Putative human liver progenitor cells in explanted liver.

BACKGROUND/AIMS: Hepatocyte progenitors have frequently been cultured from rodents but reports from human liver are rare. METHODS: Non-parenchymal cell fraction isolated from 19 explant livers (removed at orthotopic liver transplantation for acute or chronic liver disease) and histologically normal human liver was cultured. RESULTS: Proliferating epithelioid colonies were identifiable after 2-3 weeks culture as a very rare event (<1 per million cells plated) expressing mRNAs and protein antigens of mixed hepatocytic/biliary phenotype. Colony survival could be prolonged by transduction of the catalytic sub-unit of telomerase. Hepatocyte growth factor, epidermal growth factor and oncostatin M did not further enhance hepatocytic differentiation. The expression of markers associated with hepatocyte precursor status was investigated by flow cytometry. Cells expressing the stem cell-associated markers CD133 and CD117 were identified at low frequency. The proportion of cells expressing the integrin CD49f was higher in diseased liver than in normal liver, but the proportion expressing the hepatocyte growth factor receptor c-met was lower. Successful enrichment of plated populations for progenitors was not achieved. CONCLUSION: Although there is clear histological evidence of hepatocyte precursors in human explant livers, predictable culture of such cells with differentiation toward mature hepatocyte phenotype remains elusive.

The role of glycine in hepatic ischemia-reperfusion injury.

Glycine is a non-essential amino acid which is cheap, easily available and relatively non-toxic. It is composed of a single carbon attached to an amino and a carboxyl group, with a molecular weight of 75. It is involved in the production of bile, nucleic acids, porphyrins and creatine phosphate. It is part of the normal human diet and is used clinically, as an irrigant solution in urological and gynaecological procedures. Glycine has broad spectrum anti-inflammatory, cytoprotective and immunomodulatory properties whose therapeutic role has largely been un-investigated. Since the demonstration of its cytoprotective effect on hypoxic cultured renal tubule cells, further research has established its mechanism of anti-inflammatory action, which depends on stimulation of glycine sensitive chloride channel receptors on the cell membrane. The mechanism of non-specific cytoprotective effect which is present even in chloride and calcium free media is not clear. However glycine is currently being used experimentally, in human liver transplant recipients and has been shown to be beneficial in animal models of ischemia-reperfusion injury (IRI) in liver and several other organs. This review addresses the properties of glycine, its mechanism of action and its role in modulating IRI with special reference to the liver, with the aim of stimulating translational research into the potential role of glycine as a pharmaceutical agent.

IL-10 gene therapy is therapeutic for dextran sodium sulfate-induced murine colitis.

The transfer of genes encoding immunoregulatory proteins is a promising new strategy in the treatment of intestinal inflammation. Previous work has demonstrated that daily systemic interleukin (IL)-10 therapy is able to prevent disease onset in animal models of colitis but is not sufficient to treat established disease. This study investigates the therapeutic efficacy of an adenovirus encoding IL-10 (AdvmuIL-10) in the treatment of experimental colitis. Colitis was induced in BALB/c mice by the addition of dextran sodium sulfate to the drinking water for 7 days. A single systemic injection of AdvmuIL-10, empty cassette vector (Adv0), or saline vehicle was administered on day 4 after the onset of colitis. The addition of DSS to the drinking water led to an acute, dose-dependent colitis. A single injection of AdvmuIL-10 led to a marked reduction in both stool markers of inflammation (IL-1beta, IL-6, and TNFRII) and serum IL-6. Furthermore, the histological colitis score was significantly reduced in mice receiving AdvmuIL-10 compared to controls (4.9 +/- 1.1 Vs 9.1 +/- 1.2, respectively; P < 0.05). A single systemic injection of AdvmuIL-10 is therapeutic in mice with established DSS colitis. Gene therapy strategies using adenoviral vectors encoding IL-10 may prove to be a potent therapy for chronic inflammation of the colon such as Crohn's disease.

Characteristics of murine histidinaemia and its potential for genetic manipulation.

BACKGROUND: Histidinaemia is an autosomal recessive disorder affecting the hepatic enzyme histidine ammonia lyase (histidase) resulting in elevated plasma and urinary histidine and is prototypic of a series of hepatic cytosolic enzyme defects. AIMS: To characterise the physiology of murine histidinaemia with respect to histidine excretion and catabolism, and explore the potential for manipulating cellular and whole body histidase metabolism by gene transfer. MATERIALS AND METHODS: We studied his/his mice which have a G to A substitution in the gene encoding histidase, using both in vitro transduction of isolated hepatocytes by lipofection with wild-type histidase cDNA, and in vivo transduction of whole liver using a retroviral construct. RESULTS AND CONCLUSION: Histidase cDNA expression restored histidase activity in vivo and in vitro towards normal levels, demonstrated both at the cellular level and by whole body metabolic studies, establishing the potential of this model for the development of new gene therapeutic approaches.

Characterization of long-term survival of syngeneic hepatocytes in rat peritoneum.

Hepatocyte transplantation is a potential therapy for both acute and chronic hepatic insufficiency and also for treatment of inborn errors of metabolism affecting the liver. The peritoneum is one site for implantation and has several advantages: cells implanted there can be easily identified and observed, and it has a relatively large capacity. Long-term survival using "pure" hepatocytes in the peritoneum have been disappointing. We hypothesized that cotransplantation of hepatocytes with nonparenchymal cells would help maintain differentiated hepatocyte function. Rat liver cells transplanted intraperitoneally into August rats were sacrificed at 7 days, 1, 3, 6, 9, and 12 months and analyzed for presence, basal proliferation, and functionality of hepatocytes. To demonstrate that ectopic hepatocytes remained susceptible to exogenous growth factors affecting cell proliferation, rats 9 and 12 months after transplantation were stimulated with tri-iodothyronine and KGF. Hepatocytes were identified 7 days to >12 months, by H&E and immunohistochemically, as ectopic islands in the omental fat. Functionality was confirmed by glycogen deposition. Basal proliferation in 7-day rats was 28.0 +/- 10/1000 hepatocytes in ectopic islands (cf. 5.70 +/- 2.7/1000 in recipient liver). Proliferation in ectopic islands was greater than host liver. Growth factor-stimulated proliferation in ectopic islands induced a 70-fold increase in DNA synthesis. In conclusion, hepatocytes transplanted with nonparenchymal cells survive, proliferate, and function in the peritoneum of normal rats, and respond to exogenous growth stimuli. Their survival and proliferation in the presence of a normal functioning liver has implications for the potential use of the peritoneal site clinically for supplementation of liver function in metabolic disorders.

Local delivery of adenoviral vectors encoding murine interleukin 10 induces colonic interleukin 10 production and is therapeutic for murine colitis.

INTRODUCTION: Interleukin 10 knockout (IL-10-/-) mice spontaneously develop a Th1 T cell mediated colitis with many similarities to Crohn's disease. Daily injections of IL-10 are unable to induce remission in mice with established disease. In contrast, we have shown previously that intravenous administration of adenoviral vectors encoding IL-10 (AdvmuIL-10) induces hepatic IL-10 release and leads to long term disease suppression with profound systemic immunoregulatory changes. AIMS: To determine whether rectal delivery of AdvmuIL-10 induces localised colonic IL-10 expression without systemic immune suppression, and assess its therapeutic efficacy in IL-10-/- mice with established colitis. RESULTS: A single rectal infusion of 5 x 10(8) PFU AdvmuIL-10 to 10 week IL- 10-/- mice resulted in a median level of 27.3 pg/mg IL-10 in colonic homogenates harvested one week later. IL-10-/- mice with established colitis treated with an enema of 5 x 10(8) PFU AdvmuIL-10 entered clinical and histological remission whereas empty cassette adenovirus (Adv0) or phosphate buffered saline (PBS) treated mice developed progressive disease. After four weeks, the histological score of AdvmuIL-10 treated mice (4.4 (1.5)) was significantly lower than that of Adv0 (11.1 (1.1); p<0.001) and PBS (10.9 (1.0); p<0.01) treated controls. In addition, the stool concentration of IL-1beta over the four week experiment was significantly higher in mice treated with saline or Adv0 than in those treated with AdvmuIL-10 (p<0.01). CONCLUSION: Local AdvmuIL-10 therapy reverses colitis in IL-10-/- mice without the systemic effects seen after intravenous administration. Gene therapy strategies using adenoviral vectors encoding immunoregulatory cytokines may prove to be a potent approach to the treatment of chronic inflammatory diseases such as Crohn's disease.

Role of cell adhesion molecules in leukocyte recruitment in the liver and gut.

This article reviews the evidence that adhesion molecules are critical in leukocyte recirculation and pathogenesis of diseases affecting the closely related tissues of the liver and gut, which offer novel opportunities for treatment.

Local delivery of adenoviral vectors encoding murine interleukin 10 induces colonic interleukin 10 production and is therapeutic for murine colitis.

INTRODUCTION: Interleukin 10 knockout (IL-10-/-) mice spontaneously develop a Th1 T cell mediated colitis with many similarities to Crohn's disease. Daily injections of IL-10 are unable to induce remission in mice with established disease. In contrast, we have shown previously that intravenous administration of adenoviral vectors encoding IL-10 (AdvmuIL-10) induces hepatic IL-10 release and leads to long term disease suppression with profound systemic immunoregulatory changes. AIMS: To determine whether rectal delivery of AdvmuIL-10 induces localised colonic IL-10 expression without systemic immune suppression, and assess its therapeutic efficacy in IL-10-/- mice with established colitis. RESULTS: A single rectal infusion of 5 x 10(8) PFU AdvmuIL-10 to 10 week IL-10-/- mice resulted in a median level of 27.3 pg/mg IL-10 in colonic homogenates harvested one week later. IL-10-/- mice with established colitis treated with an enema of 5 x 10(8) PFU AdvmuIL-10 entered clinical and histological remission whereas empty cassette adenovirus (Adv0) or phosphate buffered saline (PBS) treated mice developed progressive disease. After four weeks, the histological score of AdvmuIL-10 treated mice (4.4 (1.5)) was significantly lower than that of Adv0 (11.1 (1.1); p<0.001) and PBS (10.9 (1.0); p<0.01) treated controls. In addition, the stool concentration of IL-1 beta over the four week experiment was significantly higher in mice treated with saline or Adv0 than in those treated with AdvmuIL-10 (p<0.01). CONCLUSION: Local AdvmuIL-10 therapy reverses colitis in IL-10-/- mice without the systemic effects seen after intravenous administration. Gene therapy strategies using adenoviral vectors encoding immunoregulatory cytokines may prove to be a potent approach to the treatment of chronic inflammatory diseases such as Crohn's disease.

Characterization of Long-Term Survival of Syngeneic Hepatocytes in Rat Peritoneum.

Hepatocyte transplantation is a potential therapy for both acute and chronic hepatic insufficiency and also for treatment of inborn errors of metabolism affecting the liver. The peritoneum is one site for implantation and has several advantages: cells implanted there can be easily identified and observed, and it has a relatively large capacity. Long-term survival using "pure" hepatocytes in the peritoneum have been disappointing. We hypothesized that cotransplantation of hepatocytes with nonparenchymal cells would help maintain differentiated hepatocyte function. Rat liver cells transplanted intraperitoneally into August rats were sacrificed at 7 days, 1, 3, 6, 9, and 12 months and analyzed for presence, basal proliferation, and functionality of hepatocytes. To demonstrate that ectopic hepatocytes remained susceptible to exogenous growth factors affecting cell proliferation, rats 9 and 12 months after transplantation were stimulated with tri-iodothyronine and KGF. Hepatocytes were identified 7 days to >12 months, by H&E and immunohistochemically, as ectopic islands in the omental fat. Functionality was confirmed by glycogen deposition. Basal proliferation in 7-day rats was 28.0 ± 10/1000 hepatocytes in ectopic islands (cf. 5.70 ± 2.7/1000 in recipient liver). Proliferation in ectopic islands was greater than host liver. Growth factor-stimulated proliferation in ectopic islands induced a 70-fold increase in DNA synthesis. In conclusion, hepatocytes transplanted with nonparenchymal cells survive, proliferate, and function in the peritoneum of normal rats, and respond to exogenous growth stimuli. Their survival and proliferation in the presence of a normal functioning liver has implications for the potential use of the peritoneal site clinically for supplementation of liver function in metabolic disorders.

Non-surgical treatment of hepatocellular carcinoma.

Primary hepatocellular cancer is a disease with a poor prognosis for which there is little consensus on treatment and a paucity of comparative trials. The coexistence of cancer with cirrhosis complicates treatment, and also confers a high risk for the development of further tumours. Surgery, either by hepatic resection or orthotopic liver transplantation, is only a feasible option in a minority of patients. This article surveys the non-surgical approaches to the treatment of hepatocellular cancers-local ablation techniques, arterial embolization with and without chemotherapy, conventional chemotherapy and hormonal modulation, and targeted and external irradiation.

Review article: the immunoregulatory cytokine interleukin-10--a therapy for Crohn's disease?

The gastrointestinal tract serves as a barrier between the host and the vast array of foreign antigens that are contained within its lumen. The mucosal immune system must balance two opposing functions: to mount an immune response to pathogens, whilst maintaining tolerance to antigens derived from commensal bacteria and food. This balance is regulated by both cellular interactions and the release of soluble mediators called cytokines. Diseases such as ulcerative colitis and Crohn's disease are characterized by alterations in the balance of pro-inflammatory and regulatory cytokines. Interleukin-10 is a regulatory cytokine which inhibits both antigen presentation and subsequent pro-inflammatory cytokine release. In addition, there is evidence that it promotes the formation of antigen-specific regulatory T-cell clones. The pivotal role played by interleukin-10 within the mucosal immune system is demonstrated both by the chronic ileocolitis that develops in gene-targeted interleukin-10 knock-out mice, and by its therapeutic efficacy in several animal models of colitis. However, trials of daily systemic interleukin-10 administration in patients with Crohn's disease have reported only a modest clinical response. Advances in the analysis of functional polymorphisms in the interleukin-10 gene may allow therapy to be targeted to patients who will respond. Finally, therapeutic strategies utilizing gene therapy may enhance mucosal delivery and increase therapeutic response.

Hormone replacement therapy may improve hepatic function in women with Turner's syndrome.

OBJECTIVE: Women with Turner's syndrome (TS) have recently been shown to be at an increased risk of developing chronic liver disease. There has been some concern that oestrogen replacement therapy may exacerbate hepatic dysfunction. The aim of this study was to assess hepatic function in women with TS and to determine the effect of oral oestradiol valerate on liver enzymes. DESIGN AND PATIENTS: A retrospective review of liver enzymes of 80 women with TS, followed by a prospective study looking at serum liver enzyme concentrations in 20 women with TS following 3 months on and off hormone replacement therapy (HRT) (oestradiol valerate, 2 mg/levonorgestril 75 microg). MEASUREMENTS: Liver enzymes (gamma glutamyl transferase, aspartate transaminase and alkaline phosphatase), albumin and bilirubin were measured on and off HRT. Viral hepatitis serology and liver autoantibodies were tested in patients with abnormal liver function. RESULTS: Thirty-five out of 80 women (44%) had elevated serum liver enzyme concentrations. Two women (2.5%) had a mildly raised serum bilirubin, but protein synthesis was normal in all subjects. HRT resulted in a significant fall in all liver enzymes (P < 0.05) but did not affect serum protein concentrations CONCLUSIONS: Women with Turner's syndrome often have elevated liver enzymes. Oestrogen/progestagen therapy using oestradiol valerate improves liver function in this group of patients. The mechanisms behind this are unclear.

The prevention and treatment of murine colitis using gene therapy with adenoviral vectors encoding IL-10.

IL-10-deficient (IL-10(-/-)) mice develop colitis with many similarities to Crohn's disease. Daily IL-10 injections have a short systemic half-life and are unable to induce complete remission in IL-10(-/-) mice with established disease. In this paper, we investigate the duration, potency, and immunogenicity of gene therapy using an adenoviral vector encoding murine IL-10 (AdvmuIL-10). A single systemic injection of AdvmuIL-10 was sufficient not only to prevent the onset of colitis for at least 10 wk but also to induce clinical and histological remission in mice with established disease. In addition, AdvmuIL-10 diminished the systemic manifestations of disease, including elevated acute-phase proteins, as well as the local consequences of inflammation such as raised stool IL-1beta concentrations. Both IL-10 protein and the effects of secreted IL-10 were detectable for 10 wk after AdvmuIL-10 injection. Furthermore, the immunoregulatory effect of a single AdvmuIL-10 injection was manifest both by a reduction in TNF-alpha, IFN-gamma, and RANTES release from stimulated splenocyte cultures, and also by a change in the proportion of CD45RB(high/low) lymphocytes in the spleen compared with control mice. The delivery of AdvmuIL-10 resulted in a significantly diminished host antiadenoviral response compared with control adenoviral vectors. Thus, gene therapy strategies using adenoviral vectors encoding immunoregulatory and antiinflammatory cytokines may prove to be a potent approach for the treatment of chronic inflammatory disease. Antiinflammatory cytokine expression protects against immune responses directed at gene vectors.

Tri-iodothyronine and a deleted form of hepatocyte growth factor act synergistically to enhance liver proliferation and enable in vivo retroviral gene transfer via the peripheral venous system.

Retroviral vectors integrate into the target cell genome in a stable manner and therefore offer the potential for permanent correction of the genetic diseases that affect the liver. These vectors, however, usually require cell division to occur in order to allow provirus entry into the nucleus. We have explored clinically acceptable methods to improve the efficiency of retroviral gene transfer to the liver, which avoid the need for liver damage. Tri-iodothyronine (T3) and recombinant hepatocyte growth factor have previously been used to induce hepatocyte proliferation in rat livers and allow in vivo retroviral gene transfer. We investigated the combined effects of these growth factors, with their differing mechanisms of action, on hepatocyte proliferation in vivo and assessed their effectiveness in priming cells for retroviral gene transfer. During the phase of hepatocyte proliferation retrovirus was administered via either the portal or tail vein. Acting synergistically, T3 and a truncated form of recombinant hepatocyte growth factor (dHGF) induced 30% of hepatocytes in normal rat liver to enter DNA synthesis at 24 h. This increased proliferation enabled the liver to be transduced in vivo by retroviral vectors via either the portal or peripheral venous system, achieving transduction efficiencies of 6.9 +/- 1.6% and 4.3 +/- 0.4% respectively. Thus, the liver can be simply and conveniently transduced in vivo with integrating vectors, introduced via the peripheral venous system during a wave of growth factor-induced proliferation, pointing the way to clinically applicable gene transfer techniques.

Animal models of acute hepatic failure.

The understanding and treatment of acute hepatic failure has developed rapidly over the last 40 years reducing morbidity and mortality from this syndrome. Progress has been made by the study of animal models that reflect the clinical, biochemical and histological pattern of the syndrome seen in man. This is of increasing importance with the use of therapeutic intervention, liver transplantation and the use of extra-corporeal liver support devices. This review examines and critically appraises the various approaches to the study of acute hepatic failure in animal models, including both surgical and pharmacological approaches.

Synergistic growth factors enhance rat liver proliferation and enable retroviral gene transfer via a peripheral vein.

BACKGROUND & AIMS: Genetic diseases reflecting abnormal hepatocyte function are potentially curable through gene therapy. Retroviral vectors offer the potential for permanent correction of such conditions. These vectors generally require cell division to occur to allow provirus entry into the nucleus, initiated in many experimental protocols by partial hepatectomy. We have explored methods to improve the efficiency of retroviral gene transfer that avoid the need for liver damage. METHODS: Triiodothyronine (T3) and keratinocyte growth factor (KGF) were used to induce hepatic proliferation in rats. The effects of intraportal and peripheral administration of a modified retrovirus that encoded the Lac Z gene during growth factor-induced liver hyperplasia were analyzed. RESULTS: T3 initiated hepatocyte proliferation midzonally; after KGF, proliferation was more diffuse. Optimal concentrations of T3 and KGF acted synergistically to induce proliferation in 61% of hepatocytes in the intact liver. This enabled in vivo hepatocyte transduction, leading to gene expression by up to 7.3% of hepatocytes after intraportal retroviral vector administration and 7. 1% after peripheral venous administration. CONCLUSIONS: T3 and KGF act synergistically to induce hepatocyte proliferation in undamaged liver. The liver can be simply transduced with integrating vectors via the peripheral venous system during a wave of growth factor-induced proliferation.

Resistance of three immortalized human hepatocyte cell lines to acetaminophen and N-acetyl-p-benzoquinoneimine toxicity.

BACKGROUND/AIMS: Acetaminophen toxicity in hepatocytes is attributed to generation of the toxic metabolite N-acetyl-p-benzoquinoneimine, leading to depletion of intracellular glutathione, alteration of redox potential and ultimately, cellular necrosis. We aimed to determine the effect of acetaminophen and N-acetyl-p-benzoquinoneimine on three human hepatocyte cell lines HH25, HH29 and HHY41, and for comparison, on primary rat hepatocytes, a cell type that is relatively resistant to acetaminophen-induced toxicity. METHODS: We investigated the effect of incubation of rat hepatocytes and 3 hepatocyte cell lines with acetaminophen or N-acetyl-p-benzoquinoneimine on LDH release, glutathione status, mitochondrial function, CYP1A activity, albumin synthesis and DNA content. RESULTS: We demonstrated that HH25, HH29 and HHY41 are resistant to the toxic effects of acetaminophen under conditions that induce cytotoxicity in rat primary hepatocytes, as indicated by maintenance of glutathione levels and basal LDH release. Incubation with N-acetyl-p-benzoquinoneimine caused a dose-dependent cytotoxicity in rat hepatocytes. Under comparable conditions N-acetyl-p-benzoquinoneimine had no effect on any of the hepatocyte cell lines. Nevertheless, when culturing the cells for a further 48 h, a decrease in glutathione levels, albumin synthesis, CYP1A activity, DNA content and mitochondrial function was apparent. CONCLUSION: HH25, HH29 and HHY41 cells are highly resistant to acetaminophen and N-acetyl-p-benzoquinoneimine-induced toxicity. They tolerate a much higher concentration of both toxins for a longer period of time compared to rat primary hepatocytes. These results are of relevance in the use of these cell lines to investigate acetaminophen hepatotoxicity, and may be of importance in the choice of cells for use in bioartificial liver support systems.

Review article: liver support systems in acute hepatic failure.

The treatment of acute hepatic failure has developed rapidly over the last 40 years, reducing morbidity and mortality from this syndrome. Whilst this has been partly attributed to significant improvements in the specialist medical management of these patients, advances in surgical techniques and pharmaceutical developments have led to the establishment of successful liver transplantation programmes, which have improved mortality significantly. This review will examine the clinical impact of alternative methods that have been used to provide extra-corporeal hepatic support. Non-biological, bio- logical and hybrid hepatic extra-corporeal support will be explored, offering a comprehensive historical overview and an appraisal of present and future advances.

Cerebral proton and phosphorus-31 magnetic resonance spectroscopy in patients with subclinical hepatic encephalopathy.

BACKGROUND/AIMS: In vivo magnetic resonance spectroscopy can be used to study cerebral metabolism non-invasively. We aimed to correlate 1H and 31P magnetic resonance spectral abnormalities in the brains of patients with subclinical hepatic encephalopathy. METHODS: Eighteen patients were studied at 1.5T, with combined 1H and 31P magnetic resonance spectra obtained from multiple voxels in the cerebral cortex and basal ganglia. Peak area ratios of choline, glutamine/glutamate, relative to creatine in the 1H spectra and percentage phosphomonoesters, phosphodiesters and betaNTP signals relative to total 31P signals in the 31P spectra were measured. RESULTS: Six patients did not complete the full examination - 31P results are available from 12 patients only. Relative to creatine, there were reductions in choline and elevations in glutamine/glutamate, varying across the brain with choline significantly reduced in occipital cortex (p<0.05) and glutamine/glutamate most significantly elevated in temporo-parietal cortex (p<0.0001). Percentage phosphomonoester (p<0.05), phosphodiester (p<0.05) and betaNTP (p<0.005) signals were significantly decreased in basal ganglia spectra. No correlation was found between the magnitude of 1H and 31P MRS changes, except between percentage phosphodiester decrease and glutamine/glutamate to creatine increase in occipital cortex. CONCLUSION: The results of this study point to a multifactorial aetiology for this condition.