ABSTRACT
BACKGROUND: Clinical use of porcine cell-based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow-up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. METHODS: Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center-BAL (AMC-BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real-time polymerase chain reaction assay has been used. RESULTS: Eight patients who received a liver transplant after AMC-BAL treatment are still alive under long-term pharmacological immunosuppression. The current clinical follow-up ranges from 5.6 to 8.7 yr after BAL treatment. A new q-real-time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥ 5 copies per 1 × 10(5) PBMCs. The linear dynamic range was from 5 × 10(0) to 5 × 10(6) copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. CONCLUSION: Up to 8.7 yr after exposure to treatment with porcine liver cell-based BAL, no PERV infection has been found in long-term immunosuppressed patients and in HCWs by a new highly sensitive and specific q-real-time PCR assay.
Subject(s)
Endogenous Retroviruses/pathogenicity , Immunocompromised Host , Liver, Artificial/virology , Retroviridae Infections/etiology , Transplantation, Heterologous/adverse effects , Animals , DNA, Viral/blood , Endogenous Retroviruses/genetics , Humans , Immunosuppressive Agents/therapeutic use , Liver Transplantation/adverse effects , Liver Transplantation/immunology , Swine , Transplantation, Heterologous/immunologyABSTRACT
Ischemia-reperfusion injury (IRI) causes up to 10% of early liver failures in humans and can lead to a higher incidence of acute and chronic rejection. So far, very few studies have investigated wide gene expression profiles associated with the IRI process. The discovery of novel genes activated by IRI might lead to the identification of potential target genes for the prevention or treatment of the injury. In our study, we compared gene expression levels in reperfused livers (RL group) vs. the basal values before retrieval from the donor (basal liver [BL] group) using oligonucleotide array technology. We examined 10 biopsies from 5 livers, analyzing approximately 33,000 genes represented on the Affymetrix HG-U133APlus 2.0 oligonucleotide arrays (Affymetrix, Santa Clara, CA). About 13,000 individual genes were considered expressed in at least 1 condition. A total of 795 genes whose expression is significantly modified by ischemia-reperfusion in human liver transplantation were identified in this study. Some of them are likely to be completely activated by IRI, as they are not expressed in basal livers. The supervised gene expression analysis revealed that at least 12% of the genes involved in the apoptotic process, 12.5% of the genes involved in inflammatory processes, and 22.5% of the genes encoding for heat shock proteins are differentially expressed in RL samples vs. BL samples. Furthermore, IRI induces the upregulation of some genes' coding for adhesion molecules and integrins. In conclusion, we have identified a relevant amount of early genes regulated in the human liver after 7-9 hours of cold ischemia and 2 hours from reperfusion, many of them not having been described before in this process. Their analyses may help us to better understand the pathophysiology of IRI and to characterize potential target genes for the prevention or treatment of the liver injury in order to increase the number of patients that successfully undergo transplantation.
Subject(s)
Gene Expression Profiling/methods , Gene Expression Regulation , Liver Diseases/genetics , Liver Diseases/therapy , Liver Transplantation/methods , Reperfusion Injury/genetics , Reperfusion Injury/pathology , Adult , Aged , Aged, 80 and over , Apoptosis , Biopsy , Down-Regulation , Female , Humans , Inflammation , Ischemia/pathology , Liver/pathology , Liver Transplantation/adverse effects , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Up-RegulationABSTRACT
BACKGROUND: Currently a number of bioartificial livers (BAL) based on porcine liver cells have been developed as a treatment to bridge acute liver failure patients to orthotopic liver transplantation or liver regeneration. These xenotransplantation related treatments hold the risk of infection of treated patients by porcine endogenous retrovirus (PERV) released from the porcine cells, as in vitro infection experiments and transplantations in immunocompromised mice have shown that PERV is able to infect human cells. The Academic Medical Center (AMC)-BAL, unlike other BALs, is characterized by direct contact between porcine liver cells and human plasma, and might therefore be permissive for PERV transfer. METHODS: Prior to a clinical phase I trial, human plasma perfused through the AMC-BAL was investigated for PERV DNA and RNA. Moreover productive infectivity was analyzed by exposing the plasma to HEK-293 cells that were subsequently tested for PERV DNA, PERV RNA and reverse transcriptase activity. RESULTS: Although PERV DNA was detected in the perfused plasma, no productive infectivity was detected. Consequently fourteen patients were treated with the AMC-BAL and monitored for PERV transmission. Immediately after treatment the plasma of the patients was positive for PERV DNA, most probably due to porcine liver cell lysis. The PERV DNA was cleared within 2 weeks post-treatment and no PERV RNA was detected. No productive infectivity in human embryonic kidney (HEK)-293 cells exposed to plasma of treated patients was detectable. CONCLUSION: To conclude, no release of infective PERV particles from the AMC-BAL was observed. Therefore we consider the AMC-BAL as safe, however careful surveillance of patients will be continued.
Subject(s)
Endogenous Retroviruses/isolation & purification , Liver, Artificial/virology , Plasmapheresis/adverse effects , Retroviridae Infections/diagnosis , Swine/surgery , Swine/virology , Adult , Animals , Cell Line , Endogenous Retroviruses/physiology , Follow-Up Studies , Humans , Middle Aged , Retroviridae Infections/virologyABSTRACT
Ischemia-reperfusion injury, a clinical problem during cardiac surgery, involves worsened adenosine trisphosphate (ATP) generation and damage to the heart. We studied carbon monoxide (CO) pretreatment, proven valuable in rodents but not previously tested in large animals, for its effects on pig hearts subjected to cardiopulmonary bypass with cardioplegic arrest. Hearts of CO-treated pigs showed significantly higher ATP and phosphocreatine levels, less interstitial edema, and apoptosis of cardiomyocytes and required fewer defibrillations after bypass. We conclude that treatment with CO improves the energy status, prevents edema formation and apoptosis, and facilitates recovery in a clinically relevant model of cardiopulmonary bypass surgery.
Subject(s)
Carbon Monoxide/pharmacology , Cardiopulmonary Bypass , Cardiotonic Agents/pharmacology , Heart/drug effects , Myocardial Ischemia/drug therapy , Myocardial Reperfusion Injury/drug therapy , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosis/drug effects , Carbon Monoxide/therapeutic use , Cardiopulmonary Bypass/adverse effects , Cardiotonic Agents/therapeutic use , Edema/prevention & control , Electric Countershock , Energy Metabolism/drug effects , Female , Guanosine Triphosphate/metabolism , Myocardial Ischemia/metabolism , Myocardial Ischemia/therapy , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , NAD/metabolism , NADP/metabolism , Oxidation-Reduction , Sus scrofaABSTRACT
Recently a phase I clinical trial has been started in Italy to bridge patients with acute liver failure (ALF) to orthotopic liver transplantation (OLT) by the AMC-bioartificial liver (AMC-BAL). The AMC-BAL is charged with 10 x 10(9) viable primary porcine hepatocytes isolated from a specified pathogen-free (SPF) pig. Here we report a patient with ALF due to acute HBV infection. This patient was treated for 35 h by two AMC-BAL treatments and was bridged to OLT. There was improvement of biochemical and clinical parameters during the treatment. No severe adverse events were observed during treatment and follow-up of 15 months after hospital discharge. Possible porcine endogenous retrovirus (PERV) activity could not be detected in the patient's blood or blood cells up to 12 months after treatment.
Subject(s)
Hepatitis B/surgery , Liver Failure, Acute/surgery , Liver Transplantation/instrumentation , Liver Transplantation/methods , Liver, Artificial/trends , Adult , Ammonia/blood , Animals , Bilirubin/blood , Female , Hepatocytes/cytology , Hepatocytes/immunology , Hepatocytes/transplantation , Humans , Lactic Acid/blood , Liver, Artificial/standards , Prothrombin/metabolism , Retroviridae/immunology , Specific Pathogen-Free Organisms , Sus scrofa , Transaminases/blood , Treatment OutcomeABSTRACT
Recently a phase I clinical trial has been started in Italy to bridge patients with acute liver failure (ALF) to orthotopic liver transplantation (OLT) by the AMC-bioartificial liver (AMC-BAL). The AMC-BAL is charged with 10 × 109 viable primary porcine hepatocytes isolated from a specified pathogen-free (SPF) pig. Here we report a patient with ALF due to acute HBV infection. This patient was treated for 35 h by two AMC-BAL treatments and was bridged to OLT. There was improvement of biochemical and clinical parameters during the treatment. No severe adverse events were observed during treatment and follow-up of 15 months after hospital discharge. Possible porcine endogenous retrovirus (PERV) activity could not be detected in the patient's blood or blood cells up to 12 months after treatment.