Identification of bipotential progenitor cells in human liver development

Intermediate filament proteins have been reported to be expressed in a cell lineage-specific manner during morphogenesis. We studied the expression of cytokeratin (CK)14, CK19, and vimentin and of the hepatocyte-specific HepPar1 antigen during the development of human liver. Nineteen fetal livers (gestational ages 4 to 40 weeks), 3 normal infant livers, and 3 normal adult livers were studied by immunoperoxidase staining of paraffin sections with monoclonal anti-CK19, anti-vimentin, and HepPar1 antibodies and polyclonal anti-CK14 antibodies. Double-immunostaining for CK14 and CK19 as well as bile duct cytokeratin and HepPar1 antigen was also done. CK19 and HepPar1 antigen were the first markers detected in immature progenitor cells of the liver primordium at 4 weeks' gestation. During subsequent liver development, the progenitor cells expressed HepPar1 antigen, CK14, and CK19, from 8 to 14 weeks' gestation. As hepatocyte differentiation progressed, expression of HepPar1 antigen increased, and CK14 and CK19 were abrogated from hepatoblasts at 14 to 16 weeks' gestation. In contrast, as progenitor cells transformed into ductal plate cells, CK19 expression increased and persisted in differentiated bile ducts, whereas CK14 and HepPar1 antigen were lost. Vimentin was detected in ductal plate and biliary epithelial cells from 9 to 36 weeks' gestation, but not in hepatoblasts or hepatocytes. Double-immunostaining confirmed coexpression of CK14 and CK19 in the progenitor cells for a short time (8 to 14 weeks' gestation) during early development. Double immunostaining for bile duct CK and HepPar1 antigen clearly demonstrated the divergence of the hepatocyte and bile duct epithelial cell lineages. Our findings suggest that hepatic progenitor cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Commitment of the HepPar1+CK19+ progenitor cells to either hepatocyte or bile duct epithelial cell lineages results in increased expression of one marker and loss of the other marker. These characteristics clearly identify bipotential hepatic progenitor cells in the developing human liver.     

Effects of fetal calf serum and disruption of cadherin function on the formation of bile canaliculi between hepatocytes

The polarization of hepatocytes to form a connected network of bile canaliculi (BC) is necessary for the function of the liver. Hepatocyte polarization may be controlled by soluble factors and/or physical interactions between cells. Monolayer cultures of embryonic chicken hepatocytes in DMEM supplemented with ornithine, dexamethasone, and insulin express BC-specific antigens for at least 7 days. However, BC-specific antigen expression is lost within 3 days of culture initiation in DMEM containing 10% fetal calf serum. The dedifferentiating effects of fetal calf serum (FCS) can be reversed. Furthermore, cultures in medium containing ornithine, dexamethasone, insulin, and 10% FCS appear identical to cultures grown in 10% FCS alone. Thus FCS contains a soluble inhibitor of hepatocyte polarization. Aggregate cultures grown in suspension maintain hepatocyte polarization for 10-12 days. This may be due to the increased cell-cell contact between hepatocytes in aggregate culture or to more normal contact with the extracellular matrix. We have evaluated the role of cadherin-mediated interactions on hepatocyte polarization. Anti-E-cadherin Fab' fragments disrupted the formation of long networks of BC in monolayer cultures but did not stop polarized expression of BC-specific antigens. The BC antigens in anti-E-cadherin-treated cells were concentrated in small areas between cells and were present at lower levels uniformly on the cell surface. These results indicate that E-cadherin is required for the formation of extended BC networks, but that other factors are responsible for maintaining the synthesis and localization of BC-specific antigens.     

Inducibility of enzyme activities associated with the cytochrome P-450 1A family, ethoxyresorufin O-deethylase, and methoxyresorufin O-demethylase in human hepatocyte lines derived from normal liver tissue

Three human hepatocyte cultures have been developed from specimens of normal human liver, in each case from an infant or child, by coculture with liver epithelial cells from 6-day-old rat pups in a complex growth medium. In the established cultures hepatocytes predominate and maintain typical hepatocellular morphology by light microscopy and albumin secretion into supernatant medium. The activity of ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) basally and after treatment with polycyclic aromatic hydrocarbons was measured spectrofluorometrically in cell homogenates from each culture. Very low levels of EROD and MROD activity were found in each culture without induction [EROD: 1.78 +/- 0.71 (mean +/- SE) pmol/min/mg protein; MROD: 1.33 +/- 0.10 pmol/min/mg protein]. After treatment with 10 microM dibenz (a,h)anthracene x 48 hr, EROD and MROD activities rose approximately 20- to 50-fold. When the basal and induced enzyme activities were remeasured 2 months later, results were essentially the same. Incubation with 10 microM benz(a)anthracene x 48 hr also led to induction of EROD and MROD activities. We believe that these cultures can be regarded as human hepatocyte lines, which conserve human hepatic polycyclic aromatic hydrocarbon-inducible P-450s, most likely including P-450 1A2.     

Hepatocyte volume as an indicator of hepatic functional reserve in cirrhotic patients with liver tumours

Using computed tomography (CT), measurements of whole liver volume have been used for the assessment of pre-operative functional reserve in cirrhotics. However, measurements of hepatocyte volume, which exclude stromal fibrous tissue, are considered to more directly reflect hepatic functional reserve. We investigated the relationship between total hepatocyte volume and each of the parameters of conventional liver function. Indocyanine green (ICG) tests and blood analyses for the assessment of liver function were performed prior to surgery in cirrhotic patients with liver tumours. Pre-operative liver volume was determined by integrating images of each liver area obtained by CT. Liver area was measured by an image processing program that traced the profile of the liver image while excluding the tumorous area. Sections of normal tissue stained by the haematoxylin-eosin method, were obtained from the resected liver. Using these sections, a hepatocyte area: whole tissue area ratio was calculated using the image processing program, by tracing the profiles of the hepatocyte nodules. The total volume of hepatocytes was then calculated by multiplying the liver volume by this ratio. The hepatocyte volume per unit bodyweight was significantly correlated with ICG tests and with many other parameters of normal liver function. However, the liver volume per unit bodyweight was correlated only with the plasma ICG disappearance rate and with the blood platelet count. These observations suggest that the functional reserve of the cirrhotic liver is assessed more precisely by hepatocyte volume than by liver volume.     

Immunolocalization of OV-6, a putative progenitor cell marker in human fetal and diseased pediatric liver

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.     

Transport of folates and antifolates in liver

The characteristics and mechanisms of hepatic transport of folates and antifolate cancer drugs, for example, methotrexate, have been studied in perfused liver, isolated hepatocytes (in both freshly isolated cells and in primary cell culture), and membrane vesicles isolated from the basolateral membrane. Both naturally occurring folates and antifolates are taken up by the perfused liver and secreted into bile by apparently active processes, since these compounds are concentrated in liver and bile compared with the perfusate. Transport of the naturally occurring folate 5-methyltetrahydrofolate in isolated hepatocytes and basolateral membrane vesicles is via cotransport with hydrogen ions, is electroneutral, and is inhibitable by other reduced folates and by methotrexate. Transport of methotrexate is by a multispecific anion carrier, is electrogenic, and is not inhibitable by reduced folates (e.g., 5-methyl- and 5-formyltetrahydrofolate). Thus, the hepatocyte has separate systems for uptake of the naturally occurring, reduced folates and for the 4-amino-substituted antifolates.     

Metabolic evaluation of cultured porcine hepatocyte monolayers in human plasma from hepatic failure patients--basic study on development of a bioreactor in hybrid artificial liver

In order to develop a functional bioreactor for hybrid artificial liver, function of cultured porcine hepatocyte monolayers in human plasma from hepatic failure patients (group III, n = 5) was investigated. Culture media, Leibovitz L-15 medium (group I, n = 7) and normal human plasma (group II, n = 3), were used as controls. Morphologically, no degeneration of porcine hepatocytes in hepatic failure plasma was observed for 5 days in culture. Levels of ureogenesis showed no significant difference against the controls at day 1, 2, 5 in culture, but the level at day 3 was significantly higher than that of group II. Levels of gluconeogenesis showed a close tendency as those of ureogenesis, but the level at day 3 was significantly lower than that of group I. Levels of intracellular DNA contents, showing between 1.85 +/- 0.39 and 1.35 +/- 0.05 microgram/cm2, were compatible with those of controls during first three days in culture, but level of group III at day 5 was significantly higher than that of group I. After incubation of porcine hepatocyte in hepatic failure plasma, the amount of valine, leucine, isoleucine, glutamine, arginine, and citrulline was significantly decreased. Elevated phenylalanine and tyrosine were also decreased, but Fischer's ratio, the ratio of branched chain amino acid against aromatic amino acids, was not significantly increased. Data obtained by this investigation showed that cultured porcine hepatocytes held proper hepatic function in the hepatic failure plasma. It is concluded that culture porcine hepatocyte monolayers were a promising candidate for a bioreactor of a hybrid artificial liver.     

A family of gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from gram-negative bacteria

Differentiation of biliary epithelial cells from hepatic endodermal cells of the mouse embryo was examined with a special attention to the role of the connective tissue. When the whole liver primordium of the 9.5-day mouse embryo was cultured in vitro for 5 days, the endodermal cells differentiated into mature hepatocytes expressing carbamoylphosphate synthetase I (CPSI) and accumulating glycogen. Intrahepatic bile duct cells and connective tissue were poorly developed in this culture. However, when the hepatic endoderm was recombined with the 4-day embryonic chick lung mesenchyme and cultured in vitro, the endodermal cells differentiated into many ductal epithelial cells as well as mature hepatocytes with abundant connective tissue development. These results suggest that the ducts might be bile ducts, and that connective tissue is very important for bile duct development. In addition, this in vitro culture system might be useful for the study of mechanisms of bile duct differentiation and congenital biliary atresia.     

Efficient induction of hepatocyte spheroids in a suspension culture using a water-soluble synthetic polymer as an artificial matrix

The preparation of hepatocyte spheroids by adding a water-soluble synthetic polymer as an artificial matrix was performed in a cell suspension system. Cell-aggregation was promoted without cytotoxicity by adding Eudragit (a copolymer of methacrylic acid and methylmethacrylate) to the culture medium. Spheroid-like cell aggregates, whose liver functions were enhanced, were effectively formed in the presence of 0.1% Eudragit, independent of the cultural substratum. Moreover, the mass preparation of spheroids could be achieved with a high production yield by means of a suspension culture in a spinner flask. In this case, the polymer protected the cells from damage due to agitation. The spheroids induced with Eudragit expressed high liver functions, such as albumin secretion, ammonia removal, and urea synthesis. On histological observation, the spheroids showed a well-developed cell adhesion apparatus and bile canaliculi. In addition, a higher calcium ion concentration in the cells of spheroids was observed compared with in monolayer cells.     

Cytotoxicity of bile in human Hep G2 cells and in primary cultures of rat hepatocytes

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.     

Sinusoidal endothelial cell damage by activated macrophages in rat liver necrosis

BACKGROUND: Massive hepatic necrosis caused by fibrin deposition in the hepatic sinusoids develops with hepatic macrophage activation in rats given endotoxin after administration of heat-killed Corynebacterium parvum. Targeted cells of such macrophages were investigated. METHODS: In C. parvum-treated rats, the pathological appearance of liver cells was serially measured in serum following endotoxin administration and compared with the appearance in the perfusate during closed liver perfusion with endotoxin. RESULTS: Serum activities of tumor necrosis factor, purine nucleoside phosphorylase present in both hepatocytes and sinusoidal endothelial cells, and levels of alanine aminotransferase were higher after 30 minutes, 1 hour, and 3 hours, respectively. Pretreatment of rats with gadolinium chloride, an inhibitor of macrophage function, reduced this liver injury. Although alanine aminotransferase activity remained almost unchanged in the liver perfusate, purine nucleoside phosphorylase activity increased. This increase was reduced when rats were pretreated with gadolinium chloride. There was sinusoidal endothelial cell damage around hepatic macrophages in the liver perfused with endotoxin. CONCLUSIONS: Activated hepatic macrophages may cause sinusoidal endothelial cell damage leading to hepatocyte necrosis in rats given C. parvum and endotoxin.     

Cooperation of Ito cells and hepatocytes in the deposition of an extracellular matrix in vitro

Cellular and molecular mechanisms involved in the deposition of extracellular matrix components in both normal and fibrotic liver are still poorly understood. We have investigated the influence of cooperation between Ito cells and hepatocytes in matrix deposition in vitro. Immunoprecipitation of radiolabeled proteins from media of 5-day-old Ito cell primary cultures showed that these cells secreted high levels of the major basement membrane components, ie, collagen IV, laminin, and entactin/nidogen. By immunocytochemistry, precursors of basement membrane components were found intracellularly, but only scarce deposits were seen around the cells. When hepatocytes were added to 2-day-old Ito cell primary cultures, they established close contacts with Ito cells in less than 24 hours and expressed ZO-1, a tight junction-associated protein not detectable in standard hepatocyte culture. Cytochemistry analysis revealed an abundant extracellular matrix deposited over hepatocyte cords and between hepatocytes and Ito cells. Immunocytochemistry studies showed that this matrix contained laminin, fibronectin, and collagens proIII and IV. These data indicate that a high level of matrix protein synthesis by liver cells in vitro is not sufficient to induce extracellular matrix deposition, and that cell-cell interactions are strongly involved in this process. Hepatocyte/Ito cell co-culture, which may reflect the actual situation in vivo, represents a useful tool for studying liver fibrogenesis.     

Ductular reaction after submassive necrosis in humans. Special emphasis on analysis of ductular hepatocytes

The ductular reaction to acute submassive necrosis was studied in human livers removed at the time of orthotopic liver transplantation. Single, double, and triple immunohistochemical labeling in combination with morphometry was used to analyze the phenotype and proliferative and apoptotic rates of various epithelial cell compartments. These were divided on the basis of immunohistochemistry and morphology into three subtypes: 1) CK19+/AE1+ mature bile duct epithelium, 2) HEP-PAR+ mature hepatocytes (HEPs), and 3) CK19+/AE1+ ductular hepatocyte (DH) cells lying at the interface between the portal tract connective tissue and the hepatic lobules. Cycling cells were defined as those showing Ki-67+ (MIB-1) nuclear labeling. Apoptotic cells were identified with in situ labeling using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling assay. Special emphasis was placed on DHs that appeared at the interface between the portal tracts and hepatic lobules. During the recovery phase from submassive hepatic necrosis, subtraction of the rate of cell death from the proliferative index shows that all of the epithelial compartments experience a net increase in the number of cells. The highest proliferation rate occurs in the DHs, which is significantly (P < 0.0001) higher than the proliferation rate seen in either the HEP or bile duct epithelium compartments. Immunohistochemical analysis of the highly proliferative DH compartment shows it to be a heterogeneous population with unique phenotypic features. Like epithelial cells in the ductal plate of fetal liver and cholangiocarcinomas, DHs are positioned on a laminin-rich matrix and focally express vimentin and Lewis(x) and show up-regulation of bcl-2 and type IV collagenase. However, unlike ductal plate cells, DHs are CD34 and alpha-fetoprotein negative. Although a subpopulation of DHs share phenotypic features with mature bile duct epithelium (AE1/cytokeratin 19 and type IV collagenase positive) or HEP (HEP-PAR, albumin, and alpha-1-antitrypsin positive), they are also clearly separate from both populations; DHs are negative or only weakly stain for glutathione-S-transferase-pi and are type IV collagenase positive. Moreover, occasional DHs also co-expressed HEP-PAR or alpha-1-antitrypsin and AE1, indicative of both hepatocyte and ductular differentiation. These findings suggest that DHs seen in human livers after submassive necrosis may represent a transient amplifying population arising from a progenitor population located in or near the canals of Herring. In addition, injured hepatocytes can express cytokeratin 19 and AE1, which normally are biliary intermediate filaments.     

The effect of donor and recipient age on engraftment of tissue-engineered liver

A novel treatment for end-stage liver disease using heterotopic hepatocyte transplantation on biodegradable polymers has been investigated. Survival and repopulation of adequate cell mass to replace hepatic function has been the principal difficulty of this method. Hence the authors have begun to investigate the role of donor and recipient age on the efficiency of hepatocyte transplantation. Lewis rats were used as donors and recipients. Hepatocytes were isolated with a collagenase digestion, both for the adult and fetal livers (17 days estimated gestational age). After digestion, the hepatocytes were seeded onto 95% porous poly-(L)-lactic acid matrices. The polymer-cell constructs with adult or fetal cells were then implanted between mesenteric leaves of three different recipient groups: adults (approximately 200 g), 2-week, and 4-week neonates (two to five animals per group, depending on litter size). The specimens were harvested at 4 weeks, stained with Hematoxylin and Eosin (H&E), and the cell area of each specimen (24 sections per group) was quantitated using morphometric analysis. Results were statistically analyzed using an unpaired, two-tailed Student's t test. At 4 weeks, all specimens showed survival of groups of hepatocytes, especially along the periphery of the polymers and near blood vessels. The hepatocyte cell area for the six groups was calculated in square micrometers: the adult cells transplanted into adult recipients, 0.16 x 10(5) microns2; fetal cells into adults, 0.47 x 10(5) microns2; adult into 4-week neonates, 1.17 x 10(5) microns2; fetal into 4-week neonates, 4.54 x 10(5) microns2; adult into 2-week neonates, 2.98 x 10(5) microns2, and fetal into 2-week neonates, 5.81 x 10(5) microns2. In all three recipient groups, the area of fetal hepatocytes was approximately two to three times the area of the adult hepatocytes (P < .05 for 2-week and 4-week neonatal recipients, P = .06 for adult recipients). Also, as the recipient age decreased, there was an increase in the hepatocyte cell area (P < .05 for fetal or adult groups). The authors conclude that fetal hepatocytes heterotopically transplanted have a significant survival advantage over adult hepatocytes, independent of recipient age. The authors further conclude that the neonatal environment is more favorable than the adult environment for implantation of hepatocytes.     

Origin of the central cells of erythroblastic islands in fetal mouse liver: ultrahistochemical studies of membrane-bound glycoconjugates

To clarify the origin of the central cells in hepatic erythroblastic islands, glycoconjugates on the surface of cellular constituents in fetal mice liver were ultrahistochemically examined using lectin staining. At 11 days of gestation, the cells derived from mesenchyme in fetal liver, including sinusoidal macrophages, endothelial cells, and erythropoietic cells, bound Griffonia simplicifolia isoagglutinin I-B4 (GS-I-B4), but hepatocytes lacked binding sites for the isolectin. Scavenger macrophages in the hepatic cords at 13 days of gestation and the central cells in the erythroblastic islands at 15 days of gestation also bound GS-I-B4. Hepatocytes, however, exhibited no GS-I-B4 binding site at any gestational day. At 11 days of gestation, none of the cells in fetal liver had binding sites for soybean agglutinin (SBA), but cells derived from mesenchyme acquired these binding sites at 13 days of gestation. The central cells in the erythroblastic islands also bound SBA, but hepatocytes did not bind the lectin at all. The central cells in the erythroblastic islands can be considered to belong to a mesenchymal cell lineage, and primitive sinusoidal macrophages at 11 days of gestation are possible precursors of these central cells.     

Apoptosis and liver fibrosis: antifibrotic strategies

Hepatic fibrosis is a frequent response of the liver and is similar to parenchymal wound healing in other tissues. Apoptosis has been described in different models of liver fibrosis. Hepatic stellate cells are the main source of extracellular matrix. At present, one can speculate that inhibition of apoptosis is responsible for activation and proliferation of hepatic stellate cells. Thus, the inhibition of hepatic stellate cell apoptosis could be a target for antifibrotic strategies. Until now, no drugs have been clearly shown to be effective in reducing specifically the development of hepatic fibrosis. However, serious candidates are presently under studies in clinical trials, including especially alpha interferon and phosphatidylcholine.     

Molecular regulation of sinusoidal liver bile acid transporters during cholestasis

Impairment of the hepatic transport of bile acids and other organic anions will result in the clinically important syndrome of cholestasis. Cloning of a number of specific hepatic organic anion transporters has enabled studies of their molecular regulation during cholestasis. The best characterized transport system is a 50-51 kDa sodium-dependent taurocholate cotransporting polypeptide (ntcp), which mediates the sodium-dependent uptake of conjugated bile acids at the sinusoidal plasma membrane of hepatocytes. Under physiologic conditions and after depletion of biliary constituents, ntcp remains constitutively expressed throughout the liver acinus. However, both function and expression of ntcp are rapidly down-regulated in rat liver in various models of experimental cholestasis, such as cholestasis induced by common bile duct ligation, estrogen, endotoxin or cytokine treatment. In addition to ntcp, the sinusoidal organic anion transporting polypeptide oatp-1 is also down-regulated at the protein and steady-state mRNA levels in estrogen-cholestasis, but does not affect sodium-independent uptake of taurocholate. The regulation of a recently cloned member of the organic anion transporter family (oatp-2), which is highly expressed in liver, remains to be studied under cholestatic conditions.     

The role of bile salt composition in liver pathology of mdr2 (-/-) mice: differences between males and females

BACKGROUND/AIMS: The mouse mdr2 gene encodes a P-glycoprotein expressed in the canalicular membrane of the hepatocyte. Mice in which this gene has been inactivated (mdr2 -/-) show a defect in biliary phospholipid and cholesterol secretion and develop non-suppurative cholangitis. We hypothesized that secretion of bile salts without lipids initiates this liver disease. METHODS: To delineate the pathologic process, mdr2 (-/-) mice were fed different bile salt-supplemented diets for 22 weeks after weaning. Aspects of liver pathology including eosinophilic bodies, portal inflammation, ductular proliferation, mitotic activity and fibrosis were semi-quantitatively scored. RESULTS: It was observed that liver pathology was more severe in female than in male mice when fed a purified control diet. This correlated with a more hydrophobic bile salt composition of female vs. male bile. When increasing amounts of cholate were added to the diet (0.01% and 0.1%), the secretion of taurocholate increased and this was accompanied by a more severe liver pathology. At the high dose of cholate (0.1%), the bile salt compositions of male and female mice became similar, as did the severity of the histological score. Addition of cholate to the diet did not induce liver pathology in (+/+) mice. Addition of ursodeoxycholate to the diet (0.5%) led to a near complete replacement of biliary bile salts by tauroursodeoxycholate and this reduced pathology and dissipated the difference between males and females. CONCLUSIONS: These observations support our hypothesis that liver pathology in the mdr2 (-/-) mouse is caused by bile salts and depends on the hydrophobicity c.q. cytotoxicity of biliary bile salts.     

Expression of c-Met correlates with grade of malignancy in human astrocytic tumours: an immunohistochemical study

The construction of the liver parenchyma throughout fetal development depends on the elaboration of intercellular contacts between epithelial cells and between epithelial and mesenchymal cells. During this time, the spatial distribution of cytokeratins in hepatocytes shows a striking evolution as demonstrated by confocal microscopy and image analysis. In the early stages of fetal rat development, the liver is mainly a hematopoietic organ and hepatocytes represent fewer than 40% of all liver cells. At this time, cytokeratin filaments are scarce and are randomly distributed inside the cytoplasm. A coexpression of desmin and cytokeratin is found in some cells. Intercellular contacts between epithelial and mesenchymal cells are more numerous than between epithelial cells. Later in development, hepatocytes are arranged in a "muralium duplex" architecture (two-cell-thick sheets). Contacts between hepatocytes become more numerous and bile canaliculi become well developed. The density of cytokeratin filaments increases and appears to be very high near the bile canaliculi. In adult liver, hepatocytes are arranged in a "muralium simplex" architecture. Cytokeratin filaments show a symmetrical distribution in relation to the nuclear region. The highest density of filaments is found near the cytoplasmic membrane. Variations of the spatial distribution of intermediate filaments throughout hepatocyte differentiation were investigated in a pilot study using computerized image analysis. We found significant differences between the filament networks in fetal and adult hepatocytes.