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We have developed a culture model to assess antifibrotic drugs using normal human liver myofibroblasts (HLMFs) obtained from 31 subjects. Activation was evaluated in terms of α-smooth muscle actin (α-SMA) and collagen 1 (Coll1) expression using RT-PCR, and proliferation as the uptake of 5-ethynil-2'-deoxyuridine. Under analysis of variance, between-subject differences accounted for 70% of all variability and inter-experiment differences for 30%. The sensitivity of the model was determined by quantifying the effects in terms of relative expression, which were 0.74±0.03 for cyclosporine A (CsA) and 2.4±0.10 for transforming growth factor-beta (TGF-β) (P<0.0001 vs no treatment) for α-SMA expression. Inter-subject variations in α-SMA and Coll1 expression enabled the classification of subjects as potentially low or high fibrosers. Finally, we observed that pirfenidone (which has beneficial effects in vivo) significantly reduced the expressions of α-SMA and Coll1, whereas the angiotensin-converting enzyme inhibitor losartan (which has no effect in vivo) had no significant effect. Our model may thus detect the antifibrotic properties of drugs. Antifibrotic drugs with promising clinical relevance could possibly be selected using a bank of HLMFs from high fibrosers.

Hepatocellular carcinoma (HCC) displays a striking resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Therefore, the characterization of pharmacological agents that overcome this resistance may provide new therapeutic modalities for HCC. Here, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors could restore TRAIL sensitivity in hepatoma cells. To this aim, the effects of two GSK-3 inhibitors, lithium and SB-415286, were analyzed on TRAIL apoptotic signaling in human hepatoma cell lines in comparison with normal hepatocytes. We observed that both inhibitors sensitized hepatoma cells, but not normal hepatocytes, to TRAIL-induced apoptosis by enhancing caspase-8 activity and the downstream recruitment of the mitochondrial machinery. GSK-3 inhibitors also stabilized p53 and the down-regulation of p53 by RNA interference abolished the sensitizing effect of lithium on caspase-3 activation. Concomitantly, GSK-3 inhibitors strongly activated c-Jun N-terminal kinases (JNKs). The pharmacological inhibition of JNKs with AS601245 or SP600125 resulted in an earlier and stronger induction of apoptosis indicating that activated JNKs transduced protective signals and provided an anti-apoptotic balance to the pro-apoptotic effects of GSK-3 inhibitors. These findings demonstrate that GSK-3 exerts a negative and complex constraint on TRAIL apoptotic signaling in hepatoma cells, which can be greatly alleviated by GSK-3 inhibitors. Therefore, GSK-3 inhibitors may open new perspectives to enhance the anti-tumor activity of TRAIL in HCC.

Background: This study evaluated the survival and functions of encapsulated porcine hepatocytes after intraperitoneal allotransplantation and xenotransplantation without immunosuppression., Methods: Isolated porcine hepatocytes were encapsulated in AN 69 polymer capsules (45.10(6)/capsule) and transplanted intraperitoneally in 12 rats and 12 pigs. Fifteen, 30, and 60 days after transplantation, capsules were removed and the viability and morphology of explanted hepatocytes were examined under light and electronic microscopy. The potential to produce albumin was assessed by evaluating the level of albumin messenger RNA, using semiquantitative reverse transcription-polymerase chain reaction. 6beta-Hydroxylase activity was measured by high-performance liquid chromatography. In addition, cytochrome P450 3A proteins were detected by Western blot only in allogeneic hepatocytes., Results: Similar results were observed after allotransplantation and xenotransplantation. Histologic studies showed that hepatocytes were well-preserved and arranged in cords for up to 30 days. The expression of porcine albumin gene was maintained up to 15 days. 6beta-Hydroxylase activity was 2.5-fold lower at day 15 than in freshly encapsulated hepatocytes, which were not transplanted. In allogeneic hepatocytes, the expression of CYP 3A protein was detected up to 60 days after transplantation., Conclusions: Encapsulated porcine hepatocytes remain viable and functional for at least 15 days after allotransplantation and xenotransplantation without immunosuppression. The demonstration of maintained hepatic functions in transplanted porcine hepatocytes up to 15 days is a first step toward application in the treatment of acute liver failure.

Background: An implantable bioartificial liver (BAL) using xenogeneic isolated hepatocytes may be an alternative method to orthotopic liver transplantation for treatment of acute liver failure. The purpose of this study was to demonstrate that not only fresh but also cryopreserved porcine hepatocytes could be used in a BAL to prevent death after the onset of acute liver failure in rats., Methods: Acute liver failure was induced by two-stage 95% hepatectomy. At the time of completion of liver resection, 100 rats were assigned to undergo or not undergo transplantation into the peritoneum of 4 meters of hollow fibers filled with 60 million either fresh or cryopreserved porcine hepatocytes, or syngeneic hepatocytes, or culture medium, or of 60 million nonencapsulated cryopreserved porcine hepatocytes without immunosuppressive therapy. Survival rates at 7 days were compared between the different groups., Results: In the control groups of hepatectomized animals not receiving encapsulated hepatocytes, 69-79% of the rats died from acute liver failure. The mortality rate was reduced to 15% (2 of 13) in rats receiving fresh porcine hepatocytes (P<0.01), 25% (4 of 16) in rats transplanted with either cryopreserved or syngeneic hepatocytes (P<0.05). Survival rates were maintained when hollow fibers were explanted > or =4 days after hepatectomy. In surviving rats, the weight of the remnant native liver increased with time and returned to the initial weight after 1 month., Conclusions: The implantable BAL using xenogeneic porcine hepatocytes was able in preventing death from acute liver failure without immunosuppressive therapy. Encapsulated cryopreserved hepatocytes were as effective as fresh hepatocytes.

Objectives: To determine the viability and differentiation of human hepatocytes immunoprotected by encapsulation and transplanted in rats without immunosuppression., Methods: Freshly isolated human hepatocytes were encapsulated in hollow fibers and transplanted in the peritoneal cavity of immunocompetent rats. The fibers were explanted for analysis at D3, D7 and D14 following transplantation. Morphological features under light and electron microscopy and gene expression were compared to those of non-transplanted encapsulated hepatocytes (D0). Human cytochrome P450 3A and albumin mRNAs were quantified by Northern blot. Cytochrome P450 3A proteins were detected by Western blot and cytochrome P450 3A enzyme activity was assessed by measuring the formation of 6beta-hydroxytestosterone by high performance liquid chromatography., Results: Transplanted hepatocytes were more than 60 % viable and exhibited morphological criteria of hepatocytic differentiation up to D7. Albumin and cytochrome P450 3A transcripts were also detected up to D14. At D3 and D7, albumin mRNA levels were of 30 %, compared to control D0 hepatocytes, while cytochrome P450 3A5 and cytochrome P450 3A4 mRNA levels were 65 % and 0 %, respectively. Cytochrome P450 3A immunoreactivity was detected by Western blot up to D14 and 6beta-hydroxylase activity was 17 % at D3 compared to D0, supporting with disappearance of cytochrome P450 3A4 mRNA., Conclusions: Human hepatocytes remain viable for a short period, following encapsulation and intraperitoneal transplantation in rat. Other experimental conditions need to be tested to prevent or delay a decrease in hepatocyte specific gene expression.

Background: To evaluate new therapies for human fulminant hepatic failure, a suitable large animal model is needed. The purpose of this study was to develop a reversible surgical model of acute hepatic liver failure by transient ischemia in pigs., Materials and Methods: Under general anesthesia, an end-to-side portacaval shunt was performed in 17 pigs and tape was laid around the hepatoduodenal ligament. Two days after construction of the functional portacaval shunt, 13 ambulant pigs underwent transient total liver ischemia by tightening of the tape around the hepatoduodenal ligament for 5.5 h. During ischemia, 10% glucose was continuously infused intravenously to prevent hypoglycemia., Results: Ten animals (77%) died with hepatic coma after a mean duration of 22.5 +/- 1.9 h. The 3 remaining animals survived more than 5 days and were sacrificed. In dying animals, encephalopathy was observed 14 +/- 1.7 h after the onset of ischemia. During ischemia, similar progressive decrease of fibrinogen, platelets, prothrombin time, and factors V and VII activities was observed in dying and surviving animals. Just before death, mean prothrombin and factors V and VII activities were respectively 22 +/- 2, 21 +/- 4.4, and 24 +/- 5%. At 22 h, plasma ammonia and lactate levels were respectively 705 +/- 93 micromol/L and 10.5 +/- 0.4 mmol/L in dying animals and 249 +/- 75 micromol/L and 2.9 +/- 0.1 mmol/L in surviving animals (P < 0.01). Estimation of the percentage liver cells necrosed was 74 +/- 4.7% in the survivors and 86 +/- 5.5% in animals who died of hepatic coma (NS)., Conclusions: This model is reproducible and reversible and should allow the quantitative evaluation of new technologies, such as bioartificial liver, for the support of hepatic failure in humans., (Copyright 2000 Academic Press.)

Objective: To demonstrate that a bioartificial liver, using allogeneic or xenogeneic hepatocytes protected from rejection by a semipermeable membrane, could prevent death from acute liver failure., Summary Background Data: An implantable bioartificial liver using isolated hepatocytes could be an alternative to orthotopic liver transplantation to treat patients with acute liver failure. It could serve either as a bridge until liver transplantation or as the main treatment until recovery of the native liver. However, allogeneic or xenogeneic hepatocytes that could be used in clinical applications are spontaneously rejected., Methods: Acute liver failure was induced in rats by 95% liver resection. Twenty-five million hepatocytes harvested in rats (allogeneic) or guinea pigs (xenogeneic) were encapsulated in a semipermeable membrane to protect them from rejection. The hollow fibers containing hepatocytes were transplanted into the peritoneum of recipient rats. Survival rates were compared between rats transplanted or not with hepatocytes., Results: In groups not transplanted with viable hepatocytes, 73% to 93% of rats died after 95% liver resection. The mortality rate was reduced to 39% in rats transplanted with allogeneic hepatocytes and 36% in rats transplanted with xenogeneic hepatocytes. The bioartificial liver could be removed 1 month after transplantation, when regeneration of the native liver was complete. Allogeneic and xenogeneic hepatocytes remained viable., Conclusions: The implantable bioartificial liver was able to prevent death in this model of acute liver failure. This could be an important step toward clinical application of the method.

The general aim is to prepare a bioartificial liver to treat acute hepatic failure using allo- and xenogeneic hepatocytes, immunoprotected by macroencapsulation and transplanted into the peritoneal cavity. The goal of this study was to prepare a large amount of isolated porcine hepatocytes, to encapsulate them within biocompatible membranes for transplant in allo- and xenogeneic combinations and to examine the viability and functionality of the cells 6 weeks later. Hepatocyte isolation was performed in 12 kg pigs (n = 15) by dissociation of the liver with collagenase D (1 g) without oxygenation. Encapsulation of the hepatocyte suspension (10(7)/mL) was performed in hydrogel membranes AN69; hollow fibers (2 m x 0.8 mm) and flaskes (1.8 cm), and transplanted to Yucatan pigs (n = 4) and Lewis rats (n = 12). Six weeks later, they were removed to study the cell viability by histological examination, and the production of albumin by immunonephelometry. The rate of isolated hepatocytes was 38 +/- 5 x 10(9)/mL by liver of pig and the mean viability was 93 +/- 2%. Six weeks after transplantation, hepatocytes were viable, organized in lobules, and showed conserved albumin production. The same results were observed for allogenic and xenogeneic combinations. In conclusion, this method of liver dissociation allowed for preparation of a large amount of isolated hepatocytes from a single pig liver, theoretically sufficient to treat a patient with acute liver failure. Hydrogel membranes were well tolerated and allowed immunoprotection without immunosuppression. Transplanted hepatocytes remained functional. This work is an important step in progress toward clinical application.

The aim of this study was to develop in rats, a model of acute hepatic failure that mimics human fulminant hepatitis and to study the effect on survival of transplantation of isolated hepatocytes. This study showed that the mortality after 90% hepatectomy was reduced by the sole correction of hypoglycemia. On the other hand, 95% hepatectomy appeared as a reliable and reproductable model, associated with a period of hepatic coma, a deep disorder of coagulation, and a high mortality rate (> 80%) despite correction of hypoglycemia. In this model of acute hepatic failure, intraperitoneal transplantation of encapsulated isolated hepatocytes significantly reduced the mortality rate from 80% in the control groups to 31% in the transplanted group. A complete regeneration of the native liver was observed in all rats surviving. All animals survived after explantation of the hollow fibers. Well preserved hepatocytes were observed in hollow fibers two months after transplantation.

A new high-water-content (83%) and highly permeable anionic polyelectrolyte hydrogel was obtained by phase inversion of a polymer solution containing 6% polyacrylonitrile-sodium methallylsulphonate, 91% dimethylsulphoxide and 3% physiological saline solution. Hydrogel-based hollow fibres (HFs) were fabricated with a co-extrusion apparatus in collaboration with Hospal (France). HFs have an internal diameter of 800 microns and a wall thickness of 100 microns. Experimental results demonstrated that hydrogel-based HFs were permeable to albumin (mol. wt 69,000) and human immunoglobulin G (150,000), but were impermeable to immunoglobulins A (170,000) and M (900,000) after 24 h of diffusion. In vitro, the viability of isolated rat hepatocytes injected into the HFs was 64 +/- 6% after 10 d versus 30 +/- 5% for hepatocytes cultured in Petri dishes (P = 0.0001). Under these conditions, the amount of albumin released by encapsulated hepatocytes was 12 +/- 3 micrograms/24 h/10(6) cells at day 10, whereas at that time no albumin was released by hepatocytes cultured in Petri dishes. In vivo, histological study of hydrogel HFs implanted up to 6 wk in the peritoneum of rats revealed a low inflammatory tissue reaction without giant multinucleate cells in the foreign tissue, which decreased after the third week. The survival rate of encapsulated hepatocytes was over 85% 45 d after transplantation in the peritoneum of syngeneic Lewis rats. Therefore, this hydrogel demonstrates highly favourable properties for encapsulation of hepatocytes with regard to its biocompatibility, permeability and ability to maintain hepatocytes in a functional state for prolonged periods.

Unlabelled: Hepatocyte transplantation could be an alternative to whole liver transplantation. Allogeneic hepatocytes are rejected if transplanted without immunosuppression. The aim of this study was to transplant allogeneic hepatocytes in the peritoneum and to protect them from rejection by encapsulation in a new semi-permeable membrane., Methods: rats hepatocytes were encapsulated in hydrogel-based hollow fibers, obtained from AN69 polymer, before being transplanted into the peritoneum of rats. Outcome of allogeneic hepatocytes encapsuled in hollow fibers was compared to that of free allogeneic hepatocytes. Cell viability was assessed by erythrosine exclusion, morphology by electronic microscopy and function by albumin release., Results: Up to 90 days, viability of allogeneic hepatocytes in hollow fibers was above 80%. The morphology remained normal at electronic microscopy. Albumin release was 16.5 +/- 0.3 (day 15), 14.2 +/- 2.0 (day 30), 8.8 +/- 0.1 (day 60) and 11.4 +/- 0.3 mg/24h/10(6) hepatocytes (day 90). Free hepatocytes did not survive at day 15., Conclusion: Viability and function of encapsulated allogeneic hepatocytes were maintained up to 90 days after transplantation, without immunosuppression.

Hepatocytes normally express few major histocompatibility complex (MHC) class I and no MHC class II molecules, a phenomenon which could explain their low immunogenicity. However, in pathological situations, such as allograft rejection and cholestasis, hepatocytes strongly express MHC class I molecules and their immunogenicity could be different. The aim of this study was to assess the role of MHC expression on the immunogenicity of hepatocytes in vivo. Hepatocytes were obtained from normal and cholestatic DA rats by whole-liver perfusion with EDTA. Cholestasis was induced by ligation-section of the common bile duct. MHC expression on hepatocytes was assessed by cytofluorimetry after labelling with monoclonal antibodies against MHC class I and class II antigens. The percentage of hepatocytes expressing MHC class I was 9.8 +/- 2.2% in normal rats and 77.2 +/- 3.3% in cholestatic rats (P = 2 x 10(-4)); MHC class II expression was present on 1 +/- 0.5% of normal hepatocytes and 0.4% +/- 0.1% of cholestatic hepatocytes (P > 0.05). Lewis rats received a DA or Wistar-Furth heart allograft 7 days after intravenous injection of 2 x 10(7) hepatocytes from normal or cholestatic DA rats. The DA heart allograft was rejected in 6.3 +/- 0.4 days in Lewis controls, 8.8 +/- 1.1 days (N.S.) in Lewis recipients that received normal DA hepatocytes and 17.6 +/- 3.0 days (P = 2 x 10(-4)) in Lewis recipients that received hepatocytes from cholestatic DA rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this work was to evaluate the effect of intrasplenic hepatocellular transplantation on hepatic encephalopathy in an experimental model of chronic liver failure induced by end-to-side portacaval shunt in the rat. Inbred male Wistar Furth rats were divided into three groups: rats subjected to portacaval shunt (n = 10), rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10) and sham-operated rats (n = 10). Behavior tests were performed in a blind fashion at 3 wk, at 2 mo and at 3 mo after surgery. Spontaneous activity and nose-poke exploration by individual rats were studied in automated open field boxes equipped with infrared cells. Each cell beam interruption was automatically recorded on a microcomputer and transformed into a score index (counts/hour). Plasma levels of amino acids, ammonia and total biliary acids were measured. Portacaval shunt rats showed reduced spontaneous activity and nose-poke exploration scores. Intrasplenic hepatocellular transplantation significantly increased spontaneous activity after 2 mo and improved nose-poke exploration after 3 wk. At 3 mo, spontaneous activity and nose-poke exploration in portacaval shunt/intrasplenic hepatocellular transplantation rats were not significantly different from those of sham rats. Increases in plasma ammonia levels after portacaval shunt were not corrected. Amino acid imbalance and bile acid concentration in plasma were partially corrected by intrasplenic hepatocellular transplantation. These data show that intrasplenic hepatocellular transplantation can correct the neurological symptoms of hepatic encephalopathy in an experimental model of chronic liver failure and suggest that intrasplenic hepatocellular transplantation might be of therapeutic interest in chronic liver failure.

The aim of this work was to assess the effect of intrasplenic liver cell transplantation (ILCT) on hepatic insufficiency induced by a terminolateral portocaval shunt (PCS) in rats. Thirty syngenic Wistar Furth rats were divided up into three groups: (a) rats with PCS (n = 10); (b) rats with PCS then ILCT of 10(7) liver cells isolated from the livers of syngenic rats (n = 10); (c) operated control rats (n = 10). Double-blind behavior tests were carried out two weeks, two months and six months after surgery. The spontaneous motor activity and the exploring activity of each rat were studied in automated cages fitted with infrared diodes. Each interruption of the infrared beam was automatically recorded by a computer and converted into an activity score (number/hour). The spontaneous motor activity and the exploring activity were poor in the rats with PCS. The ILCT significantly increased the spontaneous motor activity and the exploring activity 2 months and 3 weeks after transplantation, respectively. Three months after transplantation, the spontaneous motor activity and the exploring activity in the PCS/ILCT group were not significantly different from those of the control rats. This study shows that ILCT can correct the neurological signs of hepatic encephalopathy in an experimental model of chronic hepatic insufficiency, and suggests that ILCT may produce therapeutic benefits in chronic hepatic insufficiency.