Greg Nowak

Improving the techniques for human hepatocyte transplantation: Report from a consensus meeting in London

On September 6 and 7, 2009 a meeting was held in London to identify and discuss what are perceived to be current roadblocks to effective hepatocyte transplantation as it is currently practiced in the clinics and, where possible, to offer suggestions to overcome the blocks and improve the outcomes for this cellular therapy. Present were representatives of most of the active clinical hepatocyte transplant programs along with other scientists who have contributed substantial basic research to this field. Over the 2-day sessions based on the experience of the participants, numerous roadblocks or challenges were identified, including the source of cells for the transplants and problems with tracking cells following transplantation. Much of the discussion was focused on methods to improve engraftment and proliferation of donor cells posttransplantation. The group concluded that, for now, parenchymal hepatocytes isolated from donor livers remain the best cell source for transplantation. It was reported that investigations with other cell sources, including stem cells, were at the preclinical and early clinical stages. Numerous methods to modulate the immune reaction and vascular changes that accompany hepatocyte transplantation were proposed. It was agreed that, to obtain sufficient levels of repopulation of liver with donor cells in patients with metabolic liver disease, some form of liver preconditioning would likely be required to enhance the engraftment and/or proliferation of donor cells. It was reported that clinical protocols for preconditioning by hepatic irradiation, portal vein embolization, and surgical resection had been developed and that clinical studies using these protocols would be initiated in the near future. Participants concluded that sharing information between the groups, including standard information concerning the quality and function of the transplanted cells prior to transplantation, clinical information on outcomes, and standard preconditioning protocols, would help move the field forward and was encouraged.

Liver Transplantation for Familial Amyloidotic Polyneuropathy (FAP): A Single‐Center Experience Over 16 Years

Orthotopic liver transplantation (LTx) is currently the only available treatment that has been proven to halt the progress of familial amyloidotic polyneuropathy (FAP). The aim of this study was to assess mortality and symptomatic response to LTx for FAP. All 86 FAP patients transplanted at our hospital between April 1990 and November 2005 were included in the study. Five patients underwent retransplantation. The 1‐, 3‐ and 5‐year patient survival rates in patients transplanted during 1996–2005 were 94.6%, 92.3% and 92.3%, respectively, a significant difference from the rates of 76.7%, 66.7% and 66.7%, respectively, during 1990–1995 (p = 0.0003). Multivariate analysis revealed that the age at the time of LTx (≥40 years), duration of the disease (≥7 years) and modified body mass index (mBMI) (<600) were independent prognostic factors for patient survival. A halt in the progress of symptoms was noted in most patients, but only a minority experienced an improvement after LTx. To optimize the posttransplant prognosis, LTx should be performed in the early stages of the disease, and close post‐LTx monitoring of heart function by echocardiography and of heart arrhythmia by Holter ECG is mandatory.

Hepatocyte transplantation for inherited metabolic diseases of the liver.

Inherited metabolic diseases of the liver are characterized by deficiency of a hepatic enzyme or protein often resulting in life‐threatening disease. The remaining liver function is usually normal. For most patients, treatment consists of supportive therapy, and the only curative option is liver transplantation. Hepatocyte transplantation is a promising therapy for patients with inherited metabolic liver diseases, which offers a less invasive and fully reversible approach. Procedure‐related complications are rare. Here, we review the experience of hepatocyte transplantation for metabolic liver diseases and discuss the major obstacles that need to be overcome to establish hepatocyte transplantation as a reliable treatment option in the clinic.

Liver transplantation in the Nordic countries – An intention to treat and post-transplant analysis from The Nordic Liver Transplant Registry 1982–2013

Abstract Aim and background. The Nordic Liver Transplant Registry (NLTR) accounts for all liver transplants performed in the Nordic countries since the start of the transplant program in 1982. Due to short waiting times, donor liver allocation has been made without considerations of the model of end-stage liver disease (MELD) score. We aimed to summarize key outcome measures and developments for the activity up to December 2013. Materials and methods. The registry is integrated with the operational waiting-list and liver allocation system of Scandiatransplant (www.scandiatransplant.org) and accounted at the end of 2013 for 6019 patients out of whom 5198 were transplanted. Data for recipient and donor characteristics and relevant end-points retransplantation and death are manually curated on an annual basis to allow for statistical analysis and the annual report. Results. Primary sclerosing cholangitis, acute hepatic failure, alcoholic liver disease, primary biliary cirrhosis and hepatocellular carcinoma are the five most frequent diagnoses (accounting for 15.3%, 10.8%, 10.6%, 9.3% and 9.0% of all transplants, respectively). Median waiting time for non-urgent liver transplantation during the last 10-year period was 39 days. Outcome has improved over time, and for patients transplanted during 2004–2013, overall one-, five- and 10-year survival rates were 91%, 80% and 71%, respectively. In an intention-to-treat analysis, corresponding numbers during the same time period were 87%, 75% and 66%, respectively. Conclusion. The liver transplant program in the Nordic countries provides comparable outcomes to programs with a MELD-based donor liver allocation system. Unique features comprise the diagnostic spectrum, waiting times and the availability of an integrated waiting list and transplant registry (NLTR).

Elevated Numbers of FcγRIIIA+ (CD16+) Effector CD8 T Cells with NK Cell-Like Function in Chronic Hepatitis C Virus Infection

CTL are crucial in the defense against viral infections. In the course of investigating peripheral blood and intrahepatic CD8 T cells in patients with chronic hepatitis C virus (HCV) infection, we observed a significant population of CD8 T cells expressing the FcγRIIIA (CD16) receptor. This observation led us to characterize these cells with respect to their phenotype and function in a cohort of patients with chronic HCV infection as well as in healthy blood donors. On average, 10% of peripheral blood CD8 T cells from HCV-infected patients expressed CD16 compared with only a few percent in healthy donors. CD16+ CD8 T cells displayed a late-stage effector phenotype with high levels of perforin. These cells exhibited a restricted TCR profile suggesting underlying clonal expansion. Stimulation of CD16 on CD8 T cells evoked a vigorous response similar to that of CD16 stimulation in NK cells. Our data suggest that CD8 T cells, during chronic HCV infection in humans, continue to differentiate beyond defined stages of terminal effector cells, acquiring CD16 and NK cell-like functional properties.

Microdialysis monitoring of porcine liver metabolism during warm ischemia with arterial and portal clamping

Early detection of vascular complications following liver surgery is crucial. In the present study, intrahepatic microdialysis was used for continuous monitoring of porcine liver metabolism during occlusion of either the portal vein or the hepatic artery. Our aim was to assess whether microdialysis can be used to detect impaired vascular inflow by metabolic changes in the liver. Changes in metabolite concentrations in the hepatic interstitium were taken as markers for metabolic changes. After laparotomy, microdialysis catheters were introduced directly into the liver, enabling repeated measurements of local metabolism. Glucose, lactate, pyruvate, and glycerol were analyzed at bedside every 20 minutes, and the lactate/pyruvate ratio was calculated. In the arterial clamping group, the glucose, lactate, glycerol, and lactate/pyruvate ratio significantly increased during the 2‐hour vessel occlusion and returned to baseline levels during the 3‐hour reperfusion. In the portal occlusion group and in the control group, the measured metabolites were stable throughout the experiment. Our findings show that liver metabolism, as reflected by changes in the concentrations of glucose, lactate, and glycerol and in the lactate/pyruvate ratio, is markedly affected by occlusion of the hepatic artery. Surprisingly, portal occlusion resulted in no major metabolic changes. In conclusion, the microdialysis technique can detect and monitor arterial vascular complications of liver surgery, whereas potential metabolic changes in the liver induced by portal occlusion were not seen in the current study. Microdialysis may thus be suitable for use in liver surgery to monitor intrahepatic metabolic changes. Liver Transpl 15:280–286, 2009.

De Novo Donor-Specific HLA Antibody Formation in Two Patients With Crigler-Najjar Syndrome Type I Following Human Hepatocyte Transplantation With Partial Hepatectomy Preconditioning

Clinical hepatocyte transplantation is hampered by low engraftment rates and gradual loss of function resulting in incomplete correction of the underlying disease. Preconditioning with partial hepatectomy improves engraftment in animal studies. Our aim was to study safety and efficacy of partial hepatectomy preconditioning in clinical hepatocyte transplantation. Two patients with Crigler‐Najjar syndrome type I underwent liver resection followed by hepatocyte transplantation. A transient increase of hepatocyte growth factor was seen, suggesting that this procedure provides a regenerative stimulus. Serum bilirubin was decreased by 50%, and presence of bilirubin glucuronides in bile confirmed graft function in both cases; however, graft function was lost due to discontinuation of immunosuppressive therapy in one patient. In the other patient, serum bilirubin gradually increased to pretransplant concentrations after ≈600 days. In both cases, loss of graft function was temporally associated with emergence of human leukocyte antigen donor‐specific antibodies (DSAs). In conclusion, partial hepatectomy in combination with hepatocyte transplantation was safe and induced a robust release of hepatocyte growth factor, but its efficacy on hepatocyte engraftment needs to be evaluated with additional studies. To our knowledge, this study provides the first description of de novo DSAs after hepatocyte transplantation associated with graft loss.

Continuous Assessment of Intrahepatic Metabolism by Microdialysis During and After Portal Triad Clamping

BACKGROUND Ischemia-reperfusion injury is a major concern with portal triad clamping (PTC) in liver surgery. Microdialysis allows continuous intraoperative monitoring of tissue metabolism in the liver. Our aim was to evaluate the feasibility of microdialysis as a tool to assess the intrahepatic metabolic effects of PTC in patients undergoing liver resection. METHODS Eleven patients who underwent liver resection were subjected to intrahepatic microdialysis. Dialysis fluid samples were collected before, during, and after a 20-min period of PTC. Glucose, lactate, pyruvate (markers of ischemia), and glycerol (marker of cell membrane damage) were analyzed and the lactate/pyruvate ratio was calculated. RESULTS During PTC, intrahepatic glucose, lactate, and glycerol increased from 9.1±2.2 to 14.5±2.4 mM, from 2.2±0.3 to 5.8±0.5 mM, and from 63±14 to 142±28 μM, respectively. Pyruvate was unchanged, resulting in an increased lactate/pyruvate ratio (from 39±10 to 104±32). During initial reperfusion, glucose further increased to 16.4±2.9 mM. Pyruvate increased after reperfusion (from 93±18 to 138±23 μM), while lactate was stable, resulting in a normalized lactate/pyruvate ratio. Glycerol continued to increase during initial reperfusion. CONCLUSIONS PTC was associated with considerable intrahepatic metabolic alterations with anaerobic metabolism, increased glycogenolysis, and cellular membrane damage resulting in increased levels of glucose, lactate, glycerol, and lactate/pyruvate ratio. Microdialysis is easy to use and allows continuous monitoring of intrahepatic metabolism during liver surgery.

Transplantation With Livers From Deceased Donors Older Than 75 Years

Background The availability of donor organs limits the number of patients in need who are offered liver transplantation. Measures to expand the donor pool are crucial to prevent on-list mortality. The aim of this study was to evaluate the use of livers from deceased donors who were older than 75 years. Methods Fifty-four patients who received a first liver transplant (D75 group) from 2001 to 2011 were included. Donor and recipient data were collected from the Nordic Liver Transplant Registry and medical records. The outcome was compared with a control group of 54 patients who received a liver graft from donors aged 20 to 49 years (D20-49 group). Median donor age was 77 years (range, 75-86 years) in the D75 group and 41 years (range, 20-49 years) in the D20-49 group. Median recipient age was 59 years (range, 31-73 years) in the D75 group and 58 years (range, 31-74 years) in the D20-49 group. Results The 1-, 3-, and 5-year patient/graft survival values were 87/87%, 81/81%, and 71/67% for the D75 group and 88/87%, 75/73%, and 75/73% for the D20-49 group, respectively. Patient (P = 0.89) and graft (P = 0.79) survival did not differ between groups. The frequency of biliary complications was higher in the D75 group (29.6/13%, P = 0.03). Conclusions Selected livers from donors over age 75 years should not be excluded based on age, which does not compromise patient or graft survival despite a higher frequency of biliary complications.

Generation of hepatocyte-like cells from in vitro transdifferentiated human fetal pancreas.

Although the appearance of hepatic foci in the pancreas has been described in animal experiments and in human pathology, evidence for the conversion of human pancreatic cells to liver cells is still lacking. We therefore investigated the developmental plasticity between human embryonic pancreatic cells and liver cells. Cells were isolated and expanded from 7–8-week-old human fetal pancreata (HFP) and were characterized for the absence and presence of pancreatic and hepatic markers. In vitro expanded HFP were treated with fibroblast growth factor 2 (FGF2) and dexamethasone (DX) to induce a liver phenotye in the cells. These treated cells in various passages were further studied for their capacity to be functional in hepatic parenchyma following retrorsine-induced injury in nude C57 black mice. Amylase- and EPCAM-positive-enriched cells isolated from HFP and treated with FGF2 and DX lost expression of pancreatic markers and gained a liver phenotype. Hepatic differentiation was based on the expression (both at the mRNA and protein level) of liver markers albumin and cytokeratin 19. When transplanted in vivo into nude mice treated with retrorsine, both cell types successfully engrafted and functionally differentiated into hepatic cells expressing human albumin, glycogen, dipeptidyl peptidase, and γ-glutamyltranspeptidase. These data indicate that human fetal pancreatic cells have a capacity to alter their gene expression profile in response to exogenous treatment with FGF2 and DX. It may be possible to generate an unlimited supply of hepatocytes in vitro for cell therapy.