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Human Hepatocytes (human + hepatocyte)
Kinds of Human Hepatocytes Terms modified by Human Hepatocytes Selected AbstractsDose-dependent Induction of Cytochrome P450 (CYP) 3A4 and Activation of Pregnane X Receptor by TopiramateEPILEPSIA, Issue 12 2003Srikanth C. Nallani Summary:,Purpose: In clinical studies, topiramate (TPM) was shown to cause a dose-dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription. Methods: Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 ,M) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 ,M). The rate of testosterone 6,-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay. Results: Compared with controls, TPM (50,500 ,M),treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 ,M) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50,500 ,M) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 ,M) caused a 6-fold activation. Conclusions: The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used. [source] Inflammatory cytokines modulate chemokine production patterns of HepG2 cells toward initially inclined directionHEPATOLOGY RESEARCH, Issue 5 2009Tomohiko Ohashi Aim:, Human hepatocytes are known to express an array of inflammatory cytokines and chemokines. In this study, we examined the potential roles of hepatocytes in regulating immune responses in the liver, by assessing the induction of Th1- or Th2-specific chemokines in HepG2 cells after various inflammatory stimulations. Methods:, HepG2 cells were stimulated with IL-1,, IFN-,, IL-4, IL-10, and/or CCL2, harvested at several time points, and served for the analyses of cytokine/chemokine mRNA expressions by semi-quantitative RT-PCR. Results:, (i) IL-1, up-regulated mRNA levels of CXCL8, CXCL10, and CCL2. IFN-, increased those of CXCL9, CXCL10, and CCL5, while IL-4 or IL-10 had no effect. (ii) Addition of IL-4 to the culture of IFN-,-stimulated cells, down-regulated CXCL9 and CXCL10 mRNA levels. (iii) Addition of IFN-, to the culture of IL-1,-stimulated cells, further up-regulated CXCL9 and CXCL10 mRNA levels. Addition of IL-4 decreased CXCL8 and CXCL10 levels, and increased CCL2 level in IL-1,-stimulated cells. (iv) CCL2 induced IL-4 mRNA expression. Conclusions:, IFN-, augmented mRNA expression of Th1-specific chemokines (CXCL9 and CXCL10) in HepG2 cells. IL-4 had no effect on those of Th2-spesific chemokines (CCL17 and CCL22); however, it was supposed to augment Th2 response indirectly through the induction of CCL2 under the inflammatory condition. The findings suggest that hepatocytes have ability to promote immune responses in the liver toward the direction, initially determined by the cytokine balances in the local inflammatory region. [source] Natural killer cells suppress full cycle HCV infection of human hepatocytesJOURNAL OF VIRAL HEPATITIS, Issue 12 2008S.-H. Wang Summary., The role of natural killer (NK) cells in controlling hepatitis C virus (HCV) infection and replication has not been fully delineated. We examined NK cell-mediated noncytolytic effect on full cycle HCV infection of human hepatocytes. Human hepatocytes (Huh7.5.1 cells) co-cultured with NK cells or treated with supernatants (SN) from NK cells cultures had significantly lower levels of HCV RNA and protein than control cells. This NK cell-mediated anti-HCV activity could be largely abolished by antibody to interferon-gamma (IFN-,). The investigation of the mechanisms for NK cell-mediated anti-HCV activity showed that NK SN-treated hepatocytes expressed higher levels of IFN-,/, than the control cells. NK SN also enhanced IFN regulatory factor-3 and 7 expression in the hepatocytes. In addition, NK SN enhanced the expression of signal transducer and activator of transcription 1 and 2, the nuclear factors that are essential for the activation of IFN-mediated antiviral pathways. These data provide direct evidence at cellular and molecular levels that NK cells have a key role in suppressing HCV infection of and replication in human hepatocytes. [source] Tumorigenic study on hepatocytes coexpressing SV40 with RasMOLECULAR CARCINOGENESIS, Issue 4 2006Beicheng Sun Abstract A model of neoplastic transformation by the combination of SV40 large T antigen (LT), SV40 small T antigen (ST), oncogenic Ras, and human telomerase reverse trasncriptase subunit (hTERT) has become established and replicated in primary human fibroblasts, however, there is no report on human hepatocytes. Here we use cell transplantation model, and show that transplantation of human hepatocytes of HL-7702 and HL-7703 expressing Ha-RasV12 and SV40 LT into subrenal capsule of immunodeficient mice results in fully malignant tumors, in contrast to conventional subcutaneous injections where tumors fail to develop. In GM-847 cell study, we have found that hTERT is not required for tumorigenic growth in subrenal capsule transplantation, however, it is required in subcutaneous injection assay. These results demonstrate that Human hepatocytes can be transformed under kidney capsule by coexpressing SV40 LT and Ha-RasV12, neither hTERT nor protein phosphatase 2A (PP2A) inhibition are required for malignant transformation, a gene which increases cell survival in the subcutaneous injection model is not required for tumorigenic growth in subrenal capsule. © 2005 Wiley-Liss, Inc. [source] A Smart Nanoprobe Based On Fluorescence-Quenching PEGylated Nanogels Containing Gold Nanoparticles for Monitoring the Response to Cancer TherapyADVANCED FUNCTIONAL MATERIALS, Issue 6 2009Motoi Oishi Abstract A biocompatible, caspase-3-responsive, and fluorescence-quenching smart apoptosis nanoprobe based on a PEGylated nanogel that contains gold nanoparticles (GNPs) (fluorescence quenchers) in the cross-linked polyamine gel core and fluorescein isothiocyanate (FITC)-labeled DEVD peptides at the tethered PEG chain ends is prepared for monitoring the cancer response to therapy. FITC,DEVD,nanogel,GNP shows very little fluorescence in the absence of activated caspase-3 (normal cells) through the fluorescence resonance energy transfer (FRET) process between the GNPs and the FITC molecules, while pronounced fluorescence signals are observed in apoptotic cells because of the cleavage of the DEVD peptide by activated caspase-3 present in the cells, which results in the release of FITC molecules. Thus, remarkable quenching and dequenching of fluorescence signals in response to activated caspase-3 is observed. Apoptotic cells are detected in human hepatocyte (HuH-7) multicellular tumor spheroids (MCTSs), a commonly used three-dimensional in vitro model mimicking the in vivo biology of tumors, as early as one day post-treatment with staurosporine, an apoptosis-inducing agent; while growth inhibition (i.e., change in size) of the HuH-7 MCTSs is only observed after a delay of three days (i.e., on day 4). This demonstrates the effectiveness of the FITC,DEVD,nanogel,GNP probe as a smart nanoprobe for real-time monitoring as well as a more rapid assessment of the early response to cancer therapy. [source] Strain-dependent viral dynamics and virus-cell interactions in a novel in vitro system supporting the life cycle of blood-borne hepatitis C virus,HEPATOLOGY, Issue 3 2009Hussein Hassan Aly We developed an in vitro system that can be used for the study of the life cycle of a wide variety of blood-borne hepatitis C viruses (HCV) from various patients using a three-dimensional hollow fiber culture system and an immortalized primary human hepatocyte (HuS-E/2) cell line. Unlike the conventional two-dimensional culture, this system not only enhanced the infectivity of blood-borne HCV but also supported its long-term proliferation and the production of infectious virus particles. Both sucrose gradient fractionation and electron microscopy examination showed that the produced virus-like particles are within a similar fraction and size range to those previously reported. Infection with different HCV strains showed strain-dependent different patterns of HCV proliferation and particle production. Fluctuation of virus proliferation and particle production was found during prolonged culture and was found to be associated with change in the major replicating virus strain. Induction of cellular apoptosis was only found when strains of HCV-2a genotype were used for infection. Interferon-alpha stimulation also varied among different strains of HCV-1b genotypes tested in this study. Conclusion: These results suggest that this in vitro infection system can reproduce strain-dependent events reflecting viral dynamics and virus-cell interactions at the early phase of blood-borne HCV infection, and that this system can allow the development of new anti-HCV strategies specific to various HCV strains. (HEPATOLOGY 2009.) [source] Titration of hepatitis B virus infectivity in the sera of pre-acute and late acute phases of HBV infection: Transmission experiments to chimeric mice with human liver repopulated hepatocytesJOURNAL OF MEDICAL VIROLOGY, Issue 12 2008Ayako Tabuchi Abstract Studies of hepatitis B virus (HBV) infection in non-human primates such as chimpanzees are no longer possible due to ethical considerations and the endangered status of chimpanzees since April 2007 in Japan. A human hepatocyte transplanted chimeric mouse was used to characterize HBV infectivity in serial stages of acute infection. Chimeric mice were inoculated intravenously with serum samples obtained from an experimentally infected chimpanzee with HBV. Sera from the pre-acute phases (i.e., rump-up viremia prior to anti-HBc) and late acute phases (i.e., declining phase of HBsAg and anti-HBcAb positive) were collected from the chimpanzees 57 and 244 days after inoculation. These sera contained 2.6,×,106 and 2.8,×,106 copies/ml of HBV DNA, respectively. Three chimeric mice inoculated intravenously with 100 µl of pre-acute serum (equivalent to 100 copy of HBV DNA) developed an HBV infection. The three chimeric mice that received 100 µl of pre-acute serum (equivalent to 101 copies of HBV DNA), developed high levels of serum HBV DNA. None of the three chimeric mice inoculated with 100 µl of 1:104 dilution (equivalent to 101 copies of HBV DNA) of late-acute serum was infected, while only one of three chimeric mice inoculated with 100 µl of 1:103 dilution (equivalent to 102 copies of HBV DNA) of late-acute serum developed an HBV infection. Based on these results, chimeric mice can be used as animal models for the study of HBV infectivity, pathogenesis and control. The results show that pre-acute phase HBV serum is about 100-times more infectious than late acute phase serum. J. Med. Virol. 80:2064,2068, 2008. © 2008 Wiley-Liss, Inc. [source] Growth potential of adult hepatocytes in mammals: Highly replicative small hepatocytes with liver progenitor-like traitsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2007Katsutoshi Yoshizato The liver is one of the few organs that is capable of completely regenerating itself without using a stem cell population. When damaged, growth factors and cytokines are released, stimulating terminally differentiated adult hepatocytes and making them re-enter the cell cycle. We have been developing a series of studies on the growth potential of rat and human hepatocytes to identify a population of hepatocytes that is responsible for the regeneration of the injured liver. For this purpose, we established an appropriate culture method for hepatocytes by which growth and differentiation capacities are practically examined under various experimental conditions. This in vitro assay system allows us to identify small hepatocytes that are prominently replicative compared to large hepatocytes. Non-parenchymal cells play critical roles in the proliferation of small hepatocytes. These hepatocytes are present in both rat and human liver and are located in portal regions there. Phenotypic features were examined at morphological and gene/protein levels in detail, which showed the phenotypic plasticity in vitro. Mammalian liver includes a population of small hepatocytes in normal adults with a minute occupancy rate. We speculate that small hepatocytes play a role in regenerating the injured liver and in compensating for apoptotic hepatocytes in the physiological turnover of hepatocytes. [source] Hepatotoxicity assay using human hepatocytes trapped in microholes of a microfluidic deviceELECTROPHORESIS, Issue 18 2010Ju Hun Yeon Abstract Hepatocytes have been used for in vitro hepatotoxicity assays because of their ability to sustain intact liver-specific functions. Here, we demonstrate a hepatotoxicity assay system using primary human hepatocytes trapped in microholes of a microfluidic device, providing a microscale in vivo liver-like environment. We performed microfluidic hepatotoxicity assays of several drugs, including acetaminophen, verapamil, diclofenac, and benzopyrene, all of which are known to specifically affect hepatic function. The drug sensitivities in hepatocytes and HepG2 cells were measured by calculating the live cell fraction at various drug concentrations. The results indicated that hepatocytes were more sensitive to these drugs than HepG2 cells. The lethal concentration 50 values for all drugs tested were similar to those from the in vitro toxicity data with human hepatocytes obtained from the literature. Furthermore, we developed a mathematical hepatotoxicity model based on the time-dependent cell death profiles measured by our device. This novel assay system enabled us to analyze in vivo -like hepatotoxicity in a microfluidic device by exploiting microstructures to mimic the microenvironment of the liver. [source] Synergistic genotoxicity caused by low concentration of titanium dioxide nanoparticles and p,p,-DDT in human hepatocytesENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 3 2010Yun Shi Abstract The use of titanium dioxide nanoparticles (nano-TiO2) for the degradation of dichlorodiphenyltrichloroethane (p,p,-DDT) increases the risk of exposure to trace nano-TiO2 and p,p,-DDT mixtures. The interaction of p,p,-DDT and nano-TiO2 at low concentrations may alter toxic response relative to nano-TiO2 or p,p,-DDT alone. In this work, the combined genotoxicity of trace nano-TiO2 and p,p,-DDT on human embryo L-02 hepatocytes without photoactivation was studied. Nano-TiO2 (0.1 g/L) was mixed with 0.01,1 mmol/L p,p,-DDT to determine adsorption isotherms. L-02 cells were exposed to different levels of p,p,-DDT (0, 0.001, 0.01, and 0.1 ,mol/L) and nano-TiO2 (0, 0.01, 0.1, and 1 ,g/mL) respectively. The adsorption of p,p,-DDT by nano-TiO2 was approximately 0.3 mmol/g. Cell viability, apoptosis, and DNA double strand breaks were similar among all test groups. Nano-TiO2 alone (0.01,1 ,g/mL) increased the levels of oxidative stress and oxidative DNA adducts (8-OHdG), but it did not induce DNA breaks or chromosome damage. Addition of trace nano-TiO2 with trace p,p,-DDT synergistically enhanced genotoxicity via increasing oxidative stress, oxidative DNA adducts, DNA breaks, and chromosome damage in L-02 cells. Low concentrations of nano-TiO2 and p,p,-DDT increased oxidativestress by reactive oxygen species (ROS) formation and lipid oxidation. Oxidative stress is a major pathway for DNA and chromosome damage. Dose-dependent synergistic genotoxicity induced by combined exposure of trace p,p,-DDT and nano-TiO2 suggests a potential environmental risk of nano-TiO2 assisted photocatalysis. Environ. Mol. Mutagen., 2010. © 2009 Wiley-Liss, Inc. [source] Dose-dependent Induction of Cytochrome P450 (CYP) 3A4 and Activation of Pregnane X Receptor by TopiramateEPILEPSIA, Issue 12 2003Srikanth C. Nallani Summary:,Purpose: In clinical studies, topiramate (TPM) was shown to cause a dose-dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription. Methods: Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 ,M) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 ,M). The rate of testosterone 6,-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay. Results: Compared with controls, TPM (50,500 ,M),treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 ,M) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50,500 ,M) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 ,M) caused a 6-fold activation. Conclusions: The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used. [source] A reversibly immortalized human hepatocyte cell line as a source of hepatocyte-based biological supportADDICTION BIOLOGY, Issue 4 2001Naoya Kobayashi The application of hepatocyte transplantation (HTX) is increasingly envisioned for temporary metabolic support during acute liver failure and provision of specific liver functions in inherited liver-based metabolic diseases. Compared with whole liver transplantation, HTX is a technically simple procedure and hepatocytes can be cryopreserved for future use. A major limitation of this form of therapy in humans is the worldwide shortage of human livers for isolating an adequate number of transplantable human hepatocyes when needed. Furthermore, the numbers of donor livers available for hepatocyte isolation is limited by competition for their use in whole organ transplantation. Considering the cost of hepatocyte isolation and the need for immediate preparation of consistent and functional cells, it is unlikely that human hepatocytes can be obtained on such a scale to treat a large number of patients with falling liver functions. The utilization of xenogenic hepatocytes will result in additional concerns regarding transmission of infectious pathogens and immunological and physiological incompatibilities between animals and humans. An attractive alternative to primary human hepatocytes is the use of tightly regulated human hepatocyte cell lines. Such cell lines can provide the advantages of unlimited availability, sterility and uniformity. We describe here methods for creating transplantable human hepatocyte cell lines using currently available cell cultures and gene transfer technology. [source] Biotransformation in vitro of the 22R and 22S epimers of budesonide by human liver, bronchus, colonic mucosa and skinFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 1 2001Julio Cortijo The pharmacological effects of glucocorticoids are greatly influenced by their pharmacokinetic properties. In the present report, the in vitro biotransformation of the 22R and 22S epimers of the topical steroid budesonide was studied in the S-9 fraction of human liver, bronchus, skin and colonic mucosa. The disappearance of unchanged epimers of budesonide was measured during 90 min of incubation by high performance liquid chromatography. The rate of disappearance was high in human liver while little biotransformation occurred in bronchial tissue and colonic mucosa, and none was detected in the skin. A marked decay of the initial concentration of unchanged budesonide epimers was noticed after 2 h incubation in cultured human hepatocytes, while only a small decrease was observed after 24 h incubation in cultured human airway smooth muscle cells and BEAS-2B cells. The 22R epimer of budesonide suffered greater in vitro biotransformation than the 22S epimer in human hepatic, bronchial and colonic tissues. These findings extend those of other studies, and confirm that the high therapeutic ratio of budesonide is due to negligible local biotransformation combined with high level of liver metabolism for locally absorbed budesonide. [source] Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection,HEPATOLOGY, Issue 1 2010Nabora Soledad Reyes de Mochel Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV),induced pathogenesis. Studies have suggested that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increases the generation of superoxide and hydrogen peroxide close to the hepatocyte nucleus and that this source of ROS is reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 4 (Nox4). Huh7 human hepatoma cells and telomerase-reconstituted primary human hepatocytes, transfected or infected with virus-producing HCV strains of genotypes 2a and 1b, were examined for messenger RNA (mRNA), protein, and subcellular localization of Nox proteins along with the human liver. We found that genotype 2a HCV induced persistent elevations of Nox1 and Nox4 mRNA and proteins in Huh7 cells. HCV genotype 1b likewise elevated the levels of Nox1 and Nox4 in telomerase-reconstituted primary human hepatocytes. Furthermore, Nox1 and Nox4 proteins were increased in HCV-infected human liver versus uninfected liver samples. Unlike Nox1, Nox4 was prominent in the nuclear compartment of these cells as well as the human liver, particularly in the presence of HCV. HCV-induced ROS and nuclear nitrotyrosine could be decreased with small interfering RNAs to Nox1 and Nox4. Finally, HCV increased the level of transforming growth factor beta 1 (TGF,1). TGF,1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGF,1. Conclusion: HCV induced a persistent elevation of Nox1 and Nox4 and increased nuclear localization of Nox4 in hepatocytes in vitro and in the human liver. Hepatocyte Nox proteins are likely to act as a persistent, endogenous source of ROS during HCV-induced pathogenesis. Hepatology 2010 [source] Primary hepatocyte culture supports hepatitis C virus replication: A model for infection-associated hepatocarcinogenesis,HEPATOLOGY, Issue 6 2010Krishna Banaudha Analysis of progressive changes in hepatic gene expression that underlie hepatocarcinogenesis following hepatitis C virus (HCV) infection require examination of long-term cultures of normally differentiating primary human hepatocytes. We report a culture system of primary hepatocytes that support productive replication of infectious HCV. Hepatic functions were analyzed by reverse-transcription polymerase chain reaction amplification of total cell RNA from cultures maintained in serum-free defined medium for up to 190 days. Sustained hepatic function was assessed by expression of albumin, alpha-fetoprotein, cytochrome P4502E1, cytokeratin-18, type-1 collagen, transforming growth factor-beta 1, matrix metalloproteinase-2 (MMP-2), MMP-13, and interferon alpha-receptors 1 and 2. Normally differentiated human primary hepatocytes supported productive replication of infectious clones of HCV genotypes 1a, 1b, and 2a; virus infection was inhibited by antibodies against CD81 virus entry factor. Virus released into the culture media of HCV-infected primary hepatocytes repeatedly passage to naïve hepatocytes. Replication of the three HCV genotypes shows interferon sensitivity observed in natural infections. Conclusion: Sustained cultures of physiologic host cells for the propagation of infectious HCV strains should accelerate studies of host response to HCV infection and progressive liver disease. Hepatology 2010;51:1922,1932 [source] S -adenosylmethionine regulates dual-specificity mitogen-activated protein kinase phosphatase expression in mouse and human hepatocytes,HEPATOLOGY, Issue 6 2010Maria Lauda Tomasi Increased mitogen-activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2) and the dual-specificity MAPK phosphatase MKP-1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S -adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin-like and caspase-like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin-like and caspase-like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. Conclusion: DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration. HEPATOLOGY 2010 [source] Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway,HEPATOLOGY, Issue 5 2010Nitika Arora Gupta Glucagon-like peptide 1 (GLP-1) is a naturally occurring peptide secreted by the L cells of the small intestine. GLP-1 functions as an incretin and stimulates glucose-mediated insulin production by pancreatic , cells. In this study, we demonstrate that exendin-4/GLP-1 has a cognate receptor on human hepatocytes and that exendin-4 has a direct effect on the reduction of hepatic steatosis in the absence of insulin. Both glucagon-like peptide 1 receptor (GLP/R) messenger RNA and protein were detected on primary human hepatocytes, and receptor was internalized in the presence of GLP-1. Exendin-4 increased the phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK-1), AKT, and protein kinase C , (PKC-,) in HepG2 and Huh7 cells. Small interfering RNA against GLP-1R abolished the effects on PDK-1 and PKC-,. Treatment with exendin-4 quantitatively reduced triglyceride stores compared with control-treated cells. Conclusion: This is the first report that the G protein,coupled receptor GLP-1R is present on human hepatocytes. Furthermore, it appears that exendin-4 has the same beneficial effects in vitro as those seen in our previously published in vivo study in ob/ob mice, directly reducing hepatocyte steatosis. Future use for human nonalcoholic fatty liver disease, either in combination with dietary manipulation or other pharmacotherapy, may be a significant advance in treatment of this common form of liver disease. (HEPATOLOGY 2010) [source] A role for the pregnane X receptor in flucloxacillin-induced liver injury,HEPATOLOGY, Issue 5 2010Elise Andrews Drug-induced liver injury (DILI) due to flucloxacillin is a rare but serious complication of treatment. There is some evidence that flucloxacillin is a human pregnane X receptor (PXR) agonist. This study was designed to investigate the relevance of PXR to flucloxacillin toxicity and to identify genes changing in expression in response to flucloxacillin. Changes in gene expression in human hepatocytes after treatment with 500 ,M flucloxacillin for 72 hours were examined by expression microarray analysis. The ability of flucloxacillin to act as a PXR agonist was investigated with reporter gene experiments. Flucloxacillin DILI cases (n = 51), drug-exposed controls without toxicity (n = 64), and community controls (n = 90) were genotyped for three common PXR polymorphisms. Luciferase reporter assays were used to assess the significance of a promoter region PXR polymorphism. Seventy-two probe sets representing 50 different genes showed significant changes in expression of 1.2-fold or higher. Most genes showing changes greater than 3-fold were known to be rifampicin-responsive, and this suggested a PXR-dependent mode of regulation. Using a luciferase-everted repeat separated by 6 base pairs element construct, we confirmed that flucloxacillin was a PXR agonist. We found a difference in the distribution of a PXR polymorphism (rs3814055; C-25385T) between flucloxacillin DILI cases and controls with the CC genotype associated with an increased risk of disease (odds ratio = 3.37, 95% confidence interval = 1.55-7.30, P = 0.0023). Reporter gene experiments showed lower promoter activity for the C allele than the T allele. Conclusion: Flucloxacillin is a PXR agonist at pharmacologically relevant concentrations, and a functionally significant upstream PXR polymorphism is a risk factor for flucloxacillin-induced DILI. Hepatology 2010 [source] Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2,CD81,Claudin-1 associations,HEPATOLOGY, Issue 4 2010Sophie E. Krieger The tight junction protein claudin-1 (CLDN1) has been shown to be essential for hepatitis C virus (HCV) entry,the first step of viral infection. Due to the lack of neutralizing anti-CLDN1 antibodies, the role of CLDN1 in the viral entry process is poorly understood. In this study, we produced antibodies directed against the human CLDN1 extracellular loops by genetic immunization and used these antibodies to investigate the mechanistic role of CLDN1 for HCV entry in an infectious HCV cell culture system and human hepatocytes. Antibodies specific for cell surface,expressed CLDN1 specifically inhibit HCV infection in a dose-dependent manner. Antibodies specific for CLDN1, scavenger receptor B1, and CD81 show an additive neutralizing capacity compared with either agent used alone. Kinetic studies with anti-CLDN1 and anti-CD81 antibodies demonstrate that HCV interactions with both entry factors occur at a similar time in the internalization process. Anti-CLDN1 antibodies inhibit the binding of envelope glycoprotein E2 to HCV permissive cell lines in the absence of detectable CLDN1-E2 interaction. Using fluorescent-labeled entry factors and fluorescence resonance energy transfer methodology, we demonstrate that anti-CLDN1 antibodies inhibit CD81-CLDN1 association. In contrast, CLDN1-CLDN1 and CD81-CD81 associations were not modulated. Taken together, our results demonstrate that antibodies targeting CLDN1 neutralize HCV infectivity by reducing E2 association with the cell surface and disrupting CD81-CLDN1 interactions. Conclusion: These results further define the function of CLDN1 in the HCV entry process and highlight new antiviral strategies targeting E2-CD81-CLDN1 interactions. (HEPATOLOGY 2010.) [source] CD56+ T cells inhibit hepatitis C virus replication in human hepatocytes,HEPATOLOGY, Issue 3 2009Li Ye CD56+ T cells are abundant in liver and play an important role in defense against viral infections. However, the role of CD56+ T cells in control of hepatitis C virus (HCV) infection remains to be determined. We investigated the noncytolytic anti-HCV activity of primary CD56+ T cells in human hepatocytes. When HCV Japanese fulminant hepatitis-1 (JFH-1),infected hepatocytes were co-cultured with CD56+ T cells or incubated in media conditioned with CD56+ T cell culture supernatants (SN), HCV infectivity and replication were significantly inhibited. The antibodies to interferon (IFN)-, or IFN-, receptor could largely block CD56+ T cell,mediated anti-HCV activity. Investigation of mechanism(s) responsible for CD56+ T cell,mediated noncytolytic anti-HCV activity showed that CD56+ T SN activated the multiple elements of janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and enhanced the expression of IFN regulatory factors (IRFs) 1, 3, 7, 8, and 9, resulting in the induction of endogenous IFN-,/, expression in hepatocytes. Moreover, CD56+ T SN treatment inhibited the expression of HCV-supportive micro RNA (miRNA)-122 and enhanced the levels of anti-HCV miRNA-196a in human hepatocytes. Conclusion: These findings provide direct in vitro evidence at cellular and molecular levels that CD56+ T cells may have an essential role in innate immune cell,mediated defense against HCV infection. (HEPATOLOGY 2009.) [source] Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep,HEPATOLOGY, Issue 6 2007Jason Chamberlain Alternative methods to whole liver transplantation require a suitable cell that can be expanded to obtain sufficient numbers required for successful transplantation while maintaining the ability to differentiate into hepatocytes. Mesenchymal stem cells (MSCs) possess several advantageous characteristics for cell-based therapy and have been shown to be able to differentiate into hepatocytes. Thus, we investigated whether the intrahepatic delivery of human MSCs is a safe and effective method for generating human hepatocytes and whether the route of administration influences the levels of donor-derived hepatocytes and their pattern of distribution throughout the parenchyma of the recipient's liver. Human clonally derived MSCs were transplanted by an intraperitoneal (n = 6) or intrahepatic (n = 6) route into preimmune fetal sheep. The animals were analyzed 56,70 days after transplantation by immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry. The intrahepatic injection of human MSCs was safe and resulted in more efficient generation of hepatocytes (12.5% ± 3.5% versus 2.6% ± 0.4%). The animals that received an intrahepatic injection exhibited a widespread distribution of hepatocytes throughout the liver parenchyma, whereas an intraperitoneal injection resulted in a preferential periportal distribution of human hepatocytes that produced higher amounts of albumin. Furthermore, hepatocytes were generated from MSCs without the need to first migrate/lodge to the bone marrow and give rise to hematopoietic cells. Conclusion: Our studies provide evidence that MSCs are a valuable source of cells for liver repair and regeneration and that, by the alteration of the site of injection, the generation of hepatocytes occurs in different hepatic zones, suggesting that a combined transplantation approach may be necessary to successfully repopulate the liver with these cells. (HEPATOLOGY 2007.) [source] Increased hepatotoxicity of tumor necrosis factor,related apoptosis-inducing ligand in diseased human liver,HEPATOLOGY, Issue 5 2007Xandra Volkmann Tumor necrosis factor,related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells but not in most normal cells and has therefore been proposed as a promising antitumor agent. Recent experiments suggested that isolated primary human hepatocytes but not monkey liver cells are susceptible to certain TRAIL agonists, raising concerns about the use of TRAIL in cancer treatment. Whether TRAIL indeed exerts hepatotoxicity in vivo and how this is influenced by chemotherapeutic drugs or liver disease are completely unknown. Employing different forms of recombinant TRAIL, we found that the cytokine can induce proapoptotic caspase activity in isolated human hepatocytes. However in marked contrast, these different TRAIL preparations induced little or no cytotoxicity when incubated with tissue explants of fresh healthy liver, an experimental model that may more faithfully mimic the in vivo situation. In healthy liver, TRAIL induced apoptosis only when combined with histone deacetylase inhibitors. Strikingly, however, TRAIL alone triggered massive apoptosis accompanied by caspase activation in tissue explants from patients with liver steatosis or hepatitis C viral infection. This enhanced sensitivity of diseased liver was associated with an increased expression of TRAIL receptors and up-regulation of proapoptotic Bcl-2 proteins. Conclusion: These results suggest that clinical trials should be performed with great caution when TRAIL is combined with chemotherapy or administered to patients with inflammatory liver diseases. (HEPATOLOGY 2007.) [source] Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury,HEPATOLOGY, Issue 2 2007Calivarathan Latchoumycandane Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Leflunomide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP),catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH2 -terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-XL inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. Conclusion: Leflunomide afforded protection against APAP-induced hepatotoxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabilization. (HEPATOLOGY 2007.) [source] Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes,HEPATOLOGY, Issue 4 2006Sean E. Doyle Interleukin-28A (IL-28A), IL-28B and IL-29 are a family of class II cytokines that stimulate antiviral responses through a heterodimeric receptor that is distinct from the type I interferon (IFN) receptor. To better understand how this newly described family of cytokines regulates the antiviral state, we compared various cellular responses elicited by IL-29 and IFN-,. Here we show that these cytokines stimulate similar patterns of signal transducer and activator of transcription 1 (STAT-1), -2, -3, and -5 phosphorylation and nearly identical patterns of gene expression when analyzed in two distinct cell types by microarray analysis. Interestingly, the IL-29 receptor is preferentially expressed on primary hepatocytes within normal liver and pegylated forms of IL-29 and IFN-, induced equivalent 2,5, oligoadenylate synthetase (OAS) and MX1 gene expression in this cell type. Pegylated IL-29 also produced a significant reduction in human hepatitis B and hepatitis C viral load in vitro and reduced the cytopathic effect caused by the fully replicating flavivirus, West Nile virus. In conclusion, IL-29 and IFN-, stimulate identical antiviral responses despite their utilization of different receptors. This fact, combined with significant receptor expression in hepatitis virus-infected livers, suggests that IL-29 may have therapeutic value against chronic viral hepatitis in human patients. (HEPATOLOGY 2006;44:896,906.) [source] Hepatocytes as cytotoxic effector cells can induce cell death by CD95 ligand-mediated pathway,HEPATOLOGY, Issue 6 2006Clifford S. Guy The liver plays an increasingly recognized role in the host's immune responses. The direct contribution of hepatocytes as effector cells to local immunity, pathogen containment, and liver disease is not determined. This in vitro study examined whether hepatocytes can eliminate other cells via a CD95 ligand (CD95L or FasL)/CD95 (Fas),mediated mechanism and whether this cytotoxic activity can be modulated by cytokines such as interferon gamma (IFN-,) or tumor necrosis factor alpha (TNF-,). We have found that normal woodchuck and human hepatocytes, both cultured and primary freshly isolated, as well as human HepG2 cells, intrinsically transcribe not only CD95 but also CD95L when examined by reverse transcription-polymerase chain reaction (RT-PCR) assays. The functional competence of CD95L, which was detectable in hepatocytes and HepG2 cells by Western blotting, was confirmed in bioassays by induction of apoptosis of CD95-bearing P815 and LS102.9 cell targets and validated by inhibition of the cell killing with CD95 antagonistic antibody or with a general caspase inhibitor. Furthermore, exposure of cultured hepatocytes to IFN-, or their stable transfection with IFN-, cDNA or TNF-, cDNA increased hepatocyte CD95L/CD95,mediated cell killing. In conclusion, hepatocytes express both CD95L and CD95 and they can induce death of other cells by a CD95L-dependent mechanism. IFN-, and, to a lesser extent, TNF-, can enhance hepatocyte CD95L-mediated cytotoxicity. This suggests that the local cytokine environment may modulate the hepatocyte contribution to liver immunity. (HEPATOLOGY 2006;43:1231,1240.) [source] Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication,HEPATOLOGY, Issue 6 2006Marianne Bonvin Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-,) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3BL inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3BS and A3C had no effect. A3BL and A3BS were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3BL, but not A3BS, induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3BL, A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-, in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV. (HEPATOLOGY 2006;43:1364,1374.) [source] Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes,HEPATOLOGY, Issue 2 2006Yaakov Nahmias Low-density lipoprotein (LDL) is an important carrier of plasma cholesterol and triglycerides whose concentration is regulated by the liver parenchymal cells. Abnormal LDL regulation is thought to cause atherosclerosis, while viral binding to LDL has been suggested to facilitate hepatitis C infection. Primary hepatocytes quickly lose the ability to clear LDL during in vitro culture. Here we show that the coculture of hepatocytes with liver sinusoidal endothelial cells (LSEC) significantly increases the ability of hepatocytes to uptake LDL in vitro. LDL uptake does not increase when hepatocytes are cocultured with other cell types such as fibroblasts or umbilical vein endothelial cells. We find that LSECs induce the hepatic expression of the LDL receptor and the epidermal growth factor receptor. In addition, while hepatocytes in single culture did not take up hepatitis C virus (HCV)-like particles, the hepatocytes cocultured with LSECs showed a high level of HCV-like particle uptake. We suggest that coculture with LSECs induces the emergence of a sinusoidal surface in primary hepatocytes conducive to the uptake of HCV-like particles. In conclusion, our findings describe a novel model of polarized hepatocytes in vitro that can be used for the study of LDL metabolism and hepatitis C infection. (HEPATOLOGY 2006;43:257,265.) [source] Reversibly immortalized human hepatocytes: An eternal fountain of liver support?HEPATOLOGY, Issue 2 2000Chandan Guha M.D., Ph.D. No abstract is available for this article. [source] Effects of HCV proteins in current HCV transgenic modelsHEPATOLOGY RESEARCH, Issue 2 2010Jian Jiao Hepatits C virus (HCV) is an enveloped virus with positive-sense single-stranded RNA genome that causes both acute and persistent infections associated with chronic hepatitis, cirrhosis and hepatocellular carcinoma, which needs fully functional human hepatocytes for its development. Due to the strict human tropism of HCV, only human and higher primates such as chimpanzees have been receptive to HCV infection and development, cognition about pathophysiololgy and host immune responses of HCV infection is limited by lacking of simple laboratory models of infection for a long time. During the past decade, gene transfer approaches have been helpful to the understanding of the molecular basis of human disease. Transgenic cell lines, chimeric and transgenic animal models were developed and had been demonstrated their invaluable benefits. This review focuses on the existing HCV transgenic models and summarize the relative results about probable pathophysical changes induced by HCV proteins. [source] Bezafibrate induces multidrug-resistance P-Glycoprotein 3 expression in cultured human hepatocytes and humanized livers of chimeric miceHEPATOLOGY RESEARCH, Issue 7 2007Junichi Shoda Aim and Methods: , A decreased function of multidrug-resistance 3 P-glycoprotein (MDR3), limiting the rate of biliary phospholipid secretion, predisposes individuals to cholestasis and/or cholangitis. Fibrates induce the expression of mdr2 (homolog of human MDR3) in rodents. To investigate the effects of bezafibrate (BF) on the expression levels of MDR3 in cultured human hepatocytes and human livers, the amount of protein and subcellular localization of MDR3 was assessed in HepG2 cells treated with BF and humanized livers of BF-treated chimeric mice. Results:, In HepG2 cells, while treatment with BF did not increase the protein levels of MDR3, the treatment caused a redistribution of MDR3 in the bile canaliculi. In humanized livers of chimeric mice, oral administration of BF induced a large increase in the protein amount of MDR3 and its redistribution in the bile canaliculi. Moreover, the modulatory effects of BF on key factors involved in hepatic cholesterol and bile acid metabolism in human subjects were traced in the humanized livers of BF-treated chimeric mice. Conclusion:, BF causes an induction of MDR3 expression in human livers. This provides a rationale for the beneficial role of BF in improving cholestasis and/or cholangitis associated with defective MDR3 expression and function in various types of cholestatic hepatobiliary diseases. [source] | ||||||||||||||||||||||||||||||||||