Andrea Schlegel

HOPE for human liver grafts obtained from donors after cardiac death

BACKGROUND & AIMS Due to ethical rules in most countries, long ischemia times are unavoidable prior to organ procurement of donors without a heartbeat, which can cause early graft failure after liver transplantation or late biliary strictures. Hypothermic oxygenated machine perfusion, used prior to graft implantation, may rescue these high risk organs. METHODS Eight patients with end stage liver diseases received human livers, obtained after controlled cardiac death (Maastricht category III), with a median donor warm ischemia time of 38 min, followed by a standard cold flush and static storage at 4 °C. Hypothermic oxygenated perfusion (HOPE) was applied for 1-2h prior to implantation through the portal vein. The HOPE-perfusate was cooled at 10 °C and oxygenated (pO2 60 kPa) using an ECOPS device (Organ Assist®). Perfusion pressure was maintained below 3 mmHg. RESULTS Each machine perfused liver graft disclosed excellent early function after transplantation. The release of liver enzymes and kidney function, as well as ICU and hospital stays were comparable or better than in matched liver grafts from brain death donors. No evidence of intrahepatic biliary complications could be documented within a median follow up of 8.5 months. CONCLUSIONS This is the first report on cold machine perfusion of human liver grafts obtained after cardiac arrest and subsequent transplantation. Application of HOPE appears well tolerated, easy-to-use, and protective against early and later injuries.

Protective mechanisms of end-ischemic cold machine perfusion in DCD liver grafts

BACKGROUND & AIMS The aim of this study was to identify protective mechanisms of cold machine perfusion in liver grafts donated after cardiac death. METHODS Pig livers exposed to 60-min warm ischemia were cold stored for 7 h or treated after 6-h cold storage with 1-h hypothermic oxygenated perfusion (HOPE) through the portal vein. Different physical (perfusion pressure) and chemical (oxygen, mitochondrial transition pore inhibition) parameters were analyzed during machine perfusion to dissect key steps of mechanism. RESULTS HOPE treatment led to a significant slowdown of mitochondrial respiration rate during 1-h machine perfusion. After reperfusion following low pressure HOPE, mitochondrial injury, nuclear injury, Kupffer cell activation and endothelial injury were significantly improved, as tested on an isolated liver perfusion model. In contrast, machine perfusion with deoxygenated perfusate showed no protection from hepatocyte injury and Kupffer cell activation. However, endothelial injury was also prevented by low pressure machine perfusion in the absence of oxygen. Perfusion with higher pressure provoked endothelial damage and Kupffer cell activation. CONCLUSIONS The mechanisms of protection by hypothermic machine perfusion appear to be at least twofold. First, oxygenation under hypothermic conditions protects from mitochondrial and nuclear injury by downregulation of mitochondrial activity before reperfusion. Second, cold perfusion itself, under low pressure conditions, prevents endothelial damage, independently of oxygen.

ALPPS: from human to mice highlighting accelerated and novel mechanisms of liver regeneration.

Objectives:To develop a reproducible animal model mimicking a novel 2-staged hepatectomy (ALPPS: Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy) and explore the underlying mechanisms. Background:ALPPS combines portal vein ligation (PVL) with liver transection (step I), followed by resection of the deportalized liver (step II) within 2 weeks after the first surgery. This approach induces accelerated hypertrophy of the liver remnant to enable resection of massive tumor load. To explore the underlying mechanisms, we designed the first animal model of ALPPS in mice. Methods:The ALPPS group received 90% PVL combined with parenchyma transection. Controls underwent either transection or PVL alone. Regeneration was assessed by liver weight and proliferation-associated molecules. PVL-treated mice were subjected to splenic, renal, or pulmonary ablation instead of hepatic transection. Plasma from ALPPS-treated mice was injected into mice after PVL. Gene expression of auxiliary mitogens in mouse liver was compared to patients after ALPPS or PVL. Results:The hypertrophy of the remnant liver after ALPPS doubled relative to PVL, whereas mice with transection alone disclosed minimal signs of regeneration. Markers of hepatocyte proliferation were 10-fold higher after ALPPS, when compared with controls. Injury to other organs or ALPPS-plasma injection combined with PVL induced liver hypertrophy similar to ALPPS. Early initiators of regeneration were significantly upregulated in human and mice. Conclusions:ALPPS in mice induces an unprecedented degree of liver regeneration, comparable with humans. Circulating factors in combination with PVL seem to mediate enhanced liver regeneration, associated with ALPPS.

Hypothermic oxygenated perfusion (HOPE) protects from biliary injury in a rodent model of DCD liver transplantation

BACKGROUND & AIMS The use of livers from donors after cardiac arrest (DCD) is increasing in many countries to overcome organ shortage. Due to additional warm ischemia before preservation, those grafts are at higher risk of failure and bile duct injury. Several competing rescue strategies by machine perfusion techniques have been developed with, however, unclear effects on biliary injury. We analyze the impact of an end-ischemic Hypothermic Oxygenated PErfusion (HOPE) approach applied only through the portal vein for 1h before graft implantation. METHODS Rat livers were subjected to 30-min in situ warm ischemia, followed by subsequent 4-h cold storage, mimicking DCD-organ procurement and conventional organ transport. Livers in the HOPE group underwent also passive cold storage for 4h, but were subsequently machine perfused for 1h before implantation. Outcome was tested by liver transplantation (LT) at 12h after implantation (n=10 each group) and after 4 weeks (n=10 each group), focusing on early reperfusion injury, immune response, and later intrahepatic biliary injury. RESULTS All animals survived after LT. However, reperfusion injury was significantly decreased by HOPE treatment as tested by hepatocyte injury, Kupffer cell activation, and endothelial cell activation. Recipients receiving non-perfused DCD livers disclosed less body weight gain, increased bilirubin, and severe intrahepatic biliary fibrosis. In contrast, HOPE treated DCD livers were protected from biliary injury, as detected by cholestasis parameter and histology. CONCLUSIONS We demonstrate in a DCD liver transplant model that end-ischemic hypothermic oxygenated perfusion is a powerful strategy for protection against biliary injury.

The use of fatty liver grafts in modern allocation systems: risk assessment by the balance of risk (BAR) score.

Objective:To integrate the amount of hepatic steatosis in modern liver allocation models. Background:The aim of this study was to combine the 2 largest liver transplant databases (United States and Europe) in 1 comprehensive model to predict outcome after liver transplantation, with a novel focus on the impact of the presence of steatosis in the graft. Methods:We adjusted the balance of risk (BAR) score for its application to the European Liver Transplant Registry (ELTR) database containing 11,942 patients. All liver transplants from ELTR and United Network for Organ Sharing with recorded liver biopsies were then combined in one survival analysis in relation to the presence of graft micro- (n = 9,677) and macrosteatosis (n = 11,516). Results:Microsteatosis, regardless of the amount, was associated with a similar relationship between mortality and BAR score as nonsteatotic livers. Low-grade macrosteatotic liver grafts (⩽30% macrosteatosis) resulted in 5-year graft-survival rates of 60% or more up to BAR 18, comparable to nonsteatotic grafts. However, use of moderate or severely steatotic liver grafts (>30% macrosteatosis) resulted in acceptable outcome only if the cumulative risk at transplant was low, that is, BAR score of 9 or less. Conclusions:Microsteatotic or 30% or less macrosteatotic liver grafts can be used safely up to BAR score of 18 or less, but liver grafts with more than 30% macrosteatotis should be used with risk adjustment, that is, up to BAR score of 9 or less.

Warm vs. cold perfusion techniques to rescue rodent liver grafts

BACKGROUND & AIMS A variety of liver perfusion techniques have been proposed to protect liver grafts prior to implantation. We compared hypothermic and normothermic oxygenated perfusion techniques in a rat liver transplant model, using higher risk grafts obtained after cardiac arrest (DCD). METHODS Rat livers were subjected to 30 or 60 min in situ warm ischemia, without application of heparin. Livers were excised and stored for 4 h at 4°C, mimicking DCD organ procurement, followed by conventional organ transport. In experimental groups, DCD liver grafts received a 4 h normothermic oxygenated perfusion through the portal vein and the hepatic artery instead of cold storage. The perfusate consisted of either full blood or leukocyte-depleted blood (normothermic groups). Other livers underwent hypothermic oxygenated perfusion (HOPE) for 1 h after warm ischemia and 4 h cold storage (HOPE group). Liver injury was assessed during machine perfusion and after isolated liver reperfusion, and by orthotopic liver transplantation (OLT). RESULTS DCD livers, subjected to normothermic perfusion, disclosed reduced injury and improved survival compared to cold storage after limited warm ischemia of 30 min (70%; 7/10), but failed to protect from lethal injury in grafts exposed to 60 min warm ischemia (0%; 0/10). This finding was consistent with Kupffer and endothelial cell activation in cold stored and normothermic perfused livers. In contrast, HOPE protected from hepatocyte and non-parenchymal cell injury and led to 90% (9/10) and 63% (5/8) animal survival after 30 and 60 min of donor warm ischemia, respectively. CONCLUSIONS This is the first evidence that HOPE is superior to normothermic oxygenated perfusion in a clinically relevant model through modulation of the innate immunity and endothelial cell activation.

Hypothermic Oxygenated Perfusion (HOPE) downregulates the immune response in a rat model of liver transplantation.

Objective:To evaluate the impact of a novel oxygenated perfusion approach on rejection after orthotopic liver transplantation (OLT). Background:Hypothermic oxygenated perfusion (HOPE) was designed to prevent graft failure after OLT. One of the mechanisms is downregulation of Kupffer cells (in situ macrophages). We, therefore, designed experiments to test the effects of HOPE on the immune response in an allogeneic rodent model of nonarterialized OLT. Methods:Livers from Lewis rats were transplanted into Brown Norway rats to induce liver rejection in untreated recipients within 4 weeks. Next, Brown Norway recipients were treated with tacrolimus (1 mg/kg), whereas in a third group, liver grafts from Lewis rats underwent HOPE or deoxygenated machine perfusion for 1 hour before implantation, but recipients received no immunosuppression. In a last step, low-dose tacrolimus treatment (0.3 mg/kg) was assessed with and without HOPE. Results:Allogeneic OLT without immunosuppression led to death within 3 weeks after nonarterialized OLT due to severe acute rejection. Full-dose tacrolimus prevented rejection, whereas low-dose tacrolimus led to graft fibrosis within 4 weeks. HOPE treatment without immunosuppression also protected from lethal rejection. The combination of low-dose tacrolimus and 1-hour HOPE resulted in 100% survival within 4 weeks without any signs of rejection. Conclusions:We demonstrate that allograft treatment by HOPE not only protects against preservation injury but also impressively downregulates the immune system, blunting the alloimmune response. Therefore, HOPE may offer many beneficial effects, not only to rescue marginal grafts but also by preventing rejection and the need for immunosuppression.

Low platelet counts after liver transplantation predict early posttransplant survival: The 60‐5 criterion

Platelets play a critical role in liver injury and regeneration. Thrombocytopenia is associated with increases in postoperative complications after partial hepatectomy, but it is unknown whether platelet counts could also predict outcomes after transplantation, a procedure that is often performed in thrombocytopenic patients. Therefore, the aim of this study was to evaluate whether platelet counts could be indicators of short‐ and long‐term outcomes after liver transplantation (LT). Two hundred fifty‐seven consecutive LT recipients (January 2003‐December 2011) from our prospective database were analyzed. Preoperative and daily postoperative platelet counts were recorded until postoperative day 7 (POD7). Univariate and multivariate analyses were performed to assess whether low perioperative platelet counts were a risk factor for postoperative complications and graft and patient survival. The median pretransplant platelet count was 88 × 109/L [interquartile range (IQR) = 58‐127 × 109/L]. The lowest platelet counts occurred on POD3: the median was 56 × 109/L (IQR = 41‐86 × 109/L). Patients with low platelet counts on POD5 had higher rates of severe (grade IIIb/IV) complications [39% versus 29%, odds ratio (OR) = 1.09 (95% CI = 1.1‐3.3), P = 0.02] and 90‐day mortality [16% versus 8%, OR = 2.25 (95% CI = 1.0‐5.0), P = 0.05]. In the multivariate analysis, POD5 platelet counts < 60 × 109/L were identified as an independent risk factor for grade IIIb/IV complications [OR = 1.96 (95% CI = 1.07‐3.56), P = 0.03)], graft survival [hazard ratio (HR) = 2.0 (95% CI = 1.1‐3.6), P = 0.03)], and patient survival [HR = 2.2 (95% CI = 1.1‐4.6), P = 0.03)]. The predictive value of platelet counts for graft and patient survival was lost in patients who survived 90 days. In conclusion, after LT, platelet counts < 60 × 109/L on POD5 (the 60‐5 criterion) are an independent factor associated with severe complications and early graft and patient survival. These findings may help us to develop protective strategies or specific interventions for high‐risk patients. Liver Transpl 20:147‐155, 2014.

Role of hypothermic machine perfusion in liver transplantation.

Machine liver perfusion has significantly evolved during the last ten years to optimize extended criteria liver grafts and to address the worldwide organ shortage. This review gives an overview on available ex vivo and in vivo data on hypothermic machine liver perfusion. We discuss also possible protective pathways and show most recent clinical applications of hypothermic machine liver perfusion in human.

Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver

Host immune response to viral vectors, persistence of nonintegrating vectors, and sustained transgene expression are among the major challenges in gene therapy. To overcome these hurdles, we successfully used minicircle (MC) naked‐DNA vectors devoid of any viral or bacterial sequences for the long‐term treatment of murine phenylketonuria, a model for a genetic liver defect. MC‐DNA vectors expressed the murine phenylalanine hydroxylase (Pah) complementary DNA (cDNA) from a liver‐specific promoter coupled to a de novo designed hepatocyte‐specific regulatory element, designated P3, which is a cluster of evolutionary conserved transcription factor binding sites. MC‐DNA vectors were subsequently delivered to the liver by a single hydrodynamic tail vein (HTV) injection. The MC‐DNA vector normalized blood phenylalanine concomitant with reversion of hypopigmentation in a dose‐dependent manner for more than 1 year, whereas the corresponding parental plasmid did not result in any phenylalanine clearance. MC vectors persisted in an episomal state in the liver consistent with sustained transgene expression and hepatic PAH enzyme activity without any apparent adverse effects. Moreover, 14‐20% of all hepatocytes expressed transgenic PAH, and the expression was observed exclusively in the liver and predominately around pericentral areas of the hepatic lobule, while there was no transgene expression in periportal areas. Conclusion: This study demonstrates that MC technology offers an improved safety profile and has the potential for the genetic treatment of liver diseases. (Hepatology 2014;60:1035–1043)