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Mammalian cells take up exogenous DNA very inefficiently. However, in the absence of viral vectors, DNA can be transfected into cells by co-precipitation with calcium phosphate and usually also with carrier DNA or by lipofection or electroporation. Such DNA can be expressed efficiently by cells. Alternatively, direct injection can also result in uptake and expression of transgenes. Without carefully designed means to target DNA specifically to integrate into the host genome, the vast majority of internalised DNA remains extra-chromosomal and is degraded. The likely fate of DNA which in low levels may contaminate vaccines derived from mammalian cell lines, will also be destruction. There is a theoretical risk of DNA integration events with random sequences of donor-derived DNA but the probability of that leading to serious adverse effects to the host is extremely small.

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Notch signaling is an evolutionarily conserved mechanism, used to regulate cell fate decisions. Four Notch receptors have been identified in man (Notch-1 to -4). In this study, semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to examine the expression pattern of Notch receptor genes in whole adult human liver and isolated liver cell preparations. All 4 receptors were expressed in the adult liver, with no significant differences in the levels of Notch-1, -2, and -4 messenger RNA (mRNA) between normal and diseased liver. However, Notch-3 expression appeared to be increased in diseased tissue. The distribution of Notch-1 and -4 in normal tissue was similar, with Notch-1 also detectable at low levels in the sinusoidal endothelium. Notch-2 expression was more widely distributed, and detectable in hepatocytes, medium-sized bile ducts, and the sinusoidal endothelium. Notch-3 expression was seen on hepatocytes, with weaker expression detectable in portal veins, hepatic arteries, and the sinusoids. In normal liver tissue Notch-1, -2, and -3 were found to be coexpressed on bile duct epithelium; however, with the exception of Notch-3 in primary sclerosing cholangitis (PSC) livers, expression was absent on proliferating ductules in all disease states examined. Interestingly, the expression of Notch-2 and -3 was associated with numerous small vessels within the portal tract septa of diseased tissue. The absence of Notch receptor expression on proliferating bile ductules and its presence on neovessels suggests that Notch signaling may be important for normal bile duct formation and the aberrant neovascularization seen in diseased liver tissue.

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Although the control of biliary ductular morphogenesis has received some attention particularly using isolated rat biliary epithelial cell models, the regulation of human bile duct formation is not well defined. In the present study, using a 3-dimensional culture model comprising primary human biliary epithelial cells (BECs) and coculture with primary human hepatocytes, we have sought to define the factors involved. We have shown that primary human BECs can be expanded on collagen gels in the absence of growth factors or serum. When plated in high density in double collagen gels, BECs established 3-dimensional structures that subsequently developed into well differentiated polarized luminal ducts. This morphogenic response occurred in the absence of hepatocyte growth factor (HGF) and epidermal growth factor. Strikingly, the addition of growth factors (in the presence of serum) resulted in loss of polarity although the cells retained growth responses to both factors. Coculture of BECs with autologous human hepatocytes enhanced the ability of low-density BECs to undergo ductulogenesis. This effect was mimicked by addition of conditioned medium from previous hepatocyte-BEC cocultures. These findings indicate that for human biliary ductular morphogenesis, epithelial cell-cell interactions are required but that mesenchymally derived factors such as HGF may not be important.

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BACKGROUND & AIMS: Recent reports suggest that after bone marrow transplantation into rodents and humans, hematopoietic stem cells migrate into the liver and give rise to oval cells, hepatocytes, and biliary epithelial cells. We investigated this hypothesis further in the human liver using the hematopoietic markers c-kit and CD34. METHODS: Immunofluorescence confocal microscopy was performed using cytokeratin 19 (CK-19; biliary cell marker) with either c-kit or CD34. Immunomagnetic separation was then used to select c-kit- or CD34-positive cells. After attachment, cells were cultured for up to 7 days, and their growth and phenotypic characteristics were examined. RESULTS: In cirrhotic tissue, c-kit- or CD34-positive cells were located in the portal tracts surrounding bile ducts. Occasionally c-kit- (but not CD34-) positive cells that coexpressed CK-19 were observed integrated into bile ducts. In vitro, immunoisolated c-kit or CD34 cells gave rise to colonies of at least 2 morphologies expressing CK-19 or CD31 (endothelial cell marker). CD34- or c-kit-positive cells with similar properties were also isolated from normal liver. CONCLUSIONS: These findings indicate that cells present in human liver that express the markers c-kit or CD34 have the capacity to differentiate into biliary epithelial cell lineage and may therefore represent human biliary epithelial progenitor cells.

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OBJECTIVE: To determine whether human placenta secretes hepatocyte growth factor (HGF) and could influence fetal liver development. METHODS: Expression of HGF and c-met mRNA in paired samples of first- and second-trimester fetal liver and placenta was compared using a quantitative ribonuclease protection assay. Serum HGF concentration in 30 samples of paired umbilical and maternal blood from term pregnancies was evaluated using an enzyme-linked immunosorbent assay. RESULTS: HGF and c-met mRNA were expressed at similar levels in liver and placenta, with expression increasing from 9 to 16 weeks' gestation. Median serum HGF values were 1.4 ng/mL (maternal venous), 1.2 ng/mL (cord venous), and 1.3 ng/mL (cord arterial). The maternal venous HGF levels were significantly higher than fetal venous levels (P =.02). CONCLUSIONS: This study does not support the hypothesis that the placenta secretes HGF, because maternal serum levels were higher than fetal and there was no significant difference between umbilical arterial and venous samples. Fetal liver expresses abundant HGF mRNA during the first and second trimester and expression increases in line with receptor (c-met) expression, suggesting that hepatic growth and development are independent of placental HGF.

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A transient induction of S phase DNA synthesis is a common feature of non-genotoxic rodent hepatocarcinogens when administered in vivo. In the present study the ability of phenobarbitone (PB) to induce S phase DNA synthesis in primary cultures of rat hepatocytes was investigated. In the absence of serum or growth factors PB was not a mitogen per se. However, stimulation of S phase DNA synthesis by epidermal growth factor (EGF) was enhanced by co-culture with PB. This effect was both time and concentration dependent. The lowest concentration of PB that significantly enhanced the effect of EGF was 10 microM and the effect was maximal at 1.0 mM. At a concentration of 2.0 mM PB no longer enhanced EGF-induced S phase DNA synthesis. Hepatocyte cultures pretreated with PB (0.1 mM) for 2 days were more responsive to the induction of S phase DNA synthesis by EGF for the subsequent 2 days. Despite the inhibition of PB enhancement of S phase DNA synthesis by the antioxidant dimethylthiourea, reduced glutathione was not depleted by PB treatment nor were oxidized glutathione or lipid peroxides elevated. Western blotting analysis showed that PB had no effect on epidermal growth factor receptor (EGFR) autophosphorylation per se after 1 and 48 h culture, enhanced sensitization of EGFR therefore does not appear to contribute to the enhancement of S phase DNA synthesis by PB. In contrast, treatment of hepatocytes with PB for 12 h resulted in a small but statistically significant activation of p42/44 MAP kinase activity and activation of protein kinase C, as measured by redistribution of enzyme activity from a soluble to a particulate compartment of hepatocytes. Therefore, PB-mediated changes in protein kinase activity may contribute to the potentiation this compound affords.

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HGF-activator (HGF-A) is a circulating serine protease known to be responsible for activation of hepatocyte growth factor (HGF). Active HGF is thought to be an important regulator of trophoblast growth. In vitro, HGF-A is produced via proteolytic cleavage of its zymogen by thrombin. Immunocytochemistry and Western immunoblotting were performed using human placental tissue from all three trimesters with an antibody that recognizes both HGF-A and its zymogen. Western immunoblotting revealed a 97 kDa band equivalent to the zymogen in placenta from all three trimesters. A smaller 34 kDa band equivalent to HGF-A was only seen in first and second trimester placenta. The anti-HGF-A/zymogen antibody demonstrated immunostaining in placental villi and membranes throughout gestation. Within first trimester villi immunostaining was strongest within the syncytio- and cytotrophoblast layers, but was also seen within stromal and endothelial cells. Likewise, in third trimester placenta the syncytio-cytotrophoblast layer showed the strongest immunoreactivity. In vitro, HGF can induce trophoblast DNA synthesis and the localization of HGF-A to the peri-villous trophoblast layer (which expresses c-met, the HGF receptor) suggests that it may be responsible for activation of pro-HGF at this site. This adds further weight to the hypothesis that HGF in vivo is an important regulator of trophoblast growth.

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It has recently been shown that reactive bile ductules display neuroendocrine features, including immunoreactivity for the neural cell adhesion molecule (NCAM). In this study we have compared the immunohistochemical expression of NCAM with that of HEA-125 (biliary specific) and LKM-1 (hepatocyte specific) and other markers relevant to morphogenesis (Bcl-2, EMA) and cell proliferation (Ki-67) in cryostat sections from different chronic liver diseases and from fetal livers at different gestational ages. In parallel, viable NCAM-positive ductular cells were purified from collagenase digests of cirrhotic livers by immunomagnetic separation and characterized by immunocytochemistry and transmission electron microscopy. We demonstrated that reactive ductules with atypical morphology coexpressed NCAM and Bcl-2 and were found mainly in congenital diseases associated with ductal plate malformation and in primary cholangiopathies. On the contrary, reactive ductules with typical morphology were negative for NCAM/Bcl-2 and positive for EMA. Reactive ductules coexpressing NCAM/Bcl-2 were negative for the proliferation marker Ki-67 and appeared to be directly connected with periportal hepatocytes. In fetal livers NCAM/Bcl-2 was transiently expressed during the early developmental stages of ductal plate (10-16 weeks) and started to disappear as the ductal plate began duplicating. NCAM-positive ductal plate cells were Ki-67 negative, becoming positive in duplicated segments. Thus the histogenesis of ductular reactive cells seems to recapitulate the early stages of biliary ontogenesis. In primary cholangiopathies and ductal plate malformations, these cells do not appear to maturate further, and thus abundant ductular structures coexist with vanishing mature ducts. These NCAM-positive ductular cells were immunopurified from patients with chronic cholestatic liver diseases and showed ultrastructural features consistent with a less differentiated phenotype than mature cholangiocytes. These isolated cells represent a useful model for in vitro studies.

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Phenobarbitone (PB) treatment of mice causes a decrease in the growth factor responsiveness of hepatocytes. Here, epidermal growth factor receptor (EGFR) expression and receptor autophosphorylation was determined in hepatocytes isolated from control and PB-treated mice. There was a decrease in the level of EGFR expression in hepatocytes isolated from mice following PB administration when compared to controls. EGF caused an approximate 20-fold increase of the 170 kD phosphotyrosine band in control hepatocytes, which was inhibited by the EGFR specific tyrosine kinase inhibitor 4, 5-dianilinopthalamide. Following PB treatment, the degree of basal receptor phosphorylation (in the absence of EGF) was significantly greater and therefore the fold rise in EGFR phosphorylation in isolated hepatocytes was lower than in controls. However, the overall extent of EGF-induced receptor phosphorylation was not diminished in hepatocytes isolated from PB-treated mice. Therefore the reduction in responsiveness to growth factors seen in hepatocytes ex vivo or the cessation of proliferation observed in vivo following PB administration is unlikely to be attributed to a decrease in ligand binding and subsequent receptor autophosphorylation.

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The stem cell factor (SCF)/c-kit ligand/receptor system has been implicated in stem (oval) cell activation following liver injury in the rat. The aim of this study was to determine the role of the SCF/c-kit system in pediatric human liver during acute and chronic liver injury. Tissue was obtained from hepatectomy specimens of patients undergoing liver transplantation for extrahepatic biliary atresia (EHBA) and fulminant hepatic failure (FHF). Specific expression of mRNA for c-kit and beta-actin was measured by ribonuclease protection and by immunohistochemistry to localize c-kit in tissue sections. Expression of c-kit was detected at relatively consistent levels in normal and cirrhotic (EHBA) livers. However, in FHF, c-kit mRNA levels were elevated in 3 of 6 specimens. Immunolocalization highlighted the presence of small numbers of c-kit-positive cells in the portal tracts of normal livers with increased numbers in cirrhotic livers. The highest c-kit staining, however, was observed in FHF, in which, in addition to the cells in the portal tracts, discrete c-kit-positive cells were also found integrated into bile ducts. Colocalization studies demonstrated some of the c-kit-positive cells to be of mast cell, leukocyte, and hematopoietic cell origin. However, there remained a subset that was also negative for these markers. The up-regulation of c-kit receptor expression in diseased livers suggests an involvement of this receptor/ligand system in hepatic repair mechanisms, and we speculate that c-kit-positive cells may represent a hepatic progenitor cell population. The origin and growth/differentiation potential of these c-kit-positive cells is under investigation.

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The drug tamoxifen shows evidence of genotoxicity, and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and these arise through metabolism at the alpha-position to give an ester which reacts with DNA. (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-but-1-en e (idoxifene) is an analogue of tamoxifen in which formation of DNA adducts is greatly reduced; we could not detect any adducts in the DNA of cultured rat hepatocytes treated with 10 microM idoxifene, after analysis by the 32P-post-labelling method. The metabolite (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3-but en-2-ol (alpha-hydroxyidoxifene) gave adducts in rat hepatocytes, but far fewer than the corresponding tamoxifen metabolite. In human hepatocytes, neither idoxifene nor tamoxifen induced detectable levels of DNA adducts. We prepared the alpha-acetoxy ester of idoxifene as a model for the ultimate reactive metabolite formed in rat liver. It was less reactive than alpha-acetoxytamoxifen, as might be expected on mechanistic grounds. It reacted with DNA in the same way, to give adducts which were probably N2-alkyldeoxyguanosines, but to a lower extent. All these results indicate that idoxifene is much less genotoxic than tamoxifen, and should therefore be a safer drug.

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Severe intrauterine growth restriction (IUGR) is characterized by abnormal placentation. Mouse gene knockout studies show that an absence of either hepatocyte growth factor (HGF) or its receptor, c-met, leads to intrauterine death secondary to severe IUGR with deficient placentation. In this study, immunocytochemistry localized HGF protein throughout placental villi across gestation, whereas c-met protein was localized only to the perivillous trophoblast and vascular endothelium. Within the IUGR placentae, a reduction in HGF immunostaining within the villous stroma was observed. HGF mRNA was strongly expressed in the perivascular tissue around the stem villous arteries throughout gestation, with weaker expression within the villous stroma and the terminal villi. c-met mRNA expression was limited to the perivillous trophoblast, particularly in the first trimester, with only a faint hybridization signal from the villous stroma. Placental mRNA expression was examined quantitatively using a ribonuclease protection assay: HGF and c-met mRNA expression increased from the first to the second trimester, reaching a zenith before decreasing again through the third trimester to term. HGF mRNA levels were significantly reduced in the IUGR placentae (P = 0.036), whereas c-met mRNA expression was within the normal range for gestation. These findings suggest that HGF derived from the perivascular tissue of stem villous arteries may play an important role in controlling normal villous development. Whereas reduced expression of HGF within IUGR placentae does not prove a causative link with abnormal villous development, the association lends support to this possibility.

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The existence of progenitor (stem) cells in the human liver remains a matter of debate. In rodent models of hepatocarcinogenesis and injury, oval cells proliferate in the periportal regions of the portal tracts and are suggested to derive from a stem cell compartment, because they are capable of differentiating into hepatocytes or biliary epithelial cells. In this study, the rat oval cell marker, OV-6 has been used to investigate the hypothesis that there are stem cells present in fetal and pediatric human liver. The pattern of OV-6 expression was compared with the established adult biliary cell markers human epithelial antigen-125 (HEA-125) and cytokeratin-19 (CK-19). In normal pediatric liver (n = 7), bile ducts and ductules were immunostained with CK-19 and HEA-125, whereas OV-6 staining was consistently negative. In fetal tissue (n = 10), ductal plate cells, primitive bile ducts, and hepatoblasts were stained with CK-19 and HEA-125 although only some of the ductal plate cells and hepatoblasts were OV-6 positive. In biliary atresia (n = 6) and 1, anti-trypsin deficiency (1,AT) (n = 4), CK-19 and HEA-125 immunostained ductular proliferative cells that tended to form finely anastomosing ductules, whereas OV-6 staining was found more on discrete cells confined to portal tract margins. Additionally, in diseased liver, OV-6 was strongly positive in hepatocyte lobules with greatest intensity in the periseptal regions. This widespread hepatocyte OV-6 positivity suggests that the antibody may identify cells of a less differentiated phenotype (transitional hepatocytes) that have replaced the mature cells. Therefore, it is proposed that in human liver, OV-6 is recognizing cells with a progenitor stem cell-like phenotype with the capacity to differentiate into OV-6 positive ductular cells or lobular hepatocytes.

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The application of primary hepatocytes in hybrid artificial liver systems has been hampered by the gradual loss of differentiated morphology and function in vitro. Therefore, we have established a coculture model of autologous human hepatocytes and biliary epithelial cells (BEC) in collagen gel in the presence of hepatotrophic growth factors. Furthermore, we examined the effect of hepatocyte cell perfusion in a women multicompartment capillary membrane system. Normal hepatocytes isolated from human liver produced albumin for more than 2 weeks in serum-free media, and were further stimulated by conditioned medium. When cocultured with BEC, albumin secretion was greatly enhanced, suggesting that cellular interactions promote tissue-specific differentiation. When perfused in bioreactors, albumin levels were maintained at steady state for longer than 2 weeks. These data indicate that differentiation of primary hum hepatocytes can be maintained by coculture interactions and three-dimensional hybrid organ devices, providing appropriate growth factors and matrix for tissue regeneration.

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To assess DNA damage caused by lipid peroxidation due to copper and iron storage disorders in the human liver, the formation of the etheno adducts 1,N6-ethenodeoxyadenosine (epsilon dA) and 3,N4-ethenodeoxycytine (epsilon dC) was measured in liver DNA from normal subjects and from patients with Wilson's disease (WD) and primary hemochromatosis. The mean epsilon dA and epsilon dC levels per 10(9) parent nucleotides in normal liver were 19.3 +/- 4.9 and 27.5 +/- 10.0, respectively. The mean epsilon dA and epsilon dC levels per 10(9) parent nucleotides in WD were 61.03 +/- 7.95 and 91.50 +/- 36.02, and in primary hemochromatosis, they were 46.62 +/- 32.83 and 64.32 +/- 11.55, respectively, two to three times higher than those in the normal liver. The etheno adduct levels were highly correlated with the copper content of the liver in the normal and WD samples. This study demonstrates for the first time the formation of promutagenic etheno adducts in humans in association with copper and iron storage-induced lipid peroxidation. Thus, the etheno adducts are implicated as initiating DNA damage in copper/iron-induced carcinogenesis in humans and should also be explored as biomarkers in disease progression and prevention trials.

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