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Sample records for hepatic cell line

  1. Hepatitis C virus infection of cholangiocarcinoma cell lines

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

    2015-01-01

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV re

  2. Lipoprotein profiles of hepatic cell lines at various stages of differentiation.

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    Sasaki, Akira; Kimura, Fumiko; Miura, Mizuho; Toshima, Gen; Takahashi, Junichiro; Maruya, Sayuri; Kobayashi, Masayuki; Hata, Keishi

    2017-02-01

    We studied the lipoprotein profiles of human hepatic cells at various stages of differentiation. The production of three major classes of lipoproteins, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), was detected in three well-differentiated human hepatoma cell lines and primary human hepatocytes; however, these lipoproteins were not detected in the culture medium in which undifferentiated hepatoma cell lines were grown. Reverse transcription polymerase chain reaction analysis demonstrated that the expression levels of apolipoprotein A1 (ApoA1), ApoB100, and microsomal triglyceride transfer protein (MTP) were markedly lower in the undifferentiated hepatoma cell lines than in the well-differentiated hepatoma cell lines and primary hepatocytes. These results indicate that apolipoprotein synthesis, and triglyceride-transport by MTP might be rate-limiting steps in lipoprotein production in mature hepatic cells.

  3. Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection

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

    2010-01-01

    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

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

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

    2011-01-01

    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

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

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

    2015-01-01

    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.

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

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

    2001-01-01

    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.``

  7. Inhibitory effect of kaolin minerals compound against hepatitis C virus in Huh-7 cell lines.

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    Ali, Liaqat; Idrees, Muhammad; Ali, Muhammad; Hussain, Abrar; Ur Rehman, Irshad; Ali, Amjad; Iqbal, Syed Abbas; Kamel, Eyad Hassan

    2014-04-17

    Hepatitis C virus (HCV) is estimated to infect 200 million individuals in the globe, including approximately 10 million in Pakistan causing both acute and chronic hepatitis. The standard treatment against HCV is pegylated interferon therapy in combination with a nucleoside analogue ribavirin. In addition, several herbal extracts and phytochemicals derivatives are used traditionally in the treatment of liver diseases as well as HCV infection. The present study determines the inhibitory effect of kaolin minerals compound against hepatitis C virus in Huh-7 cell lines. Huh-7 cell lines were used for the in vitro HCV replication by using HCV positive sera from different patients with known HCV genotypes and viral titer/load. Total RNA was extracted from these infected cells and was quantified by real-time polymerase chain reaction (Real-time PCR). The viral titer was compared with the control samples to determine the anti-HCV activity of kaolin derived compounds. Kaolin is a group of clay minerals, with the chemical composition Al2 Si2O5 (OH)4. The results showed promising effectiveness of local kaolin derived anti-HCV compounds by causing 28% to 77% decrease in the HCV titer, when applied to infected Huh-7 cell lines. This study provides the basis for future work on these compounds especially to determine the specific pathway and mechanism for inhibitory action in the replicon systems of viral hepatitis. Kaolin mineral derivatives show promising inhibitory effects against HCV genotypes 3a and 1a infection, which suggests its possible use as complementary and alternative medicine for HCV viral infection.

  8. 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.

  9. Novel hepatitis C virus reporter replicon cell lines enable efficient antiviral screening against genotype 1a.

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    Robinson, Margaret; Yang, Huiling; Sun, Siu-Chi; Peng, Betty; Tian, Yang; Pagratis, Nikos; Greenstein, Andrew E; Delaney, William E

    2010-08-01

    The hepatitis C virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-HCV compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (approximately 70% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh-7 cells. In this report, we describe the selection of highly permissive Huh-7 cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support the replication of multiple genotype 1a replicons (including the H77 and SF9 strains), are significantly more permissive to genotype 1a HCV replication than parental Huh7-Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing of the nonstructural region of 12 stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes.

  10. Definition of the transcription initiation site of human plasminogen gene in liver and non hepatic cell lines.

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

    1990-12-31

    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.

  11. Permissiveness of human hepatocellular carcinoma cell lines for hepatitis C virus entry and replication.

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    Coto-Llerena, Mairene; Koutsoudakis, George; Boix, Loreto; López-Oliva, Juan Manuel; Caro-Pérez, Noelia; Fernández-Carrillo, Carlos; González, Patricia; Gastaminza, Pablo; Bruix, Jordi; Forns, Xavier; Pérez-Del-Pulgar, Sofía

    2017-07-24

    Hepatitis C virus (HCV) is a globally prevalent pathogen and is associated with high death rates and morbidity. Since its discovery in 1989, HCV research has been impeded by the lack of a robust infectious cell culture system and thus in vitro studies on diverse genetic backgrounds are hampered because of the limited number of hepatoma cell lines which are able to support different aspects of the HCV life cycle. In the current study, we sought to expand the limited number of permissive cells capable of supporting the diverse phases of the HCV life cycle. Initially, we screened a panel of new hepatoma-derived cell lines, designated BCLC-1, -2, -3, -4, -5, -6, -9 and -10 cells, for their ability to express essential HCV receptors and subsequently to support HCV entry by using the well-characterized HCV pseudoparticle system (HCVpp). Apart from BCLC-9, all BCLC cell lines were permissive for HCVpp infection. Next, BCLC cells were subjected to short- and long-term HCV RNA replication studies using HCV subgenomic replicons. Interestingly, only BCLC-1, -5 and -9 cells, supported short-term HCV RNA replication, but the latter were excluded from further studies since they were refractory for HCV entry. BCLC-1, -5 were able to support long-term HCV replication too; yet BCLC-5 cells supported the highest long-term HCV RNA replication levels. Furthermore, cured BCLC-5 clones from HCV subgenomic replicon, showed increased permissiveness for HCV RNA replication. Strikingly, we were unable to detect endogenous BCLC-5 miR122 expression - an important HCV host factor- and as expected, the exogenous expression of miR122 in BCLC-5 cells increased their permissiveness for HCV RNA replication. However, this cell line was unable to produce HCV infectious particles despite ectopic expression of apolipoprotein E, which in other hepatoma cell lines has been shown to be sufficient to enable the HCV secretion process, suggesting a lack of other host cellular factor(s) and/or the presence of

  12. Exposure to human immunodeficiency virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types.

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    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

    2016-12-01

    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.

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

    2013-01-01

    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.

  14. 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.

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

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    Miyamae, Yusaku, E-mail: ymiyamae@lif.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Nishito, Yukina; Nakai, Naomi [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Nagumo, Yoko; Usui, Takeo [Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki 305-8572 (Japan); Masuda, Seiji; Kambe, Taiho [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Nagao, Masaya, E-mail: mnagao@kais.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan)

    2016-08-12

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

  16. Urate Oxidase Knockdown Decreases Oxidative Stress in a Murine Hepatic Cell Line

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    Beth M. Cleveland

    2009-01-01

    Full Text Available Humans, birds, and some primates do not express the uric acid degrading enzyme urate oxidase (UOX and, as a result, have plasma uric acid concentrations higher than UOX expressing animals. Although high uric acid concentrations are suggested to increase the antioxidant defense system and provide a health advantage to animals without UOX, knockout mice lacking UOX develop pathological complications including gout and kidney failure. As an alternative to the knockout model, RNA interference was used to decrease UOX expression using stable transfection in a mouse hepatic cell line (ATCC, FL83B. Urate oxidase mRNA was reduced 66% (p < 0.05 compared to wild type, as measured by real time RT-PCR. To determine if UOX knockdown resulted in enhanced protection against oxidative stress, cells were challenged with hexavalent chromium (Cr(VI or 3-morpholinosydnonimine hydrochloride (SIN-1. Compared to wild type, cells with UOX knockdown exhibited a 37.2 ± 3.5% reduction (p < 0.05 in the electron spin resonance (ESR signal after being exposed to Cr(VI and displayed less DNA fragmentation (p < 0.05 following SIN-1 treatment. Cell viability decreased in wild type cells (p < 0.05, but not cells with UOX knockdown, after treatment with SIN-1. These results are consistent with an increased intracellular uric acid concentration and an increased defense against oxidative stress.

  17. Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

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    LIANG Xiaohong; SUN Wensheng; GAO Lifen; MA Chunhong; HAN Lihui; CHEN Youhai

    2005-01-01

    The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study preliminary molecular mechanisms for its effects. In order to set up a model in vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test,caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL receptors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 μg/L TRAIL was 41.4%±7.2%, significantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly confirms the effects of HBx on TRAIL- induced apoptosis from two different points and it is not related with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.

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

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

    2007-01-01

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

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

    2016-01-15

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

  20. Biological effects of extract from newborn porcine liver on hepatocytes, hepatic stellate cells, and hepatoma cell line

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective: Porcine liver extract has been shown to be effective in the clinical treatment of severe hepatitis. The aim of the present study was to study its antifibrotic as well as immune regulatory effect in vitro. Methods: Hepatocytes, hepatic stellate cells (HSCs), hepatoma cell line (HepG2) and human peripheral blood mononuclear cells (PMNCs) were studied with respect to proliferation, extracellular matrix production and apoptotic activities by proliferation assay, radioimmunoassay, gene transfection, reporter gene analysis and flow cytometry, respectively. Results: A strong stimulatory proliferation effect was observed in hepatocytes, and an inhibitory effect was found in HSCs. Hyaluronic acid (HA) production and reporter gene activities driven by various α1(Ⅰ) procollagen gene promoters in HSC-T6 were significantly decreased after treatment with the extract. Fluo-Anexin V binding apoptotic HepG2 cells were more prominent in the presence of 60 μg/ml extract. More CD4+/CD69+ positive T lymphocytes existed in the presence of the extract. Conclusion: Porcine liver extract is effective for antifibrogenesis via hepatocyte regeneration, HSC and hepatoma cell inhibition in vitro. The elevation of active T lymphocytes is helpful for immune surveillance. Fine mapping of the extract is necessary in order to get definite molecules which are essential in all described functions.

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

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    Nidal Ghosheh

    2016-01-01

    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.

  2. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

    Science.gov (United States)

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

    2016-01-01

    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

  3. Hepatitis C virus E2 protein induce reactive oxygen species (ROS)-related fibrogenesis in the HSC-T6 hepatic stellate cell line.

    Science.gov (United States)

    Ming-Ju, Hsieh; Yih-Shou, Hsieh; Tzy-Yen, Chen; Hui-Ling, Chiou

    2011-01-01

    Chronic infection of hepatitis C virus (HCV) leads to hepatic fibrosis and subsequently cirrhosis, although the underlying mechanisms have not been established. Previous studies have indicated that the binding of HCV E2 protein and CD81 on the surface of hepatic stellate cells (HSCs) lead to the increased protein level and activity of matrix metallopeptidase (MMP) 2, indicating that E2 may involve in the HCV-induced fibrosis. This study was designed to investigate the involvement of HCV E2 protein in the hepatic fibrogenesis. Results showed that E2 protein may promote the expression levels of α-smooth muscle actin (α-SMA) and collagen α(I). Furthermore, several pro-fibrosis or pro-inflammatory cytokines, including transforming growth factor (TGF)-β1, connective tissue growth factor (CTGF), interleukin (IL)-6 and IL-1β, were significantly increased in E2 transfected-HSC cell lines, while the expression of MMP-2 are also considerably increased. Moreover, the significant increases of CTGF and TGF-β1 in a stable E2-expressing Huh7 cell line were also observed the same results. Further molecular studies indicated that the impact of E2 protein on collagen production related to higher production of ROS and activated Janus kinase (JAK)1, JAK2 and also enhance the activation of ERK1/2 and p38, while catalase and inhibitors specific for JAK, ERK1/2, and p38 abolish E2-enhanced expression of collagen α(I). Taken together, this study indicated that E2 protein involve in the pathogenesis of HCV-mediated fibrosis via an up-regulation of collagen α(I) and oxidative stress, which is JAK pathway related.

  4. Dynamic microRNA profiles of hepatic differentiated human umbilical cord lining-derived mesenchymal stem cells.

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    Lina Cui

    Full Text Available Despite the extensive hepatic differentiation potential of human umbilical cord lining-derived mesenchymal stem cells (hUC-MSC, little is known about the molecular mechanisms of hUC-MSC differentiation. At the post-transcriptional level, microRNAs are key players in the control of cell fate determination during differentiation. In this study, we aimed to identify microRNAs involved in the hepatic differentiation of hUC-MSCs. After successfully isolating hUC- MSCs, we induced hepatocyte formation in vitro with growth factors. After 26 days of induction, hUC-MSCs could express hepatocyte-specific genes, synthesize urea and glycogen and uptake low-density lipoprotein. Cellular total RNA from hUC-MSCs and hepatic differentiated hUC-MSCs was collected at 7 time points, including 2 days, 6 days, 10 days, 14 days, 22 days and 26 days, for microRNA microarray analysis. Dynamic microRNA profiles were identified that did not overlap or only partially overlapped with microRNAs reported to be involved in human liver development, hepatocyte regeneration or hepatic differentiation of liver-derived progenitor cells. A total of 61 microRNAs among 1205 human and 144 human viral microRNAs displayed consistent changes and were altered at least 2-fold between hUC-MSCs and hepatic differentiated hUC-MSCs. Among these microRNAs, 25 were over-expressed; this over-expression occurred either gradually or increased sharply and was maintained at a high level. A total of 36 microRNAs were under-expressed, with an expression pattern similar to that of the over-expressed microRNAs. The expression of the altered expressed microRNAs was also confirmed by quantitative reverse-transcription polymerase chain reaction. We also found that microRNAs involved in hepatic differentiation were not enriched in hepatocyte or hepatocellular carcinoma cells and can potentially target liver-enriched transcription factors and genes. The elucidation of the microRNA profile during the hepatic

  5. Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines

    Science.gov (United States)

    Chandhok, Gursimran; Horvath, Judit; Aggarwal, Annu; Bhatt, Mohit; Zibert, Andree; Schmidt, Hartmut HJ

    2016-01-01

    AIM: To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture. METHODS: The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined. RESULTS: Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability. CONCLUSION: The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines. PMID:27122662

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

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    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.

  7. Chemical and biological insights into uranium-induced apoptosis of rat hepatic cell line

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Fang; You, Yong [University of South China, College of Hunan Province, Key Laboratory of Tumor Cellular and Molecular Pathology, Hengyang (China); Du, Ke-Jie [University of South China, School of Chemistry and Chemical Engineering, Hengyang (China); Fang, Zhen [Anhui Normal University, College of Chemistry and Materials Science, Wuhu (China); Wen, Ge-Bo [University of South China, College of Hunan Province, Key Laboratory of Tumor Cellular and Molecular Pathology, Hengyang (China); University of South China, Laboratory of Protein Structure and Function, Hengyang (China); Lin, Ying-Wu [University of South China, School of Chemistry and Chemical Engineering, Hengyang (China); University of South China, Laboratory of Protein Structure and Function, Hengyang (China)

    2015-05-15

    Uranium release into the environment is a threat to human health, and the mechanisms of cytotoxicity caused by uranium are not well-understood. To improve our understanding in this respect, we herein evaluated the effects of uranium exposure on normal rat hepatic BRL cells. As revealed by scanning electron microscopy and transmission electron microscope analysis, uranyl nitrate was found to be transformed into uranyl phosphate particles in the medium and taken up by BRL cells in an endocytotic uptake manner, which presumably initiates apoptosis of the cell, although soluble uranyl ion may also be toxic. The apoptosis of BRL cells upon uranium exposure was also confirmed by both the acridine orange and ethidium bromide double staining assay and the Annexin V/propidium iodide double staining assay. Further studies revealed that uranium induced the loss of mitochondrial membrane potential in a dose-dependent manner. Moreover, the uranium-induced apoptosis was found to be associated with the activation of caspase-3, caspase-8 and caspase-9, indicating both a mitochondria-dependent signaling pathway and a death receptor pathway by a crosstalk. This study provides new chemical and biological insights into the mechanism of uranium toxicity toward hepatic cells, which will help seek approaches for biological remediation of uranium. (orig.)

  8. Induction of hepatic CYP3A enzymes by pregnancy-related hormones: studies in human hepatocytes and hepatic cell lines.

    Science.gov (United States)

    Papageorgiou, Ioannis; Grepper, Susan; Unadkat, Jashvant D

    2013-02-01

    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.

  9. Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines.

    Science.gov (United States)

    Ye, Fei; Xin, Zhongshuai; Han, Wei; Fan, Jingjing; Yin, Bin; Wu, Shuzhen; Yang, Wei; Yuan, Jiangang; Qiang, Boqin; Sun, Wei; Peng, Xiaozhong

    2015-01-01

    Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.

  10. Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines.

    Directory of Open Access Journals (Sweden)

    Fei Ye

    Full Text Available Chronic hepatitis C virus (HCV infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1, Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1, carboxylesterase 1 (CES1, vimentin, Proteasome activator complex subunit1 (PSME1, and Cathepsin B (CTSB were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.

  11. Effects of hepatitis B virus on p53 expression in hepatoma cell line SMMU-7721

    Institute of Scientific and Technical Information of China (English)

    Jian-Hui Qu; Ming-Hua Zhu; Jing Lin; Can-Rong Ni; Fang-Mei Li; Zhi Zhu; Guan-Zhen Yu

    2005-01-01

    AIM: To investigate the contribution of HBV in the development of hepatocarcinoma by examining the effects of HBV on p53 function in SMMU-7721 cell line.METHODS: Plasmid pCvlVp53 was transfected or cotransfected with pCMVHBVa (wild-type HBV) or PCMVHBVb (mutation type HBV) into the hepatoma cell line SMMU-7721 by lipofectamine. Apoptosis cells were labeled with annexin V-FITC and confirmed by flow cytometry. Reporter plasmid PG13-CAT or p21-1uc was cotransfected, respectively, into each group to determine the transactivation activity of p53 and its effect on p21 promoter. Western blot was performed to observe p53 expression in hepatoma cell line of each group.RESULTS: The group transfected with pCMVp53 alone exhibited higher luciferase activity and higher apoptosis rate, otherwise, the p53 expression and reporter activity of PG13-CAT or P21-luc as well as cell apoptosis rate were obviously higher in the group cotransfected of pCMVp53with pCMVHBVa, but not in the other cotransfected group.CONCLUSION: Transient transfection of HBV into the SMMU-7721 cell line can enhance p53 expression and its effects on development of hepatocarcinoma.

  12. New models of hepatitis E virus replication in human and porcine hepatocyte cell lines

    Science.gov (United States)

    Hepatitis E virus (HEV) causes acute, enterically-transmitted hepatitis. It is associated with large epidemics in tropical and subtropical regions where it is endemic or with sporadic cases in non-endemic regions. Unlike other hepatitis viruses, HEV has several animal reservoirs. Phylogenetic studie...

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

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    Wang W

    2013-09-01

    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. Hepatitis B virus down-regulates expressions of MHC class I molecules on hepatoplastoma cell line.

    Science.gov (United States)

    Chen, Yongyan; Cheng, Min; Tian, Zhigang

    2006-10-01

    Chronic HBV infection is associated with a 100-fold high risk of developing hepatocellular carcinoma. Tumor recognition is of the most importance during the immune surveillance process that prevents cancer development in humans. In the present study, the expressions of MHC class I molecules on hepatoplastoma cell line HepG2.2.15 were investigated to indicate the possible effects of HBV on the immune recognition during HBV-associated hepatocellular carcinoma. It was found that the expressions of MHC class I molecules HLA-ABC, HLA-E and MICA were much lower in HepG2.2.15 cells compared with HepG2 cells. The expressing HBV in human hepatoplastoma cell line significantly down-regulated the expressions of MHC class I molecules. Additionally, it was observed that in murine chronic HBsAg carriers the expression of classical MHC-I molecule on hepatocytes was down-regulated. These results demonstrated that HBV might affect the immune recognition during HBV-associated hepatocellular carcinoma such as the recognition of CD8+ T, NK-CTL and NK cells and prevent the immune surveillance against tumors. However, the effects of HBV down-regulation of MHC class I molecules on the target cells in vivo should be further studied.

  15. Propagation of Hepatitis B Virus in a Rat Hepatoma Cell Line Stably Transfected with Human Annexin-V

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    Seyed Mohammad Jazayeri

    2007-09-01

    Full Text Available Background and Aims: Hepatitis B virus (HBV displays a distinct hepatotropism and a narrow host range in vivo. However, very little is known about the interaction of HBV with its host cells, mainly because of difficulties in the development of suitable tissue culture system. We present here confirmatory evidence of a putative role of annexin-V in HBV infection. Methods: HBV from both human sera and from culture supernatants from HepG2 2.15 cells were used to infect FTO9.1 cells (a rat hepatoma cell line transfected with a construct containing human annexin-V. Cells and culture supernatants were assayed at various times post-infection by immunofluorescent microscopy (HBcAg staining in nucleus, and by HBV cccDNA-specific PCR. Supernatants from these initially infected cells were then used to infect fresh FTO9.1 cells with a similar outcome to primary infection. Results: Core and surface gene PCRs were positive on days 2, 5 and following transfer experiments. cccDNA-specific PCR confirmed internalisation of the virus into the nucleus. HBcAg fluorescence showed nuclear staining on days 2, 5 and following transfer experiments. Addition of recombinant annexin-V and DMSO to the cell culture medium resulted in a greater efficiency of infection. Later washes were negative for HBV-DNA, ruling out contamination of the cells by external HBV particles. Conclusions: This cell line does appear to be useful in the study of the early stages of HBV infection, but requires further evaluation.

  16. Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5.

    Science.gov (United States)

    Li, Tian-Cheng; Yoshizaki, Sayaka; Yang, Tingting; Kataoka, Michiyo; Nakamura, Tomofumi; Ami, Yasushi; Yuriko, Suzaki; Takeda, Naokazu; Wakita, Takaji

    2016-02-02

    A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV.

  17. Altered expression profiles of microRNAs in a stable hepatitis B virus-expressing cell line

    Institute of Scientific and Technical Information of China (English)

    LIU Yan; ZHAO Jian-Jun; WANG Chun-mei; LI Mian-yang; HAN Ping; WANG Lin; CHENG Yong-qian; Fabien Zoulim; MA Xu; XU Dong-ping

    2009-01-01

    Background MicroRNAs (miRNAs) are highly conserved small non-coding RNAs of 18-25 nucleotides (nt) that mediate post-transcriptional gene regulation. Hepatitis B virus (HBV) can cause either acute or chronic hepatitis B, and is a high risk factor for liver cirrhosis and hepatocellular carcinoma. Some mammalian viruses have been shown to modulate the expression of host cellular miRNAs. However, interactions between the HBV and the host cellular miRNAs are largely unknown. Methods miRNA microarray and Northern blotting analysis were used to compare the expression profile of cellular miRNAs of a stable HBV-expressing cell line HepG2.2.15 and its parent cell line HepG2. mRNA microarray assay and the miRanda program were used to predict the miRNA targets. A flow cytometric assay was further used to investigate the expression of human leukocyte antigen (HLA)-A. Results Eighteen miRNAs were differentially expressed between the two cell lines. Among them, eleven were up-regulated and seven were down-regulated in HepG2.2.15 cells. Northern blotting analysis confirmed that the expression of miR-181a, miR-181b, miR-200b and miR-146a were up-regulated and the expression of miR-15a was down-regulated, which was in consistent with the results of the microarray analysis. Furthermore, some putative miRNA targets were predicted and verified to be linked with mRNA expression. The 3'-UTR of HLA-A gene had one partially complementary site for miR-181a and miR-181a might down-regulate the expression of HLA-A. Conclusion HBV replication modulates the expression of host cellular miRNAs, which may play a role in the pathogenesis of HBV-related liver diseases.

  18. Hepatic progenitor cell lines from allyl alcohol-treated adult rats are derived from gamma-irradiated mouse STO cells.

    Science.gov (United States)

    Zhang, Mingjun; Sell, Stewart; Leffert, Hyam L

    2003-01-01

    In attempts to recharacterize several markers of putative rat liver progenitor cells, single-stage reverse transcription-polymerase chain reaction (RT-PCR) analyses failed to confirm the reported immunochemical detection of albumin, alpha(1)-fetoprotein, and cytochrome P450-1A2 in the clonal line, 3(8)#21, and the cloned derivative, 3(8)#21-EGFP (enhanced green fluorescent protein). Undetectable expression occurred whether or not both lines were cultured on or off feeder layers of gamma-irradiated mouse embryonic STO (SIM [Sandoz inbred Swiss mouse] thioguanine-resistant ouabain-resistant) cells. PCR amplification of liver progenitor cell chromosomal (rat and mouse Pigr, rat INS1, mouse INS2) and mitochondrial (rat and mouse COX1) genes revealed only mouse sequences. Further analyses of rat and mouse COX1 sequences in cells from untampered storage vials of all 11 reported liver progenitor cell lines and strains revealed only mouse sequences. In addition, uniquely similar metaphase spreads were observed in STO, 3(8)#21, and 3(8)#21-EGFP cells. The combined results suggest that the previously reported "rat" liver progenitor cell lines were most likely generated during early derivation in cell culture from gamma-radiation-resistant or ineffectively irradiated mouse STO cells used as the feeder layers. These findings reveal new types of artifacts encountered in cocultures of tissue progenitor cells and feeder layer cell lines, and they sound a cautionary note: phenotypic and genotypic properties of feeder layers should be well-characterized before and during coculture with newly derived stem cells and clonal derivatives.

  19. Transient replication of Hepatitis C Virus sub-genomic RNA in murine cell lines is enabled by miR-122 and varies with cell passage.

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    Patricia A Thibault

    Full Text Available Hepatitis C Virus (HCV is a serious global health problem, infecting almost 3% of the world's population. The lack of model systems for studying this virus limit research options in vaccine and therapeutic development, as well as for studying the pathogenesis of chronic HCV infection. Herein we make use of the liver-specific microRNA miR-122 to render mouse cell lines permissive to HCV replication in an attempt to develop additional model systems for the identification of new features of the virus and its life cycle. We have determined that some wild-type and knockout mouse cell lines--NCoA6 and PKR knockout embryonic fibroblasts--can be rendered permissive to transient HCV sub-genomic RNA replication upon addition of miR-122, but we did not observe replication of full-length HCV RNA in these cells. However, other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122, and in fact, different NCoA6 and PKR knockout cell line passages and isolates from the same mice demonstrated varying permissiveness phenotypes and eventually complete loss of permissiveness. When we tested knockdown of NCoA6 and PKR in Huh7.5 cells, we saw no substantial impact in sub-genomic HCV replication, which we would expect if these genes were inhibitory to the virus' life cycle. This leads us to conclude that along with the influence of specific gene knockouts there are additional factors within the cell lines that affect their permissiveness for HCV replication; we suggest that these may be epigenetically regulated, or modulated by cell line immortalization and transformation.

  20. Evaluation of cytochrome P450 activity in vitro, using dermal and hepatic microsomes from four species and two keratinocyte cell lines in culture.

    Science.gov (United States)

    Rolsted, Kamilla; Kissmeyer, Anne-Marie; Rist, Gerda Marie; Hansen, Steen Honoré

    2008-01-01

    The Cytochrome P450 (CYP450) enzymes are expressed in the skin, and despite a low activity, as compared to the hepatic counterpart, a role during transdermal delivery of a drug cannot be excluded. Additionally, the enzymes may play a role in local toxicity, and further knowledge of dermal CYP450 activity can contribute to elucidate this issue. To achieve this, a cocktail of six selective CYP450 probe substrates were incubated with dermal and hepatic microsomes isolated from mouse, rat, minipig and man. Different species were used to evaluate if a reliable substitute for human tissue was possible. Further, the hepatic microsomes were included in this study, to estimate if the hepatic CYP450 activity is predictive of dermal CYP450 activity. The CYP450 activity was determined in two keratinocyte cell lines as well, as this in vitro model is desirable due to the ease in handling, among other factors. Overall, the metabolism found in the dermal microsomes was very low, and major differences were observed between species. When comparing the activities in dermal and hepatic microsomes, the qualitative pattern was to some extent similar within species, but also a number of differences were observed. The CYP450 metabolic activity in the two keratinocyte cell lines was not comparable to metabolism in the human dermal microsomes.

  1. Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

    DEFF Research Database (Denmark)

    Jacobsen, Kari Stougaard; Nielsen, Kirstine Overgaard; Nordmann Winther, Thilde;

    2016-01-01

    BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially...... of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes...... identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model....

  2. Regulation of Glutathione in a Rat Diploid Hepatic Epithelial Cell Line

    Science.gov (United States)

    1990-06-01

    Toxicology 2: 213-240. (1979). 9. Kosower , N. and Kosower , E., Glutathione Status of Cells. International Review of Cytology 54: 109-160. (1978). 10. Stenius...Cycloheximide. Eur. J. Biochem. 73: 287-296, (1977). 89. Kosower , N., Vanderhoff, G. and Kosower , E.M., The Effects of glutathione disulfide on initiation of...protein synthesis. Biochimca Et Biophysica ACTA 272:623-637, (1972). 114 90. Zehavi-WIllner, T., Kosower , E.M., Hunt, T. and Kosower , N., The effects of

  3. Establishment of a Novel Permissive Cell Line for the Propagation of Hepatitis C Virus by Expression of MicroRNA miR122

    Science.gov (United States)

    Kambara, Hiroto; Fukuhara, Takasuke; Shiokawa, Mai; Ono, Chikako; Ohara, Yuri; Kamitani, Wataru

    2012-01-01

    The robust cell culture systems for hepatitis C virus (HCV) are limited to those using cell culture-adapted clones (HCV in cell culture [HCVcc]) and cells derived from the human hepatoma cell line Huh7. However, accumulating data suggest that host factors, including innate immunity and gene polymorphisms, contribute to the variation in host response to HCV infection. Therefore, the existing in vitro systems for HCV propagation are not sufficient to elucidate the life cycle of HCV. A liver-specific microRNA, miR122, has been shown to participate in the efficient replication of HCV. In this study, we examined the possibility of establishing a new permissive cell line for HCV propagation by the expression of miR122. A high level of miR122 was expressed by a lentiviral vector placed into human liver cell lines at a level comparable to the endogenous level in Huh7 cells. Among the cell lines that we examined, Hep3B cells stably expressing miR122 (Hep3B/miR122) exhibited a significant enhancement of HCVcc propagation. Surprisingly, the levels of production of infectious particles in Hep3B/miR122 cells upon infection with HCVcc were comparable to those in Huh7 cells. Furthermore, a line of “cured” cells, established by elimination of HCV RNA from the Hep3B/miR122 replicon cells, exhibited an enhanced expression of miR122 and a continuous increase of infectious titers of HCVcc in every passage. The establishment of the new permissive cell line for HCVcc will have significant implications not only for basic HCV research but also for the development of new therapeutics. PMID:22114337

  4. Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1.

    Directory of Open Access Journals (Sweden)

    Kosuke Tsukamoto

    Full Text Available BACKGROUND: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293 for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. CONCLUSIONS/SIGNIFICANCE: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

  5. Characterization of P1 promoter activity of the -galactoside 2,6-sialyltransferase I gene (siat 1) in cervical and hepatic cancer cell lines

    Indian Academy of Sciences (India)

    Lorena Milflores-Flores; Lourdes Millán-Pérez; Gerardo Santos-López; Julio Reyes-Leyva; Verónica Vallejo-Ruiz

    2012-06-01

    The level of -galactoside 2,6-sialyltransferase I (ST6Gal I) mRNA, encoded by the gene siat1, is increased in malignant tissues. Expression is regulated by different promoters – P1, P2 and P3 – generating three mRNA isoforms H, X and YZ. In cervical cancer tissue the mRNA isoform H, which results from P1 promoter activity, is increased. To study the regulation of P1 promoter, different constructs from P1 promoter were evaluated by luciferase assays in cervical and hepatic cell lines. Deletion of a fragment of 1048 bp (−89 to +24 bp) increased 5- and 3-fold the promoter activity in C33A and HepG2 cell lines, respectively. The minimal region with promoter activity was a 37 bp fragment in C33A cells. The activity of this region does not require the presence of an initiator sequence. In HepG2 cells the minimal promoter activity was detected in the 66 bp fragment. Sp1 (−32) mutation increased the promoter activity only in HepG2 cells. HNF1 mutation decreased promoter activity in HepG2 cell line but not in C33A cells. We identified a large region that plays a negative regulation role. The regulation of promoter activity is cell type specific. Our study provides new insights into the complex transcriptional regulation of siat1 gene.

  6. Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

    Directory of Open Access Journals (Sweden)

    Anne Frentzen

    2011-04-01

    Full Text Available Hepatitis C virus (HCV is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

  7. Cystathionine β-synthase-derived hydrogen sulfide regulates lipopolysaccharide-induced apoptosis of the BRL rat hepatic cell line in vitro.

    Science.gov (United States)

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

    2012-11-01

    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.

  8. Hepatitis B Virus Down-Regulates Expressions of MHC Class ⅠMolecules on Hepatoplastoma Cell Line

    Institute of Scientific and Technical Information of China (English)

    Yongyan Chen; Min Cheng; ,Zhigang Tian

    2006-01-01

    Chronic HBV infection is associated with a 100-fold high risk of developing hepatocellular carcinoma. Tumor recognition is of the most importance during the immune surveillance process that prevents cancer development in humans. In the present study, the expressions of MHC class Ⅰ molecules on hepatoplastoma cell line HepG2.2.15 were investigated to indicate the possible effects of HBV on the immune recognition during HBV-associated hepatocellular carcinoma. It was found that the expressions of MHC class Ⅰ molecules HLA-ABC, HLA-E and MICA were much lower in HepG2.2.15 cells compared with HepG2 cells. The expressing HBV in human hepatoplastoma cell line significantly down-regulated the expressions of MHC class Ⅰ molecules. Additionally, it was observed that in murine chronic HBsAg carriers the expression of classical MHC-Ⅰ molecule on hepatocytes was down-regulated. These results demonstrated that HBV might affect the immune recognition during HBV-associated hepatoceilular carcinoma such as the recognition of CD8+ T, NK-CTL and NK cells and prevent the immune surveillance against tumors. However, the effects of HBV down-regulation of MHC class Ⅰ molecules on the target cells in vivo should be further studied.

  9. Evaluation of hepatitis B virus replication and proteornic analysis of HepG2.2.15 cell line after cyclosporine A treatment

    Institute of Scientific and Technical Information of China (English)

    Hai-yang XIE; Wei-liang XIA; Chun-chao ZHANG; Li-ming WU; Hao-feng JI; Yu CHENG; Shu-sen ZHENG

    2007-01-01

    Aim: The effect of cyclosporine A (CsA) on hepatitis B virus (HBV) replication was investigated, and proteomics expression differentiation after CsA treatment was studied in order to provide clues to explore the effect of CsA on HBV replication. Methods: Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cytotoxicity of CsA. The HBV replication level in the HBV genomic DNA transfected HepG2.2.15 cell line was determined by an ELISA analysis of hepatitis B surface antigens (HBsAg) and Hepatitis B e antigens (HBeAg) in culture supernatant, while the intracellular HBV DNA replication level was ana-lyzed by slot blot hybridization. Two-dimensional electrophoresis was used to investigate the alteration of protein expression in HepG2.2.15 after CsA treatment in vitro. The differentially-expressed proteins were identified by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with an online database search. Results: CsA was able to inhibit the expression of HBsAg, HBeAg, and HBV DNA replication in vitro in a dose-dependent manner. A proteomics analysis indicated that the expression of 17 proteins changed signifi-cantly in the CsA treatment group compared to the control group. Eleven of the 17 proteins were identified, including the overexpression of eukaryotic translation initiation factors (elF) 3k, otubain 1, 14.3.3 protein, elF2-1α, elF5A, and the tyrosine 3/tryptophan 5-mono-oxygenase activation protein in CsA-treated HepG2.2.15 cells. The down.regulation of the ferritin light subunit, erythrocyte cytosolic protein of 51 kDa (ECP-51), stathmin l/oncoprotein, adenine phosphoribosyl-transferase, and the position of a tumor protein, translationally-controlled 1, was shifted, suggesting it had undergone posttranslational modifications. Conclusion: Our study identified the inhibitory effect of CsA on HBV replication, and found that a group of proteins may be responsible for this inhibitory effect.

  10. In vitro evaluation of silver nanoparticles cytotoxicity on Hepatic cancer (Hep-G2) cell line and their antioxidant activity: Green approach for fabrication and application.

    Science.gov (United States)

    Kumar, Brajesh; Smita, Kumari; Seqqat, Rachid; Benalcazar, Karen; Grijalva, Marcelo; Cumbal, Luis

    2016-06-01

    In this article, biosynthesis of silver nanoparticles (AgNPs) using Andean Mora (Rubus glaucus Benth.) leaf has been reported. Different analytical techniques including UV-vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used for the characterization of AgNPs. The initial appearance of color change with the intense surface plasmon resonance (SPR) bands around 440-455 in UV-visible spectra revealing the formation of AgNPs. The TEM image showed the AgNPs to be anisotropic, quasi-spherical in shape with sizes in the range of 12-50nm. On the other hand, XRD studies revealed the formation of face-centered cubic structure for AgNPs. The surface modified AgNPs showed no cytotoxicity at the concentration ranging from 0.01μM to 1.0μM on the Hepatic cancer (Hep-G2) cell line and observed antioxidant efficacy >70% at the concentration 0.05mM/0.20mL against 1, 1-diphenyl-2-picrylhydrazyl. From the results obtained it is suggested that AgNPs could be used effectively in future drug delivery systems and other biomedical concerns. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Proteomic analysis of cell proliferation in a human hepatic cell line (HL-7702) induced by perfluorooctane sulfonate using iTRAQ

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Ruina; Zhang, Hongxia [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Guo, Xuejiang [State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029 (China); Cui, Qianqian; Wang, Jianshe [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Dai, Jiayin, E-mail: daijy@ioz.ac.cn [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China)

    2015-12-15

    Highlights: • PFOS stimulates cell proliferation of human liver cell line (HL-7702). • Differential expressed proteins are identified by iTRAQ. • Most of differential proteins caused by PFOS are related to cell proliferation. • Up-regulation of cyclin/cdk by PFOS plays a role in driving cells into cell cycle. - Abstract: Perfluorooctane sulfonate (PFOS) is a commonly used and widely distributed perfluorinated compound proven to cause adverse health outcomes. However, how PFOS affects liver cell proliferation is not well understood. In this experiment, we exposed a human liver cell line (HL-7702) to 50 μM PFOS for 48 h and 96 h. We identified 52 differentially expressed proteins using a quantitative proteomic approach. Among them, 27 were associated with cell proliferation, including hepatoma-derived growth factor (Hdgf) and proliferation biomarkers Mk167 (Ki67) and Top2α. Results from MTT, cell counting, and cell cycle analysis showed low-dose PFOS (<200 μM) stimulated HL-7702 cell viability at 48 h and 96 h, reduced the G0/G1 percentage, and increased the S + G2/M percentage. Moreover, levels of Cyclin D1, Cyclin E2, Cyclin A2, Cyclin B1 and their partner Cdks were elevated, and the expression of regulating proteins like c-Myc, p53, p21 waf/cip1 and Myt1, as well as the phosphorylation levels of p-Wee1(S642), p-Chk1(S345) and p-Chk2(T68), were disturbed. We hypothesized that low-dose PFOS stimulated HL-7702 proliferation by driving cells into G1 through elevating cyclins/cdks expression, and by promoting cell cycle progression through altering other regulating proteins. This research will shed light on the mechanisms behind PFOS-mediated human hepatotoxicity.

  12. The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

    Science.gov (United States)

    1995-04-13

    pH-dependent membrane fusion in other virus systems such as Semliki Forest virus and vesicular stomatitis virus ( Matlin , et al., 1982; Helenius...poliovirus, can infect normally insusceptible cells via the Fc receptor . Viral . 192 : 568-577 . Matlin , K. S ., H. Reggis, A. Helenius and K. Simons

  13. Storage of cell lines.

    Science.gov (United States)

    Parker, Katharine A

    2011-01-01

    The successful storage of cell lines depends upon many factors, including the condition of the cells to be frozen and the experience of the operator. Attempting to freeze down unhealthy, contaminated or poorly labelled cells can have huge implications for a research laboratory. This chapter outlines the importance of good record keeping, vigilant monitoring, aseptic technique, and high-quality reagents in the successful storage and downstream propagation of cell lines.

  14. Advances in studies on hepatic stem cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    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.

  15. Influence of captopril on the cellular uptake and toxic potential of microcystin-LR in non-hepatic adhesive cell lines.

    Science.gov (United States)

    Teneva, Ivanka; Klaczkowska, Dorota; Batsalova, Tsvetelina; Kostova, Zhivka; Dzhambazov, Balik

    2016-03-01

    Microcystin-LR (MC-LR) is a toxin produced by various cyanobacterial strains. Its cytotoxicity is due to inhibition of the protein phosphatases PP1 and PP2A, resulting in hyperphosphorylation of a number of functional and cytoskeletal proteins. To penetrate through the plasma membrane, MC-LR needs specific transporters such the organic anion-transporting polypeptides (OATP) that are highly expressed on the hepatocytes. Hence, our goal was to investigate the role of the membrane transport proteins for the cytotoxic effect of MC-LR on adhesive cell lines different from hepatocytes. We have used three cell lines--A549 (human lung carcinoma), SK-Hep-1 (human liver adenocarcinoma), FL (human amniotic normal cells), and two inhibitors of the OATP (cyclosporine A and captopril). To examine the cytotoxic effect of MC-LR we applied MTT and Neutral Red assays. In addition, a fluorescent staining of the mitochondria by JC-1 was performed. A dose-dependent cytotoxic effect was observed for the three cell lines, as this effect was most pronounced in A549. No cytotoxicity was detected when the captopril was added 2 h before treatment of the cells with MC-LR. Addition of captopril to the cells 2 h after treatment with MC-LR leads to enhancement of the cytotoxic effect. Reduced mitochondrial membrane potential after treatment with MC-LR was detected in the three cell lines, compared to untreated control cells. Results from the NR-cytotoxicity assay indicated that MC-LR does not affect the lysosomes. Captopril is an effective inhibitor of both OATP influx membrane transport proteins and the P-gp efflux pumps involved in the transport of MC-LR. It protects the cells from toxic effects of the cyanotoxin MC-LR. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Establishment of a robust hepatitis C virus replicon cell line over-expressing P-glycoprotein that facilitates analysis of P-gp drug transporter effects on inhibitor antiviral activity.

    Science.gov (United States)

    Hernandez, Dennis; Falk, Paul; Yu, Fei; Zhai, Guangzhi; Quan, Yong; Faria, Teresa; Cao, Kai; Scola, Paul; McPhee, Fiona

    2013-01-01

    P-glycoprotein (P-gp) is an active efflux pump affecting the pharmacokinetic (PK) profiles of drugs that are P-gp substrates. The Caco-2 bi-directional assay is widely used to identify drug-P-gp interactions in vitro. For molecules exhibiting non-classical drug properties however, ambiguous results limit its use in lead optimization. The goal of this study was to develop a robust cell-based assay system to directly measure the role of P-gp-driven efflux in reducing the potency of hepatitis C virus (HCV) replication inhibitors. Vinblastine (Vin) was employed to select for a Vin-resistant HCV replicon (313-11) from the parental cell line (377-2). The 313-11 cell line was >50-fold resistant to Vin and over-expressed P-gp, as determined by Western immunoblots. Increased expression of P-gp was mediated by up-regulation of the MDR1 transcript. The reduced potency of different classes of HCV replication inhibitors in the 313-11 P-gp cell line was restored in the presence of known P-gp inhibitors. Addition of the P-gp inhibitor, tariquidar, increased the uptake of a radiolabeled HCV replication inhibitor by 14-fold in the 313-11 replicon cell line. Finally, a positive correlation was demonstrated between potency in the 313-11 replicon and the bi-directional Caco-2 efflux ratio for a panel of HCV protease inhibitors. In conclusion, a robust P-gp HCV replicon cell-based assay has been developed to measure the effect of the P-gp efflux pump on the potency of different classes of HCV replication inhibitors. This system establishes a direct correlation between antiviral activity and the effect of P-gp efflux in a single cell line.

  17. Nano-SiO2 induces apoptosis via activation of p53 and Bax mediated by oxidative stress in human hepatic cell line.

    Science.gov (United States)

    Ye, Yiyi; Liu, Jianwen; Xu, Jianhe; Sun, Lijuan; Chen, Mingcang; Lan, Minbo

    2010-04-01

    Nanoparticles such as nano-SiO(2) are increasingly used in food, cosmetics, diagnosis, imaging and drug delivery. However, toxicological data of nano-SiO(2) on hepatic cells in vitro and their detailed molecular mechanisms still remain unclear. In order to assess toxicity of nano-SiO(2), L-02 cells were exposed to 0.2, 0.4 and 0.6 mg/ml of SiO(2) colloids (21, 48 and 86 nm) for 12, 24, 36 and 48h. Lactate dehydrogenase released from damaged cells were quantified, cellular ultrastructural organization was observed, and the levels of reactive oxygen species (ROS), lipid peroxidation and glutathione were measured. Apoptosis induced by 21 nm SiO(2) was characterized by annexin V-FITC/PI staining and DNA ladder assay. Furthermore, apoptosis related proteins such as p53, Bax and Bcl-2 were analyzed by using western blot analysis. Our data indicated that nano-SiO(2) caused cytotoxicity in size, dose and time dependent manners. Oxidative stress and apoptosis were induced by exposure to 21 nm SiO(2). Moreover, the expression of p53 and Bax was increased in time and dose dependent patterns, whereas the expression of Bcl-2 was not significantly changed. In conclusion, ROS-mediated oxidative stress, the activation of p53 and up-regulation of Bax/Bcl-2 ratio are involved in mechanistic pathways of 21 nm SiO(2) induced apoptosis in L-02 cells.

  18. Salvianolic Acid B Inhibits ERK and p38 MAPK Signaling in TGF-β1-Stimulated Human Hepatic Stellate Cell Line (LX-2 via Distinct Pathways

    Directory of Open Access Journals (Sweden)

    Zhigang Lv

    2012-01-01

    Full Text Available Salvianolic acid B (SA-B is water-soluble component of Radix Salvia miltiorrhiza. The previous work indicated that SA-B can inhibit MAPK and Smad signaling in activated hepatic stellate cells (HSCs to perform anti-fibrotic activity Lv et al. 2010. However, some studies have shown that there is cross-talk between MAPK and Smad in certain cell types. Thus, the anti-fibrotic action of SA-B may be through the cross-talk. In order to clarify the mechanism of SA-B further, we knocked down Smad in LX-2 cells (SRV4 via RNAi, and then added TGF-β1, and PD98059 or SB203580 and SA-B. The levels of p-MEK and p-p38 were inhibited by SA-B in SRV4 independent of TGF-β1. The expression of Col I and α-SMA in SRV4 could be reduced by SA-B independent TGF-β1. SB203580 had not significant effect on p-MEK in SRV4 stimulated by TGF-β1. The levels of p-MEK in SRV4 were not increased significantly after TGF-β1 stimulation. PD98059 had no effect on the levels of p-p38 in SRV4 irrespective of TGF-β1. In conclusion, SA-B inhibits the synthesis of Col I in LX-2 cells independent of TGF-β1 stimulation, and the anti-fibrotic effect of SA-B is due to direct inhibition of p38 signaling and inhibition the cross-talk of Smad to ERK signaling.

  19. Salvianolic Acid B Inhibits ERK and p38 MAPK Signaling in TGF-β1-Stimulated Human Hepatic Stellate Cell Line (LX-2) via Distinct Pathways

    Science.gov (United States)

    Lv, Zhigang; Xu, Lieming

    2012-01-01

    Salvianolic acid B (SA-B) is water-soluble component of Radix Salvia miltiorrhiza. The previous work indicated that SA-B can inhibit MAPK and Smad signaling in activated hepatic stellate cells (HSCs) to perform anti-fibrotic activity Lv et al. 2010. However, some studies have shown that there is cross-talk between MAPK and Smad in certain cell types. Thus, the anti-fibrotic action of SA-B may be through the cross-talk. In order to clarify the mechanism of SA-B further, we knocked down Smad in LX-2 cells (SRV4) via RNAi, and then added TGF-β1, and PD98059 or SB203580 and SA-B. The levels of p-MEK and p-p38 were inhibited by SA-B in SRV4 independent of TGF-β1. The expression of Col I and α-SMA in SRV4 could be reduced by SA-B independent TGF-β1. SB203580 had not significant effect on p-MEK in SRV4 stimulated by TGF-β1. The levels of p-MEK in SRV4 were not increased significantly after TGF-β1 stimulation. PD98059 had no effect on the levels of p-p38 in SRV4 irrespective of TGF-β1. In conclusion, SA-B inhibits the synthesis of Col I in LX-2 cells independent of TGF-β1 stimulation, and the anti-fibrotic effect of SA-B is due to direct inhibition of p38 signaling and inhibition the cross-talk of Smad to ERK signaling. PMID:21860657

  20. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

    2014-01-01

    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  1. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

    2014-01-01

    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  2. Hepatic Giant Cell Arteritis and Polymyalgia Rheumatica

    Directory of Open Access Journals (Sweden)

    Donald R Duerksen

    1994-01-01

    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.

  3. Effect of melanoma cells on proliferation and migration of activated hepatic stellate cells in vitro.

    Science.gov (United States)

    Meyer, Theresa; Koch, Andreas; Ebert, Eva-Vanessa; Czech, Barbara; Mueller, Martina; Bosserhoff, Anja; Lang, Sven Arke; Hellerbrand, Claus

    2017-04-01

    Melanoma is a highly aggressive tumor of the skin. The clinical outcome is determined by the presence or absence of metastases, and the liver is a common site of distant metastases. Hepatic metastasis is causing activation of hepatic stellate cells (HSC), which form the stroma of hepatic metastases and are increasingly recognized as a crucial component of the pro- metastatic liver microenvironment. Most studies have focused on the effects of HSC on (metastasizing) tumor cells. Here, we aimed to analyze functional in vitro effects of conditioned medium (CM) of twelve different human melanoma cell lines on LX2 cells and HSC(htert) cells, two well established human activated HSC cell lines. CM from melanoma cells significantly induced HSC proliferation and acted as chemoattractant for HSC in Boyden chamber assays. The CM effects significantly varied between different HSC as well as melanoma cells. Interestingly, CM from melanoma cell lines derived from melanoma metastases (WM239A, WM9, WM1158, WM1232, 451Lu and 1205Lu) had a stronger effect on proliferation of HSC(htert) cells than CM derived from primary melanoma tumors (SbCl2, WM3211, WM35, WM278, WM1366 and WM793). Moreover, we observed a significant correlation between the chemoattractive effects of CM from the different melanoma cells on HSC(htert) and LX2 cells. In contrast, the melanoma CM effects on the proliferation of the two HSC lines did not show a significant correlation. In summary, our data indicate that melanoma cells metastasizing to the liver have the potential to attract HSC and to induce HSC proliferation, respectively. Still, it appears that melanoma effects on HSC migration and proliferation are mediated via different soluble factors indicating the complexity of melanoma-HSC interaction. Furthermore, the intensity of at least some functional effects varies between different human tumor cells and HSC which may point to mechanisms explaining diverse hepatic metastasis in melanoma patients

  4. Directed hepatic differentiation from embryonic stem cells

    OpenAIRE

    Chen, Xuesong; Zeng, Fanyi

    2011-01-01

    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...

  5. 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

    2004-01-01

    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.

  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: yzhang@wehi.edu.au [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: tienpo@sun.im.ac.cn [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China)

    2013-06-07

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

  7. Hepatitis C virus host cell interactions uncovered

    DEFF Research Database (Denmark)

    Gottwein, Judith; Bukh, Jens

    2007-01-01

      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...

  8. Hepatitis C virus host cell interactions uncovered

    DEFF Research Database (Denmark)

    Gottwein, Judith; Bukh, Jens

    2007-01-01

      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...

  9. Regulatory T cells in viral hepatitis

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

    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.

  10. Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy.

    Science.gov (United States)

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

    2017-01-01

    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.

  11. Regenerative cells for transplantation in hepatic failure.

    Science.gov (United States)

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

    2012-01-01

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

  12. 守宫硫酸多糖对肝癌SMMC-7721细胞分化和增殖的影响%Effects of Gekko Sulfated Polysaccharide on the Proliferation and Differentiation in Hepatic Cancer Cell Line SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    辛亮; 吴雄志; 谢广茹

    2011-01-01

    目的:研究守宫硫酸多糖(Gepsin)对肝癌SMMC-7721细胞分化和增殖的影响,并进一步探讨其可能的机制.方法:将不同浓度的Gepsin加入对数生长的SMMC-7721细胞进行培养,在不同的时间利用台盼兰染色观察细胞的增殖情况.同时,收集细胞培养上清液,利用联合放免法,以溴钾酚绿法分别观察Gepsin对甲胎蛋白(AFP)、白蛋白(ALB)分泌的影响;利用酶联免疫吸附试验法观察Gepsin对转化生长因子(TGF)-β1、血管内皮生长因子(VEGF)分泌的影响;利用光镜观察Gepsin对SMMC-7721细胞形态的影响.结果:随着处理组Gepsin的浓度升高,SMMC-7721细胞的增殖明显受到抑制;而对细胞活率无明显影响.Gepsin可增加培养上清中的ALB分泌量,降低AFP分泌量.光学显微镜下可见加入Gepsin后细胞的形态由圆形变为纺锤形.Gepsin对培养上清分泌的VEGF无影响,但可使TGF-β1增加.结论:Gepsin可明显抑制肝癌细胞SMMC-7721的增殖,诱导SMMC-7721细胞分化.其机制可能是通过上调TGF-β1来诱导肝癌细胞分化实现的.%Objective: To investigate the effect of Gekko sulfated polysaccharide (Gepsin) on the proliferation and differentiation in hepatic cancer cell line SMMC-7721. Methods: SMMC-7721 cells were cultured in RPM-1640 medium with different concentrations of Gepsin. Trypan blue stain was used to determine the cell proliferations in different time points. The cell culture supernatant was collected to observe the effect of Gepsin on alpha-fetoprotein (AFP) and albumin (ALB) by bromocresol green-immunology. Levels of transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) influenced by Gepsin were detected using enzyme-linked immunosorbent test. The effect of Gepsin on the morphology of SMMC-7721 was examined by light microscopy. Results: The proliferation of SMMC-7721 cells was significantly inhibited with the increased concentrations of Gepsin, but no effect on the

  13. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

  14. Suppressor of Cytokine Signaling-3 (SOCS-3) Induces Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Expression in Hepatic HepG2 Cell Line*

    Science.gov (United States)

    Ruscica, Massimiliano; Ricci, Chiara; Macchi, Chiara; Magni, Paolo; Cristofani, Riccardo; Liu, Jingwen; Corsini, Alberto; Ferri, Nicola

    2016-01-01

    The suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway activated by proinflammatory cytokines, including the tumor necrosis factor-α (TNF-α). SOCS3 is also implicated in hypertriglyceridemia associated to insulin resistance. Proprotein convertase subtilisin kexin type 9 (PCSK9) levels are frequently found to be positively correlated to insulin resistance and plasma very low density lipoprotein (VLDL) triglycerides concentrations. The present study aimed to investigate the possible role of TNF-α and JAK/STAT pathway on de novo lipogenesis and PCSK9 expression in HepG2 cells. TNF-α induced both SOCS3 and PCSK9 in a concentration-dependent manner. This effect was inhibited by transfection with siRNA anti-STAT3, suggesting the involvement of the JAK/STAT pathway. Retroviral overexpression of SOCS3 in HepG2 cells (HepG2SOCS3) strongly inhibited STAT3 phosphorylation and induced PCSK9 mRNA and protein, with no effect on its promoter activity and mRNA stability. Consistently, siRNA anti-SOCS3 reduced PCSK9 mRNA levels, whereas an opposite effect was observed with siRNA anti-STAT3. In addition, HepG2SOCS3 express higher mRNA levels of key enzymes involved in the de novo lipogenesis, such as fattyacid synthase, stearoyl-CoA desaturase (SCD)-1, and apoB. These responses were associated with a significant increase of SCD-1 protein, activation of sterol regulatory element-binding protein-1c (SREBP-1), accumulation of cellular triglycerides, and secretion of apoB. HepG2SOCS3 show lower phosphorylation levels of insulin receptor substrate 1 (IRS-1) Tyr896 and Akt Ser473 in response to insulin. Finally, insulin stimulation produced an additive effect with SOCS3 overexpression, further inducing PCSK9, SREBP-1, fatty acid synthase, and apoB mRNA. In conclusion, our data candidate PCSK9 as a gene involved in lipid metabolism regulated by proinflammatory cytokine TNF-α in a SOCS3-dependent manner. PMID:26668321

  15. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    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

  16. Endothelial cells are damaged by autophagic induction before hepatocytes in Con A-induced acute hepatitis.

    Science.gov (United States)

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

    2010-08-01

    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.

  17. 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.

  18. Links between human LINE-1 retrotransposons and hepatitis virus-related hepatocellular carcinoma

    Science.gov (United States)

    Honda, Tomoyuki

    2016-05-01

    Hepatocellular carcinoma (HCC) accounts for approximately 80% of liver cancers, the third most frequent cause of cancer mortality. The most prevalent risk factors for HCC are infections by hepatitis B or hepatitis C virus. Findings suggest that hepatitis virus-related HCC might be a cancer in which LINE-1 retrotransposons, often termed L1, activity plays a potential role. Firstly, hepatitis viruses can suppress host defense factors that also control L1 mobilization. Secondly, many recent studies also have indicated that hypomethylation of L1 affects the prognosis of HCC patients. Thirdly, endogenous L1 retrotransposition was demonstrated to activate oncogenic pathways in HCC. Fourthly, several L1 chimeric transcripts with host or viral genes are found in hepatitis virus-related HCC. Such lines of evidence suggest a linkage between L1 retrotransposons and hepatitis virus-related HCC. Here, I briefly summarize current understandings of the association between hepatitis virus-related HCC and L1. Then, I discuss potential mechanisms of how hepatitis viruses drive the development of HCC via L1 retrotransposons. An increased understanding of the contribution of L1 to hepatitis virus-related HCC may provide unique insights related to the development of novel therapeutics for this disease.

  19. 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

    2006-01-01

    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.

  20. Regulated expression of erythropoietin by two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  1. Natural killer cells in hepatitis C: Current progress.

    Science.gov (United States)

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

    2016-01-28

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

  2. 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

    2009-01-01

    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.

  3. [Hepatic cell transplantation. Technical and methodological aspects].

    Science.gov (United States)

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

    2010-03-01

    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. Copyright 2009 AEC. Published by Elsevier Espana. All rights reserved.

  4. Hepatic progenitor cells in human liver tumor development

    Institute of Scientific and Technical Information of China (English)

    Louis Libbrecht

    2006-01-01

    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.

  5. Natural killer cells in hepatitis B virus infection.

    Science.gov (United States)

    Wu, Shao-fei; Wang, Wen-jing; Gao, Yue-qiu

    2015-01-01

    Natural killer cells are a unique type of lymphocytes with cytotoxic capacity, and play important roles against tumors and infections. Recently, natural killer cells have been increasingly valued in their effects in hepatitis B virus infection. Since hepatitis B virus is not cytopathic, the subsequent antiviral immune responses of the host are responsible for sustaining the liver injury, which may result in cirrhosis and even hepatocellular carcinoma. Many studies have confirmed that natural killer cells participate in anti-hepatitis B virus responses both in the early phase after infection and in the chronic phase via cytolysis, degranulation, and cytokine secretion. However, natural killer cells play dichotomic roles: they exert antiviral and immunoregulatory functions whilst contribute to the pathogenesis of liver injury. Here, we review the roles of natural killer cells in hepatitis B virus infection, introducing novel therapeutic strategies for controlling hepatitis B virus infection via the modulation of natural killer cells. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

  6. Recent Advances in Hepatitis C Virus Cell Entry

    Directory of Open Access Journals (Sweden)

    Jean Dubuisson

    2010-03-01

    Full Text Available More than 170 million patients worldwide are chronically infected with hepatitis C virus (HCV. Prevalence rates range from 0.5% in Northern European countries to 28% in some areas of Egypt. HCV is hepatotropic, and in many countries chronic hepatitis C is a leading cause of liver disease including fibrosis, cirrhosis and hepatocellular carcinoma. HCV persists in 50–85% of infected patients, and once chronic infection is established, spontaneous clearance is rare. HCV is a member of the Flaviviridae family, in which it forms its own genus. Many lines of evidence suggest that the HCV life cycle displays many differences to that of other Flaviviridae family members. Some of these differences may be due to the close interaction of HCV with its host’s lipid and particular triglyceride metabolism in the liver, which may explain why the virus can be found in association with lipoproteins in serum of infected patients. This review focuses on the molecular events underlying the HCV cell entry process and the respective roles of cellular co-factors that have been implied in these events. These include, among others, the lipoprotein receptors low density lipoprotein receptor and scavenger receptor BI, the tight junction factors occludin and claudin-1 as well as the tetraspanin CD81. We discuss the roles of these cellular factors in HCV cell entry and how association of HCV with lipoproteins may modulate the cell entry process.

  7. 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

    2011-01-01

    AIM:To investigate intraperitoneal transplantation of microencapsulated hepatic-like cells from human umbilical cord blood for treatment of hepatic failure in rats.METHODS:CD34+ cells in umbilical cord blood cells were isolated by magnetic cell sorting.In the in vitro experiment,sorted CD34+ cells were amplified and induced into hepatic-like cells by culturing with a combination of fibroblast growth factor 4 and hepatocyte growth factor.Cultures without growth factor addition served as controls.mRNA and protein levels for hepatic- like cells were analyzed by reverse transcriptionpolymerase chain reaction,immunohistochemistry and immunofluorescence.In the in vivo experiment,the hepatic-like cells were encapsulated and transplanted into the abdominal cavity of acute hepatic failure (AHF) rats at 48 h after D-galactosamine induction of acute hepatic failure.Transplantation with PBS and unencapsulated hepatic-like cells served as controls.The mortality rate,hepatic pathological changes and serum biochemical indexes were determined.The morphology and structure of microcapsules in the greater omentum were observed.RESULTS:Human albumin,alpha-fetoprotein and GATA-4 mRNA and albumin protein positive cells were found among cultured cells after 16 d.Albumin level in culture medium was significantly increased after culturing with growth factors in comparison with culturing without growth factor addition (P < 0.01).Compared with the unencapsulated group,the mortality rate of the encapsulated hepatic-like cell-transplanted group was significantly lower (P < 0.05).Serum biochemical parameters,alanine aminotransferase,aspartate aminotransferase and total bilirubin in the encapsulated group were significantly improvement compared with the PBS control group (P < 0.01).Pathological staining further supported these findings.At 1-2 wk post-transplantation,free microcapsules with a round clear structure and a smooth surface were observed in peritoneal lavage fluid,surviving cells

  8. 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.

  9. 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)

    2003-01-01

    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.

  10. Development of mouse hepatocyte lines permissive for hepatitis C virus (HCV.

    Directory of Open Access Journals (Sweden)

    Hussein Hassan Aly

    Full Text Available The lack of a suitable small animal model for the analysis of hepatitis C virus (HCV infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. Human and mouse harbor a comparable system for antiviral type I interferon (IFN induction and amplification, which regulates viral infection and replication. Using hepatocytes from knockout (ko mice, we determined the critical step of the IFN-inducing/amplification pathways regulating HCV replication in mouse. The results infer that interferon-beta promoter stimulator (IPS-1 or interferon A receptor (IFNAR were a crucial barrier to HCV replication in mouse hepatocytes. Although both IFNARko and IPS-1ko hepatocytes showed a reduced induction of type I interferons in response to viral infection, only IPS-1-/- cells circumvented cell death from HCV cytopathic effect and significantly improved J6JFH1 replication, suggesting IPS-1 to be a key player regulating HCV replication in mouse hepatocytes. We then established mouse hepatocyte lines lacking IPS-1 or IFNAR through immortalization with SV40T antigen. Expression of human (hCD81 on these hepatocyte lines rendered both lines HCVcc-permissive. We also found that the chimeric J6JFH1 construct, having the structure region from J6 isolate enhanced HCV replication in mouse hepatocytes rather than the full length original JFH1 construct, a new finding that suggests the possible role of the HCV structural region in HCV replication. This is the first report on the entry and replication of HCV infectious particles in mouse hepatocytes. These mouse hepatocyte lines will facilitate establishing a mouse HCV infection model with multifarious applications.

  11. Pure Red Cell Aplasia Caused by Acute Hepatitis A

    OpenAIRE

    Lee, Tae Heon; Oh, Suk Joong; Hong, Soojung; Lee, Kyu Bek; Park, Hyosoon; Woo, Hee-Yeon

    2011-01-01

    Pure red cell aplasia is characterized as a normocytic anemia associated with reticulocytopenia and the absence of erythroblasts in the bone marrow. Pure red cell aplasia can be induced by various causes such as thymoma, connective tissue disease, viral infection, lymphoma, and adverse drug reactions. There have been only a few reports of pure red cell aplasia associated with acute viral hepatitis A. In Korea, no case of pure red cell aplasia caused by acute hepatitis A has yet been reported....

  12. Establishment and characterization of rat portal myofibroblast cell lines.

    Directory of Open Access Journals (Sweden)

    Michel Fausther

    Full Text Available The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC and portal fibroblasts (PF. In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myofibroblasts and their contribution to the progression of liver fibrosis.

  13. Differential effects on apoptosis induction in hepatocyte lines by stable expression of hepatitis B virus X protein

    Institute of Scientific and Technical Information of China (English)

    Nicola Fiedler; Ellen Quant; Ludger Fink; Jianguang Sun; Ralph Schuster; Wolfram H Gerlich; Stephan Schaefer

    2006-01-01

    AIM: Hepatitis B virus protein X (HBx) has been shown to be weakly oncogenic in vitro. The transforming activities of HBx have been linked with the inhibition of several functions of the tumor suppressor p53. We have studied whether HBx may have different effects on p53 depending on the cell type.METHODS: We used the human hepatoma cell line HepG2 and the immortalized murine hepatocyte line AML12 and analyzed stably transfected clones which expressed physiological amounts of HBx. P53 was induced by UV irradiation.RESULTS: The p53 induction by UV irradiation was unaffected by stable expression of HBx. However, the expression of the cyclin kinase inhibitor p21waf/cip/sdi which gets activated by p53 was affected in the HBx transformed cell line AML12-HBx9, but not in HepG2.In AML-HBx9 cells, p21waf/cip/sdi-protein expression and p21waf/cip/sdi transcription were deregulated. Furthermore,the process of apoptosis was affected in opposite ways in the two cell lines investigated. While stable expression of HBx enhanced apoptosis induced by UV irradiation in HepG2-cells, apoptosis was decreased in HBx transformed AML12-HBx9. P53 repressed transcription from the HBV enhancer I, when expressed from expression vectors or after induction of endogenous p53 by UV irradiation.Repression by endogenous p53 was partially reversible by stably expressed HBx in both cell lines.CONCLUSION: Stable expression of HBx leads to deregulation of apoptosis induced by UV irradiation depending on the cell line used. In an immortalized hepatocyte line HBx acted anti-apoptotic whereas expression in a carcinoma derived hepatocyte line HBx enhanced apoptosis.

  14. Molluscan cells in culture: primary cell cultures and cell lines.

    Science.gov (United States)

    Yoshino, T P; Bickham, U; Bayne, C J

    2013-06-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  15. Novel matrine derivative MD-1 attenuates hepatic fibrosis by inhibiting EGFR activation of hepatic stellate cells.

    Science.gov (United States)

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

    2016-09-01

    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.

  16. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

    Science.gov (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.

    2017-01-01

    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. PMID:28134251

  17. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver.

    Science.gov (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

    2017-01-30

    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.

  18. Using X-ray in-line phase-contrast imaging for the investigation of nude mouse hepatic tumors.

    Science.gov (United States)

    Tao, Qiang; Li, Dongyue; Zhang, Lu; Luo, Shuqian

    2012-01-01

    The purpose of this paper is to report the noninvasive imaging of hepatic tumors without contrast agents. Both normal tissues and tumor tissues can be detected, and tumor tissues in different stages can be classified quantitatively. We implanted BEL-7402 human hepatocellular carcinoma cells into the livers of nude mice and then imaged the livers using X-ray in-line phase-contrast imaging (ILPCI). The projection images' texture feature based on gray level co-occurrence matrix (GLCM) and dual-tree complex wavelet transforms (DTCWT) were extracted to discriminate normal tissues and tumor tissues. Different stages of hepatic tumors were classified using support vector machines (SVM). Images of livers from nude mice sacrificed 6 days after inoculation with cancer cells show diffuse distribution of the tumor tissue, but images of livers from nude mice sacrificed 9, 12, or 15 days after inoculation with cancer cells show necrotic lumps in the tumor tissue. The results of the principal component analysis (PCA) of the texture features based on GLCM of normal regions were positive, but those of tumor regions were negative. The results of PCA of the texture features based on DTCWT of normal regions were greater than those of tumor regions. The values of the texture features in low-frequency coefficient images increased monotonically with the growth of the tumors. Different stages of liver tumors can be classified using SVM, and the accuracy is 83.33%. Noninvasive and micron-scale imaging can be achieved by X-ray ILPCI. We can observe hepatic tumors and small vessels from the phase-contrast images. This new imaging approach for hepatic cancer is effective and has potential use in the early detection and classification of hepatic tumors.

  19. Using X-ray in-line phase-contrast imaging for the investigation of nude mouse hepatic tumors.

    Directory of Open Access Journals (Sweden)

    Qiang Tao

    Full Text Available The purpose of this paper is to report the noninvasive imaging of hepatic tumors without contrast agents. Both normal tissues and tumor tissues can be detected, and tumor tissues in different stages can be classified quantitatively. We implanted BEL-7402 human hepatocellular carcinoma cells into the livers of nude mice and then imaged the livers using X-ray in-line phase-contrast imaging (ILPCI. The projection images' texture feature based on gray level co-occurrence matrix (GLCM and dual-tree complex wavelet transforms (DTCWT were extracted to discriminate normal tissues and tumor tissues. Different stages of hepatic tumors were classified using support vector machines (SVM. Images of livers from nude mice sacrificed 6 days after inoculation with cancer cells show diffuse distribution of the tumor tissue, but images of livers from nude mice sacrificed 9, 12, or 15 days after inoculation with cancer cells show necrotic lumps in the tumor tissue. The results of the principal component analysis (PCA of the texture features based on GLCM of normal regions were positive, but those of tumor regions were negative. The results of PCA of the texture features based on DTCWT of normal regions were greater than those of tumor regions. The values of the texture features in low-frequency coefficient images increased monotonically with the growth of the tumors. Different stages of liver tumors can be classified using SVM, and the accuracy is 83.33%. Noninvasive and micron-scale imaging can be achieved by X-ray ILPCI. We can observe hepatic tumors and small vessels from the phase-contrast images. This new imaging approach for hepatic cancer is effective and has potential use in the early detection and classification of hepatic tumors.

  20. 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

    2004-01-01

    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.

  1. 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

    2001-01-01

    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.

  2. Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells.

    Science.gov (United States)

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

    2012-07-31

    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.

  3. Synthetic Polymer with a Structure-Driven Hepatic Deposition and Curative Pharmacological Activity in Hepatic Cells

    DEFF Research Database (Denmark)

    Riber, Camilla Frich; Halling Folkmar Andersen, Anna; Anegaard Rolskov, Lærke

    2017-01-01

    Synthetic polymers make strong contributions as tools for delivery of biological drugs and chemotherapeutics. The most praised characteristic of polymers in these applications is complete lack of pharmacological function such as to minimize the side effects within the human body. In contrast......, and pharmacokinetic properties not observed in close structural analogues. Specifically, PEAA reveals capacity to bind to albumin with ensuing natural hepatic deposition in vivo and exhibits concurrent inhibitory activity against the hepatitis C virus and inflammation in hepatic cells. Our findings provide a view...... on synthetic polymers as curative, functional agents and present PEAA as a unique biomedical tool with applications related to health of the human liver....

  4. Comparison of the effect of interferon on two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Crespi, M.; Schoub, B.D.; Lyons, S.F.; Chiu, M.N. (University of the Witwatersrand, Johannesburg (South Africa). Dept. of Virology)

    1985-06-01

    Two human hepatoma cell lines, the PLC/PRF/5 and the Mahlavu cells, which differ in their production of the hepatitis B surface antigen (HBsAg), responded differently to interferon (IFN). After IFN treatment both cell lines were able to inhibit Sindbis virus replication. Oligo A synthetase (E enzyme) could be activated in the PLC/PRF/5 cells although they were not sensitive to exogenous 2 - 5 oligoadenylic acid (2 - 5 A). In contrast, the Mahlavu cells were sensitive to exogenous 2 - 5 A, but unable to activate the E enzyme. Both cell lines were unable to stimulate phosphorylation of the exogenous initiator factor eIF-2.

  5. Derivation and characterization of hepatic progenitor cells from human embryonic stem cells.

    Science.gov (United States)

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

    2009-07-31

    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.

  6. Bioenergetic Changes during Differentiation of Human Embryonic Stem Cells along the Hepatic Lineage

    DEFF Research Database (Denmark)

    Hopkinson, Branden M; Madsen, Claus Desler; Kalisz, Mark

    2017-01-01

    of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction...

  7. Detection of anti-liver cell membrane antibody using a human hepatocellular carcinoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Lobo-Yeo, A.; McSorley, C.; McFarlane, B.M.; Mieli-Vergani, G.; Mowat, A.P.; Vergani, D.

    1989-02-01

    A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of /sup 125/I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens.

  8. 膈下逐瘀汤加减方作用人肝癌细胞SMMC-7721蛋白质组学研究%The proteomics research of human hepatic carcinoma cell line SMMC-7721 on the effects of Gexia Zhuyu decoction pharmacological serum

    Institute of Scientific and Technical Information of China (English)

    李华成; 王建刚; 费新应; 汪建平

    2015-01-01

    Objective:To analyze the differential expression protein of human hepatic carcinoma cell line SMMC-7721 under the Gexia Zhuyu decoction( GXZY) pharmacological serum, in order to find the anti-tumor target of GXZY.Methods:Subcultured human hepatic car-cinoma cell line SMMC-7721 were randomly divided into GXZY pharmacological serum groups and blank drug serum group, using two-dimen-sional gel electrophoresis, MALDI-TOF-MS to analyze the differential expression protein.Re sults:High expression of 33 protein spots was found in each of the groups, 6 proteins were identified by mass spectrometry analysis.Expression of 5 protein spots in GXZY pharmacological serum group decreased(AHCY,PGAM1,STMN1, HSP90AB1, VAT1) and 1 increased (TPM4).Conclusion:There are differential expres-sion proteins of human hepatic carcinoma cell line SMMC-7721 in GXZY pharmacological serum, these differential expression proteins may be the therapeutic targets of GXZY.%目的:运用蛋白质组学的研究方法,分析膈下逐瘀汤加减方( GXZY)含药血清作用人肝癌细胞SMMC-7721的差异蛋白质表达情况,以期寻找GXZY抗肿瘤作用靶点。方法:将人肝癌细胞SMMC-7721传代培养后,随机分为GXZY血清组以及空白对照组,应用双向凝胶电泳、MALDI-TOF-MS鉴定分析差异蛋白质表达情况。结果:GXZY血清组和空白对照组的蛋白电泳图谱上各有33个蛋白质点高表达,经质谱分析鉴定出6个蛋白质点,GXZY血清组有5个蛋白质点AHCY、PGAM1、STMN1、HSP90AB1、VAT-1表达下调,1个蛋白质点TPM4表达上调。结论:GXZY含药血清处理的人肝癌细胞SMMC-7721蛋白质质谱表达有差异,这些差异蛋白质可能是GXZY治疗的作用靶点。

  9. Efficacy of ribavirin against malignant glioma cell lines

    Science.gov (United States)

    OGINO, AKIYOSHI; SANO, EMIKO; OCHIAI, YUSHI; YAMAMURO, SHUN; TASHIRO, SHINYA; YACHI, KAZUNARI; OHTA, TAKASHI; FUKUSHIMA, TAKAO; OKAMOTO, YUTAKA; TSUMOTO, KOUHEI; UEDA, TAKUYA; YOSHINO, ATSUO; KATAYAMA, YOICHI

    2014-01-01

    Ribavirin (1-β-D-ribofuranosy-1,2,4-triazole-3-carboxamide) has been widely administered as an antiviral agent against RNA and DNA viruses. Ribavirin, in combination with interferon, has predominantly been applied in the treatment of the hepatitis C virus infection and its potential antitumor efficacy has recently become a point of interest. The aim of the present study was to evaluate the effect of ribavirin on the growth of malignant glioma cells, to identify novel predictive genes in malignant glioma cells (by analyzing gene expression profiles) and to assess the influence of ribavirin on the cell cycle of malignant glioma cells. The present study evaluated the antitumor efficacy of ribavirin against various malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13). After culturing the cells in ribavirin-containing culture medium (final concentration, 0–1,000 μM) for 72 h, the viable proliferated cells were harvested and counted. The half maximal inhibitory concentration of ribavirin, with regard to the growth of the malignant glioma cell lines, was determined from the concentration of ribavirin required for 50% growth inhibition in comparison to the untreated control cells. Furthermore, the current study identified the genes in which the gene expression levels correlated with the ribavirin sensitivity of the malignant glioma cells lines, using a high-density oligonucleotide array. Finally, cell cycle analysis was performed on the U-87MG cell line. It was identified that ribavirin inhibited the growth of all of the malignant glioma cell lines in a dose-dependent manner, although the ribavirin sensitivity varied between each cell line. Of the extracted genes, PDGFRA demonstrated the strongest positive correlation between gene expression level and ribavirin sensitivity. Cell cycle analysis of the U-87MG cell line demonstrated that ribavirin treatment induces G0/G1 arrest and thus may be an effective agent for inhibiting malignant

  10. 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.

  11. Regulation of gene expression in hepatic cells by the mammalian Target of Rapamycin (mTOR.

    Directory of Open Access Journals (Sweden)

    Rosa H Jimenez

    Full Text Available BACKGROUND: We investigated mTOR regulation of gene expression by studying rapamycin effect in two hepatic cell lines, the non-tumorigenic WB-F344 cells and the tumorigenic WB311 cells. The latter are resistant to the growth inhibitory effects of rapamycin, thus providing us with an opportunity to study the gene expression effects of rapamycin without confounding effects on cell proliferation. METHODOLOGY/PRINCIPAL FINDINGS: The hepatic cells were exposed to rapamycin for 24 hr. Microarray analysis on total RNA preparations identified genes that were affected by rapamycin in both cell lines and, therefore, modulated independent of growth arrest. Further studies showed that the promoter regions of these genes included E-box-containing transcription factor binding sites at higher than expected rates. Based on this, we tested the hypothesis that c-Myc is involved in regulation of gene expression by mTOR by comparing genes altered by rapamycin in the hepatic cells and by c-Myc induction in fibroblasts engineered to express c-myc in an inducible manner. Results showed enrichment for c-Myc targets among rapamycin sensitive genes in both hepatic cell lines. However, microarray analyses on wild type and c-myc null fibroblasts showed similar rapamycin effect, with the set of rapamycin-sensitive genes being enriched for c-Myc targets in both cases. CONCLUSIONS/SIGNIFICANCE: There is considerable overlap in the regulation of gene expression by mTOR and c-Myc. However, regulation of gene expression through mTOR is c-Myc-independent and cannot be attributed to the involvement of specific transcription factors regulated by the rapamycin-sensitive mTOR Complex 1.

  12. JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells.

    Science.gov (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

    2008-02-01

    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.

  13. Hepatitis C virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor-7 and signal transducer and activator of transcription (STAT 1 protein expression.

    Directory of Open Access Journals (Sweden)

    Amy E L Stone

    Full Text Available Plasmacytoid Dendritic Cells (pDCs represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs through pattern recognition receptors (PRR. PRR/PAMP interactions trigger signaling events that induce interferon (IFN production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL IFNs in response to HCV RNA. Extracellular HCV core protein (Core is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.

  14. 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

    2007-01-01

    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.

  15. Difference in membrane repair capacity between cancer cell lines and a normal cell line

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

    2016-01-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane...... repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p cell line (p membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher...

  16. 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.

  17. Hepatic differentiation of embryonic stem cells by murine fetal liver mesenchymal cells.

    Science.gov (United States)

    Ishii, Takamichi; Yasuchika, Kentaro; Ikai, Iwao

    2013-01-01

    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.

  18. 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.

  19. Pentoxifylline: A first line treatment option for severe alcoholic hepatitis and hepatorenal syndrome?

    Institute of Scientific and Technical Information of China (English)

    Stelios F Assimakopoulos; Konstantinos C Thomopoulos; Chrisoula Labropoulou-Karatza

    2009-01-01

    Although favourable results of pentoxifylline (PTX) used in treatment of severe alcoholic hepatitis patients with a Maddrey discriminant function score ≥ 32 have been previously reported, it is not currently recommended as a first line treatment for alcoholic hepatitis owing to lack of evidence for its efficacy as compared to the standard treatment with corticosteroids. In a very recent issue of World Journal of Gastroenterology, Dr. De BK and colleagues compared for the first time the two treatment modalities head to head in a randomized controlled study, demonstrating the advantage of PTX over corticosteroids in terms of patients' survival and risk-benefit profile. The advantage of PTX over corticosteroids in survival of patients with severe alcoholic hepatitis was found to be related to the prevention of hepatorenal syndrome in their study. This study raises the question of the use of PTX as a standard treatment for severe alcoholic hepatitis. Considering the fact that PTX presented a spectacular efficiency in prevention of hepatorenal syndrome in their study as well as that previous studies have shown that this effect is possibly related to a primary renoprotective action because it is irrelevant of tumor necrosis factor-α synthesis inhibition or improved liver function, we tempted to speculate that PXT might be an effective option for prevention and/or treatment of hepatorenal syndrome complicating other forms of advanced liver disease. This attractive theory remains to be elucidated by pressing future studies in view of the lack of effective treatment modalities for hepatorenal syndrome.

  20. Epigenetic regulation of hepatic stellate cell activation and liver fibrosis.

    Science.gov (United States)

    El Taghdouini, Adil; van Grunsven, Leo A

    2016-12-01

    Chronic liver injury to hepatocytes or cholangiocytes, when left unmanaged, leads to the development of liver fibrosis, a condition characterized by the excessive intrahepatic deposition of extracellular matrix proteins. Activated hepatic stellate cells constitute the predominant source of extracellular matrix in fibrotic livers and their transition from a quiescent state during fibrogenesis is associated with important alterations in their transcriptional and epigenetic landscape. Areas covered: We briefly describe the processes involved in hepatic stellate cell activation and discuss our current understanding of alterations in the epigenetic landscape, i.e DNA methylation, histone modifications and the functional role of non-coding RNAs that accompany this key event in the development of chronic liver disease. Expert commentary: Although great progress has been made, our understanding of the epigenetic regulation of hepatic stellate cell activation is limited and, thus far, insufficient to allow the development of epigenetic drugs that can selectively interrupt liver fibrosis.

  1. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    Science.gov (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

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

    Science.gov (United States)

    Vestentoft, Peter Siig

    2013-05-01

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

  3. TGF-β1/SMAD SIGNALING PATHWAY MEDIATES p53-DEPENDENT APOPTOSIS IN HEPATOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To determine whether transforming growth factor betal ( TGF-β1 )/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines. Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study. TGF-β31-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay. For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements. After transfection, cells were treated with TGF-β1, then assayed for luciferase activity. Results The apoptosis rate of HepG2 cell lines (48.51% ± 8.21% ) was significantly higher than control (12. 72% ±2. 18%, P <0. 05 ). But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines. The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4. 38) was significantly higher than control (1.00, P <0. 05). But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control. Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines. Smad4 is a central mediator of TGF-β1 signaling transdution pathway. TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.

  4. Giant cell hepatitis and autoimmune hemolytic anemia after chickenpox.

    Science.gov (United States)

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

    2010-12-01

    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.

  5. Quasispecies of Hepatitis C Virus Participate in Cell-Specific Infectivity.

    Science.gov (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

    2017-03-22

    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.

  6. Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds.

    Science.gov (United States)

    Yamazoe, Taiji; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    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.

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

    Science.gov (United States)

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

    2012-07-05

    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- (PDGFR- ), we constructed targeted sterically stable liposomes (SSLs) modified by a cyclic peptide (pPB) with affinity for the PDGFR- to deliver interferon (IFN)- to HSCs. The pPB-SSL-IFN- 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- mostly accumulated in the liver until 24 h. Furthermore, the pPB-SSL-IFN- 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- were less than those treated with SSL-IFN- , IFN- and the control group. In vitro pPB-SSL-IFN- was also more effective in suppressing activated HSC proliferation and inducing apoptosis of activated HSCs. Thus the data suggest that pPB-SSL-IFN- might be a more effective anti-fibrotic agent and a new opportunity for clinical therapy of hepatic fibrosis.

  8. Acute seronegative hepatitis C manifesting itself as adult giant cell hepatitis--a case report and review of literature.

    Science.gov (United States)

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

    2003-08-01

    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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  9. File list: ALL.Liv.20.AllAg.Hepatic_Stellate_Cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.20.AllAg.Hepatic_Stellate_Cells hg19 All antigens Liver Hepatic Stellate Ce...lls SRX100919 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Liv.20.AllAg.Hepatic_Stellate_Cells.bed ...

  10. File list: ALL.Liv.10.AllAg.Hepatic_Stellate_Cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Liv.10.AllAg.Hepatic_Stellate_Cells hg19 All antigens Liver Hepatic Stellate Ce...lls SRX100919 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Liv.10.AllAg.Hepatic_Stellate_Cells.bed ...

  11. Effect of hepatitis B virus X gene on apoptosis and immune molecules of renal tubular epithelial cells

    Institute of Scientific and Technical Information of China (English)

    王轩

    2013-01-01

    Objective To investigate the effect of hepatitis B virus X(HBX)gene on apoptosis and immune moleculesof human proximal renal tubular epithelial cell line(HK-2).Methods The eukaryotic vector pcDNA3.1-myc-HBX containing HBX gene was transiently transfected into

  12. Inhibition of Hepatitis C Virus-Like Particle Binding to Target Cells by Antiviral Antibodies in Acute and Chronic Hepatitis C

    Science.gov (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.

    2004-01-01

    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

  13. Treatment of hepatic encephalopathy by on-line hemodiafiltration: a case series study

    Directory of Open Access Journals (Sweden)

    Sugiyama Mitsugi

    2010-05-01

    Full Text Available Abstract Background It is thought that a good survival rate of patients with acute liver failure can be achieved by establishing an artificial liver support system that reliably compensates liver function until the liver regenerates or a patient undergoes transplantation. We introduced a new artificial liver support system, on-line hemodiafiltration, in patients with acute liver failure. Methods This case series study was conducted from May 2001 to October 2008 at the medical intensive care unit of a tertiary care academic medical center. Seventeen consecutive patients who admitted to our hospital presenting with acute liver failure were treated with artificial liver support including daily on-line hemodiafiltration and plasma exchange. Results After 4.9 ± 0.7 (mean ± SD on-line hemodiafiltration sessions, 16 of 17 (94.1% patients completely recovered from hepatic encephalopathy and maintained consciousness for 16.4 ± 3.4 (7-55 days until discontinuation of artificial liver support (a total of 14.4 ± 2.6 [6-47] on-line hemodiafiltration sessions. Significant correlation was observed between the degree of encephalopathy and number of sessions of on-line HDF required for recovery of consciousness. Of the 16 patients who recovered consciousness, 7 fully recovered and returned to society with no cognitive sequelae, 3 died of complications of acute liver failure except brain edema, and the remaining 6 were candidates for liver transplantation; 2 of them received living-related liver transplantation but 4 died without transplantation after discontinuation of therapy. Conclusions On-line hemodiafiltration was effective in patients with acute liver failure, and consciousness was maintained for the duration of artificial liver support, even in those in whom it was considered that hepatic function was completely abolished.

  14. False leukemia-lymphoma cell lines: an update on over 500 cell lines.

    Science.gov (United States)

    Drexler, H G; Dirks, W G; Matsuo, Y; MacLeod, R A F

    2003-02-01

    Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

  15. 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

    2009-01-01

    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.

  16. Regulatory T Cells in Chronic Hepatitis B Virus Infection

    NARCIS (Netherlands)

    J.N. Stoop (Jeroen Nicolaas)

    2007-01-01

    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

  17. 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

    2015-01-01

    Kupffer cells play a central role in the pathogenesis of alcoholic hepatitis(AH). It is believed that alcohol increases the gut permeability that results in raised levels of serum endotoxins containing lipopolysaccharides(LPS). LPS binds to LPS-binding proteins and presents it to a membrane glycoprotein called CD14, which then activates Kupffer cells via a receptor called 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.

  18. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    1993-01-01

    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)

    2010-01-01

    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. 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

    2004-01-01

    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

  1. 丙型肝炎病毒非结构蛋白3稳定细胞系的建立%Establish Stable Cell Line to Express Non-structural(NS)protein 3 of Hepatitis C Virus

    Institute of Scientific and Technical Information of China (English)

    倪念亲; 王娟娟; 陈进; 李子博

    2011-01-01

    目的 利用丙型肝炎病毒非结构基因NS3的真核表达载体(pCMV-Tag1-NS3)在HEK293细胞中进行表达.方法 通过酶切分析和DNA序列分析鉴定pCMV-Tag1-NS3重组载体构建正确后,通过脂质体介导转染HEK293细胞,用RT-PCR和Western blotting证实丙型肝炎非结构蛋白NS3基因(HCV-NS3)表达.结果 RTPCR结果 显示HCV-NS3基因的mRNA在转染的HKK293细胞中表达,Western blotting结果 也显示蛋白质能正确表达.结论 成功构建了实现HCV-NS3过表达的稳定HEK293细胞系,为获得HCV非结构蛋白3的重组蛋白以及早期诊断试剂的研制与筛选药物提供了基础.%Objective To construct the expression vector of pCMV- Tagl - NS3, and to express it in HEK293 cells.Methods After the recombinant vector was identified by enzyme digestion and DNA sequence analysis, the pCMV Tag1 -NS3 was transfected into HEK293 cell by using Lipofectamine reagent. In addition, RT - PCR and Western blotting were used to detect the mRNA and protein expression efficiencies, respectively. Results The mRNA level expression of HCV- NS3 was shown in pCMV- Tagl - NS3 transfectant by RT- PCR analysis. Western blot also showed strong expression of HCV-NS3 protein in pCMV- Tagl - NS3 transfectant. Conclusions This stable cell line represents the experimental basis for further studies of HCV infection and diagnosis and may provide a model for screening inhibitor of HCV- NS3.

  2. Early hepatic stellate cell activation predicts severe hepatitis C recurrence after liver transplantation.

    Science.gov (United States)

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

    2005-10-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Claus Kordes

    2013-02-01

    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.

  4. Virus Discovery Using Tick Cell Lines

    Science.gov (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

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

    Energy Technology Data Exchange (ETDEWEB)

    Otani, Satoshi [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Kakinuma, Sei, E-mail: skakinuma.gast@tmd.ac.jp [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Department for Liver Disease Control, Tokyo Medical and Dental University, Tokyo (Japan); Kamiya, Akihide [Institute of Innovative Science and Technology, Tokai University, Isehara (Japan); Goto, Fumio; Kaneko, Shun; Miyoshi, Masato; Tsunoda, Tomoyuki; Asano, Yu; Kawai-Kitahata, Fukiko; Nitta, Sayuri; Nakata, Toru; Okamoto, Ryuichi; Itsui, Yasuhiro; Nakagawa, Mina; Azuma, Seishin [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Asahina, Yasuhiro [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Department for Liver Disease Control, Tokyo Medical and Dental University, Tokyo (Japan); Yamaguchi, Tomoyuki [Division of Stem Cell Therapy, Institute of Medical Science, The University of Tokyo, Tokyo (Japan); Koshikawa, Naohiko [Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Tokyo (Japan); Seiki, Motoharu [Medical School, Kanazawa University, Kanazawa (Japan); Nakauchi, Hiromitsu [Division of Stem Cell Therapy, Institute of Medical Science, The University of Tokyo, Tokyo (Japan); and others

    2016-01-22

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

  6. Clinical associations of hepatic stellate cell (HSC) hyperplasia.

    Science.gov (United States)

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

    2014-07-01

    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.

  7. Congenital hepatic fibrosis, liver cell carcinoma and adult polycystic kidneys.

    Science.gov (United States)

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

    1977-06-01

    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.

  8. Hepatitis

    Science.gov (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 ...

  9. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    1997-01-01

    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

  10. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    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.

  11. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    1992-01-01

    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

  12. 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

    2013-01-01

    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

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

    OpenAIRE

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

    2010-01-01

    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.

  14. 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

    2007-01-01

    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.

  15. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

    Science.gov (United States)

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

  16. 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

    2002-01-01

    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.

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

    Science.gov (United States)

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

    2012-07-01

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

  18. Apicobasal Polarity Controls Lymphocyte Adhesion to Hepatic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Natalia Reglero-Real

    2014-09-01

    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.

  19. Effect of Kruppel-like factor 4 on Notch pathway in hepatic stellate cells.

    Science.gov (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

    2016-12-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    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.

  1. 锌原卟啉ZnPP Ⅸ对Bel/FU肝癌耐药细胞株p-糖蛋白及化疗敏感性的影响%ZnPP Ⅸ's effect on p-glycoprotein expression and chemosensitivity of drug-resistant hepatic carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    张隽开; 王忠裕; 巩鹏; 罗海峰; 牟国煜

    2011-01-01

    目的:评估锌原卟啉(ZnPP)Ⅸ对Bel/FU肝癌耐药细胞株P-糖蛋白(p-glycoprotein,p-GP)的影响,探讨ZnPP Ⅸ作为化疗耐药逆转剂可能性及相关作用机制.方法:不同浓度的ZnPP Ⅸ作用于Bel/FU肝癌化疗耐药细胞株,MTT法检测细胞株对化疗药物的半数抑制浓度(IC50)、Western blot检测p-GP表达量、罗丹明潴留试验检测p-GP药物外排功能.结果:ZnPP Ⅸ作用于Bel/FU细胞诱导24 h后,化疗药物IC50值均显著降低,P-GP表达量明显降低(P均<0.01),p-GP药物外排功能减弱.结论:ZnPP Ⅸ可通过影响p-GP蛋白质水平的表达,减弱P-GP药物外排功能,使肝癌耐药细胞株的化疗敏感性降低;ZnPP Ⅸ可作为一种潜在的化疗耐药逆转剂.%Objective: To evaluate ZnPP IX's effect on the expression of p-glycoprotein (P-GP)and the chemosensitivity of drug-resistant hepatic carcinoma Bel/Fu cell line,and to explore the relevant regulating mechanism. Methods: After Bel/FU cells being induced by different gradient concentrations of ZnPP IX for 24 h, MTT assay was carried out to detect the sensitivity of Bel/Fu:P-glycoprotein protein level was determined by Western blot analysis; Rhodamine123 retention fluorescent intensity measured by flow cytometry was used to detect the function of p-GP. Results:After Bel/FU cells being dealt with ZnPP IX for 24 h,there were significant differences in 50% inhibiting concentration (IC50) of chemical drugs between control groups and contrast group. IC50 of control groups decreased obviously. Meanwhile, p-GP protein level expression in the cells of control groups were observably lower than that in contrast group. The curve of Rhodamine123 retention fluorescent intensity in the control cells shift to the right, which indicated that the efflux pump function of p-GP was weakened. Conclusions: ZnPP IX can increase the chemosensitivity of Bel/FU cell line,which relates with its action in down-regulation of P-glycoprotein expression. ZnPP IX may be

  2. Discovery of Aptamer Ligands for Hepatic Stellate Cells Using SELEX.

    Science.gov (United States)

    Chen, Zhijin; Liu, Hao; Jain, Akshay; Zhang, Li; Liu, Chang; Cheng, Kun

    2017-01-01

    Insulin like growth factor II receptor (IGFIIR) is a transmembrane protein overexpressed in activated hepatic stellate cells (HSCs), which are the major target for the treatment of liver fibrosis. In this study, we aim to discover an IGFIIR-specific aptamer that can be potentially used as a targeting ligand for the treatment and diagnosis of liver fibrosis. Systematic evolution of ligands by exponential enrichment (SELEX) was conducted on recombinant human IGFIIR to identify IGFIIR-specific aptamers. The binding affinity and specificity of the discovered aptamers to IGFIIR and hepatic stellate cells were studied using flow cytometry and Surface Plasmon Resonance (SPR). Aptamer-20 showed the highest affinity to recombinant human IGFIIR protein with a Kd of 35.5 nM, as determined by SPR. Aptamer-20 also has a high affinity (apparent Kd 45.12 nM) to LX-2 human hepatic stellate cells. Binding of aptamer-20 to hepatic stellate cells could be inhibited by knockdown of IGFIIR using siRNA, indicating a high specificity of the aptamer. The aptamer formed a chimera with an anti-fibrotic PCBP2 siRNA and delivered the siRNA to HSC-T6 cells to trigger silencing activity. In Vivo biodistribution study of the siRNA-aptamer chimera also demonstrated a high and specific uptake in the liver of the rats with CCl4-induced liver fibrosis. These data suggest that aptamer-20 is a high-affinity ligand for antifibrotic and diagnostic agents for liver fibrosis.

  3. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    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.

    Science.gov (United States)

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

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Asako Murayama

    2016-06-01

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

  6. Wnt/β-catenin signaling cell-autonomously converts non-hepatic endodermal cells to a liver fate

    Directory of Open Access Journals (Sweden)

    Juhoon So

    2012-07-01

    Wnt/β-catenin signaling plays multiple roles in liver development including hepatoblast proliferation and differentiation, hepatocyte differentiation, and liver zonation. A positive role for Wnt/β-catenin signaling in liver specification was recently identified in zebrafish; however, its underlying cellular mechanisms are unknown. Here, we present two cellular mechanisms by which Wnt/β-catenin signaling regulates liver specification. First, using lineage tracing we show that ectopic hepatoblasts, which form in the endoderm posterior to the liver upon activation of Wnt/β-catenin signaling, are derived from the direct conversion of non-hepatic endodermal cells, but not from the posterior migration of hepatoblasts. We found that endodermal cells at the 4–6th somite levels, which normally give rise to the intestinal bulb or intestine, gave rise to hepatoblasts in Wnt8a-overexpressing embryos, and that the distribution of traced endodermal cells in Wnt8a-overexpressing embryos was similar to that in controls. Second, by using an endoderm-restricted cell-transplantation technique and mosaic analysis with transgenic lines that cell-autonomously suppress or activate Wnt/β-catenin signaling upon heat-shock, we show that Wnt/β-catenin signaling acts cell-autonomously in endodermal cells to induce hepatic conversion. Altogether, these data demonstrate that Wnt/β-catenin signaling can induce the fate-change of non-hepatic endodermal cells into a liver fate in a cell-autonomous manner. These findings have potential application to hepatocyte differentiation protocols for the generation of mature hepatocytes from induced pluripotent stem cells, supplying a sufficient amount of hepatocytes for cell-based therapies to treat patients with severe liver diseases.

  7. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  8. Hepatic drug metabolizing profile of Flinders Sensitive Line rat model of depression.

    Science.gov (United States)

    Kotsovolou, Olga; Ingelman-Sundberg, Magnus; Lang, Matti A; Marselos, Marios; Overstreet, David H; Papadopoulou-Daifoti, Zoi; Johanson, Inger; Fotopoulos, Andrew; Konstandi, Maria

    2010-08-16

    The Flinders Sensitive Line (FSL) rat model of depression exhibits some behavioral, neurochemical, and pharmacological features that have been reported in depressed patients and has been very effective in screening antidepressants. Major factor that determines the effectiveness and toxicity of a drug is the drug metabolizing capacity of the liver. Therefore, in order to discriminate possible differentiation in the hepatic drug metabolism between FSL rats and Sprague-Dawley (SD) controls, their hepatic metabolic profile was investigated in this study. The data showed decreased glutathione (GSH) content and glutathione S-transferase (GST) activity and lower expression of certain major CYP enzymes, including the CYP2B1, CYP2C11 and CYP2D1 in FSL rats compared to SD controls. In contrast, p-nitrophenol hydroxylase (PNP), 7-ethoxyresorufin-O-dealkylase (EROD) and 16alpha-testosterone hydroxylase activities were higher in FSL rats. Interestingly, the wide spread environmental pollutant benzo(alpha)pyrene (B(alpha)P) induced CYP1A1, CYP1A2, CYP2B1/2 and ALDH3c at a lesser extend in FSL than in SD rats, whereas the antidepressant mirtazapine (MIRT) up-regulated CYP1A1/2, CYP2C11, CYP2D1, CYP2E1 and CYP3A1/2, mainly, in FSL rats. The drug also further increased ALDH3c whereas suppressed GSH content in B(alpha)P-exposed FSL rats. In conclusion, several key enzymes of the hepatic biotransformation machinery are differentially expressed in FSL than in SD rats, a condition that may influence the outcome of drug therapy. The MIRT-induced up-regulation of several drug-metabolizing enzymes indicates the critical role of antidepressant treatment that should be always taken into account in the designing of treatment and interpretation of insufficient pharmacotherapy or drug toxicity.

  9. Stem cell characteristics in prostate cancer cell lines.

    NARCIS (Netherlands)

    Pfeiffer, M.J.; Schalken, J.A.

    2010-01-01

    BACKGROUND: Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE: Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell pr

  10. Matrine induces the hepatic differentiation of WB-F344 rat hepatic progenitor cells and inhibits Jagged 1/HES1 signaling.

    Science.gov (United States)

    Yang, Zhiyun; Wang, Li; Wang, Xianbo

    2016-10-01

    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.

  11. Hepatic stem cells: existence and origin

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

    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.

  12. Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells.

    Science.gov (United States)

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

    2017-03-15

    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

  13. Inverse association between hepatic stellate cell apoptosis and fibrosis in chronic hepatitis C virus infection.

    Science.gov (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

    2009-02-01

    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.

  14. 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

    2009-01-01

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

  15. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  16. 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.

    Science.gov (United States)

    Coffin, C S; Osiowy, C; Gao, S; Nishikawa, S; van der Meer, F; van Marle, G

    2015-04-01

    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.

  17. Hepatitis C virus core protein induces fibrogenic actions of hepatic stellate cells via toll-like receptor 2.

    Science.gov (United States)

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

    2011-09-01

    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.

  18. The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways.

    Science.gov (United States)

    Youns, Mаhmoud; Abdel Halim Hegazy, Wael

    2017-01-01

    Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes.

  19. The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways

    Science.gov (United States)

    Youns, Mаhmoud; Abdel Halim Hegazy, Wael

    2017-01-01

    Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes. PMID:28052097

  20. Stem cells for hepatic regeneration: the role of adipose tissue derived mesenchymal stem cells.

    Science.gov (United States)

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

    2010-06-01

    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.

  1. Target hepatic artery regional chemotherapy and bevacizumab perfusion in liver metastatic colorectal cancer after failure of first-line or second-line systemic chemotherapy.

    Science.gov (United States)

    Chen, Hui; Zhang, Ji; Cao, Guang; Liu, Peng; Xu, Haifeng; Wang, Xiaodong; Zhu, Xu; Gao, Song; Guo, Jianhai; Zhu, Linzhong; Zhang, Pengjun

    2016-02-01

    Colorectal cancer liver metastasis (CRLM) is a refractory disease after failure of first-line or second-line chemotherapy. Bevacizumab is recommended as first-line therapy for advanced colorectal cancer, but is unproven in CRLM through the hepatic artery. We report favorable outcomes with targeted vessel regional chemotherapy (TVRC) for liver metastatic gastric cancer. TVRC with FOLFOX and bevacizumab perfusion through the hepatic artery was attempted for CRLM for efficacy and safety evaluation. In a single-institution retrospective observational study, 246 patients with CRLM after at least first-line or second-line failure of systemic chemotherapy received TVRC with FOLFOX (i.e. oxaliplatin, leucovorin, and 5-fluorouracil). Of 246 patients, 63 were enrolled into two groups: group 1 (n=30) received bevacizumab and TVRC following tumor progression during previous TVRC treatments; group 2 (n=33) received TVRC plus bevacizumab for CRLM on initiating TVRC. There were no significant differences in the median survival time (14.7 vs. 13.2 months, P=0.367), although the median time to progression was significant (3.3 vs. 5.5 months, P=0.026) between groups. No severe adverse events related to TVRC plus bevacizumab perfusion occurred. Target vessel regional chemotherapy with FOLFOX plus bevacizumab perfusion through the hepatic artery was effective and safe in CRLM. The optimal combination of TVRC and bevacizumab needs further confirmation in future phase II-III clinical trials.

  2. Cell Culture Models for the Investigation of Hepatitis B and D Virus Infection

    Directory of Open Access Journals (Sweden)

    Eloi R. Verrier

    2016-09-01

    Full Text Available 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.

  3. Cell Culture Models for the Investigation of Hepatitis B and D Virus Infection

    Science.gov (United States)

    Verrier, Eloi R.; Colpitts, Che C.; Schuster, Catherine; Zeisel, Mirjam B.; Baumert, Thomas F.

    2016-01-01

    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

  4. Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

    Science.gov (United States)

    Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

    2013-07-19

    Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

  5. WWOX induces apoptosis and inhibits proliferation of human hepatoma cell line SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    Ben-Shun Hu; Jing-Wang Tan; Guo-Hua Zhu; Dan-Feng Wang; Xian Zhou; Zhi-Qiang Sun

    2012-01-01

    AIM:To investigate the effects of the WWOX gene on the human hepatic carcinoma cell line SMMC-7721.METHODS:Full-length WWOX cDNA was amplified from normal human liver tissues.Full-length cDNA was subcloned into pEGFP-N1,a eukaryotic expression vector.After introduction of the WWOX gene into cancer cells using liposomes,the WWOX protein level in the cells was detected through Western blotting.Cell growth rates were assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays.Cell cycle progression and cell apoptosis were measured by flow cytometry.The phosphorylated protein kinase B (AKT)and activated fragments of caspase-9 and caspase-3 were examined by Western blotting analysis.RESULTS:WWOX significantly inhibited cell proliferation,as evaluated by the MTT and colony formation assays.Cells transfected with WWOX showed significantly higher apoptosis ratios when compared with cells transfected with a mock plasmid,and overexpression of WWOX delayed cell cycle progression from G1 to S phase,as measured by flow cytometry.An increase in apoptosis was also indicated by a remarkable activation of caspase-9 and caspase-3 and a dephosphorylation of AKT (Thr308 and Ser473) measured with Western blotting analysis.CONCLUSION:Overexpression of WWOX induces apoptosis and inhibits proliferation of the human hepatic carcinoma cell line SMMC-7721.

  6. Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo

    Institute of Scientific and Technical Information of China (English)

    PF Lalor; WK Lai; SM Curbishley; S Shetty; DH Adams

    2006-01-01

    The hepatic sinusoids are lined by a unique population of hepatic sinusoidal endothelial cells (HSEC), which is one of the first hepatic cell populations to come into contact with blood components. However, HSEC are not simply barrier cells that restrict the access of bloodborne compounds to the parenchyma. They are functionally specialised endothelial cells that have complex roles, including not only receptor-mediated clearance of endotoxin, bacteria and other compounds, but also the regulation of inflammation, leukocyte recruitment and host immune responses to pathogens. Thus understandlng the differentiation and function of HSEC is critical for the elucidation of liver biology and pathophysiology. This article reviews methods for isolating and studying human hepatic endothelial cell populations using in vitro models. We also discuss the expression and functions of phenotypic markers, such as the presence of fenestrations and expression of VAP-1, Stabilin-1, L-SIGN, which can be used to identify sinusoidal endothelium and to permit discrimination from vascular and lymphatic endothelial cells.

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

    Directory of Open Access Journals (Sweden)

    Ziad Hassoun

    2000-01-01

    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.

  8. 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

    2004-01-01

    @@ 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

  9. Altered T cell costimulation during chronic hepatitis B infection.

    Science.gov (United States)

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

    2009-01-01

    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.

  10. Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3+, CD14+ and CD19+

    OpenAIRE

    2013-01-01

    Background Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection:...

  11. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

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

    Science.gov (United States)

    Yanagida, Ayaka; Nakauchi, Hiromitsu; Kamiya, Akihide

    2016-01-01

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

  13. 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

    2006-01-01

    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 congenit.al 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.

  14. RNA-seq based transcriptome analysis of hepatitis E virus (HEV) and hepatitis B virus (HBV) replicon transfected Huh-7 cells.

    Science.gov (United States)

    Jagya, Neetu; Varma, Satya Pavan Kumar; Thakral, Deepshi; Joshi, Prashant; Durgapal, Hemlata; Panda, Subrat Kumar

    2014-01-01

    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.

  15. Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II, and SMRV in human and other primate cell lines.

    Science.gov (United States)

    Uphoff, Cord C; Denkmann, Sabine A; Steube, Klaus G; Drexler, Hans G

    2010-01-01

    The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections.

  16. Activated hepatic stellate cells in liver cirrhosis. A morphologic and morphometrical study.

    Science.gov (United States)

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

    2004-01-01

    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.

  17. 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

    2007-01-01

    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

  18. 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

    2011-01-01

    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.

  19. Anti-hepatic fibrosis effects of a novel turtle shell decoction by inhibiting hepatic stellate cell proliferation and blocking TGF-β1/Smad signaling pathway in rats.

    Science.gov (United States)

    Bai, Ganping; Yan, Guohe; Wang, Guojian; Wan, Ping; Zhang, Ronghua

    2016-11-01

    Hepatic fibrosis (HF), a wound-healing response to a variety of chronic stimuli, is characterized by the excessive synthesis of extracellular matrix (ECM) proteins by hepatic stellate cells (HSC) and eventually the development of hepatic cirrhosis. Turtle shell pill (TSP) is a common traditional Chinese medicine used for preventing and treating HF and early hepatic cirrhosis, but its side-effects and the shortage of ingredients limit its clinical application. In addition, its mechanism of action is not clear. In the present study, we first improved the original formula of TSP to produce a novel turtle shell decoction (NTSD) with less toxicity and easier accessible materials. In a carbon tetrachloride (CCl4)-induced HF rat model, we observed that NTSD and TSP had similar effects on the improvement of liver functions in rats, including a decrease in serum alanine amino transferase (ALT) and aspartate amino transferase (AST) serum concentrations and increased albumin content in addition to a marked attenuation of CCl4-induced liver damage and fibrosis. NTSD containing rat serum inhibited rat liver stellate cell line HSC-T6 cell proliferation and induced cell apoptosis in vitro. Moreover, the NTSD treatment significantly decreased the transforming growth factor beta 1 (TGF-β1) and Smad3 gene expression and increased inhibitory Smad7 gene expression in liver tissues of HF rats, suggesting that NTSD inhibited the ECM expression of HSC by downregulating the TGF-β1/Smad signaling pathway. The results of our rat model study revealed that NTSD showed good in vitro and in vivo anti-HF effects via proliferation inhibition and the induction of apoptosis of HSCs and blocked the TGF-β1/Smad signaling pathway.

  20. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  1. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  2. B cell: T cell interactions occur within hepatic granulomas during experimental visceral leishmaniasis.

    Directory of Open Access Journals (Sweden)

    John W J Moore

    Full Text Available Hepatic resistance to Leishmania donovani infection in mice is associated with the development of granulomas, in which a variety of lymphoid and non-lymphoid populations accumulate. Although previous studies have identified B cells in hepatic granulomas and functional studies in B cell-deficient mice have suggested a role for B cells in the control of experimental visceral leishmaniasis, little is known about the behaviour of B cells in the granuloma microenvironment. Here, we first compared the hepatic B cell population in infected mice, where ≈60% of B cells are located within granulomas, with that of naïve mice. In infected mice, there was a small increase in mIgM(lomIgD(+ mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production. Using 2-photon microscopy to quantify the entire intra-granuloma B cell population, in conjunction with the adoptive transfer of polyclonal and HEL-specific BCR-transgenic B cells isolated from L. donovani-infected mice, we demonstrated that B cells accumulate in granulomas over time in an antigen-independent manner. Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment. These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site.

  3. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (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

    2007-12-21

    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.

  4. Expression of Hepatitis B Virus Surface Antigen Gene in Cultured Cells by Using Recombinant Plasmid Vectors

    OpenAIRE

    Siddiqui, Aleem

    1983-01-01

    By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.

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

    Directory of Open Access Journals (Sweden)

    Phil Jun Lee

    2015-01-01

    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.

  6. Hepatitis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    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

  7. Glucocorticoids Have Opposing Effects on Liver Fibrosis in Hepatic Stellate and Immune Cells.

    Science.gov (United States)

    Kim, Kang Ho; Lee, Jae Man; Zhou, Ying; Harpavat, Sanjiv; Moore, David D

    2016-08-01

    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.

  8. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

    2011-01-01

    incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size......The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds...

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

    OpenAIRE

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

    2012-01-01

    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.

  10. Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Solimene A.C.C.

    2001-01-01

    Full Text Available The LISP-I human colorectal adenocarcinoma cell line was isolated from a hepatic metastasis at the Ludwig Institute, São Paulo, SP, Brazil. The objective of the present study was to isolate morphologically different subpopulations within the LISP-I cell line, and characterize some of their behavioral aspects such as adhesion to and migration towards extracellular matrix components, expression of intercellular adhesion molecules and tumorigenicity in vitro. Once isolated, the subpopulations were submitted to adhesion and migration assays on laminin and fibronectin (crucial proteins to invasion and metastasis, as well as to anchorage-independent growth. Two morphologically different subpopulations were isolated: LISP-A10 and LISP-E11. LISP-A10 presents a differentiated epithelial pattern, and LISP-E11 is fibroblastoid, suggesting a poorly differentiated pattern. LISP-A10 expressed the two intercellular adhesion molecules tested, carcinoembryonic antigen (CEA and desmoglein, while LISP-E11 expressed only low amounts of CEA. On the other hand, adhesion to laminin and fibronectin as well as migration towards these extracellular matrix proteins were higher in LISP-E11, as expected from its poorly differentiated phenotype. Both subpopulations showed anchorage-independent growth on a semi-solid substrate. These results raise the possibility that the heterogeneity found in the LISP-I cell line, which might have contributed to its ability to metastasize, was due to at least two different subpopulations herein identified.

  11. 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.

  12. A bioinformatics analysis of the cell line nomenclature.

    Science.gov (United States)

    Sarntivijai, Sirarat; Ade, Alexander S; Athey, Brian D; States, David J

    2008-12-01

    Cell lines are used extensively in biomedical research, but the nomenclature describing cell lines has not been standardized. The problems are both linguistic and experimental. Many ambiguous cell line names appear in the published literature. Users of the same cell line may refer to it in different ways, and cell lines may mutate or become contaminated without the knowledge of the user. As a first step towards rationalizing this nomenclature, we created a cell line knowledgebase (CLKB) with a well-structured collection of names and descriptive data for cell lines cultured in vitro. The objectives of this work are: (i) to assist users in extracting useful information from biomedical text and (ii) to highlight the importance of standardizing cell line names in biomedical research. This CLKB contains a broad collection of cell line names compiled from ATCC, Hyper CLDB and MeSH. In addition to names, the knowledgebase specifies relationships between cell lines. We analyze the use of cell line names in biomedical text. Issues include ambiguous names, polymorphisms in the use of names and the fact that some cell line names are also common English words. Linguistic patterns associated with the occurrence of cell line names are analyzed. Applying these patterns to find additional cell line names in the literature identifies only a small number of additional names. Annotation of microarray gene expression studies is used as a test case. The CLKB facilitates data exploration and comparison of different cell lines in support of clinical and experimental research. The web ontology file for this cell line collection can be downloaded at http://www.stateslab.org/data/celllineOntology/cellline.zip.

  13. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  14. On the ontology based representation of cell lines.

    Directory of Open Access Journals (Sweden)

    Matthias Ganzinger

    Full Text Available Cell lines are frequently used as highly standardized and reproducible in vitro models for biomedical analyses and assays. Cell lines are distributed by cell banks that operate databases describing their products. However, the description of the cell lines' properties are not standardized across different cell banks. Existing cell line-related ontologies mostly focus on the description of the cell lines' names, but do not cover aspects like the origin or optimal growth conditions. The objective of this work is to develop an ontology that allows for a more comprehensive description of cell lines and their metadata, which should cover the data elements provided by cell banks. This will provide the basis for the standardized annotation of cell lines and corresponding assays in biomedical research. In addition, the ontology will be the foundation for automated evaluation of such assays and their respective protocols in the future. To accomplish this, a broad range of cell bank databases as well as existing ontologies were analyzed in a comprehensive manner. We identified existing ontologies capable of covering different aspects of the cell line domain. However, not all data fields derived from the cell banks' databases could be mapped to existing ontologies. As a result, we created a new ontology called cell culture ontology (CCONT integrating existing ontologies where possible. CCONT provides classes from the areas of cell line identification, origin, cell line properties, propagation and tests performed.

  15. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  16. Copper ions stimulate the proliferation of hepatic stellate cells via oxygen stress in vitro.

    Science.gov (United States)

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

    2013-02-01

    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.

  17. 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

    2005-01-01

    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.

  18. [Hepatic cell transplantation: a new therapy in liver diseases].

    Science.gov (United States)

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

    2010-07-01

    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. Copyright 2009 AEC. Published by Elsevier Espana. All rights reserved.

  19. Characteristics of HCV replication and expression in a cultured human liver carcinoma cell line in vitro

    Institute of Scientific and Technical Information of China (English)

    SONG Zhi-qing; HAO Fei; MIN Feng; LIU Dao-jian

    2001-01-01

    Objective: To establish a cell culture system to support HCV long-term replication in vitro. Methods: A human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from chronic hepatitis C patient. Cells and supernatant of the culture medium were harvested at various time-phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supematant were examined with RT-PCR, in situ hybridization and immunohistochemistry respectively. Results: It was found that the intracellular HCV RNA was first detected on the 2nd day after culture, and then could be intermittently detected in both cells and supernatant over a period of at least 3 months after culture. HCV NS3, CP10 antigens were expressed in the cells. The fresh cells could be infected with the supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to peripheral blood mononuclear cells (PBMCs) was also observed. Conclusion: Our findings suggest that the human liver carcinoma cell line7721 is not only susceptible to HCV but also can support its long replication in vitro. This cell line with HCV infection in vitro can serve as a useful tool for the study of the mechanism of HCV infection and replication, the evaluation of antiviral agents, and the primary selection of neutralization assays and HCV vaccine development.

  20. Transcriptome-based repurposing of apigenin as a potential anti-fibrotic agent targeting hepatic stellate cells

    Science.gov (United States)

    Hicks, Daniel F.; Goossens, Nicolas; Blas-García, Ana; Tsuchida, Takuma; Wooden, Benjamin; Wallace, Michael C.; Nieto, Natalia; Lade, Abigale; Redhead, Benjamin; Cederbaum, Arthur I; Dudley, Joel T.; Fuchs, Bryan C.; Lee, Youngmin A.; Hoshida, Yujin; Friedman, Scott L.

    2017-01-01

    We have used a computational approach to identify anti-fibrotic therapies by querying a transcriptome. A transcriptome signature of activated hepatic stellate cells (HSCs), the primary collagen-secreting cell in liver, and queried against a transcriptomic database that quantifies changes in gene expression in response to 1,309 FDA-approved drugs and bioactives (CMap). The flavonoid apigenin was among 9 top-ranked compounds predicted to have anti-fibrotic activity; indeed, apigenin dose-dependently reduced collagen I in the human HSC line, TWNT-4. To identify proteins mediating apigenin’s effect, we next overlapped a 122-gene signature unique to HSCs with a list of 160 genes encoding proteins that are known to interact with apigenin, which identified C1QTNF2, encoding for Complement C1q tumor necrosis factor-related protein 2, a secreted adipocytokine with metabolic effects in liver. To validate its disease relevance, C1QTNF2 expression is reduced during hepatic stellate cell activation in culture and in a mouse model of alcoholic liver injury in vivo, and its expression correlates with better clinical outcomes in patients with hepatitis C cirrhosis (n = 216), suggesting it may have a protective role in cirrhosis progression.These findings reinforce the value of computational approaches to drug discovery for hepatic fibrosis, and identify C1QTNF2 as a potential mediator of apigenin’s anti-fibrotic activity. PMID:28256512

  1. Glial fibrillary acidic protein as an early marker of hepatic stellate cell activation in chronic and posttransplant recurrent hepatitis C.

    Science.gov (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

    2008-06-01

    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.

  2. Adult Langerhans Cell Histiocytosis with Hepatic and Pulmonary Involvement

    Directory of Open Access Journals (Sweden)

    Bruno Araujo

    2015-01-01

    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.

  3. Adult Langerhans Cell Histiocytosis with Hepatic and Pulmonary Involvement

    Science.gov (United States)

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

    2015-01-01

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

  4. 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

    2014-01-01

    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.

  5. File list: InP.Liv.50.AllAg.Hepatic_Stellate_Cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  13. The citrus fruit flavonoid naringenin suppresses hepatic glucose production from Fao hepatoma cells.

    Science.gov (United States)

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

    2009-02-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    Nasser Semmo; Paul Klenerman

    2007-01-01

    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.

  15. A Neuroblastoma × Glioma Hybrid Cell Line with Morphine Receptors

    Science.gov (United States)

    Klee, Werner A.; Nirenberg, Marshall

    1974-01-01

    A neuroblastoma × glioma hybrid cell line with well-developed neural properties was found that has high-affinity morphine receptors. The average cell contains approximately 3 × 106 receptors. In contrast, parent cells and other neuroblastoma or hybrid cell lines tested had few or no morphine receptors. PMID:4530316

  16. 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

    2006-01-01

    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.

  17. RELATIONSHIP BETWEEN SOMATOSTATIN RECEPTORS AND ACTIVATION OF HEPATIC STELLATE CELL

    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

    2004-01-01

    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.

  18. 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

    2004-01-01

    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.

  19. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

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

    Science.gov (United States)

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

    2016-04-11

    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.

  1. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌

    2003-01-01

    Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

  2. Cryopreserved hepatic progenitor cells derived from human embryonic stem cells can arrest progression of liver fibrosis in rats.

    Science.gov (United States)

    Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra

    2016-10-01

    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.

  3. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (anti n, D/sub 0/) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL60 promyelocytic leukemia; K562 erythroleukemia; 45 acute lymphocytic leukemia; and 176 acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  4. Molluscan cells in culture: primary cell cultures and cell lines

    OpenAIRE

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  5. 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

    2004-01-01

    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

  6. Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

    Science.gov (United States)

    Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari

    2017-05-01

    The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

  7. Different Responses of Two Highly Permissive Cell Lines Upon HCV Infection

    Institute of Scientific and Technical Information of China (English)

    Honghe Chen; Rongjuan Pei; Xinwen Chen

    2013-01-01

    The construction of the first infectious clone JFH-1 speeds up the research on hepatitis C virus (HCV).However,Huh7 cell line was the only highly permissive cell line for HCV infection and only a few clones were fully permissive.In this study,two different fully permissive clones of Huh7 cells,Huh7.5.1 and Huh7-Lunet-CD81 (Lunet-CD81) cells were compared for their responses upon HCV infection.The virus replication level was found slightly higher in Huh7.5.1 cells than that in Lunet-CD81 cells.Viability of Huh7.5.1 cells but not of Lunet-CD81 cells was reduced significantly after HCV infection.Further analysis showed that the cell cycle of infected Huh7.5.1 cells was arrested at G1 phase.The G1/S transition was blocked by HCV infection in Huh7.5.1 cells as shown by the cell cycle synchronization analysis.Genes related to cell cycle regulation was modified by HCV infection and gene interaction analysis in GeneSpring GX in Direct Interactions mode highlighted 31 genes.In conclusion,the responses of those two cell lines were different upon HCV infection.HCV infection blocked G1/S transition and cell cycle progress,thus reduced the cell viability in Huh7.5.1 cells but not in Lunet-CD81 cells.Lunet-CD81 cells might be suitable for long term infection studies of HCV.

  8. 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

    2013-01-01

    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.

  9. 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

    2012-01-01

    Recognition and management of gastrointestinal and hepatic complications of hematopoietic stem cell transplantation has gained increasing importance as indications and techniques of transplantation have expanded in the last few years.The transplant recipient is at risk for several complications including conditioning chemotherapy related toxicities,infections,bleeding,sinusoidal obstruction syndrome,acute and chronic 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.

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

    Directory of Open Access Journals (Sweden)

    Joseph Ignatius Irudayam

    2015-12-01

    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.

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

    Science.gov (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

    2015-12-01

    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.

  12. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.

  13. Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Li-Hsuen Chen; Chia-Yu Hsu; Ching-Feng Weng

    2006-01-01

    AIM: Thioacetamide (TAA) has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death. METHODS: The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAAinduced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAAinduced clone 9 cells were measured by Western blot. RESULTS: The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died (MTT assay) within 24 h after 100 mg/L TAA was applied. Apoptotic cell percentage (TUNEL assay) and caspase 3 activities were highest after 100 mg/L TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspasedependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment.CONCLUSION: These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents,perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.

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

    DEFF Research Database (Denmark)

    Xiao, Fei; Fofana, Isabel; Heydmann, Laura

    2014-01-01

    Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies......-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....

  15. 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

    2006-01-01

    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.

  16. Hepatic fat is not associated with beta-cell function or postprandial free fatty acid response

    NARCIS (Netherlands)

    Rijkelijkhuizen, J.M.; Doesburg, T.; Girman, C.J.; Mari, A.; Rhodes, T.; Gastaldelli, A.; Nijpels, M.G.A.A.M.; Dekker, J.M.

    2009-01-01

    We evaluated the association of hepatic fat with beta-cell function estimated from the oral glucose tolerance test. In addition, we tested the hypothesis that postprandial free fatty acid (FFA) suppression after a meal tolerance test (MTT) is linked to hepatic fat. Individuals with normal glucose

  17. Nuclear Receptor PPARα Agonist Wy-14,643 Ameliorates Hepatic Cell Death in Hepatic IKKβ-Deficient Mice.

    Science.gov (United States)

    Kim, Taehyeong; Wahyudi, Lilik Duwi; Gonzalez, Frank J; Kim, Jung-Hwan

    2017-09-01

    Inhibitor of nuclear factor kappa-B kinase beta (IKKβ) plays a critical role in cell proliferation and inflammation in various cells by activating NF-κB signaling. However, the interrelationship between peroxisome proliferator-activated receptor α (PPARα) and IKKβ in cell proliferation is not clear. In this study, we investigated the possible role of PPARα in the hepatic cell death in the absence of IKKβ gene using liver-specific Ikkb-null (Ikkb(F/F-AlbCre)) mice. To examine the function of PPARα activation in hepatic cell death, wild-type (Ikkb(F/F)) and Ikkb(F/F-AlbCre) mice were treated with PPARα agonist Wy-14,643 (0.1% w/w chow diet) for two weeks. As a result of Wy-14,643 treatment, apoptotic markers including caspase-3 cleavage, poly (ADP-ribose) polymerase (PARP) cleavage and TUNEL-positive staining were significantly decreased in the Ikkb(F/F-AlbCre) mice. Surprisingly, Wy-14,643 increased the phosphorylation of p65 and STAT3 in both Ikkb and Ikkb(F/F-AlbCre) mice. Furthermore, BrdU-positive cells were significantly increased in both groups after treatment with Wy-14,643. Our results suggested that IKKβ-derived hepatic apoptosis could be altered by PPARα activation in conjunction with activation of NF-κB and STAT3 signaling.

  18. 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

    2005-01-01

    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

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

    Science.gov (United States)

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

    2015-02-01

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

  20. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  1. Growth and Development Symposium: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19.

    Science.gov (United States)

    Talbot, N C; Caperna, T J; Garrett, W M

    2013-01-01

    Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent

  2. HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

    Science.gov (United States)

    El-Awady, Mostafa K; Tabll, Ashraf A; El-Abd, Yasmine S; Bahgat, Mahmoud M; Shoeb, Hussein A; Youssef, Samar S; Din, Noha G Bader El; Redwan, El-Rashdy M; El-Demellawy, Maha; Omran, Moataza H; El-Garf, Wael T; Goueli, Said A

    2006-01-01

    AIM: To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naïve cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively. RESULTS: The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION: HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells. PMID:16937465

  3. HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

    Institute of Scientific and Technical Information of China (English)

    Mostafa K El-Awady; Moataza H Omran; Wael T El-Garf; Said A Goueli; Ashraf A Tabll; Yasmine S El-Abd; Mahmoud M Bahgat; Hussein A Shoeb; Samar S Youssef; Noha G Bader El Din; El-Rashdy M Redwan; Maha El-Demellawy

    2006-01-01

    AIM: To establish a cell culture system with longterm replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro. METHODS: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively. RESULTS: The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION: HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

  4. DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells.

    Science.gov (United States)

    Olaso, E; Ikeda, K; Eng, F J; Xu, L; Wang, L H; Lin, H C; Friedman, S L

    2001-11-01

    Type I collagen provokes activation of hepatic stellate cells during liver injury through mechanisms that have been unclear. Here, we tested the role of the discoidin domain tyrosine kinase receptor 2 (DDR2), which signals in response to type I collagen, in this pathway. DDR2 mRNA and protein are induced in stellate cells activated by primary culture or in vivo during liver injury. The receptor becomes tyrosine phosphorylated in response to either endogenous or exogenous type I collagen, whereas its expression is downregulated during cellular quiescence induced by growth on Matrigel. We developed stellate cell lines stably overexpressing either wild-type DDR2, a constitutively active chimeric DDR2 receptor (Fc-DDR2), a truncated receptor expressing the extracellular domain, or a kinase-dead DDR2 Cells overexpressing DDR2 showed enhanced proliferation and invasion through Matrigel, activities that were directly related to increased expression of active matrix metalloproteinase 2 (MMP-2). These data show that DDR2 is induced during stellate cell activation and implicate the phosphorylated receptor as a mediator of MMP-2 release and growth stimulation in response to type I collagen. Moreover, type I collagen-dependent upregulation of DDR2 expression establishes a positive feedback loop in activated stellate cells, leading to further proliferation and enhanced invasive activity.

  5. Immunoreactive hepatic stellate cells in biopsy material in children with chronic hepatitis B. The first report in pediatric patients.

    Science.gov (United States)

    Łotowska, Joanna M; Lebensztejn, Dariusz M

    2015-09-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Adiponectin regulates aquaglyceroporin expression in hepatic stellate cells altering their functional state.

    Science.gov (United States)

    Tardelli, Matteo; Moreno-Viedma, Veronica; Zeyda, Maximilian; Itariu, Bianca K; Langer, Felix B; Prager, Gerhard; Stulnig, Thomas M

    2017-01-01

    Obesity is a major risk factor for liver fibrosis and tightly associated with low levels of adiponectin. Adiponectin has antifibrogenic activity protecting from liver fibrosis, which is mainly driven by activated hepatic stellate cells (HSC). Aquaporins are transmembrane proteins that allow the movement of water and, in case of aquaglyceroporins (AQPs), of glycerol that is needed in quiescent HSC for lipogenesis. Expression of various AQPs in liver is altered by obesity; however, the mechanisms through which obesity influences HSCs activation and AQPs expression remain unclear. This study aimed to identify obesity-associated factors that are related to HSC AQPs expression activation and lipid storage. Correlations between serum adipokine levels and hepatic AQPs gene expression were analyzed from a cohort of obese patients. AQP and fibrotic gene expression was determined in a HSC line (LX2) and in a hepatocyte cell line (HepG2) after stimulation with adiponectin using quantitative real-time polymerase chain reaction. We found that serum adiponectin significantly correlated with liver AQP3, AQP7, AQP9 gene expressions. In vitro, adiponectin induced upregulation of AQP3 gene and AQP3 protein expression in human HSCs, but not in hepatocytes, while AQP7, AQP9 remained undetectable. Accordingly, HSC stimulated with adiponectin increased glycerol uptake, lipogenic gene expression, and lipid storage while downregulating activation/fibrosis markers. These findings demonstrate that adiponectin is a potent inhibitor of HSC activation and induces AQPs expression. Thus, low serum levels of adiponectin could be a mechanism how obesity affects the functional state of HSC, thereby contributing to obesity-associated liver fibrosis. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  8. POTENTIAL CELL LINE TOXICITY OF ENVIRONMENTAL NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Mohan Durga

    2012-01-01

    Full Text Available In India, the unprecedented growth rate and urbanization along with the rapid increase in motor vehicle activity and industrialization are contributing to high levels of urban air pollution. The population is mainly exposed to high air pollution concentrations, where motor vehicle emissions constitute the main source of fine and ultrafine particles. Motor exhaust emissions is a mixture of gases and Particulate Matter (PM. Diesel and petrol fuels in vehicles produce combustion-derived particles as a result of combustion. Vehicle exhaust particles are the main constituents of environmental nanoparticles. In the present investigation, environmental nanoparticles such as Diesel Exhaust Particles (DEP and Petrol Exhaust Particles (PEP were collected from on-road vehicles using a specially designed collection chamber. The surface morphology of the collected particles was analyzed through Transmission Electron Microscope (TEM, and the elemental mapping was performed through EDAX analysis. Results indicated the presence of nanometer-size particles in both the categories of vehicle exhaust. These small-size particles of respirable range can enter the respiratory tract of humans and get deposited in the lungs and cause various effects inside the human body. The aim of this study is to assess the cytotoxicity of the collected Diesel Exhaust Nanoparticles (DENPs and Petrol Exhaust Nanoparticles (PENPs. Cytotoxicity endpoint, such as IC50 (50% Inhibitory Concentration, was determined after a 24-h exposure. Results of this study indicated that all five cell lines were sensitive to these vehicle exhaust nanoparticles at varying levels.

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

    Science.gov (United States)

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

    2016-08-01

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

  10. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines.

    Science.gov (United States)

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

    2016-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Fei Xiao

    2014-05-01

    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.

  12. Hepatic differentiation of porcine embryonic stem cells for translational research of hepatocyte transplantation.

    Science.gov (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

    2015-04-01

    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.

  13. Apelin mediates the induction of profibrogenic genes in human hepatic stellate cells.

    Science.gov (United States)

    Melgar-Lesmes, Pedro; Casals, Gregori; Pauta, Montserrat; Ros, Josefa; Reichenbach, Vedrana; Bataller, Ramon; Morales-Ruiz, Manuel; Jimenez, Wladimiro

    2010-11-01

    Apelin is a peptide with relevant functions in angiogenesis and inflammation. Recent studies have demonstrated that apelin is overexpressed in hepatic stellate cells (HSCs) of cirrhotic rats. Moreover, patients with cirrhosis show high circulating levels of this peptide. We evaluated the role of endogenous apelin system in fibrogenesis-related gene induction in human HSCs. Messenger expression and immunolocalization of apelin were analyzed in human cirrhotic liver and in control samples. Apelin expression was analyzed in a human HSC line (LX-2) under hypoxic conditions or in the presence of proinflammatory or profibrogenic stimuli. LX-2 cells were stimulated with apelin, and a selected profile of fibrogenesis-related genes was quantified. In vivo inactivation of apelin was analyzed in the liver of fibrotic rats after administrating specific blockers of the molecules triggering apelin induction. Apelin was overexpressed in HSCs from human cirrhotic liver. Neither hypoxia nor proinflammatory substances induced the expression of apelin in LX-2. By contrast, both profibrogenic molecules angiotensin II (AII) and endothelin-1 (ET-1) enhanced apelin expression in these cells. Apelin increased the synthesis of collagen-I and platelet-derived growth factor receptor β (PDGFRβ) in LX-2. AII and ET-1 stimulated collagen-I and PDGFRβ expression, and this induction was drastically reduced when apelin receptor was blocked in these cells. In accordance, AII or ET-1 receptor antagonists reduced the hepatic synthesis of apelin, collagen-I, and PDGFRβ in fibrotic rats. apelin mediates some of the fibrogenic effects triggered by AII and ET-1, thus suggesting that apelin could be an important mediator of fibrogenesis in human liver disease.

  14. Gene expression profiles of human liver cells mediated by hepatitis B virus X protein

    Institute of Scientific and Technical Information of China (English)

    Wei-ying ZHANG; Fu-qing XU; Chang-liang SHAN; Rong XIANG; Li-hong YE; Xiao-dong ZHANG

    2009-01-01

    Aim: To demonstrate the gene expression profiles mediated by hepatitis B virus X protein (HBx), we characterized the molecular features of pathogenesis associated with HBx in a human liver cell model.Methods: We examined gene expression profiles in L-O2-X cells, an engineered L-O2 cell line that constitutively expresses HBx, relative to L-O2 cells using an Agilent 22 K human 70-mer oligonucleotide microarray representing more than 21,329 unique, well-characterized Homo sapiens genes, Western blot analysis and RNA interference (RNAi) targeting HBx mRNA validated the overexpression of proliferating cell nuclear antigen (PCNA) and Bcl-2 in L-O2-X cells. Meanwhile, the BrdU incorporation assay was used to test cell proliferation mediated by upregulated cyclooxygenase-2 (COX-2).Results: The microarray showed that the expression levels of 152 genes were remarkably altered; 82 of the genes were upregulated and 70 genes were downregulated in L-O2-X cells. The altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes. PCNA and Bcl-2 were upregulated in L-O2-X cells. Furthermore, we found that COX-2 upregulation in L-O2-X cells enhanced proliferation using the BrdU incorporation assay, whereas indomethacin (an inhibitor of COX-2) abolished the promotion.Conclusion: Our findings provide new evidence that HBx is able to regulate many genes that may be involved in the car-cinogenesis. These regulated genes mediated by HBx may serve as molecular targets for the prevention and treatment of hepatocellular carcinoma.

  15. 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

    2004-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Hepatitis C virus core protein induces apoptosis-like caspase independent cell death

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    Gregor Michael

    2009-12-01

    Full Text Available Abstract Background Hepatitis C virus (HCV associated liver diseases may be related to apoptotic processes. Thus, we investigated the role of different HCV proteins in apoptosis induction as well as their potency to interact with different apoptosis inducing agents. Methods and Results The use of a tightly adjustable tetracycline (Tet-dependent HCV protein expression cell system with the founder osteosarcoma cell line U-2 OS allowed switch-off and on of the endogenous production of HCV proteins. Analyzed were cell lines expressing the HCV polyprotein, the core protein, protein complexes of the core, envelope proteins E1, E2 and p7, and non-structural proteins NS3 and NS4A, NS4B or NS5A and NS5B. Apoptosis was measured mainly by the detection of hypodiploid apoptotic nuclei in the absence or presence of mitomycin C, etoposide, TRAIL and an agonistic anti-CD95 antibody. To further characterize cell death induction, a variety of different methods like fluorescence microscopy, TUNEL (terminal deoxynucleotidyl transferase (TdT-catalyzed deoxyuridinephosphate (dUTP-nick end labeling assay, Annexin V staining, Western blot and caspase activation assays were included into our analysis. Two cell lines expressing the core protein but not the total polyprotein exerted a strong apoptotic effect, while the other cell lines did not induce any or only a slight effect by measuring the hypodiploid nuclei. Cell death induction was caspase-independent since it could not be blocked by zVAD-fmk. Moreover, caspase activity was absent in Western blot analysis and fluorometric assays while typical apoptosis-associated morphological features like the membrane blebbing and nuclei condensation and fragmentation could be clearly observed by microscopy. None of the HCV proteins influenced the apoptotic effect mediated via the mitochondrial apoptosis pathway while only the core protein enhanced death-receptor-mediated apoptosis. Conclusion Our data showed a caspase

  18. Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

    Science.gov (United States)

    The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

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

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    Reto Eggenschwiler

    2011-01-01

    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.

  20. Intracellular Glutathione Depletion by Oridonin Leads to Apoptosis in Hepatic Stellate Cells

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    Liang-Mou Kuo

    2014-03-01

    Full Text Available Proliferation of hepatic stellate cells (HSCs plays a key role in the pathogenesis of liver fibrosis. Induction of HSC apoptosis by natural products is considered an effective strategy for treating liver fibrosis. Herein, the apoptotic effects of 7,20-epoxy-ent-kaurane (oridonin, a diterpenoid isolated from Rabdosia rubescens, and its underlying mechanisms were investigated in rat HSC cell line, HSC-T6. We found that oridonin inhibited cell viability of HSC-T6 in a concentration-dependent manner. Oridonin induced a reduction in mitochondrial membrane potential and increases in caspase 3 activation, subG1 phase, and DNA fragmentation. These apoptotic effects of oridonin were completely reversed by thiol antioxidants, N-acetylcysteine (NAC and glutathione monoethyl ester. Moreover, oridonin increased production of reactive oxygen species (ROS, which was also inhibited by NAC. Significantly, oridonin reduced intracellular glutathione (GSH level in a concentration- and time-dependent fashion. Additionally, oridonin induced phosphorylations of extracellular signal-regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38 mitogen-activated protein kinase (MAPK. NAC prevented the activation of MAPKs in oridonin-induced cells. However, selective inhibitors of MAPKs failed to alter oridonin-induced cell death. In summary, these results demonstrate that induction of apoptosis in HSC-T6 by oridonin is associated with a decrease in cellular GSH level and increase in ROS production.

  1. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

    Energy Technology Data Exchange (ETDEWEB)

    Iyer, Soumya C, E-mail: chidambaram.soumya@gmail.com [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Kannan, Anbarasu [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Gopal, Ashidha [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Devaraj, Niranjali [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Halagowder, Devaraj [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India)

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy.

  2. Mannan binding lectin-associated serine protease 1 is induced by hepatitis C virus infection and activates human hepatic stellate cells.

    Science.gov (United States)

    Saeed, A; Baloch, K; Brown, R J P; Wallis, R; Chen, L; Dexter, L; McClure, C P; Shakesheff, K; Thomson, B J

    2013-11-01

    Mannan binding lectin (MBL)-associated serine protease type 1 (MASP-1) has a central role in the lectin pathway of complement activation and is required for the formation of C3 convertase. The activity of MASP-1 in the peripheral blood has been identified previously as a highly significant predictor of the severity of liver fibrosis in hepatitis C virus (HCV) infection, but not in liver disease of other aetiologies. In this study we tested the hypotheses that expression of MASP-1 may promote disease progression in HCV disease by direct activation of hepatic stellate cells (HSCs) and may additionally be up-regulated by HCV. In order to do so, we utilized a model for the maintenance of primary human HSC in the quiescent state by culture on basement membrane substrate prior to stimulation. In comparison to controls, recombinant MASP-1 stimulated quiescent human HSCs to differentiate to the activated state as assessed by both morphology and up-regulation of HSC activation markers α-smooth muscle actin and tissue inhibitor of metalloproteinase 1. Further, the expression of MASP-1 was up-regulated significantly by HCV infection in hepatocyte cell lines. These observations suggest a new role for MASP-1 and provide a possible mechanistic link between high levels of MASP-1 and the severity of disease in HCV infection. Taken together with previous clinical observations, our new findings suggest that the balance of MASP-1 activity may be proinflammatory and act to accelerate fibrosis progression in HCV liver disease.

  3. 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: ytagawa@bio.titech.ac.jp [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)

    2010-12-17

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

  4. Construction and characterization of a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B%类泛素FAT10高表达肝癌细胞株Hep3B酵母双杂交cDNA文库的构建

    Institute of Scientific and Technical Information of China (English)

    余新; 刘天; 德洪波; 李国惠; 邵江华

    2011-01-01

    AIM: To construct a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B.METHODS: Total RNA was prepared from Hep3B cells and used to purify poly (A) mRNA.Double-stranded cDNA was synthesized from the purified mRNA, ligated to EcoR Ⅰ adaptor,digested with EcoR Ⅰ/Xho Ⅰ enzymes, and then cloned into the pGADT7 vector.The recombinant vector was transformed into E.coli DH10B to obtain a primary cDNA library.The primary library was amplified and used to determine the size of cDNA inserts through enzyme digestion.RESULTS: The primary cDNA library contained 1.03 × 106independent clones.The titer of the cDNA library was estimated to be 2.50 × 106 cfu/mL, and that of the amplified library was 3.60 × 109 cfu/mL.The size of the inserts varied from 0.5 to 3.5 kb, with an average value of about 2.0 kb.CONCLUSION: A yeast two-hybrid cDNA library has been successfully generated from FAT10-over-expressing Hep3B ceils and can be used for future screening of proteins interacting with FAT10.%目的:构建类泛素FAT10高表达肝癌细胞株Hep3B的酵母双杂交用cDNA文库.方法:从肝癌细胞Hep3B中提取总RNA,分离mRNA.利用反转录酶M-MLV与Oligo(dT)AnchorPrimer合成1 Strand cDNA,用E.coli DNAPolymerase与E.coli DNA Ligase将RNA链置换成DNA链,合成2 Strand cDNA.将双链cDNA与EcoR Ⅰ Adaptor连接,然后用EcoR Ⅰ /Xho Ⅰ进行酶切.使用Spin Column除去短链cDNA与pGADT7载体连接,转化入E.coli DH10B,建成原始文库.然后对其进行扩增并随机挑取单菌落,酶切鉴定重组子插入片段大小.结果:提取的总RNA降解少且分子完整;RNA纯度高,相对分子质量为400-5000 bp;成功合成双链cDNA,均符合建库要求:库容量达到1.03×10克隆,原始文库滴度为2.50×10 cfu/L,扩增后的文库滴度为3.60×10 cfu/L.插入片段大小分布为0.5-3.5 kb,平均长度约为2.0 kb.结论:所构建文库的各项指标均达到要求,为筛选FAT10作用蛋白奠定了重要基础.

  5. Detection of Human Hepatocarcinoma Cell Line PLC/PRF/5 Genome Hepatitis b Virus DNA Integration with Alu-PCR%Alu-PCR检测人肝癌细胞株PLC/PRF/5基因组中乙型肝炎病毒DNA的整合

    Institute of Scientific and Technical Information of China (English)

    邱爽; 张会英

    2015-01-01

    目的 应用Alu-PCR方法检测人肝癌细胞株PLC/PRF/5 基因组中乙型肝炎病毒( HBV) DNA的整合. 方法 提取经培养扩增的人肝癌细胞株PLC/PRF/5基因组DNA,根据Alu-PCR方法经过3轮PCR反应扩增潜在的HBV DNA和人基因组DNA整合片段. 琼脂糖凝胶电泳观察PCR扩增产物片段,切取并纯化整合阳性的电泳条带,对纯化产物进行核酸测序,得到整合片段的核苷酸序列. 结果 经琼脂糖凝胶电泳检测,用Alu-PCR方法能够从PLC/PRF/5 细胞株中扩增得到4 条HBV DNA整合序列,经测序后与比对其中3条整合序列能够定位于人染色体03p21.31、05p15.33、12q13.12~q14.1. 结论 Alu-PCR可以准确测定肝细胞中HBV DNA的整合,为研究HBV DNA在肝细胞中的整合研究提供了一个简单、经济的方法.%Objective In this research with the method of Alu -PCR we investigate the integration of hepatitis B virus ( HBV) DNA in human hepatocarcinoma cell line (PLC/PRF/5) genome DNA.Methods We at first extracted the genome DNA from PLC/PRF/5 cells, and then the potential integration fragments of HBV DNA and human genome DNA were amplified with according to the Alu -PCR after three rounds PCR .The Alu-PCR amplification products were observated with agarose gel electrophoresis , then integration positive electrophoresis bandings were chipped and purified for nucleic acid sequencing .At last he bioinformatics information was acquired by blast online.Results Through agarose gel electrophoresis after Alu -PCR amplification, we got four potential integration bindings , among which we got three integration sequences of HBV DNA in human genome DNA .These integration sequences could be individually located in the human chromosome of 03p21.31, 05p15.33, 12q13 and 12-q14.1.Conclusion With Alu-PCR we can accurately measure the integration of HBV DNA in human genome DNA , and Alu-PCR can be a a convenience and economic method in the study of HBV DNA ′s integration in human genome

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

    Science.gov (United States)

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

    2015-10-01

    Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Hepatitis C virus and host cell lipids: an intimate connection.

    Science.gov (United States)

    Alvisi, Gualtiero; Madan, Vanesa; Bartenschlager, Ralf

    2011-01-01

    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.

  8. Hepatitis B Virus Splice-Generated Protein Induces T-Cell Responses in HLA-Transgenic Mice and Hepatitis B Virus-Infected Patients▿

    Science.gov (United States)

    Mancini-Bourgine, Maryline; Bayard, Florence; Soussan, Patrick; Deng, Qiang; Lone, Yu-Chun; Kremsdorf, Dina; Michel, Marie-Louise

    2007-01-01

    Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8+ and CD4+ human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression. PMID:17360751

  9. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1997-01-01

    . The multitarget single hit model was applied to calculate the cellular radiosensitivity (D0), the capacity for sublethal damage repair (Dq), and the extrapolation number (n). Values for alpha and beta were determined from best-fit curves according to the linear-quadratic model and these values were applied...... to calculate the surviving fraction after 2-Gy irradiation (SF2). RESULTS: In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines...

  10. Authentication of the R06E Fruit Bat Cell Line

    Directory of Open Access Journals (Sweden)

    Ingo Jordan

    2012-05-01

    Full Text Available Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus. To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery.

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

    Directory of Open Access Journals (Sweden)

    Sören V Siegmund

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

  12. Valproic Acid Increases the Hepatic Differentiation Potential of Salivary Gland Cells.

    Science.gov (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

    2015-01-01

    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.

  13. Fetal hepatic progenitors support long-term expansion of hematopoietic stem cells.

    Science.gov (United States)

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

    2013-05-01

    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. 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

    2001-01-01

    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.

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

    Science.gov (United States)

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

    1995-02-01

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

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

    Science.gov (United States)

    Della Loggia, Paolo; Cremonini, Laura

    2002-12-01

    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. The vagal nerve stimulates activation of the hepatic progenitor cell compartment via muscarinic acetylcholine receptor type 3.

    Science.gov (United States)

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

    2002-08-01

    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.

  18. Effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29

    Institute of Scientific and Technical Information of China (English)

    Chang-Wen Yu; Bi-Sheng Zhu

    2016-01-01

    Objective:To explore effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Methods: MTT and flow cytometry were adopted for detecting the effect of exogenous leptin on cell cycle of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Results: Leptin with mass concentration (0 ng/mL, 5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could stimulate the growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29; exogenous leptin with mass concentration (5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could inhibit cell growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29 after 72 h; among which, inhibiting effects of cell line-SGC7901 and cell line-HT-29 were the most significant under the effect of exogenous leptin with mass concentration-200 ng/mL.Conclusion:Within a certain concentration and action time, exogenous leptin can stimulate the growth of gastric adenocarcinoma cell line and colon cancer cell line, and then promot the tumor cell proliferation and/or inhibit the tumor cell apoptosis.

  19. 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

    2006-01-01

    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.

  20. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    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.

  1. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  2. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning

    2010-01-01

    As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties we

  3. Beryllium-stimulated apoptosis in macrophage cell lines.

    Science.gov (United States)

    Sawyer, R T; Fadok, V A; Kittle, L A; Maier, L A; Newman, L S

    2000-08-21

    In vitro stimulation of bronchoalveolar lavage cells from patients with chronic beryllium disease (CBD) induces the production of TNF-alpha. We tested the hypothesis that beryllium (Be)-stimulated TNF-alpha might induce apoptosis in mouse and human macrophage cell lines. These cell lines were selected because they produce a range of Be-stimulated TNF-alpha. The mouse macrophage cell line H36.12j produces high levels of Be-stimulated TNF-alpha. The mouse macrophage cell line P388D.1 produces low, constitutive, levels of TNF-alpha and does not up-regulate Be-stimulated TNF-alpha production. The DEOHS-1 human CBD macrophage cell line does not produce constitutive or Be-stimulated TNF-alpha. Apoptosis was determined by microscopic observation of propidium iodide stained fragmented nuclei in unstimulated and BeSO(4)-stimulated macrophage cell lines. BeSO(4) induced apoptosis in all macrophage cell lines tested. Beryllium-stimulated apoptosis was dose-responsive and maximal after 24 h of exposure to 100 microM BeSO(4). In contrast, unstimulated and Al(2)(SO(4))(3)-stimulated macrophage cell lines did not undergo apoptosis. The general caspase inhibitor BD-fmk inhibited Be-stimulated macrophage cell line apoptosis at concentrations above 50 microM. Our data show that Be-stimulated macrophage cell line apoptosis was caspase-dependent and not solely dependent on Be-stimulated TNF-alpha levels. We speculate that the release of Be-antigen from apoptotic macrophages may serve to re-introduce Be material back into the lung microenvironment, make it available for uptake by new macrophages, and thereby amplify Be-stimulated cytokine production, promoting ongoing inflammation and granuloma maintenance in CBD.

  4. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  5. Identification of genotype 4 Hepatitis E virus binding proteins on swine liver cells

    Directory of Open Access Journals (Sweden)

    Yang Shixing

    2011-10-01

    Full Text Available Abstract Hepatitis E virus (HEV is a zoonotic pathogen of which several species of animal were reported as reservoirs. Swine stands out as the major reservoir for HEV infection in humans, as suggested by the close genetic relationship of swine and human virus and cross-species infection of HEV. Up to now, the mechanism of cross-species infection of HEV from swine to humans is still unclear. This study sought to identify receptor element for genotype 4 HEV on swine liver cells using the viral overlay protein binding assay (VOPBA technique and Mass Spectrometry fingerprinting. A single virus binding band with natural molecular weight about 55 kDa was observed, and mass spectrometry revealed that this virus binding band contained 31 different proteins. Infection inhibition assay suggested that this 55 kDa protein could prevent HEV from infecting its susceptible A549 cell line, which was further confirmed by the HEV genome detecting in the inoculated cells. Further research should be performed to elucidate the accurate receptor of HEV on the swine liver cells.

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

    Institute of Scientific and Technical Information of China (English)

    Linda; Hammerich; Frank; Tacke

    2015-01-01

    Myeloid derived suppressor cells(MDSC) are a heterogeneous population of immune cells that are potent suppressors of immune responses. MDSC emerge in various compartments in the body, such as blood, 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. Motoneuron differentiation of immortalized human spinal cord cell lines.

    Science.gov (United States)

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  8. 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

    2009-01-01

    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.

  9. Performance and Serum Hepatic Enzymes of Hy-Line W-36 Laying Hens Intoxicated with Dietary Carbon Tetrachloride

    Directory of Open Access Journals (Sweden)

    Hadavi A

    2015-12-01

    Full Text Available An experiment was conducted to study the effects of carbon tetrachloride (CCl4 on post-peak performance and serum enzymes of Hy-Line W-36 laying hens from 32-36 weeks of age. The experiment was carried out with a total of 192 laying hens in a completely randomized block design. During the experiment laying hens were allocated to 4 groups consisted of T1 no CCl4 as control diet, T2, T3 and T4 control diet supplemented with 1, 3 and 5 mL CCl4/100 g diet, respectively. Each experimental group was divided into 6 blocks of 8 hens each. Egg production, cracked egg percentage and feed intake were recorded weekly. Blood samples were taken from wing veins of hens at the middle and end of the experiment to measure serum hepatic enzymes of alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase. Data showed that in comparison with the control group, the inclusion of CCl4 to the diets had no significant effect on performance parameters. However, by increasing the level of CCl4, egg production was linearly decreased and feed intake was linearly increased (P < 0.05. The effect of CCl4 on cracked eggs was significant and this effect was linearly increased (P < 0.05. Dietary supplementation of 3 and 5 mL CCl4 elevated the serum concentration of hepatic enzymes of alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase, linearly (P < 0.0001. In conclusion, the dietary supplementation of CCl4 has the ability to decrease the performance and egg quality. CCl4 is also a potent hepatic toxicity inducer and may damage liver hepatocytes. Therefore, the level of 3 mL CCl4 was assigned as the one had the maximum negative effect on serum hepatic enzymes concentration (maximum liver damage alongside the minimum negative effect on laying hen performance for further studies.

  10. 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

    2009-01-01

    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.

  11. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... working with cells derived from one individual or animal species, only to eventually learn that the cells..., morphology, pathologic or disease-state, hybrid or mixed culture, feeder cells, date of origin, etc), the STR... National Institute of Standards and Technology Identification of Human Cell Lines Project AGENCY: National...

  12. 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

    2005-01-01

    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.

  13. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1–60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  14. 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.

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

    Science.gov (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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Further characterization of the first seminoma cell line TCam-2.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; Hersmus, Remko; van Gurp, Ruud J H L M; van de Geijn, Gert-Jan M; van Drunen, Ellen; Beverloo, H Berna; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Kitazawa, Sohei; van Zoelen, E Joop; van Roozendaal, Kees; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2008-03-01

    Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

  18. Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Gui-jun YAN; Zheng GU; Jian WANG; Jia-ke TSO

    2004-01-01

    Objective To establish C57BL/6J embryonic stem (ES) cell lines with potential germline contribution Methods ES cells were isolated from blastocyst inner cell mass of C57BL/6J mice, and cultured for 15 passages, and then injected into blastococels of lCR mice blastocysts to establish chimeric mice.Results Three ES cell lines (mC57ESl,mC57ES3, mC57ES7) derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker "stage-specific embryonic antigen-1" and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC5 7ES1 cells consis tently differentiated into derivatives of all three embryonic germ layers. When mC57ES1cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained.One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

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

    Directory of Open Access Journals (Sweden)

    Chisari Francis V

    2006-10-01

    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.

  20. Chronic hepatitis C and persistent occult hepatitis C virus infection are characterized by distinct immune cell cytokine expression profiles.

    Science.gov (United States)

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

    2009-08-01

    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.

  1. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor

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    Nora Freyer

    2016-08-01

    Full Text Available The hepatic differentiation of human induced pluripotent stem cells (hiPSC holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3. Differentiation into definitive endoderm (DE was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP, a marker for DE, was significantly (p < 0.05 higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH. CYP2B6 activities were significantly (p < 0.05 higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18, and hepatocyte nuclear factor 4-alpha (HNF4A at the end of the differentiation process. In addition, cytokeratin 19 (CK19 staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture

  2. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor

    Science.gov (United States)

    Freyer, Nora; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Schrade, Petra; Bachmann, Sebastian; Damm, Georg; Seehofer, Daniel; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

    2016-01-01

    Abstract The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture

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

    OpenAIRE

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

    2001-01-01

    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...

  4. Deriving cell lines from zebrafish embryos and tumors.

    Science.gov (United States)

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  5. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  6. Normal human pluripotent stem cell lines exhibit pervasive mosaic aneuploidy.

    Directory of Open Access Journals (Sweden)

    Suzanne E Peterson

    Full Text Available Human pluripotent stem cell (hPSC lines have been considered to be homogeneously euploid. Here we report that normal hPSC--including induced pluripotent--lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY and fluorescent in situ hybridization (FISH of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation.

  7. Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    CAO Xian-ying; QI Zhi-tao; DAI Hong-lian; YAN Yu-hua; LI Shi-pu

    2003-01-01

    Stable and single-dispersed HAP nanoparticles were synthesized with chemical method assisted by ultrasonic treatment.HAP nanoparticles were surveyed by AFM and Zataplus.The effect on the Bel-7402 human hepatoma cell lines treated with HAP nanoparticles was investigated by the MTT methods and observation of morphology,and the mechanism was studied in changes of cell cycle and ultrastructure.The result shows that inhibition of HAP nanoparticles on the Bel-7402 human hepatoma cell lines is obviously in vitro.HAP nanoparticles the entered cancer cytoplasm,and cell proliferation is stopped at G1 phase of cell cycle,thus,cancer cells die directly.

  8. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  9. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  10. Generation of mesenchymal stem cell lines from murine bone marrow.

    Science.gov (United States)

    Sreejit, P; Dilip, K B; Verma, R S

    2012-10-01

    Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages.

  11. Gastrin gene expression and regulation in rat islet cell lines.

    Science.gov (United States)

    Brand, S J; Wang, T C

    1988-11-15

    Gastrin gene expression was observed in two permanent rat insulinoma (RIN) cell lines derived from a rat insulinoma. Gastrin expression was selective; highest expression was seen in a cell line which did not express other islet cell hormones. Gastrin mRNA transcription initiated from the same promoter as antral gastrin mRNA. DNA transfection studies with a gastrin chloramphenicol acetyltransferase chimeric gene showed higher expression in gastrin-expressing RIN cells than non-gastrin-expressing islet cells. This implies that gastrin-expressing RIN cells selectively express a trans-acting transcriptional activator which binds to cis-acting regulatory sequences within the 5'-flanking DNA sequence and first exon of the gastrin gene. The gastrin peptide precursor synthesized in these RIN cell lines is subject to the same repertoire of posttranslational modifications within the cell's secretory apparatus (endoproteolytic cleavage, tyrosine sulfation, and C-terminal amidation) as seen in antral G cells. Gastrin mRNA levels in these RIN cells were selectively increased by increasing the extracellular calcium concentration. Membrane depolarization also stimulated gastrin mRNA levels, probably through activation of voltage-sensitive calcium channels. Thus, these gastrin-expressing RIN cell lines provide permanent cell lines useful in analyzing the cellular regulation of gastrin gene expression.

  12. The possible role of NS3 protease activity of hepatitis C virus on fibrogenesis and miR-122 expression in hepatic stellate cells.

    Science.gov (United States)

    Khanizadeh, S; Ravanshad, M; Hosseini, S Y; Davoodian, P; Zadeh, A N; Sabahi, F; Sarvari, J; Khanlari, Z; Hasani-Azad, M

    The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors. The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression. However, the possible correlation between miR-122 levels and fibrosis state has been less investigated. Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line. Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1). Moreover, the transforming growth factor beta (TGF-β) levels in the supernatants of transfected cells were evaluated by ELISA. The gene expression analysis of fibrotic genes and TGF-β cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001). The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function. These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122.

  13. Fetal liver hepatic progenitors are supportive stromal cells for hematopoietic stem cells.

    Science.gov (United States)

    Chou, Song; Lodish, Harvey F

    2010-04-27

    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.

  14. Merocyanine 540 and Photofrin II as photosensitizers for in vitro killing of duck hepatitis B virus and human hepatoma cells

    Science.gov (United States)

    Lin, Tsung-I.; Shien, Yong-Shau; Kao, Ming-Chien

    1994-03-01

    The feasibility of using merocyanine 540 (MC 540) and Photofrin II (PII) as effective photodynamic therapeutic (PDT) agents for killing hepatoma cells and duck hepatitis B virus (DHBV) in vitro was investigated. Cultured duck hepatocytes infected with DHBV and hepatoma cells, Hep 3B and HCC 36, were used as models. MC 540 and PII effectively inhibits the DHBV growth by 90 - 99% in a dose- and light-dependent manner. Photodynamic killing of MC 540 in the two hepatoma cell lines results in 94 - 99% growth inhibition. However, both photosensitizers exhibit dark cytotoxicity (37 - 56%). The present results suggest that MC 540 and PII could be promising and effective photodynamic agents for killing HBV and hepatoma cells.

  15. 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

    2015-01-01

    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....

  16. JKT-1 is not a human seminoma cell line.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; van Gurp, Ruud J H L M; van Drunen, Ellen; Beverloo, H Berna; Lau, Yun-Fai Chris; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Hatakeyama, Shingo; Ohyama, Chikara; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2007-08-01

    The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded.

  17. Investigation of radiosensitivity gene signatures in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    John S Hall

    Full Text Available Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2 by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median was investigated using Affymetrix GeneChip Exon 1.0ST (cervix or U133A Plus2 (head and neck arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4% were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI, and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.

  18. Cell culture systems for the hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Gilles Duverlie; Czeslaw Wychowski

    2007-01-01

    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.

  19. Antiviral therapy of hepatitis C as curative treatment of indolent B-cell lymphoma

    Science.gov (United States)

    Merli, Michele; Carli, Giuseppe; Arcaini, Luca; Visco, Carlo

    2016-01-01

    The association of hepatitis C virus (HCV) and B-cell non-Hodgkin lymphomas (NHL) has been highlighted by several epidemiological and biological insights; however the most convincing evidence is represented by interventional studies demonstrating the capability of antiviral treatment (AT) with interferon (IFN) with or without ribavirin to induce the regression of indolent lymphomas, especially of marginal-zone origin. In the largest published retrospective study (100 patients) the overall response rate (ORR) after first-line IFN-based AT was 77% (44% complete responses) and responses were sustainable (median duration of response 33 mo). These results were confirmed by a recent meta-analysis on 254 patients, demonstrating an ORR of 73%. Moreover this analysis confirmed the highly significant correlation between the achievement of viral eradication sustained virological response (SVR) and hematological responses. Two large prospective studies demonstrated that AT is associated with improved survival and argue in favor of current guidelines’ recommendation of AT as preferential first-line option in asymptomatic patients with HCV-associated indolent NHL. The recently approved direct-acting antiviral agents (DAAs) revolutionized the treatment of HCV infection, leading to SVR approaching 100% in all genotypes. Very preliminary data of IFN-free DAAs therapy in indolent HCV-positive NHL seem to confirm their activity in inducing lymphoma regression.

  20. Interleukin 17-producing γδT cells promote hepatic regeneration in mice.

    Science.gov (United States)

    Rao, Raghavendra; Graffeo, Christopher S; Gulati, Rishabh; Jamal, Mohsin; Narayan, Suchithra; Zambirinis, Constantinos P; Barilla, Rocky; Deutsch, Michael; Greco, Stephanie H; Ochi, Atsuo; Tomkötter, Lena; Blobstein, Reuven; Avanzi, Antonina; Tippens, Daniel M; Gelbstein, Yisroel; Van Heerden, Eliza; Miller, George

    2014-08-01

    Subsets of leukocytes synergize with regenerative growth factors to promote hepatic regeneration. γδT cells are early responders to inflammation-induced injury in a number of contexts. We investigated the role of γδT cells in hepatic regeneration using mice with disruptions in Tcrd (encodes the T-cell receptor δ chain) and Clec7a (encodes C-type lectin domain family 7 member a, also known as DECTIN1). We performed partial hepatectomies on wild-type C57BL/6, CD45.1, Tcrd(-/-), or Clec7a(-/-) mice. Cells were isolated from livers of patients and mice via mechanical and enzymatic digestion. γδT cells were purified by fluorescence-activated cell sorting. In mice, partial hepatectomy up-regulated expression of CCL20 and ligands of Dectin-1, which was associated with recruitment and activation of γδT cells and their increased production of interleukin (IL)-17 family cytokines. Recruited γδT cells induced production of IL-6 by antigen-presenting cells and suppressed expression of interferon gamma by natural killer T cells, promoting hepatocyte proliferation. Absence of IL-17-producing γδT cells or deletion of Dectin-1 prevented development of regenerative phenotypes in subsets of innate immune cells. This slowed liver regeneration and was associated with reduced expression of regenerative growth factors and cell cycle regulators. Conversely, exogenous administration of IL-17 family cytokines or Dectin-1 ligands promoted regeneration. More broadly, we found that γδT cells are required for inflammatory responses mediated by IL-17 and Dectin-1. γδT cells regulate hepatic regeneration by producing IL-22 and IL-17, which have direct mitogenic effects on hepatocytes and promote a regenerative phenotype in hepatic leukocytes, respectively. Dectin-1 ligation is required for γδT cells to promote hepatic regeneration. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  1. Development of a cell line from Echinococcus granulosus germinal layer.

    Science.gov (United States)

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  2. [Decontamination of continual cell lines spontaneously infected with mycoplasmas].

    Science.gov (United States)

    Machatková, M; Jurmanová, K; Snejdar, V

    1986-07-01

    The continual cell lines of bovine kidneys MDBK and AUBEK, and porcine kidneys RPD and IBRS, spontaneously infected with Mycoplasma arginini and Acholeplasma laidlawii, were decontaminated by the method of selective elimination. Two elimination procedures were modified to be used for the decontamination: one based on the reduction of infection by the light treatment of the cultures, the other based on the selection of mycoplasma-free cell population through cell clonation. On the basis of a long-continued control of the cell clones a methodical procedure of the preparation of mycoplasma-free cell lines was worked out.

  3. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Tet-control system is developed to tightly control target gene expression in mammalian cells by using the regulatory elements of tetracycline-repressor of the transposor Tn10 from E.Coli.We have transfected reverse tetracycline-controlled transactivator gene (rtTA) into genome of Jurkat cells and established two Jurkat tet-on cell lines.Induction of luciferase reporter activity with doxycycline,a tetracycline derivative,is dose-dependent with a peak value of 32-fold increment.Establishment of Jurkat tet-on cell lines greatly facilitates quantitative studies on target gene functions in the cells.

  4. 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

    2010-01-01

    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,...

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

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    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...

  6. 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

    2010-01-01

    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...

  7. Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

    Indian Academy of Sciences (India)

    Hans Gerdes; Abul Elahi; Quanguang Chen; Suresh C. Jhanwar

    2000-01-01

    We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients $(P = 0.03)$. We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses around the p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of the p53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of the nm23 gene, four with concurrent losses of the p53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines with nm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss of nm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (< 20

  8. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    Science.gov (United States)

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  9. Sensitivity of Hodgkin's lymphoma cell lines to the cell cycle inhibitor roscovitine.

    Science.gov (United States)

    Foell, Juergen L; Max, Daniela; Giersberg, Corinna; Korholz, Dieter; Staege, Martin S

    2008-01-01

    The prognosis of patients with Hodgkin's lymphoma (HL) has improved in recent decades. However, not all patients can be cured and the development of alternative treatment strategies is necessary. Gene expression in HL cell lines was analyzed using DNA microarrays and both conventional and quantitative reverse transcriptase-polymerase chain reaction. Sensitivity of HL cell lines to the cell cycle inhibitor roscovitine was assessed in vitro. All HL cell lines express high levels of cyclin D2. Treatment of HL cells with roscovitine induced cell death in some cell lines whereas other cell lines were resistant to roscovitine. Roscovitine-sensitive cell lines were characterized by expression of T-cell markers and expressed high levels of the unusual cytokine interleukin-26. Roscovitine is a cytotoxic drug for a subpopulation of HL cells and might be an interesting agent for the treatment of patients with HL.

  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. Hepatic lipogenesis gene expression in two experimental egg-laying lines divergently selected on residual food consumption

    Directory of Open Access Journals (Sweden)

    Bordas André

    2000-03-01

    Full Text Available Abstract Two Rhode Island Red egg-laying lines have been divergently selected on residual food intake (low intake R- line, high intake R+ line for 19 generations. In addition to direct response, correlated responses have altered several other traits such as carcass adiposity and lipid contents of several tissues, the R+ animals being leaner than the R- ones. In a search for the biological origin of the differences observed in fat deposit, the hepatic mRNA amounts of genes involved in lipid metabolism were investigated. No difference was found between lines for mRNA levels of ATP citrate-lyase, acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and CCAAT/enhancer binding protein α, a transcription factor acting on several lipogenesis genes. The genes coding for stearoyl-CoA desaturase and apolipoprotein A1 displayed significantly lower mRNA levels in the R+ cockerels compared to the R-. All together these mRNA levels explained 40% of the overall variability of abdominal adipose tissue weight, suggesting an important role of both genes in the fatness variability.

  12. Apoptotic effect of noscapine in breast cancer cell lines.

    Science.gov (United States)

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  13. Differential effects of bisphosphonates on breast cancer cell lines

    NARCIS (Netherlands)

    Verdijk, R.; Franke, H.R.; Wolbers, F.; Vermes, I.

    2007-01-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancers cells in virtro. In this study, six bisphosphonates were administered to three breast caner cell lines. Cell proliferation was measured by quantification of th expressio of Cyclin D1 mRNA. Apoptosis was determined by flow cytome

  14. Transcription profiles of non-immortalized breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Holland James F

    2006-04-01

    Full Text Available Abstract Background Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Methods Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs were used in addition to commercially-available normal breast epithelial cells (HMECs, established breast cancer cell lines (T-est and established normal breast cells (N-est. The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. Results According to Significance Analysis of Microarray (SAM and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p Conclusion The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research.

  15. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

  16. Sertraline induces endoplasmic reticulum stress in hepatic cells.

    Science.gov (United States)

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

    2014-08-01

    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.

  17. Activated effects of parathyroid hormone-related protein on human hepatic stellate cells.

    Directory of Open Access Journals (Sweden)

    Fen-Fen Liang

    Full Text Available BACKGROUND & AIMS: After years of experiments and clinical studies, parathyroid hormone-related protein(PTHrP has been shown to be a bone formation promoter that elicits rapid effects with limited adverse reaction. Recently, PTHrP was reported to promote fibrosis in rat kidney in conjunction with transforming growth factor-beta1 (TGF-β1, which is also a fibrosis promoter in liver. However, the effect of PTHrP in liver has not been determined. In this study, the promoting actions of PTHrP were first investigated in human normal hepatic stellate cells (HSC and LX-2 cell lines. METHODS: TGF-β1, alpha-smooth muscle actin (α-SMA, matrix metalloproteinase 2 (MMP-2, and collagen I mRNA were quantified by real-time polymerase chain reaction (PCR after HSCs or LX-2 cells were treated with PTHrP(1-36 or TGF-β1. Protein levels were also assessed by western-blot analysis. Alpha-SMA were also detected by immunofluorescence, and TGF-β1 secretion was measured with enzyme-linked immunosorbent assay (ELISA of HSC cell culture media. RESULTS: In cultured human HSCs, mRNA and protein levels of α-SMA, collagen I, MMP-2, and TGF-β1 were increased by PTHrP treatment. A similar increasing pattern was also observed in LX-2 cells. Moreover, PTHrP significantly increased TGF-β1 secretion in cultured media from HSCs. CONCLUSIONS: PTHrP activated HSCs and promoted the fibrosis process in LX-2 cells. These procedures were probably mediated via TGF-β1, highlighting the potential effects of PTHrP in the liver.

  18. Activated effects of parathyroid hormone-related protein on human hepatic stellate cells.

    Science.gov (United States)

    Liang, Fen-Fen; Liu, Cui-Ping; Li, Li-Xuan; Xue, Min-Min; Xie, Fang; Guo, Yu; Bai, Lan

    2013-01-01

    After years of experiments and clinical studies, parathyroid hormone-related protein(PTHrP) has been shown to be a bone formation promoter that elicits rapid effects with limited adverse reaction. Recently, PTHrP was reported to promote fibrosis in rat kidney in conjunction with transforming growth factor-beta1 (TGF-β1), which is also a fibrosis promoter in liver. However, the effect of PTHrP in liver has not been determined. In this study, the promoting actions of PTHrP were first investigated in human normal hepatic stellate cells (HSC) and LX-2 cell lines. TGF-β1, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), and collagen I mRNA were quantified by real-time polymerase chain reaction (PCR) after HSCs or LX-2 cells were treated with PTHrP(1-36) or TGF-β1. Protein levels were also assessed by western-blot analysis. Alpha-SMA were also detected by immunofluorescence, and TGF-β1 secretion was measured with enzyme-linked immunosorbent assay (ELISA) of HSC cell culture media. In cultured human HSCs, mRNA and protein levels of α-SMA, collagen I, MMP-2, and TGF-β1 were increased by PTHrP treatment. A similar increasing pattern was also observed in LX-2 cells. Moreover, PTHrP significantly increased TGF-β1 secretion in cultured media from HSCs. PTHrP activated HSCs and promoted the fibrosis process in LX-2 cells. These procedures were probably mediated via TGF-β1, highlighting the potential effects of PTHrP in the liver.

  19. Effect of ethanol on pro-apoptotic mechanisms in polarized hepatic cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Chronic ethanol consumption is associated with serious and potentially fatal alcohol-related liver injuries such as hepatomegaly, alcoholic hepatitis and cirrhosis. Moreover,it has been documented that the clinical progression of alcohol-induced liver damage may be associated with an increase in hepatocellular death that involves apoptotic mechanisms. Although much information has been learned about the clinical manifestations associated with alcohol-related diseases, the search continues for a better understanding of the molecular and/or cellular mechanisms by which ethanol exerts its deleterious effects such as the induction of pro-apoptotic mechanisms and related cell damaging events. As part of the effort to enhance our understanding of those particular cellular pathways and mechanisms associated with ethanol toxicity, researchers over the years have utilized a variety of model systems. Recently, work has come forth demonstrating the utility of a hybrid cell line (WIF-B) as a cell culture model system for the study of alcohol-associated alterations in hepatocellular mechanisms. Success with such emerging model systems could aid in the development of potential therapeutic treatments for the prevention of alcoholinduced apoptotic cell death that may ultimately serve as a significant target in delaying the onset and/or progression of clinical symptoms of alcohol-mediated liver disease. This review article summarizes the current understanding of ethanol-mediated modifications in cell survival and thus the promotion of pro-apoptotic events with emphasis on analyses made in various experimental model systems, particularly the more recently characterized WIF-B cell system.

  20. Human hepatocyte growth factor (hHGF-modified hepatic oval cells improve liver transplant survival.

    Directory of Open Access Journals (Sweden)

    Zhu Li

    Full Text Available Despite progress in the field of immunosuppression, acute rejection is still a common postoperative complication following liver transplantation. This study aims to investigate the capacity of the human hepatocyte growth factor (hHGF in modifying hepatic oval cells (HOCs administered simultaneously with orthotopic liver transplantation as a means of improving graft survival. HOCs were activated and isolated using a modified 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH model in male Lewis rats. A HOC line stably expressing the HGF gene was established following stable transfection of the pBLAST2-hHGF plasmid. Our results demonstrated that hHGF-modified HOCs could efficiently differentiate into hepatocytes and bile duct epithelial cells in vitro. Administration of HOCs at the time of liver transplantation induced a wider distribution of SRY-positive donor cells in liver tissues. Administration of hHGF-HOC at the time of transplantation remarkably prolonged the median survival time and improved liver function for recipients compared to these parameters in the other treatment groups (P<0.05. Moreover, hHGF-HOC administration at the time of liver transplantation significantly suppressed elevation of interleukin-2 (IL-2, tumor necrosis factor-α (TNF-α and interferon-γ (IFN-γ levels while increasing the production of IL-10 and TGF-β1 (P<0.05. HOC or hHGF-HOC administration promoted cell proliferation, reduced cell apoptosis, and decreased liver allograft rejection rates. Furthermore, hHGF-modified HOCs more efficiently reduced acute allograft rejection (P<0.05 versus HOC transplantation only. Our results indicate that the combination of hHGF-modified HOCs with liver transplantation decreased host anti-graft immune responses resulting in a reduction of allograft rejection rates and prolonging graft survival in recipient rats. This suggests that HOC-based cell transplantation therapies can be developed as a means of treating severe liver

  1. Phenotypes and karyotypes of human malignant mesothelioma cell lines.

    Directory of Open Access Journals (Sweden)

    Vandana Relan

    Full Text Available BACKGROUND: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. METHODS: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. RESULTS: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. CONCLUSION: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of

  2. Antitumor effects of interleukin-18 gene-modified hepatocyte cell line on implanted liver carcinoma

    Institute of Scientific and Technical Information of China (English)

    冷建杭; 张立煌; 姚航平; 曹雪涛

    2003-01-01

    Objective To investigate the antitumor effects of intrasplenically transplanted interleukin-18 (IL-18) gene-modified hepatocytes on murine implanted liver carcinoma.Methods Embryonic murine hepatocyte cell line (BNL-CL2) was transfected with a recombinant adenovirus encoding IL-18 and used as delivery cells for IL-18 gene transfer. Two cell lines, BNL-LacZ and BNL-CL2, were used as controls. One week after intrasplenic injection of C26 cells (colon carcinoma line), tumor-bearing syngeneic mice underwent the intrasplenic transplantation of IL-18 gene-modified hepatocyte cell line and were divided into treatment group (BNL IL-18) and control groups (BNL-LacZ and BNL-CL2 ). Two weeks later, the serum levels of IL-18, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in the implanted liver carcinoma-bearing mice were assayed, the cytotoxicity of murine splenic cytotoxic T-lymphocytes (CTLs) was measured, and the morphology of the hepatic tumors was studied to evaluate the antitumor effects of the approach. Results In the treatment group, the serum levels of IL-18, IFN-γ, TNF-α and NO increased significantly. The splenic CTL activity increased markedly (P<0.01) , accompanied by a substantial decrease in tumor volume and the percentage of tumor area and prolonged survival of liver carcinomo-being mice.Conclusions In vivo IL-18 expression by ex vivo manipulated cells with IL-18 recombinant adenovirus is able to exert potent antitumor effects by inducing a predominantly T-cell-helper type 1 (Th1) immune response. Intrasplenic transplantation of adenovirus-mediated IL-18 gene-modified hepatocytes could be used as a targeting treatment for implanted liver carcinoma.

  3. Investigation of the selenium metabolism in cancer cell lines.

    Science.gov (United States)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan; Andresen, Lars; Skov, Søren; Gammelgaard, Bente

    2011-02-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 μM were incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein binding. Correlations between cell death induction and the Se compounds transformations could not be demonstrated.

  4. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  5. Determinants of intrinsic radiosensitivity of mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Radford, I.R. [Peter MacCallum Cancer Institute, East Melbourne, VIC (Australia). Research Division

    1998-12-31

    Differences in the radiosensitivity of normal and cancerous cells could arise in various ways. Although there is no compelling data to support the view, the currently prevailing opinion is that differences in radiosensitivity are related to differences in some aspect of enzymatic DNA repair. A test of the importance of possible differences in enzymatic DNA repair in determining relative radiosensitivity would be to compare lethality in cells containing equivalent numbers of DNA lesions. Six cell lines were used in these studies: two Chinese hamster (CHO and V79) and a monkey (Vero) fibroblast-like line, a mouse melanoma line (B16-F1), and a rat (RUC-2) and a human (SQ-20B) carcinoma line. This group of cell lines displays a wide range of sensitivities to external beam low-LET radiation, ranging from the relatively radiosensitive B16-F1 and Vero lines through to the highly radioresistant RUC-2 line. However, it is important to note that none of the lines has a demonstrated defect in enzymatic DNA repair and that all appear to die by necrosis following a lethal radiation insult. Despite having significantly different radiosensitivities, CHO and V79 cells showed comparable responses to DNA-associated {sup 125}I-decays with D{sub o} values of around 65. More surprisingly, the radiosensitive B16-F1 line and the radioresistant RUC-2 line both had responses with D{sub o} values of around 133 {sup 125}I-decays. The factor of two difference between the D{sub o} values for these two pairs of cell lines is probably attributable to CHO and V79 cells being pseudo-diploid whereas B 16-F1 and RUC2 appear to have derived from tetraploid cells. The generality of the above result, for DNA lesions of different quality, was tested by comparing the sensitivities of CHO and V79 cells to DNA-associated {sup 3}H-decays. Again, consistent with the {sup 125}I-decay data, there was no significant difference in the D{sub o} values for these lines. Our {sup 3}H- and {sup 125}I-decay data are

  6. 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

    2006-01-01

    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.

  7. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  8. Different mechanisms of mitotic instability in cancer cell lines

    National Research Council Canada - National Science Library

    Klein, Alexandra; Zang, Klaus D; Steudel, Wolf-Ingo; Urbschat, Steffi

    2006-01-01

    ..., 17, and 18 and observed three different phenomena. i) Five of six glioma cell lines showed a bipolar spindle but displayed a chromosome-specific malsegregation of all chromosomes studied with high but significantly different frequencies...

  9. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2017-01-01

    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

  12. 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

    2003-01-01

    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.

  13. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud

    2010-04-01

    Full Text Available Abstract Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus decoloratus, Rhipicephalus (Boophilus microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG as cryoprotectant was compared with dimethylsulfoxide (DMSO supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B. microplus, R. (B. decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B. decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B. microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B. decoloratus, R. (B. microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.

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

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    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...

  15. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    OpenAIRE

    Mehta, Rajvi H.

    2014-01-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF...

  16. Efficacy comparison between hepatic arterial infusion chemotherapy plus systemic chemotherapy used as first-line and non-first-line treatments for the patients of colorectal cancers with unresectable hepatic metastases

    Institute of Scientific and Technical Information of China (English)

    Ping Chen; Bei Zhang; Guifang Guo; Liangping Xia; Huijuan Qiu

    2014-01-01

    Objective:The combination of hepatic arterial chemotherapy (HAIC) and systemic chemotherapy (SYC) has potential ef ect on colorectal cancer (CRC) patients with unresectable hepatic metastasis. The aim of this retrospective study was to investigate the ef icacy and safety of this combined therapeutic regimen on Chinese patients based on single institute experiences. Methods:Al 54 patients of this retrospective analysis were diagnosed with CRC with unresectable liver metas-tasis and received combined HAIC and SYC. Among the patients, 23 of them received HAIC plus SYC when they developed liver metastases as first-line treatment (Group 1), and 31 patients received HAIC plus SYC as non-first-line treatment (Group 2). The dif erent ef icacy in two groups was analyzed by SPSS 19.0. Results:The overal response rate (ORR) were 52.2%and 25.8%respectively in Groups 1 and 2 (P=0.047), and the disease control rate (DCR) were 65.2%and 35.5%respec-tively in Groups 1 and 2 (P=0.031). The median progression-free survival (PFS) were 6.8 and 3.3 months (P=0.002), the median hepatic progression-free survival (H-PFS) were 8.8 and 3.7 months (P=0.001), and the median overal survival (OS) were 18.8 and 13.7 months (P=0.121) in Groups 1 and 2, respectively. No fatal reaction was observed and no significant dif erence of adverse reaction was found in two groups. Grade 3/4 toxic ef ects included neutropenia (9.7%in Group 2 only), gastrointestinal reaction (8.7%in Group 1 and 6.5%in Group 2), stomatitis (6.5%in Group 2 only) and hyperbilirubinemia (4.3%in Group 1 only). Conclusion:HAIC combined with SYC showed promising ef icacy and safe profiles on CRC patients with unresectable liver metastases.

  17. Expressional patterns of chaperones in ten human tumor cell lines

    Directory of Open Access Journals (Sweden)

    Slavc Irene

    2004-12-01

    Full Text Available Abstract Background Chaperones (CH play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression. Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. Results A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. Conclusions The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity.

  18. Establishment of a new bovine leukosis virus producing cell line.

    Science.gov (United States)

    Beier, D; Riebe, R; Blankenstein, P; Starick, E; Bondzio, A; Marquardt, O

    2004-11-01

    Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

  19. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    Science.gov (United States)

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

  20. A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors.

    Science.gov (United States)

    Jaffrelo, Loic; Chabas, Sandrine; Reigadas, Sandrine; Pflieger, Aude; Wychowski, Czeslaw; Rumi, Julie; Ventura, Michel; Toulmé, Jean-Jacques; Staedel, Cathy

    2008-09-01

    We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5' end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.

  1. Establishment and characterization of a novel osteosarcoma cell line: CHOS.

    Science.gov (United States)

    Liu, Yunlu; Feng, Xiaobo; Zhang, Yukun; Jiang, Hongyan; Cai, Xianyi; Yan, Xinxin; Huang, Zengfa; Mo, Fengbo; Yang, Wen; Yang, Cao; Yang, Shuhua; Liu, Xianzhe

    2016-12-01

    Osteosarcoma has a well-recognized bimodal distribution, with the first peak in adolescence and another in the elderly age-group. The elderly patients have different clinical features and a poorer prognosis as compared to adolescents. To better understand the biological features of osteosarcoma in the elderly population, we established a new human osteosarcoma cell line from a 58-year-old man with primary chondroblastic osteosarcoma. After 6 months of continuous culture in vitro for over 50 passages, an immortalized cell line CHOS was established. The cell line was well-characterized by cytogenetic, biomarker, functional, and histological analyses. The CHOS cells exhibited a spindle-shaped morphology and a doubling time of 36 h. Cytogenetic analysis of CHOS cells revealed the loss of chromosome Y and the gain of chromosome 12. Quantitative real-time polymerase chain reaction (RT-PCR), Western blotting and/or immunofluorescence revealed the expression of chondroblastic, mesenchymal and tumor metastasis markers in the CHOS cells. Compared with the osteosarcoma cell line, the CHOS cells were found to be more sensitive to cisplatin and doxorubicin, but were resistant to methotrexate. The cell line was highly tumorigenic and maintained the histological characteristics and invasive nature of the original tumor. Furthermore, on immunohistochemical analysis, the xenografts and metastases were found to co-express collagen II, aggrecan, vimentin and S100A4 that resembled the original tumor cells. Our results indicate, the potential of CHOS cell line to serve as a useful tool for further studies on the molecular biology of osteosarcoma, especially in the elderly patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2116-2125, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  2. Cytotoxic effect of oxaloacetate on HepG2-human hepatic carcinoma cells via apoptosis and ROS accumulation.

    Science.gov (United States)

    Jiao, Y; Ji, L; Kuang, Y; Yang, Q

    2017-01-01

    Oxaloacetate (OA) is one of the intermediates of the Krebs cycle. In addition to its role in energy production, OA may have other effects on the cell. We report here that OA could have a cell type dependent cytotoxic effect on the human hepatic carcinoma cell line HepG2 through induction of apoptosis and reactive oxygen species (ROS) accumulation. In our study, OA decreased the viability and colony formation of HepG2 cells and induced cell death. Caspase-3 activity was increased, the pro-apoptotic protein Bax was up-regulated, and the anti-apoptotic protein Bcl-2 was down-regulated in OA-treated HepG2 cells indicating that apoptosis through the intrinsic pathway was involved in the cell death. The ROS level in OA-treated HepG2 cells was increased. The anti-oxidant N-acetylcysteine (NAC) and glutathione (GSH) prevented the OA-induced decrease in cell but did not alter the enhanced apoptotic Bax/Bcl-2 mRNA ratio. These results suggest that the OA-induced apoptosis of HepG2 cell is not driven by oxidative damage and at least two distinct mechanisms, one mediated by ROS and one involving apoptosis, result in the cytotoxic effects of OA on HepG2 cells. These studies expand the biological functional repertoire of OA and provide a mechanism by which hepatocellular carcinoma may be targeted by OA.

  3. Epigallocatechin gallate inhibits HBV DNA synthesis in a viral replication - inducible cell line

    Institute of Scientific and Technical Information of China (English)

    Wei He; Li-Xia Li; Qing-Jiao Liao; Chun-Lan Liu; Xu-Lin Chen

    2011-01-01

    AIM: To analyze the antiviral mechanism of Epigallocatechin gallate (EGCG) against hepatitis B virus (HBV) replication. METHODS: In this research, the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG. Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hepatitis B virus e antigen (HBeAg) and hepatitis B virus surface antigen (HBsAg) in the supernatant were detected by enzyme-linked immunosorbent assay. Precore mRNA and pregenomic RNA (pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction (PCR) assay. The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay. HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay. RESULTS: When HepG2.117 cells were grown in the presence of EGCG, the expression of HBeAg was suppressed, however, the expression of HBsAg was not affected. HBV precore mRNA level was also downregulated by EGCG, while the transcription of precore mRNA was not impaired. The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent, however, HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment, indicating that EGCG targets only replicative intermediates of DNA synthesis. CONCLUSION: In HepG2.117 cells, EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.

  4. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad(1.17 Gy)), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad(1.65 Gy)), erythroleukemia; 45 (n . 1.1, D0 . 147 rad(1.47 Gy)), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad(0.76 Gy)), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  5. Effect of failures and repairs on multiple cell production lines

    Energy Technology Data Exchange (ETDEWEB)

    Legato, P.; Bobbio, A.; Roberti, L.

    1989-01-01

    This paper examines a production line composed of multiple stages, or cells, which are passed in sequential order to arrive to the final product. Two possible coordination disciplines are considered, namely: the classical tandem arrangement of sequential working centers with input buffer and the kanban scheme, considered the Japanese shop floor realization of the Just-In-Time (JIT) manifacturing approach. The production line is modelled and analysed by means of Stochastic Petri Nets (SPN). Finally an analysis is made of the possibility that the working cells can incur failure/repair cycles perturbing the production flow of the line and thus reduce performance indices.

  6. 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

    2006-01-01

    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

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

    Science.gov (United States)

    Fischler, Björn; Priftakis, Peter; Sundin, Mikael

    2016-06-01

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

  8. 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

    2015-01-01

    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

  9. Antitumor Activity of Propolis on Differantiated Cancer Cell Lines

    OpenAIRE

    , Neşe Ersöz Gülçelik, Dilara Zeybek, Fige; Zeybek, Dilara; Kaymaz, Figen; Gencay, Ömür; Salih, Bekir; Asan, Esin; Sorkun, Kadriye; Usman, Aydan

    2014-01-01

    Propolis is a natural bee product with several pharmacological activities. Nowadays, it is also investigated for its antitumor properties. There are contraversies on the antitumor activity of propolis, not all tumour cells seem to respond to propolis treatment. The aim of our study is to evaluate the activity of propolis on differantiated thyroid cancer cell lines. Tyripan blue test and MTT assay were performed to evaluate the cell viability of B-CPAP cells after propolis treatment and compar...

  10. Antitumor Activity of Propolis on Differantiated Cancer Cell Lines

    OpenAIRE

    , Neşe Ersöz Gülçelik, Dilara Zeybek, Fige

    2012-01-01

    Propolis is a natural bee product with several pharmacological activities. Nowadays, it is also investigated for its antitumor properties. There are contraversies on the antitumor activity of propolis, not all tumour cells seem to respond to propolis treatment. The aim of our study is to evaluate the activity of propolis on differantiated thyroid cancer cell lines. Tyripan blue test and MTT assay were performed to evaluate the cell viability of B-CPAP cells after propolis treatment and compar...

  11. On-line characterization of a hybridoma cell culture process.

    Science.gov (United States)

    Zhou, W; Hu, W S

    1994-06-20

    The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    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.

  13. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  14. Exometabolom analysis of breast cancer cell lines: Metabolic signature.

    Science.gov (United States)

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-08-21

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach.

  15. Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ritter Peter R

    2010-03-01

    Full Text Available Abstract Background Taurolidine (TRD represents an anti-infective substance with anti-neoplastic activity in many malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by TRD simultaneously in different malignant cell lines. Materials and methods Five different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma, AsPC-1/pancreas and BxPC-3/pancreas were incubated with increasing concentrations of TRD (100 μM, 250 μM and 1000 μM for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis (Propidiumiodide/AnnexinV staining. Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the radical scavenger N-Acetylcystein (NAC and the Gluthation depleting agent BSO to examine the contribution of caspase activation and reactive oxygen species in TRD induced cell death. Results All cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and BSO co-treatment were highly different among cell lines - suggesting a cell line specific involvement of ROS in TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly among the cell lines. Conclusion This is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.

  16. Thyroid hormone negatively regulates CDX2 and SOAT2 mRNA expression via induction of miRNA-181d in hepatic cells

    Energy Technology Data Exchange (ETDEWEB)

    Yap, Chui Sun; Sinha, Rohit Anthony [Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 8, College Road, Singapore 169857 (Singapore); Ota, Sho; Katsuki, Masahito [Department of Molecular Endocrinology, Tohoku University Graduate School of Medicine, Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Yen, Paul Michael, E-mail: paul.yen@duke-nus.edu.sg [Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 8, College Road, Singapore 169857 (Singapore)

    2013-11-01

    Highlights: •Thyroid hormone induces miR-181d expression in human hepatic cells and mouse livers. •Thyroid hormone downregulates CDX2 and SOAT2 (or ACAT2) via miR-181d. •miR-181d reduces cholesterol output from human hepatic cells. -- Abstract: Thyroid hormones (THs) regulate transcription of many metabolic genes in the liver through its nuclear receptors (TRs). Although the molecular mechanisms for positive regulation of hepatic genes by TH are well understood, much less is known about TH-mediated negative regulation. Recently, several nuclear hormone receptors were shown to downregulate gene expression via miRNAs. To further examine the potential role of miRNAs in TH-mediated negative regulation, we used a miRNA microarray to identify miRNAs that were directly regulated by TH in a human hepatic cell line. In our screen, we discovered that miRNA-181d is a novel hepatic miRNA that was regulated by TH in hepatic cell culture and in vivo. Furthermore, we identified and characterized two novel TH-regulated target genes that were downstream of miR-181d signaling: caudal type homeobox 2 (CDX2) and sterol O-acyltransferase 2 (SOAT2 or ACAT2). CDX2, a known positive regulator of hepatocyte differentiation, was regulated by miR-181d and directly activated SOAT2 gene expression. Since SOAT2 is an enzyme that generates cholesteryl esters that are packaged into lipoproteins, our results suggest miR-181d plays a significant role in the negative regulation of key metabolic genes by TH in the liver.

  17. Characterisation of thyroid medullary carcinoma TT cell line.

    Science.gov (United States)

    Zabel, M; Grzeszkowiak, J

    1997-01-01

    TT cell line is the best known stabilized cell line derived from the human medullary thyroid carcinoma. The ultrastructural characteristics of these cells include well developed rough endoplasmic reticulum, a prominent Golgi apparatus and a considerable number of secretory granules. Numerous hormones were immunocytochemically demonstrated in TT cells of which calcitonin and calcitonin gene-related peptide (CGRP) are the products of the same gene but an alternative RNA processing. TT cells were found to produce some other hormones as well, namely ACTH, neurotensin, enkephalin, PTHrP, gastrin-releasing peptide (GRP), serotonin but also functional proteins of the chromogranin group, synaptophysin, NSE, calbindin and tyrosine hydroxylase. Some marker proteins have been detected in the cytosol (CEA) and in the cytoskeleton (alpha-tubulin, cytokeratin). The influence of numerous factors on the secretory activity of these cells has been demonstrated so far, including effects of 1,25-dihydroxycholecalciferol, glucocorticoids, sex steroids, cAMP, gastrin-releasing peptide, sodium butyrate, phorbol esters, ionomycin and forskolin. The investigators performed on the TT cell line demonstrate that this is the most reliable model system for the human parafollicular cells developed so far, in comparison to other cell lines derived from the medullary carcinoma of the thyroid.

  18. The hepatic progenitor cell niche in man and dog

    NARCIS (Netherlands)

    Schotanus, B.A.

    2011-01-01

    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

  19. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  20. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

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

    Science.gov (United States)

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

    2011-01-01

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

  2. Expression of MicroRNA miR-122 Facilitates an Efficient Replication in Nonhepatic Cells upon Infection with Hepatitis C Virus

    Science.gov (United States)

    Fukuhara, Takasuke; Kambara, Hiroto; Shiokawa, Mai; Ono, Chikako; Katoh, Hiroshi; Morita, Eiji; Okuzaki, Daisuke; Maehara, Yoshihiko; Koike, Kazuhiko

    2012-01-01

    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

  3. Genotyping hepatitis C viruses from Southeast Asia by a novel line probe assay that simultaneously detects core and 5′ untranslated regions

    NARCIS (Netherlands)

    S. Noppornpanth (Suwanna); B.L. Haagmans (Bart); E. Sablon (Erwin); K. de Nys (Kathy); T.X. Lien (Truong Xuang); J. Brouwer (Jan); M. van Brussel (Marianne); S.L. Smits (Saskia); Y. Poovorawan (Yong); A.D.M.E. Osterhaus (Albert)

    2006-01-01

    textabstractHepatitis C viruses (HCVs) display a high level of sequence diversity and are currently divided into six genotypes. A line probe assay (LiPA), which targets the 5′ untranslated region (5′UTR) of the HCV genome, is widely used for genotyping. However, this assay cannot distinguish many ge

  4. 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

  5. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes.

    Science.gov (United States)

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M Cecilia T; Wolf, Don; Mitalipov, Shoukhrat

    2008-03-01

    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.

  6. Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies

    Science.gov (United States)

    FAUVELLE, CATHERINE; ZAHID, MUHAMMAD NAUMAN; TUREK, MARINE; HEYDMANN, LAURA; CURY, KARINE; HAYER, JULIETTE; COMBET, CHRISTOPHE; COSSET, FRANÇOIS–LOÏC; PIETSCHMANN, THOMAS; HIET, MARIE–SOPHIE; BARTENSCHLAGER, RALF; HABERSETZER, FRANÇOIS; DOFFOËL, MICHEL; KECK, ZHEN–YONG; FOUNG, STEVEN K. H.; ZEISEL, MIRJAM B.; STOLL–KELLER, FRANÇOISE; BAUMERT, THOMAS F.

    2017-01-01

    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

  7. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration.

    Science.gov (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

    2015-12-25

    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.

  8. Comparison of seven cell lines derived from human gastric carcinomas.

    Science.gov (United States)

    Motoyama, T; Hojo, H; Watanabe, H

    1986-01-01

    In an attempt to elucidate various histological features of gastric cancers, seven human gastric adenocarcinomas were studied in vitro and in nude mice. Growth pattern of each cultured cell line in vitro corresponded well to the histological type of parent tumor. The cell lines, MKN7, MKN74, and MKN28 derived from differentiated carcinomas showed morphological characteristics of intestinal differentiation in cell polarity and microvilli with core-filaments in vitro as well as in nude mice. However, they gradually diminished the characteristics in course of time. The cell lines, MKN 45 and OKAJIMA, derived from undifferentiated carcinomas, had natures of not only ordinary gastric mucosa but also intestinal metaplastic mucosa. They seem to have multipotentiality for differentiation, and preserved well the natures for long periods of culture. The KWS-I cell line composed of undifferentiated cells in vitro displayed the potential for differentiation in nude mice. However, the differentiation of KATO-III cells derived from a signet-ring cell carcinoma was suppressed in nude mice. The common abnormality of chromosome was not found, and the growth rate in vitro was not dependent on the histological type of parent tumor.

  9. Transportation characteristics of nolatrexed in three tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    LI Yi-lei; ZHAO Ai-guo; WU Shu-guang

    2002-01-01

    Objective:To investigate the association of the transportation characteristics of nolatrexed in tumor cells with its drug sensitivity. Methods: The sensitivity of 3 tumor cell lines, C6, SRS82 and LoVo, to nolatrexed were determined by growth inhibition study. After exposure to 20 μmol/L nolatrexed at different time intervals ranging from 0 to 30 min, or to nolatrexed at different concentrations ranging from 0 to 40μmol/L for 10 min, the intracellular drug concentration was measured using high-performance liquid chromatography. Results: C6 was the most sensitive cell line among the three, with sensitivity 6. 8-fold and 13.8-fold those of SRS-82 and LoVo cells respectively. Transportation of nolatrexed in the 3 cell lines were qualitatively similar, which rapidly achieved steady-state within 5 min, and linear relationship between the intracellular and extracellular drug concentration was observed. The intracellular steady-state level achieved in C6 was significantly higher than those in the other two cell lines, the latter having comparable levels. Conclusion: Nolatrexed enters the cell very quickly and different transport capacities are involved in the generation of varied sensitivity to nolatrexed in tumor cells.

  10. Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines

    Directory of Open Access Journals (Sweden)

    Travert Carine

    2010-10-01

    Full Text Available Abstract Background Worldwide used pesticides containing different adjuvants like Roundup formulations, which are glyphosate-based herbicides, can provoke some in vivo toxicity and in human cells. These pesticides are commonly found in the environment, surface waters and as food residues of Roundup tolerant genetically modified plants. In order to know their effects on cells from liver, a major detoxification organ, we have studied their mechanism of action and possible protection by precise medicinal plant extracts called Dig1. Methods The cytotoxicity pathways of four formulations of glyphosate-based herbicides were studied using human hepatic cell lines HepG2 and Hep3B, known models to study xenobiotic effects. We monitored mitochondrial succinate dehydrogenase activity and caspases 3/7 for cell mortality and protection by Dig1, as well as cytochromes P450 1A1, 1A2, 3A4 and 2C9 and glutathione-S-transferase to approach the mechanism of actions. Results All the four Roundup formulations provoke liver cell death, with adjuvants having stronger effects than glyphosate alone. Hep3B are 3-5 times more sensitive over 48 h. Caspases 3/7 are greatly activated in HepG2 by Roundup at non-cytotoxic levels, and some apoptosis induction by Roundup is possible together with necrosis. CYP3A4 is specifically enhanced by Roundup at doses 400 times less than used in agriculture (2%. CYP1A2 is increased to a lesser extent together with glutathione-S-transferase (GST down-regulation. Dig 1, non cytotoxic and not inducing caspases by itself, is able to prevent Roundup-induced cell death in a time-dependant manner with an important efficiency of up to 89%, within 48 h. In addition, we evidenced that it prevents Caspases 3/7 activation and CYP3A4 enhancement, and not GST reduction, but in turn it slightly inhibited CYP2C9 when added before Roundup. Conclusion Roundup is able to provoke intracellular disruption in hepatic cell lines at different levels, but a

  11. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    Energy Technology Data Exchange (ETDEWEB)

    Sherwood, J.B.; Shouval, D.

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion.

  12. Cytomegalovirus-Driven Adaptive-Like Natural Killer Cell Expansions Are Unaffected by Concurrent Chronic Hepatitis Virus Infections

    Directory of Open Access Journals (Sweden)

    David F. G. Malone

    2017-05-01

    Full Text Available Adaptive-like expansions of natural killer (NK cell subsets are known to occur in response to human cytomegalovirus (CMV infection. These expansions are typically made up of NKG2C+ NK cells with particular killer-cell immunoglobulin-like receptor (KIR expression patterns. Such NK cell expansion patterns are also seen in patients with viral hepatitis infection. Yet, it is not known if the viral hepatitis infection promotes the appearance of such expansions or if effects are solely attributed to underlying CMV infection. In sizeable cohorts of CMV seropositive hepatitis B virus (HBV, hepatitis C virus (HCV, and hepatitis delta virus (HDV infected patients, we analyzed NK cells for expression of NKG2A, NKG2C, CD57, and inhibitory KIRs to assess the appearance of NK cell expansions characteristic of what has been seen in CMV seropositive healthy individuals. Adaptive-like NK cell expansions observed in viral hepatitis patients were strongly associated with CMV seropositivity. The number of subjects with these expansions did not differ between CMV seropositive viral hepatitis patients and corresponding healthy controls. Hence, we conclude that adaptive-like NK cell expansions observed in HBV, HCV, and/or HDV infected individuals are not caused by the chronic hepatitis infections per se, but rather are a consequence of underlying CMV infection.

  13. 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.

    2012-01-01

    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

  14. Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines.

    Science.gov (United States)

    Knutsen, Turid; Padilla-Nash, Hesed M; Wangsa, Danny; Barenboim-Stapleton, Linda; Camps, Jordi; McNeil, Nicole; Difilippantonio, Michael J; Ried, Thomas

    2010-03-01

    In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer.

  15. Guidelines for the use of cell lines in biomedical research.

    Science.gov (United States)

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-09-09

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

  16. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  17. 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

    OpenAIRE

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

    2011-01-01

    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...

  18. Up-regulation of interleukin-22 mediates liver fibrosis via activating hepatic stellate cells in patients with hepatitis C.

    Science.gov (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

    2015-05-01

    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.

  19. Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-mediated Inflammatory Cell Infiltration.

    Science.gov (United States)

    Piao, Chunmei; Cai, Lun; Qiu, Shulan; Jia, Lixin; Song, Wenchao; Du, Jie

    2015-04-24

    Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor β, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors.

  20. 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

    2007-01-01

    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.

  1. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-jun; WANG Bin; YAN Zhi-yong; QIAN Dong-meng; SONG Xu-xia; Ding Shou-yi; BAI Zhi-qiang

    2007-01-01

    Objective: To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods: Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 (Immobilized Mental Affinity Capture) and WCX2 (Weak Cation Exchange) chips on the SELDI-TOF-MS ProteinChip reader. Results: Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion: Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

  2. Persistence of a monosomic cell line in a fetus with mosaic trisomy 8.

    Science.gov (United States)

    Turchetti, Daniela; Pompilii, Eva; Magrini, Elisabetta; Bonasoni, Maria Paola; Pittalis, Maria Carla; Segata, Maria; Pession, Annalisa; Santini, Donatella; Pilu, Gianluigi; Seri, Marco

    2011-11-01

    We report on a fetus presenting with an increased nuchal translucency, in which chorionic villus sampling led to the diagnosis of mosaic trisomy 8. Ultrasound scan performed at 15(+6) weeks revealed bilateral cleft lip and palate, flat facial profile, and arrhinia. Pregnancy was terminated at 16(+6); postmortem examination showed additional findings including hypospadias, bilateral renal dysplasia, and focal portal fibrosis of the liver. In order to confirm the presence of trisomy 8, FISH analysis was performed in abnormal renal and hepatic tissue, which, unexpectedly, showed a higher fraction of cells with only one fluorescent probe signal (43% and 23%, respectively), if compared with normal fetal liver and kidney (3-10%). This finding is consistent with the survival in this fetus of a monosomic cell line after mitotic non-disjunction, which is in contrast with what is generally thought about mosaic trisomy genesis. We hypothesize that the possible persistence of the monosomic cell line, in addition to the variable distribution of aneuploid cells in the body tissues, could explain the high heterogeneity of mosaic trisomy 8 phenotype.

  3. Establishment, immortalisation and characterisation of pteropid bat cell lines.

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    Gary Crameri

    Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

  4. Reconstruction of endometrium from human endometrial side population cell lines.

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    Irene Cervelló

    Full Text Available Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC population recently identified by several groups using the side population (SP technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP cell lines (ICE 1-7: four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3 and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN. Phenotype analysis corroborated their epithelial (CD9+ or stromal (vimentin+ cell origin and mesenchymal (CD90+, CD73+ and CD45⁻ attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα or progesterone receptor (PR. The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.

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

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    Yanli Li

    2016-09-01

    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.

  6. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation

    Science.gov (United States)

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

    2016-01-01

    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

  7. 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

    2001-01-01

    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.

  8. Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway.

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    Qian Wang

    Full Text Available Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

  9. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    Science.gov (United States)

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.

    2011-01-01

    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

  10. 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

    2005-01-01

    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.

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

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    Yang Bi

    2014-10-01

    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.

  12. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations.

    Science.gov (United States)

    Meeth, Katrina; Wang, Jake Xiao; Micevic, Goran; Damsky, William; Bosenberg, Marcus W

    2016-09-01

    The remarkable success of immune therapies emphasizes the need for immune-competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here, we describe the Yale University Mouse Melanoma (YUMM) lines, a comprehensive system of mouse melanoma cell lines that are syngeneic to C57BL/6, have well-defined human-relevant driver mutations, and are genomically stable. This will be a useful tool for the study of tumor immunology and genotype-specific cancer biology.

  13. Ultrastructural changes in hepatic sinusoidal endothelial cells acutely exposed to colloidal iron.

    Science.gov (United States)

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

    2003-07-01

    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.

  14. Retinoic acid alleviates Con A-induced hepatitis and differentially regulates effector production in NKT cells.

    Science.gov (United States)

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

    2012-07-01

    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