Human hepatocyte transplantation for liver disease: current status and future perspectives.

Liver transplantation is the accepted treatment for patients with acute liver failure and liver-based metabolic disorders. However, donor organ shortage and lifelong need for immunosuppression are the main limitations to liver transplantation. In addition, loss of the native liver as a target organ for future gene therapy for metabolic disorders limits the futuristic treatment options, resulting in the need for alternative therapeutic strategies. A potential alternative to liver transplantation is allogeneic hepatocyte transplantation. Over the last two decades, hepatocyte transplantation has made the transition from bench to bedside. Standardized techniques have been established for isolation, culture, and cryopreservation of human hepatocytes. Clinical hepatocyte transplantation safety and short-term efficacy have been proven; however, some major hurdles-mainly concerning shortage of donor organs, low cell engraftment, and lack of a long-lasting effect-need to be overcome to widen its clinical applications. Current research is aimed at addressing these problems, with the ultimate goal of increasing hepatocyte transplantation efficacy in clinical applications.

Bilirubin, a physiological antioxidant, can improve cryopreservation of human hepatocytes.

The availability of cryopreserved hepatocytes is required for a more widespread use of hepatocyte transplantation, but human hepatocytes are easily damaged during freezing-thawing. Here, preincubation with unconjugated bilirubin, a physiological antioxidant, resulted in increased viability and function of hepatocytes (as determined by trypan blue exclusion, mitochondrial succinate dehydrogenases activity, urea synthesis, and cytochrome P450 1A/2) compared with cells incubated without the pigment. These findings suggest that unconjugated bilirubin may be used as cryoprotectant in clinical hepatocyte transplantation.

Human hepatocyte transplantation: state of the art.

Hepatocyte transplantation is making its transition from bench to bedside for liver-based metabolic disorders and acute liver failure. Over eighty patients have now been transplanted world wide and the safety of the procedure together with medium-term success has been established. A major limiting factor in the field is the availability of good quality cells as hepatocytes are derived from grafts that are deemed unsuitable for transplantation. Alternative sources of cell, including stem cells may provide a sustainable equivalent to primary hepatocytes. There is also a need to develop techniques that will improve the engraftment, survival and function of transplanted hepatocytes. Such developments may allow hepatocyte transplantation to become an accepted and practical alternative to liver transplantation in the near future.

Hepatocyte transplantation followed by auxiliary liver transplantation--a novel treatment for ornithine transcarbamylase deficiency.

We report the first successful use of hepatocyte transplantation as a bridge to subsequent auxiliary partial orthotopic liver transplantation (APOLT) in a child antenatally diagnosed with severe ornithine transcarbamylase (OTC) deficiency. A total of 1.74 x 10(9) fresh and cryopreserved hepatocytes were administered intraportally into the liver over a period of 6 months. Immunosuppression was with tacrolimus and prednisolone. A sustained decrease in ammonia levels and a gradual increase in serum urea were observed except during episodes of sepsis in the first 6 months of life. The patient was able to tolerate a normal protein intake and presented a normal growth and neurological development. APOLT was successfully performed at 7 months of age. We conclude that hepatocyte transplantation can be used in conjunction with APOLT as an effective treatment for severe OTC-deficient patients, improving neurodevelopmental outcomes.

Characterization of p53 mutations in colorectal liver metastases and correlation with clinical parameters.

The presence and type of mutations of the p53 tumor suppressor gene were determined in 40 patients undergoing curative hepatic resection for metastatic colorectal carcinoma. This represents the largest series in the literature on the screening of p53 mutations for liver metastases. The analysis was performed in exons 5-9 by denaturing gradient gel electrophoresis followed by direct sequencing. Forty-five percent of tumors showed mutation in p53, and this was observed only in exons 5-8. Mutations at codon positions 167, 196, 204, 213, 245, 281, 282, 286, and 306; deletion of codon 251 and of the first nucleotide of codon 252; and Leu residue (CTC) insertion downstream codon 252 are reported for the first time in colorectal liver metastasis. Mutations at codon positions 163, 248, and 273 have been reported previously. Correlation of p53 status with clinical parameters showed that patients with mutated p53 had a statistically higher number of lesions when compared with patients with wild-type p53 (P<0.050). In particular, of patients with mutated p53, 41% had three or more metastases compared with 14% of patients with wild-type p53. Synchronous metastases were present in 70% of the patients with p53 mutations and in only 29% of patients with wild-type p53 (P<0.025). In addition, patients with p53 mutations are more likely to develop recurrence (73%) compared with patients with wild-type p53 (33%; P<0.001). Other factors considered, including preoperative carcinoembryonic antigen level, bilobar distribution, and size of the lesion(s), did not show significant correlation with p53 status. These results suggest that p53 status might be an important prognostic indicator to predict the pattern and likelihood of treatment failure after hepatic resection.

Effects of serum from patients with acute liver failure due to paracetamol overdose on human hepatocytes in vitro.

OBJECTIVE: Our goal was to investigate the effects of serum from patients with acute liver failure due to paracetamol (acetaminophen) overdose on the function of human hepatocytes in vitro. METHODS: Freshly isolated human hepatocytes plated on collagen-coated culture plates were, incubated (24 hours 37 degrees C) in medium containing pooled human sera (0%-80%) obtained from normal individuals or from patients with acute liver failure due to paracetamol overdose. The effects of the sera on cell function were assessed using MTT, [14C]-leucine incorporation, and cytochrome P450 (CYP1A1/2) activity assays. RESULTS: The overall cellular metabolic activity was significantly greater at all concentrations after exposure to acute liver failure serum compared to normal serum. There were no significant differences in the decreases produced by pooled acute liver failure and normal sera at concentrations up to 80% on the [14C]-leucine incorporation or CYP1A1/2 activity. CONCLUSION: The overall cell function/activity of human hepatocytes was not impaired in vitro on exposure to serum from patients with acute liver failure due to paracetamol overdose.

E1B-deleted adenovirus (dl1520) gene therapy for patients with primary and secondary liver tumors.

Clinical studies were performed with a recombinant mutant adenovirus with an E1B 55-kDa deletion, dl1520, to assess its toxicity and efficacy in patients with irresectable primary and secondary liver tumors. A phase I study showed that dl1520 was well tolerated when administered directly intratumorally, intraarterially, or intravenously up to a dose of 3 x 10(11) PFU. Ultrastructural examination of tissue showed the presence of adenovirus in cell cytoplasm around the nucleus and revealed two dissimilar end points of cell death after virus infection: a preapoptotic sequence and necrosis. A phase II study showed that the combination of dl1520 and 5-fluorouracil (5-FU), when infused into the hepatic artery, was well tolerated. Further improvement in the recombinant vector design will be needed in order to achieve better clinical response.

Clinical short-term results of radiofrequency ablation in primary and secondary liver tumors.

BACKGROUND: Radiofrequency ablation (RFA) is emerging as a new therapeutic method for management of solid tumors. We report here our experience in the use of this technique for management of primary and secondary unresectable liver cancers. METHODS: Thirty-five patients with liver cancers were considered not suitable for curative resection at presentation: 8 with primary hepatocellular carcinoma ([HCC] 6 HCC and 2 fibrolamellar); 27 with metastatic liver cancer (17 colorectal carcinoma and 10 others). They were treated either with radiofrequency heat ablation (Radionics Europe N.V., Wettdren, Belgium) alone percutaneously and/or intraoperatively or in conjunction with surgical resections. The quality of RFA was based on the subjective feeling of whether the tumor was completely destroyed or not. The effectiveness of RFA was assessed according to clinical findings, radiographic images, and tumor markers at follow-up. RESULTS: In 8 primary liver cases, 4 patients with a high level of alpha fetoprotein (AFP) benefited from the RFA with a 83.3% to 99.7% reduction of AFP. One with fibrolamellar hepatocellular carcinoma died 2 months after an incomplete percutaneous RFA from recurrence. The rest all had stable disease at the time of follow-up (mean 10.4 months). In patients with colorectal liver metastases, there were 4 deaths: 1 patient died postoperatively on the 30th day from a severe chest infection having shown a considerable reduction of carcinoembryonic antigen level (CEA, 8 versus 36 microg/L); 3 died from local and systemic disease, 1 at 12 months and 2 at 1 month, having had an incomplete RFA. The others had stable disease at follow-up (mean 7.6 months). Five patients underwent liver resections successfully with the application of RFA for residual lesions in the remaining contralateral lobe. In 10 patients with other liver tumors, 7 patients had stable disease at follow-up (mean 13.4 months); 1 patient had evidence of local and systemic recurrence 10 months after surgical resections with the intraoperative RFA and 2 patients died of systemic recurrence of disease 3 and 6 months after RFA alone. Two patients had liver resections in conjunction with the intraoperative RFA. The mean follow-up in our series was 8.5 months. CONCLUSION: Radiofrequency heat ablation is useful as a primary treatment for unresectable liver cancers. The procedure can be used to treat the small residual tumor load in the contralateral lobe following liver resection in those considered unresectable at the first presentation. This new therapeutic strategy seems to increase surgical resectability in patients judged unresectable.

Direct DNA Injection (p53) into HCC Tumors.

Liver tumors, specially HCC, are among the most common malignancies in the world, and their annual world incidence is about 250,000 cases, with a male to female ratio 4:1 (1). HCC is one of the most important neoplasms in tropical and subtropical regions, particularly among the sub-Saharan African black population and ethnic Chinese (2). The prognosis is very poor, and patients with advanced tumors are unlikely to survive 3 mo (3). Most HCC cases are beyond radical resection when detected. All other forms of the currently available therapies are rarely beneficial (2).

Clinical Protocol for p53 Gene Therapy for Liver Tumors.

This chapter is intended to help other workers with the preparation of human gene therapy proposals. What follows is an abridged version of a protocol describing the use of gene replacement with p53 for liver tumors. This was submitted to the Gene Therapy Advisory Committee (GTAC) of the Department of Health (United Kingdom). This is the first trial to be approved by GTAC for gene therapy of liver tumors in humans.

p53 Plasmid Preparation and Techniques for Analysis of Gene Transfer and Expression.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer death worldwide and is especially prevalent in certain areas of Africa and Asia. The most important etiological factor is infection with the hepatitis B or C virus. Treatment is generally unsatisfactory as the majority of patients are not suitable for surgical resection and chemotherapy is not particularly effective.

Results of resection for hilar cholangiocarcinoma with analysis of prognostic factors.

BACKGROUND/AIMS: Resection for hilar cholangiocarcinoma remains a challenging procedure and recent published results continue to show that few patients are cured of their disease. The objective of this review was to determine the results of radical resection and to identify factors associated with long-term survival. METHODOLOGY: Retrospective review of resection for hilar cholangiocarcinoma in 29 consecutive patients with statistical analysis of prognostic factors, including p53 status. RESULTS: The mortality and morbidity rates were 6.9% and 34%, respectively. The overall 5-year survival was 20% with the median survival being 16 months. Univariate analysis identified the following factors associated with poor survival; involved lymph nodes, vascular invasion, advanced tumor stage, positive tumor margins, and p53 mutation. However, none of these factors was associated with poor survival by multivariate analyses. CONCLUSIONS: Radical resection for hilar cholangiocarcinoma can be performed with acceptable morbidity and mortality, but most patients succumb to their disease. p53 status may be a helpful adjunct to routine pathological staging.

Analysis of adipokine concentrations in paediatric non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in children. It is important to distinguish children with more severe disease or steatohepatitis (NASH) from those with the less severe simple steatosis (SS) as prognosis differs. The importance of adipokines in the evolution of NASH is well recognized. OBJECTIVE: As adipokines are important in mediating inflammation, they may also be useful biomarkers of disease. METHODS: Plasma from 40 children (30 boys), median age 13.4 years, with liver biopsy-proven NAFLD was analysed. Liver biopsies were scored using the NAFLD activity score and compared with adipokine concentrations. RESULTS: Median body mass index z-score was 2.12 with a median homeostasis model of assessment- insulin resistance of 4.08. Resistin was lower in NASH than in SS (P = 0.03). Monocyte chemoattractant protein 1 (MCP-1) was also lower in NASH (P = 0.04). MCP-1 was higher in children with severe fibrosis (P = 0.008) with an area under the receiver operating characteristic curve (AUROC) of 0.76. Plasminogen activator inhibitor 1 (PAI-1) was also higher in this group (P = 0.011) with an AUROC of 0.78. There were no significant differences in leptin, adiponectin, adipsin, interleukin (IL) 6, IL10 or tumour necrosis factor α between groups. CONCLUSION: PAI-1 MCP-1 and resistin were differentially expressed with increasing severity of NAFLD. Though it is unlikely that this profile alone would serve as a biomarker of disease, differences found may contribute to understanding the role of these mediators in NAFLD.

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus.

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Hepatocyte transplantation for metabolic disorders, experience at King's College hospital and review of literature.

Hepatocyte transplantation is emerging as a potential treatment for liver based metabolic disorders and acute liver failure. To date, clinical studies have shown that hepatocyte transplantation could be used as a "bridge" to liver transplantation in a few patients with acute liver failure and has changed the phenotype of metabolic patients in terms of reducing the severity of illness. For many years, research studies have been carried out to optimise conditions for a) hepatocyte isolation in order to obtain the highest possible number of viable hepatocytes isolated from various sources of unused donor liver tissues, and b) cryopreservation and storage of hepatocytes for immediate cell availability in emergency cases. In this review, we summarise the worldwide clinical experience in hepatocyte transplantation for liver based metabolic disorders including the experience of our centre at King's College Hospital, London (United Kingdom). We briefly comment on the possible future developments and improvements needed in this field.

Detection of adenovirus and initiation of apoptosis in hepatocellular carcinoma cells after Ad-p53 treatment.

Transcription of the p53 gene can regulate progression of apoptosis in a wide variety of tissues. Three categories of human hepatocyte culture have been used to show the initiation of apoptosis after treatment with p53-bearing adenovirus. Chang liver cells are derived from normal liver tissue and express native p53, whereas hepatocellular carcinoma (HCC)-derived cell lines were Hep3B (p53-deleted) and PLC/PRF/5 (p53-mutant). Cultures were infected with Ad-p53 (15 particles per cell; 36 hours), and after treatment, morphological changes in all cell categories were observed by electron microscopy. Infection was evident in the cytoplasm of all treated cell types: after entry across the plasma membrane viruses translocated and came to rest surrounding and adjacent to nuclei, cytoplasm proximal to nuclear membranes became dense with virus- and membrane-derived debris, but intact viruses did not enter nuclei. Apoptosis, recognized morphologically by characteristic chromatin and cytoplasmic condensation, occurred more frequently in HCC-derived cells, and the ultimate fate of apoptotic bodies was phagocytosis and degradation by neighboring cells.

Wild-type p53 induces apoptosis in Hep3B through up-regulation of bax expression.

We demonstrated that introduction and expression of wild-type p53 gene in the human hepatocellular carcinoma cell line, Hep3B, resulted in up-regulation of both p21WAF1/CIP1 and bax gene expression and apoptosis. This cell line contains integrated hepatitis B virus sequences and lacks the expression of both p53 and retinoblastoma tumor suppressor genes because of deletions. Our results suggest that whereas an increased level of bax expression mediates apoptosis, an increased level of p21WAF1/CIP1 expression does not induce arrest of cell growth, presumably because of the deletion of the retinoblastoma gene. This study also confirms reported observations that p53 is a tumor suppressor gene, which induces apoptosis in malignant cells that lack normal p53 activity because of mutation, deletion, or inactivation of the gene by the presence of oncogenic viral proteins.

Overexpression of hepatic prothymosin alpha, a novel marker for human hepatocellular carcinoma.

Identification of gene products exclusively or abundantly expressed in cancer may yield novel tumour markers. We recently isolated a number of cDNA clones, including alpha-prothymosin, from rat hepatocellular carcinoma (HCC) using a subtraction-enhanced display technique. Alpha-Prothymosin is involved in cell proliferation and is regulated by the oncogene c-myc in vitro. In the present study, we analysed alpha-prothymosin gene expression and its correlation with c-myc in patients with HCC, cirrhosis and adenoma and in normal controls. Hepatic alpha-prothymosin messenger RNA (mRNA) levels were two- to 9.2-fold higher in tumoral tissues than in adjacent non-tumoral tissues in 14 of 17 patients with HCC, regardless of coexisting cirrhosis and viral hepatitis. No marked difference in alpha-prothymosin mRNA levels was present in patients with adenoma and hepatic cirrhosis and in healthy controls. The c-myc mRNA amounts were two- to fivefold increased in 11 of 17 patients with HCC and correlated significantly with those of alpha-prothymosin (P < 0.001). In situ hybridization revealed that increased alpha-prothymosin mRNA was localized in the tumour nodules of the patients with HCC. These data suggest that overexpression of alpha-prothymosin in HCC patients, correlated with c-myc, is possibly involved in the tumorigenic process and may be a novel molecular marker for human HCC.