Sample records for cell derived hepatic

  1. Derivation and characterization of hepatic progenitor cells from human embryonic stem cells. (United States)

    Zhao, Dongxin; Chen, Song; Cai, Jun; Guo, Yushan; Song, Zhihua; Che, Jie; Liu, Chun; Wu, Chen; Ding, Mingxiao; Deng, Hongkui


    The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

  2. Novel matrine derivative MD-1 attenuates hepatic fibrosis by inhibiting EGFR activation of hepatic stellate cells. (United States)

    Feng, Yi; Ying, Hai-Yan; Qu, Ying; Cai, Xiao-Bo; Xu, Ming-Yi; Lu, Lun-Gen


    Matrine (MT), the effective component of Sophora flavescens Ait, has been shown to have anti-inflammation, immune-suppressive, anti-tumor, and anti-hepatic fibrosis activities. However, the pharmacological effects of MT still need to be strengthened due to its relatively low efficacy and short half-life. In the present study, we report a more effective thio derivative of MT, MD-1, and its inhibitory effects on the activation of hepatic stellate cells (HSCs) in both cell culture and animal models. Cytological experiments showed that MD-1 can inhibit the proliferation of HSC-T6 cells with a half-maximal inhibitory concentration (IC50) of 62 μmol/L. In addition, MD-1 more strongly inhibits the migration of HSC-T6 cells compared to MT and can more effectively induce G0/G1 arrest and apoptosis. Investigating the biological mechanisms underlying anti-hepatic fibrosis in the presence of MD-1, we found that MD-1 can bind the epidermal growth factor receptor (EGFR) on the surface of HSC-T6 cells, which can further inhibit the phosphorylation of EGFR and its downstream protein kinase B (Akt), resulting in decreased expression of cyclin D1 and eventual inhibition of the activation of HSC-T6 cells. Furthermore, in rats with dimethylnitrosamine (DMN)-induced hepatic fibrosis, MD-1 slowed the development and progression of hepatic fibrosis, protecting hepatic parenchymal cells and improving hepatic functions. Therefore, MD-1 is a potential drug for anti-hepatic fibrosis.

  3. Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure.

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    Biju Parekkadan

    Full Text Available Modulation of the immune system may be a viable alternative in the treatment of fulminant hepatic failure (FHF and can potentially eliminate the need for donor hepatocytes for cellular therapies. Multipotent bone marrow-derived mesenchymal stem cells (MSCs have been shown to inhibit the function of various immune cells by undefined paracrine mediators in vitro. Yet, the therapeutic potential of MSC-derived molecules has not been tested in immunological conditions in vivo. Herein, we report that the administration of MSC-derived molecules in two clinically relevant forms-intravenous bolus of conditioned medium (MSC-CM or extracorporeal perfusion with a bioreactor containing MSCs (MSC-EB-can provide a significant survival benefit in rats undergoing FHF. We observed a cell mass-dependent reduction in mortality that was abolished at high cell numbers indicating a therapeutic window. Histopathological analysis of liver tissue after MSC-CM treatment showed dramatic reduction of panlobular leukocytic infiltrates, hepatocellular death and bile duct duplication. Furthermore, we demonstrate using computed tomography of adoptively transferred leukocytes that MSC-CM functionally diverts immune cells from the injured organ indicating that altered leukocyte migration by MSC-CM therapy may account for the absence of immune cells in liver tissue. Preliminary analysis of the MSC secretome using a protein array screen revealed a large fraction of chemotactic cytokines, or chemokines. When MSC-CM was fractionated based on heparin binding affinity, a known ligand for all chemokines, only the heparin-bound eluent reversed FHF indicating that the active components of MSC-CM reside in this fraction. These data provide the first experimental evidence of the medicinal use of MSC-derived molecules in the treatment of an inflammatory condition and support the role of chemokines and altered leukocyte migration as a novel therapeutic modality for FHF.

  4. Stem cells for hepatic regeneration: the role of adipose tissue derived mesenchymal stem cells. (United States)

    Ishikawa, Tetsuya; Banas, Agnieszka; Hagiwara, Keitaro; Iwaguro, Hideki; Ochiya, Takahiro


    Severe hepatic dysfunctions including hepatic cirrhosis and hepatocarcinoma are life-threatening conditions for which effective medical treatments are needed. With the only effective treatment to date being orthotropic liver transplantation, alternative approaches are needed because of the limited number of donors and the possibility of immune-rejection. One alternative is regenerative medicine, which holds promise for the development of a cell-based therapy enabling hepatic regeneration through transplantation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) or hepatocyte-like cells generated from AT-MSCs. When compared with embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, the use of AT-MSCs as regenerative cells would be advantageous in regard to ethical and safety issues since AT-MSCs are somatic cells and have the potential to be used without in vitro culture. These autologous cells are immuno-compatible and exhibit controlled differentiation and multi-functional abilities and do not undergo post-transplantation rejection or unwanted differentiation such as formation of teratomas. AT-MSC-based therapies may provide a novel approach for hepatic regeneration and hepatocyte differentiation and thereby support hepatic function in diseased individuals.

  5. Cryopreserved hepatic progenitor cells derived from human embryonic stem cells can arrest progression of liver fibrosis in rats. (United States)

    Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra


    Hepatocytes generated from human embryonic stem cells (hESCs) are considered to be an excellent candidate for restoring the liver function deficiencies. We have earlier standardized a three-step differentiation protocol to generate functional hepatocyte-like cells (HLCs) from hESCs, which expressed the major hepatic markers. We have also found that the HLCs remain stable and functional even after extended period of in vitro culture and cryopreservation. In the present study, we have aimed to investigate the therapeutic potential of cryopreserved-thawed hESC-derived hepatic progenitor cells following transplantation in carbon tetrachloride-induced fibrotic rat livers. Significant therapeutic effects, including improved hepatic histology and normal serum biochemistry of hepatic enzymes along with increased survival rate, were observed in the cell transplanted rats. This result is an encouraging indication to develop methods for clinical application of hESC-derived hepatic lineage cells.

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

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    Linda; Hammerich; Frank; Tacke


    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, bonemarrow or spleen, especially in conditions of cancer, infections or inflammation. MDSC usually express CD11 b, CD33, and low levels of human leukocyte antigen-DR in humans or CD11 b 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.

  7. Amniotic-fluid-derived mesenchymal stem cells overexpressing interleukin-1 receptor antagonist improve fulminant hepatic failure.

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    Yu-Bao Zheng

    Full Text Available Uncontrolled hepatic immunoactivation is regarded as the primary pathological mechanism of fulminant hepatic failure (FHF. The major acute-phase mediators associated with FHF, including IL-1β, IL-6, and TNF-α, impair the regeneration of liver cells and stem cell grafts. Amniotic-fluid-derived mesenchymal stem cells (AF-MSCs have the capacity, under specific conditions, to differentiate into hepatocytes. Interleukin-1-receptor antagonist (IL-1Ra plays an anti-inflammatory and anti-apoptotic role in acute and chronic inflammation, and has been used in many experimental and clinical applications. In the present study, we implanted IL-1Ra-expressing AF-MSCs into injured liver via the portal vein, using D-galactosamine-induced FHF in a rat model. IL-1Ra expression, hepatic injury, liver regeneration, cytokines (IL-1β, IL-6, and animal survival were assessed after cell transplantation. Our results showed that AF-MSCs over-expressing IL-1Ra prevented liver failure and reduced mortality in rats with FHF. These animals also exhibited improved liver function and increased survival rates after injection with these cells. Using green fluorescent protein as a marker, we demonstrated that the engrafted cells and their progeny were incorporated into injured livers and produced albumin. This study suggests that AF-MSCs genetically modified to over-express IL-1Ra can be implanted into the injured liver to provide a novel therapeutic approach to the treatment of FHF.

  8. Ionone Derivatives from the Mycelium of Phellinus linteus and the Inhibitory Effect on Activated Rat Hepatic Stellate Cells. (United States)

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


    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.

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

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    Shiow-Chyn Huang


    Full Text Available 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.

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



    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.

  11. Identification of potential biomarkers of hepatitis B-induced acute liver failure using hepatic cells derived from human skin precursors. (United States)

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


    Besides their role in the elucidation of pathogenic processes of medical and pharmacological nature, biomarkers can also be used to document specific toxicological events. Hepatic cells generated from human skin-derived precursors (hSKP-HPC) were previously shown to be a promising in vitro tool for the evaluation of drug-induced hepatotoxicity. In this study, their capacity to identify potential liver-specific biomarkers at the gene expression level was investigated with particular emphasis on acute liver failure (ALF). To this end, a set of potential ALF-specific biomarkers was established using clinically relevant liver samples obtained from patients suffering from hepatitis B-associated ALF. Subsequently, this data was compared to data obtained from primary human hepatocyte cultures and hSKP-HPC, both exposed to the ALF-inducing reference compound acetaminophen. It was found that both in vitro systems revealed a set of molecules that was previously identified in the ALF liver samples. Yet, only a limited number of molecules was common between both in vitro systems and the ALF liver samples. Each of the in vitro systems could be used independently to identify potential toxicity biomarkers related to ALF. It seems therefore more appropriate to combine primary human hepatocyte cultures with complementary in vitro models to efficiently screen out potential hepatotoxic compounds.

  12. Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-γ expression. (United States)

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


    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.

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

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    Xiaoling Zhou


    Full Text Available 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.

  14. Expression and the role of myeloid-derived suppressor cells in the peripheral blood in patients with chronic hepatitis B

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    Objective To investigate the correlation between the frequency of myeloid-derived suppressor cells(MDSC)and the frequency of regulatory T cells(Treg)in the peripheral blood in patients with chronic hepatitis B(CHB)and its clinical significance.Methods A total of 45 CHB patients including 23 mild-to-moderate CHB patients,22severe CHB patients,and 15 healhy control cytometry

  15. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection. (United States)

    Lin, Chih-Lang; Chien, Rong-Nan; Lin, Shi-Ming; Ke, Po-Yuan; Lin, Chen-Chun; Yeh, Chau-Ting


    Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

  16. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection.

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    Chih-Lang Lin

    Full Text Available Occult hepatitis B virus (HBV infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg. Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2. Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

  17. Functional Characteristics of Reversibly Immortalized Hepatic Progenitor Cells Derived from Mouse Embryonic Liver

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    Yang Bi


    Full Text Available Background/Aims: Liver is a vital organ and retains its regeneration capability throughout adulthood, which requires contributions from different cell populations, including liver precursors and intrahepatic stem cells. To overcome the mortality of hepatic progenitors (iHPs in vitro, we aim to establish reversibly immortalized hepatic progenitor cells from mouse embryonic liver. Methods and Results: Using retroviral system to stably express SV40 T antigen flanked with Cre/LoxP sites, we establish a repertoire of iHP clones with varied differentiation potential. The iHP cells maintain long-term proliferative activity and express varied levels of progenitor markers (Pou5f1/Oct4 and Dlk and hepatocyte markers (AFP, Alb and ApoB. Five representative iHP clones express hepatic/pancreatic transcription factors HNF3α/Foxa1, HNF3β/Foxa2, and HNF4α/MODY1. Dexamethasone is shown to promote the expression of hepatocyte markers AFP and TAT, along with ICG-uptake and glycogen storage functions in the iHP clones. Cre-mediated removal of SV40 T antigen reverses the proliferative activity of iHP cells. When iHP cells are subcutaneously implanted in athymic nude mice, no tumor formation is observed for up to 8 weeks. Conclusions: We demonstrate that the established iHP cells are stable, reversible, and non-tumorigenic hepatic progenitor-like cells, which should be valuable for studying liver organogenesis, metabolic regulations, and hepatic lineage-specific differentiation.

  18. Activated hepatic stellate cells promote liver cancer by induction of myeloid-derived suppressor cells through cyclooxygenase-2 (United States)

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


    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

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

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    Srilatha Badaboina


    Full Text Available 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.

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

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    Ruchi Sharma


    Full Text Available 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.

  1. Hepatic ischemia and reperfusion injury in the absence of myeloid cell-derived COX-2 in mice.

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    Sergio Duarte

    Full Text Available Cyclooxygenase-2 (COX-2 is a mediator of hepatic ischemia and reperfusion injury (IRI. While both global COX-2 deletion and pharmacologic COX-2 inhibition ameliorate liver IRI, the clinical use of COX-2 inhibitors has been linked to increased risks of heart attack and stroke. Therefore, a better understanding of the role of COX-2 in different cell types may lead to improved therapeutic strategies for hepatic IRI. Macrophages of myeloid origin are currently considered to be important sources of the COX-2 in damaged livers. Here, we used a Cox-2flox conditional knockout mouse (COX-2-M/-M to examine the function of COX-2 expression in myeloid cells during liver IRI. COX-2-M/-M mice and their WT control littermates were subjected to partial liver ischemia followed by reperfusion. COX-2-M/-M macrophages did not express COX-2 upon lipopolysaccharide stimulation and COX-2-M/-M livers showed reduced levels of COX-2 protein post-IRI. Nevertheless, selective deletion of myeloid cell-derived COX-2 failed to ameliorate liver IRI; serum transaminases and histology were comparable in both COX-2-M/-M and WT mice. COX-2-M/-M livers, like WT livers, developed extensive necrosis, vascular congestion, leukocyte infiltration and matrix metalloproteinase-9 (MMP-9 expression post-reperfusion. In addition, myeloid COX-2 deletion led to a transient increase in IL-6 levels after hepatic reperfusion, when compared to controls. Administration of celecoxib, a selective COX-2 inhibitor, resulted in significantly improved liver function and histology in both COX-2-M/-M and WT mice post-reperfusion, providing evidence that COX-2-mediated liver IRI is caused by COX-2 derived from a source(s other than myeloid cells. In conclusion, these results support the view that myeloid COX-2, including myeloid-macrophage COX-2, is not responsible for the hepatic IRI phenotype.

  2. Vγ4 γδ T cell-derived IL-17A negatively regulates NKT cell function in Con A-induced fulminant hepatitis. (United States)

    Zhao, Na; Hao, Jianlei; Ni, Yuanyuan; Luo, Wei; Liang, Ruifang; Cao, Guangchao; Zhao, Yapu; Wang, Puyue; Zhao, Liqing; Tian, Zhigang; Flavell, Richard; Hong, Zhangyong; Han, Jihong; Yao, Zhi; Wu, Zhenzhou; Yin, Zhinan


    Con A-induced fulminant hepatitis is a well-known animal model for acute liver failure. However, the role of γδ T cells in this model is undefined. In this report, using TCR δ(-/-) mice, we demonstrated a protective role of γδ T cells in Con A-induced hepatitis model. TCR δ(-/-) mice showed significantly decreased levels of IL-17A and IL-17F in the Con A-treated liver tissue, and reconstitution of TCR δ(-/-) mice with wild-type (Wt), but not IL-17A(-/-), γδ T cells significantly reduced hepatitis, strongly suggesting a critical role of IL-17A in mediating the protective effect of γδ T cells. Interestingly, only Vγ4, but not Vγ1, γδ T cells exerted such a protective effect. Furthermore, depletion of NKT cells in TCR δ(-/-) mice completely abolished hepatitis, and NKT cells from Con A-challenged liver tissues of TCR δ(-/-) mice expressed significantly higher amounts of proinflammatory cytokine IFN-γ than those from Wt mice, indicating that γδ T cells protected hepatitis through targeting NKT cells. Finally, abnormal capacity of IFN-γ production by NKT cells of TCR δ(-/-) mice could only be downregulated by transferring Wt, but not IL-17(-/-), Vγ4 γδ T cells, confirming an essential role of Vγ4-derived IL-17A in regulating the function of NKT cells. In summary, our report thus demonstrated a novel function of Vγ4 γδ T cells in mediating a protective effect against Con A-induced fulminant hepatitis through negatively regulating function of NKT cells in an IL-17A-dependent manner, and transferring Vγ4 γδ T cells may provide a novel therapeutic approach for this devastating liver disease.

  3. Tumor-induced CD11b(+) Gr-1(+) myeloid-derived suppressor cells exacerbate immune-mediated hepatitis in mice in a CD40-dependent manner. (United States)

    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


    Immunosuppressive CD11b(+) Gr-1(+) myeloid-derived suppressor cells (MDSCs) accumulate in the livers of tumor-bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune-mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α-galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor-free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b(+) Gr-1(+) cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN-γ-dependent upregulation of CD40 on hepatic CD11b(+) Gr-1(+) cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor-induced CD11b(+) Gr-1(+) MDSCs as well as enhanced reactive oxygen species (ROS)-mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con 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 ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor-induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40-dependent manner.

  4. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line (United States)

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G.; Wenzel, Jürgen J.; Johne, Reimar


    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown. PMID:27690085

  5. Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells

    Institute of Scientific and Technical Information of China (English)

    SHI Cheng; SHEN Huan; JIANG Wei; SONG Zhi-hua; WANG Cheng-yan; WEI Li-hui


    Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background,which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use,especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability.Methods Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propogate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells.Results We generated a new Chinese human embryonic stem cells line,CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines:normal morphology,karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line.Conclusions This newly established Chinese cell line,CH1,which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells,will provide

  6. Hepatitis C virus core proteins derived from different quasispecies of genotype 1b inhibit the growth of Chang liver cells

    Institute of Scientific and Technical Information of China (English)

    Xue-Bing Yan; Lei Mei; Xia Feng; Mei-Rong Wan; Zhi Chen; Nicole Pavia; Christian Brechot


    AIM: To investigate the influence of different quasispecies of hepatitis C virus (HCV) genotype 1b core protein on growth of Chang liver cells.METHODS: Three eukaryotic expression plasmids (pEGFP-N1/core) that contained different quasispecies truncated core proteins of HCV genotype 1b were constructed. These were derived from tumor (T) and nontumor (NT) tissues of a patient infected with HCV and C191 (HCV-J6). The core protein expression plasmids were transiently transfected into Chang liver cells. At different times, the cell cycle and apoptosis was assayed by flow cytometry, and cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) assay.RESULTS: The proportion of S-phase Chang liver cells transfected with pEGFP-N1/core was significantly lower than that of cells transfected with blank plasmid at three different times after transfection (all P NT > C191), and apoptosis was increased in cells transfected with pEGFP-N1/core as the transfection time increased (72 h > 48 h > 24 h).CONCLUSION: These results suggest that HCV genotype 1b core protein induces apoptosis, and inhibits cellcycle progression and proliferation of Chang liver cells.Different quasispecies core proteins of HCV genotype 1b might have some differences in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.

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

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    Renwei Huang


    Full Text Available 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.

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

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    Danna Ye


    Full Text Available 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.

  9. Role of RhoA in platelet-derived growth factor-BB-induced migration of rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    LI Lei; LI Jing; WANG Ji-yao; YANG Chang-qing; JIA Ming-lei; JIANG Wei


    Background Although the migration of hepatic stellate cells (HSCs) is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases (especially RhoA) in platelet-derived growth factor (PDGF)-BB-induced migration of HSCs.Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases (RhoA, Rac1 and Cdc42) within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active (CA, Q63L) or dominant negative (DN, T19N) RhoA mutants. Data were analyzed using SPSS 16.0 software. Student's t test was used to analyze differences between two groups and one-way analysis of variance (ANOVA) was used among multiple groups.Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic (direct) and chemotactic (indirect) stimulation by PDGF-BB. PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Rac1 and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls.Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of

  10. Effects of lamivudine on the function of dendritic cells derived from patients with chronic hepatitis B virus infection

    Institute of Scientific and Technical Information of China (English)

    Peng-Yuan Zheng; Dong-Yun Zhang; Gao-Feng Lu; Ping-Chang Yang; Yuan-Ming Qi; Bai-Sheng Wang


    AIM: To investigate if the nucleoside analogue lamivudine (LAM), a potent inhibitor of HBV replication,could restore the function of dendritic cells derived from patients with chronic hepatitis B (CHB) in an Asian population.METHODS: Dendritic cells (DCs) derived from mononuclearcytes of patients with chronic HBV infection were cultured in the presence of IL-4, granulocytemacrophage colony-stimulating factors (GM-CSF) and gradient concentrations of LAM (0-2 mmol/L). Cell morphology was observed under light microscopy. Cell surface molecules, including HLA-DR, CD80, CD83,and CD1α, were analyzed with flow cytometry. The concentrations of IL-6 and IL-12 in the supernatant were assayed by ELISA. T cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT).RESULTS: The expression of CD1α on DC treated with 0.5 mmol/L LAM (LAM-DC 0.5 mmol/L) was significantly higher than that of DC untreated with LAM (54.1 ± 4.21vs 33.57 ± 3.14, P < 0.05), and so was the expression of CD83 (20.24 ± 2.51 vs 12.83 ± 2.12, P < 0.05) as well as the expression of HLA-DR (74.5 ± 5.16 vs 52.8 ±2.51, P < 0.05). Compared with control group, LAM-DC group (0.5 mmol/L) secreted significantly more IL-12 (910± 91.5 vs 268 ± 34.3 pg/mL, P < 0.05), had lower levels of IL-6 in the culture supernatant (28 ± 2.6 vs 55 ± 7.36pg/mL, P < 0.05), markedly enhanced the stimulatory capacity in the allogeneic mixed leukocyte reaction (MLR)(1.87 ± 0.6 vs 1.24 ± 0.51, P < 0.05).CONCLUSION: The lower expression of phenotypic molecules and impaired allogeneic mixed lymphocyte reaction function of dendritic cells derived from patients with HBV infection could be restored in vitro by incubation with LAM.

  11. Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

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    Silvia Berardis

    Full Text Available Adult-derived human liver stem/progenitor cells (ADHLSC are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity. ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays. Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.

  12. Aggravation of viral hepatitis by platelet-derived serotonin. (United States)

    Lang, Philipp A; Contaldo, Claudio; Georgiev, Panco; El-Badry, Ashraf Mohammad; Recher, Mike; Kurrer, Michael; Cervantes-Barragan, Luisa; Ludewig, Burkhard; Calzascia, Thomas; Bolinger, Beatrice; Merkler, Doron; Odermatt, Bernhard; Bader, Michael; Graf, Rolf; Clavien, Pierre-Alain; Hegazy, Ahmed N; Löhning, Max; Harris, Nicola L; Ohashi, Pamela S; Hengartner, Hans; Zinkernagel, Rolf M; Lang, Karl S


    More than 500 million people worldwide are persistently infected with hepatitis B virus or hepatitis C virus. Although both viruses are poorly cytopathic, persistence of either virus carries a risk of chronic liver inflammation, potentially resulting in liver steatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific T cells are a major determinant of the outcome of hepatitis, as they contribute to the early control of chronic hepatitis viruses, but they also mediate immunopathology during persistent virus infection. We have analyzed the role of platelet-derived vasoactive serotonin during virus-induced CD8(+) T cell-dependent immunopathological hepatitis in mice infected with the noncytopathic lymphocytic choriomeningitis virus. After virus infection, platelets were recruited to the liver, and their activation correlated with severely reduced sinusoidal microcirculation, delayed virus elimination and increased immunopathological liver cell damage. Lack of platelet-derived serotonin in serotonin-deficient mice normalized hepatic microcirculatory dysfunction, accelerated virus clearance in the liver and reduced CD8(+) T cell-dependent liver cell damage. In keeping with these observations, serotonin treatment of infected mice delayed entry of activated CD8(+) T cells into the liver, delayed virus control and aggravated immunopathological hepatitis. Thus, vasoactive serotonin supports virus persistence in the liver and aggravates virus-induced immunopathology.

  13. In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus. (United States)

    Lew, Y Y; Michalak, T I


    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 (approximately 10(3) 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.

  14. Role of myeloid-derived suppressor cells in amelioration of experimental autoimmune hepatitis following activation of TRPV1 receptors by cannabidiol.

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    Venkatesh L Hegde

    Full Text Available BACKGROUND: Myeloid-derived suppressor cells (MDSCs are getting increased attention as one of the main regulatory cells of the immune system. They are induced at sites of inflammation and can potently suppress T cell functions. In the current study, we demonstrate how activation of TRPV1 vanilloid receptors can trigger MDSCs, which in turn, can inhibit inflammation and hepatitis. METHODOLOGY/PRINCIPAL FINDINGS: Polyclonal activation of T cells, following injection of concanavalin A (ConA, in C57BL/6 mice caused acute hepatitis, characterized by significant increase in aspartate transaminase (AST, induction of inflammatory cytokines, and infiltration of mononuclear cells in the liver, leading to severe liver injury. Administration of cannabidiol (CBD, a natural non-psychoactive cannabinoid, after ConA challenge, inhibited hepatitis in a dose-dependent manner, along with all of the associated inflammation markers. Phenotypic analysis of liver infiltrating cells showed that CBD-mediated suppression of hepatitis was associated with increased induction of arginase-expressing CD11b(+Gr-1(+ MDSCs. Purified CBD-induced MDSCs could effectively suppress T cell proliferation in vitro in arginase-dependent manner. Furthermore, adoptive transfer of purified MDSCs into naïve mice conferred significant protection from ConA-induced hepatitis. CBD failed to induce MDSCs and suppress hepatitis in the livers of vanilloid receptor-deficient mice (TRPV1(-/- thereby suggesting that CBD primarily acted via this receptor to induce MDSCs and suppress hepatitis. While MDSCs induced by CBD in liver consisted of granulocytic and monocytic subsets at a ratio of ∼2∶1, the monocytic MDSCs were more immunosuppressive compared to granulocytic MDSCs. The ability of CBD to induce MDSCs and suppress hepatitis was also demonstrable in Staphylococcal enterotoxin B-induced liver injury. CONCLUSIONS/SIGNIFICANCE: This study demonstrates for the first time that MDSCs play a

  15. Contact-dependent depletion of hydrogen peroxide by catalase is a novel mechanism of myeloid-derived suppressor cell induction operating in human hepatic stellate cells. (United States)

    Resheq, Yazid J; Li, Ka-Kit; Ward, Stephen T; Wilhelm, Annika; Garg, Abhilok; Curbishley, Stuart M; Blahova, Miroslava; Zimmermann, Henning W; Jitschin, Regina; Mougiakakos, Dimitrios; Mackensen, Andreas; Weston, Chris J; Adams, David H


    Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14(+) monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14(+)HLADR(low/-) suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14(+) monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = -0.6555, p Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.

  16. Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

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    Vinken Mathieu


    Full Text Available Abstract Background The capability of human mesenchymal stem cells (hMSC derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. Results Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4, hepatocyte growth factor (HGF, insulin-transferrin-sodium-selenite (ITS and dexamethasone] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone, however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK18 expression. Additional exposure of the cells to trichostatin A (TSA considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF-3β, alpha-fetoprotein (AFP, CK18, albumin (ALB, HNF1α, multidrug resistance-associated protein (MRP2 and CCAAT-enhancer binding protein (C/EBPα, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP-dependent activity. Conclusion hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells.

  17. Hepatic maturation of human iPS cell-derived hepatocyte-like cells by ATF5, c/EBPα, and PROX1 transduction. (United States)

    Nakamori, Daiki; Takayama, Kazuo; Nagamoto, Yasuhito; Mitani, Seiji; Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki


    Hepatocyte-like cells differentiated from human iPS cells (human iPS-HLCs) are expected to be utilized in drug development and research. However, recent hepatic characterization of human iPS-HLCs showed that these cells resemble fetal hepatocytes rather than adult hepatocytes. Therefore, in this study, we aimed to develop a method to enhance the hepatic function of human iPS-HLCs. Because the gene expression levels of the hepatic transcription factors (activating transcription factor 5 (ATF5), CCAAT/enhancer-binding protein alpha (c/EBPα), and prospero homeobox protein 1 (PROX1)) in adult liver were significantly higher than those in human iPS-HLCs and fetal liver, we expected that the hepatic functions of human iPS-HLCs could be enhanced by adenovirus (Ad) vector-mediated ATF5, c/EBPα, and PROX1 transduction. The gene expression levels of cytochrome P450 (CYP) 2C9, 2E1, alpha-1 antitrypsin, transthyretin, Na+/taurocholate cotransporting polypeptide, and uridine diphosphate glucuronosyl transferase 1A1 and protein expression levels of CYP2C9 and CYP2E1 were upregulated by ATF5, c/EBPα, and PROX1 transduction. These results suggest that the hepatic functions of the human iPS-HLCs could be enhanced by ATF5, c/EBPα, and PROX1 transduction. Our findings would be useful for the hepatic maturation of human iPS-HLCs.

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

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    Stephanie C Burke Schinkel

    Full Text Available 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.

  19. Prostaglandin E2 inhibits platelet-derived growth factor-stimulated cell proliferation through a prostaglandin E receptor EP2 subtype in rat hepatic stellate cells. (United States)

    Koide, Shigeki; Kobayashi, Yoshimasa; Oki, Yutaka; Nakamura, Hirotoshi


    Prostaglandin (PG) E2 inhibits hepatic stellate cell (HSC) mitogenesis. PGE-specific receptors are divided into four subtypes that are coupled either to Ca2+ mobilization (EP1 and EP3) or to the stimulation of adenyl cyclase (EP2 and EP4). The aims of the current study were to identify PGE receptor subtypes in cultured rat HSC and to examine which PGE receptor subtype(s) mediates the inhibitory effect of PGE2 on platelet-derived growth factor (PDGF)-stimulated proliferation. Reverse transcription-polymerase chain reaction analysis was performed to detect PGE receptor subtype mRNA expression. Cell proliferation was determined by measuring [3H]thymidine incorporation, and intracellular cyclic AMP was measured by radioimmunoassay. Cultured rat HSC expressed mRNAs for all four subtypes of PGE receptor. PGE2- and EP2-selective agonist produced dose-dependent inhibitory effects on PDGF-stimulated proliferation. Neither EP1-, EP3-, nor EP4-selective agonists showed any inhibitory effect. An adenylate cyclase inhibitor strongly blunted the inhibition of DNA synthesis elicited by PGE2 and the EP2 agonist. The EP2 agonist generated higher and more prolonged increases in intracellular cyclic AMP than the EP4 agonist. Activation of the PGE EP2 receptor has an antiproliferative effect in HSC that may be mediated by cyclic AMP-related signal transduction pathways.

  20. Hepatitis C virus infection of cholangiocarcinoma cell lines

    NARCIS (Netherlands)

    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.


    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 re

  1. Inhibitory effect of isothiocyanate derivant targeting AGPS by computer-aid drug design on proliferation of glioma and hepatic carcinoma cells. (United States)

    Zhu, Yu; Li, Wen-Ming; Zhang, Ling; Xue, Jing; Zhao, Meng; Yang, Ping


    Lipids metabolism was involved in the process of many types of tumor and alkylglycerone phosphate synthase (AGPS) was considered implicated in tumor process. Benzyl isothiocyanate (BITC) showed the inhibitory effect of tumor and AGPS activity, therefore, we screened a group of small molecular compound based on BITC by computer-aid design targeting AGPS and the results showed that the derivants could suppress the proliferation, the expression of tumor related genes such as survivin and Bcl-2, and the level of ether lipids such as lysophosphatidic acid ether (LPAe) and platelet activating factor ether (PAFe); however, the activity of caspase-3/8 was improved in glioma U87MG and hepatic carcinoma HepG2 cells in vitro.

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

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    Yang Xu


    Full Text Available 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.

  3. In Situ Transplantation of Alginate Bioencapsulated Adipose Tissues Derived Stem Cells (ADSCs via Hepatic Injection in a Mouse Model.

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    Mong-Jen Chen

    Full Text Available Adipose tissue derived stem cells (ADSCs transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model.The ADSCs isolated from human alpha 1 antitrypsin (M-hAAT transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one. Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two.Our results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted

  4. Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy. (United States)

    Pareja, Eugenia; Gómez-Lechón, María José; Tolosa, Laia


    Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

  5. Innate Immune Responses in Viral Hepatitis: the role of Kupffer cells and liver-derived monocytes in shaping intrahepatic immunity in mice using the LCMV infection model

    NARCIS (Netherlands)

    D. Movita (Dowty)


    markdownabstract__Abstract__ This study was performed to elucidate the immunological role of the liver in viral hepatitis. The immune functions of the liver are shaped by the intrahepatic cells present during steady state condition, as well as the recruited immune cells during liver inflammation.

  6. Hepatic progenitor cells in human liver tumor development

    Institute of Scientific and Technical Information of China (English)

    Louis Libbrecht


    In recent years, the results of several studies suggest that human liver tumors can be derived from hepatic progenitor cells rather than from mature cell types.The available data indeed strongly suggest that most combined hepatocellular-cholangiocarcinomas arise from hepatic progenitor cells that retained their potential to differentiate into the hepatocytic and biliary lineages.Hepatic progenitor cells could also be the basis for some hepatocellular carcinomas and hepatocellular adenomas, although it is very difficult to determine the origin of an individual hepatocellular carcinoma. There is currently not enough data to make statements regarding a hepatic progenitor cell origin of cholangiocarcinoma.The presence of hepatic progenitor cell markers and the presence and extent of the cholangiocellular component are factors that are related to the prognosis of hepatocellular carcinomas and combined hepatocellularcholangiocarcinomas, respectively.

  7. Hepatic differentiation of embryonic stem cells by murine fetal liver mesenchymal cells. (United States)

    Ishii, Takamichi; Yasuchika, Kentaro; Ikai, Iwao


    Hepatocytes derived from embryonic stem cells (ESCs) are a potential cell source for regenerative medicine. However, it has been technically difficult to differentiate ESCs into mature hepatocytes because the definitive growth factors and molecular mechanisms governing hepatocyte differentiation have not yet been well defined. The CD45(-)CD49f(+/-)Thy1(+)gp38(+) mesenchymal cells that reside in murine fetal livers induce hepatic progenitor cells to differentiate into mature hepatocytes by direct cell-cell contact. Utilizing these cells, we employ a two-step procedure for hepatic maturation of ESCs: first, ESCs are differentiated into endodermal cells or hepatic progenitor cells, and second, ESC-derived endodermal cells are matured into functional hepatocytes by coculture with murine fetal liver mesenchymal cells. The ESC-derived hepatocyte-like cells possess hepatic functions, including ammonia removal activity, albumin secretion ability, glycogen synthesis and storage, and cytochrome P450 enzymatic activity.

  8. Analysis of Monocytic and Granulocytic Myeloid-Derived Suppressor Cells Subsets in Patients with Hepatitis C Virus Infection and Their Clinical Significance

    Directory of Open Access Journals (Sweden)

    Gang Ning


    Full Text Available Myeloid-derived suppressor cells (MDSCs have been shown to inhibit T-cell responses in many diseases, but, in hepatitis C virus (HCV infected patients, MDSCs are still poorly studied. In this assay, we investigated the phenotype and frequency of two new populations of MDSCs denoted as monocytic and granulocytic MDSCs (M-MDSCs and G-MDSCs in HCV infected patients and analyzed their clinical significance in these patients respectively. We found that the frequency of CD14+HLA-DR-/low cells (M-MDSCs from HCV infected patients (mean ± SE, 3.134% ± 0.340% was significantly increased when compared to healthy controls (mean ± SE, 1.764% ± 0.461% (Z = −2.438, P = 0.015, while there was no statistical difference between the frequency of HLA-DR-/lowCD33+CD11b+CD15+ (G-MDSCs of HCV infected patients and healthy donors (0.201% ± 0.038% versus 0.096% ± 0.026%, P > 0.05, which suggested that HCV infection could cause the proliferation of M-MDSCs instead of G-MDSCs. Besides, we found that the frequency of M-MDSCs in HCV infected patients had certain relevance with age (r = 0.358, P = 0.003; patients older than 40 years old group (mean ± SE, 3.673% ± 0.456% had a significantly higher frequency of M-MDSCs than that of age less than 40 years old group (mean ± SE, 2.363% ± 0.482% (Z = −2.685, P = 0.007. The frequency of M-MDSCs, however, had no correlation with HCV RNA loads, aspartate aminotransferase (AST, alanine aminotransferase (ALT, and the level of liver inflammation degree.

  9. Transplantation of microencapsulated umbilical-cord-bloodderived hepatic-like cells for treatment of hepatic failure

    Institute of Scientific and Technical Information of China (English)

    Fang-Ting Zhang; Hui-Juan Wan; Ming-Hua Li; Jing Ye; Mei-Jun Yin; Chun-Qiao Huang; Jie Yu


    inside microcapsules were found by trypan blue staining,but some fibrous tissue around microcapsules was also detected in the greater omentum of encapsulated group by hematoxylin and eosin staining.CONCLUSION:Transplantation of microencapsulated hepatic-like cells derived from umbilical cord blood cells could preliminarily alleviate the symptoms of AHF rats.

  10. Advances in studies on hepatic stem cells

    Institute of Scientific and Technical Information of China (English)


    The question whether hepatic stem cells exist or not has been debated for several decades. Current researches confirm that there are hepatic stem cells in the liver. Oval cells, putative bipotential hepatic stem cells, are probably located within canals of Hering, portal tracts or branches of biliary trees. Bone marrow is a potential source of oval cells, indicating that there exists a close relationship between liver and hematopoiesis in adulthood. Hepatic stem cells are able to proliferate in vitro and can be induced to differentiate into hepatocytes. This will provide a promising approach of cell transplantation, tissue engineering and gene therapy for liver diseases. In this review, the evidence of their presence, origin, identification, proliferation in vitro, differentiation by induction, application prospects of hepatic stem cells and future directions for the field are discussed.

  11. Cystathionine β-synthase-derived hydrogen sulfide regulates lipopolysaccharide-induced apoptosis of the BRL rat hepatic cell line in vitro. (United States)

    Yan, Jun; Teng, Feixiang; Chen, Weiwei; Ji, Yinglei; Gu, Zhenyong


    Hydrogen sulfide (H(2)S), is a member of the novel family of endogenous gaseous transmitters, termed "gasotransmitters exhibiting diverse physiological activities, and is generated in mammalian tissues mainly by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) in conjunction with cysteine (aspartate) aminotranferase (CAT). The distributions of these enzymes are species- and tissue-specific. The liver, as the main organ that generates H(2)S in vivo, functions in biotransformation and metabolism. However, the liver is vulnerable to damage from internal and external factors, including inflammatory mediators, drugs and poisons. The present study evaluated the endogenous CBS-H(2)S synthesis regulating lipopolysaccharide (LPS)-induced apoptosis of hepatic cells. The rat hepatic cell line, BRL, was incubated with LPS for various time periods to establish a cell-damage model. Incubation with LPS resulted in a significant increase in CBS expression and H(2)S production. It also stimulated apoptosis and decreased the mitochondrial membrane potential. Pretreatment with the CBS inhibitor aminooxyacetic acid (AOAA) or CBS small interfering RNA (siRNA) decreased LPS-enhanced H(2)S production. Notably, apoptosis increased for a short period and then decreased gradually, while the mitochondrial membrane potential demonstrated the opposite trend. These results showed that endogenous CBS-H(2)S synthesis demonstrated early anti-apoptotic activity and subsequent pro-apoptotic activity in LPS-induced apoptosis. These results suggest a new approach for developing novel drugs for this condition.

  12. Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Ning-Li Chai; Qiang Fu; Hui Shi; Chang-Hao Cai; Jun Wan; Shi-Ping Xu; Ben-Yan Wu


    AIM:To investigate the potential mechanism of ArgGly-Asp (RGD) peptide-labeled liposome loading oxymatrine (OM) therapy in CCl4-induced hepatic fibrosis in METHODS:We constructed a rat model of CCl4-induced hepatic fibrosis and treated the rats with different formulations of OM.To evaluate the antifibrotic effect of OM,we detected levels of alkaline phosphatase,hepatic histopathology (hematoxylin and eosin stain and Masson staining) and fibrosis-related gene expression of matrix metallopeptidase (MMP)-2,tissue inhibitor of metalloproteinase (TIMP)-1 as well as type Ⅰ procollagen via quantitative real-time polymerase chain reaction.To detect cell viability and apoptosis of hepatic stellate cells (HSCs),we performed 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay and flow cytometry.To reinforce the combination of oxymatrine with HSCs,we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM,and its targeting of HSCs was examined by fluorescent microscopy.RESULTS:OM attenuated CCl4-induced hepatic fibrosis,as defined by reducing serum alkaline phosphatase (344.47 ± 27.52 U/L vs 550.69 ± 43.78 U/L,P < 0.05),attenuating liver injury and improving collagen deposits (2.36% ± 0.09% vs 7.70% ± 0.60%,P < 0.05) and downregulating fibrosis-related gene expression,that is,MMP-2,TIMP-1 and type Ⅰ procollagen (P < 0.05).OM inhibited cell viability and induced apoptosis of HSCs in vitro.RGD promoted OM targeting of HSCs and enhanced the therapeutic effect of OM in terms of serum alkaline phosphatase (272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L,P < 0.05),liver injury,collagen deposits (0.26% ± 0.09% vs 2.36% ± 0.09%,P < 0.05) and downregulating fibrosis-related gene expression,that is,MMP-2,TIMP-1 and type Ⅰ procollagen (P < 0.05).Moreover,in vitro assay demonstrated that RGD enhanced the effect of OM on HSC viability and apoptosis.CONCLUSION:OM attenuated hepatic fibrosis by

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

    Institute of Scientific and Technical Information of China (English)

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


    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

  14. Hepatic Giant Cell Arteritis and Polymyalgia Rheumatica

    Directory of Open Access Journals (Sweden)

    Donald R Duerksen


    Full Text Available Polymyalgia rheumatica (PMR is a clinical syndrome of the elderly characterized by malaise, proximal muscle aching and stiffness, low grade fever, elevated erythrocyte sedimentation rare and the frequent association with temporal giant cell arteritis. The authors describe a case of PMR associated with hepatic giant cell arteritis. This lesion has been described in two other clinical reports. The distribution of the arteritis may be patchy; in this report, diagnosis was made with a wedge biopsy performed after an initial nonspecific percutaneous liver biopsy. The authors review the spectrum of liver involvement in PMR and giant cell arteritis. Hepatic abnormalities respond to systemic corticosteroids, and patients with hepatic arteritis have a good prognosis.

  15. Directed hepatic differentiation from embryonic stem cells


    Chen, Xuesong; Zeng, Fanyi


    The liver is the largest internal organ in mammals, and is important for the maintenance of normal physiological functions of other tissues and organs. Hepatitis, cirrhosis, liver cancer and other chronic liver diseases are serious threats to human health, and these problems are compounded by a scarcity of liver donors for transplantation therapies. Directed differentiation of embryonic stem cells to liver cells is a promising strategy for obtaining hepatocytes that can be used for cell trans...

  16. Genetic and Chemical Correction of Cholesterol Accumulation and Impaired Autophagy in Hepatic and Neural Cells Derived from Niemann-Pick Type C Patient-Specific iPS Cells

    Directory of Open Access Journals (Sweden)

    Dorothea Maetzel


    Full Text Available Niemann-Pick type C (NPC disease is a fatal inherited lipid storage disorder causing severe neurodegeneration and liver dysfunction with only limited treatment options for patients. Loss of NPC1 function causes defects in cholesterol metabolism and has recently been implicated in deregulation of autophagy. Here, we report the generation of isogenic pairs of NPC patient-specific induced pluripotent stem cells (iPSCs using transcription activator-like effector nucleases (TALENs. We observed decreased cell viability, cholesterol accumulation, and dysfunctional autophagic flux in NPC1-deficient human hepatic and neural cells. Genetic correction of a disease-causing mutation rescued these defects and directly linked NPC1 protein function to impaired cholesterol metabolism and autophagy. Screening for autophagy-inducing compounds in disease-affected human cells showed cell type specificity. Carbamazepine was found to be cytoprotective and effective in restoring the autophagy defects in both NPC1-deficient hepatic and neuronal cells and therefore may be a promising treatment option with overall benefit for NPC disease.

  17. Transplantation of human umbilical cord-derived mesenchymal stem cells improves hepatic fibrosis in rats with carbon etrachloride-induced hepatic cirrhosis%人脐带源间充质干细胞移植改善四氯化碳诱导肝硬化大鼠的肝纤维化

    Institute of Scientific and Technical Information of China (English)

    刘英; 施占立; 赵宗泽; 郭胜男; 徐金凯; 李东杰


    背景:干细胞移植作为一种全新的肝硬化治疗方法的可行性和有效性,在国内外文献中报道极少.目的:观察人脐带源间充质干细胞移植对四氯化碳诱导肝硬化大鼠肝纤维化的影响.方法:32 只Wistar 大鼠随机分为2 组,对照组经尾静脉内注射生理盐水,肝硬化组采用四氯化碳植物油皮下注射8 周制成肝硬化大鼠模型,再随机分为3 组,盐水对照组、人脐带源间充质干细胞移植组分别经尾静脉内注射生理盐水和人脐带源间充质干细胞混悬液,8 周肝硬化组无其他干预.结果与结论:对照组血清碱性磷酸酶水平明显低于其他3 组(P 0.05),与8 周肝硬化组和盐水对照组相比明显升高(P 0.05), but they were significantly higher compared with 8-week hepatic cirrhosis and normal saline group (P < 0.05). In the 8-week hepatic cirrhosis group, a large amount of hyperplastic collagenous fiber was presented in the liver tissue and the frame of the pseudolobuli formed. The normal saline group and 8-week hepatic cirrhosis group exhibited similar histological changes. Scattered green antinuclear antibody-positive cells in the liver were only observed in the hUCMSCs group. The amount of hyperplastic collagenous fiber was significantly lower in the hUCMSCs transplantation group than in the normal saline group. These findings suggest that transplantation of human umbilical cord-derived mesenchymal stem cells via the tail vein can obviously improve hepatic fibrosis in rats with carbon etrachloride- induced hepatic cirrhosis.

  18. Hepatitis C virus host cell interactions uncovered

    DEFF Research Database (Denmark)

    Gottwein, Judith; Bukh, Jens


      Insights into virus-host cell interactions as uncovered by Randall et al. (1) in a recent issue of PNAS further our understanding of the hepatitis C virus (HCV) life cycle, persistence, and pathogenesis and might lead to the identification of new therapeutic targets. HCV persistently infects 180...... million individuals worldwide, causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The only approved treatment, combination therapy with IFN- and ribavirin, targets cellular pathways (2); however, a sustained virologic response is achieved only in approximately half of the patients...... treated. Therefore, there is a pressing need for the identification of novel drugs against hepatitis C. Although most research focuses on the development of HCV-specific antivirals, such as protease and polymerase inhibitors (3), cellular targets could be pursued and might allow the development of broad...

  19. Regulatory T cells in viral hepatitis

    Institute of Scientific and Technical Information of China (English)

    Eva Billerbeck; Tobias B(o)ttler; Robert Thimme


    The pathogenesis and outcome of viral infections are significantly influenced by the host immune response.The immune system is able to eliminate many viruses in the acute phase of infection. However, some viruses,like hepatitis C virus (HCV) and hepatitis B virus (HBV),can evade the host immune responses and establish a persistent infection. HCV and HBV persistence is caused by various mechanisms, like subversion of innate immune responses by viral factors, the emergence of T cell escape mutations, or T cell dysfunction and suppression.Recently, it has become evident that regulatory T cells may contribute to the pathogenesis and outcome of viral infections by suppressing antiviral immune responses.Indeed, the control of HCV and HBV specific immune responses mediated by regulatory T cells may be one mechanism that favors viral persistence, but it may also prevent the host from overwhelming T cell activity and liver damage. This review will focus on the role of regulatory T cells in viral hepatitis.

  20. Diagnostic value of platelet derived growth factor-BB, transforming growth factor-β1,matrix metalloproteinase-1, and tissue inhibitor of matrix metalloproteinase-1 in serum and peripheral blood mononuclear cells for hepatic fibrosis

    Institute of Scientific and Technical Information of China (English)

    Bin-Bin Zhang; Wei-Min Cai; Hong-Lei Weng; Zhong-Rong Hu; Jun Lu; Min Zheng; Rong-Hua Liu


    AIM: Noninvasive diagnosis of hepatic fibrosis has become the focus because of the limited biopsy, especially in the surveillance of treatment and in screening hepatic fibrosis.Recently, regulatory elements involved in liver fibrosis, such as platelet derived growth factor-BB (PDGF-BB), transforming growth factor-β1 (TGF-β1), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), have been studied extensively. To determine whether these factors or enzymes could be used as the indices for the diagnosis of hepatic fibrosis, we investigated them by means of receiver operating characteristic (ROC) curve.METHODS: Serum samples from sixty patients with chronic viral hepatitis B and twenty healthy blood donors were assayed to determine the level of PDGF-BB, TGF-β1, MMP-1, and TIMP-1 with ELISA, and HA, PCIII, C-IV, and LN level with RIA. The message RNA (mRNA) expression of TIMP-1 and MMP-1 in peripheral blood mononuclear cells (PBMCs) was detected by RT-PCR and Northern blot hybridization. Liver biopsy was performed in all patients.The biopsy samples were histopathologically examined. The trial was double-blind controlled.RESULTS: The serum level of PDGF-BB, TIMP-1, the ratio of TIMP-1 and MMP-1 (TIMP-1/MMP-1), mRNA expression of TIMP-1 (TIMP-1mRNA), and the ratio of TIMP-1mRNA and MMP-1mRNA (TIMP-1mRNA/MMP-1mRNA) in patients was significantly higher than those in the healthy blood donors (t=2.514-11.435, P=0.000-0.016). The serum level of PDGF-BB, TIMP-1, TIMP-1/MMP-1, and TIMP-1mRNA was positively correlated with fibrosis stage and inflammation grade (r=0.239-0.565, P=0.000-0.033), while the serum level of MMP-1 was negatively correlated with fibrosis stage and inflammation grade, and TIMP-1mRNA/MMP-1mRNA was positively correlated with inflammation grade. Through the analysis by ROC curve, serum PDGF-BB was the most valuable marker, and its sensitivity was the highest among the nine indices. The markers with the highest

  1. Regenerative cells for transplantation in hepatic failure. (United States)

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


    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.

  2. Generation and In Vitro Expansion of Hepatic Progenitor Cells from Human iPS Cells. (United States)

    Yanagida, Ayaka; Nakauchi, Hiromitsu; Kamiya, Akihide


    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.

  3. Hepatitis

    Institute of Scientific and Technical Information of China (English)


    2008312 Impact of hepatitis B virus infection on the activity of hematopoietic stem cell.SHI Yanmei(石雁梅),et al.Dept Infect Dis,1st Clin Coll,Harbin Med Univ,Harbin 150001.Chin J Infect Dis 2008;26(4):197-201.Objective To study the impact of hepatitis B virus (HBV)infection on the activity of cord hematopoieticstem cells.Methods CD34+cells were isolated from healthy human cord blood by mini MACS.Cells were

  4. Pluripotent stem cell-derived hepatocyte-like cells. (United States)

    Schwartz, R E; Fleming, H E; Khetani, S R; Bhatia, S N


    Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are

  5. The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B

    Directory of Open Access Journals (Sweden)

    Young Hee Joung


    Full Text Available Disease prevention through vaccination is considered to be the greatest contribution to public health over the past century. Every year more than 100 million children are vaccinated with the standard World Health Organization (WHO-recommended vaccines including hepatitis B (HepB. HepB is the most serious type of liver infection caused by the hepatitis B virus (HBV, however, it can be prevented by currently available recombinant vaccine, which has an excellent record of safety and effectiveness. To date, recombinant vaccines are produced in many systems of bacteria, yeast, insect, and mammalian and plant cells. Among these platforms, the use of plant cells has received considerable attention in terms of intrinsic safety, scalability, and appropriate modification of target proteins. Research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over 30 diseases, most frequently HepB and influenza. More inspiring, approximately 12 plant-made antigens have already been tested in clinical trials, with successful outcomes. In this study, the latest information from the last 10 years on plant-derived antigens, especially hepatitis B surface antigen, approaches are reviewed and breakthroughs regarding the weak points are also discussed.

  6. The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B (United States)

    Joung, Young Hee; Park, Se Hee; Moon, Ki-Beom; Jeon, Jae-Heung; Cho, Hye-Sun; Kim, Hyun-Soon


    Disease prevention through vaccination is considered to be the greatest contribution to public health over the past century. Every year more than 100 million children are vaccinated with the standard World Health Organization (WHO)-recommended vaccines including hepatitis B (HepB). HepB is the most serious type of liver infection caused by the hepatitis B virus (HBV), however, it can be prevented by currently available recombinant vaccine, which has an excellent record of safety and effectiveness. To date, recombinant vaccines are produced in many systems of bacteria, yeast, insect, and mammalian and plant cells. Among these platforms, the use of plant cells has received considerable attention in terms of intrinsic safety, scalability, and appropriate modification of target proteins. Research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over 30 diseases, most frequently HepB and influenza. More inspiring, approximately 12 plant-made antigens have already been tested in clinical trials, with successful outcomes. In this study, the latest information from the last 10 years on plant-derived antigens, especially hepatitis B surface antigen, approaches are reviewed and breakthroughs regarding the weak points are also discussed. PMID:27754367

  7. The improving effects on hepatic fibrosis of interferon-γ liposomes targeted to hepatic stellate cells (United States)

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


    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.

  8. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting. (United States)

    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


    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.

  9. Construct hepatic analog by cell-matrix controlled assembly technology

    Institute of Scientific and Technical Information of China (English)

    LIU Haixia; YAN Yongnian; WANG Xiaohong; CHENG Jie; LIN Feng; XIONG Zhuo; Wu Rendong


    A mixture of hepatic cells and chitosan/gelatin solution was deposited to construct a hepatic analog by way of layer-by-layer deposition technique using a home-made devise. The size and cell concentration of the analogs can be controlled freely. Approximately 90% of the hepatic cells remained viable under 0.2 Mpa extrusion pressure. Cultured in vitro 8 weeks before animal test, hepatic cells in structure maintained their phenotype and kept proliferating, and albumin and other secretion of the cells increased. Cords and hepaton-like structures were observed after culture for 20 d. These results indicate that hepatic cells could be assembled directly into a 3D viable structure and expanded to form a hepatic organoid. This accomplishment is considered to be an interesting means for the fabrication of liver replacements.

  10. Hepatic differentiation of porcine embryonic stem cells for translational research of hepatocyte transplantation. (United States)

    Park, K M; Hussein, K H; Ghim, J H; Ahn, C; Cha, S H; Lee, G S; Hong, S H; Yang, S; Woo, H M


    Porcine embryonic stem cells (ES) are considered attractive preclinical research tools for human liver diseases. Although several studies previously reported generation of porcine ES, none of these studies has described hepatic differentiation from porcine ES. The aim of this study was to generate hepatocytes from porcine ES and analyze their characteristics. We optimized conditions for definitive endoderm induction and developed a 4-step hepatic differentiation protocol. A brief serum-free condition with activin A efficiently induced definitive endoderm differentiation from porcine ES. The porcine ES-derived hepatocyte-like cells highly expressed hepatic markers including albumin and α-fetoprotein, and displayed liver characteristics such as glycogen storage, lipid production, and low-density lipoprotein uptake. For the first time, we describe a highly efficient protocol for hepatic differentiation from porcine ES. Our findings provide valuable information for translational liver research using porcine models, including hepatic regeneration and transplant studies, drug screening, and toxicology.

  11. Signal molecule-mediated hepatic cell communication during liver regeneration

    Institute of Scientific and Technical Information of China (English)

    Zhen-Yu Zheng; Shun-Yan Weng; Yan Yu


    Liver regeneration is a complex and well-orchestrated process, during which hepatic cells are activated to produce large signal molecules in response to liver injury or mass reduction. These signal molecules, in turn, set up the connections and cross-talk among liver cells to promote hepatic recovery. In this review, we endeavor to summarize the network of signal molecules that mediates hepatic cell communication in the regulation of liver regeneration.

  12. Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles

    DEFF Research Database (Denmark)

    Russell, R S; Kawaguchi, K; Meunier, J-C


    . Retrovirus-based HCV-pseudotyped viruses (HCVpp; genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide were generated. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid...

  13. Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells. (United States)

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


    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.

  14. [Hepatic cell transplantation. Technical and methodological aspects]. (United States)

    Pareja, Eugenia; Martínez, Amparo; Cortés, Miriam; Bonora, Ana; Moya, Angel; Sanjuán, Fernando; Gómez-Lechón, M José; Mir, José


    Hepatic cell transplantation consists of grafting already differentiated cells such as hepatocytes. Human hepatocytes are viable and functionally active. Liver cell transplantation is carried out by means of a 3-step method: isolation of hepatocytes from donor liver rejected for orthotopic transplantation, preparing a cell suspension for infusion and, finally, hepatocytes are implanted into the recipient. There are established protocols for the isolation of human hepatocytes from unused segments of donor livers, based on collagenase digestion of cannulated liver tissue at 37 degrees C. The hepatocytes can be used fresh or cryopreserved. Cryopreservation of isolated human hepatocytes would then be available for planned use. In cell transplant, the important aspects are: infusion route, number of cells, number of infusions and viability of the cells. The cells are infused into the patient through a catheter inserted via portal vein or splenic artery. Liver cell transplantation allows liver tissue to be used that would, otherwise, be discarded, enabling multiple patients to be treated with hepatocytes from a single tissue donor.

  15. Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells. (United States)

    Devhare, Pradip B; Sasaki, Reina; Shrivastava, Shubham; Di Bisceglie, Adrian M; Ray, Ranjit; Ray, Ratna B


    Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of nonparenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate (LX2) cells were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo), and the expression of fibrosis-related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased the expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and target SOCS3 in HSC, which in turn activates the STAT3-mediated transforming growth factor β (TGF-β) signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in sera of chronic HCV patients with fibrosis compared to healthy volunteers and non-HCV-related liver disease patients with fibrosis. Together, our results demonstrated that miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating the SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome-mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection.IMPORTANCE HCV-associated liver fibrosis is a critical step for end-stage liver disease progression. However, the molecular mechanisms for hepatic stellate-cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role for miR-19a carried through the exosomes in intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrate the role of exosomal miR-19a in activation of the STAT3-TGF-β pathway in HSC. This study contributes to the

  16. 体外诱导脂肪源性干细胞向类肝细胞的定向分化%Directional differentiation of adipose tissue-derived stem cells into hepatic-like cells in vitro by procedure-induction

    Institute of Scientific and Technical Information of China (English)

    刘剑; 李立; 冉江华; 张升宁; 邵剑春


    BACKGROUND: How to establish effective stable hepatic cell differentiation schedule, to purify or rapidly amplify stable hepatic-like cells needs to be solved before adipose tissue-derived stem cells (ADSCs) were used to treat liver diseases. OBJECTIVE: To establish sequencing induction system of ADSCs transforming into hepatic-like cells.METHODS: After isolated and purified the Lewis rat ADSCs, the surface marks of ADSCs were identified by flow cytometry. The rat ADSCs were transformed into the hepatic-like cells in the procedure-culture system by tris-step including hepatocyte growth factor (FGF), fibroblast growth factor-4, acid fibroblast growth factor and oncostatin M cytokine. RESULTS AND CONCLUSION: After rat ADSCs were induced at 7, 14 and 21 days, the expression of albumin (ALB), alpha fetoprotein (AFP) and CK18 mRNA was determined and fortified over time. Hepatic-like cells had ALB synthesis function. Metabolism of ammonia and urea synthesis occurred and lasted after 9-12 days. Results suggest ADSCs were successfully differentiated into hepatic-like cells in vitro after procedure-induction.%背景:用脂肪源性干细胞治疗肝脏疾病之前,如何建立有效稳定的肝细胞分化诱导方案,纯化并快速扩增性能稳定的类肝细胞等问题亟待解决.目的:建立大鼠脂肪源性干细胞转化为类肝细胞的程序化诱导体系.方法:分离纯化Lewis大鼠脂肪源性干细胞,流式细胞仪鉴定其表面标志,分3个阶段加入含有肝细胞生长因子、成纤维细胞生长因子4、酸性成纤维细胞生长因子、制瘤素M细胞因子的诱导培养体系,使脂肪源性干细胞向肝细胞转化.结果与结论:大鼠脂肪源性干细胞诱导7,14,21 d后,细胞阳性表达 ALB、AFP、CK18mRNA,表达量随诱导时间延长而增强,类肝细胞具有白蛋白合成功能.氨代谢和尿素的合成功能在9~12 d出现并持续存在.结果表明脂肪源性干细胞体外分段诱导可成功转化为类肝细胞.

  17. Inhibitory effects of caffeic acid phenethyl ester derivatives on replication of hepatitis C virus.

    Directory of Open Access Journals (Sweden)

    Hui Shen

    Full Text Available Caffeic acid phenethyl ester (CAPE has been reported as a multifunctional compound. In this report, we tested the effect of CAPE and its derivatives on hepatitis C virus (HCV replication in order to develop an effective anti-HCV compound. CAPE and CAPE derivatives exhibited anti-HCV activity against an HCV replicon cell line of genotype 1b with EC50 values in a range from 1.0 to 109.6 µM. Analyses of chemical structure and antiviral activity suggested that the length of the n-alkyl side chain and catechol moiety are responsible for the anti-HCV activity of these compounds. Caffeic acid n-octyl ester exhibited the highest anti-HCV activity among the tested derivatives with an EC50 value of 1.0 µM and an SI value of 63.1 by using the replicon cell line derived from genotype 1b strain Con1. Treatment with caffeic acid n-octyl ester inhibited HCV replication of genotype 2a at a similar level to that of genotype 1b irrespectively of interferon signaling. Caffeic acid n-octyl ester could synergistically enhance the anti-HCV activities of interferon-alpha 2b, daclatasvir, and VX-222, but neither telaprevir nor danoprevir. These results suggest that caffeic acid n-octyl ester is a potential candidate for novel anti-HCV chemotherapy drugs.

  18. Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. (United States)

    Kajiwara, Masatoshi; Aoi, Takashi; Okita, Keisuke; Takahashi, Ryosuke; Inoue, Haruhisa; Takayama, Naoya; Endo, Hiroshi; Eto, Koji; Toguchida, Junya; Uemoto, Shinji; Yamanaka, Shinya


    Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, the in vitro directed differentiation of human pluripotent stem cells into mature hepatocytes remains challenging. Little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. In the current study, we developed an improved hepatic differentiation protocol and compared 28 hiPSC lines originated from various somatic cells and derived using retroviruses, Sendai viruses, or episomal plasmids. This comparison indicated that the origins, but not the derivation methods, may be a major determinant of variation in hepatic differentiation. The hiPSC clones derived from peripheral blood cells consistently showed good differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts showed poor differentiation. However, when we compared hiPSCs from peripheral blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. These data underscore the importance of donor differences when comparing the differentiation propensities of hiPSC clones.

  19. Immunohistochemical study of hepatic oval cells in human chronic viral hepatitis

    Institute of Scientific and Technical Information of China (English)

    Xiong Ma; De Kai Qiu; Yan Shen Peng


    AIM To detect immunohistochemically the presence of oval cells in chronic viral hepatitis with antibody against c-kit.METHODS We detected oval cells in paraffin-embedded liver sections of 3 normal controls and 26 liver samples from patients with chronic viral hepatitis, using immunohistochemistry with antibodies against c-kit, π-class glutathione Stransferase ( Tr-GST ) and cytokeratins 19(CK19).RESULTS Oval cells were not observed in normal livers. In chronic viral hepatitis, hepatic oval cells were located predominantly in the periportal . region and fibrosis septa,characterized by an ovoid nucleus, small size,and scant cytoplasm. Antibody against stem cell factor receptor, c-kit, had higher sensitivity and specificity than π-GST and CK19. About 50% -70% of c-kit positive oval cells were stained positively for either π-GST or CK19.CONCLUSION Oval cells are frequently detected in human livers with chronic viral hepatitis, suggesting that oval cell proliferation is associated with the liver regeneration in this condition.

  20. Hepatic zonation of carbon and nitrogen fluxes derived from glutamine and ammonia transformations

    Directory of Open Access Journals (Sweden)

    Constantin Jorgete


    Full Text Available Abstract Background Glutaminase predominates in periportal hepatocytes and it has been proposed that it determines the glutamine-derived nitrogen flow through the urea cycle. Glutamine-derived urea production should, thus, be considerably faster in periportal hepatocytes. This postulate, based on indirect observations, has not yet been unequivocally demonstrated, making a direct investigation of ureogenesis from glutamine highly desirable. Methods Zonation of glutamine metabolism was investigated in the bivascularly perfused rat liver with [U-14C]glutamine infusion (0.6 mM into the portal vein (antegrade perfusion or into the hepatic vein (retrograde perfusion. Results Ammonia infusion into the hepatic artery in retrograde and antegrade perfusion allowed to promote glutamine metabolism in the periportal region and in the whole liver parenchyma, respectively. The results revealed that the space-normalized glutamine uptake, indicated by 14CO2 production, gluconeogenesis, lactate production and the associated oxygen uptake, predominates in the periportal region. Periportal predominance was especially pronounced for gluconeogenesis. Ureogenesis, however, tended to be uniformly distributed over the whole liver parenchyma at low ammonia concentrations (up to 1.0 mM; periportal predominance was found only at ammonia concentrations above 1 mM. The proportions between the carbon and nitrogen fluxes in periportal cells are not the same along the liver acinus. Conclusions In conclusion, the results of the present work indicate that the glutaminase activity in periportal hepatocytes is not the rate-controlling step of the glutamine-derived nitrogen flow through the urea cycle. The findings corroborate recent work indicating that ureogenesis is also an important ammonia-detoxifying mechanism in cells situated downstream to the periportal region.

  1. Efficient differentiation of embryonic stem cells into hepatic cells in vitro using a feeder-free basement membrane substratum.

    Directory of Open Access Journals (Sweden)

    Nobuaki Shiraki

    Full Text Available The endoderm-inducing effect of the mesoderm-derived supportive cell line M15 on embryonic stem (ES cells is partly mediated through the extracellular matrix, of which laminin α5 is a crucial component. Mouse ES or induced pluripotent stem cells cultured on a synthesized basement membrane (sBM substratum, using an HEK293 cell line (rLN10-293 cell stably expressing laminin-511, could differentiate into definitive endoderm and subsequently into pancreatic lineages. In this study, we investigated the differentiation on sBM of mouse and human ES cells into hepatic lineages. The results indicated that the BM components played an important role in supporting the regional-specific differentiation of ES cells into hepatic endoderm. We show here that knockdown of integrin β1 (Itgb1 in ES cells reduced their differentiation into hepatic lineages and that this is mediated through Akt signaling activation. Moreover, under optimal conditions, human ES cells differentiated to express mature hepatocyte markers and secreted high levels of albumin. This novel procedure for inducing hepatic differentiation will be useful for elucidating the molecular mechanisms controlling lineage-specific fates during gut regionalization. It could also represent an attractive approach to providing a surrogate cell source, not only for regenerative medicine, but also for pharmaceutical and toxicologic studies.

  2. Hepatic stem cells: existence and origin

    Institute of Scientific and Technical Information of China (English)

    Ying Zhang; Xue-Fan Bai; Chang-Xing Huang


    Stem cells are not only units of biological organization,responsible for the development and the regeneration oftissue and organ systems, but also are units in evolution bynatural selection. It is accepted that there is stem cellpotential in the liver. Like most organs in a healthy adult,the liver maintains a perfect balance between cell gain andloss. It has three levels of cells that can respond to loss ofhepatocytes: (1) Mature hepatocytes, which proliferate afternormal liver tissue renewal, less severe liver damage, etc;they are numerous, unipotent, "committed" and respondrapidly to liver injury. (2) Oval cells, which are activated toproliferate when the liver damage is extensive and chronic,or if proliferation of hepatocytes is inhibited; they lie withinor immediately adjacent tothe canal of Hering (CoH); theyare less numerous, bipotent and respond by longer, but stilllimited proliferation. (3) Exogenous liver stem cells, whichmay derive from circulating hematopoietic stem cells (HSCs)or bone marrow stem cells; they respond to allyl alcoholinjury or hepatocarcinogenesis; they are multipotent, rare,but have a very long proliferation potential. They make amore significant contribution to regeneration, and evencompletely restore normal function in a murine model ofhereditary tyrosinaemia. How these three stem cellpopulations integrate to achieve a homeostatic balanceremains enigmatic. This review focuses on the location,activation, markers of the three candidates of liver stemcell, and the most importantly, therapeutic potential ofhepatic stem cells.

  3. Adipose-Derived Stem Cells

    DEFF Research Database (Denmark)

    Toyserkani, Navid Mohamadpour; Quaade, Marlene Louise; Sheikh, Søren Paludan


    Emerging evidence has shown that adipose tissue is the richest and most accessible source of mesenchymal stem cells. Many different therapies for chronic wounds exist with varying success rates. The capacity of adipose-derived stem cells (ASCs) to promote angiogenesis, secrete growth factors......, regulate the inflammatory process, and differentiate into multiple cell types makes them a potential ideal therapy for chronic wounds. The aim of this article was to review all preclinical trials using ASCs in problem wound models. A systematic search was performed and 12 studies were found where different...

  4. Epigenetic Changes during Hepatic Stellate Cell Activation.

    Directory of Open Access Journals (Sweden)

    Silke Götze

    Full Text Available 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.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.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.

  5. Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope. (United States)

    Bonino, F; Heermann, K H; Rizzetto, M; Gerlich, W H


    Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d. Images PMID:3701932

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

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


    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.

  7. 脂肪间充质干细胞经肝动脉移植治疗晚期肝病%Adipose-derived mesenchymal stem cell transplantation via the hepatic artery for the treatment of advanced liver diseases

    Institute of Scientific and Technical Information of China (English)

    郭宪立; 刘跃; 周利敏; 胡玥


    BACKGROUND:Stem cel transplantation is a promising treatment for advanced liver diseases, and adipose-derived mesenchymal stem cels are a hot topic folowing bone marrow mesenchymal stem cels. OBJECTIVE:To explore the therapeutic effect of adipose-derived mesenchymal stem cels transplantationvia the hepatic artery on advanced liver diseases in rats. METHODS:Forty-five rats were randomized into three groups, 15 rats in each group: control group, model group and transplantation group. Rat models of liver cirrhosis were made in the latter two groups through subcutaneous injection of carbon tetrachloride. Then, 1 mL of CFSE-labeled adipose-derived mesenchymal stem cels was infusedvia the hepatic artery in the transplantation group, and the same volume of normal saline was infused in the model group. Control group had no treatment. Pathological changes, liver function and degree of hepatic fibrosis were observed in the three groups at 4 weeks after treatment. RESULTS AND CONCLUSION:After transplantation, green fluorescence-labeled adipose-derived mesenchymal stem cels were seen in the liver of rats. Hematoxylin-eosin staining and Masson staining showed unclear hepatic lobule structure in the model group with the formation of false lobules, cel cloudy sweling and loose, some degeneration and necrosis, and inflammatory cel infiltration; in the control group, there was nothing abnormal in the liver tissues of rats in the control group; in the transplantation group, the pathological changes of the rat liver were better than those in the model group, but worse than those in the control group. Compared with the model group, the level of serum albumin was higher in the control and transplantation group (P < 0.05), and the levels of bilirubin, aminotransferase and type IV colagen were lower in the control and transplantation group (P < 0.05). Thus, it can be seen that adipose-derived mesenchymal stem cel transplantation can improve liver function and reduce liver fibrosis in

  8. The Current Immune Function of Hepatic Dendritic Cells

    Institute of Scientific and Technical Information of China (English)

    Willy Hsu; Shang-An Shu; Eric Gershwin; Zhe-Xiong Lian


    While only a small percentage of the liver as dendritic cells, they play a major role in the regulation of liver immunity. Four major types of dendritic cell subsets include myeloid CD8α-B220-, lymphoid CD8α+B220-,plasmacytoid CD8α-B220+, and natural killer dendritic cell with CD8α-B220-NK1.1+ phenotype. Although these subsets have slightly different characteristics, they are all poor na(i)ve T cell stimulators. In exchange for their reduced capacity for allostimulation, hepatic DCs are equipped with an enhanced ability to secrete cytokines in response to TLR stimulation. In addition, they have increased level of phagocytosis. Both of these traits suggest hepatic DC as part of the innate immune system. With such a high rate of exposure to the dietary and commensal antigens, it is important for the hepatic DCs to have an enhanced innate response while maintaining a tolerogenic state to avoid chronic inflammation. Only upon secondary infectivity does the hepatic DC activate memory T cells for rapid eradication of recurring pathogen. On the other hand, overly tolerogenic characteristics of hepatic DC may be responsible for the increase prevalence of autoimmunity or liver malignancies.

  9. Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection (United States)

    Chen, Zhilei; Simeon, Rudo; Chockalingam, Karuppiah; Rice, Charles M.


    The present study describes the creation and characterization of a hepatoma cell line, n4mBid, that supports all stages of the hepatitis C virus (HCV) life cycle and strongly reports HCV infection by a cell-death phenotype. The n4mBid cell line is derived from the highly HCV-permissive Huh-7.5 hepatoma cell line and contains a modified Bid protein (mBid) that is cleaved and activated by the HCV serine protease NS3-4A. N4mBid exhibited a 10–20 fold difference in cell viability between the HCV-infected and mock-infected states, while the parental Huh-7.5 cells showed <2 fold difference under the same conditions. The pronounced difference in n4mBid cell viability between the HCV- and mock-infected states in a 96-well plate format points to its usefulness in cell survival-based high-throughput screens for anti-HCV molecules. The degree of cell death was found to be proportional to the intracellular load of HCV. HCV-low n4mBid cells, expressing an anti-HCV short hairpin RNA, showed a significant growth advantage over naïve cells and could be rapidly enriched after HCV infection, suggesting the possibility of using n4mBid cells for the cell survival-based selection of genetic anti-HCV factors. PMID:20188762

  10. Host cell kinases and the hepatitis C virus life cycle. (United States)

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


    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.

  11. Hepatitis C virus and host cell lipids: an intimate connection. (United States)

    Alvisi, Gualtiero; Madan, Vanesa; Bartenschlager, Ralf


    Hepatitis C virus (HCV) is a major human pathogen, persistently infecting more than 170 million individuals worldwide. The recent establishment of fully permissive culture systems allowed unraveling the close link between host cell lipids and HCV, at each step of the viral replication cycle. HCV entry is triggered by the timely coordinated interaction of virus particles with cell surface receptors, including the low-density lipoprotein receptor. Viral RNA replication strictly depends on fatty acids and cholesterol biosynthesis. This process occurs on modified intracellular membranes, forming a membranous web. Their biogenesis is induced by the viral nonstructural proteins (NS) 4B and NS5A and requires the activity of cellular lipid kinases belonging to the phosphatidylinositol-4-kinase III family. A hallmark of HCV-induced membranes is thus the presence of phosphatidylinositol-4-phosphate (PI4P), which is synthesized by these kinases. Intriguingly, certain recently identified HCV dependency factors selectively bind to PI derivatives, suggesting a crucial role for PIPs in viral RNA replication and assembly. The latter occurs on the surface of lipid droplets and is tightly connected to the very low density lipoprotein pathway leading to the formation of unique lipoviro particles. Thus, HCV exploits lipid metabolism in many ways and may therefore serve as a model system to gain insights into membrane biogenesis, lipid droplet formation and lipid trafficking.

  12. Definition of the transcription initiation site of human plasminogen gene in liver and non hepatic cell lines. (United States)

    Malgaretti, N; Bruno, L; Pontoglio, M; Candiani, G; Meroni, G; Ottolenghi, S; Taramelli, R


    We have mapped the cap site of the human plasminogen mRNA by primer extension and PCR techniques and found that it is located at position -161 relative to the first ATG, 97 bases upstream to the 5' end of the previously isolated cDNA clone. Seven human hepatic and non hepatic cell lines and fresh liver cells were tested for human plasminogen mRNA expression: the liver and the liver derived HepG2 cell line represent the major site of plasminogen RNA synthesis while the other cell lines (Hep3B, HeLa, IMR, 293 CaCo and SW626) show much lower levels.

  13. Giant cell hepatitis and autoimmune hemolytic anemia after chickenpox. (United States)

    Baran, Maşallah; Özgenç, Funda; Berk, Ömer; Gökçe, Demir; Kavakli, Kaan; Yilmaz, Funda; Şen, Sait; Yağci, Raşit Vural


    Autoimmune hemolytic anemia with giant cell hepatitis is a distinct entity in children. It is usually fatal with progressive liver disease. Immunosuppressive treatment with conventional drugs offers some response; however, it is usually only temporary. Alternative therapeutic options with monoclonals have been reported with promising remission of the disease. We report a case with autoimmune hemolytic anemia+giant cell hepatitis after varicella infection. She was resistant to standard immunosuppressive combinations, and rescue therapy with rituximab was used. Remission was not achieved with the drug and the child died with septic complication.

  14. Influence of serum collected from rat perfused with compound Biejiaruangan drug on hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Shun-Gen Guo; Wei Zhang; Tao Jiang; Min Dai; Lu-Fen Zhang; Yi-Chun Meng; Li-Yun Zhao; Jian-Zhao Niu


    AIM: To observe the effect of compound Biejiaruangan decoction (CBJRGC) (composite prescription of Carapax trionycis for softening the liver) on proliferation, activation,excretion of collagen and cytokine of hepatic stellate cells (HSCs) and to find the mechanism of prevention and treatment of hepatic fibrosis by CBJRGC.METHODS: Using MTT, immunohistochemistry and image analysis technology, the related indexes for proliferation,activation, excretion of collagen and cytokine of hepatic stellate cells were detected in 24 h, 48 h, and 72 h after adminstration of different dosages of CBJRGC.RESULTS: Statistical analysis showed that serum collected from rat perfused with CBJRGC could restrain the proliferation of HSC in 48 h and 72 h especially in high and medium dosage groups, markedly decrease the expression of desmin, synapsin and platelet derived growth factor (PDGF) in HSC in 24 h, 48 h and 72 h, as well as the expression of α-SMA, collagen Ⅲ, TIMP and TGFβ1 in 48 h and 72 h, decrease the excretion of collagen Ⅰ in 72 h.CBJRGC serum had no significant effect on collagens Ⅰ, Ⅲ and TIMP in 24 h.CONCLUSION: CBJRGC serum has a good curative effect on hepatic fibrosis. Its main mechanism may be related to the following factors. The drug serum can restrain the proliferation and activation of HSC, decrease the number of activated HSC and the total number of HSC, the excretion of collagens Ⅰ, Ⅲ, enhance the degradation of collagen and restore the balance of synthesis and degradation of collagen,inhibit the expression of transforming growth factor β1 (TGFβ1) and platelet derived growth factor (PDGF) in HSC,block and delay the process of hepatic fibrosis. Synapsin is a new marker of activation of HSC, which provides a theoretical and testing basis for neural regulation in the developing process of hepatic fibrosis.

  15. Acute seronegative hepatitis C manifesting itself as adult giant cell hepatitis--a case report and review of literature. (United States)

    Kryczka, Wiesław; Walewska-Zielecka, Bozena; Dutkiewicz, Ewa


    Adult giant cell hepatitis (AGCH) is a rare event and only about 100 cases have been reported within the last 20 years. The AGCH has been observed in association with viral infection, drug reactions or autoimmune disorders but in many cases its etiology remains unclear. AGCH manifests clinically as severe form of hepatitis histologically characterized by diffuse giant cell transformation of hepatocytes. We report the case of a 39-yr-old man with acute community-acquired hepatitis without previous pathology of the liver. Laboratory data revealed slight hypergammaglobulinemia and high titer of anti-smooth-muscle antibody with negative serology of hepatotropic viruses and absence of other known causes of hepatitis. Preliminary diagnosis of autoimmune hepatitis was established, additionally confirmed by excellent clinical and biochemical improvement during corticosteroid treatment. A liver biopsy showed the typical findings of panlobular syncytial giant cell hepatitis and positive HCV-RNA both in serum and liver. The above verified the diagnosis of acute type C hepatitis manifested histologically as adult giant cell hepatitis. After three months of treatment we withdrew corticosteroids as spontaneous clearance of HCV occurred and the lack of autoantibodies in serum as well as significant improvement of liver histology was ascertained. Within 30 months of the follow-up we have not observed biochemical and immunological abnormalities and control liver biopsy has shown no signs of hepatitis.

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  18. Hepatitis

    Institute of Scientific and Technical Information of China (English)


    930140 Hepatocyte stimulator peptide and itsclinical significance in viral hepatitis.ZHOUWeiping(周卫平),et al.Instit Viral Hepatitis,Chongqing Med Univ,630010.Chin J InternMed 1992;31(10):626-628.Hepatocyte stimulator peptide(HSP)is anewly developed hepatic stimulator substance.Its monoclonal antibodies have been obtained inour laboratory.In this study,HSP was deter-mined in the sera of 315 subjects including pa-

  19. Hepatitis

    Institute of Scientific and Technical Information of China (English)


    2010349 Relationships between serum hepatitis B virus load in mothers,free maternal DNA in peripheral blood of newborns and hepatitis B virus infection of newborns. WEI Junni(魏俊妮),et al. Dept Epidemiol,Shanxi Med Univ,Taiyuan 030001. Chin J Infect Dis 2010;28(5):297-300. Objective To study the relationships between serum hepatitis B virus (HBV) DNA level

  20. Alcoholic hepatitis: The pivotal role of Kupffer cells

    Institute of Scientific and Technical Information of China (English)

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


    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 tolllike 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.

  1. Regulatory T Cells in Chronic Hepatitis B Virus Infection

    NARCIS (Netherlands)

    J.N. Stoop (Jeroen Nicolaas)


    textabstractWorldwide 400 million people suffer from chronic hepatitis B virus (HBV) infection and approximately 1 million people die annually from HBV-related disease. To clear HBV, an effective immune response, in which several cell types and cytokines play a role, is important. It is known that p

  2. Basis study on the model of hepatitis B-Vitro cell culture

    Institute of Scientific and Technical Information of China (English)

    Zheng-mingHUANG; Xin-boYANG; Wen-binCAO; Hong-yanCHEN; He-zhiLIU; ZhuangLI


    AIM: To explore and set up many kinds of experimental model of hepatitis B in order to provide varies methods for application study on drugs to prevent and to cure hepatitis B. METHODS: According to the disorder and characters of hepatitis B, we used the models of duck primary hepatocytes which were infected duck hepatitis B virus (DHBV), the human hepatocellular carcinoma (cell 2215, Hep G2) which was transferred with hepatitis Bvirus and rats primary hepatocytes cultured with CCl4 in vitro

  3. Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Xiao-Ping Chen; Wan-Guang Zhang; Feng Zhang; Shuai Xiang; Han-Hua Dong; Lei Zhang


    AIM: To elucidate the interaction between nonparenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover.CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.

  4. Early hepatic stellate cell activation predicts severe hepatitis C recurrence after liver transplantation. (United States)

    Gawrieh, Samer; Papouchado, Bettina G; Burgart, Lawrence J; Kobayashi, Shogo; Charlton, Michael R; Gores, Gregory J


    Only a subset of hepatitis C virus (HCV)-infected patients develop progressive hepatic fibrosis after liver transplantation (LT). Hepatic stellate cell (HSC) activation is a pivotal step in hepatic fibrosis and precedes clinically apparent fibrosis. We determined whether early HSC activation, measured in 4-month protocol post-LT biopsies, is predictive of subsequent development of more histologically severe recurrence of HCV. Early (4 month) post-LT HSC activation, as measured by alpha-smooth muscle actin (alpha-SMA) staining, was determined in liver biopsies from recipients with severe (fibrosis score > or = 2, n = 13) and with mild (fibrosis score of 0, n = 13) recurrence of HCV at one-year post-LT. Immunohistochemical staining for alpha-smooth muscle actin (alpha-SMA) was used to generate HSC activation scores (regional and total). Total HSC activation scores at 4 months were similar in patients with severe and mild HCV recurrence (3.9 +/- 2.0 vs. 2.7 +/- 2.2, P = 0.2). Regional HSC activation, assessed as parenchymal (zones 1, 2, and 3) or mesenchymal (portal tracts and fibrous septa), was different between the study groups, with higher mesenchymal scores predictive of progression. No patients in the mild recurrence group had detectable mesenchymal alpha-SMA staining vs. 46% (6/13) of patients with severe recurrence (P HCV or HSC-targeted therapy.

  5. Inhibition of Hepatitis C Virus-Like Particle Binding to Target Cells by Antiviral Antibodies in Acute and Chronic Hepatitis C (United States)

    Steinmann, Daniel; Barth, Heidi; Gissler, Bettina; Schürmann, Peter; Adah, Mohammed I.; Gerlach, J. Tilman; Pape, Gerd R.; Depla, Erik; Jacobs, Dirk; Maertens, Geert; Patel, Arvind H.; Inchauspé, Geneviève; Liang, T. Jake; Blum, Hubert E.; Baumert, Thomas F.


    Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies. PMID:15308699

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

    Energy Technology Data Exchange (ETDEWEB)

    Zan, Yanlu [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Zhang, Yuxia, E-mail: [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Tien, Po, E-mail: [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China)


    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.

  7. Clinical associations of hepatic stellate cell (HSC) hyperplasia. (United States)

    Mounajjed, Taofic; Graham, Rondell P; Sanderson, Schuyler O; Smyrk, Thomas C


    Hepatic stellate cell (HSC) hyperplasia has been principally attributed to hypervitaminosis A. There are sporadic reports of HSC hyperplasia in other conditions such as chronic biliary disease and hepatitis C, but clinical associations of this entity have not been studied in detail. We aimed to investigate the clinical associations of HSC hyperplasia aside from hypervitaminosis A. We identified 34 patients whose liver histology showed HSC hyperplasia. We reviewed the liver samples; additional histologic findings in addition to HSC hyperplasia were consolidated into a histologic diagnosis. We collected clinical, laboratory, and radiologic data; the histologic diagnosis was combined with this data to reach an "overall diagnosis." Four patients had hypervitaminosis A (all native livers). In native livers (n = 24), HSC hyperplasia also occurred in association with drug-induced hepatitis [n = 6, niacin was the most common inducing agent (n = 3)], reactive hepatitis (n = 4), chronic hepatitis C (n = 4), autoimmune hepatitis (n = 3), steatohepatitis (n = 1), chronic biliary disease (n = 1), and portal venopathy (n = 1). In liver allografts (n = 10), HSC hyperplasia was seen in protocol biopsies without other significant abnormalities (n = 5), chronic biliary disease (n = 4), and acute cellular rejection (n = 1). All patients used medications (total of 99) and 82 % were on multiple medications. HSC hyperplasia is an uncommon and relatively nonspecific finding that most commonly occurs in multimedicated patients, often in the absence of hypervitaminosis A. Associated conditions include drug toxicity (such as niacin), post-liver transplant setting, reactive hepatitis (due to systemic illness or inflammatory disorders of the gastrointestinal tract), and chronic liver disease.

  8. Hepatic Stellate Cells Support Hematopoiesis and are Liver-Resident Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Claus Kordes


    Full Text Available Background/Aims: Hematopoiesis can occur in the liver, when the bone marrow fails to provide an adequate environment for hematopoietic stem cells. Hepatic stellate cells possess characteristics of stem/progenitor cells, but their contribution to hematopoiesis is not known thus far. Methods: Isolated hepatic stellate cells from rats were characterized with respect to molecular markers of bone marrow mesenchymal stem cells (MSC and treated with adipocyte or osteocyte differentiation media. Stellate cells of rats were further co-cultured with murine stem cell antigen-1+ hematopoietic stem cells selected by magnetic cell sorting. The expression of murine hematopoietic stem cell markers was analyzed by mouse specific quantitative PCR during co-culture. Hepatic stellate cells from eGFP+ rats were transplanted into lethally irradiated wild type rats. Results: Desmin-expressing stellate cells were associated with hematopoietic sites in the fetal rat liver. Hepatic stellate cells expressed MSC markers and were able to differentiate into adipocytes and osteocytes in vitro. Stellate cells supported hematopoietic stem/progenitor cells during co-culture similar to bone marrow MSC, but failed to differentiate into blood cell lineages after transplantation. Conclusion: Hepatic stellate cells are liver-resident MSC and can fulfill typical functions of bone marrow MSC such as the differentiation into adipocytes or osteocytes and support of hematopoiesis.

  9. Synthesis of C-4-Substituted Steviol Derivatives and Their Inhibitory Effects against Hepatitis B Virus. (United States)

    Lin, Shwu-Jiuan; Su, Ta-Chi; Chu, Chin-Nan; Chang, Yi-Chih; Yang, Li-Ming; Kuo, Yu-Cheng; Huang, Tsurng-Juhn


    ent-13-Hydroxykaur-16-ene-19-N-butylureide (6) was one of 33 synthesized C-4-substituted steviol derivatives that were evaluated for their effects on hepatitis B virus (HBV) surface antigen (HBsAg) secretion. The IC50 (16.9 μM) and SI (57.7) values for inhibiting HBV DNA replication of compound 6 were greater than those of the reference compound, lamivudine (3-TC; IC50: 107.5 μM; SI: 22.0). Thus, the anti-HBV mechanism of 6 was investigated, and it specifically inhibited viral gene expression and reduced viral DNA levels, as well as potently attenuated all of the viral promoter activity of HBV-expressing Huh7 cells. Examination of cellular signaling pathways found that 6 inhibited the activities of the nuclear factor (NF)-κB- and activator protein (AP)-1 element-containing promoters, but had no effects on AP-2 or interferon-stimulated response element (ISRE)-containing promoters in HBV-expressing cells. Meanwhile, it significantly eliminated NF-κB and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling-related protein levels and inhibited their phosphorylation in HBV-transfected Huh7 cells. The inhibitory potency of 6 against HBV DNA replication was reversed by cotransfecting the NF-κB p65 expression plasmid. Using the MAPK-specific activator anisomycin also reversed the inhibitory effect of 6 on viral DNA replication. The present findings suggest that the anti-HBV mechanism of 6 is partly mediated through the NF-κB and MAPK signaling pathways.

  10. Forced expression of Hnf1b/Foxa3 promotes hepatic fate of embryonic stem cells. (United States)

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


    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.

  11. Hepatitis

    Institute of Scientific and Technical Information of China (English)


    970349 Primary structure and variability of partialsequences in nonstructural gene 5 region of hepatitis Gvirus, CHANG Jinhong(常锦红), et al. Hepatol Instis,People’s Hosp, Beijing Med Univ, Beijing, 100044. NatlMed J China 1997; 77(3): 178-182. Objective: To sequence partial genome of hepatitis G

  12. Hepatitis

    Institute of Scientific and Technical Information of China (English)


    2009209 Effects of chronic hepatitis B virus infection on human hepatic cytochrome P450 2C9.ZHO Fuping(周福平),et al.Dept Infect Dis,Shanghai Changzheng Hosp,Shanghai 200003.Chin J Infect Dis,2009;27(2):94-98.

  13. Hepatitis (United States)

    ... inflammation of the liver.” This inflammation can be caused by a wide variety of toxins, drugs, and metabolic diseases, as well as infection. There are at least 5 hepatitis viruses. Hepatitis A is contracted when a child eats food or drinks water that is contaminated with the virus or has ...

  14. Hepatitis

    Institute of Scientific and Technical Information of China (English)


    920691 The determination of serum hepa-titis B virus DNA by polymerase chain rea-ction in hepatitis B patients treated withalpha-interferon. XU. Jianye(徐建业), et al.Centr Lab, Chongqing Cancer Instit, 630030.Chin J Intern Med, 1992; 31(5): 278-280. To clarify the status of HBV in serum of

  15. Congenital hepatic fibrosis, liver cell carcinoma and adult polycystic kidneys. (United States)

    Manes, J L; Kissane, J M; Valdes, A J


    In reviewing the literature, we found no liver cell carcinoma (LCC) or well-documented adult polycystic kidneys (APK) associated with congenital hepatic fibrosis (CHF). We report a 69-year-old man with CHF, LCC, APK, duplication cyst of distal portion of stomach, two calcified splenic artery aneurysms, myocardial fibrosis and muscular hypertrophy of esophagus. The LCC was grossly predunculated and microscopically showed prominent fibrosis and hyaline intracytoplasmic inclusions in the tumor cells.

  16. Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis


    Deng, Xin; Liang, Jian; LIN, ZHI-XIU; Wu, Fa-Sheng; Zhang, Ya-ping; Zhang, Zhi-Wei


    AIM: To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.

  17. mIL-2R, T cell subsets & hepatitis C

    Institute of Scientific and Technical Information of China (English)

    Chao-Pin Li; Ke-Xia Wang; Jian Wang; Bo-Rong Pan


    AIM: To study the levels of membrane interleukin-2 receptor(mIL-2R ) and T cell subsets in peripheral bloodmononuclear cells (PBMC) from patients with hepatitis Cand their role in the pathogenesis of hepatitis C.METHODS: The levels of mlL-2R and T cells subsets in PBMCWere detected by biotin- streptstividin (BSA) technique beforeand after stimulation with PHA in 203 patients with hepatitis Cwith HCV-RNA( + ), anti-HCV( + ), anti-HCV(-).RESULTS: The total expressive levels of mlL-2R before andafter stimulation with PHA(0.03 ± 0.01, 0.03 ± 0.02, 0.04 ± 0.02, 0.36±0.03), and Tcell subsets in PBMC (0.62±0.06,0.37 ± 0.05, 0.35 ± 0.07) were all lower in patients withhepatitis C than those in normal controls (0.66 ± 0.07, 0.41± 0.06, 0.31 ± 0.05, P < 0.01 ). Among the patients, thelevels of mlL-2R were lower in silence than those in situationof PHA inducting (P< 0.01). However, the levels of mlL-2Rwere similar in acute hepatitis C to that in chronic hepatitis C(P>0.05). The levels of CD3+, CD4+, CD4 +/CD8+ Were lov erand CD8 + was higher in patients with acute and chronichepatitis C with anti-HCV( + ) than those in normal controls (0.62±0.06, 0.37±0.05, 0.35±0.07, 1.18±0.30, 0.61±0.07, 0.37±0.05, 1.39±0.33, 0.31±0.05, P<0.05-P<0.01).CONCLUSION: The cellular immunity is obviously changed inpatients with hepatitis C. The levels of mlL-2R end activationof T cells am closely associated with chronicity of hepatitis C.

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


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


    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...

  19. Apicobasal Polarity Controls Lymphocyte Adhesion to Hepatic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Natalia Reglero-Real


    Full Text Available 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.

  20. Intrahepatic transplantation of hepatic oval cells for fulminant hepatic failure in rats

    Institute of Scientific and Technical Information of China (English)

    Chen-Xuan Wu; Qi Zou; Zheng-Yan Zhu; Ying-Tang Gao; Yi-Jun Wang


    AIM:To evaluate the effect of intrahepatic transplantation of hepatic oval cells (HOC) on fulminant hepatic failure (FHF) in rats. METHODS:HOC obtained from rats were labeled wi th green fluocescent protein (GFP) or 5, 6- carboxyfluorescein diacetate succinmidyl ester (CFDASE). Cell fluorescence was observed under fluorescent microscope at 6, 24, 48 and 72 h after labeling. CFDASE labeled HOC (5 × 106 cells each rat) were injected into livers of rats with FHF induced by D-galactosamine. Serum albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBil) levels were measured at different time points. Liver function of rats was examined on days 3, 7, 14 and 21 after HOC transplantation. RESULTS:The positive rate of GFP and CFDA-SE labeled HOC was 10% and 90%, respectively, with no significant change in cell viabilities. The survival rate was higher in HOC transplantation group than in control group, especially 48 (9/15 vs 6/15) and 72 h (9/15 vs 4/15) after HOC transplantation. The serum ALT, AST and TBil levels were decreased while the serum Alb level was increased after HOC transplantation. Fluorescence became faded and diffused in liver tissues, suggesting that proliferation and differentiation occur in transplanted HOC. CONCLUSION:CFDA-SE is superior to GFP in labeling HOC, although fluorescence intensity is decreased progressively with cell division. HOC transplantation can improve the liver function and increase the survival rate of recipients.

  1. Hepatic Bel-7402 Cell Proliferation on Different Phospholipid Surfaces

    Institute of Scientific and Technical Information of China (English)


    Phospholipids are believed to be important biomaterials.However, limited information is available on their cytocompatibilities.The objective of this study is to evaluate the effects of different phospholipids on the proliferation of hepatic Bel-7402 cells by comparing the adhesion, viability and proliferation of Bel-7402 cells cultured on different phospholipid surfaces.The cell adhesion, determined by counting the number of adhered cells to the surface, indicated that the cell adhesion was enhanced on charged phospolipid membranes.The cell viability evaluated by MTT[3 (4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium-bromide] showed that cells cultured on charged phospholipids have greater viability than those cultured on the control, while cells cultured on neutral phospholipids showed lower viability.The cell cycle analysis using flow cytometry demonstrated that S phase entry increased on charged phospholipids, while S phase entry decreased on neutral phospholipids.The results suggested that charged phospholipids, especially positively charged phospholipids, show better cytocompatibilities than neutral phospholipids to hepatic Bel-7402 cell.

  2. Monocyte-Derived Suppressor Cells in Transplantation. (United States)

    Ochando, Jordi; Conde, Patricia; Bronte, Vincenzo

    Myeloid-derived suppressor cells (MDSC) are cells of myeloid origin with enhanced suppressive function. They are negative regulators of the immune responses and comprise a heterogeneous mixture of immunosuppressive cells of monocytic (M-MDSC) and granulocytic (G-MDSC) origin. A more recent nomenclature proposes the term "suppressive monocyte derived cells" (suppressive MCs) to define CSF1/CSF2-dependent mouse suppressor cells that develop from common monocyte progenitors (cMoPs) after birth. Here, we review the literature about monocytic-derived cells with demonstrated suppressor function in vitro and in vivo within the context of solid organ transplantation.

  3. Hepatitis

    Institute of Scientific and Technical Information of China (English)


    2005226 Characteristics of peripheral blood T lymphocyte subsets in hepatitis B patients. FAN Zhen-ping(范振平),et al. Center Bio Ther, Instit Infect Dis, 302 Hosp Chin PLA, Beijing 100039. World Chin J Digestol, 2005;13(2): 194-197. Objective: To characterize the T-lymphocyte subsets in peripheral blood of patients with acute and chronic hepatitis B, and to explore their relations with the disease state. Methods: Peripheral blood

  4. Successful Interferon Therapy Reverses Enhanced Hepatic Progenitor Cell Activation in Patients with Chronic Hepatitis C. (United States)

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


    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.

  5. [Hepatic cell transplantation: a new therapy in liver diseases]. (United States)

    Pareja, Eugenia; Cortés, Miriam; Martínez, Amparo; Vila, Juan José; López, Rafael; Montalvá, Eva; Calzado, Angeles; Mir, José


    Liver transplantation has been remarkably effective in the treatment in patients with end-stage liver disease. However, disparity between solid-organ supply and increased demand is the greatest limitation, resulting in longer waiting times and increase in mortality of transplant recipients. This situation creates the need to seek alternatives to orthotopic liver transplantation.Hepatocyte transplantation or liver cell transplantation has been proposed as the best method to support patients. The procedure consists of transplanting individual cells to a recipient organ in sufficient quantity to survive and restore the function. The capacity of hepatic regeneration is the biological basis of hepatocyte transplantation. This therapeutic option is an experimental procedure in some patients with inborn errors of metabolism, fulminant hepatic failure and acute and chronic liver failure, as a bridge to orthotopic liver transplantation. In the Hospital La Fe of Valencia, we performed the first hepatocyte transplantation in Spain creating a new research work on transplant program.

  6. Patient-Derived Antibody Targets Tumor Cells (United States)

    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.

  7. Inhibitory effect of tanshinone IIA on rat hepatic stellate cells.

    Directory of Open Access Journals (Sweden)

    Ya-Wei Liu

    Full Text Available Anti-inflammation via inhibition of NF-κB pathways in hepatic stellate cells (HSCs is one therapeutic approach to hepatic fibrosis. Tanshinone IIA (C19H18O3, Tan IIA is a lipophilic diterpene isolated from Salvia miltiorrhiza Bunge, with reported anti-inflammatory activity. We tested whether Tan IIA could inhibit HSC activation.The cell line of rat hepatic stellate cells (HSC-T6 was stimulated with lipopolysaccharide (LPS (100 ng/ml. Cytotoxicity was assessed by MTT assay. HSC-T6 cells were pretreated with Tan IIA (1, 3 and 10 µM, then induced by LPS (100 ng/ml. NF-κB activity was evaluated by the luciferase reporter gene assay. Western blotting analysis was performed to measure NF-κB-p65, and phosphorylations of MAPKs (ERK, JNK, p38. Cell chemotaxis was assessed by both wound-healing assay and trans-well invasion assay. Quantitative real-time PCR was used to detect gene expression in HSC-T6 cells.All concentrations of drugs showed no cytotoxicity against HSC-T6 cells. LPS stimulated NF-κB luciferase activities, nuclear translocation of NF-κB-p65, and phosphorylations of ERK, JNK and p38, all of which were suppressed by Tan IIA. In addition, Tan IIA significantly inhibited LPS-induced HSCs chemotaxis, in both wound-healing and trans-well invasion assays. Moreover, Tan IIA attenuated LPS-induced mRNA expressions of CCL2, CCL3, CCL5, IL-1β, TNF-α, IL-6, ICAM-1, iNOS, and α-SMA in HSC-T6 cells.Our results demonstrated that Tan IIA decreased LPS-induced HSC activation.

  8. Vaccination with dendritic cells pulsed with hepatitis C pseudo particles induces specific immune responses in mice

    Institute of Scientific and Technical Information of China (English)

    Kilian Weigand; Franziska Voigt; Jens Encke; Birgit Hoyler; Wolfgang Stremmel; Christoph Eisenbach


    AIM:To explore dendritic cells (DCs) multiple functions in immune modulation.METHODS:We used bone-marrow derived dendritic cells from BALB/c mice pulsed with pseudo particles from the hepatitis C virus to vaccinate naive BALB/c mice.Hepatitis C virus (HCV) pseudo particles consist of the genotype 1b derived envelope proteins E1 and E2,covering a non-HCV core structure.Thus,not a single epitope,but the whole "viral surface" induces immunogenicity.For vaccination,mature and activated DC were injected subcutaneously twice.RESULTS:Humoral and cellular immune responses measured by enzyme-linked immunosorbent assay and interferon-gamma enzyme-linked immunosorbent spot test showed antibody production as well as T-cells directed against HCV.Furthermore,T-cell responses confirmed two highly immunogenic regions in E1 and E2 outside the hypervariable region 1.CONCLUSION:Our results indicate dendritic cells as a promising vaccination model for HCV infection that should be evaluated further.

  9. Inverse association between hepatic stellate cell apoptosis and fibrosis in chronic hepatitis C virus infection. (United States)

    Gonzalez, S A; Fiel, M I; Sauk, J; Canchis, P W; Liu, R-C; Chiriboga, L; Yee, H T; Jacobson, I M; Talal, A H


    Perisinusoidal hepatic stellate cells (HSC) are the principal fibrogenic cells in the liver. In animal models, HSC apoptosis is the predominant clearance mechanism of activated HSC, although data evaluating whether the same processes occur in humans are limited. We conducted a cross-sectional study to evaluate the association between HSC apoptosis and fibrosis stage in subjects with chronic hepatitis C virus (HCV) infection (n = 44) and HCV-negative controls with normal liver histology (n = 9). We used immunohistochemical techniques to identify activated (alpha-smooth muscle actin+), proliferative (Ki-67+) and apoptotic (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end-labelling+) HSC in liver biopsy specimens from all subjects. The same pathologist enumerated positive cells per high-power field (HPF, x 200) in 20 periportal/lobular areas. HSC apoptosis was decreased in HCV-positive subjects compared with controls (median 0.4, range 0.0-3.1 vs 1.1, 0.2-3.5 cells/HPF, P = 0.02). Among HCV-positive subjects, HSC apoptosis was decreased in those with moderate to advanced fibrosis (P = 0.04) compared with those with mild fibrosis. By multivariate analysis, HSC apoptosis decreased by an average of 0.14 cells/HPF (95% confidence interval 0.01-0.28 cells/HPF) per increase in fibrosis stage (P = 0.04). While the number of activated and proliferative HSC was significantly increased in HCV-infected subjects compared with that in uninfected controls, the numbers of these cells did not differ between HCV-infected subjects with mild vs moderate/advanced fibrosis. In conclusion, the number of apoptotic HSC was significantly decreased in HCV-infected subjects with advanced fibrosis. In chronic HCV infection, inhibition of HSC apoptosis may be one mechanism by which fibrosis progresses.

  10. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration. (United States)

    Hu, Chenxia; Li, Lanjuan


    Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

  11. Hepatitis

    Institute of Scientific and Technical Information of China (English)


    2008449 A cross-sectional survey of occult hepatitis B virus infection in HIV-infected patients. MA Jianxin(马建新), et al.Dept Infect Dis, Shanghai Public Health Clin Center, Shanghai 201508. Chin J Intern Med 2008;47(7):574-577. Objective To assess the prevalence of occult HBV infection in HIV-infected patients.

  12. Toll-like receptor-4 expression by hepatic progenitor cells and biliary epithelial cells in HCV-related chronic liver disease. (United States)

    Vespasiani-Gentilucci, Umberto; Carotti, Simone; Onetti-Muda, Andrea; Perrone, Giuseppe; Ginanni-Corradini, Stefano; Latasa, Maria U; Avila, Matias A; Carpino, Guido; Picardi, Antonio; Morini, Sergio


    Notwithstanding numerous evidences implicating toll-like receptor-4 (TLR4) in the pathogenesis of chronic hepatitis C virus (HCV) infection, the localization and level of TLR4 expression in the liver of patients with hepatitis C have never been investigated. We aimed to evaluate, by means of immunohistochemistry and real-time PCR (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection. Fifty patients who had undergone liver biopsy and 11 patients transplanted because of chronic HCV infection, and 12 controls free of liver disease, were included in the study. Each case was analyzed by immunohistochemistry for TLR4, α-smooth muscle actin and cytokeratin-7 (CK-7), and a subgroup of patients and all controls by rt-PCR for TLR4. Immunohistochemistry for α-smooth muscle actin was used to derive a score of activation of hepatic stellate cells and portal/septal myofibroblasts, while immunohistochemistry for CK-7 was used to evaluate and count hepatic progenitor cells, interlobular bile ducts and intermediate hepatocytes. In patients, the parenchymal elements responsible for the highest TLR4 level of expression were hepatic progenitor cells and biliary epithelial cells of interlobular bile ducts. Double-labeling experiments between anti-TLR4 and anti-CK7, anti-CD133, anti-CD44, anti-neural cell adhesion molecule, anti-epithelial cell adhesion molecule and anti-sex determining region Y-box 9, confirmed these findings. TLR4-positive hepatic progenitor cells and interlobular bile ducts were significantly correlated with the stage of liver disease (PHCV-related infection.

  13. A Case of Giant Cell Hepatitis Recurring after Liver Transplantation and Treated with Ribavirin

    Directory of Open Access Journals (Sweden)

    Ziad Hassoun


    Full Text Available A patient who underwent orthotopic liver transplantation for giant cell hepatitis with cirrhosis and in whom giant cell hepatitis recurred twice after orthotopic liver transplantation is reported. He was treated with ribavirin with an excellent result. The literature on this subject is reviewed. This observation clearly confirms the efficacy of ribavirin for the treatment of giant cell hepatitis, thus providing evidence for its viral origin.

  14. Expression of AT1amRNA in rat hepatic stellate cells and its effects on cell growth collagen production

    Institute of Scientific and Technical Information of China (English)

    张艺军; 杨希山; 吴平生; 廖贵清; 杨国平; 张晓峰; 陈晓清


    @@ Activated hepatic stellate cells (HSCs) play important roles in hepatic fibrosis. Studies on HSCs activation in vitro have shown that this process is regulated by a wide variety of growth factors and cytokines.1 Recent data indicate that AngⅡ is responsible for the mechanisms of myocardial fibrosis and kidney fibrosis; but there are only few reports on hepatic fibrosis.2-8

  15. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.


    Nagy, A.; Rossant, J.; Nagy, R.; Abramow-Newerly, W; Roder, J C


    Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and...

  16. Altered T cell costimulation during chronic hepatitis B infection. (United States)

    Barboza, Luisa; Salmen, Siham; Peterson, Darrell L; Montes, Henry; Colmenares, Melisa; Hernández, Manuel; Berrueta-Carrillo, Leidith E; Berrueta, Lisbeth


    T-cell response to hepatitis B virus (HBV) is vigorous, polyclonal and multi-specific in patients with acute hepatitis who ultimately clear the virus, whereas it is narrow and inefficient in patients with chronic disease, where inappropriate early activation events could account for viral persistence. We investigated the induction of activation receptors and cytokine production in response to HBcAg and crosslinking of CD28 molecules, in CD4+ cells from a group of chronically infected patients (CIP) and naturally immune subjects (NIS). We demonstrated that CD4+ cells from CIP did not increase levels of CD40L and CD69 following stimulation with HBcAg alone or associated to CD28 crosslinking, in contrast to subjects that resolved the infection (p<0.01). Furthermore, CD4+ cells from CIP produced elevated levels of IL-10 in response to HBcAg. These results suggest that a predominant inhibitory environment may be responsible for altered T cell costimulation, representing a pathogenic mechanism for viral persistence.

  17. Antithrombin reduces reperfusion-induced hepatic metastasis of colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Masanao Kurata; Kenji Okajima; Toru Kawamoto; Mitsuhiro Uchiba; Nobuhiro Ohkohchi


    AIM: To examine whether antithrombin (AT) could prevent hepatic ischemia/reperfusion (I/R)-induced hepatic metastasis by inhibiting tumor necrosis factor (TNF)-α-induced expression of E-selectin in rats.METHODS: Hepatic I/R was induced in rats and mice by clamping the left branches of the portal vein and the hepatic artery. Cancer cells were injected intrasplenically.The number of metastatic nodules was counted on day 7after I/R. TNF-α and E-selectin mRNA in hepatic tissue,serum fibrinogen degradation products and hepatic tissue levels of 6-keto-PGF1α, a stable metabolite of PGI2,were measured.RESULTS: AT inhibited increases in hepatic metastasis of tumor cells and hepatic tissue mRNA levels of TNF-αand E-selectin in animals subjected to hepatic I/R.Argatroban, a thrombin inhibitor, did not suppress any of these changes. Both AT and argatroban inhibited I/R-induced coagulation abnormalities. I/R-induced increases of hepatic tissue levels of 6-keto-PGF1αwere significantly enhanced by AT. Pretreatment with indomethacin completely reversed the effects of AT.Administration of OP-2507, a stable PGI2 analog, showed effects similar to those of AT in this model. Hepatic metastasis in AT-deficient mice subjected to hepatic I/R was significantly increased compared to that observed in wild-type mice. Administration of AT significantly reduced the number of hepatic metastases in congenital AT-deficient mice.CONCLUSION: AT might reduce I/R-induced hepatic metastasis of colon cancer cells by inhibiting TNF-α-induced expression of E-selectin through an increase in the endothelial production of PGI2. These findings also raise the possibility that AT might prevent hepatic metastasis of tumor cells if administered during the resection of liver tumors.

  18. Activated hepatic stellate cells in liver cirrhosis. A morphologic and morphometrical study. (United States)

    Carpino, Guido; Franchitto, Antonio; Morini, Sergio; Corradini, Stefano Ginanni; Merli, Manuela; Gaudio, Eugenio


    Hepatic stellate cells have been considered the most important cell-type involved in hepatic fibrogenesis. Proliferation and differentiation of hepatic stellate cells into myofibroblast-like cells has been related to the development of liver fibrosis. The alpha-actin expressed by hepatic stellate cells was considered a marker of their activation to myofibroblast-like cell. The aim of this study is to evaluate the changes in morphology, distribution, percentage and alpha-smooth muscle actin expression of hepatic stellate cells in normal and cirrhotic livers, and to correlate activated hepatic stellate cells with the progression of fibrosis. Human liver biopsies (n=121) were divided in five groups: 1) normal livers (controls); 2) cirrhosis post-HCV hepatitis; 3) cirrhosis post-HBV hepatitis; 4) non viral related cirrhosis; 5) recurrent HCV hepatitis after orthotopic liver transplantation. Samples immunostained with anti alpha-smooth muscle actin antibody by immunoperoxidase method were semi-quantitatively evaluated. Liver fibrosis was quantified by computer image analysis on specimens stained with Masson's trichrome. In normal adult livers stellate cells were very rarely stained for alpha-smooth muscle actin. In cirrhotic livers, a strongly enhanced percentage of stellate cells expressing alpha-smooth muscle actin was detected in cirrhotic fragments with respect to the control group, with a significant correlation between alpha-smooth muscle actin positive stellate cells and the volume fraction of fibrosis. Moreover, liver biopsies of recurrent hepatitis revealed an increased number of activated stellate cells compared to normal livers, and intermediate volume fraction of fibrosis. These results confirmed that a direct correlation existed between activated stellate cells and the progression of fibrosis. Alpha-smooth muscle actin confirmed to be a reliable marker of hepatic stellate cells activation also in precocious stages of the disease.

  19. Hepatic compartmentalization of exhausted and regulatory cells in HIV/HCV-coinfected patients. (United States)

    Barrett, L; Trehanpati, N; Poonia, S; Daigh, L; Sarin, S Kumar; Masur, H; Kottilil, S


    Accelerated intrahepatic hepatitis C virus (HCV) pathogenesis is likely the result of dysregulation within both the innate and adaptive immune compartments, but the exact contribution of peripheral blood and liver lymphocyte subsets remains unclear. Prolonged activation and expansion of immunoregulatory cells have been thought to play a role. We determined immune cell subset frequency in contemporaneous liver and peripheral blood samples from chronic HCV-infected and HIV/HCV-coinfected individuals. Peripheral blood mononuclear cells (PBMC) and biopsy-derived liver-infiltrating lymphocytes from 26 HIV/HCV-coinfected, 10 chronic HCV-infected and 10 HIV-infected individuals were assessed for various subsets of T and B lymphocytes, dendritic cell, natural killer (NK) cell and NK T-cell frequency by flow cytometry. CD8(+) T cells expressing the exhaustion marker PD-1 were increased in HCV-infected individuals compared with uninfected individuals (P = 0.02), and HIV coinfection enhanced this effect (P = 0.005). In the liver, regulatory CD4(+) CD25(+) Foxp3(+) T cells, as well as CD4(+) CD25(+) PD1(+) T cells, were more frequent in HIV/HCV-coinfected than in HCV-monoinfected samples (P HIV infection (P ≤ 0.005 for all). Low CD8(+) expression was observed only in PD-1(+) CD8(+) T cells from HCV-infected individuals and healthy controls (P = 0.002) and was associated with enhanced expansion of exhausted CD8(+) T cells when exposed in vitro to PHA or CMV peptides. In conclusion, in HIV/HCV coinfection, ongoing HCV replication is associated with increased regulatory and exhausted T cells in the periphery and liver that may impact control of HCV. Simultaneous characterization of liver and peripheral blood highlights the disproportionate intrahepatic compartmentalization of immunoregulatory T cells, which may contribute to establishment of chronicity and hepatic fibrogenesis in HIV coinfection.

  20. Immunohistochemical characterization of hepatic stem cell-related cells in developing human liver

    Institute of Scientific and Technical Information of China (English)

    XU Jun; HU Yong; WANG Jian; ZHOU Ji; ZHANG Taiping; YU Hongyu


    Little is known about the expression characteristics of the various kinds of possible markers in hepatic stem cells(HSCs)and other HSC-related cells in human fetal liver in various developmental stages.It is significant to investigate the immunohistochemical expression for better understanding of the origin,difierentiation and migration of HSCs in the developing human liver.H-E staining and immunohistochemical methods were used to observe the expression of hepatic/cholangiocellular differentiation markers(AFF,GST-π,CK7,CK19)and hematopoietic stem cell markers(CD34 and c-kit)in several kinds of HSC-related cells in thirty cases of fetal liver samples (4-35 weeks after pregnancy).AFP expression appears in fetal hepatocytes at four weeks'gestation.It Deaks at 16-24 weeks'gestation and decreases gradually afterwards.Finally,weak signals were only found in some ductal plate cells and a few limiting plate cells.GST-π was detected in hepatic cord cells from the sixth week and in the ductal plate cells from the eighth week.Twenty-six weeks later,only some ductal plate cells and a few limiting plate cells show positive signals.CK19 expression peaks during the 6th-11th weeks in hepatic cord cells and decreases gradually afterwards,except for the ductal plates.CK7 expression was limited in the ductal plate cells and bile ducts cells from the 14th week.CD34 and c-kit were detected at the eighth week in some ductal plate cells and a few mononuclear cells in the hepatic cords/mesenchymal tissue of portal areas.After 21 weeks.CD34 and c-kit were found only in ductal plate cells and a few mononuclear cells in the hepatic mesenchymal tissue of portal areas.Fetal hepatocytes at 4-16 weeks'gestation are mainly constituted by HSCs characterized with bi-potential differentiation capacity.At 16 weeks'gestation,most hepatic cord cells begin to differentiate into hepatocytes and abundant HSCs remain in ductal plate(the origin site of Hering canals).It is also indicated mat the

  1. Human embryonic stem cell derivation and directed differentiation. (United States)

    Trounson, A


    Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much

  2. Hepatic stellate cells and innate immunity in alcoholic liver disease

    Institute of Scientific and Technical Information of China (English)

    Yang-Gun Suh; Won-Il Jeong


    Constant alcohol consumption is a major cause of chronic liver disease, and there has been a growing concern regarding the increased mortality rates worldwide. Alcoholic liver diseases (ALDs) range from mild to more severe conditions, such as steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The liver is enriched with innate immune cells (e.g. natural killer cells and Kupffer cells) and hepatic stellate cells (HSCs), and interestingly, emerging evidence suggests that innate immunity contributes to the development of ALDs (e.g. steatohepatitis and liver fibrosis). Indeed, HSCs play a crucial role in alcoholic steatosis via production of endocannabinoid and retinol metabolites. This review describes the roles of the innate immunity and HSCs in the pathogenesis of ALDs, and suggests therapeutic targets and strategies to assist in the reduction of ALD.

  3. Liver stem cell-derived β-cell surrogates for treatment of type 1 diabetes☆ (United States)

    Yang, Li-Jun


    Consistent with the common embryonic origin of liver and pancreas as well the similar glucose-sensing systems in hepatocytes and pancreatic β-cells, it should not be surprising that liver stem cells/hepatocytes can transdifferentiate into insulin-producing cells under high-glucose culture conditions or by genetic reprogramming. Persistent expression of the pancreatic duodenal homeobox-1 (Pdx1) transcription factor or its super-active form Pdx1-VP16 fusion protein in hepatic cells reprograms these cells into pancreatic β-cell precursors. In vitro culture at elevated glucose concentrations or in vivo exposure to a hyperglycemia are required for further differentiation and maturation of liver-derived pancreatic β-cell precursor into functional insulin-producing pancreatic β-like cells. Under appropriate conditions, multiple pancreatic transcription factors can work in concert to reprogram liver stem/adult liver cells into functional insulin-producing cells. If such autologous liver-derived insulin-producing cells can be made to escape the type 1 diabetes-associated autoimmunity, they may serve as a valuable cell source for future cell replacement therapy without the need for life-long immunosuppression. PMID:16890895

  4. Hepatitis

    Institute of Scientific and Technical Information of China (English)


    2008079 Relationship of HBV genotype and bcp and pc mutations with HBV DNA rebound after lamivudine therapy. SU Minghua(苏明华), et al. Dept Infect Dis Clin Hosp, Guangxi Med Univ, Nanning 530027. World Chin J Digestol 2007;15(33):3507-3513. Objective To investigate the relationship of HBV gene mutations with HBV DNA rebound after lamivudine therapy. Methods Twenty-seven hepatitis B patients with HBV DNA rebound after

  5. Reconstitution of the Entire Hepatitis C Virus Life Cycle in Nonhepatic Cells


    Da Costa, Daniel; Turek, Marine; Felmlee, Daniel,; Girardi, Erika; Pfeffer, Sébastien; Long, Gang; Bartenschlager, Ralf; Zeisel, Mirjam,; Baumert, Thomas,


    International audience; Hepatitis C virus (HCV) is a human hepatotropic virus, yet the relevant host factors restricting HCV infection to hepatocytes are only partially understood. We demonstrate that exogenous expression of defined host factors reconstituted the entire HCV life cycle in human non-hepatic 293T cells. This study shows robust HCV entry, RNA replication, and production of infectious virus in human non-hepatic cells, and highlights key host factors required for liver tropism of HCV.

  6. Adipose-Derived Stem Cells

    NARCIS (Netherlands)

    Gathier, WA; Türktas, Z; Duckers, HJ


    Until recently bone marrow was perceived to be the only significant reservoir of stem cells in the body. However, it is now recognized that there are other and perhaps even more abundant sources, which include adipose tissue. Subcutaneous fat is readily available in most patients, and can easily be

  7. Enhanced antioxidant capacity of dental pulp-derived iPSC-differentiated hepatocytes and liver regeneration by injectable HGF-releasing hydrogel in fulminant hepatic failure. (United States)

    Chiang, Chih-Hung; Wu, Wai-Wah; Li, Hsin-Yang; Chien, Yueh; Sun, Cho-Chin; Peng, Chi-Hsien; Lin, Alex Tong-Long; Huang, Chi-Shuan; Lai, Ying-Hsiu; Chiou, Shih-Hwa; Hung, Shuen-Iu; Chang, Yuh-Lih; Lan, Yuan-Tzu; Liu, Dean-Mo; Chien, Chian-Shiu; Huo, Teh-Ia; Lee, Shou-Dong; Wang, Chien-Ying


    Acute hepatic failure (AHF) is a severe liver injury leading to sustained damage and complications. Induced pluripotent stem cells (iPSCs) may be an alternative option for the treatment of AHF. In this study, we reprogrammed human dental pulp-derived fibroblasts into iPSCs, which exhibited pluripotency and the capacity to differentiate into tridermal lineages, including hepatocyte-like cells (iPSC-Heps). These iPSC-Heps resembled human embryonic stem cell-derived hepatocyte-like cells in gene signature and hepatic markers/functions. To improve iPSC-Heps engraftment, we next developed an injectable carboxymethyl-hexanoyl chitosan hydrogel (CHC) with sustained hepatocyte growth factor (HGF) release (HGF-CHC) and investigated the hepatoprotective activity of HGF-CHC-delivered iPSC-Heps in vitro and in an immunocompromised AHF mouse model induced by thioacetamide (TAA). Intrahepatic delivery of HGF-CHC-iPSC-Heps reduced the TAA-induced hepatic necrotic area and rescued liver function and recipient viability. Compared with PBS-delivered iPSC-Heps, the HGF-CHC-delivered iPSC-Heps exhibited higher antioxidant and antiapoptotic activities that reduced hepatic necrotic area. Importantly, these HGF-CHC-mediated responses could be abolished by administering anti-HGF neutralizing antibodies. In conclusion, our findings demonstrated that HGF mediated the enhancement of iPSC-Hep antioxidant/antiapoptotic capacities and hepatoprotection and that HGF-CHC is as an excellent vehicle for iPSC-Hep engraftment in iPSC-based therapy against AHF.

  8. LIGHT/TNFSR14 can regulate hepatic lipase expression by hepatocytes independent of T cells and Kupffer cells.

    Directory of Open Access Journals (Sweden)

    Bijoy Chellan

    Full Text Available LIGHT/TNFSF14 is a costimulatory molecule expressed on activated T cells for activation and maintenance of T cell homeostasis. LIGHT over expressed in T cells also down regulates hepatic lipase levels in mice through lymphotoxin beta receptor (LTβR signaling. It is unclear whether LIGHT regulates hepatic lipase directly by interacting with LTβR expressing cells in the liver or indirectly by activation of T cells, and whether Kupffer cells, a major cell populations in the liver that expresses the LTβR, are required. Here we report that LIGHT expression via an adenoviral vector (Ad-LIGHT is sufficient to down regulate hepatic lipase expression in mice. Depletion of Kupffer cells using clodronate liposomes had no effect on LIGHT-mediated down regulation of hepatic lipase. LIGHT-mediated regulation of hepatic lipase is also independent of LIGHT expression by T cells or activation of T cells. This is demonstrated by the decreased hepatic lipase expression in the liver of Ad-LIGHT infected recombination activating gene deficient mice that lack mature T cells and by the Ad-LIGHT infection of primary hepatocytes. Hepatic lipase expression was not responsive to LIGHT when mice lacking LTβR globally or only on hepatocytes were infected with Ad-LIGHT. Therefore, our data argues that interaction of LIGHT with LTβR on hepatocytes, but not Kupffer cells, is sufficient to down regulate hepatic lipase expression and that this effect can be independent of LIGHT's costimulatory function.

  9. Determining the cellular diversity of hepatitis C virus quasispecies by single-cell viral sequencing. (United States)

    McWilliam Leitch, E Carol; McLauchlan, John


    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.

  10. Hepatitis B Virus Replication in CD34+ Hematopoietic Stem Cells From Umbilical Cord Blood. (United States)

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


    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.

  11. Bisdemethoxycurcumin Induces Apoptosis in Activated Hepatic Stellate Cells via Cannabinoid Receptor 2

    Directory of Open Access Journals (Sweden)

    Phil Jun Lee


    Full Text Available Activated Hepatic Stellate Cells (HSCs, major fibrogenic cells in the liver, undergo apoptosis when liver injuries cease, which may contribute to the resolution of fibrosis. Bisdemethoxycurcumin (BDMC is a natural derivative of curcumin with anti-inflammatory and anti-cancer activities. The therapeutic potential of BDMC in hepatic fibrosis has not been studied thus far in the context of the apoptosis in activated HSCs. In the current study, we compared the activities of BDMC and curcumin in the HSC-T6 cell line and demonstrated that BDMC relatively induced a potent apoptosis. BDMC-induced apoptosis was mediated by a combinatory inhibition of cytoprotective proteins, such as Bcl2 and heme oxygenase-1 and increased generation of reactive oxygen species. Intriguingly, BDMC-induced apoptosis was reversed with co-treatment of sr144528, a cannabinoid receptor (CBR 2 antagonist, which was confirmed with genetic downregulation of the receptor using siCBR2. Additionally, incubation with BDMC increased the formation of death-induced signaling complex in HSC-T6 cells. Treatment with BDMC significantly diminished total intracellular ATP levels and upregulated ATP inhibitory factor-1. Collectively, the results demonstrate that BDMC induces apoptosis in activated HSCs, but not in hepatocytes, by impairing cellular energetics and causing a downregulation of cytoprotective proteins, likely through a mechanism that involves CBR2.

  12. Interferon Response in Hepatitis C Virus (HCV) Infection: Lessons from Cell Culture Systems of HCV Infection. (United States)

    Sung, Pil Soo; Shin, Eui-Cheol; Yoon, Seung Kew


    Hepatitis C virus (HCV) is a positive-stranded RNA virus that infects approximately 130-170 million people worldwide. In 2005, the first HCV infection system in cell culture was established using clone JFH-1, which was isolated from a Japanese patient with fulminant HCV infection. JFH-1 replicates efficiently in hepatoma cells and infectious virion particles are released into the culture supernatant. The development of cell culture-derived HCV (HCVcc) systems has allowed us to understand how hosts respond to HCV infection and how HCV evades host responses. Although the mechanisms underlying the different outcomes of HCV infection are not fully understood, innate immune responses seem to have a critical impact on the outcome of HCV infection, as demonstrated by the prognostic value of IFN-λ gene polymorphisms among patients with chronic HCV infection. Herein, we review recent research on interferon response in HCV infection, particularly studies using HCVcc infection systems.

  13. Variable expression of cystatin C in cultured trans-differentiating rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Axel M Gressner; Birgit Lahme; Steffen K Meurer; Olav Gressner; Ralf Weiskirchen


    AIM: To study the expression of cystatin C (CysC), its regulation by transforming growth factor-β1 (TGF-β1)and platelet-derived growth factor (PDGF) and the potential interference of CysC with TGF-β1 signaling in this special cell type.METHODS: We evaluated the CysC expression in cultured, profibrogenic hepatic stellate cells and transdifferentiated myofibroblasts by Northern and Western blotting and confocal laser scanning microscopy.RESULTS: CysC was increased significantly in the course of trans-differentiation. Both TGF-β1 and PDGFBB suppressed CysC expression. Furthermore, CysC secretion was induced by the treatment with TGF-β1.Although CysC induced an increased binding affinity of TGF-β receptor type Ⅲ (beta-glycan) as assessed by chemical cross-linking with [125I]-TGF-β1, it did not modulate TGF-β1 signal transduction as shown by evaluating the Smad2/3 phosphorylation status and [CAGA]-MLP-luciferase reporter gene assay. Interestingly,the shedding of type Ⅲ TGF-β receptor beta-glycan was reduced in CysC-treated cells. Our data indicated that CysC expression was upregulated during transdifferentiation.CONCLUSION: Increased CysC levels in the serum of patients suffering from liver diseases are at least partially due to a higher expression in activated hepatic stellate cells. Furthermore, TGF-β1 influences the secretion of CysC, highlighting a potentially important role of cysteine proteases in the progression of hepatic fibrogenesis.

  14. Copper ions stimulate the proliferation of hepatic stellate cells via oxygen stress in vitro. (United States)

    Xu, San-qing; Zhu, Hui-yun; Lin, Jian-guo; Su, Tang-feng; Liu, Yan; Luo, Xiao-ping


    This study examined the effect of copper ions on the proliferation of hepatic stellate cells (HSCs) and the role of oxidative stress in this process in order to gain insight into the mechanism of hepatic fibrosis in Wilson's disease. LX-2 cells, a cell line of human HSCs, were cultured in vitro and treated with different agents including copper sulfate, N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO) for different time. The proliferation of LX-2 cells was measured by non-radioactive cell proliferation assay. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of platelet-derived growth factor receptor β subunit (PDGFβR), ELISA to determine the level of glutathione (GSH) and oxidized glutathione (GSSG), dichlorofluorescein assay to measure the level of reactive oxygen species (ROS), and lipid hydroperoxide assay to quantify the level of lipid peroxide (LPO). The results showed that copper sulfate over a certain concentration range could promote the proliferation of LX-2 cells in a time- and dose-dependent manner. The effect was most manifest when LX-2 cells were treated with copper sulfate at a concentration of 100 μmol/L for 24 h. Additionally, copper sulfate could dose-dependently increase the levels of ROS and LPO, and decrease the ratio of GSH/GSSG in LX-2 cells. The copper-induced increase in mRNA and protein expression of PDGFβR was significantly inhibited in LX-2 cells pre-treated with NAC, a precursor of GSH, and this phenomenon could be reversed by the intervention of BSO, an inhibitor of NAC. It was concluded that copper ions may directly stimulate the proliferation of HSCs via oxidative stress. Anti-oxidative stress therapies may help suppress the copper-induced activation and proliferation of HSCs.

  15. Glial fibrillary acidic protein as an early marker of hepatic stellate cell activation in chronic and posttransplant recurrent hepatitis C. (United States)

    Carotti, Simone; Morini, Sergio; Corradini, Stefano Ginanni; Burza, Maria Antonella; Molinaro, Antonio; Carpino, Guido; Merli, Manuela; De Santis, Adriano; Muda, Andrea Onetti; Rossi, Massimo; Attili, Adolfo Francesco; Gaudio, Eugenio


    Activated alpha-smooth muscle actin (alpha-SMA)-positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. In the rat, increased GFAP expression in the acute response to injury and down-regulation in the chronic response have been observed, whereas reports concerning GFAP expression in human liver are still conflicting. We investigated the utility of GFAP compared to alpha-SMA as an immunohistochemical marker of early activated HSCs in chronic and posttransplant recurrent hepatitis C and correlated GFAP expression with vascular remodeling and fibrosis progression. With immunohistochemistry and a semiquantitative scoring system, the expression of GFAP and alpha-SMA in HSCs and the microvessel density were analyzed in biopsies from normal livers obtained from cadaveric donors [donor liver (DL); n = 21] and from livers from posttransplant hepatitis C virus recurrent hepatitis (HCV-PTR) patients (n = 19), hepatitis C virus chronic hepatitis (HCV-CH) patients, (n = 12), and hepatitis C virus cirrhosis (HCV-C) patients (n = 16). The percentage of alpha-SMA-positive HSCs was significantly higher in the HCV-PTR, HCV-CH, and HCV-C groups compared to the DL group (P HCV-PTR group compared to the DL, HCV-C (P HCV-CH (P HCV-CH group compared to the DL group (P HCV-PTR group, the percentage of GFAP-positive HSCs correlated with fibrosis progression (P HCV-CH and seems to predict fibrosis progression in HCV-PTR.

  16. Relationship between hepatitis B virus infection and hepatic metastasis in non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    Fei Gao; Lin Jia; Xiaobo Du; Yun Wang; Jianjun Han


    Objective:The purpose of the study was to explore the relationship between hepatitis B virus (HBV) infec-tion and hepatic metastasis in non-smal celllung cancer (NSCLC). Methods:Four hundred and eighty cases of NSCLC were retrospectively analyzed from January 2003 to January 2010, and the prevalence of hepatic metastasis of NSCLC in patients with and without hepatitis B virus infection were compared. Results:In the HBV carriers’ group, the prevalence of synchronous hepatic metastasis and metachronous hepatic metastasis were 13.2%and 5.9%, respectively. Meanwhile in the non-HBV group, those were 21.6%and 9.5%respectively. A significant dif erence between the two groups was found (P<0.05). Conclusion:The prevalence of synchronous hepatic metastasis and metachronous hepatic metastasis in non-smal celllung cancer with HBV infection are lower than those in non-HBV infection group. Hepatic metastasis is infrequent in HBV infected cases of NSCLC.

  17. Human skin-derived stem cells as a novel cell source for in vitro hepatotoxicity screening of pharmaceuticals. (United States)

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


    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.

  18. Adult Langerhans Cell Histiocytosis with Hepatic and Pulmonary Involvement (United States)

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


    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

  19. Adult Langerhans Cell Histiocytosis with Hepatic and Pulmonary Involvement

    Directory of Open Access Journals (Sweden)

    Bruno Araujo


    Full Text Available 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.

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  8. The citrus fruit flavonoid naringenin suppresses hepatic glucose production from Fao hepatoma cells. (United States)

    Purushotham, Aparna; Tian, Min; Belury, Martha A


    Hepatic gluconeogenesis is the major source of fasting hyperglycemia. Here, we investigated the role of the citrus fruit flavonoid naringenin, in the attenuation of hepatic glucose production from hepatoma (Fao) cells. We show that naringenin, but not its glucoside naringin, suppresses hepatic glucose production. Furthermore, unlike insulin-mediated suppression of hepatic glucose production, incubation of hepatocytes with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor Ly294002 had no effect on the ability of naringenin to suppress hepatic glucose production. Further, naringenin did not increase phosphorylation of Akt at Ser473 or, Thr308, indicating this down-stream target of PI3-kinase is also not a player in naringenin-mediated suppression of hepatic glucose production. Importantly, like the dimethylbiguanide, metformin, naringenin significantly decreased cellular ATP levels without increasing cell cytotoxicity. Together, these results suggest that the aglycone, naringenin, has a role in the attenuation of hyperglycemia and may exert this effect in a manner similar to the drug, metformin.

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

    DEFF Research Database (Denmark)

    Xiao, Fei; Fofana, Isabel; Heydmann, Laura;


    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......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...

  10. CD4+ T cell responses in hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    Nasser Semmo; Paul Klenerman


    Hepatitis C virus (HCV) infection is a major cause of liver damage, with virus-induced end-stage disease such as liver cirrhosis and hepatocellular carcinoma resulting in a high rate of morbidity and mortality worldwide. Evidence that CD4+ T cell responses to HCV play an important role in the outcome of acute infection has been shown in several studies. However, the mechanisms behind viral persistence and the failure of CD4+ T cell responses to contain virus are poorly understood. During chronic HCV infection, HCV-specific CD4+ T cell responses are relatively weak or absent whereas in resolved infection these responses are vigorous and multispecific. Persons with a T-helper type Ⅰ profile, which promotes cellular effector mechanisms are thought to be more likely to experience viral clearance, but the overall role of these cells in the immunopathogenesis of chronic liver disease is not known. To define this, much more data is required on the function and specificity of virus-specific CD4+ T cells,especially in the early phases of acute disease and in the liver during chronic infection. The role and possible mechanisms of action of CD4+ T cell responses in determining the outcome of acute and chronic HCV infection will be discussed in this review.

  11. Identification of hepatic niche harboring human acute lymphoblastic leukemic cells via the SDF-1/CXCR4 axis.

    Directory of Open Access Journals (Sweden)

    Itaru Kato

    Full Text Available In acute lymphoblastic leukemia (ALL patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc (null mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.

  12. Relationship between somatostatin receptors and activation of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 陆良勇; 尤汉宁; 徐芹芳


    Background Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs.Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation.SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immunohistochemical staining.Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc.Conclusion The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.


    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 尤汉宁; 徐芹芳; 陆良勇


    Objective To investigate the relationship between expression of somatostatin receptors (SSTRs) and activation of rat hepatic stellate cell (HSC). Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation, and then SSTR1 ~5 mRNA levels in the differentiated first passage HSCs were detected by means of reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1 ~5 in normal as well as fibrotic liver was measured by immunohistochemical staining. Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs and normal rat liver. But SSTR1~3 mRNA appeared as HSCs became wholly activated, and SSTR1 ~3 could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc Conclusion The expression of SSTR1~3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)


    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.

  15. Immunogenicity of standard and low dose vaccination using yeast-derived recombinant hepatitis B surface antigen in elderly volunteers

    NARCIS (Netherlands)

    S. de Rave (Sjoerd); R.A. Heijtink; M. Bakker-Bendik (M.); J. Boot (Jenneke); S.W. Schalm (Solko)


    textabstractThere is no conclusive evidence that age influences the response to vaccination against hepatitis B virus. We therefore studied the immunogenicity of yeast-derived rHBsAg vaccine in elderly volunteers. The study was conducted in the outpatient clinics of an academic and a regional hospit

  16. Derivation of naive human embryonic stem cells. (United States)

    Ware, Carol B; Nelson, Angelique M; Mecham, Brigham; Hesson, Jennifer; Zhou, Wenyu; Jonlin, Erica C; Jimenez-Caliani, Antonio J; Deng, Xinxian; Cavanaugh, Christopher; Cook, Savannah; Tesar, Paul J; Okada, Jeffrey; Margaretha, Lilyana; Sperber, Henrik; Choi, Michael; Blau, C Anthony; Treuting, Piper M; Hawkins, R David; Cirulli, Vincenzo; Ruohola-Baker, Hannele


    The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

  17. Modulation of Kupffer cells on hepatic drug metabolism

    Institute of Scientific and Technical Information of China (English)

    Hong Ding; Jing Tong; Shi-Cheng Wu; Deng-Ke Yin; Xian-Fen Yuan; Jian-Yuan Wu; Jun Chen; Gang-Gang Shi


    AIM: To observe the effects of Kupffer cells on hepatic drug metabolic enzymes.METHODS: Kunming mice were ip injected with GdCl310,20, 40 mg/kg to decrease the number and block the function of kupffer cells selectively. The contents of drug metabolic enzymes, cytochrome P450, NADPH-cytochrom C redutase (NADPH-C), aniline hydroxylase (ANH), aminopyrine Ndemethylase (AMD), erythromycin N-demethylase (EMD),and glutathione s-transferase (mGST) in hepatic microsome and S9-GSTpi, S9-GST in supernatant of 9 000 g were accessed 1 d after the injection. The time course of alteration of drug metabolic enzymes was observed on d 1, 3, and 6 treated with a single dose GdCl3. Mice were treated with Angelica sinensis polysaccharides (ASP) of 30, 60, 120 mg/kg, ig, qd ×6 d, respectively and the same assays were performed.RESULTS: P450 content and NADPH-C, ANH, AMD, and END activities were obviously reduced 1 d after Kupffer cell blockade. However, mGST and S9-GST activities were significantly increased. But no relationship was observed between GdCl3 dosage and enzyme activities. With single dose GdCl3 treatment, P450 content, NADPH-C, and ANH activities were further decreased following Kupffer cell blockade lasted for 6 d, by 35.7%, 50.3%, 36.5% after 3 d, and 57.9%, 57.9%, 63.2% after 6 d, respectively. On the contrary, AMD, EMD, mGST, and Sg-GST activities were raised by 36.5%, 71.9%, 23.1%, 35.7% after 3 d,and 155%, 182%, 21.5%, 33.7% after 6 d, respectively.Furthermore, the activities of drug metabolic enzymes were markedly increased after 30 mg/kg ASP treatment,and decreased significantly after 120 mg/kg ASP treatment.No change in activity of Sg-GSTpi was observed in the present study.CONCLUSION: Kupffer cells play an important role in the modulation of drug metabolic enzymes. The changes of drug metabolic enzyme activities depend on the time of kupffer cell blockade and on the degree of Kupffer cells activated. A low concentration of ASP increases the activities of drug

  18. Mesenchymal stem cells from the human umbilical cord ameliorate fulminant hepatic failure and increase survival in mice

    Institute of Scientific and Technical Information of China (English)

    Jin-Feng Yang; Hong-Cui Cao; Qiao-Ling Pan; Jiong Yu; Jun Li; Lan-Juan Li


    BACKGROUND:Cell therapy has been promising for various diseases. We investigated whether transplantation of human umbilical cord mesenchymal stem cells (hUCMSCs) has any therapeutic effects on D-galactosamine/lipopolysaccharide (GalN/LPS)-induced fulminant hepatic failure in mice. METHODS:hUCMSCs isolated from human umbilical cord were cultured and transplanted via the tail vein into severe combined immune deifciency mice with GalN/LPS-induced fulminant hepatic failure. After transplantation, the localiza-tion and differentiation of hUCMSCs in the injured livers were investigated by immunohistochemical and genetic analy-ses. The recovery of the injured livers was evaluated histologi-cally. The survival rate of experimental animals was analyzed by the Kaplan-Meier method and log-rank test. RESULTS:hUCMSCs expressed high levels of CD29, CD73, CD13, CD105 and CD90, but did not express CD31, CD79b, CD133, CD34, and CD45. Cultured hUCMSCs displayed adip-ogenic and osteogenic differentiation potential. Hematoxylin and eosin staining revealed that transplantation of hUCMSCs reduced hepatic necrosis and promoted liver regeneration. Transplantation of hUCMSCs prolonged the survival rate of mice with fulminant hepatic failure. Polymerase chain reaction for humanalu sequences showed the presence of human cells in mouse livers. Positive staining for human albumin, human alpha-fetoprotein and human cytokeratin 18 suggested the for-mation of hUCMSCs-derived hepatocyte-like cellsin vivo. CONCLUSIONS:hUCMSC was a potential candidate for stem cell based therapies. After transplantation, hUCMSCs partially repaired hepatic damage induced by GalN/LPS in mice. hUC-MSCs engrafted into the injured liver and differentiated into hepatocyte-like cells.

  19. Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells. (United States)

    Ghodsizadeh, Arefeh; Hosseinkhani, Hossein; Piryaei, Abbas; Pournasr, Behshad; Najarasl, Mostafa; Hiraoka, Yosuke; Baharvand, Hossein


    Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

  20. A Novel Human Radixin Peptide Inhibits Hepatitis C Virus Infection at the Level of Cell Entry. (United States)

    Bukong, Terence N; Kodys, Karen; Szabo, Gyongyi


    Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin-moesin-radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and their functional mechanism we analyzed the anti-HCV properties of different human derived radixin peptides. Sixteen different radixin peptides were derived, synthesized and tested. Real-time quantitative PCR, cell toxicity assay, immuno-precipitation/western blot analysis and computational resource for drug discovery software were used for experimental analysis. We found that a human radixin hinge region peptide (Peptide1) can specifically block HCV J6/JFH-1 infection of Huh7.5 cells. Peptide 1 had no cell toxicity or intracellular uptake into Huh7.5 cells. Mechanistically, the anti-HCV activity of Peptide 1 extended to disruption of HCV engagement of CD81 thereby blocking downstream SYK activation, which we have recently demonstrated to be important for effective HCV infection of target hepatocytes. Our findings highlight a novel functional class of anti-HCV agents that can inhibit HCV infection, most likely by disrupting vital viral-host signaling interactions at the level of virus entry.

  1. Hepatic Differentiation of Murine Disease-Specific Induced Pluripotent Stem Cells Allows Disease Modelling In Vitro

    Directory of Open Access Journals (Sweden)

    Reto Eggenschwiler


    Full Text Available Direct reprogramming of somatic cells into pluripotent cells by retrovirus-mediated expression of OCT4, SOX2, KLF4, and C-MYC is a promising approach to derive disease-specific induced pluripotent stem cells (iPSCs. In this study, we focused on three murine models for metabolic liver disorders: the copper storage disorder Wilson's disease (toxic-milk mice, tyrosinemia type 1 (fumarylacetoacetate-hydrolase deficiency, FAH−/− mice, and alpha1-antitrypsin deficiency (PiZ mice. Colonies of iPSCs emerged 2-3 weeks after transduction of fibroblasts, prepared from each mouse strain, and were maintained as individual iPSC lines. RT-PCR and immunofluorescence analyses demonstrated the expression of endogenous pluripotency markers. Hepatic precursor cells could be derived from these disease-specific iPSCs applying an in vitro differentiation protocol and could be visualized after transduction of a lentiviral albumin-GFP reporter construct. Functional characterization of these cells allowed the recapitulation of the disease phenotype for further studies of underlying molecular mechanisms of the respective disease.

  2. Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Joseph Ignatius Irudayam


    Full Text Available Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5, hepatoblast (day 15 and hepatocyte-like cells (day 21 were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type, the hepatic cells (days 15 and 21 had significantly higher expression of acute phase protein genes including complement factors, coagulation factors, serum amyloid A and serpins. Furthermore, hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1, IL1R1, IL1RAP, IL2RG, IL6R, IL6ST and IL10RB. These cells also produced CCL14, CCL15, and CXCL- 1, 2, 3, 16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors, CXCR4 and CXCR7, than that of hepatic cells. Sirtuin family of genes involved in aging, inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF family as well as downstream signaling factors TRAF2, TRAF4, FADD, NFKB1 and NFKBIB were differentially expressed during hepatic differentiation.

  3. Endothelial cells are damaged by autophagic induction before hepatocytes in Con A-induced acute hepatitis. (United States)

    Yang, Ming-Chen; Chang, Chih-Peng; Lei, Huan-Yao


    We have reported both T-cell-dependent and -independent hepatitis in immunocompetent and immunodeficiency mice, respectively, after intravenous injection of Con A in mice. The mode of hepatocyte cell death is different: autophagy for T-cell-independent hepatitis in contrast to apoptosis for T-cell-dependent one. In this study, we further demonstrate that liver blood vessels are the first target in both modes. The infused Con A bond to the hepatic vascular endothelial cells and cause its damage with autophagy. Before the elevation of the serum alanine aminotransferase at 6 h post-injection, the plasma leakage and hemorrhage occur at 1-3 h without inflammation. Con A induces autophagy of endothelial cells and hemorrhage that is enhanced by IFN-gamma. Using the endothelial cell line HMEC-1, a dose- and time-dependent cell death with autophagic LC3-II (microtubule-associated protein light chain 3) conversion was induced by Con A and was enhanced by IFN-gamma. In conclusion, Con A induced autophagy on hepatic endothelial cells; the damage of liver blood vessel occurs before the induction of T-cell-dependent hepatitis via apoptosis or T-cell-independent hepatitis via autophagy.

  4. Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells. (United States)

    Ignatius Irudayam, Joseph; Contreras, Deisy; Spurka, Lindsay; Ren, Songyang; Kanagavel, Vidhya; Ramaiah, Arunachalam; Annamalai, Alagappan; French, Samuel W; Klein, Andrew S; Funari, Vincent; Arumugaswami, Vaithilingaraja


    Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs) to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5), hepatoblast (day 15) and hepatocyte-like cells (day 21) were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type, the hepatic cells (days 15 and 21) had significantly higher expression of acute phase protein genes including complement factors, coagulation factors, serum amyloid A and serpins. Furthermore, hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1, IL1R1, IL1RAP, IL2RG, IL6R, IL6ST and IL10RB. These cells also produced CCL14, CCL15, and CXCL- 1, 2, 3, 16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors, CXCR4 and CXCR7, than that of hepatic cells. Sirtuin family of genes involved in aging, inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF) family as well as downstream signaling factors TRAF2, TRAF4, FADD, NFKB1 and NFKBIB were differentially expressed during hepatic differentiation.

  5. Gastrointestinal and hepatic complications of hematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

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


    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 graftversus-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.

  6. Roles of the Lipid Metabolism in Hepatic Stellate Cells Activation

    Institute of Scientific and Technical Information of China (English)

    Xin-yan Jing; Xue-feng Yang; Kai Qing; Yan Ou-Yang


    The lipids present in hepatic stellate cells (HSCs) lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Activation of HSCs is crucial to the development of fibrosis in liver disease. During activation, HSCs transform into myofibroblasts with concomitant loss of their lipid droplets and production of excessive extracellular matrix. Release of lipid droplets containing retinyl esters and triglyceride is a defining feature of activated HSCs. Accumulating evidence supports the proposal that recovering the accumulation of lipids would inhibit the activation of HSCs. In healthy liver, quiescent HSCs store 80%of total liver retinols and release them depending on the extracellular retinol status. However, in injured liver activated HSCs lose their retinols and produce a considerable amount of extracellular matrix, subsequently leading to liver fibrosis. Further findings prove that lipid metabolism of HSCs is closely associated with its activation, yet relationship between activated HSCs and the lipid metabolism has remained mysterious.

  7. KN-93, a specific inhibitor of CaMK Ⅱ inhibits human hepatic stellate cell proliferation in vitro

    Institute of Scientific and Technical Information of China (English)

    Ping An; Jun-Yong Zhu; Yan Yang; Peng Lv; Yi-Hao Tian; Ming-Kai Chen; He-Sheng Luo


    AIM: To investigate the effects of KN-93, a CaMKⅡ selective inhibitor on cell proliferation and the expression of p53 or p21 protein in human hepatic stellate ceils.METHODS: Human hepatic stellate cells (LX-2) were incubated with various concentrations (0-50 μmol/L) of KN-93 or its inactive derivative, KN-92. Cell proliferation was measured by CCK-8 assay, and the expression of two cell cycle regulators, p53 and p21, was determined by SDS-PAGE and Western blotting.RESULTS: KN-93 (5-50 μmol/L) decreased the proliferation of human hepatic stellate cells in a dosedependent manner from 81.76% (81.76% + 2.58% vs 96.63% + 2.69%, P < 0.05) to 27.15% (27.15% + 2.86% vs 96.59% + 2.44%, P < 0.01) after 24 h treatment.Incubation of 10 μmol/L KN-93 induced the cell growth reduction in a time-dependent manner from 78.27% at 8 h to 11.48% at 48 h. However, KN-92, an inactive derivative of KN-93, did not inhibit cell proliferation effectively. Moreover, analysis of cell cycle regulator expression revealed that KN-93 rather than KN-92 reduced the expression of p53 and p21.CONCLUSION: KN-93 has potent inhibitory effect on proliferation of LX-2 cells by modulating the expression of two special cell cycle regulators, p53 and p21.

  8. Familial Follicular-Cell Derived Carcinoma

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    Eun Ju eSon


    Full Text Available Follicular cell-derived well-differentiated thyroid cancer, papillary (PTC and follicular thyroid carcinomas (FTC compose 95% of all thyroid malignancies. Familial follicular cell-derived well-differentiated thyroid cancers contribute to 5% of those cases. These familial follicular cell derived carcinomas or non-medullary thyroid carcinomas (NMTC divide into two clinical-pathological groups. One group, syndromic-associated, composed by predominately non-thyroidal tumors, is comprised of Pendred syndrome, Warner syndrome, Carney complex type 1, PTEN-hamartoma tumor syndrome (Cowden disease; PHTS, familial adenomatous polyposis (FAP/Gardner syndrome. Additionally other less established links correlated to the development of follicular cell-derived tumors have also included Ataxia-teleangiectasia syndrome, McCune Albright syndrome, and Peutz-Jeghers syndrome. The subsequent group encompasses syndromes typified by non-medullary thyroid carcinomas or NMTC, as well as, pure familial (f PTC with or without oxyphilia, fPTC with multinodular goiter and fPTC with papillary renal cell carcinoma. This heterogeneous group of diseases has not a established genotype-phenotype correlation as the well-known genetic events identified in the familial C-cell-derived tumors or medullary thyroid carcinomas (MTC. Clinicians should be have the knowledge to identify the likelihood of a patient presenting with thyroid cancer having an additional underlying familial syndrome stemming from characteristics through morphological findings that would alert the pathologist to have the patient undergo subsequent molecular genetics evaluations. This review will discuss the clinical and pathological findings of the patients with familial papillary thyroid carcinoma, such as familial adenomatous polyposis, Carney complex, Werner syndrome, and Pendred syndrome and the heterogeneous group of familial papillary thyroid carcinoma.

  9. 含临床病毒株聚合酶逆转录酶区的乙肝病毒DNA稳定复制细胞系的构建%Establishment of a Stable Cell Line Replicating Hepatitis B Virus DNA Carrying the Reverse Transcriptase Region Derived from a Clinical Isolate

    Institute of Scientific and Technical Information of China (English)

    向明确; 蔡雪飞; 张文露; 黄爱龙; 胡接力


    目的 构建含有临床病毒株聚合酶逆转录酶(RT)区的乙肝病毒(HBV) DNA稳定复制细胞系.方法 采用巢式PCR从患者血清扩增HBV DNA片段,利用片段置换反应将该片段克隆到HBV DNA复制载体,并在该载体上引入新霉素抗性基因,在确认该重组DNA体外可复制后,将其转染HepG2细胞,G418筛选,采用real-time PCR结合ELISA及Southern blot检测初筛和鉴定HBV DNA稳定复制细胞系.结果 从患者血清扩增出的HBV DNA片段nt55~1654被成功置换到HBV复制质粒pLL相应区域,得到质粒p11;新霉素抗性基因表达片段被克隆到p11中HBV DNA下游,获得质粒p11-neo,Southern blot检测证实p11-neo可支持体外复制;p11-neo转染HepG2后,经筛选鉴定,获得了可支持HBV DNA稳定复制的细胞系3-10.结论 建立了含有临床病毒株聚合酶RT区的HBV DNA稳定复制细胞系,real-time PCR结合ELISA有助于HBV DNA稳定复制细胞系的快速初筛鉴定.%Objective To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Methods Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction ( FSR) , followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis. Results Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11 , leading to the plasmid pll-neo, and pll-neo was confirmed to be HBV-replication-competent. A stable

  10. Fulminant hepatic failure resulting from small-cell lung cancer and dramatic response of chemotherapy

    Institute of Scientific and Technical Information of China (English)

    Kyoichi Kaira; Atsushi Takise; Rieko Watanabe; Masatomo Mori


    Prompt treatment in tumor-associated encephalopathy may prolong survival. We describe a 69-year-old male patient who was presented with fulminant hepatic failure, secondary to small-cell lung carcinoma with rapidly progressing encephalopathy. Both symptoms remitted following chemotherapy, suggesting swift diagnosis and administration of chemotherapy to be effective in treatment of fulminant hepatic failure and encephalopathy.

  11. A New Oleanolic Acid Derivative against CCl4-Induced Hepatic Fibrosis in Rats

    Directory of Open Access Journals (Sweden)

    Hongjun Xiang


    Full Text Available A novel hepatoprotective oleanolic acid derivative, 3-oxours-oleana-9(11, 12-dien-28-oic acid (Oxy-Di-OA, has been reported. In previous studies, we found that Oxy-Di-OA presented the anti-HBV (Hepatitis B Virus activity (IC50 = 3.13 µg/mL. Remarkably, it is superior to lamivudine in the inhibition of the rebound of the viral replication rate. Furthermore, Oxy-Di-OA showed good performance of anti-HBV activity in vivo. Some studies showed that liver fibrosis may affiliate with HBV gene mutations. In addition, the anti-hepatic fibrosis activity of Oxy-Di-OA has not been studied. Therefore, we evaluated the protective effect of Oxy-Di-OA against carbon tetrachloride (CCl4-induced liver injury in rats. Daily intraperitoneally administration of Oxy-Di-OA prevented the development of CCl4-induced liver fibrosis, which was evidenced by histological study and immunohistochemical analysis. The entire experimental protocol lasted nine weeks. Oxy-Di-OA significantly suppressed the increases of plasma aspartate aminotransferase (AST and alanine aminotransferase (ALT levels (p < 0.05. Furthermore, Oxy-Di-OA could prevent expression of transforming growth factor β1 (TGF-β1. It is worth noting that the high-dose group Oxy-Di-OA is superior to bifendate in elevating hepatic function. Compared to the model group, Oxy-Di-OA in the high-dose group and low-dose group can significantly reduce the liver and spleen indices (p < 0.05. The acute toxicity test showed that LD50 and a 95% confidence interval (CIs value of Oxy-Di-OA were 714.83 mg/kg and 639.73–798.73 mg/kg via intraperitoneal injection in mice, respectively. The LD50 value of Oxy-Di-OA exceeded 2000 mg/kg via gavage in mice. In addition, a simple and rapid high performance liquid chromatography-ultraviolet (HPLC-UV method was developed and validated to study the pharmacokinetic characteristics of the compound. After single-dose oral administration, time to reach peak concentration of Oxy-Di-OA (Cmax

  12. In vivo effects of Chinese herbal recipe, Danshaohuaxian, on apoptosis and proliferation of hepatic stellate cells in hepatic fibrotic rats

    Institute of Scientific and Technical Information of China (English)

    Xiao-Xia Geng; Qin Yang; Ru-Jia Xie; Xin-Hua Luo; Bing Han; Li Ma; Cheng-Xiu Li; Ming-Liang Cheng


    AIM: To investigate the effects of Danshaohuaxian (DSHX),a Chinese herbal recipe, on the apoptosis and cell cycles of hepatic stellate cells (HSCs) in rat hepatic fibrosis and its possible mechanisms. METHODS: Seventy-six male Wistar rats were randomly divided into normal control group, hepatic fibrosis group,non-DSHX-treated group and DSHX-treated group. Except for the normal control group, rat hepatic fibrotic models were induced by subcutaneous injection of carbon tetrachloride (CCl4), drinking alcohol, giving diet of hyperlipid and hypoprotein for 8 wk. When the hepatic fibrotic models were produced, 12 rats of hepatic fibrosis group (15 rats survived, others died during the 8 wk) were sacrificed to collect blood and livers. HSCs were isolated from the other 3 rats to detect the apoptotic index (AI) and cell cycles by flow cytometry. DSHX was then given to the DSHX-treated group (1.0 g/kg, PO daily) for 8 wk. At the same time, normal control group and non-DSHX-treated group were given normal saline for 8 wk. At end of the experiment, some rats in these three groups were sacrificed to collect blood and livers, the other rats were used for HSC isolation to detect the apoptotic index (AI) and cell cycles. Then the liver index, serum hyaluronic acid (HA) and alanine aminotransferase (ALT),degree of hepatic fibrosis, urinary excretion of hydroxyproline (Hyp) and expression of collagen types Ⅰ and Ⅲ (COL Ⅰ and Ⅲ) in these four groups were detected respectively.RESULTS: Compared with the indexes of the hepatic fibrosis group and non-DSHX-treated group, the DSHX-treated group revealed a liver index of (0.0267±0.0017 vs 0.0423±0.0044, 0.0295±0.0019, P<0.05), levels of serum HA (200.78±31.71 vs316.17±78.48, 300.86±72.73, P<0.05)and ALT(93.13±5.79 vs 174.5±6.02, 104.75±6.54, P<0.01),and stage of hepatic fibrosis (1.30 vs 4.25, 2.60, P<0.01)all reduced. The urinary excretion of Hyp increased (541.09±73.39 vs 62.00±6.40, 182.44±30.83, P<0

  13. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration. (United States)

    Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M; Verma, Vikas K; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C; Shah, Vijay H


    Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.

  14. Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells. (United States)

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


    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.

  15. Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass

    Directory of Open Access Journals (Sweden)

    Mark J. Solloway


    Full Text Available Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and β-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into β-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.

  16. Hepatic Cell Apoptosis Was Triggerred by HBx Accumulation and Independent on Verapamil

    Institute of Scientific and Technical Information of China (English)

    王海平; 陈孝平; 白祥军


    Summary: In order to studythe roles of HBx and calcium inhibitor verapamil in apoptosis of human normal hepatic cells, L02-off, a pTet-off stably integrated human hepatic cell line was established,in which HBx expression was tightly induced by Doxycycline. The effect of different amounts of HBx and verapamil on apoptosis of human normal hepatic cells was detected. The study showed that apoptosis was triggered by accumulation of intracellular HBx, while verapamil had no effects on the apoptotic process. It was concluded that apoptosis mediated by HBx was dose-dependent but calcium-independent.

  17. Immunoreactive hepatic stellate cells in biopsy material in children with chronic hepatitis B. The first report in pediatric patients. (United States)

    Łotowska, Joanna M; Lebensztejn, Dariusz M


    The research objective was to identify and quantify the immunohistochemically (IHC) stained hepatic stellate cells (HSCs) in children with chronic hepatitis B (CHB), including staging (S), location in the hepatic lobule, and correlation with hepatocyte count. Retrospective morphological analysis was based on liver biopsies obtained from 70 CHB children before antiviral treatment. To determine fibrosis stage, the Batts and Ludwig scoring system was applied. Immunohistochemical examinations used monoclonal antibodies against - SMA. IHC observations in CHB children revealed a significant positive correlation between the mean number of SMA immunopositive HSCs within the hepatic lobule (r = 0.518; p biopsy specimens with intensive fibrosis, most HSCs had an elongated shape and demonstrated evidently strong immunoexpression of cytoskeletal protein - SMA. The mean counts of HSCs/100 hepatocytes (in high power field) in 4 study groups, i.e. with S-0, S-1, S-2, S-3, were 5.00; 5.98; 9.80; 12.19, respectively. Interestingly, in most groups the highest count of immunoreactive HSCs/100 hepatocytes was in the intermediate zone, indicating its high metabolic activity in liver fibrogenesis. Immunohistochemical and statistical investigations of HSCs in children with CHB showed a close positive correlation of cell count with fibrosis intensity, which may have prognostic implications in this pathology.

  18. Permissivity of primary human hepatocytes and different hepatoma cell lines to cell culture adapted hepatitis C virus.

    Directory of Open Access Journals (Sweden)

    Francois Helle

    Full Text Available Significant progress has been made in Hepatitis C virus (HCV culture since the JFH1 strain cloning. However, developing efficient and physiologically relevant culture systems for all viral genotypes remains an important goal. In this work, we aimed at producing a high titer JFH1 derived virus to test different hepatic cells' permissivity. To this end, we performed successive infections and obtained a JFH1 derived virus reaching high titers. Six potential adaptive mutations were identified (I599V in E2, R1373Q and M1611T in NS3, S2364P and C2441S in NS5A and R2523K in NS5B and the effect of these mutations on HCV replication and infectious particle production was investigated. This cell culture adapted virus enabled us to efficiently infect primary human hepatocytes, as demonstrated using the RFP-NLS-IPS reporter protein and intracellular HCV RNA quantification. However, the induction of a strong type III interferon response in these cells was responsible for HCV inhibition. The disruption of this innate immune response led to a strong infection enhancement and permitted the detection of viral protein expression by western blotting as well as progeny virus production. This cell culture adapted virus also enabled us to easily compare the permissivity of seven hepatoma cell lines. In particular, we demonstrated that HuH-7, HepG2-CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry, replication and secretion even if the efficiency was very low in PLC/PRF/5 and Hep3B cells. In contrast, we did not observe any infection of SNU-182, SNU-398 and SNU-449 hepatoma cells. Using iodixanol density gradients, we also demonstrated that the density profiles of HCV particles produced by PLC/PRF/5 and Hep3B cells were different from that of HuH-7 and HepG2-CD81 derived virions. These results will help the development of a physiologically relevant culture system for HCV patient isolates.

  19. Stemness is derived from thyroid cancer cells

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    Risheng eMa


    Full Text Available Background: One hypothesis for thyroid cancer development is its derivation from thyroid cancer stem cells (CSCs. Such cells could arise via different paths including from mutated resident stem cells within the thyroid gland or via epithelial to mesenchymal transition (EMT from malignant cells since EMT is known to confer stem-like characteristics. Methods: To examine the status of stemness in thyroid papillary cancer we employed a murine model of thyroid papillary carcinoma and examined the expression of stemness and EMT using qPCR and histochemistry in mice with a thyroid-specific knock-in of oncogenic Braf (LSL-Braf(V600E/TPO-Cre. This construct is only activated at the time of thyroid peroxidase (TPO expression in differentiating thyroid cells and cannot be activated by undifferentiated stem cells which do not express TPO.Results: There was decreased expression of thyroid specific genes such as Tg and NIS and increased expression of stemness markers such as Oct4, Rex1, CD15 and Sox2 in the thyroid carcinoma tissue from 6 week old BRAFV600E mice. The decreased expression of the epithelial marker E-cadherin and increased EMT regulators including Snail, Slug, and TGF-β1 and TGF-β3, and the mesenchymal marker vimentin demonstrated the simultaneous progression of EMT and the CSC-like phenotype. Stemness was also found in a derived cancer thyroid cell line in which overexpression of Snail caused up-regulation of vimentin expression and up regulation of stemness markers Oct4, Rex1, CD15 with enhanced migration ability of the cells. Conclusions: Our findings support our earlier hypothesis that stemness in thyroid cancer is derived via EMT rather than from resident thyroid stem cells. In mice with a thyroid-specific knock-in of oncogenic Braf (LSL-Braf(V600E/TPO-Cre the neoplastic changes were dependent on thyroid cell differentiation and the onset of stemness must have been derived from differentiated thyroid epithelial cells.

  20. Neoplasms derived from plasmacytoid dendritic cells. (United States)

    Facchetti, Fabio; Cigognetti, Marta; Fisogni, Simona; Rossi, Giuseppe; Lonardi, Silvia; Vermi, William


    Plasmacytoid dendritic cell neoplasms manifest in two clinically and pathologically distinct forms. The first variant is represented by nodular aggregates of clonally expanded plasmacytoid dendritic cells found in lymph nodes, skin, and bone marrow ('Mature plasmacytoid dendritic cells proliferation associated with myeloid neoplasms'). This entity is rare, although likely underestimated in incidence, and affects predominantly males. Almost invariably, it is associated with a myeloid neoplasm such as chronic myelomonocytic leukemia or other myeloid proliferations with monocytic differentiation. The concurrent myeloid neoplasm dominates the clinical pictures and guides treatment. The prognosis is usually dismal, but reflects the evolution of the associated myeloid leukemia rather than progressive expansion of plasmacytoid dendritic cells. A second form of plasmacytoid dendritic cells tumor has been recently reported and described as 'blastic plasmacytoid dendritic cell neoplasm'. In this tumor, which is characterized by a distinctive cutaneous and bone marrow tropism, proliferating cells derive from immediate CD4(+)CD56(+) precursors of plasmacytoid dendritic cells. The diagnosis of this form can be easily accomplished by immunohistochemistry, using a panel of plasmacytoid dendritic cells markers. The clinical course of blastic plasmacytoid dendritic cell neoplasm is characterized by a rapid progression to systemic disease via hematogenous dissemination. The genomic landscape of this entity is currently under intense investigation. Recurrent somatic mutations have been uncovered in different genes, a finding that may open important perspectives for precision medicine also for this rare, but highly aggressive leukemia.

  1. Adult liver stem cells in hepatic regeneration and cancer

    NARCIS (Netherlands)

    Nantasanti, Sathidpak


    An alternative source of livers for transplantation in patients with (genetic) liver diseases and liver failure is needed because liver donors are scarce. HPC-derived hepatocyte-like cells could be one of the options. Because dogs and humans share liver-pathologies and disease-pathways, the dog is c

  2. Hepatocyte growth factor-induced proliferation of hepatic stem-like cells depends on activation of NF-κB

    Institute of Scientific and Technical Information of China (English)

    PengYao; YiqunZhan; WangxiangXu; ChangyanLi; PeibinYue; ChengwangXu; DarongHU; ChengkuiQu; XiaomingYang


    Background/Aims: Hepatocyte growth factor (HGF) regulates proliferation of hepatic stem cells. Transcription factor nuclear factor kappa B (NF-κB) has been demonstrated as a key mediator for cell growth regulation. We investigated the role of NF-κB in HGF-mediated cellular proliferation responses in a rat liver.derived hepatic stem-like cell line WB.F344. Methods: Cell proliferation was determined by incorporation of [3H]thymidine. Phosphorylation of ERK1/2, p38 MAPK, Akt and IκBα by HGF stimulation was detected by Western blotting. NF-κB activation was determined by electrophoretic mobility shift assay and NF-κB.mediated SEAP reporter assay. NF-κB activation was inhibited by treatment with an IκBα dominant-negative vector or inhibitor BAY-11-7082. Results: We found that stimulation of WB-F344 cells with HGF promoted cell proliferation and effectively protected WB-F344 cells from apoptosis induced by TNF-α. We also observed activation of ERK1/2, p38 MAPK, Akt and NF-κB signaling pathways by HGF in WB-F344 cells. HGF-induced cell proliferation was partly blocked by pre-treatment of the cells with inhibitors against MEK1 or p38 MAPK, and completely blocked using an inhibitor for NF-κB activity.Furthermore, it was demonstrated that IκB mutant that suppressed NF-κB activity completely blocked HGF-induced cell proliferation. Conclusions: NF-κB activity is required for HGF-induced proliferation in hepatic stem-like cell line WB-F344, and this activity requires ERK1/2 and p38 MAPK pathways.

  3. Human embryonic stem cells derived by somatic cell nuclear transfer. (United States)

    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


    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.

  4. Characterization and enrichment of hepatic progenitor cells in adult rat liver

    Institute of Scientific and Technical Information of China (English)

    Ai-Lan Qin; Xia-Qiu Zhou; Wei Zhang; Hong Yu; Qin Xie


    AIM: To detect the markers of oval cells in adult rat liver and to enrich them for further analysis of characterization in vitro.METHODS: Rat model for hepatic oval cell proliferation was established with 2-acetylaminofluorene and two third partial hepatectomy (2-AAF/PH). Paraffin embedded rat liver sections from model (11 d after hepatectomy) and control groups were stained with HE and OV6, cytokeratin19 (CK19),albumin, alpha fetoprotein (AFP), connexin43, and c-kit antibodies by immunohistochemistry. Oval cell proliferation was measured with BrdU incorporation test. C-kit positive oval cells were enriched by using magnetic activated cell sorting (MACS) .The sorted oval cells were cultured in a low density to observe colony formation and to examine their characterization in vitroby immunocytochemistry and RT-PCR. RESULTS: A 2-AAF/PH model was successfully established to activate the oval cell compartment in rat liver. BrdU incorporation test of oval cell was positive. The hepatic oval cells coexpressed oval cell specific marker OV6, hepatocytemarker albumin and cholangiocyte-marker CK19. They also expressed AFP and connexin 43. C-kit, one hematopoietic stem cell receptor, was expressed in hepatic oval cells at high levels. By using c-kit antibody in conjunction with MACS,we developed a rapid oval cell isolation protocol. The sorted cells formed colony when cultured in vitro. Cells in the colony expressed albumin or CK19 or coexpressed both and BrdU incorporation test was positive. RT-PCR on colony showed expression of albumin and CK19 gene.CONCLUSION: Hepatic oval cells in the 2-AAF/PH model had the properties of hepatic stem/progenitor cells. Using MACS, we established a method to isolate oval cells. The sorted hepatic oval cells can form colony in vitro which expresses different combinations of phenotypic markers and genes from both hepatocytes and cholangiocyte lineage.

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

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    Fei Xiao


    Full Text Available 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.

  6. Modelling the Impact of Cell-To-Cell Transmission in Hepatitis B Virus (United States)


    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

  7. Genetic abolishment of hepatocyte proliferation activates hepatic stem cells.

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    Yoko Endo

    Full Text Available Quiescent hepatic stem cells (HSCs can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1, an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

  8. Three-dimensional growth as multicellular spheroid activates the proangiogenic phenotype of colorectal carcinoma cells via LFA-1-dependent VEGF: implications on hepatic micrometastasis

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    Muruzabal Francisco J


    Full Text Available Abstract Background The recruitment of vascular stromal and endothelial cells is an early event occurring during cancer cell growth at premetastatic niches, but how the microenvironment created by the initial three-dimensional (3D growth of cancer cells affects their angiogenesis-stimulating potential is unclear. Methods The proangiogenic profile of CT26 murine colorectal carcinoma cells was studied in seven-day cultured 3D-spheroids of Results Spheroid-derived CT26 cells increased vascular endothelial growth factor (VEGF secretion by 70%, which in turn increased the in vitro migration of primary cultured hepatic sinusoidal endothelium (HSE cells by 2-fold. More importantly, spheroid-derived CT26 cells increased lymphocyte function associated antigen (LFA-1-expressing cell fraction by 3-fold; and soluble intercellular adhesion molecule (ICAM-1, given to spheroid-cultured CT26 cells, further increased VEGF secretion by 90%, via cyclooxygenase (COX-2-dependent mechanism. Consistent with these findings, CT26 cancer cells significantly increased LFA-1 expression in non-hypoxic avascular micrometastases at their earliest inception within hepatic lobules in vivo; and angiogenesis also markedly increased in both subcutaneous tumors and hepatic metastases produced by spheroid-derived CT26 cells. Conclusion 3D-growth per se enriched the proangiogenic phenotype of cancer cells growing as multicellular spheroids or as subclinical hepatic micrometastases. The contribution of integrin LFA-1 to VEGF secretion via COX-2 was a micro environmental-related mechanism leading to the pro-angiogenic activation of soluble ICAM-1-activated colorectal carcinoma cells. This mechanism may represent a new target for specific therapeutic strategies designed to block colorectal cancer cell growth at a subclinical micrometastatic stage within the liver.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sakai, Hiroshi [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B51, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Tagawa, Yoh-ichi, E-mail: [Frontier Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B51, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan); Tamai, Miho [Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B51, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Motoyama, Hiroaki [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Ogawa, Shinichiro [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); McEwen Center for Regenerative Medicine, University Health Network, 190 Elizabeth Street, Toronto, Ont., Canada M5G 2C4 (Canada); Soeda, Junpei; Nakata, Takenari; Miyagawa, Shinichi [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)


    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.

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

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    Nidal Ghosheh


    Full Text Available 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.

  11. Valproic Acid Increases the Hepatic Differentiation Potential of Salivary Gland Cells. (United States)

    Petrakova, O S; Ashapkin, V V; Shtratnikova, V Y; Kutueva, L I; Vorotelyak, E A; Borisov, M A; Terskikh, V V; Gvazava, I G; Vasiliev, A V


    The studies of cell plasticity and differentiation abilities are important problems in modern cellular biology. The use of histone deacetylase inhibitor - valproic acid is a promising approach to increasing the differentiation efficiency of various cell types. In this paper we investigate the ability of mouse submandibular salivary gland cells to differentiate into the hepatic direction and the effect of valproic acid on the efficiency of this differentiation. It was shown that the gene expression levels of hepatocyte markers (Aat, Afp, G6p, Pepck, Tat, Cyp3a13) and liver-enriched transcription factors (Hnf-3α, Hnf-3β, Hnf-4α, Hnf-6) were increased after differentiation in salivary gland cells. Valproic acid increases the specificity of hepatic differentiation, reducing the expression levels of the ductal (Krt19, Hhex1, Cyp7a1) and acinar (Ptf1a) markers. After valproic acid exposure, the efficiency of hepatic differentiation also increases, as evidenced by the increase in the gene expression level of Alb and Tdo, and increase in urea production by differentiated cells. No change was found in DNA methylation of the promoter regions of the genes; however, valproic acid treatment and subsequent hepatic differentiation largely affected the histone H3 methylation of liver-enriched genes. Thus, mouse submandibular salivary gland cells are capable of effective differentiation in the hepatic direction. Valproic acid increases the specificity and efficiency of the hepatic differentiation of these cells.

  12. Hepatitis G Viral RNA Co-infection in Plasma and Peripheral Blood Mononuclear Cells in Patients with Hepatitis C

    Institute of Scientific and Technical Information of China (English)

    LI; Shuli; ZENG; Linglan; LUO; Duande; LIU; Wei; GUO; Jingsong; YANG; Xiaoming


    The incidence of the co-infection of hepatitis G virus (HGV) and hepatitis C virus(HCV) and its clinical implication was investigated and the difference in the positive rate of HGV RNA and HCV RNA between plasma and peripheral blood mononuclear cells (PBMCs) observed. By using reverse transcriptase polymerase chain reaction (RT-PCR) assay, HCV-RNA and HGV-RNA in plasma and PBMCs of 72 patients with hepatitis C was detected. It was showed that HGV RNA was positive in plasma of 11 patients, in PBMCs of 15 patients, and simultaneously in both of plasma and PBMCs of 10 patients with the co-infection rate being 22.2 %. Nine patients were both HGV RNA and HCV RNA positive in plasma, 11 patients were both HGV RNA and HCV RNA positive in PBMC, and 6 patients were both HGV RNA and HCV RNA positive in both plasma and PBMC with the positive rate being 12.4 %, 15.3 % and 8.3 % respectively. The positive rate of both HGV RNA and HCV RNA in PBMCs was higher than in plasma. It was concluded that the HGV co-infection rate in the patients with hepatitis C was 22. 2 %. Simultaneous examination of plasma and PBMC can improve clinically detectable rate.

  13. Fetal hepatic progenitors support long-term expansion of hematopoietic stem cells. (United States)

    Chou, Song; Flygare, Johan; Lodish, Harvey F


    We have developed a coculture system that establishes DLK(+) fetal hepatic progenitors as the authentic supportive cells for expansion of hematopoietic stem (HSCs) and progenitor cells. In 1-week cultures supplemented with serum and supportive cytokines, both cocultured DLK(+) fetal hepatic progenitors and their conditioned medium supported rapid expansion of hematopoietic progenitors and a small increase in HSC numbers. In 2- and 3-week cultures DLK(+) cells, but not their conditioned medium, continuously and significantly (>20-fold) expanded both hematopoietic stem and progenitor cells. Physical contact between HSCs and DLK(+) cells was crucial to maintaining this long-term expansion. Similar HSC expansion (approximately sevenfold) was achieved in cocultures using a serum-free, low cytokine- containing medium. In contrast, DLK(-) cells are incapable of expanding hematopoietic cells, demonstrating that hepatic progenitors are the principle supportive cells for HSC expansion in the fetal liver.

  14. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus (United States)

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


    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.

  15. Adipose derived stem cells and nerve regeneration

    Institute of Scientific and Technical Information of China (English)

    Alessandro Faroni; Richard JP Smith; Adam J Reid


    Injuries to peripheral nerves are common and cause life-changing problems for patients along-side high social and health care costs for society. Current clinical treatment of peripheral nerve injuries predominantly relies on sacriifcing a section of nerve from elsewhere in the body to pro-vide a graft at the injury site. Much work has been done to develop a bioengineered nerve graft, precluding sacriifce of a functional nerve. Stem cells are prime candidates as accelerators of re-generation in these nerve grafts. This review examines the potential of adipose-derived stem cells to improve nerve repair assisted by bioengineered nerve grafts.

  16. [Acute hepatitis-associated pure red cell aplasia: a case report]. (United States)

    Della Loggia, Paolo; Cremonini, Laura


    After a holiday in Egypt, a 57-year-old caucasian woman developed acute hepatitis A. The illness seemed initially to have a benign course, with a decreasing trend of hepatic enzymes and an apparent recovery. Three weeks later a relapse occurred. Recurrence of symptoms and aminotransferase elevation were associated with severe anemia; a hyporegenerative anemia was diagnosed and all laboratory findings were consistent with pure red cell aplasia. The haematologic disorder was successfully treated with red cell transfusion and glucocorticoids.

  17. Liver cancer-derived hepatitis C virus core proteins shift TGF-beta responses from tumor suppression to epithelial-mesenchymal transition.

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    Serena Battaglia

    Full Text Available BACKGROUND: Chronic hepatitis C virus (HCV infection and associated liver cirrhosis represent a major risk factor for hepatocellular carcinoma (HCC development. TGF-beta is an important driver of liver fibrogenesis and cancer; however, its actual impact in human cancer progression is still poorly known. The aim of this study was to investigate the role of HCC-derived HCV core natural variants on cancer progression through their impact on TGF-beta signaling. PRINCIPAL FINDINGS: We provide evidence that HCC-derived core protein expression in primary human or mouse hepatocyte alleviates TGF-beta responses in terms or growth inhibition or apoptosis. Instead, in these hepatocytes TGF-beta was still able to induce an epithelial to mesenchymal transition (EMT, a process that contributes to the promotion of cell invasion and metastasis. Moreover, we demonstrate that different thresholds of Smad3 activation dictate the TGF-beta responses in hepatic cells and that HCV core protein, by decreasing Smad3 activation, may switch TGF-beta growth inhibitory effects to tumor promoting responses. CONCLUSION/SIGNIFICANCE: Our data illustrate the capacity of hepatocytes to develop EMT and plasticity under TGF-beta, emphasize the role of HCV core protein in the dynamic of these effects and provide evidence for a paradigm whereby a viral protein implicated in oncogenesis is capable to shift TGF-beta responses from cytostatic effects to EMT development.

  18. Endothelial cells derived from human embryonic stem cells (United States)

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


    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.

  19. Induced pluripotent stem cell lines derived from human somatic cells. (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A


    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  20. The vagal nerve stimulates activation of the hepatic progenitor cell compartment via muscarinic acetylcholine receptor type 3. (United States)

    Cassiman, David; Libbrecht, Louis; Sinelli, Nicoletta; Desmet, Valeer; Denef, Carl; Roskams, Tania


    In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver.

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

    Institute of Scientific and Technical Information of China (English)

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


    AIM: To determine the gene expression profile data for the whole liver during development of dimethylnitrosamine (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 HSCspecific 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 hepatocytespecific 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.

  2. Expression kinetics of hepatic progenitor markers in cellular models of human liver development recapitulating hepatocyte and biliary cell fate commitment. (United States)

    Chaudhari, Pooja; Tian, Lipeng; Deshmukh, Abhijeet; Jang, Yoon-Young


    Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations.

  3. Cell culture system of a hepatitis C genotype 3a and 2a chimera

    DEFF Research Database (Denmark)


    A robust and genetically stable cell culture system for Hepatitis C Virus (HCV) genotype 3a is provided. A genotype 3a/2a (S52/JFH1) recombinant containing the structural genes (Core, E1, E2), p7 and NS2 of strain S52 was constructed and characterized in Huh7.5 cells. S52/JFH1 and J6/JFH viruses...... passaged in cell culture had comparable growth kinetics and yielded similar peak HCV RNA titers and infectivity titers. Direct genome sequencing of cell culture derived S52/JFH1 viruses identified putative adaptive mutations in Core, E2, p7, NS3, and NS5A; clonal analysis revealed that all genomes analyzed...... exhibited different combinations of these mutations. Finally, viruses resulting from transfection with RNA transcripts of five S52/JFH1 recombinants containing these combinations of putative adaptive mutations performed as efficiently as J6/JFH viruses in Huh7.5 cells and were all genetically stable after...

  4. Hepatitis B virus infects hepatic stellate cells and affects their proliferation and expression of collagen type Ⅰ

    Institute of Scientific and Technical Information of China (English)

    LIU Xuan; ZHU Sheng-tao; YOU Hong; CONG Min; LIU Tian-hui; WANG Bao-en; JIA Ji-dong


    Background Hepatitis B is at particularly high risk of fibrosis progression. Unfortunately, the mechanism of hepatic fibrogenesis induced by hepatitis B virus (HBV) has not been fully understood to date. The aim of this study was to observe whether HBV can infect hepatic stellate cells (HSCs), and to examine the effects of HBV or HBV S protein (HBs) on the proliferation and collagen type Ⅰ expression of HSCs.Methods The supernatants of HepG2.2.15 cells which contained HBV-DNA or HBs were added to LX-2 cells for 72 hours. Cell survival was determined by MTT assay. HBV particles in LX-2 cells were detected by transmission electron microscopy. The expression of HBs and HBV C protein (HBc) was determined by confocal fluorescence microscopy. The expression levels of HBV-DNA were measured by real-time PCR. The cellular collagen type Ⅰ mRNA and protein levels were quantified by reverse transcription-PCR and ELISA, respectively.Results High concentrations of HBV (1.2x105-5.0x105 copies/ml) or HBs (1.25-20 μg/ml) inhibited the proliferation of LX-2 cells, while low concentrations of HBV (1.0x103-6.2x104 copies/ml) or HBs (0.04-0.62 μg/ml) promoted the proliferation. After treating LX-2 cells with HBV for 72 hours, about 42 nm HBV-sized particles and strong expression of HBs and HBc were found in the cytoplasm of LX-2 cells. HBV-DNA in the culture medium of LX-2 cells decreased at 24 hours, rose at 48 hours and thereafter, decreased again at 72 hours. The mRNA and protein expression of cellular collagen type Ⅰ in LX-2 cells were significantly increased by HBV infection but not by recombinant HBs. Conclusions HBV and HBs affect the proliferation of HSCs; HBV can transiently infect and replicate in cultured HSCs and express HBs and HBc in vitro. Furthermore, HBV can significantly increase the expression of collagen type Ⅰ mRNA and protein in HSCs.

  5. Fibrosing cholestatic hepatitis following cytotoxic chemotherapy for small-cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    Jaime Ceballos-Viro; José M López-Picazo; José L Pérez-Gracia; Jesús J Sola; Gregorio Aisa; Ignacio Gil-Bazo


    Fibrosing cholestatic hepatitis (FCH) is a variant of viral hepatitis reported in hepatitis B virus or hepatitis C virus infected liver, renal or bone transplantation recipients and in leukemia and lymphoma patients after conventional cytotoxic chemotherapy. FCH constitutes a well-described form of fulminant hepatitis having extensive fibrosis and severe cholestasis as its most characteristic pathological findings. Here, we report a case of a 49-year-old patient diagnosed with small-cell lung cancer who developed this condition following conventional chemotherapy-induced immunosuppression. This is the first reported case in the literature of FCH after conventional chemotherapy for a solid tumor. In addition to a detailed report of the case, a physiopathological examination of this potentially life-threatening condition and its treatment options are discussed.

  6. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver. (United States)

    Gamal, Wesam; Treskes, Philipp; Samuel, Kay; Sullivan, Gareth J; Siller, Richard; Srsen, Vlastimil; Morgan, Katie; Bryans, Anna; Kozlowska, Ada; Koulovasilopoulos, Andreas; Underwood, Ian; Smith, Stewart; Del-Pozo, Jorge; Moss, Sharon; Thompson, Alexandra Inés; Henderson, Neil C; Hayes, Peter C; Plevris, John N; Bagnaninchi, Pierre-Olivier; Nelson, Leonard J


    Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.

  7. Sensitive detection of hepatocellular injury in chronic hepatitis C patients with circulating hepatocyte-derived microRNA-122. (United States)

    van der Meer, A J; Farid, W R R; Sonneveld, M J; de Ruiter, P E; Boonstra, A; van Vuuren, A J; Verheij, J; Hansen, B E; de Knegt, R J; van der Laan, L J W; Janssen, H L A


    As chronic hepatitis C patients with progressive disease can present themselves with normal ALT levels, more sensitive biomarkers are needed. MicroRNAs are newly discovered small noncoding RNAs that are stable and detectable in the circulation. We aimed to investigate the association between hepatocyte-derived microRNAs in serum and liver injury in patients with chronic hepatitis C. The hepatocyte-derived miR-122 and miR-192 were analysed in sera of 102 chronic HCV-infected patients and 24 healthy controls. Serum levels of miR-122 and miR-192 correlated strongly with ALT (R = 0.67 and R = 0.65, respectively, P chronic HCV infection (P = 0.026). Importantly, miR-122 was also superior in discriminating chronic HCV-infected patients with a normal ALT from healthy controls compared with the ALT level (AUC = 0.97 vs AUC = 0.78, P = 0.007). In conclusion, our study confirmed that liver injury is associated with high levels of hepatocyte-derived microRNAs in circulation and demonstrated that in particular miR-122 is a sensitive marker to distinguish chronic hepatitis C patients from healthy controls. More sensitive blood markers would benefit especially those patients with minor levels of hepatocellular injury, who are not identified by current screening with ALT testing.

  8. RNA Interference Targeting Leptin Gene Effect on Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    XUE Xiulan; LIN Jusheng; SONG Yuhu; SUN Xuemei; ZHOU Hejun


    To construct the specific siRNA expression vectors and investigate their effect on leptin and collagen I in HSC, which provide a new approach to the prevent and treat hepatic fibrosis. The five siRNAs against leptin gene were transcript synthesized intracellularly by expression templates of plasmid vector psiRNA-hH1neo. The recombinant leptin siRNA plasmid vectors could express in eukaryocyte , and then to evaluate them by using enzyme cutting and sequencing. The recombinant plasmids were transfected into HSCs using Lipofectamine methods respectively. The cells were selected after growing in DMEM containing 300 μg/mL G418 for about 4 weeks. Gene expression of leptin and collagen I were showed by Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Identification by enzyme cutting and sequencing showed that the leptin siRNA expression vectors were constructed successfully, and leptin siRNA could inhibit the leptin and collagen I gene expression effectively. It was concluded that RNA interference-mediated silencing of leptin gene diminished leptin and collagen I gene expression in HSCs. Furthermore, attenuated the extracellular matrix over-deposition at the same time. Leptin gene is ideal targets of gene therapy for liver fibrosis.

  9. Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis (United States)

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


    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

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

    Directory of Open Access Journals (Sweden)

    Silvia Affò

    Full Text Available 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.

  11. Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis. (United States)

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


    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.

  12. Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies (United States)



    BACKGROUND & AIMS The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion. METHODS We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture–derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors. RESULTS By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus–antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general. CONCLUSIONS We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines. PMID:22503792

  13. Exosomes Derived from Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Bo Yu


    Full Text Available The functional mechanisms of mesenchymal stem cells (MSCs have become a research focus in recent years. Accumulating evidence supports the notion that MSCs act in a paracrine manner. Therefore, the biological factors in conditioned medium, including exosomes and soluble factors, derived from MSC cultures are being explored extensively. The results from most investigations show that MSC-conditioned medium or its components mediate some biological functions of MSCs. Several studies have reported that MSC-derived exosomes have functions similar to those of MSCs, such as repairing tissue damage, suppressing inflammatory responses, and modulating the immune system. However, the mechanisms are still not fully understood and the results remain controversial. Compared with cells, exosomes are more stable and reservable, have no risk of aneuploidy, a lower possibility of immune rejection following in vivo allogeneic administration, and may provide an alternative therapy for various diseases. In this review, we summarize the properties and biological functions of MSC-derived exosomes and discuss the related mechanisms.

  14. Chronic hepatitis C and persistent occult hepatitis C virus infection are characterized by distinct immune cell cytokine expression profiles. (United States)

    Pham, T N Q; Mercer, S E; Michalak, T I


    Hepatitis C virus (HCV) replicates in immune cells in both chronic hepatitis C (CHC) and occult HCV infection, but the extent of virus replication in this compartment in these opposing infection forms varies greatly. It was unknown whether this could be linked to HCV genotype or to differences in host gene expression shaping the immune response, and whether HCV replication in immune cells is sensitive to endogenous antiviral cytokines. In this study, we uncovered that significantly greater HCV load in peripheral blood mononuclear cells (PBMC), but not in plasma, coincided with HCV genotypes 2 and 3 in CHC, but with genotype 1 in residual occult infection after clinical resolution of hepatitis C. Moreover, PBMC from individuals with occult infection transcribed significantly greater levels of IFN-alpha, IFN-gamma and TNF-alpha, but less interleukin (IL)-10 than those from CHC. In CHC, PBMC with low HCV load expressed significantly more IFN-gamma but less IL-12 than did cells with high virus content. In occult infection, HCV RNA detection in PBMC was associated with much lower IFN-alpha and IL-12 expression. Further, HCV replication in T lymphocytes could be completely eliminated by activation of endogenous IFN-gamma in CHC, but of IFN-alpha in occult infection. In conclusion, CHC and persistent occult HCV infection are characterized by clearly different profiles of antiviral cytokine response in circulating immune cells which are also different from those of healthy individuals. Higher expression of IL-10, combined with lower transcription of IFN-alpha, IFN-gamma and TNF-alpha, is associated with a more robust HCV replication in immune cells.

  15. Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells


    Liu, Yu-Tsueng; Brazas, Rob; Ganem, Don


    Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian...

  16. Cell-derived microparticles and the lung

    Directory of Open Access Journals (Sweden)

    Dario Nieri


    Full Text Available 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.

  17. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering

    DEFF Research Database (Denmark)

    Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan

    A huge shortage of liver organs for transplantation has motivated the research field of tissue engineering to develop bioartificial liver tissue and even a whole liver. The goal of NanoBio4Trans is to create a vascularized bioartificial liver tissue, initially as a liver-support system. Due...... to limitations of primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic...... differentiation of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed....

  18. Molecular evolution of hepatitis A virus in a human diploid cell line

    Institute of Scientific and Technical Information of China (English)

    Cai-Hua Tang; Jiang-Sen Mao; Shao-Ai Chai; Yong Chen; Fang-Cheng Zhuang


    AIM: To investigate the hotspots, direction, and the time course of evolution of hepatitis A virus in the process of consecutive cell culture passage in human KMB17 diploid cells.METHODS: Wild type hepatitis A virus H2w was serially propagated in KMB17 cells until passage 30, and the full-length genomes of H2w and its six chosen progenies were determined by directly sequencing RT-PCR products amplified from viral genomic RNA. Alignment comparison of sequences from H2w with its six progenies and phylogenetic analysis of the whole VP1 region from H2w, progenies of H2w, and other cell culture adapted hepatitis A virus were then carried out to obtain data on the molecular evolution of hepatitis A virus in the process of consecutive passage in KMB17 cells.RESULTS: Most of the mutations occurred by passage 5 and several hotspots related to adaptation of the virus during cell growth were observed. After that stage, few additional mutations occurred through the remaining duration of passage in KMB17 cells except for mutation in the virulence determinants, which occurred in the vicinity of passage 15. The phylogenetic analysis of the whole VP1 region suggested that the progenies of H2w evolved closely to other cell culture adapted hepatitis A virus, i.e. MBB, L-A-1, other than its progenitor H2w.CONCLUSION: Hepatitis A virus served as a useful model for studying molecular evolution of viruses in a given environment. The information obtained in this study may provide assistance in cultivating the next generation of a seed virus for live hepatitis A vaccine production.

  19. CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells. (United States)

    Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y; Tanaka, Minoru; Miyajima, Atsushi


    To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes.


    Institute of Scientific and Technical Information of China (English)

    吴朝栋; 陶其敏


    Expression vector inserted with E2/NS1 gane derived from ganotype Ⅲ Chinese isolates of HCV was transfected into mammalian cells to express E2 glycoprotein. Expressed protein was used as antigen for anti-E2 antibody detection in 19 cases of hepatitis C patients by Western blot. It was first to express E2 glycoprotein of genotype Ⅲ Chinese hepatitis C virus isolates. For anti-E2 detection, 14 cases of patients were positive of antibodies against E2(73.7%). These results indicated that E2 glycoprotein expressed in mammalian cells had good immunoganicity and cross reactivity to serum infected with genotype Ⅱ Chinese hepatitis C virus isolates.

  1. Inhibition of hepatic tumor cell proliferation in vitro and tumor growth in vivo by taltobulin,a synthetic analogue of the tripeptide hemiasterlin

    Institute of Scientific and Technical Information of China (English)

    Yogesh K Vashist; Celine Tiffon; Christoforos Stoupis; Claudio A Redaelli


    AIM:To investigate the inhibitory effects of taltobulin (HTI-286),a synthetic analogue of natural hemiasterlin derived from marine sponges, on hepatic tumor growth in vitro andin vivo.METHODS: The potential anti-proliferative effects of HTI-286 on different hepatic tumor cell lines in vitro and in vivo were examined.RESULTS:HTI-286 significantly inhibited proliferation of all three hepatic tumor cell lines (mean IC50 = 2 nmol/L± 1 nmol/L)in vitro. Interestingly, no decrease in viable primary human hepatocytes (PHH) was detected under HTI-286 exposure. Moreover, intravenous administration of HTI-286 significantly inhibited tumor growth in vivo (rat allogratt model).CONCLUSION:HTI-286 might be considered a potent promising drug in treatment of liver malignancies.HTI-286 is currently undergoing clinical evaluation in cancer patients.

  2. Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

    DEFF Research Database (Denmark)

    Ndeboko, Bénédicte; Lemamy, Guy Joseph; Nielsen, Peter E


    Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers...... hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its...... against chronic hepatitis B....

  3. Fetal liver hepatic progenitors are supportive stromal cells for hematopoietic stem cells. (United States)

    Chou, Song; Lodish, Harvey F


    Previously we showed that the ~2% of fetal liver cells reactive with an anti-CD3epsilon monoclonal antibody support ex vivo expansion of both fetal liver and bone marrow hematopoietic stem cells (HSCs); these cells express two proteins important for HSC ex vivo expansion, IGF2, and angiopoietin-like 3. Here we show that these cells do not express any CD3 protein and are not T cells; rather, we purified these HSC-supportive stromal cells based on the surface phenotype of SCF(+)DLK(+). Competitive repopulating experiments show that SCF(+)DLK(+) cells support the maintenance of HSCs in ex vivo culture. These are the principal fetal liver cells that express not only angiopoietin-like 3 and IGF2, but also SCF and thrombopoietin, two other growth factors important for HSC expansion. They are also the principal fetal liver cells that express CXCL12, a factor required for HSC homing, and also alpha-fetoprotein (AFP), indicating that they are fetal hepatic stem or progenitor cells. Immunocytochemistry shows that >93% of the SCF(+) cells express DLK and Angptl3, and a portion of SCF(+) cells also expresses CXCL12. Thus SCF(+)DLK(+) cells are a highly homogenous population that express a complete set of factors for HSC expansion and are likely the primary stromal cells that support HSC expansion in the fetal liver.

  4. Long-term efficacy of plasma-derived hepatitis B vaccine among Chinese children: a 12-year follow-up study

    Institute of Scientific and Technical Information of China (English)


    @@ INTRODUCTION To evaluate long-term efficacy of a plasma-derived hepatitis B vaccine and provide evidence for decision-making on the vaccine booster doses, we conducted a prevalent follow-up study to examine serologic changes in hepatitis markers and vaccine efficacy in 350 children from the original cohort of 513 children who participated in a randomized, double-blind and placebo-controlled trial on a plasma-derived hepatitis B vaccine in Longan County, Guangxi Autonomous Region, China, in 1982.

  5. Replication of a hepatitis C virus replicon clone in mouse cells

    Directory of Open Access Journals (Sweden)

    Chisari Francis V


    Full Text Available Abstract Background Hepatitis C Virus (HCV is a significant public health burden and small animal models are needed to study the pathology and immunobiology of the virus. In effort to develop experimental HCV mouse models, we screened a panel of HCV replicons to identify clones capable of replicating in mouse hepatocytes. Results We report the establishment of stable HCV replication in mouse hepatocyte and fibroblast cell lines using replicons derived from the JFH-1 genotype 2a consensus sequence. Viral RNA replication efficiency in mouse cells was comparable to that observed in human Huh-7 replicon cells, with negative-strand HCV RNA and the viral NS5A protein being readily detected by Northern and Western Blot analysis, respectively. Although HCV replication was established in the absence of adaptive mutations that might otherwise compromise the in vitro infectivity of the JFH-1 clone, no infectious virus was detected when the culture medium from full length HCV RNA replicating mouse cells was titrated on Huh-7 cells, suggesting that the mouse cells were unable to support production of infectious progeny viral particles. Consistent with an additional block in viral entry, infectious JFH-1 particles produced in Huh-7 cells were not able to establish detectable HCV RNA replication in naïve mouse cells. Conclusion Thus, this report expands the repertoire of HCV replication systems and possibly represents a step toward developing mouse models of HCV replication, but it also highlights that other species restrictions might continue to make the development of a purely murine HCV infectious model challenging.

  6. Derivation of induced pluripotent stem cells from pig somatic cells. (United States)

    Ezashi, Toshihiko; Telugu, Bhanu Prakash V L; Alexenko, Andrei P; Sachdev, Shrikesh; Sinha, Sunilima; Roberts, R Michael


    For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after approximately 22 days, providing an overall reprogramming efficiency of approximately 0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of approximately 17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.

  7. Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis

    Institute of Scientific and Technical Information of China (English)


    AIM:To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.METHODS: A proteomic strategy combining two-dimensional gel electrophoresis and ultraperform ance liquid chromatographyelectrospray ionizationtandem mass spectrometry (UPLCESIMS/MS) was used to study the differential expression of proteins and Western blotting was used to validate the re...

  8. MRI of magnetically labeled mesenchymal stem cells in hepatic failure model

    Institute of Scientific and Technical Information of China (English)

    Kyu; Ri; Son; Se; Young; Chung; Hyo-Cheol; Kim; Hoe; Suk; Kim; Seung; Hong; Choi; Jeong; Min; Lee; Woo; Kyung; Moon


    AIM:To track intravascularly transplanted mesenchymal stem cells (MSCs) labeled with superparamagnetic iron oxide (SPIO) by using magnetic resonance imaging (MRI) in an experimental rabbit model of hepatic failure.METHODS:Human MSCs labeled with FDA-approved SPIO particles (Feridex) were transplanted via the mes-enteric vein into rabbits (n=16) with carbon tetrachloride-induced hepatic failure.Magnetic resonance (MR) examinations were performed with a 3.0 T clinical scanner immediately before and 2 h and 1,...

  9. Hepatitis B-related events in autologous hematopoietic stem cell transplantation recipients

    Institute of Scientific and Technical Information of China (English)

    zcan; eneli; Zübeyde; Nur; zkurt; Kadir; Acar; Seyyal; Rota; Sahika; Zeynep; Aki; Zeynep; Arzu; Yegin; Münci; Yagci; Seren; zenirler; Gülsan; Türkz; Sucak


    AIM: To investigate the frequency of occult hepatitis B, the clinical course of hepatitis B virus (HBV) reactivation and reverse seroconversion and associated risk factors in autologous hematopoietic stem cell transplantation (HSCT) recipients. METHODS: This study was conducted in 90 patients undergoing autologous HSCT. Occult HBV infection was investigated by HBV-DNA analysis prior to transplantation, while HBV serology and liver function tests were screened prior to and serially after transplantation. HBV...

  10. Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis.

    Directory of Open Access Journals (Sweden)

    Ursula Manuelpillai

    Full Text Available Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl(4 twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2 × 10(6 were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively. Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl(4 treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl(4 administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl(4 demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl(4 treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl(4 alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl(4 treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established

  11. Alginate hydrogel protects encapsulated hepatic HuH-7 cells against hepatitis C virus and other viral infections.

    Directory of Open Access Journals (Sweden)

    Nhu-Mai Tran

    Full Text Available 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.

  12. Sertraline induces endoplasmic reticulum stress in hepatic cells. (United States)

    Chen, Si; Xuan, Jiekun; Couch, Letha; Iyer, Advait; Wu, Yuanfeng; Li, Quan-Zhen; Guo, Lei


    Sertraline is used for the treatment of depression, and is also used for the treatment of panic, obsessive-compulsive, and post-traumatic stress disorders. Previously, we have demonstrated that sertraline caused hepatic cytotoxicity, with mitochondrial dysfunction and apoptosis being underlying mechanisms. In this study, we used microarray and other biochemical and molecular analyses to identify endoplasmic reticulum (ER) stress as a novel molecular mechanism. HepG2 cells were exposed to sertraline and subjected to whole genome gene expression microarray analysis. Pathway analysis revealed that ER stress is among the significantly affected biological changes. We confirmed the increased expression of ER stress makers by real-time PCR and Western blots. The expression of typical ER stress markers such as PERK, IRE1α, and CHOP was significantly increased. To study better ER stress-mediated drug-induced liver toxicity; we established in vitro systems for monitoring ER stress quantitatively and efficiently, using Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP) as ER stress reporters. These in vitro systems were validated using well-known ER stress inducers. In these two reporter assays, sertraline inhibited the secretion of Gluc and SEAP. Moreover, we demonstrated that sertraline-induced apoptosis was coupled to ER stress and that the apoptotic effect was attenuated by 4-phenylbutyrate, a potent ER stress inhibitor. In addition, we showed that the MAP4K4-JNK signaling pathway contributed to the process of sertraline-induced ER stress. In summary, we demonstrated that ER stress is a mechanism of sertraline-induced liver toxicity.

  13. Enriched retinal ganglion cells derived from human embryonic stem cells (United States)

    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.


    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

  14. Derivation of epithelial-like cells from eyelid fat-derived stem cells in thermosensitive hydrogel. (United States)

    Heidari Keshel, Saeed; Rostampour, Maryam; Khosropour, Golbahar; Bandbon B, Atefehsadat; Baradaran-Rafii, Alireza; Biazar, Esmaeil


    Injectable hydrogel is one of the great interests for tissue engineering and cell encapsulation. In the study, the thermosensitive chitosan/gelatin/β-glycerol phosphate (C/G/GP) disodium salt hydrogels were designed and investigated by different analyses. The eye fat-derived stem cells were used to evaluate the biocompatibility of hydrogels based on their phenotypic profile, viability, proliferation, and attachment ability. The results show that the sol/gel transition temperature of the C/G/GP hydrogel was in the range of 31.1-33.8 °C at neutral pH value, the gelation time was shortened, and the gel strength also improved at body temperature when compared with the C/GP hydrogel. In vitro cell culture experiments with eyelid fat-derived stem cells in hydrogel showed beneficial effects on the cell phenotypic morphology, proliferation, and differentiation. Microscopic figures showed that the eyelid fat stem cell were firmly anchored to the substrates and were able to retain a normal stem cell phenotype. Immunocytochemistry (ICC) and real-time-PCR results revealed change in the expression profile of eyelid fat stem cells grown with hydrogels when compared to those grown on control in epithelial induction condition. This study indicates that using chitosan/gelatin/β-glycerol phosphate hydrogel for cell culture is feasible and may apply in minimal invasive surgery in the future.

  15. Sarcoma derived from cultured mesenchymal stem cells. (United States)

    Tolar, Jakub; Nauta, Alma J; Osborn, Mark J; Panoskaltsis Mortari, Angela; McElmurry, Ron T; Bell, Scott; Xia, Lily; Zhou, Ning; Riddle, Megan; Schroeder, Tania M; Westendorf, Jennifer J; McIvor, R Scott; Hogendoorn, Pancras C W; Szuhai, Karoly; Oseth, Leann; Hirsch, Betsy; Yant, Stephen R; Kay, Mark A; Peister, Alexandra; Prockop, Darwin J; Fibbe, Willem E; Blazar, Bruce R


    To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon-related genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

  16. Hepatic perivascular epithelioid cell tumor (PEComa): a case report with a review of literatures (United States)

    Son, Hyun-Jin; Kang, Dong Wook; Kim, Joo Heon; Han, Hyun Young; Lee, Min Koo


    Hepatic perivascular epithelioid cell tumors (PEComas) are very rare. We report a primary hepatic PEComa with a review of the literature. A 56-year-old women presented with a nodular mass detected during the management of chronic renal failure and chronic hepatitis C. Diagnostic imaging studies suggested a nodular hepatocellular carcinoma in segment 5 of the liver. The patient underwent partial hepatectomy. A brown-colored expansile mass measuring 3.2×3.0 cm was relatively demarcated from the surrounding liver parenchyma. The tumor was mainly composed of epithelioid cells that were arranged in a trabecular growth pattern. Adipose tissue and thick-walled blood vessels were minimally identified. A small amount of extramedullary hematopoiesis was observed in the sinusoidal spaces between tumor cells. Tumor cells were diffusely immunoreactive for human melanoma black 45 (HMB45) and Melan A, focally immunoreactive for smooth muscle actin, but not for hepatocyte specific antigen (HSA). PMID:28288506

  17. Back to the drawing board: Understanding the complexity of hepatic innate lymphoid cells. (United States)

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


    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.

  18. Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and trans-differentiation

    Institute of Scientific and Technical Information of China (English)

    Hong Shen; Guo-Jiang Huang; Yue-Wen Gong


    AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

  19. Paclitaxel ameliorates fibrosis in hepatic stellate cells via inhibition of TGF-β/Smad activity

    Institute of Scientific and Technical Information of China (English)


    AIM: To investigated if paclitaxel can attenuate hepatic fi brosis in rat hepatic stellate cells (RHSCs). METHODS: RHSCs were cultured in vitro and randomly assigned to four groups: normal control group (treated only with Dulbecco's Modified Eagle's Medium), Taxol group (200 nmol/L paclitaxel was added to the cell culture), transforming growth factor (TGF)-β group (5 ng/mL recombinant human TGF-β1 was added to the cell culture), and TGF-β + Taxol group. TGF-β signaling cascade and status of various extracel...

  20. Sofosbuvir and Simeprevir Treatment of a Stem Cell Transplanted Teenager With Chronic Hepatitis C Infection. (United States)

    Fischler, Björn; Priftakis, Peter; Sundin, Mikael


    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.

  1. Interaction of targeted liposomes with primary cultured hepatic stellate cells : Involvement of multiple receptor systems

    NARCIS (Netherlands)

    Adrian, Joanna Ewa; Poelstra, Klaas; Scherphof, Gerrit; Molema, Ingrid; Meijer, D.K F; Reker-Smit, Catharina; Morselt, Henriette; Kamps, Jan


    Background/Aims: In designing a versatile liposomal drug carrier to hepatic stellate cells (HSC), the interaction of mannose 6-phosphate human serum albumin (M6P-HSA) liposomes with cultured cells was studied. Methods: M6P-HSA was covalently coupled to the liposomal surface and the uptake and bindin

  2. Melatonin suppresses activation of hepatic stellate cells through ROR alpha-mediated inhibition of 5-lipoxygenase

    NARCIS (Netherlands)

    Shajari, Shiva; Laliena, Almudena; Heegsma, Janette; Jesus Tunon, Maria; Moshage, Han; Faber, Klaas Nico


    Liver fibrosis is scar tissue resulting from an uncontrolled wound-healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to prolifer

  3. Clonogenic colony-forming ability of hepatic stem cells in the spleens of mice

    Institute of Scientific and Technical Information of China (English)


    To confirm the existence of hepatic stem cells (HSCs), fetal liver cells isolated from mice on embryonic day 13 (ED13) were long-term cultured in vitro. Growth of the cells was observed intensively and characteristics were identified by immunocytochemistry. The results showed that some of the cells grew as colonies, in which some cells expressed AFP, CD34 and Albumin. Then the cells were transplanted intravenously into irradiated syngeneic mice. At day 12 a number of small hyperplasia nodules were seen in the apparently enlarged spleens of recipient mice. Moreover, some nodules were positive for AFP and CD34 and consisted of various types of cells, suggesting the very existence of hepatic stem cells in the mouse fetal liver.

  4. Overexpression of Hepatitis B Virus-binding Protein, Squamous Cell Carcinoma Antigen 1, Extends Retention of Hepatitis B Virus in Mouse Liver

    Institute of Scientific and Technical Information of China (English)

    Hong-Bin XIA; Xi-Gu CHEN


    How receptors mediate the entry of hepatitis B virus (HBV) into the target liver cells is poorly understood. Recently, human squamous cell carcinoma antigen 1 (SCCA1) has been found to mediate binding and internalization of HBV to liver-derived cell lines in vitro. In this report, we investigate if SCCA1 is able to function as an HBV receptor and mediate HBV entry into mouse liver. SCCA1 transgene under the control of Rous sarcoma virus promoter was constructed in a minicircle DNA vector that was delivered to NOD/SCID mouse liver using the hydrodynamic technique. Subsequently, HBV-positive human serum was injected intravenously. We demonstrated that approximately 30% of the mouse liver cells expressed a high level of recombined SCCA1 protein for at least 37 d. The HBV surface antigen was found to persist in mouse liver for up to 17 d. Furthermore, HBV genome also persisted in mouse liver, as determined by polymerase chain reaction, for up to 17 d, and in mouse circulation for 7 d. These results suggest that SCAA1 might serve as an HBV receptor or co-receptor and play an important role in mediating HBV entry into hepatocytes, although its role in human HBV infection remains to be determined.

  5. The role of dystroglycan in PDGF-BB-dependent migration of activated hepatic stellate cells/myofibroblasts (United States)

    Kastanis, George John; Hernandez-Nazara, Zamira; Nieto, Natalia; Rincón-Sanchez, Ana Rosa; Popratiloff, Anastas; Dominguez-Rosales, Jose Alfredo; Lechuga, Carmen G.


    Hepatic stellate cells are embedded in the loose connective tissue matrix within the space of Disse. This extracellular matrix contains several basement membrane components including laminin, but its composition changes during liver injury because of the production of extracellular matrix components found in scar tissue. These changes in extracellular matrix composition and in cell-extracellular matrix interactions may play a key role in hepatic stellate cell transdifferentiation. In this communication we used early passages of mouse hepatic stellate cells (activated HSC/myofibroblasts) to study the platelet-derived growth factor BB (PDGF-BB)-dependent expression and regulation of β-dystroglycan and its role in activated HSC/myofibroblast migration. We used Northern and Western analysis to study dystroglycan expression and confocal microscopy to investigate changes in subcellular distribution of the protein. Activated HSC migration was investigated using an in vitro wound-healing assay. PDGF-BB induced significant changes in dystroglycan regulation and subcellular distribution of the protein. Whereas steady-state levels of dystroglycan mRNA remained constant, PDGF-BB increased dystroglycan transcription but shortened the t1/2 by 50%. Moreover, PDGF-BB changed dystroglycan and α5-integrin cellular distribution. Cell migration experiments revealed that PDGF-BB-dependent migration of activated HSC/myofibroblasts was completely blocked by neutralizing antibodies to fibronectin, α5-integrin, laminin, and β-dystroglycan. Overall, these findings suggest that both laminin and fibronectin and their receptors play a key role in PDGF-BB-induced activated HSC migration. PMID:21659621

  6. The hepatic progenitor cell niche in man and dog

    NARCIS (Netherlands)

    Schotanus, B.A.


    Chronic progressive liver diseases occur frequently in humans and animals, and lead to severe dysfunction and cirrhosis. The only available treatment is liver transplantation. Due to donor liver shortage, alternatives for liver transplantation are needed. Several forms of hepatitis occurring in dogs

  7. Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle (United States)

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


    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.

  8. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Yeonhee Kim

    Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

  9. Brivanib attenuates hepatic fibrosis in vivo and stellate cell activation in vitro by inhibition of FGF, VEGF and PDGF signaling.

    Directory of Open Access Journals (Sweden)

    Ikuo Nakamura

    Full Text Available Brivanib is a selective inhibitor of vascular endothelial growth factor receptor (VEGFR and fibroblast growth factor receptor (FGFR tyrosine kinases, which are both involved in mechanisms of liver fibrosis. We hypothesized that inhibition of VEGFR and FGFR by brivanib would inhibit liver fibrosis. We therefore examined the effect of brivanib on liver fibrosis in three mouse models of fibrosis.In vivo, we induced liver fibrosis by bile duct ligation (BDL, chronic carbon tetrachloride (CCl4, and chronic thioacetamide (TAA administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs to assess the effect of brivanib on stellate cell proliferation and activation.After in vivo induction with BDL, CCl4, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF, VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.Brivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.

  10. Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation

    NARCIS (Netherlands)

    Testerink, N.; Ajat, M.A.; Houweling, M.; Brouwers, J.F.; Pully, V.V.; Manen, van H.J.; Otto, C.; Helms, J.B.; Vaandrager, A.B.


    Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed t

  11. Comparative evaluation of differentiation potential of menstrual blood- versus bone marrow-derived stem cells into hepatocyte-like cells.

    Directory of Open Access Journals (Sweden)

    Sayeh Khanjani

    Full Text Available Menstrual blood has been introduced as an easily accessible and refreshing stem cell source with no ethical consideration. Although recent works have shown that menstrual blood stem cells (MenSCs possess multi lineage differentiation capacity, their efficiency of hepatic differentiation in comparison to other stem cell resources has not been addressed so far. The aim of this study was to investigate hepatic differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs under protocols developed by different concentrations of hepatocyte growth factor (HGF and oncostatin M (OSM in combination with other components in serum supplemented or serum-free culture media. Such comparison was made after assessment of immunophenotye, trans-differentiation potential, immunogenicity and tumorigeicity of these cell types. The differential expression of mature hepatocyte markers such as albumin (ALB, cytokeratin 18 (CK-18, tyrosine aminotransferase and cholesterol 7 alpha-hydroxylase activities (CYP7A1 at both mRNA and protein levels in differentiating MenSCs was significantly higher in upper concentration of HGF and OSM (P1 compared to lower concentration of these factors (P2. Moreover, omission of serum during differentiation process (P3 caused typical improvement in functions assigned to hepatocytes in differentiated MenSCs. While up-regulation level of ALB and CYP7A1 was higher in differentiated MenSCs compared to driven BMSCs, expression level of CK-18, detected level of produced ALB and glycogen accumulation were lower or not significantly different. Therefore, based on the overall comparable hepatic differentiation ability of MenSCs with BMSCs, and also accessibility, refreshing nature and lack of ethical issues of MenSCs, these cells could be suggested as an apt and safe alternative to BMSCs for future stem cell therapy of chronic liver diseases.

  12. Loss of Discoidin Domain Receptor 2 Promotes Hepatic Fibrosis after Chronic Carbon Tetrachloride through Altered Paracrine Interactions between Hepatic Stellate Cells and Liver-Associated Macrophages


    Olaso, Elvira; ARTETA, BEATRIZ; BENEDICTO, AITOR; Crende, Olatz; Friedman, Scott L.


    Hepatic stellate cells (HSCs) interact with fibrillar collagen through the discoidin domain receptor 2 (DDR2) in acute hepatic injury, generating increased fibrosis. However, the contribution of DDR2 signaling to chronic liver fibrosis in vivo is unclear, despite its relevance to chronic human liver disease. We administered carbon tetrachloride (CCl4) to DDR2+/+ and DDR2−/− mice twice weekly, and liver tissues and isolated HSCs were analyzed. In contrast to changes seen in acute injury, after...

  13. Exposure to human immunodeficiency virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types. (United States)

    Salloum, Shadi; Holmes, Jacinta A; Jindal, Rohit; Bale, Shyam S; Brisac, Cynthia; Alatrakchi, Nadia; Lidofsky, Anna; Kruger, Annie J; Fusco, Dahlene N; Luther, Jay; Schaefer, Esperance A; Lin, Wenyu; Yarmush, Martin L; Chung, Raymond T


    Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection accelerates progressive liver fibrosis; however, the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers transforming growth factor beta-1 (TGFβ1) (mediated by reactive oxygen species [ROS]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in hepatocytes and hepatic stellate cells in monoculture; however, they do not account for cellular crosstalk that naturally occurs. We created an in vitro coculture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. Green fluorescent protein reporter cell lines driven by functional ROS (antioxidant response elements), NFκB, and mothers against decapentaplegic homolog 3 (SMAD3) promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cell lines were exposed to HIV, HCV, or HIV/HCV. Activation of the 3 pathways was measured and compared according to infection status. Extracellular matrix products (collagen type 1 alpha 1 (CoL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1)) were also measured. Both HCV and HIV independently activated TGFβ1 signaling through ROS (antioxidant response elements), NFκB, and SMAD3 in both cell lines in coculture. Activation of these profibrotic pathways was additive following HIV/HCV coexposure. This was confirmed when examining CoL1A1 and TIMP1, where messenger RNA and protein levels were significantly higher in LX2 cells in coculture following HIV/HCV coexposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells.

  14. Up-regulation of interleukin-22 mediates liver fibrosis via activating hepatic stellate cells in patients with hepatitis C. (United States)

    Wu, Li-Yuan; Liu, Shuhong; Liu, Yuan; Guo, Chaonan; Li, Hanwei; Li, Wenshu; Jin, Xueyuan; Zhang, Keming; Zhao, Ping; Wei, Lai; Zhao, Jingmin


    Interleukin-22 (IL-22) is known to play a critical role in liver immunity. However, the role of IL-22 in HCV-associated liver fibrosis is poorly understood. In this study, patients with HCV infection disclosed significant increases in peripheral numbers of IL-22-producing cells as well as in IL-22 plasma levels. In the liver, the increased intrahepatic IL-22(+) cells were positively correlated with fibrotic staging scores and clinical progression from CHC to cirrhosis. Moreover, the majority of IL-22(+) cells were located in fibrotic areas in the liver of patients with cirrhosis and co-localized with α-smooth muscle actin (α-SMA) positive hepatic stellate cells (HSCs). In vitro, administration of IL-22 was accompanied with inhibited LX-2 cell apoptosis, promoted LX-2 cell proliferation, increased expression of α-SMA, and up-regulated collagen production by LX-2 cells. Collectively, our data provide evidence that IL-22 may contribute to the fibrogenesis of HCV-associated liver fibrosis by activating HSCs.

  15. Comparison of biological characteristics of marrow mesenchymal stem cells in hepatitis B patients and normal adults

    Institute of Scientific and Technical Information of China (English)

    Liang Peng; Hua Li; Lin Gu; Xiao-Mou Peng; Yang-Su Huang; Zhi-Liang Gao


    AIM: To establish a culture system of marrow mesenchymal stem cells (MSCs) from hepatitis B patients and normal adults and to compare their biological characteristics.METHODS: MSCs were isolated from bone marrow in 34 male hepatitis B patients and 15 male normal adults and cultivated in vitro. Their biological characteristics including surface markers, shapes and appearances, growth curves, first passage time and passage generations were compared.RESULTS: Cultivation achievement ratio of hepatitis B patients was lower than that of normal adults, no statistical significance (82.35% vs 100%, P >0.05). Compared with MSCs of normal adults, MSCs of hepatitis B patients presented a statistical lower growth curve, longer first passage time (13.0 ± 1.6 d vs 11.4 ± 1.5 d, P < 0.05), fewer passaging generation numbers (10.5 ± 1.4 generations vs 12.3±1.7 generations, P < 0.05), though both shared same appearances, shapes and surface markers. MSCs in hepatitis B patients would expand, spread out and age more easily and there were more refractive particles in the cytoplasm.CONCLUSION: MSCs from hepatitis B patients can be cultured in vitro. Although their appearance, shape and surface marker are similar to those of MSCs from normal adults, there are differences in their biological characteristics.

  16. Influence of the invasion of peripheral blood mononuclear cells by hepatitis B virus on immune response of the patients with chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    XING Tong-jing; ZHANG Lian; HOU Jin-lin; ZHANG Ming-xia; YANG Jie; LUO Kang-xian


    To explore the influence of HBV invasion into peripheral blood mononuclear cells (PBMC) on the immune response of patients with chronic hepatitis B. Methods: The cytokine levels in the culture supernatant of PBMC from 56 patients with chronic hepatitis B were determined by ELISA, and PCR was employed to amplify the HBV DNA. Results: The levels of IFN-γ in patients with hepatitis B was lower than thoset of the control, but the difference was not statistically significant, while the levels of IL-4 were significantly higher than those of the control (P<0.01). The serum levels of HBV DNA were negatively correlated with that of IFN-y in culture supernatants of PBMC. Thirty-five patients positive of HBV DNA in the PBMCs were identified from 56 patients with hepatitis B,and their IFN-γ level proved to be significantly different. Conclusions: Th2 cell-mediated immune response is predominant in chronic hepatitis B which is associated with the chronicity of HBV infection. HBV invasion into the PBMCs may affect Th1 and Th2 cell-mediated immune response of the patients with chronic hepatitis B.

  17. Hepatitis B e antigen polypeptides isolated from sera of individuals infected with hepatitis B virus: comparison with HBeAg polypeptide derived from Dane particles. (United States)

    Takahashi, K; Imai, M; Gotanda, T; Sano, T; Oinuma, A; Mishiro, S; Miyakawa, Y; Mayumi, M


    Hepatitis B e antigen (HBeAg) occurs in the serum of individuals infected with hepatitis B virus both free and in association with IgG. Utilizing a succession of steps involving salt precipitation, affinity chromatography, ion-exchange chromatography and isoelectrofocusing, we isolated free and IgG-bound forms of HBeAg from the sera of infected individuals with an overall gain in specific activity of 3000-fold and 540-fold, respectively. Polypeptide profiles of purified HBeAg preparations were studied by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Both free and IgG-bound preparations revealed polypeptides with mol. wt. of 15500 (P15.5) and 16 500 (P16.5), and HBeAg activity was detected corresponding to their positions. The HBeAg polypeptides (P15.5/16.5) derived from sera were physicochemically different from the two polypeptides with HBeAg activity (P19 and P45) liberated from Dane particle cores by the conventional method involving incubation with Nonidet P40 and 2-mercaptoethanol. However, when core particles were prepared in the presence of a proteolytic enzyme, in addition to Nonidet P40 and 2-mercaptoethanol, they gave rise to HBeAg polypeptides with mol. wt. of 31000 (P31) and 15 500. Furthermore, P31 split into P15.5 when heated at 100 degrees C for 2 min. On the basis of these results, P15.5 may be assumed to be the essential polypeptide bearing HBeAg activity in the serum and also in Dane particles.

  18. Endogenous n-3 polyunsaturated fatty acids attenuate T cell-mediated hepatitis via autophagy activation

    Directory of Open Access Journals (Sweden)

    Yanli Li


    Full Text Available Omega-3 polyunsaturated fatty acids (n-3 PUFAs exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A was administered intravenously to wild-type (WT and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase (ALT activity, and inhibited production of pro-inflammatory cytokines (e.g. TNF-α, IL-6, IL-17A and IFN-γ. In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism, and could be exploited as a new therapeutic approach for autoimmune hepatitis.

  19. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation (United States)

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming


    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis. PMID:27679638

  20. The in Vitro Assessment of Biochemical Factors in Hepatocyte like Cells Derived from Umbilical Cord Blood Stem Cells

    Directory of Open Access Journals (Sweden)

    A KHoramroodi


    Full Text Available Introduction & Objective: Umbilical cord blood (UCB is a source of Hematopoietic Stem Cells (HSC and progenitor cells that can reconstitute the hematopoietic system in patients with malignant and nonmalignant disorders. Mesenchymal stem cell-derived from umbilical cord blood (UCB have been differentiated to some kind of cells, such as osteobblast, adipoblast and chondroblast in Vitro. This study examined the differentiation of Umbilical Cord Blood (UCB derived stem cells to functional hepatocytes. Materials & Methods: The present study was an experimental study which was carried out in the Payam-e-Noor University of Tehran in cooperation with Hamedan University of Medical Sciences in 2008. Umbilical cord blood (UCB was obtained from Fatemieh hospital (Hamadan, Iran. Stem cells were isolated from the cord blood by combining density gradient centrifugation with plastic adherence. When the isolated cells reached 80% confluence, they differentiated to hepatocyte like cells. The medium which was used was consists of DMEM and 10% Fetal Bovine Serum (FBS supplemented with 20 ng/mL Hepatocyte Growth Factor (HGF, 10 ng/mL basic Fibroblast Growth Factor (bFGF and 20 ng/mL Oncostatin M (OSM.The medium was changed every 3 days and stored for Albumin (ALB, Alpha Fetoprotein (AFP, Alkaline Phosphatase (ALP, and urea assay. Finally PAS stain was done to study Glycogen storage in the differentiated cell. Results: Measurement of biochemical factors in different days showed that concentration of albumin (ALB, alpha fetoprotein (AFP, alkaline phosphatase (ALP, and Urea gradually increased. Also, PAS staining showed the storage of glycogen in these cells. Conclusion: Stem cell-derived from human umbilical cord blood (HUCB is a new source of cell types for cell transplantation therapy of hepatic diseases and under certain conditions these cells can differentiate into liver cells.

  1. Alterations of mast cells and TGF-β1 on the silymarin treatment for CCl4-induced hepatic fibrosis

    Institute of Scientific and Technical Information of China (English)

    Da-Hee Jeong; Gi-Ppeum Lee; Won-Il Jeong; Sun-Hee Do; Hai-Jie Yang; Dong-Wei Yuan; Ho-Yong Park; Kyu-Jong Kim; Kyu-Shik Jeong


    AIM: Silymarin is a potent antioxidant, antiinflammatory and anti-fibrogenic agent in the liver, which is mediated by alteration of hepatic Kupffer cell function, lipid peroxidation, and collagen production. Especially, in hepatic fibrogenesis, mast cells are expressed in chronic inflammatory conditions, and promote fibroblast growth and stimulate production of the extracellular matrix by hepatic stellate cells.METHODS: We examined the inhibitory mechanism of silymarin on CCl4-induced hepatic cirrhosis in rats. At 4, 8,and 12 wk, liver tissues were examined histopathologically for fibrotic changes produced by silymarin treatment.RESULTS: In the silymarin with CCl4-treated group,increase of hepatic stellate cells and TGF-β1 production were lower than in the CCl4-treated group at early stages.Additionally, at the late fibrogenic stage, expressions of TGF-β1 were weaker and especially not expressed in hepatocytes located in peripheral areas. Moreover, the number of mast cell in portal areas gradually increased and was dependent on the fibrogenic stage, but those of CCl4+silymarin-treated group decreased significantly.CONCLUSION: Anti-fibrotic and antiinflammatory effects of silymarin were associated with activation of hepatic stellate cells through the expression of TGF-β1 and stabilization of mast cells. These results suggest that silymarin prevent hepatic fibrosis through suppression of inflammation and hypoxia in the hepatic fibrogenesis.

  2. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    R Rajkannan; E J Padma Malar


    The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.

  3. Reverse seroconversion of hepatitis B virus after hematopoietic stem cell transplantation. (United States)

    Goyama, S; Kanda, Y; Nannya, Y; Kawazu, M; Takeshita, M; Niino, M; Komeno, Y; Nakamoto, T; Kurokawa, M; Tsujino, S; Ogawa, S; Aoki, K; Chiba, S; Motokura, T; Shiratori, Y; Hirai, H


    Hepatitis B virus (HBV) reactivation in patients previously positive for hepatitis B surface antibody (HBsAb), so-called reverse seroconversion, has been considered to be a rare complication after hematopoietic stem cell transplantation (HSCT). We experienced two patients who developed reverse seroconversion among nine who were HBsAb positive and Hepatitis B core antibody (HBcAb) positive before HSCT; one after autologous bone marrow transplantation (BMT) and another after allogeneic peripheral blood stem cell transplantation (PBSCT). We reviewed the literature and considered that reverse seroconversion of HBV after HSCT is not uncommon among HBsAb positive recipients. The use of corticosteroids, the lack of HBsAb in donor, and a decrease in serum HBsAb and HBcAb levels may predict reverse seroconversion after HSCT.

  4. Retinoic acid alleviates Con A-induced hepatitis and differentially regulates effector production in NKT cells. (United States)

    Lee, Kyoo-A; Song, You Chan; Kim, Ga-Young; Choi, Gyeyoung; Lee, Yoon-Sook; Lee, Jung-Mi; Kang, Chang-Yuil


    Retinoic acid (RA) is a diverse regulator of immune responses. Although RA promotes natural killer T (NKT) cell activation in vitro by increasing CD1d expression on antigen-presenting cells (APCs), the direct effects of RA on NKT-cell responses in vivo are not known. In the present study, we demonstrated the effect of RA on the severity of Con A-induced hepatitis and molecular changes of NKT cells. First, we demonstrated that Con A-induced liver damage was ameliorated by RA. In correlation with cytokine levels in serum, RA regulated the production of IFN-γ and IL-4 but not TNF-α by NKT cells without influencing the NKT-cell activation status. However, RA did not alleviate α-GalCer-induced liver injury, even though it reduced IFN-γ and IL-4 but not TNF-α levels in serum. This regulation was also detected when liver mononuclear cells (MNCs) or NKT hybridoma cells were treated with RA in vitro. The regulatory effect of RA on NKT cells was mediated by RAR-α, and RA reduced the phosphorylation of MAPK. These results suggest that RA differentially modulates the production of effector cytokines by NKT cells in hepatitis, and the suppressive effect of RA on hepatitis varies with the pathogenic mechanism of liver injury.

  5. Ultrastructural changes in hepatic sinusoidal endothelial cells acutely exposed to colloidal iron. (United States)

    Bassett, Mark L; Dahlstrom, Jane E; Taylor, Matthew C; Koina, Mark E; Maxwell, Lesley; Francis, Douglas; Jain, Sanjiv; McLean, Allan J


    Hepatic sinusoidal endothelial cells form an important interface between the vascular system, represented by the sinusoids, and the space of Disse that surrounds the hepatocyte microvilli. This study aimed to assess the light microscopic and ultrastructural effects of acute exposure of hepatic sinusoidal endothelial cells to colloidal iron by injection of rats with iron polymaltose. Eight minutes after a single intravenous injection of iron polymaltose sinusoidal endothelial cells showed defenestration, and thickening and layering as assessed by transmission electron microscopy. Kupffer cells and stellate cells appeared activated. These changes were not observed in control animals, experiments using equivalent doses of maltose, or experiments using colloidal carbon except for Kupffer cell activation due to colloidal carbon. No significant light microscopic changes were seen in study or control animals. The findings indicate that acute exposure to colloidal iron causes changes in hepatic sinusoidal endothelial cells, stellate cells and Kupffer cells. This may be the result of a direct toxic effect of iron or increased production of reactive oxygen species. These observations suggest a possible mechanism for defenestration of sinusoidal endothelial cells in ageing and in disease states.

  6. Natural killer cells in highly exposed hepatitis C-seronegative injecting drug users. (United States)

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


    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.

  7. Immune-driven adaptation of hepatitis B virus genotype D involves preferential alteration in B-cell epitopes and replicative attenuation--an insight from human immunodeficiency virus/hepatitis B virus coinfection. (United States)

    Mondal, R K; Khatun, M; Ghosh, S; Banerjee, P; Datta, S; Sarkar, S; Saha, B; Santra, A; Banerjee, S; Chowdhury, A; Datta, S


    An important driving force behind the sequence diversity of hepatitis B virus (HBV) is viral adaptation to host immune responses. To gain an insight into the impact of host immunity on genetic diversification and properties of HBV, we characterized HBV of genotype D from treatment-naive hepatitis B e antigen-positive (EP) and hepatitis B e antigen-negative (EN) patients with chronic hepatitis B (CHB), where HBV is under stronger immune pressure, with that of HBV derived from human immunodeficiency virus (HIV)/HBV-coinfected individuals, where HIV infection has significantly weakened the immune system. Full-length sequence analysis showed that HBV heterogeneity was most extensive in EN-CHB followed by EP-CHB and HIV/HBV coinfection. The relative magnitude of non-synonymous changes within B-cell epitopes was greater than that in T-cell epitopes of HBV open reading frames (ORFs) in both EP-CHB and EN-CHB. Nine amino acid substitutions were identified in B-cell epitopes and one in a T-cell epitope of HBV in EN-CHB, most of which resulted in altered hydrophobicities, as determined using the Kyte and Doolittle method, relative to wild-type residues found in HBV from the HIV-positive group. Additionally, 19 substitutions occurred at significantly higher frequencies in non-epitope regions of HBV ORF-P in EN-CHB than HIV/HBV-coinfected patients. In vitro replication assay demonstrated that the substitutions, particularly in reverse transcriptase and RNaseH domains of ORF-P, resulted in a decline in replication capacity of HBV. Hence, our results indicate that HBV adapts to increasing immune pressure through preferential mutations in B-cell epitopes and by replicative attenuation. The viral epitopes linked to immune response identified in this study bear important implications for future HBV vaccine studies.

  8. [Effects of BaP exposure on ultrastructures of hepatic cells of Boleophthalmus pectinirostris]. (United States)

    Feng, Tao; Zheng, Weiyun; Ouyang, Gaoliang; Hong, Wanshu


    The changes of ultrastructures of hepatic cells of Boleophthalmus pectinirostris were investigated after the fish were exposed under benzo(a) pyrene in different concentrations under experimental condition. The results showed that the organelles in hepatic cells of B. pectinirostris were damaged to different extents after the fish was exposed under lower concentration of BaP (0.5 mg.L-1) for up to 7 d, in which, mitochondria and endoplasmic reticulum were the chief organelles affected by BaP exposure. While the fish was exposed under higher concentration of BaP (5 mg.L-1) for 2 h, almost all of the organelles including mitochondria and endoplasmic reticulum in hepatic cells of B. pectinirostr were affected by BaP exposure. The structures of liver cells were seriously damaged. It was demonstrated that BaP could produce multiorganalle lesions in hepatic cells of B. pectinirostris, and the severity extent of such lesions was dependent on the concentration level of BaP.

  9. Flow cytometric quantification of T cell proliferation and division kinetics in woodchuck model of hepatitis B. (United States)

    Gujar, Shashi A; Michalak, Tomasz I


    Woodchucks infected with woodchuck hepatitis virus (WHV) represent the closest natural animal model to study the immunopathogenesis of liver injury caused by essentially noncytopathic, highly human specific hepatitis B virus (HBV). The importance of antiviral T cell response in induction of hepatitis and in control of HBV replication has been demonstrated. However, the understanding of how these responses contribute to the development of different immunomorphological forms of liver disease and their outcomes remain elusive. In this study, we established and standardized a flow cytometry assay using peripheral blood mononuclear cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to assess WHV-specific and mitogen-driven T lymphocyte proliferative responses in woodchucks. The assay is of significantly greater sensitivity than the adenine incorporation assay currently used when applied to measure either WHV-specific T cell responses in acute (P measuring cell division rates. The study shows that woodchuck PBMC labeled with CFSE exhibit light scatter and fluorescence profiles compatible to those of human PBMC, allowing quantitation and deconvolution of the flow cytometric data by applying the existing analytical softwares. The availability of this novel assay should facilitate a more precise and comprehensive evaluation of hepadnavirus-specific and generalized T cell responses in experimental WHV hepatitis.

  10. Parameters influencing derivation of embryonic stem cells from murine embryos. (United States)

    Batlle-Morera, Laura; Smith, Austin; Nichols, Jennifer


    The derivation of ES cells is poorly understood and varies in efficiency between different strains of mice. We have investigated potential differences between embryos of permissive and recalcitrant strains during diapause and ES cell derivation. We found that in diapause embryos of the recalcitrant C57BL/6 and CBA strains, the epiblast failed to expand during the primary explant phase of ES cell derivation, whereas in the permissive 129 strain, it expanded dramatically. Epiblasts from the recalcitrant strains could be expanded by reducing Erk activation. Isolation of 129 epiblasts facilitated very efficient derivation of ES cell lines in serum- and feeder-free conditions, but reduction of Erk activity was required for derivation of ES cells from isolated C57BL/6 or CBA epiblasts. The results suggest that the discrepancy in ES cell derivation efficiency is not attributable merely to variable prodifferentiative effects of the extra-embryonic lineages but also to an intrinsic variability within the epiblast to maintain pluripotency.

  11. Human Stem Cell Derived Cardiomyocytes: An Alternative ... (United States)

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

  12. Stephanthraniline A suppressed CD4(+) T cell-mediated immunological hepatitis through impairing PKCθ function. (United States)

    Chen, Feng-Yang; Zhou, Li-Fei; Li, Xiao-Yu; Zhao, Jia-Wen; Xu, Shi-Fang; Huang, Wen-Hai; Gao, Li-Juan; Hao, Shu-Juan; Ye, Yi-Ping; Sun, Hong-Xiang


    Stephanthraniline A (STA), a C21 steroid isolated from Stephanotis mucronata (Blanco) Merr., was previously shown to inhibit T cells activation and proliferation in vitro and in vivo. The purpose of this study was to further evaluate the in vivo immunosuppressive activity of STA and to elucidate its potential mechanisms. The results showed that pretreatment with STA significantly attenuated concanavalin A (Con A)-induced hepatitis and reduced CD4(+) T cells activation and aggregation in hepatic tissue in mice. STA directly suppressed the activation and proliferation of Con A-induced CD4(+) T cells, and inhibited NFAT, NFκB and MAPK signaling cascades in activated CD4(+) T cells in vitro. Moreover, it was proved that STA inhibited T cells activation and proliferation through proximal T cell-receptor (TCR) signaling- and Ca(2+) signaling-independent way. The molecular docking studies predicted that STA could tight bind to PKCθ via five hydrogen. The further findings indicated STA directly inhibited PKCθ kinase activity, and its phosphorylation in activated CD4(+) T cells in vitro. Collectively, the present study indicated that STA could protect against CD4(+) T cell-mediated immunological hepatitis in mice through PKCθ and its downstream NFAT, NFκB and MAPK signaling cascades. These results highlight the potential of STA as an effective leading compound for use in the treatment of CD4(+) T cell-mediated inflammatory and autoimmune diseases.

  13. [Changes in neuropeptide Y and substance P immunoreactive nerve fibres and immunocompetent cells in hepatitis]. (United States)

    Fehér, Erzsébet


    Neuropeptide Y and substance P were thought to play a role in the function of immune cells and in amplification or elimination of the inflammatory processes. In hepatitis the number of both neuropeptide Y and substance P immunoreactive nerve fibres are increased, where the increase of neoropeptide Y is significant. A large number of lymphocytes and mast cells are also stained for neuropeptide Y and substance P. Very close associations (less than 1 µm) were observed between neuropeptide Y immunoreactive nerve fibres and immune cells stained also with neuropeptide Y. Some immune cells were also found to be immunoreactive for tumor necrosis factor-α and NF-κB. Some of the SP IR immunocells were also stained for TNF-α and nuclear factor kappaB. Based on these data it is hypothesized that neuropeptid Y and substance P released from nerve fibres and immune cells play a role in inflammation and elimination of inflammation in hepatitis.

  14. γδ T cells are indispensable for interleukin-23-mediated protection against Concanavalin A-induced hepatitis in hepatitis B virus transgenic mice. (United States)

    Meng, Ziyu; Wang, Jingya; Yuan, Yifang; Cao, Guangchao; Fan, Shuobing; Gao, Chao; Wang, Li; Li, Zheng; Wu, Xiaoli; Wu, Zhenzhou; Zhao, Liqing; Yin, Zhinan


    Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation-induced hepatitis is unknown. In this study, using HBV transgenic (HBs-Tg) and HBs-Tg T-cell receptor-δ-deficient (TCR-δ(-/-) ) mice, we found that mice genetically deficient in γδ T cells exhibited more severe liver damage upon Concanavalin A (Con A) treatment, as indicated by substantially higher serum alanine aminotransferase levels, further elevated interferon-γ (IFN-γ) levels and more extensive necrosis. γδ T-cell deficiency resulted in elevated IFN-γ in CD4(+) T cells but not in natural killer or natural killer T cells. The depletion of CD4(+) T cells and neutralization of IFN-γ reduced liver damage in HBs-Tg and HBs-Tg-TCR-δ(-/-) mice to a similar extent. Further investigation revealed that HBs-Tg mice showed an enhanced interleukin-17 (IL-17) signature. The administration of exogenous IL-23 enhanced IL-17A production from Vγ4 γδ T cells and ameliorated liver damage in HBs-Tg mice, but not in HBs-Tg-TCR-δ(-/-) mice. In summary, our results demonstrated that γδ T cells played a protective role in restraining Con A-induced hepatitis by inhibiting IFN-γ production from CD4(+) T cells and are indispensable for IL-23-mediated protection against Con A-induced hepatitis in HBs-Tg mice. These results provided a potential therapeutic approach for treating the hypersensitivity of HBV carriers to biochemical stimulation-induced liver damage.

  15. Peroxiredoxin I is important for cancer-cell survival in Ras-induced hepatic tumorigenesis. (United States)

    Han, Bing; Shin, Hye-Jun; Bak, In Seon; Bak, Yesol; Jeong, Ye-Lin; Kwon, Taeho; Park, Young-Ho; Sun, Hu-Nan; Kim, Cheol-Hee; Yu, Dae-Yeul


    Peroxiredoxin I (Prx I), an antioxidant enzyme, has multiple functions in human cancer. However, the role of Prx I in hepatic tumorigenesis has not been characterized. Here we investigated the relevance and underlying mechanism of Prx I in hepatic tumorigenesis. Prx I increased in tumors of hepatocellular carcinoma (HCC) patients that aligned with overexpression of oncogenic H-ras. Prx I also increased in H-rasG12V transfected HCC cells and liver tumors of H-rasG12V transgenic (Tg) mice, indicating that Prx I may be involved in Ras-induced hepatic tumorigenesis. When Prx I was knocked down or deleted in HCC-H-rasG12V cells or H-rasG12V Tg mice, cell colony or tumor formation was significantly reduced that was associated with downregulation of pERK pathway as well as increased intracellular reactive oxygen species (ROS) induced DNA damage and cell death. Overexpressing Prx I markedly increased Ras downstream pERK/FoxM1/Nrf2 signaling pathway and inhibited oxidative damage in HCC cells and H-rasG12V Tg mice. In this study, we found Nrf2 was transcriptionally activated by FoxM1, and Prx I was activated by the H-rasG12V/pERK/FoxM1/Nrf2 pathway and suppressed ROS-induced hepatic cancer-cell death along with formation of a positive feedback loop with Ras/ERK/FoxM1/Nrf2 to promote hepatic tumorigenesis.

  16. Relationships among hepatitis C virus, hepatocellular carcinoma, and diffuse large B cell lymphoma: A case report

    Energy Technology Data Exchange (ETDEWEB)

    Byun, Hyuk Jun; Kim, Seong Hoon [Dept. of Radiology, Daegu Fatima Hospital, Daegu (Korea, Republic of)


    Hepatitis C virus (HCV) is one of the main causes of hepatocellular carcinoma (HCC). Recent studies have reported various associations between HCV and the incidence of non-Hodgkin's lymphoma. We report the radiologic findings in a rare case of simultaneous occurrence of HCC and diffuse large B cell lymphoma in a HCV carrier.

  17. Oxymatrine could promote mesenchymal stem cell therapy in hepatic fibrosis rats:an experimental research

    Institute of Scientific and Technical Information of China (English)



    Objective To investigate whether oxymatrine (OM) could promote mesenchymal stem cell (MSC) therapy in CCl4-induced hepatic fibrosis (HF) in rats and to initially explore its mechanisms.Methods Totally 50 male SD rats were randomly divided into five groups,i.e.,nor-

  18. Mitochondrial and bioenergetic dysfunction in human hepatic cells infected with dengue 2 virus


    El-Bacha, Tatiana; Midlej, Victor; Silva, Ana Paula Pereira da; COSTA,LEANDRO SILVA DA; Benchimol, Marlene; Galina, Antonio; POIAN,ANDREA T. DA


    Mitochondrial and bioenergetic dysfunction in human hepatic cells infected with dengue 2 virus correspondence: Corresponding author. Fax: +55 21 22708647. (El-Bacha, Tatiana) (El-Bacha, Tatiana) Laboratorio de Bioquimica de Virus, Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro - RJ-Brasil--> , Av. Bauhinia n? 400 ? CCS Bloco H 2? andar--> , sala 22. Ilha do Governador--> ...

  19. Cytoskeletal Requirements for Hepatitis C Virus (HCV) RNA Synthesis in the HCV Replicon Cell Culture System


    Bost, Anne G.; Venable, Daryl; Liu, Lifei; Heinz, Beverly A.


    Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.

  20. Cytoskeletal requirements for hepatitis C virus (HCV) RNA synthesis in the HCV replicon cell culture system. (United States)

    Bost, Anne G; Venable, Daryl; Liu, Lifei; Heinz, Beverly A


    Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.

  1. Reactivation of hepatitis D virus after chemotherapy for diffuse large B cell lymphoma despite lamivudine prophylaxis

    DEFF Research Database (Denmark)

    Andersen, Ellen Sloth; Gerstoft, Jan; Weis, Nina Margrethe


    We describe a case of reactivation of hepatitis D virus (HDV) in a patient treated with chemotherapy for a diffuse large B cell lymphoma despite lamivudine prophylaxis. This case suggests that previously cleared HDV should be considered when administering chemotherapy to patients with lymphoma....

  2. FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequency

    NARCIS (Netherlands)

    Peiseler, M.; Sebode, M.; Franke, B.; Wortmann, F.; Schwinge, D.; Quaas, A.; Baron, U.; Olek, S.; Wiegard, C.; Lohse, A.W.; Weiler-Normann, C.; Schramm, C.; Herkel, J.


    BACKGROUND & AIMS: The pathogenesis of autoimmune hepatitis (AIH) is not understood, but it was suggested that AIH may be related to a numerical or functional impairment of CD4+CD25+FOXP3+ regulatory T cells (Treg), which are important mediators of immune tolerance to self-antigens. However, the rol

  3. Bile acids induce hepatic stellate cell proliferation via activation of the epidermal growth factor receptor

    NARCIS (Netherlands)

    Svegliati-Baroni, G; Ridolfi, F; Hannivoort, R; Saccomanno, S; Homan, M; De Minicis, S; Jansen, PLM; Candelaresi, C; Benedetti, A; Moshage, H


    Background B Aims: Hepatic stellate cell (HSC) proliferation is a key event in the development of liver fibrosis. In many liver diseases, HSCs are exposed to inflammatory cytokines, reactive oxygen species, and bile acids. Although inflammatory cytokines and reactive oxygen species are known to prom

  4. Changes in the balance between Treg and Th17 cells in patients with chronic hepatitis B. (United States)

    Su, Zhi-Jun; Yu, Xue-Ping; Guo, Ru-Yi; Ming, De-Song; Huang, Lv-Ye; Su, Mi-Long; Deng, Yong; Lin, Zhen-Zhong


    The purpose of this study was to explore the role of Treg cells, Th17 cells and cytokines associated with Treg/Th17 differentiation in the occurrence, development and outcome of chronic hepatitis B (CHB). To do so, we detected populations of Treg and Th17 cells and their associated cytokines in the peripheral blood of CHB patients. The populations of Treg cells (CD4(+)CD25(high)CD127(low) T cells) and Th17 cells (CD3(+)CD8(-)IL-17(+) T cells) were analyzed in 46 patients with low to moderate chronic hepatitis B (CHB-LM), 24 patients with severe chronic hepatitis B (CHB-S) and 20 healthy controls (HC) using flow cytometry. The levels of cytokines associated with Treg/Th17 differentiation, including IL-10, TGF-β1, IL-17 and IL-23, were measured by enzyme-linked immunosorbent assay (ELISA). Our study showed that the imbalance of Treg and Th17 cells might play an important role in the occurrence, development and outcome of CHB.

  5. Endoderm Induction for Hepatic and Pancreatic Diff erentiation of ES Cells

    Directory of Open Access Journals (Sweden)



    Full Text Available Hepatic and pancreatic differentiation from ES cells is of great interest for the impact that this knowledge could have on the treatment of hepatic and diabetic patients. The liver and pancreas initially develop by budding from the embryonic endoderm. Thus, the development of the endoderm represents an important step and has an integral common role in initiating the early stages of pancreatic and liver development. We know that the development of hepatocytes and insulin-producing pancreatic beta-cells from ES cells represents the culmination of a complex developmental program. However, there has been recent progress in directing ES cells to endoderm and early-stage hepatic and pancreatic progenitor cells. We here discuss the role of the microenvironment, transcriptional factors and cytokines, which have been recognized as important molecules during the major steps of the development of the liver and pancreas. We also present the most recent advances and efforts taken to produce definitive endoderm-committed ES cells for the further differentiation of hepatocyte-like and insulinproducing cells. Recent progress in the search for new sources of hepatocytes and beta-cells has opened up several possibilities for the future of new perspectives for future of new prophylactic and therapeutic possibilities for liver diseases and diabetes.

  6. Hepatic progenitor cells in human liver cirrhosis:Immunohistochemical,electron microscopic and immunofluorencence confocal microscopic findings

    Institute of Scientific and Technical Information of China (English)

    Jia-Cheng Xiao; Xiao-Long Jin; Peter Ruck; Anne Adam; Edwin Kaiserling


    AIM: To investigate whether hepatic progenitor cells (HPC),that reveal the features of oval cells in rodents and small epithelial cells (SEC) in certain human liver disease, were also found in human liver cirrhosis (HLC).METHODS: Surgical liver specimens from 20 cases of hepatitis B virus-positive HLC (15 cases containing hepatocellular carcinoma) were investigated by light microscopic immunohistochemistry (LM-IHC). Among them specimens from 15 cases were investigated by electron microscopy (EM)and those from 5 cases by immunofluorencence confocal laser scanning microscopy (ICLSM). Antibodies against cytokeratin 7 and albumin were used and single and/or double labelling were performed respectively.RESULTS: LM-IHC showed that at the margins of regenerating nodules and in the fibrous septae, a small number of cells in the proliferating bile ductules were positive for CK7 and albumin. At the EM level these HPC were morphologically similar to the SEC described previously, and also similar to the oval cells seen in experimental hepatocarcinogenesis.They were characterized by their small size, oval shape, a high nucleus/cytoplasm ratio, a low organelle content in cytoplasm, and existence of tonofilaments and intercellular junctions. ICLSM revealed that HPC expressed both cytokeratin 7 and albumin.CONCLUSION: HPC with ultrastructural and immunophenotypical features of oval cells, i.e., hepatic stem cell-like cells as noted in other liver diseases, were found in HLC. These findings further support the hypothesis that bipotent hepatic stem cells, that may give rise to biliary epithelial cells and hepatocytes, exist in human livers.

  7. Pluripotent stem cells derived from mouse and human white mature adipocytes. (United States)

    Jumabay, Medet; Abdmaulen, Raushan; Ly, Albert; Cubberly, Mark R; Shahmirian, Laurine J; Heydarkhan-Hagvall, Sepideh; Dumesic, Daniel A; Yao, Yucheng; Boström, Kristina I


    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.

  8. Comprehensive proteomic characterization of stem cell-derived extracellular matrices. (United States)

    Ragelle, Héloïse; Naba, Alexandra; Larson, Benjamin L; Zhou, Fangheng; Prijić, Miralem; Whittaker, Charles A; Del Rosario, Amanda; Langer, Robert; Hynes, Richard O; Anderson, Daniel G


    In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as in vitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering.

  9. Bone marrow-derived dendritic cells. (United States)

    Roney, Kelly


    While much is understood about dendritic cells and their role in the immune system, the study of these cells is critical to gain a more complete understanding of their function. Dendritic cell isolation from mouse body tissues can be difficult and the number of cells isolated small. This protocol describes the growth of large number of dendritic cells from the culture of mouse bone marrow cells. The dendritic cells grown in culture facilitate experiments that may require large number of dendritic cells without great expense or use of large number of mice.

  10. Hepatitis C virus core protein induces fibrogenic actions of hepatic stellate cells via toll-like receptor 2. (United States)

    Coenen, Martin; Nischalke, Hans Dieter; Krämer, Benjamin; Langhans, Bettina; Glässner, Andreas; Schulte, Daniela; Körner, Christian; Sauerbruch, Tilman; Nattermann, Jacob; Spengler, Ulrich


    Hepatic stellate cells (HSCs) represent the main fibrogenic cell type accumulating extracellular matrix in the liver. Recent data suggest that hepatitis C virus (HCV) core protein may directly activate HSCs. Therefore, we examined the influence of recombinant HCV core protein on human HSCs. Primary human HSCs and the human HSC line LX-2 were stimulated with recombinant HCV proteins core and envelope 2 protein. Expression of procollagen type I α-1, α-smooth muscle actin, cysteine- and glycine-rich protein 2, glial fibrillary acidic protein, tissue growth factor β1, matrix metalloproteinases 2 (MMP2) and 13, tissue inhibitor of metalloproteinases 1 and 2 was investigated by real-time PCR. Intracellular signaling pathways of ERK1/2, p38 and, jun-amino-terminal kinase (JNK) were analyzed by western blot analysis. Recombinant HCV core protein induced upregulation of procollagen type I α-1, α-smooth muscle actin, MMP 2 and 13, tissue inhibitor of metalloproteinases 1 and 2, tissue growth factor β1, cysteine- and glycine-rich protein 2, and glial fibrillary acidic protein mRNA expression, whereas HCV envelope 2 protein did not exert any significant effect. Blocking of toll-like receptor 2 (TLR2) with a neutralizing antibody prevented mRNA upregulation by HCV core protein confirming that the TLR2 pathway was involved. Furthermore, western blot analysis revealed HCV-induced phosphorylation of the TLR2-dependent signaling molecules ERK1/2, p38 and JNK mitogen-activated kinases. Our in vitro results demonstrate a direct effect of HCV core protein on activation of HSCs toward a profibrogenic state, which is mediated via the TLR2 pathway. Manipulating the TLR2 pathway may thus provide a new approach for antifibrotic therapies in HCV infection.

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

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe; Frøbert, Ole; Holst-Hansen, Claus


    Background: Treatment of myocardial infarction 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...... myocardial infarction 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 myocardial infarction, using a fully...... grown non-immunecompromised rat model. Methods: 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...

  12. P2X receptors regulate adenosine diphosphate release from hepatic cells. (United States)

    Chatterjee, Cynthia; Sparks, Daniel L


    Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2-4 h. Nucleotide release from hepatic cells is stimulated by the Ca(2+) ionophore, ionomycin, and by the P2 receptor agonist, 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (BzATP). Ionomycin (10 μM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10-100 μM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca(2+) levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.

  13. Transforming Growth Factor-beta signal responding in hepatic stem-like cells

    Institute of Scientific and Technical Information of China (English)

    CUI Wei


    Objective To investigate the effects of TGF-β on the expressions and distribution of phosphorated Smad2/3 and Smad7 in hepatic stem-like cells. Methods Using immunogold transmission electron microscopy, we observed the expressions and distribution of phosphorated Smad2/3, and Smad7 before and after TGF-β1 (5 ng·mL-1) treatment for 4, 8, and 24 hours in hepatic stem-like cells (WB cells). In addition, we also detected the apoptosis status after TGF-β1 stimulation by transmission electron microscopy. Results TGF-β1 stimulation can result in expression increasing of phosphorated Smad2/3 in WB cells, and reach the peak at 8 h, especially in the nuclear. After treatment with TGF-β1 for 24 h, the nuclear expression of phosphorated Smad2/3 gradually decreased. Additionally, we found that TGF-β1 treatment also contributed to increasing in protein level and alteration in cellular distribution of Smad7 (translocation from the nucleus to the cytoplasm) in WB cells. Furthermore, we observed apoptotic body in WB cells after TGF-β1 treatment for 8 h. Conclusions These results indicate that TGF-β stimulation can alter the expression and cellular distribution of phosphorated Srnad2/3 and Smad7 which are its downstream molecular, suggesting hepatic stem-like cells own intact responding to TGF-β.

  14. Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Hella Wobser; Christoph Dorn; Thomas S Weiss; Thomas Amann; Cornelius Bollheimer; Roland Büttner; Jürgen Sc(o)lmerich; Claus Hellerbrand


    Despite the initial belief that non-alcoholic fatty liver disease is a benign disorder, it is now recognized that fbrosis progression occurs in a significant number of patients. Furthermore, hepatic steatosis has been identified as a risk factor for the progression of hepatic fibrosis in a wide range of other liver diseases. Here, we established an in vitro model to study the effect of hepatic lipid accumulation on hepatic stellate cells (HSCs), the central mediators of liver fibrogenesis. Primary human hepatocytes were incubated with the saturated fatty acid palmitate to induce intracellular lipid accumulation. Subsequently, human HSCs were incubated with conditioned media (CM) from steatotic or control hepatocytes. Lipid accumulation in hepatocytes induced the release of factors that accelerated the activation and proliferation of HSC, and enhanced their resistance to apoptosis, largely mediated via activation of the PI-3-kinase pathway. Furthermore, CM from steatotic hepatocytes induced the expression of the profibrogenic genes TGF-β, tissue inhibitor of metallo-proteinase-1 (TIMP-1), TIMP-2 and matrix-metallo-proteinase-2, as well as nuclear-factor Κb-dependent MCP-1 expression in HSC. In summary, our in vitro data indicate a potential mechanism for the pathophysiological link between hepatic steatosis and fibrogenesis in vivo. Herewith, this study provides an attractive in vitro model to study the molecular mechanisms of steatosis-induced fibrogenesis, and to identify and test novel targets for antifibrotic therapies in fatty liver disease.

  15. Detection of the covalently closed circular DNA in peripheral blood mononuclear cells of hepatitis B patients and its clinical significance

    Institute of Scientific and Technical Information of China (English)



    Objective To analyze the correlation between covalently closed circular DNA(ccc DNA)in the peripheral blood mononuclear cells(PBMC)of hepatitis B virus(HBV)-infected patients and serum HBV DNA,hepatitis B surface antigen(HBsA g),hepatitis B e antigen(HBe Ag)and liver histology of hepatitis B patients,and to explore the clinical significance of HBV ccc DNA detection in PBMC.Methods One hundred and eight patients with chronic HBV infection were involved in this

  16. Hepatitis virus vaccines: present status. (United States)

    Krugman, S.


    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

  17. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)


    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  18. Profile of stress and toxicity gene expression in human hepatic cells treated with Efavirenz. (United States)

    Gomez-Sucerquia, Leysa J; Blas-Garcia, Ana; Marti-Cabrera, Miguel; Esplugues, Juan V; Apostolova, Nadezda


    Hepatic toxicity and metabolic disorders are major adverse effects elicited during the pharmacological treatment of the human immunodeficiency virus (HIV) infection. Efavirenz (EFV), the most widely used non-nucleoside reverse transcriptase inhibitor (NNRTI), has been associated with these events, with recent studies implicating it in stress responses involving mitochondrial dysfunction and oxidative stress in human hepatic cells. To expand these findings, we analyzed the influence of EFV on the expression profile of selected stress and toxicity genes in these cells. Significant up-regulation was observed with Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), which indicated metabolic stress. Several genes directly related to oxidative stress and damage exhibited increased expression, including Methalothionein 2A (MT2A), Heat shock 70kDa protein 6 (HSPA6), Growth differentiation factor 15 (GDF15) and DNA-damage-inducible transcript 3 (DDIT3). In addition, Early growth response protein 1 (EGR1) was enhanced, whereas mRNA levels of the inflammatory genes Chemokine (C-X-C motif) ligand 10 (CXCL10) and Serpin peptidase inhibitor (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1) decreased and increased, respectively. This profile of gene expression supports previous data demonstrating altered mitochondrial function and presence of oxidative stress/damage in EFV-treated hepatic cells, and may be of relevance in the search for molecular targets with therapeutic potential to be employed in the prevention, diagnosis and treatment of the hepatic toxicity associated with HIV therapy.

  19. Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering. (United States)

    Jiang, Wei-Cheng; Cheng, Yu-Hao; Yen, Meng-Hua; Chang, Yin; Yang, Vincent W; Lee, Oscar K


    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.

  20. The Therapeutic Targets of miRNA in Hepatic Cancer Stem Cells


    Sabrina Bimonte; Maddalena Leongito; Antonio Barbieri; Vitale del Vecchio; Michela Falco; Aldo Giudice; Raffaele Palaia; Vittorio Albino; Raimondo Di Giacomo; Antonella Petrillo; Vincenza Granata; Francesco Izzo


    Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide malignancy and the third leading cause of cancer death in patients. Several studies demonstrated that hepatic cancer stem cells (HCSCs), also called tumor-initiating cells, are involved in regulation of HCC initiation, tumor progression, metastasis development, and drug resistance. Despite the extensive research, the underlying mechanisms by which HCSCs are regulated remain still unclear. MicroRNAs (miRNAs) are able to r...

  1. Daily genetic profiling indicates JAK/STAT signaling promotes early hepatic stellate cell transdifferentiation

    Institute of Scientific and Technical Information of China (English)

    Ashley; M; Lakner; Cathy; C; Moore; Alyssa; A; Gulledge; Laura; W; Schrum


    AIM: To identify signaling pathways and genes that initiate and commit hepatic stellate cells (HSCs) to transdifferentiation. METHODS: Primary HSCs were isolated from male Sprague-Dawley rats and cultured on plastic for 0-10 d. Gene expression was assessed daily (quiescent to day 10 culture-activation) by real time polymerase chain reaction and data clustered using AMADA software. The significance of JAK/STAT signaling to HSC transdifferentiation was determined by treating cells with a JAK2 inhibitor. RESUL...

  2. Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death

    NARCIS (Netherlands)

    Dunning, Sandra; Rehman, Atta Ur; Tiebosch, Marjolein H.; Hannivoort, Rebekka A.; Haijer, Floris W.; Woudenberg, Jannes; van den Heuvel, Fiona A. J.; Buist-Homan, Manon; Faber, Klaas Nico; Moshage, Han


    Background: In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated i

  3. Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Waters, Katrina M. [Computational Biology and Bioinformatics, Pacific Northwest National Laboratory, Richland, WA 99354 (United States); Sontag, Ryan L. [Systems Toxicology Groups, Pacific Northwest National Laboratory, Richland, WA 99354 (United States); Weber, Thomas J., E-mail: [Systems Toxicology Groups, Pacific Northwest National Laboratory, Richland, WA 99354 (United States)


    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.

  4. JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells. (United States)

    Braiterman, Lelita T; Heffernan, Sean; Nyasae, Lydia; Johns, David; See, Alfred P; Yutzy, Rebeca; McNickle, Allison; Herman, Mira; Sharma, Arun; Naik, Ulhas P; Hubbard, Ann L


    Junctional adhesion molecule (JAM) is involved in tight junction (TJ) formation in epithelial cells. Three JAMs (A, B, and C) are expressed in rat hepatocytes, but only rat JAM-A is present in polarized WIF-B cells, a rat-human hepatic line. We used knockdown (KD) and overexpression in WIF-B cells to determine the role of JAM-A in the development of hepatic polarity. Expression of rat JAM-A short hairpin RNA resulted in approximately 50% KD of JAM-A and substantial loss of hepatic polarity, as measured by the absence of apical cysts formed by adjacent cells and sealed by TJ belts. When inhibitory RNA-resistant human JAM-A (huWT) was expressed in KD cells, hepatic polarity was restored. In contrast, expression of JAM-A that either lacked its PDZ-binding motif (huDeltaC-term) or harbored a point mutation (T273A) did not complement, indicating that multiple sites within JAM-A's cytoplasmic tail are required for the development of hepatic polarity. Overexpression of huWT in normal WIF-B cells unexpectedly blocked WIF-B maturation to the hepatic phenotype, as did expression of three huJAM-A constructs with single point mutations in putative phosphorylation sites. In contrast, huDeltaC-term was without effect, and the T273A mutant only partially blocked maturation. Our results show that JAM-A is essential for the development of polarity in cultured hepatic cells via its possible phosphorylation and recruitment of relevant PDZ proteins and that hepatic polarity is achieved within a narrow range of JAM-A expression levels. Importantly, formation/maintenance of TJs and the apical domain in hepatic cells are linked, unlike simple epithelia.


    Institute of Scientific and Technical Information of China (English)


    Objective To assess the efficacy and the immunological memory of plasma-derived hepatitis B vac- cine 11 years after the initial inoculation. Methods A randomized, double-blind and placebo-controlled trial design was used. Results The immunogenicity and protection rate of the vaccine were good 1 1 years after vaccination, how- ever, from 9 to 11 years after vaccination, the perscn year HBV infection rate showed no noticeable difference be- tween the vaccine group and placebo-controls. Furthermore, the immunological memory remained 11 years after in- oculation, but was significantly lower than that observed in the past 10 years. Conclusion Vaccine protection wanes over the years. More information is needed to define the appropriate time for vaccine booster doses.

  6. Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture. (United States)

    Yu, Mei; Peng, Betty; Chan, Katie; Gong, Ruoyu; Yang, Huiling; Delaney, William; Cheng, Guofeng


    Genotype 6 (GT6) hepatitis C virus (HCV) is prevalent in Southeast Asia and southern China, where it can constitute up to 50% of HCV infections. Despite this, no direct-acting antivirals are approved to treat GT6 HCV infection, and no cell culture systems have been described. In this study, we aimed to develop a GT6 HCV subgenomic replicon to facilitate the identification and development of new HCV therapies with pan-genotype activity. A subgenomic replicon cDNA encoding a GT6a consensus sequence plus an NS5A amino acid substitution (S232I) was synthesized. Electroporation of RNA encoding the GT6a replicon into Huh-7-derived cells consistently yielded 20 to 100 stable replicon colonies. Genotypic analyses of individual replicon colonies revealed new adaptive mutations across multiple viral nonstructural proteins. The E30V and K272R mutations in NS3 and the K34R mutation in NS4A were observed most frequently and were confirmed to enhance GT6a replicon replication in the presence of the NS5A amino acid substitution S232I. These new adaptive mutations allowed establishment of robust luciferase-encoding GT6a replicons for reproducible quantification of HCV replication, and the luciferase-encoding replicons enabled efficient determinations of antiviral activity for HCV inhibitors in a 384-well assay format. While nucleoside/nucleotide NS5B inhibitors and cyclophilin A inhibitors had similar antiviral activities against both GT6a and GT1b replicons, some nonnucleoside NS5B inhibitors, NS3 protease inhibitors, and NS5A inhibitors had less antiviral activity against GT6a replicons. In conjunction with other genotype replicons, this robust GT6a replicon system will aid in the development of pan-genotypic HCV regimens.

  7. Hepatitis A vaccine associated with autoimmune hepatitis

    Institute of Scientific and Technical Information of China (English)

    PA Berry; G Smith-Laing


    To describe a case of probable relapsing autoimmune hepatitis associated with vaccination against hepatitis A virus (HAV). A case report and review of literature were written concerning autoimmune hepatitis in association with hepatitis A and other hepatotropic viruses. Soon after the administration of formalin-inactivated hepatitis A vaccine, a man who had recently recovered from an uncharacterized but self-limiting hepatitic illness,experienced a severe deterioration (AST 1687 U/L, INR 1.4). Anti-nuclear antibodies were detectable, and liver biopsy was compatible with autoimmune hepatitis. The observation supports the role of HAV as a trigger of autoimmune hepatitis. Studies in helper T-cell activity and antibody expression against hepatic proteins in the context of hepatitis A infection are summarized, and the concept of molecular mimicry with regard to other forms of viral hepatitis and autoimmunity is briefly explored.

  8. Effect of Kruppel-like factor 4 on Notch pathway in hepatic stellate cells. (United States)

    Xue, Yin-Kai; Tan, Jun; Dou, Dong-Wei; Chen, Ding; Chen, Lu-Jia; Ren, Huan-Ping; Chen, Li-Bo; Xiong, Xin-Gao; Zheng, Hai


    The relationship between Kruppel-like factor 4 (KLF4) and the Notch pathway was determined to investigate the effect of KLF4 on the activation of hepatic stellate cells and underlying mechanisms. Fifty SPF BALB/c mice were randomly divided into two groups. A liver fibrosis model was established in 25 mice as the experimental group, and the remaining 25 mice served as controls. On the day 0, 7, 14, and 35, liver tissues were removed for immunofluorescent detection. The Notch pathway inhibitor DAPT was added to the primary original hepatic stellate cells, and KLF4 and Notch-associated factor expression was detected by qRT-PCR. Additionally, the hepatic stellate cell line LX-2 was used to establish control and experimental groups, and was cultured in vitro. LX-2 cells in the experimental groups were treated with DAPT and the Notch activator transforming growth factor-beta 1 separately, whereas those in the control group were given isotonic culture medium. After 48 h, KLF4 expression was examined by Western blotting. After transient transfection of LX-2 cells to increase KLF4, the expression of Notch factor was examined. Immunofluorescence analysis showed that, with the aggravation of liver fibrosis, the absorbance (A) values of KLF4 were decreased (day 0: 980.73±153.19; day 7: 1087.99±230.23; day 14: 390.95±93.56; day 35: 245.99±87.34). The expression of Notch pathway- related factors (Notch-1, Notch-2, and Jagged-1) in the hepatic stellate cell membrane was negatively correlated to KLF4 expression. With the increase of KLF4 expression, Notch-2 (0.73±0.13) and Jagged-1 (0.43±0.12) expression decreased, whereas Notch-1 level was not detectable. When the Notch pathway was inhibited, KLF4 levels generally increased (18.12±1.31). Our results indicate that KLF4 expression is negatively correlated to the Notch pathway in hepatic stellate cells, which may provide a reference for the treatment of hepatic fibrosis.

  9. Hepatic stellate cells secreted hepatocyte growth factor contributes to the chemoresistance of hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Guofeng Yu

    Full Text Available As the main source of extracellular matrix proteins in tumor stroma, hepatic stellate cells (HSCs have a great impact on biological behaviors of hepatocellular carcinoma (HCC. In the present study, we have investigated a mechanism whereby HSCs modulate the chemoresistance of hepatoma cells. We used human HSC line lx-2 and chemotherapeutic agent cisplatin to investigate their effects on human HCC cell line Hep3B. The results showed that cisplatin resistance in Hep3B cells was enhanced with LX-2 CM (cultured medium exposure in vitro as well as co-injection with LX-2 cells in null mice. Meanwhile, in presence of LX-2 CM, Hep3B cells underwent epithelial to mesenchymal transition (EMT and upregulation of cancer stem cell (CSC -like properties. Besides, LX-2 cells synthesized and secreted hepatic growth factor (HGF into the CM. HGF receptor tyrosine kinase mesenchymal-epithelial transition factor (Met was activated in Hep3B cells after LX-2 CM exposure. The HGF level of LX-2 CM could be effectively reduced by using HGF neutralizing antibody. Furthermore, depletion of HGF in LX-2 CM abolished its effects on activation of Met as well as promotion of the EMT, CSC-like features and cisplatin resistance in Hep3B cells. Collectively, secreting HGF into tumor milieu, HSCs may decrease hepatoma cells sensitization to chemotherapeutic agents by promoting EMT and CSC-like features via HGF/Met signaling.

  10. Inhibition of tyrosine kinase receptor Tie2 reverts HCV-induced hepatic stellate cell activation.

    Directory of Open Access Journals (Sweden)

    Samuel Martín-Vílchez

    Full Text Available BACKGROUND: Hepatitis C virus (HCV infection is a major cause of chronic liver disease (CLD and is frequently linked to intrahepatic microvascular disorders. Activation of hepatic stellate cells (HSC is a central event in liver damage, due to their contribution to hepatic renewal and to the development of fibrosis and hepatocarcinoma. During the progression of CLDs, HSC attempt to restore injured tissue by stimulating repair processes, such as fibrosis and angiogenesis. Because HSC express the key vascular receptor Tie2, among other angiogenic receptors and mediators, we analyzed its involvement in the development of CLD. METHODS: Tie2 expression was monitored in HSC cultures that were exposed to media from HCV-expressing cells (replicons. The effects of Tie2 blockade on HSC activation by either neutralizing antibody or specific signaling inhibitors were also examined. RESULTS: Media from HCV-replicons enhanced HSC activation and invasion and upregulated Tie2 expression. Notably, the blockade of Tie2 receptor (by a specific neutralizing antibody or signaling (by selective AKT and MAPK inhibitors significantly reduced alpha-smooth muscle actin (α-SMA expression and the invasive potential of HCV-conditioned HSC. CONCLUSIONS: These findings ascribe a novel profibrogenic function to Tie2 receptor in the progression of chronic hepatitis C, highlighting the significance of its dysregulation in the evolution of CLDs and its potential as a novel therapeutic target.

  11. T cell immunopathogenesis and immunotherapeutic strategies for chronic hepatitis B virus infection

    Institute of Scientific and Technical Information of China (English)

    Yukihiro Shimizu


    Hepatitis B is caused by the host immune response and T cells play a major role in the immunopathogenesis.More importantly,T cells not only destroy hepatocytes infected by hepatitis B virus (HBV),but also control HBV replication or eradicate HBV in a noncytolytic manner.Therefore,analysis of T cell immune response during acute and chronic HBV infection is important to develop a strategy for successful viral control,which could lead to immunotherapy for terminating persistent HBV infection.There have been many attempts at immunotherapy for chronic HBV infection,and some have shown promising results.High viral load has been shown to suppress antiviral immune responses and immunoinhibitory signals have been recently elucidated,therefore,viral suppression by nucleos(t)ide analogs,stimulation of antiviral immune response,and suppression of the immunoinhibitory signals must be combined to achieve desirable antiviral effects.

  12. Kupffer cells hasten resolution of liver immunopathology in mouse models of viral hepatitis.

    Directory of Open Access Journals (Sweden)

    Giovanni Sitia


    Full Text Available Kupffer cells (KCs are widely considered important contributors to liver injury during viral hepatitis due to their pro-inflammatory activity. Herein we utilized hepatitis B virus (HBV-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus to demonstrate that KCs do not directly induce hepatocellular injury nor do they affect the pathogenic potential of virus-specific CD8 T cells. Instead, KCs limit the severity of liver immunopathology. Mechanistically, our results are most compatible with the hypothesis that KCs contain liver immunopathology by removing apoptotic hepatocytes in a manner largely dependent on scavenger receptors. Apoptotic hepatocytes not readily removed by KCs become secondarily necrotic and release high-mobility group box 1 (HMGB-1 protein, promoting organ infiltration by inflammatory cells, particularly neutrophils. Overall, these results indicate that KCs resolve rather than worsen liver immunopathology.

  13. Multinucleation and cell dysfunction induced by amorphous silica nanoparticles in an L-02 human hepatic cell line

    Directory of Open Access Journals (Sweden)

    Wang W


    Full Text Available Wen Wang,1–3,* Yang Li,1–3,* Xiaomei Liu,3 Minghua Jin,3 Haiying Du,3 Ying Liu,3 Peili Huang,1,2 Xianqing Zhou,1,2 Lan Yuan,4 Zhiwei Sun1–3 1School of Public Health, Capital Medical University, Beijing, 2Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing, 3School of Public Health, Jilin University, Changchun, Jilin, 4Medical and Healthy Analysis Centre, Peking University, Beijing, People's Republic of China *These authors contributed equally to this work Abstract: Silica nanoparticles (SNPs are one of the most important nanomaterials, and have been widely used in a variety of fields. Therefore, their effects on human health and the environment have been addressed in a number of studies. In this work, the effects of amorphous SNPs were investigated with regard to multinucleation in L-02 human hepatic cells. Our results show that L-02 cells had an abnormally high incidence of multinucleation upon exposure to silica, that increased in a dose-dependent manner. Propidium iodide staining showed that multinucleated cells were arrested in G2/M phase of the cell cycle. Increased multinucleation in L-02 cells was associated with increased generation of cellular reactive oxygen species and mitochondrial damage on flow cytometry and confocal microscopy, which might have led to failure of cytokinesis in these cells. Further, SNPs inhibited cell growth and induced apoptosis in exposed cells. Taken together, our findings demonstrate that multinucleation in L-02 human hepatic cells might be a failure to undergo cytokinesis or cell fusion in response to SNPs, and the increase in cellular reactive oxygen species could be responsible for the apoptosis seen in both mononuclear cells and multinucleated cells. Keywords: silica nanoparticles, human hepatic cell L-02, multinucleation, cell cycle, cell dysfunction, apoptosis

  14. Multiple Effects of Berberine Derivatives on Colon Cancer Cells

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    Luis Miguel Guamán Ortiz


    Full Text Available 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.

  15. Exosome-associated hepatitis C virus in cell cultures and patient plasma. (United States)

    Liu, Ziqing; Zhang, Xiugen; Yu, Qigui; He, Johnny J


    Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.

  16. Involvement of hepatitis B X-interacting protein (HBXIP) in proliferation regulation of cells

    Institute of Scientific and Technical Information of China (English)

    Feng-ze WANG; Li SHA; Wei-ying ZHANG; Lian-ying WU; Ling QIAO; Nan LI; Xiao-dong ZHANG; Li-hong YE


    Aim: To investigat the effect of Hepatitis B X-interacting protein (HBXIP) on cell proliferation. Methods: A rabbit antibody against HBXIP was generated. The RNA interference (RNAi) fragment of the HBXIP gene was constructed in the pSilencer-3.0-H1 vector termed pSilencer-hbxip. Plasmids of the pcDNA3-hbxip encoding HBXIP gene and pSilencer-hbxip were transfected into human breast carcinoma MCF-7 cells, hepatoma H7402 cells, and the normal human hepatic cell line L-O2, respectively. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro- mide (MTT) assay and 5-bromo-2-deoxyuridine incorporation assay were applied to detect cell proliferation. MCF-7 cells and L-O2 cells in the cell cycle were examined by flow cytometry. The proteins involved in cell proliferation and cell cycle were investigated by Western blot. Results: Overexpression of HBXIP resulted in the promotion of proliferation of MCF-7, H7402, and L-O2 cells. Flow cytometry showed that the overexpression of HBXIP promoted the cell prolifera-tion of MCF-7 and L-O2 cells, and led to an increased cell proliferative index in MCF-7 cells (from 46.25% to 58.28%) and L-O2 cells (from 29.62% to 35.54%). Western blot showed that expression levels of c-Myc, Bcl-2, and proliferating cell nuclear antigen were upregulated in MCF-7, H7402, or L-O2 cells, whereas that of p27 was downregulated. However, the RNAi of HBXIP brought opposite results.Conclusion: One of the functions of HBXIP is its involvement in cell proliferation.

  17. Derivation of human embryonic stem cells in defined conditions. (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A


    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  18. Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds. (United States)

    Yamazoe, Taiji; Shiraki, Nobuaki; Kume, Shoen


    The induced pluripotent stem (iPS) cells of murine and human are capable to differentiate into any cell type of the body through recapitulating normal development, similarly as the embryonic stem (ES) cells. Lines of evidence support that both ES cells and iPS cells are induced to differentiate in vitro by sequential treatment of humoral cues such as growth factors and chemicals, combined with the use of certain microenvironments including extracellular matrices and scaffolds.Here, we describe the procedure to potentiate hepatic lineage cells differentiation from murine and human iPS cells, using growth factor cocktails and nanofiber scaffolds. Nanofiber scaffolds have a three-dimensional surface mimicking the fine structures of the basement membrane in vivo, allow the iPS cells to differentiate into the definitive endoderm and mature hepatocyte-like cells more efficiently than the two-dimensional conventional culture plates.

  19. Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; You-Hua Xie; Yu-Ying Kong; Ye Ye; Chun-Lin Wang; Guang-Di Li; Yuan Wang


    AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis.METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO)cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258staining, flow cytometry and DNA fragmentation analysis.RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage,chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line.CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

  20. Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1.1 + cells

    Institute of Scientific and Technical Information of China (English)

    Zhong-Jun Dong; Hai-Ming Wei; Rui Sun; Bin Gao; Zhi-Gang Tian


    AIM: To choose an appropriate methods for the isolation of hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepatic lymphocytes.METHODS: Hepatic lymphocytes were isolated from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope.Effects of digestive. enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate [(percentage of post-digestion -percentage of pre-digestion)/percentage of pre-digestion].Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h 51Cr release assay.RESULTS: NK1.1+ cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their relative change rates were about 28% and 32%,respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I:C)-treated or ConA-treated mice by mechanical dissection was not significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and γδ cells in normal C57BL/6 mice.After poly (I:C) stimulation, hepatic NK cells rose to about 35%, while NKT cells simultaneously decreased. Following ConA injection, the number of hepatic NKT cells was remarkably reduced to 3.67%. Higher ratio of intracellular IFN-γ+(68%) or TNF-α+(15%) NK1.1+ cells from poly (I:C)-treated mice was obtained using mechanical dissection method than control mice. There was no difference in viability between the mechanical dissection and the enzymatic digestion, and hepatic lymphocytes obtained with the two methods had similar cytotoxicity against YAC-1cells

  1. Alternate Reading Frame Protein (F Protein) of Hepatitis C Virus: Paradoxical Effects of Activation and Apoptosis on Human Dendritic Cells Lead to Stimulation of T Cells (United States)

    Samrat, Subodh Kumar; Li, Wen; Singh, Shakti; Kumar, Rakesh; Agrawal, Babita


    Hepatitis C virus (HCV) leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs) process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF) have been implicated in modulation of dendritic cells (DCs) and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F), whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a) were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans. PMID:24475147

  2. Alternate reading frame protein (F protein of hepatitis C virus: paradoxical effects of activation and apoptosis on human dendritic cells lead to stimulation of T cells.

    Directory of Open Access Journals (Sweden)

    Subodh Kumar Samrat

    Full Text Available Hepatitis C virus (HCV leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF have been implicated in modulation of dendritic cells (DCs and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F, whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans.

  3. Technical Challenges in the Derivation of Human Pluripotent Cells

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    Parinya Noisa


    Full Text Available It has long been discovered that human pluripotent cells could be isolated from the blastocyst state of embryos and called human embryonic stem cells (ESCs. These cells can be adapted and propagated indefinitely in culture in an undifferentiated manner as well as differentiated into cell representing the three major germ layers: endoderm, mesoderm, and ectoderm. However, the derivation of human pluripotent cells from donated embryos is limited and restricted by ethical concerns. Therefore, various approaches have been explored and proved their success. Human pluripotent cells can also be derived experimentally by the nuclear reprogramming of somatic cells. These techniques include somatic cell nuclear transfer (SCNT, cell fusion and overexpression of pluripotent genes. In this paper, we discuss the technical challenges of these approaches for nuclear reprogramming, involving their advantages and limitations. We will also highlight the possible applications of these techniques in the study of stem cell biology.

  4. Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope.

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    Ying Qi

    Full Text Available 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.

  5. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines. (United States)

    Pigni, Matteo; Ashok, Devika; Acha-Orbea, Hans


    It is notoriously difficult to obtain large quantities of non-activated dendritic cells ex vivo. For this reason, we produced and characterized a mouse model expressing the large T oncogene under the CD11c promoter (Mushi mice), in which CD8α(+) dendritic cells transform after 4 months. We derived a variety of stable cell lines from these primary lines. These cell lines reproducibly share with freshly isolated dendritic cells most surface markers, mRNA and protein expression, and all tested biological functions. Cell lines can be derived from various strains and knockout mice and can be easily transduced with lentiviruses. In this article, we describe the derivation, culture, and lentiviral transduction of these dendritic cell lines.

  6. Hepatic zonation of carbon and nitrogen fluxes derived from glutamine and ammonia transformations


    Constantin Jorgete; Suzuki-Kemmelmeier Fumie; Comar Jurandir F; Bracht Adelar


    Abstract Background Glutaminase predominates in periportal hepatocytes and it has been proposed that it determines the glutamine-derived nitrogen flow through the urea cycle. Glutamine-derived urea production should, thus, be considerably faster in periportal hepatocytes. This postulate, based on indirect observations, has not yet been unequivocally demonstrated, making a direct investigation of ureogenesis from glutamine highly desirable. Methods Zonation of glutamine metabolism was investig...

  7. Modification of histone acetylation facilitates hepatic differentiation of human bone marrow mesenchymal stem cells.

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    Xuejun Dong

    Full Text Available The multi-potentiality of mesenchymal stem cells makes them excellent options for future tissue engineering and clinical therapy, including liver injury. In this study, we investigated the effects of valproic acid (VPA, a direct inhibitor of histone deacetylase (HDAC, on the hepatic differentiation of human bone marrow mesenchymal stem cells (BMMSCs. The cells were found to differentiate into a more homogeneous hepatocyte-like population when pretreated with 5 mM VPA for 72 h. The expression of liver-specific markers was significantly upregulated in the VPA-treated group at the mRNA and protein levels. VPA treatment also significantly enhanced the hepatic functions of the differentiated cells, including glycogen storage, cytochrome P450 activity, AFP and ALB synthesis, and urea production. Further analysis showed that treatment with 5 mM of VPA for 72 h greatly improved the histones H3 and H4 acetylation. These results demonstrated that VPA could considerably improve the hepatic differentiation of human BMMSCs, probably because the chromatin-acetylated state changes upon VPA treatment through its HDAC inhibitory effect. Thus, this study provides a direct research model for producing human hepatocytes for clinical purposes.

  8. Inhibition of hepatitis B virus replication by quercetin in human hepatoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Zhikui; Cheng; Ge; Sun; Wei; Guo; Yayun; Huang; Weihua; Sun; Fei; Zhao; Kanghong; Hu


    Hepatitis B virus(HBV) infection is one of the most serious and prevalent viral diseases in the world. Although several anti-HBV drugs have been used clinically, their side and adverse effects limit treatment efficacy. Therefore, it is necessary to identify novel potential anti-HBV agents. The flavonol quercetin has shown activity against some retroviruses, but its effect on HBV remains unclear. In the present study, quercetin was incubated with Hep G2.2.15 cells, as well as Hu H-7 cells transfected with an HBV plasmid. Quercetin was shown to significantly reduce Hepatitis B surface antigen(HBs Ag) and Hepatitis B e antigen(HBe Ag), secretion and HBV genomic DNA levels in both cell lines. In addition, co-incubation with lamivudine(3TC), entecavir(ETV), or adefovir(Ade) further enhanced the quercetin-induced inhibition of HBV replication. This inhibition was partially associated with decreased heat shock proteins and HBV transcription levels. The results indicate that quercetin inhibited HBV antigen secretion and genome replication in human hepatoma cell lines, which suggests that quercetin may be a potentially effective anti-HBV agent.

  9. Ultrastructure of oval cells in children with chronic hepatitis B, with special emphasis on the stage of liver fibrosis: The first pediatric study

    Institute of Scientific and Technical Information of China (English)

    Maria Elzbieta Sobaniec-Lotowska; Joanna Maria Lotowska; Dariusz Marek Lebensztejn


    AIM: To investigate the ultrastructure of oval ceils in children with chronic hepatitis B, with special emphasis on their location in areas of collagen fibroplasia.METHODS: Morphological investigations were conducted on biopsy material obtained from 40 children,aged 3-16 years with chronic hepatitis B. The stage of fibrosis was assessed histologically using the arbitrary semiquantitative numerical scoring system proposed by Ishak et al. The material for ultrastructural investigation was fixed in glutaraldehyde and paraformaldehyde and processed for transmission-electron microscopic analysis.RESULTS: Ultrastructural examination of biopsy specimens obtained from children with chronic hepatitis B showed the presence of two types of oval cells, the hepatic progenitor cells and intermediate hepatic-like cells. These cells were present in the parenchyma and were seen most commonly in areas of intense periportal fibrosis (at least stage 2 according to Ishak et al) and in the vicinity of the limiting plate of the lobule. The activated nonparenchymal hepatic cells, i.e. transformed hepatic stellate cells and Kupffer cells were seen in close proximity to the intermediate hepatic-like cells.CONCLUSION: We found a distinct relationship between the prevalence of oval cells (hepatic progenitor cells and intermediate hepatocyte-like cells) and fibrosis stage in pediatric patients with chronic hepatitis B.

  10. Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Kumi Kimura


    Full Text Available 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.

  11. Tumor-derived lactate and myeloid-derived suppressor cells: Linking metabolism to cancer immunology. (United States)

    Husain, Zaheed; Seth, Pankaj; Sukhatme, Vikas P


    Many malignant cells produce increased amounts of lactate, which promotes the development of myeloid-derived suppressor cells (MDSCs). MDSCs, lactate, and a low pH in the tumor microenvironment inhibit the function of natural killer (NK) cells and T lymphocytes, hence allowing for disease progression. Ketogenic diets can deplete tumor-bearing animals from MDSCs and regulatory T cells, thereby improving their immunological profile.

  12. Short-term arginine deprivation results in large-scale modulation of hepatic gene expression in both normal and tumor cells: microarray bioinformatic analysis

    Directory of Open Access Journals (Sweden)

    Sabo Edmond


    Full Text Available Abstract Background We have reported arginine-sensitive regulation of LAT1 amino acid transporter (SLC 7A5 in normal rodent hepatic cells with loss of arginine sensitivity and high level constitutive expression in tumor cells. We hypothesized that liver cell gene expression is highly sensitive to alterations in the amino acid microenvironment and that tumor cells may differ substantially in gene sets sensitive to amino acid availability. To assess the potential number and classes of hepatic genes sensitive to arginine availability at the RNA level and compare these between normal and tumor cells, we used an Affymetrix microarray approach, a paired in vitro model of normal rat hepatic cells and a tumorigenic derivative with triplicate independent replicates. Cells were exposed to arginine-deficient or control conditions for 18 hours in medium formulated to maintain differentiated function. Results Initial two-way analysis with a p-value of 0.05 identified 1419 genes in normal cells versus 2175 in tumor cells whose expression was altered in arginine-deficient conditions relative to controls, representing 9–14% of the rat genome. More stringent bioinformatic analysis with 9-way comparisons and a minimum of 2-fold variation narrowed this set to 56 arginine-responsive genes in normal liver cells and 162 in tumor cells. Approximately half the arginine-responsive genes in normal cells overlap with those in tumor cells. Of these, the majority was increased in expression and included multiple growth, survival, and stress-related genes. GADD45, TA1/LAT1, and caspases 11 and 12 were among this group. Previously known amino acid regulated genes were among the pool in both cell types. Available cDNA probes allowed independent validation of microarray data for multiple genes. Among genes downregulated under arginine-deficient conditions were multiple genes involved in cholesterol and fatty acid metabolism. Expression of low-density lipoprotein receptor was

  13. Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

    Institute of Scientific and Technical Information of China (English)

    Zhi-Qiang Song; Fei Hao; Feng Min; Qiao-Yu Ma; Guo-Dong Liu


    AIM To establish a cell culture system with long-term replication of hepatitis C virus in vitro.``METHODS Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RT-PCR, in situ hybridization and immunohistochemistry respectively.``RESULTS The intracellular HCV RNA was first detected on d 2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3, CP10antigens could be observed in cells. The fresh cells could be infected by supematant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed.``CONCLUSION The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.``

  14. Quasispecies of Hepatitis C Virus Participate in Cell-Specific Infectivity. (United States)

    Fukuhara, Takasuke; Yamamoto, Satomi; Ono, Chikako; Nakamura, Shota; Motooka, Daisuke; Mori, Hiroyuki; Kurihara, Takeshi; Sato, Asuka; Tamura, Tomokazu; Motomura, Takashi; Okamoto, Toru; Imamura, Michio; Ikegami, Toru; Yoshizumi, Tomoharu; Soejima, Yuji; Maehara, Yoshihiko; Chayama, Kazuaki; Matsuura, Yoshiharu


    It is well documented that a variety of viral quasispecies are found in the patients with chronic infection of hepatitis C virus (HCV). However, the significance of quasispecies in the specific infectivity to individual cell types remains unknown. In the present study, we analyzed the role of quasispecies of the genotype 2a clone, JFH1 (HCVcc), in specific infectivity to the hepatic cell lines, Huh7.5.1 and Hep3B. HCV RNA was electroporated into Huh7.5.1 cells and Hep3B/miR-122 cells expressing miR-122 at a high level. Then, we adapted the viruses to Huh7 and Hep3B/miR-122 cells by serial passages and termed the resulting viruses HCVcc/Huh7 and HCVcc/Hep3B, respectively. Interestingly, a higher viral load was obtained in the homologous combination of HCVcc/Huh7 in Huh7.5.1 cells or HCVcc/Hep3B in Hep3B/miR-122 cells compared with the heterologous combination. By using a reverse genetics system and deep sequence analysis, we identified several adaptive mutations involved in the high affinity for each cell line, suggesting that quasispecies of HCV participate in cell-specific infectivity.

  15. Adipose-derived regenerative cells in patients with ischemic cardiomyopathy

    DEFF Research Database (Denmark)

    Perin, Emerson C; Sanz-Ruiz, Ricardo; Sánchez, Pedro L


    AIMS: Adipose-derived regenerative cells (ADRCs) can be isolated from liposuction aspirates and prepared as fresh cells for immediate administration in cell therapy. We performed the first randomized, placebo-controlled, double-blind trial to examine the safety and feasibility of the transendocar...

  16. Derivation, propagation and differentiation of human embryonic stem cells. (United States)

    Conley, Brock J; Young, Julia C; Trounson, Alan O; Mollard, Richard


    Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug

  17. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Genz, Berit [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Thomas, Maria [Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart (Germany); Pützer, Brigitte M. [Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Rostock (Germany); Siatkowski, Marcin; Fuellen, Georg [Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Rostock (Germany); Vollmar, Brigitte [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Abshagen, Kerstin, E-mail: [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany)


    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.

  18. Differentiation of embryoid-body cells derived from embryonic stem cells into hepatocytes in alginate microbeads in vitro

    Institute of Scientific and Technical Information of China (English)

    Sheng FANG; Yu-dong QIU; Liang MAO; Xiao-lei SHI; De-cai YU; Yi-tao DING


    Aim: Embryonic stem (ES) cells are being widely investigated as a promising source of hepatocytes with their proliferative, renewable, and pluripotent capacities. However, controlled and scalable ES cell differentiation culture into functional hepatocytes is challenging. In this study, we examined the differentiat- ing potential of embryoid-body cells derived from ES cells into hepatocytes in alginate microbeads containing exogenous growth factors in vitro. Methods: Embryoid bodies were formed from ES cells by suspension methods. Embryoid bodies cultured for 5 d were treated with trypsin-EDTA. The disaggregated cells were encapsulated in alginate microbeads and stimulated with exogenous growth factors to induce hepatic differentiation. In the course of cell differentiation, cell morphology and viability were observed, and the expression patterns of some genes of the hepatocyte were confirmed by RT-PCR. An immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK18). Hepatocyte functional assays were confirmed by the secretion of ALB and urea. Results: We showed that embryoid-body cells could maintain cell viability in alginate microbeads in vitro. We also found that directed differentiated cells expressed several hepatocyte genes including α-fetoprotein (AFP), ALB, Cyp7al, CK18, transthyretin (TTR) and tyrosine aminotransferase (TAT) and produced ALB and urea in alginate microbeads. The directed differentiated cells expressed ALB and CK18 proteins on d 14. However, embryoid-body cells could not form hepatocytes without exogenous growth factors in alginate microbeads. Conclusion: The differentiation of embryoid-body cells into hepatocytes con- taining exogenous growth factors in alginate microbeads gives rise to functional hepatocytes and may develop scalable stem cell differentiation strategies for bioartificial livers and hepatocyte transplantation.

  19. Hepatic circadian clock oscillators and nuclear receptors integrate microbiome-derived signals (United States)

    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


    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

  20. Kupffer cells modulate hepatic fatty acid oxidation during infection with PR8 influenza. (United States)

    Tarasenko, Tatyana N; Singh, Larry N; Chatterji-Len, Milani; Zerfas, Patricia M; Cusmano-Ozog, Kristina; McGuire, Peter J


    In response to infection, patients with inborn errors of metabolism may develop a functional deterioration termed metabolic decompensation. The biochemical hallmarks of this disruption of metabolic homeostasis are disease specific and may include acidosis, hyperammonemia or hypoglycemia. In a model system previously published by our group, we noted that during influenza infection, mice displayed a depression in hepatic mitochondrial enzymes involved in nitrogen metabolism. Based on these findings, we hypothesized that this normal adaptation may extend to other metabolic pathways, and as such, may impact various inborn errors of metabolism. Since the liver is a critical organ in inborn errors of metabolism, we carried out untargeted metabolomic profiling of livers using mass spectrometry in C57Bl/6 mice infected with influenza to characterize metabolic adaptation. Pathway analysis of metabolomic data revealed reductions in CoA synthesis, and long chain fatty acyl CoA and carnitine species. These metabolic adaptations coincided with a depression in hepatic long chain β-oxidation mRNA and protein. To our surprise, the metabolic changes observed occurred in conjunction with a hepatic innate immune response, as demonstrated by transcriptional profiling and flow cytometry. By employing an immunomodulation strategy to deplete Kupffer cells, we were able to improve the expression of multiple genes involved in β-oxidation. Based on these findings, we are the first to suggest that the role of the liver as an immunologic organ is central in the pathophysiology of hepatic metabolic decompensation in inborn errors of metabolism due to respiratory viral infection.

  1. Corona-directed nucleic acid delivery into hepatic stellate cells for liver fibrosis therapy. (United States)

    Zhang, Zhengping; Wang, Chunming; Zha, Yinhe; Hu, Wei; Gao, Zhongfei; Zang, Yuhui; Chen, Jiangning; Zhang, Junfeng; Dong, Lei


    Strategies to modify nanoparticles with biological ligands for targeted drug delivery in vivo have been widely studied but met with limited clinical success. A possible reason is that, in the blood circulation, serum proteins could rapidly form a layer of protein "corona" on the vehicle surface, which might block the modified ligands and hamper their targeting functions. We speculate that strategies for drug delivery can be designed based upon elegant control of the corona formation on the vehicle surfaces. In this study, we demonstrate a retinol-conjugated polyetherimine (RcP) nanoparticle system that selectively recruited the retinol binding protein 4 (RBP) in its corona components. RBP was found to bind retinol, and direct the antisense oligonucleotide (ASO)-laden RcP carrier to hepatic stellate cells (HSC), which play essential roles in the progression of hepatic fibrosis. In both mouse fibrosis models, induced by carbon tetrachloride (CCl4) and bile duct ligation (BDL), respectively, the ASO-laden RcP particles effectively suppressed the expression of type I collagen (collagen I), and consequently ameliorated hepatic fibrosis. Such findings suggest that this delivery system, designed to exploit the power of corona proteins, can serve as a promising tool for targeted delivery of therapeutic agents for the treatment of hepatic fibrosis.

  2. Hepatosplenic Gamma/DeltaT-Cell Lymphoma Masquerading as Alcoholic Hepatitis and Methadone Withdrawal

    Directory of Open Access Journals (Sweden)

    H.A. Lopez Morra


    Full Text Available Hepatosplenic gamma/delta T-cell lymphoma is a rare neoplasm of mature gamma/delta T-cells with sinusoidal infiltration of spleen, liver, and bone marrow. Patients are predominantly adolescent and young adult males and usually present with marked hepatosplenomegaly. Pancytopenia is another common finding. Despite an initial response to treatment, patients have a median survival of one to two years. In this report, we document a case of alcoholic hepatitis and methadone withdrawal masquerading unsuspected, hepatosplenic gamma/delta T-cell lymphoma with unusual CD20 positivity.

  3. Advances in Liver Regeneration: Revisiting Hepatic Stem/Progenitor Cells and Their Origin. (United States)

    Sadri, Ali-Reza; Jeschke, Marc G; Amini-Nik, Saeid


    The liver has evolved to become a highly plastic organ with extraordinary regenerative capabilities. What drives liver regeneration is still being debated. Adult liver stem/progenitor cells have been characterized and used to produce functional hepatocytes and biliary cells in vitro. However, in vivo, numerous studies have questioned whether hepatic progenitor cells have a significant role in liver regeneration. Mature hepatocytes have recently been shown to be more plastic than previously believed and give rise to new hepatocytes after acute and chronic injury. In this review, we discuss current knowledge in the field of liver regeneration and the importance of the serotonin pathway as a clinical target for patients with liver dysfunction.

  4. Upregulation of MiR-122 via Trichostatin A Treatments in Hepatocyte-like Cells Derived from Mesenchymal Stem Cells. (United States)

    Alizadeh, Effat; Eslaminejad, MohamadReza Baghaban; Akbarzadeh, Abolfazl; Sadeghi, Zohre; Abasi, Mozghan; Herizchi, Roya; Zarghami, Nosratollah


    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.

  5. Induction of hepatic CYP3A enzymes by pregnancy-related hormones: studies in human hepatocytes and hepatic cell lines. (United States)

    Papageorgiou, Ioannis; Grepper, Susan; Unadkat, Jashvant D


    CYP3A activity is induced by approximately 2-fold during the third trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17β-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from premenopausal female donors (n = 2) with the physiologic (unbound, 1× total) and the 10× total third trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5, and the respective hormone receptors (growth hormone receptor, glucocorticoid receptor, and estrogen receptor alpha). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/growth hormone and cortisol induced CYP3A activity and expression significantly more than did cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the third trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the first and second trimesters, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.

  6. A Novel Human Radixin Peptide Inhibits Hepatitis C Virus Infection at the Level of Cell Entry


    Bukong, Terence N; Kodys, Karen; Szabo, Gyongyi


    Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin–moesin–radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and...

  7. Quercetin improves insulin resistance and hepatic lipid accumulation in vitro in a NAFLD cell model


    LI, XIULI; Wang, Rong; Zhou, Na; Wang, XiaoHui; Liu, Qingyan; Bai, Yuqin; BAI, YIN; Liu, Zhijie; YANG, HUIMING; ZOU, JIHONG; Wang, Hongxia; SHI, TIEWEI


    Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver diseases in the absence of significant alcohol consumption. The aim of this study was to investigate the effect of quercetin on insulin resistance and lipid metabolic abnormalities in free fatty acid (FFA)- and insulin-induced HepG2 cell model of NAFLD, and to determine the possible underlying mechanism. Quercetin markedly improves hepatic lipid accumulation and decreases the levels of triglyceride (TG). The lipid-lower...

  8. Liver tryptase-positive mast cells and fibrosis in children with hepatic echinococcosis

    Directory of Open Access Journals (Sweden)

    Gulubova Maya


    Full Text Available The hepatic echinococcosis in children is a serious surgical problem. The aim of this study is to investigate the participation of mast cells in liver inflammatory reactions triggered by echinococcal cysts. Liver biopsy samples were collected from the tissue surrounding the cysts from 16 sick children (11 boys and 5 girls in the course of abdominal surgery and from 5 controls. Light and ultrastructural immunocytochemistry was performed using monoclonal antibody against tryptase. Light microscopical immunocytochemistry revealed abundance of tryptase-positive (MCT mast cells in the capsules of the cysts (43.58 cells/mm2. There were also observed greatly increased numbers of mast cells in portal tracts surrounding the cyst, compared to those of control biopsies (26.49 vs. 1.78 cells/mm2, p=0.0009, Mann-Whitney U test. Based on the ultrastructural appearance of tryptase-positive mast cell granules, morphological sings of activation of most of the mast cells were distinguished. In conclusion, we suggest that the accumulated and activated tryptase-positive mast cells in liver tissues surrounding the echinococcal cysts play a crucial role in modulation of the inflammatory liver response and could induce chronic inflammation and fibrogenesis, resulting in serious liver injury such as nonspecific reactive hepatitis.

  9. Identification of transforming hepatitis B virus S gene nonsense mutations derived from freely replicative viruses in hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Shiu-Feng Huang

    Full Text Available BACKGROUND & AIMS: The correlation between chronic hepatitis B virus (HBV infection and hepatocellular carcinoma (HCC has been well-established. But the roles of viral factor remain uncertain. Only HBV X gene and nonsense mutations of S gene (C-terminal truncation of HBV surface protein have been demonstrated to have transforming activity. Whether they play a significant role in hepatocarcinogenesis is still uncertain. METHODS: Twenty-five HBV-related HCC patients were positive for hepatitis B core antigen (HBcAg in the cancerous parts of their HCC liver tissues by immunohistochemistry studies, and had available tissue for whole HBV genome sequence analysis. The results were compared with 25 gender and age-matched HBcAg negative HCCs. Plasmids encoding HBV S gene nonsense mutations identified from HBcAg (+ HCC tissue were constructed to investigate their cell proliferation, transformation activity and the oncogenic potentials by xenograft study and in vivo migration assay. RESULTS: HBcAg (+ HCC patients were significantly associated with cirrhosis and small tumor size (≦2 cm when compared with HBcAg (- HCC patients. Southern blot analyses revealed freely replicative forms of HBV in the cancerous parts of HBcAg(+ HCC. Three nonsense mutations of S gene (sL95*, sW182*, and sL216* were identified in the HBcAg(+ HCC tumor tissues. sW182* and sL216* were recurrently found in the 25 HBcAg (- HCC tumor tissue, too. Functional studies of the above 3 non-sense mutations all demonstrated higher cell proliferation activities and transformation abilities than wild type S, especially sW182*. Tumorigenicity analysis by xenograft experiments and in vitro migration assay showed potent oncogenic activity of sW182* mutant. CONCLUSIONS: This study has demonstrated potent oncogenic activity of nonsense mutations of HBV S gene, suggesting they may play an important role in hepatocarcinogenesis.

  10. Murine junctional adhesion molecules JAM-B and JAM-C mediate endothelial and stellate cell interactions during hepatic fibrosis. (United States)

    Hintermann, Edith; Bayer, Monika; Ehser, Janine; Aurrand-Lions, Michel; Pfeilschifter, Josef M; Imhof, Beat A; Christen, Urs


    Classical junctional adhesion molecules JAM-A, JAM-B and JAM-C influence vascular permeability, cell polarity as well as leukocyte recruitment and immigration into inflamed tissue. As the vasculature becomes remodelled in chronically injured, fibrotic livers we aimed to determine distribution and role of junctional adhesion molecules during this pathological process. Therefore, livers of naïve or carbon tetrachloride-treated mice were analyzed by immunohistochemistry to localize all 3 classical junctional adhesion molecules. Hepatic stellate cells and endothelial cells were isolated and subjected to immunocytochemistry and flow cytometry to determine localization and functionality of JAM-B and JAM-C. Cells were further used to perform contractility and migration assays and to study endothelial tubulogenesis and pericytic coverage by hepatic stellate cells. We found that in healthy tissue, JAM-A was ubiquitously expressed whereas JAM-B and JAM-C were restricted to the vasculature. During fibrosis, JAM-B and JAM-C levels increased in endothelial cells and JAM-C was de novo generated in myofibroblastic hepatic stellate cells. Soluble JAM-C blocked contractility but increased motility in hepatic stellate cells. Furthermore, soluble JAM-C reduced endothelial tubulogenesis and endothelial cell/stellate cell interaction. Thus, during liver fibrogenesis, JAM-B and JAM-C expression increase on the vascular endothelium. More importantly, JAM-C appears on myofibroblastic hepatic stellate cells linking them as pericytes to JAM-B positive endothelial cells. This JAM-B/JAM-C mediated interaction between endothelial cells and stellate cells stabilizes vessel walls and may control the sinusoidal diameter. Increased hepatic stellate cell contraction mediated by JAM-C/JAM-C interaction may cause intrahepatic vasoconstriction, which is a major complication in liver cirrhosis.

  11. LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis (United States)

    Odena, Gemma; Chen, Jiegen; Lozano, Juan Jose; Altamirano, Jose; Rodrigo-Torres, Daniel; Affo, Silvia; Morales-Ibanez, Oriol; Matsushita, Hiroshi; Zou, Jian; Dumitru, Raluca; Caballeria, Juan; Gines, Pere; Arroyo, Vicente; You, Min; Rautou, Pierre-Emmanuel; Valla, Dominique; Crews, Fulton; Seki, Ekihiro; Sancho-Bru, Pau; Bataller, Ramon


    Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development. PMID:27752144

  12. Balancing Ethical Pros and Cons of Stem Cell Derived Gametes. (United States)

    Segers, Seppe; Mertes, Heidi; de Wert, Guido; Dondorp, Wybo; Pennings, Guido


    In this review we aim to provide an overview of the most important ethical pros and cons of stem cell derived gametes (SCD-gametes), as a contribution to the debate about reproductive tissue engineering. Derivation of gametes from stem cells holds promising applications both for research and for clinical use in assisted reproduction. We explore the ethical issues connected to gametes derived from embryonic stem cells (both patient specific and non-patient specific) as well as those related to gametes derived from induced pluripotent stem cells. The technology of SCD-gametes raises moral concerns of how reproductive autonomy relates to issues of embryo destruction, safety, access, and applications beyond clinical infertility.

  13. 0Adipose-derived stem cells: Implications in tissue regeneration

    Institute of Scientific and Technical Information of China (English)

    Wakako; Tsuji; J; Peter; Rubin; Kacey; G; Marra


    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 differ-entiate 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 dam-aged by injury and diseases. This article reviews the implications of ASCs in tissue regeneration.

  14. A novel feeder-free culture system for human pluripotent stem cell culture and induced pluripotent stem cell derivation.

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    Sanna Vuoristo

    Full Text Available 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.

  15. Methods of Liver Stem Cell Therapy in Rodents as Models of Human Liver Regeneration in Hepatic Failure. (United States)

    Hashemi Goradel, Nasser; Darabi, Masoud; Shamsasenjan, Karim; Ejtehadifar, Mostafa; Zahedi, Sarah


    Cell therapy is a promising intervention for treating liver diseases and liver failure. Different animal models of human liver cell therapy have been developed in recent years. Rats and mice are the most commonly used liver failure models. In fact, rodent models of hepatic failure have shown significant improvement in liver function after cell infusion. With the advent of stem-cell technologies, it is now possible to re-programme adult somatic cells such as skin or hair-follicle cells from individual patients to stem-like cells and differentiate them into liver cells. Such regenerative stem cells are highly promising in the personalization of cell therapy. The present review article will summarize current approaches to liver stem cell therapy with rodent models. In addition, we discuss common cell tracking techniques and how tracking data help to direct liver cell therapy research in animal models of hepatic failure.

  16. Distribution specificity of polarized populations of T helper cells in patients with chronic hepatitis B virus infection

    Institute of Scientific and Technical Information of China (English)

    JIANG Rong-long; FENG Xiao-rong; LU Qiao-sheng; LUO Kang-xian; FU Ning


    Objective: To investigate the roles of the polarized populations of T helper cells isolated from the peripheral blood mononuclear cells (PBMCs) of individuals with chronic hepatitis B virus (HBV) infection. Methods: PBMCs from patients with chronic HBV infection were separated routinely, stimulated by PMA, ionomycin and monensin, and the production of IL-4, IFN-γ and TGF-β by CD4+ T cells was observed by flow cytometry(FACS). Results: The percentages of the T cells producing IFN-γ, IL-4 or TGF-β ranged from 2.3% to 18.6%, 1.1% to 8.7% and 0.7% to 7.1% respectively among CD4+ cells from non-infected individuals. The majority of CD4+ T cells in PBMCs from individuals with chronic HBV infection were Th0 cells. The proportion of Th1 cells in patients with active chronic hepatitis B was higher than that in patients at inactive stage of the disease (P<0.05), indicating a significant elevation of Thl cells with the hepatic inflammation activity. The percentage of Th2 cells in individuals with HBV infection was higher than that in controls (P<0.05),but showed no difference between different patients (P>0.05). The percentage of Th3 cells was higher in asymptomatic HBV carriers than that in patients with chronic hepatitis B and in healthy controls (P<0.05). Conclusions: Th1-type cytokines are related with hepatic inflammation activity of chronic hepatitis B, and Th2 cells may be associated with the persistence of HBV infection. Th3 cells cooperating with Th2 cells are likely to function as negative immunoregulator, and may be responsible for the immune tolerance state of chronic HBV infection.

  17. Expression of MicroRNA miR-122 Facilitates an Efficient Replication in Nonhepatic Cells upon Infection with Hepatitis C Virus (United States)

    Fukuhara, Takasuke; Kambara, Hiroto; Shiokawa, Mai; Ono, Chikako; Katoh, Hiroshi; Morita, Eiji; Okuzaki, Daisuke; Maehara, Yoshihiko; Koike, Kazuhiko


    Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly. PMID:22593164

  18. Matrine induces the hepatic differentiation of WB-F344 rat hepatic progenitor cells and inhibits Jagged 1/HES1 signaling. (United States)

    Yang, Zhiyun; Wang, Li; Wang, Xianbo


    Matrine is a Chinese medicine, which is widely utilized for the attenuation of liver injuries and promotion of liver regeneration. It was previously observed that the in vivo administration of matrine promoted oval cell‑mediated liver regeneration in a rat model, suggesting that this compound may affect the differentiation of hepatic progenitor cells. The present study aimed to determine the mechanisms underlying this observation and to investigate the effect of matrine on the differentiation of the WB‑F344 rat hepatic progenitor cell line. Matrine was administered to rats, and rat serum was collected. WB‑F344 cells were cultured in the presence or absence of the rat serum for 24‑72 h, and the effects on cell viability and proliferation were assessed using acridine orange/propidium iodide staining and a 3‑(4,5‑dimethylthiazol‑2‑yl) ‑2,5‑diphenyltetrazolium bromide assay. The expression of albumin (ALB, a hepatocyte marker) and the notch signaling pathway ligand, Jagged 1, were assessed using immunohistochemistry and western blotting, and the mRNA transcription of ALB, Jagged 1 and hairy and enhancer of split‑1 (HES1, another notch signaling ligand) were measured using reverse transcription‑polymerase chain reaction analysis. The results showed that proliferation of the WB‑F344 cells was inhibited by matrine serum in a concentration‑ and time‑dependent manner. Matrine serum downregulated Jagged 1 and HES1, and upregulated ALB, indicating the induction of WB‑F344 cell differentiation. The effects of matrine serum were reversed by supplementing the culture medium with 0.1 mol/l parathyroid hormone, a Notch signaling pathway activator. In conclusion, matrine induced hepatic differentiation of the hepatic progenitor cells, likely by inhibiting the Jagged 1/HES1 signaling pathway.

  19. T cell responses to hepatitis B surface antigen are detectable in non-vaccinated individuals

    Institute of Scientific and Technical Information of China (English)

    Martin R Weihrauch; Michael von Bergwelt-Baildon; Milos Kandic; Martin Weskott; Winfried Klamp; Joachim R(o)sier; Joachim L Schultze


    AIM: To evaluate, whether humoral hepatitis-B-vaccine non-responders also fail to mount a T cell response and to compare these results to normal vaccinees.METHODS: Fourty-seven health care employees were enrolled in this study including all available nonresponders (n = 13) with an anti-HBsAg titer 1000 kU/L as controls.PBMC from all subjects were analyzed by IFN-γ and IL-4 ELISPOT assays for the presence of hepatitis B surface antigen (HBsAg) reactive T cells.RESULTS: Non-responders and low-responders had no or only very limited T cell responses, respectively.Individuals responding to vaccination with the induction of a high anti-HBsAg titer showed a strong T cell response after the third vaccination.Surprisingly, these individuals showed response even before the first vaccination.T cell response to control antigens and mitogens was similar in all groups.CONCLUSION: Our data suggest that there is no general immune deficiency in non-/low-responders.Thus,we hypothesize that the induction of anti-HBsAg responses by vaccination is significantly dependent on the pre-existing T cell repertoire against the specific antigen rather than the presence of a general T cell defect.

  20. Transcriptomic and proteomic analysis of human hepatic stellate cells treated with natural taurine. (United States)

    Liang, Jian; Deng, Xin; Wu, Fa-Sheng; Tang, Yan-Fang


    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.

  1. Autologous Stem Cells Transplantation in Egyptian Patients with Liver Cirrhosis on Top of Hepatitis C Virus (United States)

    Al Tayeb, Hoda; El Dorry, Ahmed; Amer, Nehad; Mowafy, Nadia; Zimaity, Maha; Bayoumy, Essam; Saleh, Shereen A.


    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

  2. Graptopetalum paraguayense ameliorates chemical-induced rat hepatic fibrosis in vivo and inactivates stellate cells and Kupffer cells in vitro.

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    Li-Jen Su

    Full Text Available 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 (CCl(4-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 CCl(4-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.

  3. Dedifferentiated adipocyte-derived progeny cells (DFAT cells)


    Wei, Shengjuan; Zan, Linsen; Hausman, Gary J.; Rasmussen, Theodore P; Bergen, Werner G.; Dodson, Michael V.


    Analyses of mature adipocytes have shown that they possess a reprogramming ability in vitro, which is associated with dedifferentiation. The subsequent dedifferentiated fat cells (DFAT cells) are multipotent and can differentiate into adipocytes and other cell types as well. Mature adipocytes can be easily obtained by biopsy, and the cloned progeny cells are homogeneous in vitro. Therefore, DFAT cells (a new type of stem cell) may provide an excellent source of cells for tissue regeneration, ...

  4. Inhibitory effects of prostaglandin E1 on activation of hepatic stellate cells in rabbits with schistosomiasis

    Institute of Scientific and Technical Information of China (English)

    Wei-Long Zou; Zhen Yang; Yun-Jin Zang; Dong-Jian Li; Zhi-Peng Liang; Zhong-Yang Shen


    BACKGROUND:Liver ifbrosis is the result of an imbalance between synthesis and degradation of extracellular matrix proteins of the liver. At the cellular and molecular levels, this progressive process is mainly characterized by activation of hepatic stellate cells (HSCs). Schistosoma japonica is one of the most prevalent causes of liver ifbrosis in China. It is characterized by hepatocyte damage, inlfammation, and chronic parasite egg-induced granuloma formation leading to ifbrosis. This study aimed to investigate the inhibitory effects of prostaglandin E1 (PGE1) on activation of HSCs and the alteration of type Ⅰ and Ⅲ collagen in rabbits with schistosomiasis. The study may promote the clinical application of praziquantel and PGE1 as a combined therapy to reverse hepatic ifbrosis caused by schistosomiasis. METHODS: Rabbits were percutaneously infected with cercaria of S. japonicum. Seven rabbits were subjected to intravenous injections of PGE1 (2.5 μg/kg daily) from days 60 to 120 after infection. The ultrastructural changes in activated HSCs were observed under transmission electron microscopy. The expression ofα-smooth muscle actin (α-SMA) was detected by immunohistochemistry. Fibril-forming collagens were detected by picrosirius staining. RESULTS: Activation of HSCs was a characteristic alteration in schistosome-induced hepatic ifbrosis. The expression of contraction-related α-SMA and the content of collagens were increased. Exogenous PGE1 markedly inhibited the activation of HSCs and reduced the expression of α-SMA around the hepatic sinusoids (P CONCLUSIONS:Activation of HSCs may play a key role in the progress of schistosome-induced hepatic ifbrosis. PGE1 effectively protects rabbit liver from ifbrosis, at least in part by inhibiting the activation of HSCs.

  5. Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture. (United States)

    Anzai, Kazuya; Chikada, Hiromi; Tsuruya, Kota; Ida, Kinuyo; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tesuya; Kamiya, Akihide


    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.

  6. Generation and characterization of functional cardiomyocytes derived from human T cell-derived induced pluripotent stem cells.

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    Tomohisa Seki

    Full Text Available Induced pluripotent stem cells (iPSCs have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.

  7. High level expression of the capsid protein of hepatitis E virus in diverse eukaryotic cells using the Semliki Forest virus replicon. (United States)

    Torresi, J; Meanger, J; Lambert, P; Li, F; Locarnini, S A; Anderson, D A


    The capsid protein of hepatitis E virus (HEV) is encoded by open reading frame 2 (ORF 2) and exhibits variable processing when expressed in insect and COS cells, but nothing is known of its processing in cells relevant to its replication. The full-length ORF 2 protein was expressed at high levels in mammalian cells by insertion of ORF 2 in the Semliki Forest virus (SFV) replicon to generate rSFV/HEV ORF 2K. Expression of the capsid protein was detected readily by metabolic labelling and indirect immunofluorescence in BHK-21 cells transfected with RNA transcripts derived from rSFV/HEV ORF 2K. ORF 2 protein was also expressed at high levels in cells of diverse origin, including liver-derived cell lines Huh7 and HepG2, following infection with recombinant virus derived from cotransfection of BHK-21 cells with the rSFV/HEV ORF 2K and helper SFV replicon RNAs. The addition of hypertonic KCl during metabolic labelling reduced the level of host cell protein synthesis and enhanced the detection of intermediates in ORF 2 protein processing. The wide host range and high level expression directed by SFV replicon particles has particular utility in the analysis of cell-specific factors in the protein processing and assembly of non-cultivable viruses such as HEV.

  8. Challenges for the therapeutic use of pluripotent stem derived cells

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    Magda eForsberg


    Full Text Available Human embryonic stem cells (hESC and induced pluripotent stem cells (hiPSC are an attractive cell source for regenerative medicine. These cells can be expanded to vast numbers and can be differentiated to many cell types to generate pluripotent stem cells (PSC derived therapeutic cells. These cells are desired for cell transplantations. Cell replacement is promising, but it has many challenges. The challenge of introduction of exogenous cells in a recipient requires addressing several different topics; the immunological response and possible rejection, cleanliness, exclusion of tumor formation and functionality of the PSC derived therapeutic cells. Immunological rejection can be addressed with immunomodulation of the cells and the recipient. Cleanliness can be optimized using good manufacturing practice (GMP quality systems. Tumor formation requires the removal of any PSC remaining after differentiation. At last, the functionality of the cells must be tested in in-vitro and in animal models. After addressing these challenges, precise strategies are developed to monitor the status of the cells at different times and in case of undesired results, corresponding counteracting strategies must exist before any clinical attempt.

  9. Promoted differentiation of cynomolgus monkey ES cells into hepatocyte-like cells by co-culture with mouse fetal liver-derived cells

    Institute of Scientific and Technical Information of China (English)

    Ko Saito; Masahide Yoshikawa; Yukiteru Ouji; Kei Moriya; Mariko Nishiofuku; Shigehiko Ueda; Noriko Hayashi; Shigeaki Ishizaka; Hiroshi Fukui


    AIM:To explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes.METHODS:Mouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLCassisted differentiation, spontaneous differentiation,and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4,hepatocyte growth factor (HGF), oncostatin M (OSM),and dexamethasone.RESULTS:The mRNA expression of α-fetoprotein,albumin (ALB), α-1-antitrypsin, and hepatocyte nuclear factor 4α was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes,was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Tmmunocytochemical analysis revealed an increased ratio of ALS-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storage and urea synthesis were also demonstrated.CONCLUSION:MFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.

  10. Chronic hepatitis B serum promotes apoptotic damage in human renal tubular cells

    Institute of Scientific and Technical Information of China (English)

    Cun-Liang Deng; Xin-Wen Song; Hai-Jun Liang; Chen Feng; Yun-Jian Sheng; Ming-Yong Wang


    AIM: To investigate the effect of the serum of patients with chronic hepatitis B (CHB) on apoptosis of renal tubular epithelial cells in vitro and to study the role of hepatitis B virus (HBV) and transforming growth factor-β1 (TGF-β1) in the pathogenesis of hepatitis B virus associated glomerulonephritis (HBV-GN).METHODS: The levels of serum TGF-β1 were measured by specific enzyme linked immunosorbent assay (ELISA) and HBV DNA was tested by polymerase chain reaction (PCR) in 44 patients with CHB ,and 20 healthy persons as the control. The normal human kidney proximal tubular cell (HK-2) was cultured together with the sera of healthy persons, CHB patients with HBV-DNA negative(20 cases) and HBV-DNA positive (24 cases) for up to 72 h. Apoptosis and Fas expression of the HK-2 were detected by flow cytometer.RESULTS: The apoptosis rate and Fas expression of HK-2 cells were significantly higher in HBV DNA positive serum group 19.01±5.85% and 17.58±8.35%, HBV DNA negative serum group 8.12±2.80% and 6.96 ± 2.76% than those in control group 4.25±0.65% and 2.33 ± 1.09%, respectively (P < 0.01). The apoptosis rate and Fas expression of HK-2 in HBV DNA positive serum group was significantly higher than those in HBV DNA negative serum (P < 0.01). Apoptosis rate of HK-2 cells in HBV DNA positive serum group was positively correlated with the level of HBV-DNA (r = 0.657). The level of serum TGF-β1 in CHB group was 163.05 ± 91.35 μg/L, significantly higher as compared with 81.40 ± 40.75 μg/L in the control group (P < 0.01).CONCLUSION: The serum of patients with chronic hepatitis B promotes apoptotic damage in human renal tubular cells by triggering a pathway of Fas up-regulation. HBV and TGF-β1 may play important roles in the mechanism of hepatitis B virus associated glomerulonephritis.

  11. Blockade of Tim-3 Pathway Ameliorates Interferon-γ Production from Hepatic CD8+ T Cells in a Mouse Model of Hepatitis B Virus Infection

    Institute of Scientific and Technical Information of China (English)

    Ying Ju; Nan Hou; Xiaoning Zhang; Di Zhao; Ying Liu; Jinjin Wang; Fang Luan; Wei Shi; Faliang Zhu; Wensheng Sun; Lining Zhang; Chengjiang Gao; Lifen Gao; Xiaohong Liang; Chunhong Ma


    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.

  12. Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells. (United States)

    Kassmer, Susannah H; Bruscia, Emanuela M; Zhang, Ping-Xia; Krause, Diane S


    Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM.

  13. Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

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    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 (SPRI)


    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.

  14. Cell culture systems for the hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Gilles Duverlie; Czeslaw Wychowski


    Since the discovery of HCV in 1989, the lack of a cell culture system has hampered research progress on this important human pathogen. No robust system has been obtained by empiric approaches, and HCV cell culture remained hypothetical until 2005. The construction of functional molecular clones has served as a starting point to reconstitute a consensus infectious cDNA that was able to transcribe infectious HCV RNAs as shown by intrahepatic inoculation in a chimpanzee. Other consensus clones have been selected and established in a human hepatoma cell line as replicons, i.e. self-replicating subgenomic or genomic viral RNAs. However, these replicons did not support production of infectious virus. Interestingly, some full-length replicons could be established without adaptive mutations and one of them was able to replicate at very high levels and to release virus particles that are infectious in cell culture and in vivo. This new cell culture system represents a major breakthrough in the HCV field and should enable a broad range of basic and applied studies to be achieved.

  15. Hepatitis B virus (HBV) variants fluctuate in paired plasma and peripheral blood mononuclear cells among patient cohorts during different chronic hepatitis B (CHB) disease phases. (United States)

    Coffin, C S; Osiowy, C; Gao, S; Nishikawa, S; van der Meer, F; van Marle, G


    Hepatitis B virus is classically considered a hepatotropic virus but also infects peripheral blood mononuclear cells. Chronic hepatitis B has different disease phases modulated by host immunity. We compared HBV variability, drug resistance and immune escape mutations in the overlapping HBV polymerase/surface gene in plasma and peripheral blood mononuclear cells in different disease phases. Plasma and peripheral blood mononuclear cells were isolated from 22 treatment naïve patient cohorts (five inactive, six immune-active, nine HBeAg negative and two immune-tolerant). HBV was genotyped via line probe assay, hepatitis B surface antigen titres were determined by an in-house immunoassay, and HBV DNA was quantified by kinetic PCR. The HBV polymerase/surface region, including full genome in some, was PCR-amplified and cloned, and ~20 clones/sample were sequenced. The sequences were subjected to various mutational and phylogenetic analyses. Clonal sequencing showed that only three of 22 patients had identical HBV genotype profiles in both sites. In immune-active chronic hepatitis B, viral diversity in plasma was higher compared with peripheral blood mononuclear cells. Mutations at residues, in a minority of clones, associated with drug resistance, and/or immune escape were found in both compartments but were more common in plasma. Immune escape mutations were more often observed in the peripheral blood mononuclear cells of immune-active CHB carriers, compared with other disease phases. During all CHB disease phases, differences exist between HBV variants found in peripheral blood mononuclear cells and plasma. Moreover, these data indicate that HBV evolution occurs in a compartment and disease phase-specific fashion.

  16. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells. (United States)

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza


    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.

  17. Cell Culture Models for the Investigation of Hepatitis B and D Virus Infection (United States)

    Verrier, Eloi R.; Colpitts, Che C.; Schuster, Catherine; Zeisel, Mirjam B.; Baumert, Thomas F.


    Chronic hepatitis B virus (HBV) and hepatitis D virus (HDV) infections are major causes of liver disease and hepatocellular carcinoma worldwide. Despite the presence of an efficient preventive vaccine, more than 250 million patients are chronically infected with HBV. Current antivirals effectively control but only rarely cure chronic infection. While the molecular biology of the two viruses has been characterized in great detail, the absence of robust cell culture models for HBV and/or HDV infection has limited the investigation of virus-host interactions. Native hepatoma cell lines do not allow viral infection, and the culture of primary hepatocytes, the natural host cell for the viruses, implies a series of constraints restricting the possibilities of analyzing virus-host interactions. Recently, the discovery of the sodium taurocholate co-transporting polypeptide (NTCP) as a key HBV/HDV cell entry factor has opened the door to a new era of investigation, as NTCP-overexpressing hepatoma cells acquire susceptibility to HBV and HDV infections. In this review, we summarize the major cell culture models for HBV and HDV infection, discuss their advantages and limitations and highlight perspectives for future developments. PMID:27657111

  18. Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential. (United States)

    Matsumoto, Taro; Kano, Koichiro; Kondo, Daisuke; Fukuda, Noboru; Iribe, Yuji; Tanaka, Nobuaki; Matsubara, Yoshiyuki; Sakuma, Takahiro; Satomi, Aya; Otaki, Munenori; Ryu, Jyunnosuke; Mugishima, Hideo


    When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.

  19. Tetramethylpyrazine Inhibits Activation of Hepatic Stellate Cells through Hedgehog Signaling Pathways In Vitro

    Directory of Open Access Journals (Sweden)

    Jue Hu


    Full Text Available Background and Aim. Tetramethylpyrazine (TMP, a major alkaloid isolated from Ligusticum chuanxiong, has been reported in hepatic fibrosis models. However, the action mechanism remains unclear. In the present study, effects of tetramethylpyrazine (TMP against hepatic stellate cell (HSC activation as well as the possible mechanisms were evaluated. Methods. Western blot assay was used to detect TMP effects on protein expression of Smo, Patched, Hhip, and Gli and to investigate the effects of TMP on Cyclin D1, Cyclin E1, CDK2, Bcl-2, Bax, and caspase expression with cyclopamine supplementation. Results. Our results showed that TMP significantly inhibits the expression of Cyclin D1, Cyclin E1, and Cyclin-dependent kinase CDK2 and changes the HSC cycle by inhibiting the proliferation of HSC. Moreover, TMP has also been shown to decrease the expression of Bcl-2 and increase the expression of Bax in HSC-T6 cells. Furthermore, TMP can inhibit the expression of connective tissue growth factor (CTGF, and the inhibitory effect was intensified after the application of joint treatment with TMP and cyclopamine. Conclusion. TMP may be an effective Hh signaling pathway inhibitor for hepatic fibrosis treatment.

  20. Potential cellular receptors involved in hepatitis C virus entry into cells

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    Muellhaupt Beat


    Full Text Available Abstract Hepatitis C virus (HCV infects hepatocytes and leads to permanent, severe liver damage. Since the genomic sequence of HCV was determined, progress has been made towards understanding the functions of the HCV-encoded proteins and identifying the cellular receptor(s responsible for adsorption and penetration of the virus particle into the target cells. Several cellular receptors for HCV have been proposed, all of which are associated with lipid and lipoprotein metabolism. This article reviews the cellular receptors for HCV and suggests a general model for HCV entry into cells, in which lipoproteins play a crucial role.

  1. Regulation of Hepatic Stellate Cells and Fibrogenesis by Fibroblast Growth Factors

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    Justin D. Schumacher


    Full Text Available Fibroblast growth factors (FGFs are a family of growth factors critically involved in developmental, physiological, and pathological processes, including embryogenesis, angiogenesis, wound healing, and endocrine functions. In the liver, several FGFs are produced basally by hepatocytes and hepatic stellate cells (HSCs. Upon insult to the liver, expression of FGFs in HSCs is greatly upregulated, stimulating hepatocyte regeneration and growth. Various FGF isoforms have also been shown to directly induce HSC proliferation and activation thereby enabling autocrine and paracrine regulation of HSC function. Regulation of HSCs by the endocrine FGFs, namely, FGF15/19 and FGF21, has also recently been identified. With the ability to modulate HSC proliferation and transdifferentiation, targeting FGF signaling pathways constitutes a promising new therapeutic strategy to treat hepatic fibrosis.

  2. Interleukin-8 derived from local tissue-resident stromal cells promotes tumor cell invasion. (United States)

    Welte, Gabriel; Alt, Eckhard; Devarajan, Eswaran; Krishnappa, Srinivasalu; Jotzu, Constantin; Song, Yao-Hua


    The aim of this study is to evaluate the role of adipose tissue resident stromal cells on tumor cell invasion. Our data show that a subpopulation of adipose tissue derived stromal cells expressing Nestin, NG2, α-smooth muscle actin and PDGFR-α migrate toward the cancer cells. Microarray analysis revealed the upregulation of IL-8 in the migrated cells. We demonstrated that stromal cell derived IL-8 promote the invasion and the anchorage-independent growth of cancer cells. We conclude that human breast cancer cells attract a subpopulation of stromal cells that secrete IL-8 to promote tumor cell invasion in a paracrine fashion.

  3. Derivation of the human embryonic stem cell line RCM1

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    P.A. De Sousa


    Full Text Available 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.

  4. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine (United States)

    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...

  5. Hepatic Stellate Cells Improve Engraftment of Human Primary Hepatocytes: A Preclinical Transplantation Study in an Animal Model. (United States)

    Dusabineza, Ange-Clarisse; Najimi, Mustapha; van Hul, Noémi; Legry, Vanessa; Khuu, Dung Ngoc; van Grunsven, Leo A; Sokal, Etienne; Leclercq, Isabelle A


    Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether cotransplantation of hepatocytes with hepatic stellate cells (HSCs) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSCs (quiescent or after culture activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin-positive (huAlb(+)) hepatocytes were found scattered. When cotransplanted in a mixture with HSCs or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2- to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSCs increased the number of hepatocytes entrapped in the host liver at an early time point posttransplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks posttransplantation, we found no accumulation of αSMA(+)-activated HSCs or collagen deposition. To follow the fate of transplanted HSCs, HSCs derived from GFP(+) mice were injected into GFP(-) littermates: 17 h posttransplant, GFP(+) HSCs were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Coculture with HSCs improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSCs. In vivo, cotransplantation of hepatocytes with HSCs into a healthy liver recipient does not generate fibrosis, but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing.

  6. Dedifferentiated adipocyte-derived progeny cells (DFAT cells) (United States)

    Wei, Shengjuan; Zan, Linsen; Hausman, Gary J; Rasmussen, Theodore P; Bergen, Werner G; Dodson, Michael V


    Analyses of mature adipocytes have shown that they possess a reprogramming ability in vitro, which is associated with dedifferentiation. The subsequent dedifferentiated fat cells (DFAT cells) are multipotent and can differentiate into adipocytes and other cell types as well. Mature adipocytes can be easily obtained by biopsy, and the cloned progeny cells are homogeneous in vitro. Therefore, DFAT cells (a new type of stem cell) may provide an excellent source of cells for tissue regeneration, engineering and disease treatment. The dedifferentiation of mature adipocytes, the multipotent capacity of DFAT cells and comparisons and contrasts with mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPS) are discussed in this review. PMID:23991357

  7. Focal adhesion kinase (FAK) mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV)-infected cells. (United States)

    Alisi, Anna; Arciello, Mario; Petrini, Stefania; Conti, Beatrice; Missale, Gabriele; Balsano, Clara


    Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs.

  8. Platelet-derived stromal cell-derived factor-1 is required for the transformation of circulating monocytes into multipotential cells.

    Directory of Open Access Journals (Sweden)

    Noriyuki Seta

    Full Text Available BACKGROUND: We previously described a primitive cell population derived from human circulating CD14(+ monocytes, named monocyte-derived multipotential cells (MOMCs, which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s derived from circulating CD14(- cells. The present study was conducted to identify factors that induce MOMC differentiation. METHODS: We cultured CD14(+ monocytes on fibronectin in the presence or absence of platelets, CD14(- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. RESULTS: The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1's critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14(+CXCR4(high cell population. CONCLUSION: The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs.

  9. Hepatic failure caused by plasma cell infiltration in multiple Myeloma

    Institute of Scientific and Technical Information of China (English)

    Fadi E Rahhal; Robert R Schade; Asha Nayak; Teresa A Coleman


    Although plasma cell infiltration is not rare in autopsy of patients with multiple myeloma (MM), it is very rarely detected in living patients. This is because MM rarely causes significant liver dysfunction that requires further evaluation. A 49-year-old man presented with acute renal failure and was diagnosed with kappa light chain MM stage ?B. Thalidomide and dexamethasone were initiated. The patient developed a continuous increase in bilirubin that led to severe cholestasis. A liver biopsy revealed plasma cell infiltration. He then rapidly progressed to liver failure and died. Treatment options are limited in MM with significant liver dysfunction.Despite new drug therapies in MM, those patients with rapidly progressive liver failure appear to have a dismal outcome.

  10. Derivation and application of pluripotent stem cells for regenerative medicine. (United States)

    Wang, Jiaqiang; Zhou, Qi


    Pluripotent stem cells (PSCs) are cells that can differentiate into any type of cells in the body, therefore have valuable promise in regenerative medicine of cell replacement therapies and tissue/organ engineering. PSCs can be derived either from early embryos or directly from somatic cells by epigenetic reprogramming that result in customized cells from patients. Here we summarize the methods of deriving PSCs, the various types of PSCs generated with different status, and their versatile applications in both clinical and embryonic development studies. We also discuss an intriguing potential application of PSCs in constructing tissues/organs in large animals by interspecies chimerism. All these emerging findings are likely to contribute to the breakthroughs in biological research and the prosperous prospects of regenerative medicine.

  11. Pretreatment Hepatoprotective Effect of the Marine Fungus Derived from Sponge on Hepatic Toxicity Induced by Heavy Metals in Rats

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    Nehad M. Abdel-Monem


    Full Text Available 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.

  12. Rat hepatic stellate cells alter the gene expression profile and promote the growth, migration and invasion of hepatocellular carcinoma cells. (United States)

    Wang, Zhi-Ming; Zhou, Le-Yuan; Liu, Bin-Bin; Jia, Qin-An; Dong, Yin-Ying; Xia, Yun-Hong; Ye, Sheng-Long


    The aim of the present study was to examine the effects of activated hepatic stellate cells (HSCs) and their paracrine secretions, on hepatocellular cancer cell growth and gene expression in vitro and in vivo. Differentially expressed genes in McA-RH7777 hepatocellular carcinoma (HCC) cells following non-contact co-culture with activated stellate cells, were identified by a cDNA microarray. The effect of the co-injection of HCC cells and activated HSCs on tumor size in rats was also investigated. Non-contact co-culture altered the expression of 573 HCC genes by >2-fold of the control levels. Among the six selected genes, ELISA revealed increased protein levels of hepatic growth factor, matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9). Incubation of HCC cells with medium conditioned by activated HSCs significantly increased the proliferation rate (Pprofile of HCC cells and affected their growth, migration and invasiveness. The results from the present study indicate that the interaction between the activated HSCs and HCC has an important role in the development of HCC.

  13. Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen

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    Arnaud Carpentier


    Full Text Available The establishment of protocols to differentiate human pluripotent stem cells (hPSCs including embryonic (ESC and induced pluripotent (iPSC stem cells into functional hepatocyte-like cells (HLCs creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli.

  14. Metformin-mediated Bambi expression in Hepatic Stellate Cells induces pro-survival Wnt/β-catenin signaling


    Subramaniam, Nanthakumar; Sherman, Mara H.; Rao, Renuka; Wilson, Caroline; Coulter, Sally; Atkins, Annette R.; Evans, Ronald M.; Liddle, Christopher; Downes, Michael


    Adenosine monophosphate-activated protein kinase (AMPK) regulates lipid, cholesterol and glucose metabolism in specialized metabolic tissues, such as muscle, liver and adipose tissue. Agents that activate AMPK, such as metformin and AICAR, have beneficial effects on liver glucose and lipid metabolism. Additionally, AMPK activation in proliferating hepatic stellate cells (HSCs) induces growth arrest and inhibits hepatic fibrosis. As metformin and AICAR act in different ways to achieve their ef...

  15. Viral replication modulated by synthetic peptide derived from hepatitis B virus X protein

    Institute of Scientific and Technical Information of China (English)

    Chang-Zheng Song; Qing-Wei Wang; Chang-Cheng Song; Zeng-Liang Bai


    AIM: A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We reported it here for the first time not only to highlight the biohazard issue and safety importance for viral peptide vaccine, but also to explore the effect of a fully conserved peptide on HBV replication within the carboxyl terminus of HBx.METHODS: We synthesized the fully conserved peptide (CP)with nine residues, FVLGGCRHK. HBV-producing 2.2.15 cells were treated with or without 3.5 μM CP for 36 hours.Quantitative detection of viral DNA was performed by realtime PCR. HBV antigens were determined by enzyme-linked immunoadsorbent assay (ELISA). Quantitative analyses of p53 and Bax proteins were based on immunofluorescence.Flow cytometry was performed to detect cell cycle and apoptosis.RESULTS: Both extracellular and intracellular copies of HBV DNA per ml were significantly increased atter incubation with 3.5 μM of CP. HBsAg and HBeAg in the cultured medium of CP-treatment cells were as abundant as untreated control cells. CP infiuenced negatively the extracellular viral gene products, and 3.5 μM CP could significantly inhibit intracellular HBsAg expression. In response to CP, intracellular HBeAg displayed an opposite pattern to that of HBsAg, and 3.5 μM CP could efficiently increase the level of intracellular HBeAg.Flow cytometric analyses exhibited no significant changes on cell cycle, apoptosis, p53 and Bax proteins in 2.2.15 cells with or without CP.CONCLUSION: Together with the resulte generated from the synthetic peptide, we address that the conserved region,a domain of HBx, may be responsible for modulating HBV replication. As conserved peptides from infectious microbes are used as immunogens to elicit immune responses, their latent biological hazard for human beings should be evaluated.

  16. Glucocorticoids Have Opposing Effects on Liver Fibrosis in Hepatic Stellate and Immune Cells. (United States)

    Kim, Kang Ho; Lee, Jae Man; Zhou, Ying; Harpavat, Sanjiv; Moore, David D


    Liver fibrosis is a reversible wound-healing process that is protective in the short term, but prolonged fibrotic responses lead to excessive accumulation of extracellular matrix components that suppresses hepatocyte regeneration, resulting in permanent liver damage. Upon liver damage, nonparenchymal cells including immune cells and hepatic stellate cells (HSCs) have crucial roles in the progression and regression of liver fibrosis. Here, we report differential roles of the glucocorticoid receptor (GR), acting in immune cells and HSCs, in liver fibrosis. In the carbon tetrachloride hepatotoxin-induced fibrosis model, both steroidal and nonsteroidal GR ligands suppressed expression of fibrotic genes and decreased extracellular matrix deposition but also inhibited immune cell infiltration and exacerbated liver injury. These counteracting effects of GR ligands were dissociated in mice with conditional GR knockout in immune cells (GR(LysM)) or HSC (GR(hGFAP)): the impacts of dexamethasone on immune cell infiltration and liver injury were totally blunted in GR(LysM) mice, whereas the suppression of fibrotic gene expression was diminished in GR(hGFAP) mice. The effect of GR activation in HSC was further confirmed in the LX-2 HSC cell line, in which antifibrotic effects were mediated by GR ligand inhibition of Sma and mad-related protein 3 (SMAD3) expression. We conclude that GR has differential roles in immune cells and HSCs to modulate liver injury and liver fibrosis. Specific activation of HSC-GR without alteration of GR activity in immune cells provides a potential therapeutic approach to treatment of hepatic fibrosis.

  17. Fullerene derivatives protect endothelial cells against NO-induced damage

    Energy Technology Data Exchange (ETDEWEB)

    Lao Fang; Han Dong; Qu Ying; Liu Ying; Zhao Yuliang; Chen Chunying [CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 (China); Li Wei [CAS Key Laboratory for Nuclear Analytical Techniques, Institute of High Energy Physics (IHEP), Chinese Academy of Sciences, Beijing 100049 (China)], E-mail:


    Functional fullerene derivatives have been demonstrated with potent antioxidation properties. Nitric oxide (NO) is a free radical that plays a part in leading to brain damage when it is accumulated to a high concentration. The possible scavenging activity of NO by the hydroxylated fullerene derivative C{sub 60}(OH){sub 22} and malonic acid derivative C{sub 60}(C(COOH){sub 2}){sub 2} was investigated using primary rat brain cerebral microvessel endothelial cells (CMECs). Results demonstrate that sodium nitroprusside (SNP), used as an NO donor, caused a marked decrease in cell viability and an increase in apoptosis. However, fullerene derivatives can remarkably protect against the apoptosis induced by NO assault. In addition, fullerene derivatives can also prevent NO-induced depolymerization of cytoskeleton and damage of the nucleus and accelerate endothelial cell repair. Further investigation shows that the sudden increase of the intercellular reactive oxygen species (ROS) induced by NO was significantly attenuated by post-treatment with fullerene derivatives. Our results suggest that functional fullerene derivatives are potential applications for NO-related disorders.

  18. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor


    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  19. Adipose Tissue-Derived Stem Cells for Myocardial Regeneration (United States)

    Joo, Hyung Joon; Kim, Jong-Ho


    Over the past decade, stem cell therapy has been extensively studied for clinical application for heart diseases. Among various stem cells, adipose tissue-derived stem cell (ADSC) is still an attractive stem cell resource due to its abundance and easy accessibility. In vitro studies showed the multipotent differentiation potentials of ADSC, even differentiation into cardiomyocytes. Many pre-clinical animal studies have also demonstrated promising therapeutic results of ADSC. Furthermore, there were several clinical trials showing the positive results in acute myocardial infarction using ADSC. The present article covers the brief introduction, the suggested therapeutic mechanisms, application methods including cell dose and delivery, and human clinical trials of ADSC for myocardial regeneration.

  20. OVCAR-3 spheroid-derived cells display distinct metabolic profiles.

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    Kathleen A Vermeersch

    Full Text Available Recently, multicellular spheroids were isolated from a well-established epithelial ovarian cancer cell line, OVCAR-3, and were propagated in vitro. These spheroid-derived cells displayed numerous hallmarks of cancer stem cells, which are chemo- and radioresistant cells thought to be a significant cause of cancer recurrence and resultant mortality. Gene set enrichment analysis of expression data from the OVCAR-3 cells and the spheroid-derived putative cancer stem cells identified several metabolic pathways enriched in differentially expressed genes. Before this, there had been little previous knowledge or investigation of systems-scale metabolic differences between cancer cells and cancer stem cells, and no knowledge of such differences in ovarian cancer stem cells.To determine if there were substantial metabolic changes corresponding with these transcriptional differences, we used two-dimensional gas chromatography coupled to mass spectrometry to measure the metabolite profiles of the two cell lines.These two cell lines exhibited significant metabolic differences in both intracellular and extracellular metabolite measurements. Principal components analysis, an unsupervised dimensional reduction technique, showed complete separation between the two cell types based on their metabolite profiles. Pathway analysis of intracellular metabolomics data revealed close overlap with metabolic pathways identified from gene expression data, with four out of six pathways found enriched in gene-level analysis also enriched in metabolite-level analysis. Some of those pathways contained multiple metabolites that were individually statistically significantly different between the two cell lines, with one of the most broadly and consistently different pathways, arginine and proline metabolism, suggesting an interesting hypothesis about cancerous and stem-like metabolic phenotypes in this pair of cell lines.Overall, we demonstrate for the first time that metabolism

  1. An immunodominant HLA-A*1101-restricted CD8+ T-cell response targeting hepatitis B surface antigen in chronic hepatitis B patients. (United States)

    Chen, Xiaoling; Wang, Wenbo; Wang, Shufeng; Meng, Gang; Zhang, Mengjun; Ni, Bing; Wu, Yuzhang; Wang, Li


    Hepatitis B virus (HBV) infection is a worldwide public health problem. HBV-specific CD8(+) CTLs are vital for viral clearance. Identification of immunodominant CTL epitopes from HBV-associated antigens is necessary for therapeutic vaccine development. We showed that the HLA-A*1101 allele is one of the most common alleles in both healthy individuals and chronic hepatitis B (CHB) patients in the Chongqing area, China. However, less than 10% of epitopes of HBV-associated antigens have been identified in an HLA-A*1101 context. Here, we describe an immunodominant CD8(+) T-cell response targeting a hepatitis B surface antigen determinant (HBs(295-304)) restricted by HLA-A*1101 in both healthy individuals and CHB patients. Moreover, HBs(295-304) is more immunogenic for CTL induction than a known naturally HLA-A*1101-processed epitope from hepatitis B core antigen (HBc(88-96)). Therefore, the newly identified epitope, HBs(295-304), will benefit the development of immunotherapeutic approaches for HBV infection.

  2. Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells (HSC). (United States)

    Breitkopf, Katja; Roeyen, Claudia van; Sawitza, Iris; Wickert, Lucia; Floege, Jürgen; Gressner, Axel M


    The platelet-derived growth factor (PDGF) family, which regulates many physiological and pathophysiological processes has recently been enlarged by two new members, the isoforms PDGF-C and -D. Little is known about the expression levels of these new members in hepatic fibrosis. We therefore investigated by quantitative real time PCR (Taqman) the mRNA expression profiles of all four PDGF isoforms in transdifferentiating primary cultured hepatic stellate cells (HSC), an in vitro model system of hepatic fibrogenesis, either with or without stimulation of the cells with PDGF-BB or TGF-beta1. All four isoforms were expressed in HSC transdifferentiating to myofibroblast-like cells (MFB) albeit with different profiles: while PDGF-A mRNA exhibited minor fluctuations only, PDGF-B was rapidly down-regulated. In contrast, both PDGF-C and -D mRNA were strongly induced: PDGF-C up to 5 fold from day 2 to day 8 and PDGF-D up to 8 fold from day 2 to day 5 of culture. Presence of PDGF-DD in activated HSC was confirmed at the protein level by immunocytochemistry. Stimulation of HSC and MFB with PDGF-BB led to down-regulation of the new isoforms, whereas TGF-beta1 upregulated PDGF-A only. We further show that PDGF receptor-beta (PDGFR-beta) mRNA was rapidly upregulated within the first day of culture and was constantly expressed from day 2 on while the expression profile of PDGFR-alpha mRNA was very similar to that of PDGF-A during transdifferentiation. Given the dramatic changes in PDGF-C and -D expression, which may compensate for down-regulation of PDGF-B, we hypothesize that the new PDGF isoforms may fulfil specific functions in hepatic fibrogenesis.

  3. Polythiol-containing, recombinant mannosylated-albumin is a superior CD68+/CD206+ Kupffer cell-targeted nanoantioxidant for treatment of two acute hepatitis models. (United States)

    Maeda, Hitoshi; Hirata, Kenshiro; Watanabe, Hiroshi; Ishima, Yu; Chuang, Victor Tuan Giam; Taguchi, Kazuaki; Inatsu, Akihito; Kinoshita, Manabu; Tanaka, Motohiko; Sasaki, Yutaka; Otagiri, Masaki; Maruyama, Toru


    Since reactive oxygen species (ROS) derived from Kupffer cells (KC), especially CD68(+) KC, play a key role in the induction of hepatic oxidative stress and injuries, we developed a polythiolated- and mannosylated human serum albumin (SH-Man-HSA), which functions as a novel nanoantioxidant for delivering thiol to CD68(+) KC. In vitro electron paramagnetic resonance coupled with pharmacokinetics and immunohistochemical studies showed that SH-Man-HSA possessed powerful radical-scavenging activity and rapidly and selectively delivered thiols to the liver via mannose receptor (CD206) on CD68(+) cells. SH-Man-HSA significantly improved the survival rate of concanavalin-A (Con-A)-treated mice. Moreover, SH-Man-HSA exhibited excellent hepatoprotective functions, not by decreasing tumor necrosis factor or interferon-γ production that is closely associated with Con-A-induced hepatitis, but by suppressing ROS production. Interestingly, the protective effect of SH-Man-HSA was superior to N-acetyl cysteine (NAC). This could be attributed to the difference in the inhibition of hepatic oxidative stress between the two antioxidants depending on their potential for thiol delivery to the liver. Similar results were also observed for acetaminophen (APAP)-induced hepatopathy models. Flow cytometric data further confirmed that an increase in F4/80(+)/ROS(+) cells was dramatically decreased by SH-Man-HSA. The administration of SH-Man-HSA at 4 hours following a Con-A or APAP injection also exhibited a profound hepatoprotective action against these hepatitis models, whereas this was not observed for NAC. It can be concluded therefore that SH-Man-HSA has great potential for use in a rescue therapy for hepatopathy as a nanoantioxidant because of its ability to efficiently and rapidly deliver thiols to CD68(+)/CD206(+) KC.

  4. Flow cytometry assay of myeloid dendritic cells (mDCs) in peripheral blood during acute hepatitis C: Possible pathogenetic mechanisms

    Institute of Scientific and Technical Information of China (English)

    Alessandro Perrella; Oreste Perrella; Luigi Atripaldi; Pasquale Bellopede; Tommaso Patarino; Costanza Sbreglia; Giovanni Tarantino; Paolo Sorrentino; Paolo Conca; Luca Ruggiero


    AIM: To asses the expression of myeloid dendritic cells (CD11c+) subset during acute HCV hepatitis and its possible involvement in natural history of the infection.METHODS: We enrolled 11 patients with acute hepatitis C (AHC) (Group A), 10 patients with acute hepatitis A (AHA) (as infective control-Group B) and 10 healthy donors (group C) in this study. All patients underwent selective flow cytometry gating strategies to assess the peripheral number of the myeloid dendritic cells (mDCs)to understand the possible role and differences during acute hepatitis.RESULTS: Eight of 11 patients with acute HCV hepatitis did not show any increase of mDCs compared to healthy individuals, while a significant decrease of mDCs was found in absolute cell count (z=-2.37; P<0.05) and percentage (z=-2.30; P<0.05) as compared with AHA.On the contrary, The remaining three patients of the group A had a higher mDCs number and percentage as occur in group B. Interestingly, after six months, those patients did not show any increase of mDCs subset were chronically infected. while the three subjects with an increase of peripheral mDCs, as in HAV acute infection,resolved the illness.CONCLUSION: The lack of increase of mDCs during acute hepatitis C might be an important factor involved in chronicization of the infection.

  5. Long-term persistence of T cell memory to HBsAg after hepatitis B vaccination

    Institute of Scientific and Technical Information of China (English)

    Ru-Xiang Wang; Greet J. Boland; Jan van Hattum; Gijsbert C. de Gast


    AIM: To determine if the T cell memory to HBsAg can persist for a long time after hepatitis B (HB) vaccination.METHODS: Thirty one vaccine recipients who were healthcare workers (18 females and 13 males aged 34-58 years) from Utrecht University Hospital, Netherlands, and had previously Received a standard course of vaccination for hepatitis B were investigated and another 9 unvaccinated healthy volunteers from the same hospital were used as the control. Blood samples were taken just before the experiment to test serum anti-HBs levels and the subjects were classified into different groups according to their serum titers of anti-HBs and vaccination history. Their peripheral blood mononuclear cells (pBrvMc) were isolated from freshly heparinized venous blood and the proliferative response of Tlymphocytes to the recombinant hepatitis B surface antigen(HBsAg) was investigated.RESULTS: Positive serum anti-HBs was found in 61.3%(19/31) vaccine recipients and a significant in vitro lymphocyte proliferative response to recombinant HBsAg was observed in all the vaccinees with positive anti-HBs. Serum anti-HBs level ≤10 IU/L was found in 38.7% (12/31)subjects. In this study, we specially focused on lymphocyte proliferative response to recombinant HBsAg in those vaccine recipients with serum anti-HBsAg less than 10 IU/L.Most of them had Received a standard course of vaccination about 10 years before. T lymphocyte proliferative response was found positive in 7 of the 12 vaccine recipients. These results confirmed that HBsAg-specific memory T cells remained detectable in the circulation for a long time after vaccination, even when serum anti-HBs level had been undetectable.CONCLUSION: The T cell memory to HBsAg can persist for at least 10 years after HB vaccination. Further booster injection is not necessary in healthy responders to HB vaccine.

  6. Dendritic Cell-Derived Exosomes Stimulate Stronger CD8+ CTL Responses and Antitumor Immunity than Tumor Cell-Derived Exosomes

    Institute of Scientific and Technical Information of China (English)

    Siguo Hao; Ou Bai; Jinying Yuan; Mabood Qureshi; Jim Xiang


    Exosomes (EXO) derived from dendritic cells (DC) and tumor cells have been used to stimulate antitumor immune responses in animal models and in clinical trials. However, there has been no side-by-side comparison of the stimulatory efficiency of the antitumor immune responses induced by these two commonly used EXO vaccines. In this study, we selected to study the phenotype characteristics of EXO derived from a transfected EG7 tumor cells expressing ovalbumin (OVA) and OVA-pulsed DC by flow cytometry. We compared the stimulatory effect in induction of OVA-specific immune responses between these two types of EXO. We found that OVA protein-pulsed DCovA-derived EXO (EXODC) can more efficiently stimulate naive OVA-specific CD8+ T cell proliferation and differentiation into cytotoxic T lymphocytes in vivo, and induce more efficient antitumor immunity than EG7 tumor cell-derived EXO (EXOEG7). In addition, we elucidated the important role of the host DC in EXO vaccines that the stimulatory effect of EXO is delivered to T cell responses by the host DC. Therefore, DC-derived EXO may represent a more effective EXO-based vaccine in induction of antitumor immunity.

  7. Metformin-mediated Bambi expression in hepatic stellate cells induces prosurvival Wnt/β-catenin signaling. (United States)

    Subramaniam, Nanthakumar; Sherman, Mara H; Rao, Renuka; Wilson, Caroline; Coulter, Sally; Atkins, Annette R; Evans, Ronald M; Liddle, Christopher; Downes, Michael


    AMP-activated protein kinase (AMPK) regulates lipid, cholesterol, and glucose metabolism in specialized metabolic tissues, such as muscle, liver, and adipose tissue. Agents that activate AMPK, such as metformin and 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), have beneficial effects on liver glucose and lipid metabolism. In addition, AMPK activation in proliferating hepatic stellate cells (HSC) induces growth arrest and inhibits hepatic fibrosis. As metformin and AICAR act in different ways to achieve their effects, our aim was to examine the effects of AMPK activation in quiescent HSCs with these two agents on HSC function. We found that phospho-AMPK levels were markedly upregulated by both AICAR and metformin in quiescent HSCs. However, although AICAR treatment induced cell death, cells treated with metformin did not differ from untreated controls. AICAR-mediated HSC cell death was paralleled by loss of expression of the TGF-β decoy receptor Bambi, whereas metformin increased Bambi expression. Transfection of siRNA-Bambi into HSCs also induced cell death, mimicking the effects of AICAR, whereas overexpression of Bambi partially rescued AICAR-treated cells. As Bambi has previously been shown to promote cell survival through Wnt/β-catenin signaling, a reporter incorporating binding sites for a downstream target of this pathway was transfected into HSCs and was induced. We conclude that although AICAR and metformin both activate AMPK in quiescent HSCs, AICAR rapidly induced cell death, whereas metformin-treated cells remained viable. The finding that metformin increases Bambi expression and activates Wnt/β-catenin signaling provides a possible mechanistic explanation for this observation. These results suggest that AICAR and metformin may confer disease-specific therapeutic benefits.

  8. Radiation response of mesenchymal stem cells derived from bone marrow and human pluripotent stem cells


    Islam, Mohammad S; Stemig, Melissa E.; Takahashi, Yutaka; Hui, Susanta K.


    Mesenchymal stem cells (MSCs) isolated from human pluripotent stem cells are comparable with bone marrow-derived MSCs in their function and immunophenotype. The purpose of this exploratory study was comparative evaluation of the radiation responses of mesenchymal stem cells derived from bone marrow- (BMMSCs) and from human embryonic stem cells (hESMSCs). BMMSCs and hESMSCs were irradiated at 0 Gy (control) to 16 Gy using a linear accelerator commonly used for cancer treatment. Cells were harv...

  9. RNA-seq based transcriptome analysis of hepatitis E virus (HEV) and hepatitis B virus (HBV) replicon transfected Huh-7 cells. (United States)

    Jagya, Neetu; Varma, Satya Pavan Kumar; Thakral, Deepshi; Joshi, Prashant; Durgapal, Hemlata; Panda, Subrat Kumar


    Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (preplicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV.

  10. Genetic characteristics of the human hepatic stellate cell line LX-2.

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    Ralf Weiskirchen

    Full Text Available The human hepatic cell line LX-2 has been described as tool to study mechanisms of hepatic fibrogenesis and the testing of antifibrotic compounds. It was originally generated by immortalisation with the Simian Vacuolating Virus 40 (SV40 transforming (T antigen and subsequent propagation in low serum conditions. Although this immortalized line is used in an increasing number of studies, detailed genetic characterisation has been lacking. We here have performed genetic characterisation of the LX-2 cell line and established a single-locus short tandem repeat (STR profile for the cell line and characterized the LX-2 karyotype by several cytogenetic and molecular cytogenetic techniques. Spectral karyotyping (SKY revealed a complex karyotype with a set of aberrations consistently present in the metaphases analyses which might serve as cytogenetic markers. In addition, various subclonal and single cell aberrations were detected. Our study provides criteria for genetic authentication of LX-2 and offers insights into the genotype changes which might underlie part of its phenotypic features.

  11. Extracellular vesicles derived from preosteoblasts influence embryonic stem cell differentiation. (United States)

    Nair, Rekha; Santos, Lívia; Awasthi, Siddhant; von Erlach, Thomas; Chow, Lesley W; Bertazzo, Sergio; Stevens, Molly M


    Embryonic stem cells (ESCs) can differentiate into all cell types of the body and, therefore, hold tremendous promise for cell-based regenerative medicine therapies. One significant challenge that should be addressed before using ESCs in the clinic is to improve methods of efficiently and effectively directing the differentiation of this heterogeneous cell population. The work presented here examines the potential of harnessing naturally derived extracellular vesicles to deliver genetic material from mature cells to undifferentiated ESCs for the purpose of manipulating stem cell fate. Vesicles were isolated from preosteoblast cells and were found to be ∼170 nm in diameter and to express the CD40 surface marker. Multiple interactions were visualized between vesicles and ESCs using confocal microscopy, and no significant difference in cell viability was noted. Incubation with vesicles caused significant changes in ESC gene expression, including persistence of pluripotent gene levels as well as increased neurectoderm differentiation. Genetic cargo of the vesicles as well as the cells from which they were derived were examined using a small microRNA (miRNA) gene array. Interestingly, ∼20% of the examined miRNAs were increased more than twofold in the vesicles compared with preosteoblast cells. Together, these results suggest that extracellular vesicles may be utilized as a novel method of directing stem cell differentiation. Future work examining methods for controlled delivery of vesicles may improve the clinical potential of these physiological liposomes for therapeutic applications.

  12. Role of adipose-derived stem cells in wound healing. (United States)

    Hassan, Waqar Ul; Greiser, Udo; Wang, Wenxin


    Impaired wound healing remains a challenge to date and causes debilitating effects with tremendous suffering. Recent advances in tissue engineering approaches in the area of cell therapy have provided promising treatment options to meet the challenges of impaired skin wound healing such as diabetic foot ulcers. Over the last few years, stem cell therapy has emerged as a novel therapeutic approach for various diseases including wound repair and tissue regeneration. Several different types of stem cells have been studied in both preclinical and clinical settings such as bone marrow-derived stem cells, adipose-derived stem cells (ASCs), circulating angiogenic cells (e.g., endothelial progenitor cells), human dermal fibroblasts, and keratinocytes for wound healing. Adipose tissue is an abundant source of mesenchymal stem cells, which have shown an improved outcome in wound healing studies. ASCs are pluripotent stem cells with the ability to differentiate into different lineages and to secrete paracrine factors initiating tissue regeneration process. The abundant supply of fat tissue, ease of isolation, extensive proliferative capacities ex vivo, and their ability to secrete pro-angiogenic growth factors make them an ideal cell type to use in therapies for the treatment of nonhealing wounds. In this review, we look at the pathogenesis of chronic wounds, role of stem cells in wound healing, and more specifically look at the role of ASCs, their mechanism of action and their safety profile in wound repair and tissue regeneration.

  13. Skin Tissue Engineering: Application of Adipose-Derived Stem Cells (United States)

    Zimoch, Jakub; Biedermann, Thomas


    Perception of the adipose tissue has changed dramatically over the last few decades. Identification of adipose-derived stem cells (ASCs) ultimately transformed paradigm of this tissue from a passive energy depot into a promising stem cell source with properties of self-renewal and multipotential differentiation. As compared to bone marrow-derived stem cells (BMSCs), ASCs are more easily accessible and their isolation yields higher amount of stem cells. Therefore, the ASCs are of high interest for stem cell-based therapies and skin tissue engineering. Currently, freshly isolated stromal vascular fraction (SVF), which may be used directly without any expansion, was also assessed to be highly effective in treating skin radiation injuries, burns, or nonhealing wounds such as diabetic ulcers. In this paper, we review the characteristics of SVF and ASCs and the efficacy of their treatment for skin injuries and disorders.

  14. Large Scale Production of Stem Cells and Their Derivatives (United States)

    Zweigerdt, Robert

    Stem cells have been envisioned to become an unlimited cell source for regenerative medicine. Notably, the interest in stem cells lies beyond direct therapeutic applications. They might also provide a previously unavailable source of valuable human cell types for screening platforms, which might facilitate the development of more efficient and safer drugs. The heterogeneity of stem cell types as well as the numerous areas of application suggests that differential processes are mandatory for their in vitro culture. Many of the envisioned applications would require the production of a high number of stem cells and their derivatives in scalable, well-defined and potentially clinical compliant manner under current good manufacturing practice (cGMP). In this review we provide an overview on recent strategies to develop bioprocesses for the expansion, differentiation and enrichment of stem cells and their progenies, presenting examples for adult and embryonic stem cells alike.

  15. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes. (United States)

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M Cecilia T; Wolf, Don; Mitalipov, Shoukhrat


    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

  16. Dendritic cells in hepatitis C virus infection: key players in the IFNL3-genotype response. (United States)

    O'Connor, Kate S; George, Jacob; Booth, David; Ahlenstiel, Golo


    Recently, single nucleotide polymorphisms, in the vicinity of the interferon lambda 3 (IFNL3) gene have been identified as the strongest predictor of spontaneous and treatment induced clearance of hepatitis C virus (HCV) infection. Since then, increasing evidence has implicated the innate immune response in mediating the IFNL3 genotype effect. Dendritic cells (DCs) are key to the host immune response in HCV infection and their vital role in the IFNL3 genotype effect is emerging. Reports have identified subclasses of DCs, particularly myeloid DC2s and potentially plasmacytoid DCs as the major producers of IFNL3 in the setting of HCV infection. Given the complexities of dendritic cell biology and the conflicting current available data, this review aims to summarize what is currently known regarding the role of dendritic cells in HCV infection and to place it into context of what is know about lambda interferons and dendritic cells in general.

  17. Hepatic Leukemia Factor Promotes Resistance To Cell Death: Implications For Therapeutics and Chronotherapy

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    Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J.


    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.

  18. Hop bitter acids exhibit anti-fibrogenic effects on hepatic stellate cells in vitro. (United States)

    Saugspier, Michael; Dorn, Christoph; Thasler, Wolfgang E; Gehrig, Manfred; Heilmann, Jörg; Hellerbrand, Claus


    Female inflorescences of the hop plant Humulus lupulus L. contain a variety of secondary metabolites with bitter acids (BA) as quantitatively dominating secondary metabolites. The use of hops in beer brewing has a long history due to the antibacterial effects of the BA and their typical bitter taste. Furthermore, hop cones are used in traditional medicine and for pharmaceutical purposes. Recent studies indicate that BA may affect activity of the transcription factor NFκB. NFκB plays a key role in the activation process of hepatic stellate cells (HSC), which is the key event of hepatic fibrosis. The aim of this study was to investigate the effect of BA on HSC (activation) and their potential to inhibit molecular processes involved in the pathogenesis of hepatic fibrosis. HSC were isolated from murine and human liver tissue and incubated with a characterized fraction of bitter acids purified from a CO(2) hop extract. At a concentration of 25μg/ml BA started to induce LDH leakage. Already at lower concentrations BA lead to a dose dependent inhibition of HSC proliferation and inhibited IκB-α-phosphorylation, nuclear p65 translocation and binding activity in a dose dependent way (up to 10μg/ml). Accordingly, the same BA-doses inhibited the expression of pro-inflammatory and NFκB regulated genes as MCP-1 and RANTES, but did not affect expression of genes not related to NFκB signaling. In addition to the effect on activated HSC, BA inhibited the in vitro activation process of freshly isolated HSC as evidenced by delayed expression of collagen I and α-SMA mRNA and protein. Together, these findings indicate that BA inhibit NFκB activation, and herewith the activation and development of profibrogenic phenotype of HSC. Thus, bitter acids appear as potential functional nutrients for the prevention or treatment hepatic fibrosis in chronic liver disease.

  19. Two sides of one coin: massive hepatic necrosis and progenitor cell-mediated regeneration in acute liver failure. (United States)

    Weng, Hong-Lei; Cai, Xiaobo; Yuan, Xiaodong; Liebe, Roman; Dooley, Steven; Li, Hai; Wang, Tai-Ling


    Massive hepatic necrosis is a key event underlying acute liver failure, a serious clinical syndrome with high mortality. Massive hepatic necrosis in acute liver failure has unique pathophysiological characteristics including extremely rapid parenchymal cell death and removal. On the other hand, massive necrosis rapidly induces the activation of liver progenitor cells, the so-called "second pathway of liver regeneration." The final clinical outcome of acute liver failure depends on whether liver progenitor cell-mediated regeneration can efficiently restore parenchymal mass and function within a short time. This review summarizes the current knowledge regarding massive hepatic necrosis and liver progenitor cell-mediated regeneration in patients with acute liver failure, the two sides of one coin.

  20. Human induced hepatic lineage-oriented stem cells: autonomous specification of human iPS cells toward hepatocyte-like cells without any exogenous differentiation factors.

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    Tetsuya Ishikawa

    Full Text Available Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG, conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5, transporters (SULT2A1, SLC13A5, and SLCO2B1, and urea cycle-related enzymes (ARG1 and CPS1. In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the

  1. Decreased peripheral natural killer cells activity in the immune activated stage of chronic hepatitis B.

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    Yuan Li

    Full Text Available BACKGROUND & AIMS: The natural course of chronic hepatitis B virus (HBV infection is characterized by different immune responses, ranging from immune tolerant (IT to immune activated (IA stages. In our study, we investigated the natural killer (NK cells activity in patients at different immunological stages of chronic HBV infection. METHODS: Blood samples obtained from 57 HBeAg positive patients with chronic hepatitis B (CHB, including 15 patients in the immune tolerant (IT stage, 42 patients in the immune activated (IA stage, and 18 healthy individuals (HI. The analyses included flow cytometry to detect NK cells, the determination of cytokine levels as well as of surface receptor expression and cytotoxicity. RESULTS: NK cells in peripheral blood were significantly lower in patients in the IA stage of CHB compared to HI (p<0.05. Patients in the IA stage of CHB had lower levels of NK cells activating receptor NKp30 and NKG2D expression, cytokine interferon-γ (IFN-γ and tumor necrosis factor-α (TNF-α production, as compared to patients in the IT stage and HI, respectively (p<0.05. Cytotoxicity of NK cells was lower in patients in the IA stage of CHB compared to patients in the IT stage and HI, respectively (p<0.05. The level of IFN-γ but not level of TNF-α and cytotoxicity of NK cells was inversely correlated with serum HBV load in patients with CHB. Peripheral NK cells activity did not correlate with ALT level. CONCLUSION: NK cells activity was lower in CHB patients, especially in those in the IA stage.

  2. Hepatitis C virus and ethanol alter antigen presentation in liver cells

    Institute of Scientific and Technical Information of China (English)

    Natalia A Osna


    Alcoholic patients have a high incidence of hepatitis Cvirus (HCV) infection. Alcohol consumption enhances the severity of the HCV disease course and worsens the outcome of chronic hepatitis C. The accumulation of virally infected cells in the liver is related to the HCVinduced inability of the immune system to recognizeinfected cells and to develop the immune responses. This review covers the effects of HCV proteins and ethanol on major histocompatibility complex (MHC) classⅠ- and class Ⅱ-restricted antigen presentation. Here, we discuss the liver which functions as an immune privilege organ; factors, which affect cleavage and loading of antigenic peptides onto MHC classⅠand class Ⅱ in hepatocytes and dendritic cells, and the modulating effects of ethanol and HCV on antigen presentation by liver cells. Altered antigen presentation in the liver limits the ability of the immune system to clear HCV and infected cells and contributes to disease progression. HCV by itself affects dendritic cell function, switching their cytokine profile to the suppressive phenotype of interleukin-10 (IL-10) and transforming growth factor beta (TGFβ) predominance,preventing cell maturation and allostimulation capacity.The synergistic action of ethanol with HCV results in the suppression of MHC class Ⅱ-restricted antigen presentation. In addition, ethanol metabolism and HCV proteins reduce proteasome function and interferon signaling, thereby suppressing the generation of peptides for MHC classⅠ-restricted antigen presentation.Collectively, ethanol exposure further impairs antigen presentation in HCV-infected liver cells, which may provide a partial explanation for exacerbations and the poor outcome of HCV infection in alcoholics.

  3. Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2

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    Guang-yao KONG; Jun-ping ZHANG; Shuai ZHANG; Chang-liang SHAN; Li-hong YE; Xiao-dong ZHANG


    To investigate the mechanism underlying the increase of hepatoma cell proliferation by hepatitis B virus X protein (HBx).Methods:HepG2,H7402 and HepG2.2.15 cells,which constitutively replicated hepatitis B virus were used.The effects of HBx on hepatoma cell proliferation were examined using 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and MTT assay.The expression level of MEKK2 was measured using RT-PCR,Western blot and luciferase reporter gene assay.The activity of activator protein 1 (AP-1) was detected using luciferase reporter gene assay.The phosphorylation levels of JNK and c-Jun were measured using Western blot.The expression levels of HBx and MEKK2 in 11 clinical hepatocellular carcinoma (HCC) tissues were measured using real time PCR and Western blot.In addition,the expression of MEKK2 in 95 clinical HCC tissues was examined using immunohistochemistry.Results:HBx significantly enhanced HepG2-X cell proliferation.In HepG2-X,H7402-X and HepG2.2.15 cells,the expression level of MEKK2 was remarkably increased.In HepG2.2.15 cells,HBx was found to activate JNK and AP-1,which were the downstream effectors of MEKK2 in HepG2-X and HepG2.2.15 cells.In 11 clinical HCC tissues,both HBx and MEKK2 expression levels were remarkably increased,as compared to those in the corresponding peritumor tissues.In 95 clinical HCC tissues,the rate of detection of MEKK2 was 85.3%.Conclusion:HBx promotes hepatoma cell proliferation via upregulating MEKK2,which may be involved in hepatocarcinogenesis.

  4. Foetal stem cell derivation & characterization for osteogenic lineage

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    A Mangala Gowri


    Full Text Available Background & objectives: Mesencymal stem cells (MSCs derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications. Methods: Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR analysis and confirmed by sequencing using genetic analyzer. Results: Ovine foetal samples were processed to obtain mononuclear (MNC cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45 - /CD14 - was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. Interpretation & conclusions: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established

  5. Optimized protocol for derivation of human embryonic stem cell lines. (United States)

    Camarasa, María Vicenta; Galvez, Víctor Miguel; Brison, Daniel Roy; Bachiller, Daniel


    For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.

  6. Autoimmune hepatitis. (United States)

    Heneghan, Michael A; Yeoman, Andrew D; Verma, Sumita; Smith, Alastair D; Longhi, Maria Serena


    Autoimmune hepatitis is a disease of the hepatic parenchyma that can present in acute or chronic forms. In common with many autoimmune diseases, autoimmune hepatitis is associated with non-organ-specific antibodies in the context of hepatic autoimmunity. This dichotomy has made definition of a unifying hypothesis in the pathophysiology of the disease difficult, although data from the past 8 years have drawn attention to the role of regulatory T cells. Several triggers have been identified, and the disease arises in genetically susceptible individuals. Clinical and biochemical remission is achievable in up to 85% of cases. For the remaining patients, alternative immunosuppression strategies are an option. Liver transplantation provides an excellent outcome for patients with acute liver failure or complications of end-stage liver disease, including hepatocellular carcinoma. Variant or overlapping syndromes are worthy of consideration when unexpected disease features arise.

  7. Hepatitis C virus E2 protein induce reactive oxygen species (ROS)-related fibrogenesis in the HSC-T6 hepatic stellate cell