Inherited Liver Diseases Research Articles

Activity of the Bile Salt Export Pump (ABCB11) Is Critically Dependent on Canalicular Membrane Cholesterol Content

Mutations in ATP8B1 cause severe inherited liver disease. The disease is characterized by impaired biliary bile salt excretion (cholestasis), but the mechanism whereby impaired ATP8B1 function results in cholestasis is poorly understood. ATP8B1 is a type 4 P-type ATPase and is a flippase for phosphatidylserine. Atp8b1-deficient mice display a dramatic increase in the biliary extraction of cholesterol from the canalicular (apical) membrane of the hepatocyte. Here we studied the hypothesis that disproportionate cholesterol extraction from the canalicular membrane impairs the activity of the bile salt transporter, ABCB11, and as a consequence causes cholestasis. Using single pass liver perfusions, we show that not only ABCB11-mediated transport but also Abcc2-mediated transport were reduced at least 4-fold in Atp8b1 deficiency. We show that canalicular membranes of cholestatic Atp8b1-deficient mice have a dramatically reduced cholesterol to phospholipid ratio, i.e. 0.75 +/- 0.24 versus 2.03 +/- 0.71 for wild type. In vitro depletion of cholesterol from mouse liver plasma membranes using methyl-beta-cyclodextrin demonstrated a near linear relation between cholesterol content of the membranes and ATP-dependent taurocholate transport. Abcc2-mediated transport activity was not affected up to 30% of membrane cholesterol depletion but declined to negligible levels at 70% of membrane cholesterol depletion. These effects were reversible as cholesterol repletion of the liver membranes completely restored Abcb11- and Abcc2-mediated transport. Our data demonstrate that membrane cholesterol content is a critical determinant of ABCB11/ABCC2 transport activity, provide an explanation for the etiology of ATP8B1 disease, and suggest a novel mechanism protecting the canalicular membrane against luminal bile salt overload.

A new surgical approach to improve gene transfer in liver using lentiviral vectors

Purpose Metabolic inherited liver diseases are attractive targets for gene therapy. Recombinant lentiviruses are very powerful viral vectors able to infect nonmitotic cells. We wanted to develop a new surgical approach to improve gene transfer in adult liver using low viral doses. Materials and methods Adult rats were injected with 2.108 infectious particles of lentiviral vectors encoding the green fluorescent protein marker gene under control of a liver-specific promoter transthyretin. In the control group (n = 5), gene delivery was performed by inflow intraportal injection. In the surgical group (n = 5), liver was completely excluded from systemic circulation before viral injection in infrahepatic vena cava with high pressure. Results At day 9, transduction efficiency was 14.35% in the surgical group 3 and 0.39% in the control group ( P = .016). At month 2, the number of transduced hepatocytes decreased in the most part of rats, except in half of rats in the surgical group. Antibodies against green fluorescent protein were detected in all rats at month 2, except one in the surgical group. Conclusions We developed a new surgical approach allowing an efficient transduction of hepatocytes in adult rats using lentivirus at low viral doses. We have now to control the immune response to permit long-term expression of transgene.

Bone marrow transplantation results in donor-derived hepatocytes in an animal model of inherited cholestatic liver disease

Cell transplantation is a potential therapy for acquired or inherited liver diseases. Donor-derived hepatocytes (DDH) have been found in humans and mice after bone marrow transplantation (BMT) but with highly variable frequencies in different disease models. To test the effect of liver repopulation after BMT in inherited cholestatic liver diseases, spgp (sister of P-glycoprotein, or bile salt export pump, abcb11) knockout mice, a model for human progressive intrahepatic cholestasis type 2 with defects in excreting bile salts across the hepatocyte canalicular membrane, were transplanted with bone marrow cells from enhanced green fluorescent protein (EGFP) transgenic donor mice after lethal irradiation. One to 6 months later, scattered EGFP-positive DDHs with positive spgp staining were observed in the liver. These hepatocytes had been incorporated into hepatic plates and stained positively with hepatocyte-specific marker albumin. RT-PCR for the spgp gene revealed positive expression in the liver of sgsp knockout mice that had received the transplant. Bile acid analysis of bile samples showed that these mice also had higher levels of total biliary bile acid and taurocholic acid concentration than knockout mice without transplantation, indicating that BMT partially improved biliary bile acid secretion. Our results indicate that bone marrow cells could serve as a potential source for restoration of hepatic functions in chronic metabolic liver disease.

Therapeutic Potential of Adult Bone Marrow Stem Cells in Liver Disease and Delivery Approaches

Hematopoietic stem cells (HSCs) and mesenchymal stem cell (MSCs) are two main subtypes of bone marrow stem cells. Extensive studies have been carried out to investigate the therapeutic potential of BMSCs in liver disease. A number of animal and human studies demonstrated that either HSCs or MSCs could be applied to therapeutic purposes in certain liver diseases. The diseased liver may recruit migratory stem cells, particularly from the bone marrow, to generate hepatocyte-like cells either by transdifferentiation or cell fusion. Transplantation of BMSCs has therapeutic effects of restoration of liver mass and function, alleviation of fibrosis and correction of inherited liver diseases. There are still controversial results over the potential effects of BMSCs on liver diseases, and some of the discrepancies are thought to be lied in the differences of experimental protocols, differences in individual research laboratory, and the uncertainties of the techniques employed. Several potential approaches for BMSCs delivery in liver diseases have been proposed in animal studies and human trials. BMSCs can be delivered via intraportal vein, systemic infusion, intraperitoneal, intrahepatic, intrasplenic. The optimal stem cells delivery should be easy to perform, less invasive and traumatic, minimum side effects, and with high cells survival rate. In this review, we focus on the up-to-date evidence of therapeutic effects of BMSCs on liver disease, the characteristics of various delivery approaches, and the considerations for future studies.

Inherited metabolic disease of the liver

Elucidation of metabolic pathways for copper and iron improved our understanding of Wilson disease and genetic hemochromatosis. Some inherited liver diseases are now characterized by protein-folding mutations, including Gaucher disease, cystic fibrosis and ZZ alpha1-antitrypsin deficiency. Studies now focus on associations between glycogen storage disease, hepatic adenoma formation and transformation to hepatocellular carcinoma. Continued progress in the study of the diagnosis, natural history and treatment of inherited liver diseases is the subject of this review. Further understanding of metabolic pathways for iron and copper have led to a search for factors that modify phenotypic expression of Wilson disease and genetic hemochromatosis. Hepcidin plays a key role in modulating iron uptake in iron-overload disorders and new studies elucidate hepcidin regulation. For glycogen storage diseases, studies on the natural history and hepatocellular transformation necessitate tumor surveillance and possible early transplantation. A better understanding of genetic and nongenetic modifiers in ZZ alpha1-antitrypsin deficiency and other disorders of protein misfolding will improve our ability to manage these patients. Recent discoveries in iron, copper and glycogen metabolism advance our ability to diagnose and treat inherited metabolic diseases of the liver. Some of these important findings are detailed in this review.

Hepatocyte transplantation: a step forward

Purpose of review Hepatocyte transplantation has been evaluated for more than 20 years as an alternative to liver transplantation for definitive treatment of inherited liver diseases and as a metabolic support during liver failure. Although this technique is potentially useful in the treatment of a diverse spectrum of human liver diseases, several issues remain to be solved. Recent findings Potential cell sources for hepatocyte transplantation have been identified: porcine hepatocytes, fetal hepatocytes, stem cell-derived hepatocytes and immortalized hepatocytes. Moreover, innovations aimed at improving hepatocyte delivery, survival and function have recently opened up the field of liver tissue engineering, which may improve our clinical experience of hepatocyte transplantation. Summary We review historical evidence for hepatocyte transplantation as well as leading works in the field of clinical applications, different hepatic cell sources and liver tissue engineering, with special attention being paid to the conversion of laboratory work into clinical practice. We hope that this review will help in the development of a routinely effective therapy for liver diseases.

Prevention of hepatocyte allograft rejection in rats by transferring adenoviral early region 3 genes into donor cells

Hepatocyte transplantation is being evaluated as an alternative to liver transplantation for metabolic support during liver failure and for definitive treatment of inherited liver diseases. However, as with liver transplantation, transplantation of allogeneic hepatocytes requires prolonged immunosuppression with its associated untoward effects. Therefore, we explored strategies for the genetic modification of donor hepatocytes that could eliminate allograft rejection, obviating the need for immunosuppression. Products of early region 3 (AdE3) of the adenoviral genome are known to protect infected cells from immune recognition and destruction. In the present study we showed that immortalized rat hepatocytes that had been stably transduced with AdE3 before transplantation into fully MHC-mismatched rats are protected from allograft rejection. Quantitative real-time PCR analysis showed that a similar number of engrafted AdE3-transfected hepatocytes had survived in syngeneic and allogeneic recipients. AdE3 expression did not reduce expression of MHC class I on the surfaces of donor hepatocytes. Consistent with this, the in vivo cytotoxic cell-mediated alloresponse was attenuated but not abolished in recipients of AdE3-transfected allogeneic hepatocytes. In contrast, graft survival correlated with a marked reduction in cell-surface localization of Fas receptor in the transplanted cells and inhibition of Fas-mediated apoptosis, which are related to the antiapoptotic functions of the AdE3 proteins. AdE3 gene products prevent hepatocyte allograft rejection mainly by protecting the cells from the effector limb of the host immune response and could be used as a tool to facilitate allogeneic hepatocyte transplantation.

Effect of living donor liver transplantation on outcome of children with inherited liver disease and hepatocellular carcinoma

We described six children with heritable liver disease and hepatocellular carcinoma treated with living-related liver transplantation. Underlying liver diseases were type-1 tyrosinemia (three patients), progressive familial intrahepatic cholestasis type II (two patients), and Wilson's disease (one patient). Two of the tumors were found incidentally during liver transplantation. Number of nodules was 12, 15, 3, 2, and 1 (in two patients). Three patients were treated with chemotherapy before the procedure. Chemotherapy was not given to any patient after liver transplantation. The mean follow-up was 17.7 +/- 6 months (range: 7-24). All patients are tumor recurrence free. Both graft and patient survival rates are 100% at a median of 18.5 months follow-up. Physicians in charge of treating children with heritable liver disease should screen them periodically for the development of hepatocellular carcinoma. Liver transplantation may offer these children better survival rates.

175: A New Paradigm for Tissue Regeneration: Preparative Irradiation for Cell-Based Therapies as an Alternative to Organ Transplantation

Orthotopic liver transplantation (OLT) is the only curative therapy for patients with terminal liver diseases. A significant number of patients fail to get OLT due to shortage of donor livers. Hepatocyte transplantation (HT) is being explored as a therapeutic alternative to OLT for the treatment of end-stage liver diseases. However, the number of cells that can be safely transplanted into the liver and the inability of the transplanted hepatocytes to proliferate in the host liver currently limit the clinical application of this procedure. We hypothesized that a preparative regimen of hepatic irradiation (HIR), in combination with hepatic mitotic stimuli, would suppress host hepatocellular proliferation and induce post-mitotic death, thus ”making room“ for transplanted, nonirradiated hepatocytes to proliferate preferentially and repopulate the irradiated host liver. C57Bl/6 mice received HIR (0-50 Gy) to the anterior liver lobes after exposing the liver by laparotomy and shielding the posterior liver lobes and other abdominal organs, followed by an injection of a recombinant adenoviral vector expressing human hepatocyte growth factor (Ad-HGF, 5-10 x 1010 particles). One day after HIR, the mice received intrasplenic HT with 0.8-1×106 primary hepatocytes, freshly isolated from congenic, beta-galactosidase-expressing transgenic (ROSA)26 C57Bl/6 mice. Control mice received Ad-HGF alone, or HIR + Ad-LacZ with HT. Animals were sacrificed at 7 days, 1, 2, 4 and 6 months after HT and the liver sections were examined for beta-galactosidase-expressing donor cells after histochemical and hematoxylin-eosin staining. Compared to unirradiated controls, HIR augmented the engraftment of transplanted hepatocytes, with an increase in the number of integrated donor hepatocytes into liver cords by 7 days (p<0.01). There was progressive repopulation of the irradiated anterior liver lobes by transplanted hepatocytes with near-total (80-95%) replacement of the host hepatocytes by the donor cells within 6 months. In contrast, the unirradiated posterior and caudate liver lobes exhibited occasional presence of beta-galactosidase-expressing donor hepatocytes, indicating engraftment without repopulation. H&E staining demonstrated normal liver architecture without any evidence of hepatic radiation injury. Liver function tests of the transplanted animals were normal. Preparative irradiation is routinely used for bone marrow transplantation, but we are the first to use HIR to facilitate the engraftment and host liver repopulation by donor cells after HT in rodent models. HIR significantly enhanced the integration of transplanted cells in the host liver. Furthermore, partial liver irradiation enabled selective repopulation of a liver lobe by the donor cells, thereby increasing the safety of our regimen. Clinically, preparative HIR could be safely administered using radiosurgery or 3-D conformal RT and combined with systemic administration of hepatic growth factors to facilitate HT for 1) the treatment of inherited liver diseases, 2) liver failure, 3) ex vivo hepatic gene therapy, 4) rescuing patients with liver cancer, following chemo-radiation therapy, and 5) expanding human hepatocytes in animal liver for generating animal models for human-specific infections.

Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport

Progressive familial intrahepatic cholestasis type 1 (PFIC1, Byler disease, OMIM 211600) is a severe inherited liver disease caused by mutations in ATP8B1. ATP8B1 is a member of the type 4 subfamily of P-type ATPases, which are phospholipid flippases. PFIC1 patients generally develop end-stage liver disease before the second decade of life. The disease is characterized by impaired biliary bile salt excretion, but the mechanism whereby impaired ATP8B1 function results in cholestasis is unclear. In a mouse model for PFIC1, we observed decreased resistance of the hepatocanalicular membrane to hydrophobic bile salts as evidenced by enhanced biliary recovery of phosphatidylserine, cholesterol, and ectoenzymes. In liver specimens from PFIC1 patients, but not in those from control subjects, ectoenzyme expression at the canalicular membrane was markedly deficient. In isolated mouse livers Atp8b1 deficiency impaired the transport of hydrophobic bile salts into bile. In conclusion, our study shows that Atp8b1 deficiency causes loss of canalicular phospholipid membrane asymmetry that in turn renders the canalicular membrane less resistant toward hydrophobic bile salts. The loss of phospholipid asymmetry may subsequently impair bile salt transport and cause cholestasis.

Mixed hematopoietic molecular chimerism results in permanent transgene expression from retrovirally transduced hepatocytes in mice

Cytotoxic immune elimination of transduced hepatocytes may limit gene therapy for inherited liver diseases. Using beta-galactosidase as a marker gene, we studied whether creation of mixed beta-galactosidase molecular hematopoietic chimerism could induce tolerance to beta-galactosidase-transduced hepatocytes. Molecular hematopoietic chimerism was established in irradiated recipient mice by transplantation of either a mixture of wild-type and beta-galactosidase-transgenic bone marrow or autologous bone marrow stem cells that were transduced with beta-galactosidase lentiviral vectors. After transplantation, mice were hepatectomized and injected with beta-galactosidase recombinant retroviruses to transduce regenerating hepatocytes. We monitored the presence of beta-galactosidase-expressing hepatocytes as well as the appearance of anti-beta-galactosidase antibodies during the time. In control animals, anti-beta-galactosidase antibodies and cytotoxic T-lymphocyte (CTL) response developed as early as 3 weeks after gene transfer. Transduced hepatocytes disappeared concomitantly. In bone marrow transplanted mice, tolerance could be observed in a significant proportion of animals. Tolerance resulted in permanent liver transgene expression and was absent unless a chimerism above 1% was achieved, demonstrating a threshold effect. Creation of a molecular hematopoietic chimerism can result in transgene tolerance and evade immune rejection of retrovirally transduced hepatocytes. This strategy may be useful for hepatic inherited diseases in which the transgene product behaves as a non-self protein.

Molecular basis of cholestatic diseases of surgical interest

Cholestasis constitutes one of the most common and severe manifestations of acquired or inherited liver disease. When manifest in early infancy, it is often life-threatening and usually requires surgical management. In many cases, liver transplantation is the only effective therapy. Extensive knowledge about the molecular mechanisms underlying several pediatric cholestatic disorders has been gained in recent years from studies in both experimental models and clinical forms. In this review, we focus on recent contributions to the knowledge of molecular basis of main pediatric cholestatic disorders, such as biliary atresia, Alagille syndrome, and familial intrahepatic cholestasis. For some of them, putative targets of therapeutic interest, such as interferon-gamma and Farnesoid X receptor, have been proposed.

Stem-cell therapy for hepatobiliary pancreatic disease

The transplantation of pancreatic beta cells or hepatocytes represents a potential therapeutic approach for type I diabetes and inherited liver diseases, respectively. Furthermore, acquired liver diseases, particularly acute hepatic failure due to toxic or viral injury, have been treated in limited clinical trials with fetal and adult hepatocytes. However, a major limitation is the insufficient amount of beta cells and hepatocytes available for grafts. Alternative sources of these cells have yet to be determined. During the past few years, progress has been made in the development of new strategies to produce mature beta cells and hepatocytes. In this review, we outline the current state of scientific understanding and controversy regarding the properties of embryonic and adult stem cells in the field of hepatobiliary and pancreatic diseases. Our objective is to provide a framework of understanding for the challenges behind translating fundamental stem cell biology into clinical therapies.

597. Liver Tissue Engineering at Extra-Hepatic Site: A Novel Therapeutic Approach toward Hemophilia

Introduction: In response to the demand to establish new-generation treatment approaches for the inherited liver diseases including hemophilia, cell-based therapies using hepatocyte transplantation and liver tissue engineering have been highlighted. We have recently established technique to engineer stable liver tissues at extra-hepatic site (under the kidney capsule) with the potential for liver regeneration by transplanting mature hepatocytes with extracellular matrix components. Since the liver is a major contributory organ for clotting factor VIII production, we hypothesized that engineering a liver tissue in vivo would be a therapeutic option for hemophilia. The present study was aimed at assessing the therapeutic efficacy of the liver tissue engineering on the mouse model of hemophilia A.

Kupffer Cells and Not Liver Sinusoidal Endothelial Cells Prevent Lentiviral Transduction of Hepatocytes

Lentiviral vectors can stably transduce dividing and nondividing cells in vivo and are best suited to long-term correction of inherited liver diseases. Intraportal administration of lentiviral vectors expressing green fluorescent protein (Lenti-GFP) in mice resulted in a higher transduction of nonparenchymal cells than hepatocytes (7.32 +/- 3.66% vs 0.22 +/- 0.08%, respectively). Therefore, various treatments were explored to increase lentiviral transduction of hepatocytes. Lenti-GFP was injected into the common bile duct, which led to transduction of biliary epithelium and hepatocytes at low efficiency. Transient removal of the sinusoidal endothelial cell layer by cyclophosphamide to increase accessibility to hepatocytes did not improve hepatocyte transduction (0.42 +/- 0.36%). Inhibition of Kupffer cell function by gadolinium chloride led to a significant decrease in GFP-positive nonparenchymal cells (2.15 +/- 3.14%) and a sevenfold increase in GFP-positive hepatocytes compared to nonpretreated mice (1.48 +/- 2.01%). These findings suggest that sinusoidal endothelial cells do not significantly limit lentiviral transduction of hepatocytes, while Kupffer cells sequester lentiviral particles thereby preventing hepatocyte transduction. Therefore, the use of agents that inhibit Kupffer cell function may be important for lentiviral vector treatment of liver disease.

Cholestasis Familiaris Groelandica: an epidemiological, clinical and genetic study

Objectives. Accumulation of Cholestasis Familiaris Groenlandica (CFG) or progressive familial intrahepatic cholestasis type 1 (PFIC1) occurs in indigenous Inuit families in Greenland. It is an autosomal recessive inherited liver disease. From early childhood the children suffer from failure to thrive, jaundice, pruritus and enlarged liver. Affected persons generally die very young. Study design. Patients’ information has been collected from the Greenlandic death register and hospital records. Methods. Detailed genealogy including clinical description and examination if possible. Interviews of parents and relatives, linkage and DNA analysis of the probands and the closest relatives have been studied. Results. 46 affected cases from a highly inbred population have been diagnosed since 1943. The disease is caused by a missense mutation in the FIC1 gene ATP8B1, chromosome 18q21. Six affected children are alive aged 1–21 years. Among the tested relatives 220 are heterozygote. One prenatal diagnosis has been performed. Conclusion. The mutation causing Cholestasis Familiaris Groenlandica is widespread in Greenland, but accumulation is seen in certain areas. The disease is burdensome for the child, the parents and the Greenlandic society. Genetic counselling and carrier screening are strongly recommended.

Indel in the FIC1/ATP8B1 gene-a novel rare type of mutation associated with benign recurrent intrahepatic cholestasis.

Benign recurrent intrahepatic cholestasis (BRIC) is a rare inherited liver disease characterized by recurrent attacks of severe cholestasis with no progression to end stage liver disease. Patients have jaundice, however, serum gamma-glutamyltransferase and cholesterol levels remain within the normal range during the attacks. Three mutations in the familial intrahepatic cholestasis 1 (ATP8B1) gene encoding a P-type ATPase have been reported so far in patients with the autosomal recessive form of BRIC. A novel rare type insertion-deletion mutation, also called indel, was found in exon 24 of ATP8B1 in our patient together with a known missense mutation 1982T>C in exon 17. The mechanism of the indel formation is proposed and impact of the indel mutation on the function of ATP8B1 protein is discussed.

Long term transgene expression by hepatocytes transduced with retroviral vectors requires induction of immune tolerance to the transgene

Background/Aims Gene therapy for inherited liver diseases requires permanent expression of the therapeutic gene. However, in vivo liver transduction with retroviral vectors triggers an immune elimination of transduced hepatocytes. Here we investigated whether immune response could be prevented by treatment with compounds known to induce tolerance in organ transplantation: CTLA4Ig and LF-15-0195. Methods CTLA4Ig was administered either via i.p. injection of the drug or by i.m. injection of recombinant adenoviruses encoding CTLA4Ig. LF-15-0195 was administered i.p. All animals were subjected to partial hepatectomy and received β-galactosidase retroviral vectors intravenously. Appearance of anti-β-galactosidase antibodies was monitored and the number of positive hepatocytes was assessed at day 7 and at sacrifice. Results No β-galactosidase antibodies were detected as long as CTLA4Ig was detectable in serum. Short-term treatment with CTLA4Ig induced tolerance in a significant proportion of animals only at high dose (1 mg/kg). Administration of CTLA4Ig adenovectors resulted in prolonged secretion of CTLA4Ig and permanent absence of anti-β-galactosidase antibodies. LF-15-0915 administration achieved tolerance in some animals. Conclusions In conclusion, manipulation of the immune system at the time of virus delivery using clinically relevant tolerance-inducing protocols is a promising approach to achieve long term expression after retrovirus-mediated gene transfer to the liver.

329. Long-Term Transgene Expression by Hepatocytes Transduced with Retroviral Vectors Requires Induction of Immune Tolerance to the Transgene

Gene therapy of inherited liver disease requires long-term expression of the therapeutic transgene. Recombinant retroviruses are able to integrate into the transduced cell's genome and hence are convenient tools to achieve permanent transgene expression. However, we previously demonstrated that a cytotoxic immune response directed against the transgene product eliminated hepatocytes transduced in vivo using recombinant retroviruses.

726. Ultrasound Enhances Retroviral-Mediated Gene Transfer

Lentiviral vectors transduce quiescent cells, exhibit stable genomic integration, and provide long-term expression. These properties make them ideal gene delivery vehicles for the treatment of inherited liver diseases. In vivo studies using vesicular stomatits virus g protein (VSVg) pseudotyped lentiviral vectors have low transduction efficiency in hepatocytes. The broad tropism of VSVg pseudotyped lentiviral vectors present problems for in vivo studies where gene transfer should be restricted to a specific tissue and the transduction of professional antigen presenting cells (APC) may accelerate the clearance of transduced cells by the immune system. Past attempts to redirect viral tropism through modification of the viral envelope protein have proven to be unsuccessful. To improve the transduction efficiency of hepatocytes we investigated the targeting of lentiviral vectors to the liver by using an alternative envelope protein. We needed to find an envelope protein that gives high titer virus that can be concentrated without significant loss of titer. There are several viral envelope proteins reported in literature to give a liver tropism, but to date none are capable of producing the high titer, stable virus particles needed for in vivo studies.