A surgical model for isolating the pig liver in vivo for gene therapy.

Several studies report results that suggest the need of vascularization blocking for efficient gene transfer to the liver, especially in nonviral gene therapy. In this study, we describe a surgical strategy for in vivo isolation of the pig liver, resulting in a vascular watertight organ that allows the evaluation of several gene injection conditions. The hepatic artery and portal, suprahepatic and infrahepatic cava veins were dissected. Then, liver vascularization was excluded for 5-7 min. In that time, we first injected 200 ml saline solution containing the p3c-eGFP plasmid (20 µg/ml) simultaneously through two different catheters placed in the portal and cava veins, respectively. Vital constants were monitored during the surgery to assess the safety of the procedure. Basal systolic/diastolic blood pressures were 92.8/63.2 mm Hg and dropped to 40.7/31.3 mm Hg at the end of vascular exclusion; the mean basal heart rate was 58 bpm, reaching 95 bpm when the blood pressure was low. Oxygen saturation was maintained above 98% during the intervention, and no relevant changes were observed in the ECG tracing. Peak plasma AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels were observed after 24 h (151 and 57 IU, respectively). These values were higher, but not relevant, in 60 ml/s injection than in 20 ml/s injection. Efficiency of gene transfer was studied with simultaneous (cava and portal veins) injection of eGFP gene at flow rates of 20 and 60 ml/s. Liver tissue samples were collected 24 h after injection and qPCR was carried out on each lobe sample. The results confirmed the efficiency of the procedure. Gene delivery differed between 20 ml/s (9.9-31.0 eGFP DNA copies/100 pg of total DNA) and 60 ml/s injections (0.6-1.1 eGFP DNA copies/100 pg of total DNA). Gene transcription showed no significant differences between 20 ml/s (15,701.8-21,475.8 eGFP RNA copies/100 ng of total RNA) and 60 ml/s (12,014-36,371 eGFP RNA copies/100 ng of total RNA). The procedure is not harmful for animals and it offers a wide range of gene delivery options because it allows different perfusion ways (anterograde and retrograde) and different flow rates to determine the optimal conditions of gene transfer. This strategy permits the use of cell therapy and viral or non-viral liver gene therapy, especially appropriated to a wide variety of inherited or acquired diseases because of the liver's ability to produce and deliver proteins to the bloodstream.

New alternatives to the treatment of acute liver failure.

INTRODUCTION: Acute-on-chronic liver failure (ACLF) is defined as an acute deterioration of a chronic liver disease. The most effective treatment in these patients is orthotopic liver transplantation (OLT), which is highly limited by the donor shortage. The aim of this study was to increase the usefulness of hepatocyte transplantation (HT) as a bridge or alternative to OLT. METHODS: During the last 2 years, we have performed HT in 3 patients with ACLF. The diagnosis was graft cirrhosis due to hepatitis C virus in 2 of them, who were already included on waiting lists for retransplantation, and the third, unknown alcoholic cirrhosis. RESULTS: After the first HT infusion, we observed an improvement in the clinical condition in all patients, hyperammonemia, and a partial correction of the degree of encephalopathy; 1 patient was retransplanted 6 days after the first HT. DISCUSSION: The main indications for HT are inborn errors of metabolism in children. Other indications especially in adults, are acute liver failure, ACLF in patients with end-stage-liver disease who are a waiting OLT, and acute liver failure after an hepatectomy. HT may be a new treatment to improve the clinical condition in patients awaiting OLT.

Functionality of cultured human hepatocytes from elective samples, cadaveric grafts and hepatectomies.

The major possible sources of human liver for hepatocyte isolation are elective liver biopsies, cadaveric liver grafts and therapeutic liver resections. The suitability in terms of metabolic-competent hepatocyte cultures and risk/benefit of these resources has been comparatively studied. To this end, viability of isolated hepatocytes, yield of isolation procedure, hepatocyte survival during culture and CYP activities were the parameters analysed. The best results were found in hepatocytes prepared from elective biopsies, whereas a marked reduction in viability and functional competence was seen in hepatocytes from hepatectomy samples. Metabolic differences were observed in total CYP oxidative metabolism (7-ethoxycoumarin O-deethylation, total testosterone hydroxylation), as well as in CYP3A4, CYP2C9 or CYP2C19 activities (testosterone oxidations at 6beta-, 16beta- and 17-positions, respectively). Vascular control during the hepatectomy procedure influenced hepatocyte functionality: higher CYP activities were found in hepatocytes isolated from samples obtained under non-ischemic conditions or continuous vascular clamping than in those obtained under intermittent vascular clamping. In addition to cellular functionality, other criteria such as sample availability or ethical aspects should be considered. Elective biopsies have low, but not absent, surgical risk. However, the better functionality and the higher accessibility of elective liver samples in comparison to the other groups suggest this source of liver tissue as the most appropriate for cell harvesting purposes.

[A new case of hepatic adenomatosis treated with orthotopic liver transplantation]. / Un nouveau cas d'adénomatose hépatique traité par transplantation hépatique orthotopique.

Hepatic adenomatosis is a rare disease with multiple hepatic adenomas (10 or more), not associated with an history of oral contraceptive use or anabolic steroids use or with glycogen storage disease. A new case is reported in a 23 year-old woman who consulted for an abdominal mass and who had more than 50 adenomas of the liver. The suspicion of malignant transformation by the elevation of the alpha-foetoprotein, and the diffuse affectation of the liver, with minimum free parenchyma, suggested to carry out an orthotopic liver transplantation. The definitive histological examination of the surgical specimen confirmed the existence of local areas of hepatocellular carcinoma.

[Isolated hepatic transplant in patient with cystic fibrosis]. / Trasplante hepático aislado en paciente afectado de fibrosis quística.

Cystic fibrosis (CF) is a recessive autosomic disease with multiorgan, although predominantly pancreatic and pulmonary, involvement. Liver involvement is infrequent in children under the age of 5 years, but increases progressively with time. It is characterized by the development of focal biliary cirrhosis with eventual appearance of portal hypertension. During the last few years the more effective control of the pulmonary complications, which are the main cause of mortality, has led to an increase in the survival of these patients and thus the number of patients with CF and liver involvement is greater every day. In these cases, the prognosis is bad and most patients die in 4 to 5 years. Isolated liver transplantation is a recently proposed alternative for patients who have developed liver cirrhosis but who maintain acceptable pulmonary function. The case of a 14-years-old patient in whom liver transplantation was performed with good results after 8 months of follow-up is presented. Improvement in the nutritive state and pulmonary function was observed.