells undermine the allostimulatory function of liver myeloid dendritic cells via STAT3-dependent induction of IDO

    PubMed Central

    Sumpter, Tina L.; Dangi, Anil; Matta, Benjamin M.; Huang, Chao; Stolz, Donna B.; Vodovotz, Yoram; Thomson, Angus W.; Gandhi, Chandrashekhar R.

    2012-01-01

    Hepatic stellate cells (HSCs) are critical for hepatic wound repair and tissue remodeling. They also produce cytokines and chemokines that may contribute to the maintenance of hepatic immune homeostasis and the inherent tolerogenicity of the liver. The functional relationship between HSCs and the professional migratory APCs in the liver, i.e. dendritic cells (DCs), has not been evaluated. Here, we report that murine liver DCs co-localize with HSCs in vivo under normal, steady-state conditions, and cluster with HSCs in vitro. In vitro, HSCs secrete high levels of DC chemoattractants, such as MIP1α and MCP-1, as well as cytokines that modulate DC activation, including TNFα, IL-6 and IL-1β. Culture of HSCs with conventional liver myeloid (m) DCs resulted in increased IL-6 and IL-10 secretion compared to that of either cell population alone. Co-culture also resulted in enhanced expression of co-stimulatory (CD80, CD86) and co-inhibitory (B7-H1) molecules on mDCs. HSC-induced mDC maturation required cell-cell contact and could be blocked, in part, by neutralizing MIP1α or MCP-1. HSC-induced mDC maturation was dependent on activation of STAT3 in mDCs and in part on HSC-secreted IL-6. Despite up-regulation of co-stimulatory molecules, mDCs conditioned by HSCs demonstrated impaired ability to induce allogeneic T cell proliferation, which was independent of B7-H1, but dependent upon HSC-induced STAT3 activation and subsequent up-regulation of IDO. In conclusion, by promoting IDO expression, HSCs may act as potent regulators of liver mDCs and function to maintain hepatic homeostasis and tolerogenicity. PMID:22962681

  17. Hepatitis B Virus X Protein Driven Alpha Fetoprotein Expression to Promote Malignant Behaviors of Normal Liver Cells and Hepatoma Cells

    PubMed Central

    Zhu, Mingyue; Lu, Yan; Li, Wei; Guo, Junli; Dong, Xu; Lin, Bo; Chen, Yi; Xie, Xieju; Li, Mengsen

    2016-01-01

    Background: The infection of Hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma(HCC), HBV-X protein(HBx) is able to induce expression of alpha-fetoprotein(AFP) in normal liver cells, and AFP harbors a function to promote malignant transformation of normal liver cells, but the role AFP playing in malignant behaviors of HCC cells is still unclear. Methods: Fifty-six liver tissue samples were collected from the clinical patients through hepatectomy(include normal liver tissues, HBV-related hepatitis liver tissues and HBV-related HCC tissues), and diagnosis of these tissues by pathology section, expression of AFP, Ras and CXCR4 were evidenced by immunohisochemical staining and Western blotting; The proliferation of human normal liver cells line L-02 cells and human hepatoma cells line, HLE cells(non AFP-producing) were performed by MTT method; Repaired capacity of L-02 and HLE cells were compared by wound healing assay; Migration and invasion of these cells were analyzed by Transwell chamber assay; HBx expressed vectors(pcDNA3.1-HBx) were constructed and transfected into L-02 and HLE cells, effects of pcDNA3.1-HBx on the malignant behaviors were also detected by MTT, Transwell chamber assay and the expression of AFP, Ras and CXCR4 were evidenced by Western blotting. Results: we found that expression of AFP, Ras and CXCR4 in HBV-related HCC and lymph nodes metastasis tissues were significantly elevated compared with HBV-related HCC, non metastasis tissues and HBV-related hepatitis tissues; Expression of AFP, Ras and CXCR4 in HBV-related hepatitis tissues were significantly enhanced compared with normal liver tissues; The growth ratio, migratory and invasive ability, expression of AFP, Ras and CXCR4 of the cells were outstanding promoted while L-02 and HLE cells were transfected with pcDNA3.1-HBx vectors. The proliferation ratio, migration and invasion ability, and expression of Ras and CXCR4 were significantly inhibited while

  18. Hepatic circadian clock oscillators and nuclear receptors integrate microbiome-derived signals

    PubMed Central

    Montagner, Alexandra; Korecka, Agata; Polizzi, Arnaud; Lippi, Yannick; Blum, Yuna; Canlet, Cécile; Tremblay-Franco, Marie; Gautier-Stein, Amandine; Burcelin, Rémy; Yen, Yi-Chun; Je, Hyunsoo Shawn; Maha, Al-Asmakh; Mithieux, Gilles; Arulampalam, Velmurugesan; Lagarrigue, Sandrine; Guillou, Hervé; Pettersson, Sven; Wahli, Walter

    2016-01-01

    The liver is a key organ of metabolic homeostasis with functions that oscillate in response to food intake. Although liver and gut microbiome crosstalk has been reported, microbiome-mediated effects on peripheral circadian clocks and their output genes are less well known. Here, we report that germ-free (GF) mice display altered daily oscillation of clock gene expression with a concomitant change in the expression of clock output regulators. Mice exposed to microbes typically exhibit characterized activities of nuclear receptors, some of which (PPARα, LXRβ) regulate specific liver gene expression networks, but these activities are profoundly changed in GF mice. These alterations in microbiome-sensitive gene expression patterns are associated with daily alterations in lipid, glucose, and xenobiotic metabolism, protein turnover, and redox balance, as revealed by hepatic metabolome analyses. Moreover, at the systemic level, daily changes in the abundance of biomarkers such as HDL cholesterol, free fatty acids, FGF21, bilirubin, and lactate depend on the microbiome. Altogether, our results indicate that the microbiome is required for integration of liver clock oscillations that tune output activators and their effectors, thereby regulating metabolic gene expression for optimal liver function. PMID:26879573

  19. Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid.

    PubMed Central

    Saltiel, A R; Cuatrecasas, P

    1986-01-01

    Insulin binding to plasma membrane receptors results in the generation of substances that acutely mimic the actions of the hormone on certain target enzymes. Two such substances, which modulate the activity of the high-affinity cAMP phosphodiesterase (EC 3.1.4.17), have been purified from hepatic plasma membranes. The two have similar properties and activities but can be resolved by ion-exchange chromatography and high-voltage electrophoresis. They exhibit a net negative charge, even at pH 1.9, and an apparent molecular weight of approximately 1400. The generation of these substances from membranes by insulin can be reproduced by addition of a phosphatidylinositol-specific phospholipase C purified from Staphylococcus aureus. This enzyme is known to selectively hydrolyze phosphatidylinositol and release from membranes several proteins that are covalently linked to phosphatidylinositol by a glycan anchor. Both enzyme-modulating substances appear to be generated by the phosphodiesterase cleavage of a phosphatidylinositol-containing glycolipid precursor that has been characterized by thin-layer chromatography. Some of the chemical properties of these substances have been examined. They appear to be related complex carbohydrate-phosphate substances containing glucosamine and inositol. These findings suggest that insulin may activate a selective phospholipase activity that hydrolyzes a membrane phospholipid, releasing a carbohydrate-containing molecule that regulates cAMP phosphodiesterase and perhaps other insulin-sensitive enzymes. PMID:3016721

  20. Activation of NLRP3 and AIM2 inflammasomes in Kupffer cells in hepatic ischemia/reperfusion.

    PubMed

    Kim, Hyo-Yeon; Kim, Seok-Joo; Lee, Sun-Mee

    2015-01-01

    Inflammasome activation by danger signals in ischemia/reperfusion (I/R) injury is responsible for the sterile inflammatory response. Signals triggering formation and activation of the inflammasome involve the generation of oxidative stress. The aim of this study was to examine the molecular mechanisms of inflammasome activation and the involvement of reactive oxygen species in hepatic I/R. I/R induced the formation of nucleotide-binding domain leucine-rich repeat containing family pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes and the subsequent serum release of interleukin 1β. Pannexin-1 inhibitor and anti-cathepsin B antibody attenuated I/R-induced inflammasome activation and hepatic injury. The expression of the thioredoxin-interacting protein gene and the interaction between NLRP3 and the thioredoxin-interacting protein increased after I/R. Treatment with the antioxidant N-acetylcysteine significantly attenuated protein conversion of interleukin 1β after hepatic I/R. Moreover, pannexin-1 protein expression and cathepsin B release were strongly attenuated by N-acetylcysteine. The depletion of Kupffer cells with gadolinium chloride markedly decreased NLRP3 and AIM2 inflammasome expression and activation of their signaling pathways, and also reduced the level of caspase-1 protein in F4/80-positive cells. Our findings suggest that reactive-oxygen-species-mediated activation of NLRP3 and AIM2 inflammasomes leads to I/R-induced inflammatory responses in which Kupffer cells play a crucial role. PMID:25327779

  1. Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease

    PubMed Central

    Apta, Bonita; Kuravi, Sahithi J.; Yates, Clara M.; Kennedy, Amy; Odedra, Arjun; Alassiri, Mohammed; Harrison, Matthew; Martin, Ashley; Barone, Francesca; Nayar, Saba; Hitchcock, Jessica R.; Cunningham, Adam F.; Raza, Karim; Filer, Andrew; Copland, David A.; Dick, Andrew D.; Robinson, Joseph; Kalia, Neena; Walker, Lucy S. K.; Buckley, Christopher D.; Nash, Gerard B.; Narendran, Parth; Rainger, G. Ed.

    2015-01-01

    During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3.ζδ protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type-1-diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to healthy age matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Importantly, control of patient T cell trafficking is re-established by exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic I/R injury, Salmonella infection, Uveitis and Sjögren’s Syndrome, PEPITEM could reduce T cell recruitment into inflamed tissues. PMID:25894827

  2. Red blood cell-derived microparticles: An overview.

    PubMed

    Westerman, Maxwell; Porter, John B

    2016-07-01

    The red blood cell (RBC) is historically the original parent cell of microparticles (MPs). In this overview, we describe the discovery and the early history of red cell-derived microparticles (RMPs) and present an overview of the evolution of RMP. We report the formation, characteristics, effects of RMP and factors which may affect RMP evaluation. The review examines RMP derived from both normal and pathologic RBC. The pathologic RBC studies include sickle cell anemia (SCA), sickle cell trait (STr), thalassemia intermedia (TI), hereditary spherocytosis (HS), hereditary elliptocytosis (HE), hereditary stomatocytosis (HSt) and glucose-6-phosphate dehydrogenase deficiency (G6PD). PMID:27282583

  3. Human placenta-derived adherent cells induce tolerogenic immune responses.

    PubMed

    Liu, Wei; Morschauser, Andrew; Zhang, Xin; Lu, Xiaohua; Gleason, Joseph; He, Shuyang; Chen, Hong-Jung; Jankovic, Vladimir; Ye, Qian; Labazzo, Kristen; Herzberg, Uri; Albert, Vivian R; Abbot, Stewart E; Liang, Bitao; Hariri, Robert

    2014-05-01

    Human placenta-derived adherent cells (PDAC cells) are a culture expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory and anti-inflammatory properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. To elucidate the mechanisms underlying the immunoregulatory properties of PDAC cells, we investigated their effects on immune cell populations, including T cells and dendritic cells (DC) in vitro and in vivo. PDAC cells suppressed T-cell proliferation in an OT-II T-cell adoptive transfer model, reduced the severity of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis and ameliorated inflammation in a delayed type hypersensitivity response model. In vitro, PDAC cells suppressed T-cell proliferation and inhibited Th1 and Th17 differentiation. Analysis of tissues derived from PDAC cell-treated animals revealed diminished CD86 expression on splenic DC, suggesting that they can also modulate DC populations. Furthermore, PDAC cells modulate the differentiation and maturation of mouse bone marrow-derived DC. Similarly, human DC differentiated from CD14(+) monocytes in the presence of PDAC cells acquired a tolerogenic phenotype. These tolerogenic DC failed to induce allogeneic T-cell proliferation and differentiation toward Th1, but skewed T-cell differentiation toward Th2. Inhibition of cyclo-oxygenase-2 activity resulted in a significant, but not complete, abrogation of PDAC cells' effects on DC phenotype and function, implying a role for prostaglandin E2 in PDAC-mediated immunomodulation. This study identifies modulation of DC differentiation toward immune tolerance as a key mechanism underlying the immunomodulatory activities of PDAC cells. PMID:25505962

  4. Effect of cadmium on calcium transport in a human fetal hepatic cell line (WRL-68 cells).

    PubMed

    Souza, V; Bucio, L; Jay, D; Chávez, E; Gutiérrez-Ruiz, M C

    1996-08-16

    Toxic metals appear to use the transport pathways that exist for biologically essential metals. Calcium uptake in cells occurs through specific membrane channels. Since cadmium inhibits calcium uptake, this study was carried on to elucidate the mechanism of Cd interference with calcium transport using the fetal hepatic cell line WRL-68 as an in vitro model. Ca accumulation by WRL-68 cells presented an initial rapid phase, followed by a sustained phase of slower accumulation over a 60 min period. A concentration of 50 microM CdCl2 produced 39% inhibition of the uptake of CaCl2 (100 microM), while 100 microM nifedipine or verapamil decreased Ca accumulation by 35 and 63%, respectively. All Cd concentrations tested produced significant decrease in Ca uptake in a concentration-dependent manner at 1 min and thereafter, although with 10 microM CdCl2 no significant difference was found after 30 min of incubation. From the Lineweaver-Burk plot, we found that Cd exerted a competitive inhibition on Ca uptake, since there was no significant effect on the Vmax but an increased K(m). A second order rate constant of Cd inactivation of 0.061 mM-1.s-1 was determined from the course of Ca uptake during Cd inhibition. SH groups seemed to play an essential role in Ca inhibition uptake by Cd because the inhibition of Ca accumulation by 50 microM Cd was practically reversed after the addition of dithiothreitol. PMID:8814339

  5. Lost in translation: pluripotent stem cell-derived hematopoiesis

    PubMed Central

    Ackermann, Mania; Liebhaber, Steffi; Klusmann, Jan-Henning; Lachmann, Nico

    2015-01-01

    Pluripotent stem cells (PSCs) such as embryonic stem cells or induced pluripotent stem cells represent a promising cell type to gain novel insights into human biology. Understanding the differentiation process of PSCs in vitro may allow for the identification of cell extrinsic/intrinsic factors, driving the specification process toward all cell types of the three germ layers, which may be similar to the human in vivo scenario. This would not only lay the ground for an improved understanding of human embryonic development but would also contribute toward the generation of novel cell types used in cell replacement therapies. In this line, especially the developmental process of mesodermal cells toward the hematopoietic lineage is of great interest. Therefore, this review highlights recent progress in the field of hematopoietic specification of pluripotent stem cell sources. In addition, we would like to shed light on emerging factors controlling primitive and definitive hematopoietic development and to highlight recent approaches to improve the differentiation potential of PSC sources toward hematopoietic stem/progenitor cells. While the generation of fully defined hematopoietic stem cells from PSCs remains challenging in vitro, we here underline the instructive role of cell extrinsic factors such as cytokines for the generation of PSC-derived mature hematopoietic cells. Thus, we have comprehensively examined the role of cytokines for the derivation of mature hematopoietic cell types such as macrophages, granulocytes, megakaryocytes, erythrocytes, dendritic cells, and cells of the B- and T-cell lineage. PMID:26174486

  6. p53 signaling is involved in leptin-induced growth of hepatic and breast cancer cells

    PubMed Central

    Shrestha, Mohan

    2016-01-01

    Leptin, an adipokine predominantly produced from adipose tissue, is well known to induce tumor growth. However, underlying molecular mechanisms are not established yet. While p53 has long been well recognized as a potent tumor suppressor gene, accumulating evidence has also indicated its potential role in growth and survival of cancer cells depending on experimental environments. In the present study, we examined if p53 signaling is implicated in leptin-induced growth of cancer cells. Herein, we demonstrated that leptin treatment significantly increased p53 protein expression in both hepatic (HepG2) and breast (MCF-7) cancer cells without significant effect on mRNA expression. Enhanced p53 expression by leptin was mediated via modulation of ubiquitination, in particular ubiquitin specific protease 2 (USP2)-dependent manner. Furthermore, gene silencing of p53 by small interfering RNA (siRNA) suppressed leptin-induced growth of hepatic and breast cancer cells, indicating the role of p53 signaling in tumor growth by leptin. In addition, we also showed that knockdown of p53 restored suppression of caspase-3 activity by leptin through modulating Bax expression and prevented leptin-induced cell cycle progression, implying the involvement of p53 signaling in the regulation of both apoptosis and cell cycle progression in cancer cells treated with leptin. Taken together, the results in the present study demonstrated the potential role of p53 signaling in leptin-induced tumor growth. PMID:27610035

  7. p53 signaling is involved in leptin-induced growth of hepatic and breast cancer cells.

    PubMed

    Shrestha, Mohan; Park, Pil-Hoon

    2016-09-01

    Leptin, an adipokine predominantly produced from adipose tissue, is well known to induce tumor growth. However, underlying molecular mechanisms are not established yet. While p53 has long been well recognized as a potent tumor suppressor gene, accumulating evidence has also indicated its potential role in growth and survival of cancer cells depending on experimental environments. In the present study, we examined if p53 signaling is implicated in leptin-induced growth of cancer cells. Herein, we demonstrated that leptin treatment significantly increased p53 protein expression in both hepatic (HepG2) and breast (MCF-7) cancer cells without significant effect on mRNA expression. Enhanced p53 expression by leptin was mediated via modulation of ubiquitination, in particular ubiquitin specific protease 2 (USP2)-dependent manner. Furthermore, gene silencing of p53 by small interfering RNA (siRNA) suppressed leptin-induced growth of hepatic and breast cancer cells, indicating the role of p53 signaling in tumor growth by leptin. In addition, we also showed that knockdown of p53 restored suppression of caspase-3 activity by leptin through modulating Bax expression and prevented leptin-induced cell cycle progression, implying the involvement of p53 signaling in the regulation of both apoptosis and cell cycle progression in cancer cells treated with leptin. Taken together, the results in the present study demonstrated the potential role of p53 signaling in leptin-induced tumor growth. PMID:27610035

  8. Autoimmune Hepatitis: Progress from Global Immunosuppression to Personalised Regulatory T Cell Therapy

    PubMed Central

    Than, Nwe Ni; Jeffery, Hannah C.; Oo, Ye H.

    2016-01-01

    Autoimmune hepatitis (AIH) is an immune mediated liver injury. The precise aetiology of AIH is still unknown but current evidence suggests both genetic and environmental factors are involved. Breakdown in peripheral self-tolerance, and impaired functions of FOXP3+ regulatory T cell along with effector cell resistance to suppression at the tissue level seem to play an important role in AIH immunopathogenesis. AIH is predominantly a T lymphocytes driven disease but B lymphocytes are also involved in the immunopathology. Innate immune cells are crucial in the initial onset of disease and their response is followed by adaptive T (Th1, Th17, and cytotoxic T cells) and B cell responses evidenced by liver histology and peripheral blood serology. Standard treatment regimens involving steroid and immunosuppressive medications lead to global immune suppression requiring life-long therapy with many side effects. Biologic therapies have been attempted but duration of remission is short-lived. Future direction of diagnosis and treatment for AIH should be guided by “omics” and the immunology profile of the individual patient and clinicians should aim to deliver personalised medicine for their patients. Cell therapy such as infusion of autologous, antigen-specific, and liver-homing regulatory T cells to restore hepatic immune tolerance may soon be a potential future treatment for AIH patients. PMID:27446862

  9. Transcriptomic and proteomic analysis of human hepatic stellate cells treated with natural taurine.

    PubMed

    Liang, Jian; Deng, Xin; Wu, Fa-Sheng; Tang, Yan-Fang

    2013-05-01

    The aim of this study was to investigate the differential expression of genes and proteins between natural taurine (NTau)‑treated hepatic stellate cells (HSCs) and control cells as well as the underlying mechanism of NTau in inhibiting hepatic fibrosis. A microculture tetrazolium (MTT) assay was used to analyze the proliferation of NTau‑treated HSCs. Flow cytometry was performed to compare the apoptosis rate between NTau-treated and non‑treated HSCs. Proteomic analysis using a combination of 2-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) was conducted to identify the differentially expressed proteins. Microarray analysis was performed to investigate the differential expression of genes and real-time polymerase chain reaction (PCR) was used to validate the results. The experimental findings obtained demonstrated that NTau decreased HSC proliferation, resulting in an increased number of cells in the G0/G1 phase and a reduced number of cells in the S phase. Flow cytometric analysis showed that NTau-treated HSCs had a significantly increased rate of apoptosis when compared with the non‑treated control group. A total of 15 differentially expressed proteins and 658 differentially expressed genes were identified by 2DE and MS, and microarray analysis, respectively. Gene ontology (GO) functional analysis indicated that these genes and proteins were enriched in the function clusters and pathways related to cell proliferation, cellular apoptosis and oxidation. The transcriptome and proteome analyses of NTau-treated HSCs demonstrated that NTau is able to significantly inhibit cell proliferation and promote cell apoptosis, highlighting its potential therapeutic benefits in the treatment of hepatic fibrosis. PMID:23525364

  10. Autologous Stem Cells Transplantation in Egyptian Patients with Liver Cirrhosis on Top of Hepatitis C Virus

    PubMed Central

    Al Tayeb, Hoda; El Dorry, Ahmed; Amer, Nehad; Mowafy, Nadia; Zimaity, Maha; Bayoumy, Essam; Saleh, Shereen A.

    2015-01-01

    Background and Objectives Use of pluripotent stem cells is an ideal solution for liver insufficiencies. This work aims is to evaluate the safety and feasibility of autologous stem cells transplantation (SCT) in Egyptian patients of liver cirrhosis on top of hepatitis C virus (HCV). Subjects and Results 20 patients with HCV induced liver cirrhosis were divided into 2 groups. Group I: included 10 patients with liver cirrhosis Child score ≥9, for whom autologous stem cell transplantation was done using granulocyte colony stimulating factor (G-CSF) for stem cells mobilization. Separation and collection of the peripheral blood stem cells was done by leukapheresis. G-CSF mobilized peripheral blood mononuclear cells (G-CSF PB-MNCs) were counted by flow cytometry. Stem cell injection into the hepatic artery was done. Group II: included 10 patients with HCV induced liver cirrhosis as a control group. Follow up and comparison between both groups were done over a follow up period of 6 months. The procedure was well tolerated. Mobilization was successful and the total number of G-CSF PB-MNCs in the harvests ranged from 25×106 to 191×106. There was improvement in the quality of life, serum albumin, total bilirubin, liver enzymes and the Child-Pugh score of group I over the first two-three months after the procedure. Conclusion SCT in HCV induced liver cirrhosis is a safe procedure. It can improve the quality of life and hepatic functions transiently with no effect on the life expectancy or the fate of the liver cirrhosis. PMID:26634069

  11. Upregulation of MiR-122 via Trichostatin A Treatments in Hepatocyte-like Cells Derived from Mesenchymal Stem Cells.

    PubMed

    Alizadeh, Effat; Eslaminejad, MohamadReza Baghaban; Akbarzadeh, Abolfazl; Sadeghi, Zohre; Abasi, Mozghan; Herizchi, Roya; Zarghami, Nosratollah

    2016-02-01

    The miR-122 is a tissue-specific miRNA; its expression is abundant in liver. MiR-122 upregulation is crucial for differentiation, functionality, and maintenance of differentiated phenotype in hepatocytes. The improving effects of trichostatin A (TSA) on hepatic differentiation have been reported previously. The aim of this study was to determine whether TSA can affect the expression of miR-122 in hepatocyte-like cells (HLCs) generated from human adipose tissue-derived mesenchymal stem cells (hAT-MSCs). The hepatic differentiation of hAT-MSCs induced by a mixture of growth factors and cytokines either with or without TSA treatments. The functionality of HLCs generated with or without TSA and the expression levels of miR-122 were studied. The expression levels of miR-122 in TSA-treated HLCs was significantly (p < 0.05) higher than those generated by growth factors and cytokines, only. The downregulation of a-fetoprotein (AFP) levels but enhanced albumin synthesis (p < 0.05) and upregulation of liver-enriched transcription factors (LETFs) HNF4α (hepatocyte nuclear factor 4α) and HNF6 (hepatocyte nuclear factor 6) were observed in TSA-treated HLCs (p < 0.05). In conclusion, administration of TSA in hepatogenic differentiation of hAT-MSCs resulted in higher expression levels of miR-122, facilitation of differentiation, and subsequently attenuation of AFP levels. PMID:26360933

  12. Graptopetalum Paraguayense Ameliorates Chemical-Induced Rat Hepatic Fibrosis In Vivo and Inactivates Stellate Cells and Kupffer Cells In Vitro

    PubMed Central

    Su, Li-Jen; Chang, Chia-Chuan; Yang, Chih-Hsueh; Hsieh, Shur-Jong; Wu, Yi-Chin; Lai, Jin-Mei; Tseng, Tzu-Ling; Huang, Chi-Ying F.; Hsu, Shih-Lan

    2013-01-01

    Background Graptopetalum paraguayense (GP) is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN)- and carbon tetrachloride (CCl4)-induced liver injury rats. Methods Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP) by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs) and Kupffer cells, respectively, were evaluated. Results Oral administration of MGP significantly alleviated DMN- or CCl4-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA) expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression. Conclusions The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis. PMID:23335984

  13. Hepatitis B virus-specific T-cell responses during IFN administration in a small cohort of chronic hepatitis B patients under nucleos(t)ide analogue treatment.

    PubMed

    Sprinzl, M F; Russo, C; Kittner, J; Allgayer, S; Grambihler, A; Bartsch, B; Weinmann, A; Galle, P R; Schuchmann, M; Protzer, U; Bauer, T

    2014-01-01

    The effect of pegylated interferon-α (IFN) add-on therapy on HBV-specific T-cell responses was evaluated in 12 patients with stable, undetectable hepatitis B virus (HBV) load under nucleos(t)ide analogue therapy. Peripheral blood mononuclear cells were isolated at week 0, 4, 8, 12, 24 and 48 of IFN add-on therapy. Quantity and quality of circulating HBV S- and core-specific CD4 and CD8 T cells were analysed ex vivo by flow cytometry. HBV S- and core-specific CD4 T-cell numbers modestly increased within 8 weeks of IFN administration (P = 0.0391 and P = 0.0195), whereas HBV-specific CD8 T cells in general showed only minor changes under IFN add-on therapy. Functionality of HBV-specific CD4 but not CD8 T cells positively correlated with serum transaminase activity. In addition, we observed an increase in CD4 T cells producing tumour necrosis factor-α (TNFα) without antigen restimulation (P = 0.0039), which correlated with elevated transaminases. During IFN add-on therapy, two patients developed an anti-HBs seroconversion, only one of whom showed a relevant increase in HBV-specific T cells. In conclusion, IFN add-on therapy of chronic hepatitis B increased HBV-specific T-cell responses and affected a previously unrecognized TNFα-monofunctional CD4 T-cell population. Although the observed T-cell responses did not correlate with HBsAg seroconversion, we expect additional insights into the immunopathogenesis of hepatitis B, following the characterization of the newly identified TNF α-monofunctional T-cell population. PMID:24251783

  14. Enhanced hepatic differentiation of mesenchymal stem cells after pretreatment with injured liver tissue.

    PubMed

    Mohsin, Sadia; Shams, Sulaiman; Ali Nasir, Ghazanfar; Khan, Mohsin; Javaid Awan, Sana; Khan, Shaheen N; Riazuddin, Sheikh

    2011-01-01

    Liver failure represents a serious challenge for cell based therapies. Mesenchymal stem cells (MSCs) possess potential for regeneration of fibrotic liver; however, there is a dire need to improve their hepatic differentiation. This study examines a pretreatment strategy to augment the differentiation potential of MSCs towards hepatic lineage. MSCs were isolated from C57BL/6 wild type mice and were characterized by flow cytometry for CD44 (92.4%), CD90 (96.6%), CD105 (94.7%), CD45 (0.8%) and CD34 (1.4%) markers. To improve the differentiation potential of MSCs towards hepatic lineage, cells were pretreated with injured liver tissue in an in-vitro model, which resulted in high expression of albumin, cytokeratin 8, 18, TAT and HNF1α as compared to untreated MSCs. The efficacy of pretreated MSCs was evaluated by preparing in-vivo mouse model with liver fibrosis by intraperitoneal administration of CCl(4). Pretreated MSCs were transplanted in the left lateral lobe of mice with liver fibrosis and showed enhanced localization and differentiation abilities after 1 month. The expression for cytokeratin 8, 18, albumin and Bcl-xl was up-regulated and that of HGF, Bax and Caspase- 3 was down-regulated in animals transplanted with pretreated MSCs. Sirus red staining also confirmed a significant reduction in the fibrotic area in liver tissue transplanted with pretreated MSCs as compared to untreated MSCs and was concomitant with improved serum levels of bilirubin and alkaline phosphatase (ALP). Therefore, it was concluded that pretreatment with injured liver tissue augment homing and hepatic differentiation abilities of MSCs and provides an improved procedure for the treatment of liver fibrosis. PMID:20943307

  15. Differential thioredoxin reductase activity from human normal hepatic and hepatoma cell lines.

    PubMed

    Jung, Haeng-Im; Lim, Hye-Won; Kim, Byung-Chul; Park, Eun-Hee; Lim, Chang-Jin

    2004-04-30

    Thioredoxin reductase (TrxR), a component of the thioredoxin system, including thioredoxin (Trx) and NADPH, catalyzes the transfer of electrons from NADPH to Trx, acts as a reductant of disulfide-containing proteins and participates in the defense system against oxidative stresses. In this study, the regulation pattern of TrxR in the presence of various stressful reagents was compared between Chang (human normal hepatic cell) and HepG2 (human hepatoma cell) cell lines. Aluminum chloride (0.5 mM) and zinc chloride (0.5 mM) enhanced the TrxR activity in the Chang cell line to a higher degree than in the HepG2 cell line, but cupric chloride (0.2 mM) and cadmium chloride (0.1 mM) enhanced the TrxR activity in the HepG2 cell line to a greater degree. The TrxR activities in both Chang and HepG2 cell lines were similarly induced by treatment with sodium selenite (0.02 mM) and menadione (0.5 and 1.0 mM). Lipopolysaccharide (2 micro g/m1) increased the TrxR activity upto 4.02- and 2.2-fold in the Chang and HepG2 cell lines, respectively, in time-dependent manners. Hydrogen peroxide (5 mM) markedly enhanced the TrxR activity in the HepG2 cell line, but not in the Chang cell line. NO-generating sodium nitroprusside (3.0 and 6.0 mM) induced TrxR activities in both human liver cell lines. The TrxR activity was also induced in human liver cells under limited growth conditions by serum deprivation. These results imply that the TrxR activities in normal hepatic and hepatoma cell lines are subject to different regulatory responses to various stresses. PMID:15118998

  16. Anticytoproliferative effect of Vitamin C on rat hepatic stellate cell

    PubMed Central

    Su, Min; Chao, Guo; Liang, Minqing; Song, Jianhua; Wu, Ka

    2016-01-01

    This study was conducted to investigate the potential therapeutical benefit of Vitamin (VC), a potent antioxidant, on suppressing proliferation of immortalized rat liver stellate cell line (HSC-T6) in vitro, and to discuss the underlying mechanism. HSC-T6 was co-treated with different concentrations of VC (50, 100, 200 μmol/L) on designed time points. Then, cell viability was assessed by using MTT analysis, and the changes of cytomorphology was observed with apoptosis-specific TUNEL and immunohistochemical stains, as well as the intracellular target genes was determined by using RT-PCR, respectively. As the outcomes, VC-treated HSC-T6 showed significantly inhibited cell growth in a dose-dependent manner when compared to the vehicle control. Cytologically, VC increased TUNEL-labeled positive cells in cultured HSC-T6, which the cell count was greater than vehicle control. Meanwhile, VC-treated HSC-T6 showed elevated immunoreactive for TGF-β1-labeled cells. Moreover, VC contributed to down-regulated expressions of intracellular c-myc, cyclin D1, mTOR mRNAs in HSC-T6. Collectively, these preliminary findings have demonstrated that VC-mediated anti-proliferative effect on HSCs is involved in molecular mechanisms of promoting apoptosis and blocking endogenous collagenation. PMID:27398165

  17. Stem Cell-Derived Extracellular Vesicles and Immune-Modulation

    PubMed Central

    Burrello, Jacopo; Monticone, Silvia; Gai, Chiara; Gomez, Yonathan; Kholia, Sharad; Camussi, Giovanni

    2016-01-01

    Extra-cellular vesicles (EVs) are bilayer membrane structures enriched with proteins, nucleic acids, and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag)-presenting cells for instance, have been involved in both innate and acquired (or adaptive) immune responses, as Ag carriers or presenters, or as vehicles for delivering active signaling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species), and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation, and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response (IIR). Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content, and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review, we will discuss how stem cell-derived EVs may contribute toward the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system. PMID:27597941

  18. Stem Cell-Derived Extracellular Vesicles and Immune-Modulation.

    PubMed

    Burrello, Jacopo; Monticone, Silvia; Gai, Chiara; Gomez, Yonathan; Kholia, Sharad; Camussi, Giovanni

    2016-01-01

    Extra-cellular vesicles (EVs) are bilayer membrane structures enriched with proteins, nucleic acids, and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag)-presenting cells for instance, have been involved in both innate and acquired (or adaptive) immune responses, as Ag carriers or presenters, or as vehicles for delivering active signaling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species), and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation, and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response (IIR). Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content, and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review, we will discuss how stem cell-derived EVs may contribute toward the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system. PMID:27597941

  19. Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture

    PubMed Central

    Anzai, Kazuya; Chikada, Hiromi; Tsuruya, Kota; Ida, Kinuyo; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tesuya; Kamiya, Akihide

    2016-01-01

    Liver consists of parenchymal hepatocytes and other cells. Liver progenitor cell (LPC) is the origin of both hepatocytes and cholangiocytic cells. The analyses of mechanism regulating differentiation of LPCs into these functional cells are important for liver regenerative therapy using progenitor cells. LPCs in adult livers were found to form cysts with cholangiocytic characteristics in 3D culture. In contrast, foetal LPCs cannot form these cholangiocytic cysts in the same culture. Thus, the transition of foetal LPCs into cholangiocytic progenitor cells might occur during liver development. Primary CD45−Ter119−Dlk1+ LPCs derived from murine foetal livers formed ALBUMIN (ALB)+CYTOKERATIN (CK)19− non-cholangiocytic cysts within 3D culture. In contrast, when foetal LPCs were pre-cultured on gelatine-coated dishes, they formed ALB−CK19+ cholangiocytic cysts. When hepatocyte growth factor or oncostatin M, which are inducers of hepatocytic differentiation, was added to pre-culture, LPCs did not form cholangiocytic cysts. These results suggest that the pre-culture on gelatine-coated dishes changed the characteristics of foetal LPCs into cholangiocytic cells. Furthermore, neonatal liver progenitor cells were able to form cholangiocytic cysts in 3D culture without pre-culture. It is therefore possible that the pre-culture of mid-foetal LPCs in vitro functioned as a substitute for the late-foetal maturation step in vivo. PMID:27335264

  20. Changes in Biliary Levels of Arginine and its Methylated Derivatives after Hepatic Ischaemia/Reperfusion.

    PubMed

    Ferrigno, Andrea; Di Pasqua, Laura Giuseppina; Berardo, Clarissa; Rizzo, Vittoria; Richelmi, Plinio; Vairetti, Mariapia

    2016-07-01

    Arginine (Arg) can be methylated to form symmetrical dimethylarginine (SDMA) and asymmetrical dimethylarginine (ADMA), the latter an endogenous inhibitor of nitric oxide synthase (NOS). SDMA is excreted in the urine, while ADMA is mainly subjected to degradation in the liver. Arg competes with ADMA and SDMA for cellular transport across cationic amino-acid transporters (CATs). We evaluated the changes in serum, tissue and biliary levels of Arg, citrulline (Cit), ADMA and SDMA and the modifications in CATs after ischaemia-reperfusion (I/R). Male Wistar rats were subjected to 30-min. partial-hepatic ischaemia or sham-operated. After 60-min. reperfusion, the concentrations of ADMA, SDMA, Arg and Cit in serum, tissue and bile were measured. Serum levels of AST, ALT and alkaline phosphatase (AP) levels were determined. mRNA of cationic transporter 2A (CAT-2A) and 2B (CAT-2B) were also quantified. An increase in ADMA and a decrease in SDMA were observed in bile at the end of reperfusion. On the contrary, lower tissue ADMA levels and higher SDMA levels were quantified. No serum changes in ADMA and SDMA were found. A decrease in Arg and an increase of Cit were detected in serum, bile and tissue after I/R. A marked increase in AST, ALT and AP levels in serum confirmed I/R injury. A decrease in mRNA transporter CAT-2A but not in CAT-2B was detected. This study supported a biliary CAT-2B-dependent transport of ADMA and demonstrated, for the first time, that the liver is also responsible for the biliary excretion of SDMA into the bile. PMID:26663642

  1. Development and Characterization of Human Induced Pluripotent Stem Cell-Derived Cholangiocytes

    PubMed Central

    De Assuncao, Thiago M.; Sun, Yan; Jalan-Sakrikar, Nidhi; Drinane, Mary; Huang, Bing Q.; Li, Ying; Davila, Jaime I.; Wang, Ruisi; O’Hara, Steven P.; Lomberk, Gwen A.; Urrutia, Raul A.; Ikeda, Yasuhiro; Huebert, Robert C.

    2015-01-01

    Background and Aims Cholangiocytes are the target of a heterogeneous group of liver diseases, known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore, the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Methods Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a step-wise differentiation strategy toward iDCs using precise temporal exposure to key biliary morphogens and we characterized the cells using a variety of morphologic, molecular, cell biologic, functional, and in vivo approaches. Results Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC, definitive endoderm, hepatic specification, hepatic progenitors, and ultimately cholangiocytes. Immunostaining, Western blotting, and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP, form intact primary cilia, and self-assemble into duct-like structures in 3-dimensional culture. In vivo, the cells engraft within mouse liver following retrograde intra-biliary infusion. Conclusions In summary, we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation, iDCs represent a platform for in vitro disease modelling, pharmacologic testing, and individualized, cell-based, regenerative therapies for the cholangiopathies. PMID:25867762

  2. Development and characterization of human-induced pluripotent stem cell-derived cholangiocytes.

    PubMed

    De Assuncao, Thiago M; Sun, Yan; Jalan-Sakrikar, Nidhi; Drinane, Mary C; Huang, Bing Q; Li, Ying; Davila, Jaime I; Wang, Ruisi; O'Hara, Steven P; Lomberk, Gwen A; Urrutia, Raul A; Ikeda, Yasuhiro; Huebert, Robert C

    2015-06-01

    Cholangiocytes are the target of a heterogeneous group of liver diseases known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore, the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a stepwise differentiation strategy toward iDCs, using precise temporal exposure to key biliary morphogens, and we characterized the cells, using a variety of morphologic, molecular, cell biologic, functional, and in vivo approaches. Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC, definitive endoderm, hepatic specification, hepatic progenitors, and ultimately cholangiocytes. Immunostaining, western blotting, and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP, form intact primary cilia, and self-assemble into duct-like structures in three-dimensional culture. In vivo, the cells engraft within mouse liver, following retrograde intrabiliary infusion. In summary, we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation, iDCs represent a platform for in vitro disease modeling, pharmacologic testing, and individualized, cell-based, regenerative therapies for the cholangiopathies. PMID:25867762

  3. Blockade of Tim-3 Pathway Ameliorates Interferon-γ Production from Hepatic CD8+ T Cells in a Mouse Model of Hepatitis B Virus Infection

    PubMed Central

    Ju, Ying; Hou, Nan; Zhang, Xiaoning; Zhao, Di; Liu, Ying; Wang, Jinjin; Luan, Fang; Shi, Wei; Zhu, Faliang; Sun, Wensheng; Zhang, Lining; Gao, Chengjiang; Gao, Lifen; Liang, Xiaohong; Ma, Chunhong

    2009-01-01

    T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus (HBV) infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8+ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-γ production from hepatic CD8+ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8+ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection. PMID:19254478

  4. Lack of phenotypic and functional impairment in dendritic cells from chimpanzees chronically infected with hepatitis C virus.

    PubMed

    Larsson, Marie; Babcock, Ethan; Grakoui, Arash; Shoukry, Naglaa; Lauer, Georg; Rice, Charles; Walker, Christopher; Bhardwaj, Nina

    2004-06-01

    Dendritic cells (DCs), which are potent antigen-presenting cells (APCs), are used as adjuvants for the treatment of cancer and infectious diseases in human and nonhuman primates, with documented clinical efficacy. The hepatitis C virus (HCV)-chimpanzee model is the best available model for testing the immunotherapeutic effects of DCs in the setting of a chronic infection, as chimpanzees develop a persistent infection resembling that seen in humans. However, several reports have suggested that DCs derived from chronically infected individuals or nonhuman primates are functionally compromised. As a prelude to clinical studies, we evaluated whether functionally mature DCs could be generated in chimpanzee plasma by good manufacturing practice using CD14(+) mononuclear precursors from chronically infected chimpanzees. DCs generated in a medium with HCV-negative plasma and treated with a defined cocktail of cytokines or a CD40 ligand trimer matured fully, as measured by the induction of CD83 expression and the upregulation of costimulatory molecules. Furthermore, the expression of CCR7 was induced, suggesting an acquisition of migration capacity. Mature DCs were capable of stimulating allogeneic T cells, antigen-specific memory CD4(+) T cells, and HCV-specific CD8(+)-T-cell clones. In all cases, there was no evidence of HCV infection in DCs. Furthermore, these DCs maintained their phenotype and APC function after cryopreservation. Finally, no discernible differences were noted between DCs derived from HCV-infected and uninfected chimpanzees. In summary, precursor cells from HCV-infected chimpanzees are fully capable of differentiating into functional, mature DCs, which can now be reproducibly prepared for investigations of their immunotherapeutic potential in the setting of chronic HCV infection. PMID:15163708

  5. Gene screening of Wharton's jelly derived stem cells.

    PubMed

    Mechiche Alami, S; Velard, F; Draux, F; Siu Paredes, F; Josse, J; Lemaire, F; Gangloff, S C; Graesslin, O; Laurent-Maquin, D; Kerdjoudj, H

    2014-01-01

    Stem cells are the most powerful candidate for the treatment of various diseases. Suitable stem cell source should be harvested with minimal invasive procedure, found in great quantity, and transplanted with no risk of immune response and tumor formation. Fetal derived stem cells have been introduced as an excellent alternative to adult and embryonic stem cells use, but unfortunately, their degree of "stemness" and molecular characterization is still unclear. Several studies have been performed deciphering whether fetal stem cells meet the needs of regenerative medicine. We believe that a transcriptomic screening of Wharton's jelly stem cells will bring insights on cell population features. PMID:24928918

  6. Successful differentiation to T cells, but unsuccessful B-cell generation, from B-cell-derived induced pluripotent stem cells.

    PubMed

    Wada, Haruka; Kojo, Satoshi; Kusama, Chie; Okamoto, Naoki; Sato, Yorino; Ishizuka, Bunpei; Seino, Ken-ichiro

    2011-01-01

    Forced expression of certain transcription factors in somatic cells results in generation of induced pluripotent stem (iPS) cells, which differentiate into various cell types. We investigated T-cell and B-cell lineage differentiation from iPS cells in vitro. To evaluate the impact of iPS cell source, murine splenic B-cell-derived iPS (B-iPS) cells were generated after retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc). B-iPS cells were identical to embryonic stem (ES) cells and mouse embryonic fibroblast (MEF)-derived iPS cells in morphology, ES cell marker expression as well as teratoma and chimera mouse formation. Both B-iPS and MEF-derived iPS cells differentiated into lymphocytes in OP9 co-culture systems. Both efficiently differentiated into T-cell lineage that produced IFN-γ on T-cell receptor stimulation. However, iPS cells including B-iPS cells were relatively resistant to B-cell lineage differentiation. One of the reasons of the failure of B-cell lineage differentiation seemed due to a defect of Pax5 expression in the differentiated cells. Therefore, current in vitro differentiation systems using iPS cells are sufficient for inducing T-cell but not B-cell lineage. PMID:21135032

  7. A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation

    PubMed Central

    Vuoristo, Sanna; Toivonen, Sanna; Weltner, Jere; Mikkola, Milla; Ustinov, Jarkko; Trokovic, Ras; Palgi, Jaan; Lund, Riikka; Tuuri, Timo; Otonkoski, Timo

    2013-01-01

    Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines. PMID:24098444

  8. Hepatic progenitor cells, stem cells, and AFP expression in models of liver injury

    PubMed Central

    Kuhlmann, Wolf D; Peschke, Peter

    2006-01-01

    Adult hepatocytes and liver-cell progenitors play a role in restoring liver tissue after injury. For the study of progenitor cells in liver repair, experimental models included (a) surgical removal of liver tissue by partial hepatectomy; (b) acute injury by carbontetrachloride; (c) acute injury by d-galactosamine (GalN) and N-nitrosomorpholine (NNM); and (d) chemical hepatocarcinogenesis by feeding NNM in low and high doses. Serological and immunohistological detection of alpha-fetoprotein gene expression served to follow pathways of cellular differentiation. Stem cells were not required in models of surgical removal of parenchyma and in carbon tetrachloride intoxication of adult hepatocytes. In contrast, regeneration of liver occurred through biliary epithelial cells in injuries induced by GalN and NNM. These biliary epithelial cells, collectively called oval cells, are most probably derived from the canals of Hering. Proliferating bile duct cells reached a level of differentiation with reactivation of foetal genes and significant alpha-1-fetoprotein (AFP) synthesis signalling a certain degree of retrodifferentiation with potential stemness. Due to the same embryonic origin of bile ducts and hepatocytes, biliary epithelium and its proliferating progeny (oval cells) have a defined role in liver regeneration as a transit and amplification compartment. In their early proliferation stage, oval cells were heavily engaged in DNA synthesis ([3H]thymidine labelling). Pulse-chase experiments during experimental hepatocarcinogenesis exhibited their development into hepatocytes with high risk for transformation and leading to foci of altered hepatocytes. Hepatocellular carcinomas may arise either from proliferating/differentiating oval cells or from adult hepatocytes; both cell types have stem-like properties. AFP-positive and AFP-negative carcinomas occurred in the same liver. They may represent random clonal origin. The heterogeneity of phenotypic marker (AFP) correlated

  9. Khat (Catha edulis) generates reactive oxygen species and promotes hepatic cell apoptosis via MAPK activation.

    PubMed

    Abid, Morad Dirhem Naji; Chen, Juan; Xiang, Min; Zhou, Jie; Chen, Xiaoping; Gong, Feili

    2013-08-01

    A number of studies have suggested an association between khat (Catha edulis) chewing and acute liver lesions or chronic liver disease. However, little is known about the effects of khat on hepatic cells. In the current study, we investigated the mechanism behind khat-induced apoptosis in the L02 human hepatic cell line. We used cell growth inhibition assay, flow cytometry and Hoechst 33258 staining to measure hepatocyte apoptosis induced by khat. Western blot analysis was used to detect the expression levels of caspase-8 and -9, as well as those of Bax and Bcl-2. We also measured reactive oxygen species production. The results indicated that khat induced significant hepatocyte apoptosis in L02 cells. We found that khat activated caspase-8 and -9, upregulated Bax protein expression and downregulated Bcl-2 expression levels, which resulted in the coordination of apoptotic signals. Khat-induced hepatocyte apoptosis is primarily regulated through the sustained activation of the c-Jun NH2-terminal kinase (JNK) pathway and only partially via the extracellular signal-regulated kinase (ERK) cascade. Furthermore, the khat-induced reactive oxygen species (ROS) production and the activation of the ROS scavenger, N-acetyl-L-cysteine (NAC), attenuated the khat-induced activation of JNK and ERK. Our results demonstrate that khat triggers the generation of intracellular ROS and sequentially induces the sustainable activation of JNK, which in turn results in a decrease in cell viability and an increase in cell apoptosis. PMID:23708648

  10. Monocyte Heterogeneity: Consequences for Monocyte-Derived Immune Cells

    PubMed Central

    de Vries, Teun J.; Everts, Vincent

    2016-01-01

    Blood monocytes are precursors of dendritic cells, macrophages, and osteoclasts. They are a heterogeneous cell population with differences in size, phenotype, and function. Although monocytes maintain several tissue-specific populations of immune cells in homeostasis, their contribution to populations of dendritic cells, macrophages, and osteoclasts is significantly increased in inflammation. Identification of a growing number of functionally different subsets of cells within populations of monocyte-derived immune cells has recently put monocyte heterogeneity into sharp focus. Here, we summarize recent findings in monocyte heterogeneity and their differentiation into dendritic cells, macrophages, and osteoclasts. We also discuss these advances in the context of the formation of functionally different monocyte-derived subsets of dendritic cells, macrophages, and osteoclasts. PMID:27478854

  11. Diosgenin attenuates hepatic stellate cell activation through transforming growth factor-β/Smad signaling pathway

    PubMed Central

    Xie, Wei-Lin; Jiang, Rong; Shen, Xiao-Lu; Chen, Zhi-Yu; Deng, Xiao-Ming

    2015-01-01

    Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species, and it inhibited high glucose-induced renal tubular fibrosis. However, the effects of diosgenin against hepatic fibrosis remain elusive. Therefore, in this study, we investigated the effects of diosgenin on TGF-β1-induced HSCs and elucidate the possible mechanism of its anti-fibrotic effect. Our results demonstrated that diosgenin inhibited TGF-β1-induced HSC proliferation, reduced the expression of collagen I and α-smooth muscle actin (α-SMA), as well as the expression of TGF-β receptor I (TGF-β RI) and II. Moreover, diosgenin suppressed TGF-β1-induced phosphorylation of Smad3 in HSCs. In conclusion, our data demonstrate that diosgenin inhibited HSC-T6 cell proliferation and activation, at least in part, via the TGF-β1/Smad signaling pathway. These results provide that diosgenin may have potential to treat liver fibrosis. PMID:26884947

  12. Diosgenin attenuates hepatic stellate cell activation through transforming growth factor-β/Smad signaling pathway.

    PubMed

    Xie, Wei-Lin; Jiang, Rong; Shen, Xiao-Lu; Chen, Zhi-Yu; Deng, Xiao-Ming

    2015-01-01

    Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species, and it inhibited high glucose-induced renal tubular fibrosis. However, the effects of diosgenin against hepatic fibrosis remain elusive. Therefore, in this study, we investigated the effects of diosgenin on TGF-β1-induced HSCs and elucidate the possible mechanism of its anti-fibrotic effect. Our results demonstrated that diosgenin inhibited TGF-β1-induced HSC proliferation, reduced the expression of collagen I and α-smooth muscle actin (α-SMA), as well as the expression of TGF-β receptor I (TGF-β RI) and II. Moreover, diosgenin suppressed TGF-β1-induced phosphorylation of Smad3 in HSCs. In conclusion, our data demonstrate that diosgenin inhibited HSC-T6 cell proliferation and activation, at least in part, via the TGF-β1/Smad signaling pathway. These results provide that diosgenin may have potential to treat liver fibrosis. PMID:26884947

  13. Modulation of a Glycoprotein Recognition System on Rat Hepatic Endothelial Cells by Glucose and Diabetes Mellitus

    PubMed Central

    Summerfield, John A.; Vergalla, John; Jones, E. Anthony

    1982-01-01

    The cellular location and carbohydrate specificities of a glycoprotein recognition system on rat hepatic sinusoidal cells have been determined. Purified preparations of endothelial, Kupffer, and parenchymal cells were prepared by collagenase liver perfusion, centrifugation on Percoll gradients, and centrifugal elutriation. 125I-labeled agalactoorosomucoid, an N-acetylglucosamine-terminated glycoprotein, was selectively taken up in vitro by endothelial cells. Uptake was shown to be protein dependent, calcium ion dependent, and saturable, and could be described by Michaelis-Menten kinetics (apparent Km 0.29 μM; apparent maximum velocity 4.8 pmol/h per 5 × 106 cells). Uptake was inhibited not only by N-acetylglucosamine, mannose, and mannan but also by glucose, fructose, and a glucose-albumin conjugate. Inhibition by glucose was competitive over a wide range of concentrations and was almost 100% at a glucose concentration of 56 mM. Fasting and the induction of diabetes mellitus prior to isolation of cells was associated with 60% reductions in the recovery of endothelial cells. Uptake by cells isolated from fasted rats was enhanced (apparent maximum velocity 14.3 pmol/h per 5 × 106 cells without change in the apparent Km). These observations suggest that fasting is associated with a marked increase in the mean number of glycoprotein receptors per endothelial cell isolated from normal rats. This effect of fasting could be due to upregulation of glycoprotein receptors on endothelial cells or to the selective isolation of a subpopulation of endothelial cells from fasted animals that bears more glycoprotein receptors per cell than does another subpopulation of these cells. In addition, in vivo studies of the fate of intravenously administered 125I-agalactoorosomucoid indicated that its rate of disappearance from plasma, hepatic accumulation, and catabolism were slower in diabetic than in normal rats. The results suggest that modulation of a carbohydrate

  14. Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells

    PubMed Central

    Kossack, Nina; Meneses, Juanito; Shefi, Shai; Nguyen, Ha Nam; Chavez, Shawn; Nicholas, Cory; Gromoll, Joerg; Turek, Paul J; Reijo-Pera, Renee A

    2009-01-01

    Several reports have documented the derivation of pluripotent cells (multipotent germline stem cells) from spermatogonial stem cells obtained from the adult mouse testis. These spermatogonia-derived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. Data indicate that derivation may involve reprogramming of endogenous spermatogonia in culture. Here, we report the derivation of human multipotent germline stem cells (hMGSCs) from a testis biopsy. The cells express distinct markers of pluripotency, form embryoid bodies that contain derivatives of all three germ layers, maintain a normal XY karyotype, are hypomethylated at the H19 locus, and express high levels of telomerase. Teratoma assays indicate the presence of human cells 8 weeks post-transplantation but limited teratoma formation. Thus, these data suggest the potential to derive pluripotent cells from human testis biopsies but indicate a need for novel strategies to optimize hMGSC culture conditions and reprogramming. PMID:18927477

  15. Structural Phenotyping of Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2015-01-01

    Summary Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. PMID:25733020

  16. Adipose-derived stem cells: Implications in tissue regeneration

    PubMed Central

    Tsuji, Wakako; Rubin, J Peter; Marra, Kacey G

    2014-01-01

    Adipose-derived stem cells (ASCs) are mesenchymal stem cells (MSCs) that are obtained from abundant adipose tissue, adherent on plastic culture flasks, can be expanded in vitro, and have the capacity to differentiate into multiple cell lineages. Unlike bone marrow-derived MSCs, ASCs can be obtained from abundant adipose tissue by a minimally invasive procedure, which results in a high number of cells. Therefore, ASCs are promising for regenerating tissues and organs damaged by injury and diseases. This article reviews the implications of ASCs in tissue regeneration. PMID:25126381

  17. Schizophrenia patient-derived olfactory neurosphere-derived cells do not respond to extracellular reelin

    PubMed Central

    Tee, Jing Yang; Sutharsan, Ratneswary; Fan, Yongjun; Mackay-Sim, Alan

    2016-01-01

    Reelin expression is reduced in various regions in the post-mortem brain of schizophrenia patients but the exact role of reelin function in the neurobiology of schizophrenia remains elusive. Absence of reelin in knockout mouse causes inverted lamination of the neocortex due to aberrant neuronal migration. The aim of this study was to utilize patient-derived olfactory neurosphere-derived (ONS) cells to investigate whether extracellular reelin alters cell motility in schizophrenia patient-derived cells. ONS cells from nine patients were compared with cells from nine matched healthy controls. Automated high-throughput imaging and analysis were used to track motility of individual living cells on reelin-coated surfaces produced from reelin secreted into the medium by HEK293FT cells transfected with the full-length reelin plasmid pCrl. Automated assays were used to quantify intracellular cytoskeleton composition, cell morphology, and focal adhesions. Expression of reelin and components of the reelin signaling pathway were measured by western blot and flow cytometry. Reelin inhibited the motility of control cells but not patient cells, and increased the number and size of focal adhesions in control cells but not patient cells. Patient and control cells expressed similar levels of the reelin receptors and the reelin signaling protein, Dab1, but patient cells expressed less reelin. Patient cells were smaller than control cells and had less actin and acetylated α-tubulin, components of the cytoskeleton. These findings are the first direct evidence that cellular responses to reelin are impaired in schizophrenia and are consistent with the role of reelin in cytoarchitectural deficits observed in schizophrenia patient brains. PMID:27602387

  18. Schizophrenia patient-derived olfactory neurosphere-derived cells do not respond to extracellular reelin.

    PubMed

    Tee, Jing Yang; Sutharsan, Ratneswary; Fan, Yongjun; Mackay-Sim, Alan

    2016-01-01

    Reelin expression is reduced in various regions in the post-mortem brain of schizophrenia patients but the exact role of reelin function in the neurobiology of schizophrenia remains elusive. Absence of reelin in knockout mouse causes inverted lamination of the neocortex due to aberrant neuronal migration. The aim of this study was to utilize patient-derived olfactory neurosphere-derived (ONS) cells to investigate whether extracellular reelin alters cell motility in schizophrenia patient-derived cells. ONS cells from nine patients were compared with cells from nine matched healthy controls. Automated high-throughput imaging and analysis were used to track motility of individual living cells on reelin-coated surfaces produced from reelin secreted into the medium by HEK293FT cells transfected with the full-length reelin plasmid pCrl. Automated assays were used to quantify intracellular cytoskeleton composition, cell morphology, and focal adhesions. Expression of reelin and components of the reelin signaling pathway were measured by western blot and flow cytometry. Reelin inhibited the motility of control cells but not patient cells, and increased the number and size of focal adhesions in control cells but not patient cells. Patient and control cells expressed similar levels of the reelin receptors and the reelin signaling protein, Dab1, but patient cells expressed less reelin. Patient cells were smaller than control cells and had less actin and acetylated α-tubulin, components of the cytoskeleton. These findings are the first direct evidence that cellular responses to reelin are impaired in schizophrenia and are consistent with the role of reelin in cytoarchitectural deficits observed in schizophrenia patient brains. PMID:27602387

  19. Selective Expansion of Allogeneic Regulatory T Cells by Hepatic Stellate Cells: Role of Endotoxin and Implications for Allograft Tolerance

    PubMed Central

    Dangi, Anil; Sumpter, Tina L.; Kimura, Shoko; Stolz, Donna B.; Murase, Noriko; Raimondi, Giorgio; Vodovotz, Yoram; Huang, Chao; Thomson, Angus W.; Gandhi, Chandrashekhar R.

    2012-01-01

    Hepatic stellate cells (HSCs) may play an important role in hepatic immune regulation by producing numerous cytokines/chemokines, and expressing Ag-presenting and T cell co-regulatory molecules. Due to disruption of the endothelial barrier during cold-ischemic storage and reperfusion of liver grafts, HSCs can interact directly with cells of the immune system. Endotoxin (LPS), levels of which increase in liver diseases and transplantation, stimulates the synthesis of many mediators by HSCs. We hypothesized that LPS-stimulated HSCs might promote hepatic tolerogenicity by influencing naturally-occurring immunosuppressive CD4+CD25+FoxP3+ regulatory T cells (Tregs). Following their portal venous infusion, allogeneic CD4+ T cells, including Tregs, were found closely associated with HSCs, and this association increased in LPS-treated livers. In vitro, both unstimulated and LPS-stimulated HSCs up-regulated Fas (CD95) expression on conventional CD4+ T cells and induced their apoptosis in a Fas/FasL-dependent manner. By contrast, HSCs induced Treg proliferation, which required cell-cell contact, and was MHC class II-dependent. This effect was augmented when HSCs were pretreated with LPS. LPS increased the expression of MHC class II, CD80 and CD86, and stimulated the production of IL-1α, IL-1β, IL-6, IL-10 and TNFα by HSCs. Interestingly, production of IL-1α, IL-1β, IL-6 and TNFα was strongly inhibited, but that of IL-10 enhanced, in LPS-pretreated HSC/Treg co-cultures. Adoptively transferred allogeneic HSCs migrated to the secondary lymphoid tissues and induced Treg expansion in lymph nodes. These data implicate endotoxins-stimulated HSCs as important immune regulators in liver transplantation by inducing selective expansion of tolerance-promoting Tregs, and reducing inflammation and allo-immunity. PMID:22427640

  20. The genetic organization of integrated hepatitis B virus DNA in the human hepatoma cell line PLC/PRF/5.

    PubMed Central

    Koch, S; Freytag von Loringhoven, A; Kahmann, R; Hofschneider, P H; Koshy, R

    1984-01-01

    Hepatitis B virus (HBV) DNA is often found integrated in the genome of infected human liver cells and is supposed to be related to the development of primary liver carcinoma (PLC). Four clones of HBV DNA-containing sequences derived from DNA of the human PLC-derived cell line PLC/PRF/5 are discussed. The viral sequences show no intricate rearrangements excepting for a duplication and an inversion in one case, and a deletion in another. In all cases integration of the viral DNA was seen to be in a region which is single-stranded in the unintegrated HBV DNA. Sequence homologies between human and viral DNA flanking the integration sites have been detected. That may have a functional role in integration. Nucleotide sequence analyses of regions encompassing the viral-human junctions reveal open reading frames which consist of viral and/or human information. The possible expression of chimeric or cellular proteins may play a role in tumour development, and offers directions for further investigations. Images PMID:6091042

  1. Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Arasappan, Ashok; Venkatraman, Srikanth; Vibulbhan, Bancha; Yang, Weiying; Parekh, Tejal N.; Pichardo, John; Prongay, Andrew; Cheng, Kuo-Chi; Butkiewicz, Nancy; Yao, Nanhua; Madison, Vincent; Girijavallabhan, Viyyoor

    2008-06-30

    The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic {alpha}-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K*{sub i}). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K*{sub i} = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K*{sub i} = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC{sub 50} of 130 nM in a cellular replicon assay, while IC{sub 50} for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.

  2. Hepatitis E vaccines: progress and prospects.

    PubMed

    Worm, Harald Claus; Wirnsberger, Gerhard

    2004-01-01

    Hepatitis E accounts for the major part of enterally transmitted non-A, non-B hepatitis worldwide. Its agent, the hepatitis E virus (HEV), is a small, single-stranded RNA virus. Only one serotype of HEV is recognised. Infection results in protective immunity with long-lived neutralising antibodies. In developing countries with poor sanitary conditions and high population density, hepatitis E causes water-borne epidemics with substantial mortality rates in pregnant women. In addition, more than 50% of cases of acute hepatic failure and sporadic acute hepatitis are due to hepatitis E. The overall prevalence rates of antibodies to the HEV in populations native to these areas rarely exceed 25%. Hence, many individuals remain susceptible to hepatitis E infection, making hepatitis E an important public health concern. In this context, the development of an HEV vaccine is warranted. Because HEV does not grow adequately in cell cultures the development of a vaccine based on inactivated or attenuated whole-virus particles is not feasible. HEV vaccines currently under study are based on recombinant proteins derived from immunogenic parts of the HEV capsid gene. Other approaches such as DNA-based vaccines or transgenic tomatoes have also been developed. Several recombinant protein-based vaccines elicited neutralising antibodies and protective immunity in vaccinated non-human primates. One such vaccine has passed phase I trial and is currently under further evaluation in field trials. Even so, several questions remain to be answered before vaccination programmes could be implemented. PMID:15233590

  3. Dysregulation of Bmi1 promotes malignant transformation of hepatic progenitor cells.

    PubMed

    Zhang, R; Wu, W R; Shi, X D; Xu, L B; Zhu, M S; Zeng, H; Liu, C

    2016-01-01

    Adult hepatic progenitor cells (HPCs) are involved in a wide range of human liver diseases, including hepatocellular carcinoma (HCC). Bmi1 has been reported to have vital roles in stem cell self-renewal and carcinogenesis. We have previously demonstrated that Bmi1 is upregulated in HCC with bile duct tumor thrombi, a subtype of HCC characterized by profuse expression of hepatic stem cell markers. However, the function of Bmi1 in HPCs has not yet been well elucidated. The current study was designed to investigate the effects of Bmi1 on the biological properties of rat HPCs. To accomplish this, Bmi1 was silenced or enhanced in two HPC cell lines (WB-F344 and OC3) by, respectively, using either small interfering RNA against Bmi1 or a forced Bmi1 expression retroviral vector. The biological functions of Bmi1 in HPCs were investigated through cell proliferation assays, colony-formation assays, cell cycle analysis and invasion assays, as well as through xenograft-formation assays. In this study, genetic depletion of Bmi1 repressed cell proliferation, colony formation and invasion in both assessed HPC cell lines relative to controls. Conversely, forced expression of Bmi1 in two HPCs cell lines promoted cell proliferation, colony formation and invasion in vitro. Aldehyde dehydrogenase (ALDH) assay revealed a significant increase in the number of ALDH-positive cells following the forced expression of Bmi1 in HPCs. Most importantly, transplantation of forced Bmi1 expression HPCs into nude mice resulted in the formation of tumors with histological features of poorly differentiated HCC. Taken together, our findings indicate that forced expression of Bmi1 promotes the malignant transformation of HPCs, suggesting Bmi1 might be a potential molecular target for the treatment of HCC. PMID:26926789

  4. Hydrogen peroxide mediates vascular cell adhesion molecule-1 expression from interleukin-18-activated hepatic sinusoidal endothelium: implications for circulating cancer cell arrest in the murine liver.

    PubMed

    Mendoza, L; Carrascal, T; De Luca, M; Fuentes, A M; Salado, C; Blanco, J; Vidal-Vanaclocha, F

    2001-08-01

    The mechanism of intrasinusoidal arrest of circulating cancer cells, which is a critical step in liver metastasis, appears to be facilitated by tumor-derived proinflammatory factors that increase sinusoidal cell adhesion receptors for cancer cells. However, how this prometastatic microenvironment is up-regulated remains unknown. Using intrasplenically injected B16 melanoma (B16M) cells, we show that the expression of vascular cell adhesion molecule-1 (VCAM-1) significantly increased in hepatic sinusoidal endothelium (HSE) cells over physiologic baseline within the first 24 hours of metastatic cancer cell infiltration in the liver. This correlated with increased in vitro adhesion of B16M cells to HSE cells isolated from B16M cell-injected mice. In vivo VCAM-1 blockade with specific antibodies before B16M cell injection decreased sinusoidal retention of luciferase-transfected B16M cells by 85%, and metastasis development by 75%, indicating that VCAM-1 expression on tumor-activated HSE cells had a prometastatic contribution. Because VCAM-1 expression is oxidative stress-inducible, recombinant catalase was in vivo administered, resulting in a complete abrogation of both VCAM-1 expression and B16M cell adhesion increases in HSE cells isolated from B16M cell-injected mice. Catalase also abrogated the proadhesive response of HSE cells to B16M-conditioned medium (B16M-CM) in vitro, although this did not affect the concomitant release of major proinflammatory cytokines by HSE cells. HSE cells treated with B16M-CM released interleukin (IL)-18 via tumor necrosis factor-alpha (TNF-alpha)-dependent IL-1beta in vitro. In turn, H(2)O(2) production from B16M-CM-treated HSE cells was regulated by IL-18. Thus, liver-infiltrating B16M cells activated their adhesion to HSE through a sequential process involving TNF-alpha-dependent IL-1beta, which induced IL-18 to up-regulate VCAM-1 via H(2)O(2). The pivotal position of H(2)O(2) was further supported by the fact that incubation of HSE

  5. Interferon-β Mediates Signaling Pathways Uniquely Regulated in Hepatic Stellate Cells and Attenuates the Progression of Hepatic Fibrosis in a Dietary Mouse Model

    PubMed Central

    Nishimura, Kazumi; Akiyama, Hideo; Funamoto, Saeko; Izawa, Akiko; Sai, Takafumi; Kunita, Kana; Kainoh, Mie; Suzuki, Tomohiko; Kawada, Norifumi

    2015-01-01

    The results of clinical and experimental studies suggest that type I interferons (IFNs) may have direct antifibrotic activity in addition to their antiviral properties. However, the mechanisms are still unclear; in particular, little is known about the antifibrotic activity of IFN-β and how its activity is distinct from that of IFN-α. Using DNA microarrays, we demonstrated that gene expression in TWNT-4 cells, an activated human hepatic stellate cell line, was remarkably altered by IFN-β more than by IFN-α. Integrated pathway enrichment analyses revealed that a variety of IFN-β–mediated signaling pathways are uniquely regulated in TWNT-4 cells, including those related to cell cycle and Toll-like receptor 4 (TLR4) signaling. To investigate the antifibrotic activity of IFN-β and the involvement of TLR4 signaling in vivo, we used mice fed a choline-deficient l-amino acid-defined diet as a model of nonalcoholic steatohepatitis-related hepatic fibrosis. In this model, the administration of IFN-β significantly attenuated augmentation of the area of liver fibrosis, with accompanying transcriptional downregulation of the TLR4 adaptor molecule MyD88. Our results provide important clues for understanding the mechanisms of the preferential antifibrotic activity of IFN-β and suggest that IFN-β itself, as well as being a modulator of its unique signaling pathway, may be a potential treatment for patients with hepatic fibrosis in a pathogenesis-independent manner. PMID:25715168

  6. Recapitulation of the hepatitis C virus life-cycle in engineered murine cell lines

    PubMed Central

    Vogt, Alexander; Scull, Margaret A.; Friling, Tamar; Horwitz, Joshua A.; Donovan, Bridget M.; Dorner, Marcus; Gerold, Gisa; Labitt, Rachael N.; Rice, Charles M.; Ploss, Alexander

    2013-01-01

    Hepatitis C virus (HCV) remains a major medical problem. In-depth study of HCV pathogenesis and immune responses is hampered by the lack of suitable small animal models. The narrow host range of HCV remains incompletely understood. We demonstrate that the entire HCV life-cycle can be recapitulated in mouse cells. We show that antiviral signaling interferes with HCV RNA replication in mouse cells. We were able to infect mouse cells expressing human CD81 and occludin (OCLN) - the minimal set of entry factor factors required for HCV uptake into mouse cells. Infected mouse cells sustain HCV RNA replication in the presence of miR122 and release infectious particles when mouse apoE is supplied. Our data demonstrate that the barriers of HCV interspecies transmission can be overcome by engineering a suitable cellular environment and provide a blue-print towards constructing a small animal model for HCV infection. PMID:23777661

  7. Effects of β-like cell autotransplantation through hepatic arterial intervention on diabetic dogs.

    PubMed

    Mu, Yongxu; Hao, Zhiming; He, Junfeng; Yan, Ruiqiang; Liu, Haiyan; Zhang, Lei; Liu, Heming; Hu, Xiaoyan; Li, Qiming

    2016-08-01

    Exogenous insulin and EGFP genes were transduced into bone marrow mesenchymal stem cells of beagle dogs using the retroviral vector pMSCV to prepare β-like cells. These cells were autotransplanted into the liver of diabetic dogs through hepatic arterial intervention, and physiological indices before and after transplantation were monitored. Autotransplantation of β-like cells significantly improved blood sugar, insulin levels, and body mass of diabetic dogs (P < 0.01) continuously for over 80 weeks. Since the liver function remained normal and no tumors formed, this method was determined to be reliable and safe. Intrahepatic autotransplantation of β-like cells had long-term, reliable, and safe therapeutic effects on diabetic dogs. PMID:27328726

  8. Capsaicin modulates proliferation, migration, and activation of hepatic stellate cells.

    PubMed

    Bitencourt, Shanna; Mesquita, Fernanda; Basso, Bruno; Schmid, Júlia; Ferreira, Gabriela; Rizzo, Lucas; Bauer, Moises; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis; Mannaerts, Inge; van Grunsven, Leo Adrianus; Oliveira, Jarbas

    2014-03-01

    Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro. PMID:23955514

  9. Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone

    PubMed Central

    Date, Tomoko; Kato, Takanobu; Kato, Junko; Takahashi, Hitoshi; Morikawa, Kenichi; Akazawa, Daisuke; Murayama, Asako; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi

    2012-01-01

    Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones. PMID:22787209

  10. Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1).

    PubMed

    Fuller, Michael J; Callendret, Benoit; Zhu, Baogong; Freeman, Gordon J; Hasselschwert, Dana L; Satterfield, William; Sharpe, Arlene H; Dustin, Lynn B; Rice, Charles M; Grakoui, Arash; Ahmed, Rafi; Walker, Christopher M

    2013-09-10

    Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti-PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti-PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited. PMID:23980172

  11. Hepatitis B virus X protein mutants exhibit distinct biological activities in hepatoma Huh7 cells

    SciTech Connect

    Liu Xiaohong; Zhang Shuhui; Lin Jing; Zhang Shunmin; Feitelson, Mark A.; Gao Hengjun; Zhu Minghua

    2008-09-05

    The role of the hepatitis B virus X protein (HBx) in hepatocarcinogenesis remains controversial. To investigate the biological impact of hepatitis B virus x gene (HBx) mutation on hepatoma cells, plasmids expressing the full-length HBx or HBx deletion mutants were constructed. The biological activities in these transfectants were analyzed by a series of assays. Results showed that HBx3'-20 and HBx3'-40 amino acid deletion mutants exhibited an increase in cellular proliferation, focus formation, tumorigenicity, and invasive growth and metastasis through promotion of the cell cycle from G0/G1 to the S phase, when compared with the full-length HBx. In contrast, HBx3'-30 amino acid deletion mutant repressed cell proliferation by blocking in G1 phase. The expression of P53, p21{sup WAF1}, p14{sup ARF}, and MDM2 proteins was regulated by expression of HBx mutants. In conclusions, HBx variants showed different effects and functions on cell proliferation and invasion by regulation of the cell cycle progression and its associated proteins expression.

  12. Myogenic potential of adipose-tissue-derived cells.

    PubMed

    Di Rocco, Giuliana; Iachininoto, Maria Grazia; Tritarelli, Alessandra; Straino, Stefania; Zacheo, Antonella; Germani, Antonia; Crea, Filippo; Capogrossi, Maurizio C

    2006-07-15

    Adipose-tissue-derived mesenchymal stem cells can be directed towards a myogenic phenotype in vitro by the addition of specific inductive media. However, the ability of these or other adipose-tissue-associated cells to respond to ;natural' myogenic cues such as a myogenic environment has never been investigated in detail. Here, we provide evidence that a restricted subpopulation of freshly harvested adipose-tissue-derived cells possesses an intrinsic myogenic potential and can spontaneously differentiate into skeletal muscle. Conversion of adipose-tissue-derived cells to a myogenic phenotype is enhanced by co-culture with primary myoblasts in the absence of cell contact and is maximal when the two cell types are co-cultured in the same plate. Conversely, in vitro expanded adipose-tissue-derived mesenchymal stem cells require direct contact with muscle cells to generate skeletal myotubes. Finally, we show that uncultured adipose-tissue-associated cells have a high regenerative capacity in vivo since they can be incorporated into muscle fibers following ischemia and can restore significantly dystrophin expression in mdx mice. PMID:16825428

  13. Identification of a Binding Site for ASF/SF2 on an RNA Fragment Derived from the Hepatitis delta Virus Genome

    PubMed Central

    Sikora, Dorota; Zhang, Dajiang; Bojic, Teodora; Beeharry, Yasnee; Tanara, Ali; Pelchat, Martin

    2013-01-01

    The hepatitis delta virus (HDV) is a small (∼1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used. PMID:23349975

  14. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    PubMed

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments. PMID:27026484

  15. Autoimmune-Like Hepatitis: A "Hepatitic" Manifestation of Chronic Graft Versus Host Disease in Post-Stem Cell Transplant.

    PubMed

    Baniak, Nick M; Kanthan, Rani

    2016-04-01

    A 59-year-old female received a matched related donor stem cell transplant for chronic myelogenous leukemia. After being successfully treated with prednisone for chronic graft versus host disease that initially started 50 days posttransplant, she developed hepatic dysfunction during the steroid taper on day 531, as evidenced by jaundice, elevated liver enzymes, and increased bilirubin. Liver biopsy showed histology suggestive of autoimmune-like hepatitis, which is a rare manifestation of chronic "hepatitic" graft versus host disease. PMID:26464160

  16. Cardiac Repair by Embryonic Stem-Derived cells

    PubMed Central

    Rubart, M.

    2008-01-01

    Cell transplantation approaches offer the potential to promote regenerative growth of diseased hearts. It is well established that donor cardiomyocytes stably engraft into recipient hearts when injected directly into the myocardial wall. Moreover, the transplanted donor cardiomyocytes participate in a functional syncytium with the host myocardium. Thus, transplantation of donor cardiomyocytes resulted in at least partial restoration of lost muscle mass. It is also well established that embryonic stem (ES) cells differentiate into cells of ecto-, endo-, and mesodermal lineages when cultured under appropriate conditions in vitro. Robust cardiomyogenic differentiation was frequently observed in spontaneously differentiating ES cultures. Cellular, molecular and physiologic analyses indicated that ES-derived cells were bona fide cardiomyocytes, with in vitro characteristics typical for cells obtained from early stages of cardiac development. Thus, ES-derived cardiomyocytes constitute a viable source of donor cells for cell transplantation therapies. PMID:16370325

  17. Strain-Specific T-Cell Suppression and Protective Immunity in Patients with Chronic Hepatitis C Virus Infection

    PubMed Central

    Sugimoto, Kazushi; Kaplan, David E.; Ikeda, Fusao; Ding, Jin; Schwartz, Jonathan; Nunes, Frederick A.; Alter, Harvey J.; Chang, Kyong-Mi

    2005-01-01

    Hepatitis C virus (HCV) frequently persists with an apparently ineffective antiviral T-cell response. We hypothesized that some patients may be exposed to multiple HCV subtypes and that strain-specific T cells could contribute to the viral dynamics in this setting. To test this hypothesis, CD4 T-cell responses to three genotype 1a-derived HCV antigens and HCV antibody serotype were examined in chronically HCV infected (genotypes 1a, 1b, 2, 3, and 4) and spontaneously HCV recovered subjects. Consistent with multiple HCV exposure, 63% of patients infected with genotypes 2 to 4 (genotypes 2-4) and 36% of those infected with genotype 1b displayed CD4 T-cell responses to 1a-derived HCV antigens, while 29% of genotype 2-4-infected patients showed serotype responses to genotype 1. Detection of 1a-specific T cells in patients without active 1a infection suggested prior self-limited 1a infection with T-cell-mediated protection from 1a but not from non-1a viruses. Remarkably, CD4 T-cell responses to 1a-derived HCV antigens were weakest in patients with homologous 1a infection and greater in non-1a-infected patients: proportions of patients responding were 19% (1a), 36% (1b), and 63% (2-4) (P = 0.0006). Increased 1a-specific CD4 T-cell responsiveness in non-1a-infected patients was not due to increased immunogenicity or cross-reactivity of non-1a viruses but directly related to sequence divergence. We conclude that the T-cell response to the circulating virus is either suppressed or not induced in a strain-specific manner in chronically HCV infected patients and that, despite their ability to clear one HCV strain, patients may be reinfected with a heterologous strain that can then persist. These findings provide new insights into host-virus interactions in HCV infection that have implications for vaccine development. PMID:15890937

  18. Influence of the APOE genotype on hepatic stress response: Studies in APOE targeted replacement mice and human liver cells.

    PubMed

    Dose, Janina; Nebel, Almut; Piegholdt, Stefanie; Rimbach, Gerald; Huebbe, Patricia

    2016-07-01

    Apolipoprotein E (APOE) is a multifunctional plasma protein mainly acting in lipid metabolism. Human APOE is polymorphic with three major isoforms (APOE2, APOE3 and APOE4). Up to 75% of the body's APOE is produced by the liver. There is increasing evidence from studies in brain-derived cells that APOE4 affects mitochondrial function and biogenesis as well as stress and inflammatory responses - processes, whose disturbances are considered hallmarks of the ageing process. However, although the liver is the main production site of APOE, knowledge about the impact of the APOE genotype on hepatic stress response-related processes is rather limited. Therefore, we studied biomarkers of oxidative status (glutathione levels, 3-nitrotyrosine adducts, protein carbonyl concentration), ER stress (XBP1(S), BiP, DDIT3), proteasome activity, mitochondrial function (respiratory complexes, ATP levels and mitochondrial membrane potential as well as biomarkers of mitochondrial biogenesis, fission and fusion), autophagy (LC3, LAMP2A), apoptosis (BCL2, BAX, CYCS) and DNA damage in the liver of APOE targeted replacement (TR) mice and in Huh7 hepatocytes overexpressing the APOE3 and the APOE4 isoform, respectively. APOE4 mice exhibited a lower chymotrypsin-like and a higher trypsin-like proteasome activity. Levels of protein carbonyls were moderately higher in liver tissue of APOE4 vs. APOE3 mice. Other biomarkers of oxidative stress were similar between the two genotypes. Under basal conditions, the stress-response pathways investigated appeared largely unaffected by the APOE genotype. However, upon stress induction, APOE4 expressing cells showed lower levels of adenosine triphosphate (ATP) and lower mRNA levels of the ATP-generating complex V of the mitochondrial respiratory chain. Overall, our findings provide evidence for a rather low influence of the APOE genotype on the hepatic stress response processes investigated in this study. PMID:27130033

  19. CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism

    PubMed Central

    Wilhelm, Annika; Aldridge, Victoria; Haldar, Debashis; Naylor, Amy J; Weston, Christopher J; Hedegaard, Ditte; Garg, Abhilok; Fear, Janine; Reynolds, Gary M; Croft, Adam P; Henderson, Neil C; Buckley, Christopher D; Newsome, Philip N

    2016-01-01

    Introduction CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. Objective To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. Design CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248−/− and wild-type controls with carbon tetrachloride (CCl4) treatment. Results Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248−/− mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248−/− mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-β at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248−/− HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248−/− HSC was confirmed by significantly reduced c-fos expression in CD248−/− HSC compared with wild-type HSC. Conclusions Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury. PMID:26078290

  20. Apolipoprotein E, but Not Apolipoprotein B, Is Essential for Efficient Cell-to-Cell Transmission of Hepatitis C Virus

    PubMed Central

    Gondar, Virgínia; Molina-Jiménez, Francisca; Hishiki, Takayuki; García-Buey, Luisa; Koutsoudakis, George; Shimotohno, Kunitada

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) infects hepatocytes through two different routes: (i) cell-free particle diffusion followed by engagement with specific cellular receptors and (ii) cell-to-cell direct transmission mediated by mechanisms not well defined yet. HCV exits host cells in association with very-low-density lipoprotein (VLDL) components. VLDL particles contain apolipoproteins B (ApoB) and E (ApoE), which are required for viral assembly and/or infectivity. Based on these precedents, we decided to study whether these VLDL components participate in HCV cell-to-cell transmission in vitro. We observed that cell-to-cell viral spread was compromised after ApoE interference in donor but not in acceptor cells. In contrast, ApoB knockdown in either donor or acceptor cells did not impair cell-to-cell viral transmission. Interestingly, ApoB participated in the assembly of cell-free infective virions, suggesting a differential regulation of cell-to-cell and cell-free HCV infection. This study identifies host-specific factors involved in these distinct routes of infection that may unveil new therapeutic targets and advance our understanding of HCV pathogenesis. IMPORTANCE This work demonstrates that cell-to-cell transmission of HCV depends on ApoE but not ApoB. The data also indicate that ApoB is required for the assembly of cell-free infective particles, strongly suggesting the existence of mechanisms involving VLDL components that differentially regulate cell-free and cell-to-cell HCV transmission. These data clarify some of the questions regarding the role of VLDL in HCV pathogenesis and the transmission of the virus cell to cell as a possible mechanism of immune evasion and open the door to therapeutic intervention. PMID:26202245

  1. Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions and flow effects.

    PubMed

    Lee, Seung-A; No, Da Yoon; Kang, Edward; Ju, Jongil; Kim, Dong-Sik; Lee, Sang-Hoon

    2013-09-21

    We have developed a three-dimensional (3D) liver-on-a-chip to investigate the interaction of hepatocytes and hepatic stellate cells (HSCs) in which primary 3D hepatocyte spheroids and HSCs are co-cultured without direct cell-cell contact. Here, we show that the 3D liver chip offers substantial advantages for the formation and harvesting of spheroids. The most important feature of this liver chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. We also demonstrate that flow assists the formation and long-term maintenance of spheroids. Additionally, we quantitatively and qualitatively investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell-cell contacts, thereby improving liver-specific function. Spheroids derived from co-cultures exhibited improved albumin and urea secretion rates compared to mono-cultured spheroids after 9 days. Immunostaining for cytochrome P450 revealed that the enzymatic activity of spheroids co-cultured for 8 days was greater than that of mono-cultured spheroids. These results indicate that this system has the potential for further development as a unique model for studying cellular interactions or as a tool that can be incorporated into other models aimed at creating hepatic structure and prolonging hepatocyte function in culture. PMID:23657720

  2. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  3. An aqueous extract of Salacia oblonga root, a herb-derived peroxisome proliferator-activated receptor-alpha activator, by oral gavage over 28 days induces gender-dependent hepatic hypertrophy in rats.

    PubMed

    Rong, Xianglu; Kim, Moon Sun; Su, Ning; Wen, Suping; Matsuo, Yukimi; Yamahara, Johji; Murray, Michael; Li, Yuhao

    2008-06-01

    Activation of peroxisome proliferator-activated receptor (PPAR)-alpha by natural and synthetic chemicals induces hepatic hypertrophy. An aqueous extract of Salacia oblonga root (SOW) is an Ayurvedic medicine with anti-diabetic and anti-obesity properties. In the present study, it was found that SOW (100, 300 and 900mg/kg, once daily by oral gavage over a 28 day period) elicited dose-related increases in liver weight (LW) by 1.6%, 13.4% and 42.5%, respectively, and in the ratio of LW to body weight by 8.8%, 16.7% and 40.2%, respectively, in male rats. These effects were less pronounced in females. SOW selectively increased liver mass in male rats but Sudan red staining was not different, which indicates that hepatic lipid accumulation was similar in both genders. However, SOW even at the highest dosage did not influence serum ALT and AST activities in male or female rats. Moreover, SOW was found to activate PPAR-alpha in human hepatoma-derived HepG2 cells, as evidenced by the upregulation of PPAR-alpha and acyl-CoA oxidase mRNA expression. Thus, SOW-dependent PPAR-alpha activation may precede the development of the gender difference in hepatic hypertrophy; this process may be influenced by sex hormone status. PMID:18397819

  4. Exploration of acetanilide derivatives of 1-(ω-phenoxyalkyl)uracils as novel inhibitors of Hepatitis C Virus replication.

    PubMed

    Magri, Andrea; Ozerov, Alexander A; Tunitskaya, Vera L; Valuev-Elliston, Vladimir T; Wahid, Ahmed; Pirisi, Mario; Simmonds, Peter; Ivanov, Alexander V; Novikov, Mikhail S; Patel, Arvind H

    2016-01-01

    Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N(3)-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N(1) position. PMID:27406141

  5. Exploration of acetanilide derivatives of 1-(ω-phenoxyalkyl)uracils as novel inhibitors of Hepatitis C Virus replication

    PubMed Central

    Magri, Andrea; Ozerov, Alexander A.; Tunitskaya, Vera L.; Valuev-Elliston, Vladimir T.; Wahid, Ahmed; Pirisi, Mario; Simmonds, Peter; Ivanov, Alexander V.; Novikov, Mikhail S.; Patel, Arvind H.

    2016-01-01

    Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N3-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N1 position. PMID:27406141

  6. Pretreatment Hepatoprotective Effect of the Marine Fungus Derived from Sponge on Hepatic Toxicity Induced by Heavy Metals in Rats

    PubMed Central

    Abdel-Monem, Nehad M.; Abdel-Azeem, Ahmed M.; El-Ashry, El-Sayed H.; Ghareeb, Doaa A.; Nabil-adam, Asmaa

    2013-01-01

    The aim of this study was to evaluate the pretreatment hepatoprotective effect of the extract of marine-derived fungus Trichurus spiralis Hasselbr (TS) isolated from Hippospongia communis sponge on hepatotoxicity. Twenty-eight male Sprague-Dawley rats were divided into four groups (n = 7). Group I served as −ve control, group II served as the induced group receiving subcutaneously for seven days 0.25 mg heavy metal mixtures, group III received (i.p.) TS extract of dose 40 mg for seven days, and group IV served as the protected group pretreated with TS extract for seven days as a protection dose, and then treated with the heavy metal-mixture. The main pathological changes within the liver after heavy-metal mixtures administrations marked hepatic damage evidenced by foci of lobular necrosis with neutrophilic infiltration, adjacent to dysplastic hepatocytes. ALT and AST measurements show a significant increase in group II by 46.20% and 45.12%, respectively. Total protein, elevated by about 38.9% in induction group compared to the −ve control group, in contrast to albumin, decreased as a consequence of metal administration with significant elevation on bilirubin level. The results prove that TS extract possesses a hepatoprotective property due to its proven antioxidant and free-radical scavenging properties. PMID:23484129

  7. Hepatic B-cell non-Hodgkin's lymphoma of MALT type in the liver explant of a patient with chronic hepatitis C infection.

    PubMed

    Orrego, Mauricio; Guo, Linsheng; Reeder, Craig; De Petris, Giovanni; Balan, Vijayan; Douglas, David D; Byrne, Thomas; Harrison, Edwyn; Mulligan, David; Rodriguez-Luna, Hector; Moss, Adyr; Reddy, Kunam; Rakela, Jorge; Vargas, Hugo E

    2005-07-01

    B-cell non-Hodgkin's lymphoma (B-NHL) is a well-documented complication of hepatitis C virus (HCV) infection. Marginal zone (mucosa-associated lymphoid tissue; MALT) lymphomas constitute a less common type of B-NHL. In this article, we report a case of liver MALT in a cirrhotic patient, incidentally discovered after liver transplantation (LT). We discuss pertinent diagnostic and management strategies in this clinical setting. PMID:15973702

  8. Derivation of the human embryonic stem cell line RCM1.

    PubMed

    De Sousa, P A; Tye, B J; Sneddon, S; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Gardner, J; Downie, J M; Courtney, A; Brison, D R

    2016-03-01

    The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346018

  9. Fluorescent Fructose Derivatives for Imaging Breast Cancer Cells

    PubMed Central

    Levi, Jelena; Cheng, Zhen; Gheysens, Olivier; Patel, Manish; Chan, Carmel T.; Wang, Yingbing; Namavari, Mohammad; Gambhir, Sanjiv Sam

    2014-01-01

    Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells. PMID:17444608

  10. Reduced protein synthesis in schizophrenia patient-derived olfactory cells

    PubMed Central

    English, J A; Fan, Y; Föcking, M; Lopez, L M; Hryniewiecka, M; Wynne, K; Dicker, P; Matigian, N; Cagney, G; Mackay-Sim, A; Cotter, D R

    2015-01-01

    Human olfactory neurosphere-derived (ONS) cells have the potential to provide novel insights into the cellular pathology of schizophrenia. We used discovery-based proteomics and targeted functional analyses to reveal reductions in 17 ribosomal proteins, with an 18% decrease in the total ribosomal signal intensity in schizophrenia-patient-derived ONS cells. We quantified the rates of global protein synthesis in vitro and found a significant reduction in the rate of protein synthesis in schizophrenia patient-derived ONS cells compared with control-derived cells. Protein synthesis rates in fibroblast cell lines from the same patients did not differ, suggesting cell type-specific effects. Pathway analysis of dysregulated proteomic and transcriptomic data sets from these ONS cells converged to highlight perturbation of the eIF2α, eIF4 and mammalian target of rapamycin (mTOR) translational control pathways, and these pathways were also implicated in an independent induced pluripotent stem cell-derived neural stem model, and cohort, of schizophrenia patients. Analysis in schizophrenia genome-wide association data from the Psychiatric Genetics Consortium specifically implicated eIF2α regulatory kinase EIF2AK2, and confirmed the importance of the eIF2α, eIF4 and mTOR translational control pathways at the level of the genome. Thus, we integrated data from proteomic, transcriptomic, and functional assays from schizophrenia patient-derived ONS cells with genomics data to implicate dysregulated protein synthesis for the first time in schizophrenia. PMID:26485547

  11. Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B