Scot D. Henry

Hypothermic Machine Preservation in Human Liver Transplantation: The First Clinical Series

Hypothermic machine perfusion (HMP) is widely used to preserve kidneys for transplantation with improved results over cold storage (CS). To date, successful transplantation of livers preserved with HMP has been reported only in animal models. In this, the first prospective liver HMP study, 20 adults received HMP‐preserved livers and were compared to a matched group transplanted with CS livers. HMP was performed for 3–7 h using centrifugal perfusion with Vasosol® solution at 4–6°C. There were no cases of primary nonfunction in either group. Early allograft dysfunction rates were 5% in the HMP group versus 25% in controls (p = 0.08). At 12 months, there were two deaths in each group, all unrelated to preservation or graft function. There were no vascular complications in HMP livers. Two biliary complications were observed in HMP livers compared with four in the CS group. Serum injury markers were significantly lower in the HMP group. Mean hospital stay was shorter in the HMP group (10.9 ± 4.7 days vs. 15.3 ± 4.9 days in the CS group, p = 0.006). HMP of donor livers provided safe and reliable preservation in this pilot case‐controlled series. Further multicenter HMP trials are now warranted.

Hypothermic Machine Preservation Facilitates Successful Transplantation of “Orphan” Extended Criteria Donor Livers

Hypothermic machine preservation (HMP) remains investigational in clinical liver transplantation. It is widely used to preserve kidneys for transplantation with improved results over static cold storage (SCS). At our center, we have used HMP in 31 adults receiving extended criteria donor (ECD) livers declined by the originating United Network for Organ Sharing region (“orphan livers”). These cases were compared to ECD SCS cases in a matched cohort study design. Livers were matched for donor age, recipient age, cold ischemic time, donor risk index and Model for End‐Stage Liver Disease (MELD) score. HMP was performed for 3–7 h at 4–8°C using our previously published protocol. Early allograft dysfunction rates were 19% in the HMP group versus 30% in the control group (p = 0.384). One‐year patient survival was 84% in the HMP group versus 80% in the SCS group (p = NS). Post hoc analysis revealed significantly less biliary complications in the HMP group versus the SCS group (4 vs. 13, p = 0.016). Mean hospital stay was significantly shorter in the HMP group (13.64 ± 10.9 vs. 20.14 ± 11.12 days in the SCS group, p = 0.001). HMP provided safe and reliable preservation in orphan livers transplanted at our center.

Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi)

Background/aims RNA interference (RNAi), a sequence-specific gene silencing technology triggered by small interfering RNA (siRNA), represents promising new avenues for treatment of various liver diseases including hepatitis C virus (HCV) infection. In plants and invertebrates, RNAi provides an important mechanism of cellular defence against viral pathogens and is dependent on the spread of siRNA to neighbouring cells. A study was undertaken to investigate whether vector-delivered RNAi can transfer between hepatic cells in vitro and in mice, and whether this exchange could extend the therapeutic effect of RNAi against HCV infection. Methods Transmission of RNAi was investigated in culture by assessing silencing of HCV replication and expression of viral entry receptor CD81 using a human hepatic cell line and primary B lymphocytes transduced with siRNA-expressing vectors. In vivo transmission between hepatic cells was investigated in NOD/SCID mice. Involvement of exosomes was demonstrated by purification, uptake and mass spectrometric analysis. Results Human and mouse liver cells, as well as primary human B cells, were found to have the ability to exchange small RNAs, including cellular endogenous microRNA and delivered siRNA targeting HCV or CD81. The transmission of RNAi was largely independent of cell contact and partially mediated by exosomes. Evidence of RNAi transmission in vivo was observed in NOD/SCID mice engrafted with human hepatoma cells producing CD81 siRNA, causing suppression of CD81 expression in mouse hepatocytes. Conclusion Both human and mouse hepatic cells exchange small silencing RNAs, partially mediated by shuttling of exosomes. Transmission of siRNA potentially extends the therapeutic reach of RNAi-based therapies against HCV as well as other liver diseases.

Impact of Steroids on Hepatitis C Virus Replication in Vivo and in Vitro

Abstract:  Chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation. Transplantation outcome is often compromised by a rapid re‐infection of the graft. Several factors have been implicated in the increased severity of recurrence, including steroid‐based immunosuppression. Evidence suggests that steroid boluses used to treat acute rejection are associated with an increase in HCV viral load and the severity of recurrence. Two possible mechanisms for a steroid‐mediated effect on HCV viral loads can be postulated, the first being a direct effect of steroids on the virus by enhancing its replication. The second, an indirect effect due to the suppression of the HCV immune response, allows unrestricted HCV replication. To investigate the direct effect of steroids on HCV replication, dexamethasone (Dex) and prednisolone (Pred) were tested in an in vitro replicon model. HCV replication was assessed on the basis of luciferase reporter expression (luminescence) and HCV RNA (RT‐PCR). At clinically relevant concentrations (1–10 nM), treatment with both Dex and Pred did not enhance, but resulted in a slight reduction of relative luciferase activity (HCV replication), which was independent of increased cellular protein content and reduced cell proliferation. This minor reduction of HCV replication was confirmed by RT‐PCR showing more than 41% reduction in HCV RNA levels. In conclusion, despite clinical evidence that the use of steroids aggravates recurrence of HCV, our in vitro study suggests that there is no direct stimulatory effect of steroids on the replication of HCV. As such, the increased viral loads after high‐dose steroid treatment are more likely due to a downregulation of the immune response. In such patients, a dampened immune response allows viruses like HCV to replicate free of immune‐mediated killing of their host cells. When a change occurs, such as a tapering or an alteration of immunosuppressant drugs, the immune system reinitiates and vigorously attempts to control the virus, resulting in acceleration of liver damage. Therefore, either steroid avoidance or maintaining low levels, coupled with a slow tapering of corticosteroids, may be beneficial to HCV‐infected transplantation recipients.

Combined antiviral activity of interferon-α and RNA interference directed against hepatitis C without affecting vector delivery and gene silencing

The current standard interferon-alpha (IFN-α)-based therapy for chronic hepatitis C virus (HCV) infection is only effective in approximately half of the patients, prompting the need for alternative treatments. RNA interference (RNAi) represents novel approach to combat HCV by sequence-specific targeting of viral or host factors involved in infection. Monotherapy of RNAi, however, may lead to therapeutic resistance by mutational escape of the virus. Here, we proposed that combining lentiviral vector-mediated RNAi and IFN-α could be more effective and avoid therapeutic resistance. In this study, we found that IFN-α treatment did not interfere with RNAi-mediated gene silencing. RNAi and IFN-α act independently on HCV replication showing combined antiviral activity when used simultaneously or sequentially. Transduction of mouse hepatocytes in vivo and in vitro was not effected by IFN-α treatment. In conclusion, RNAi and IFN-α can be effectively combined without cross-interference and may represent a promising combinational strategy for the treatment of hepatitis C.

Liver Defatting: An Alternative Approach to Enable Steatotic Liver Transplantation

Macrovesicular steatosis in greater than 30% of hepatocytes is a significant risk factor for primary graft nonfunction due to increased sensitivity to ischemia reperfusion (I/R) injury. The growing prevalence of hepatic steatosis due to the obesity epidemic, in conjunction with an aging population, may negatively impact the availability of suitable deceased liver donors. Some have suggested that metabolic interventions could decrease the fat content of liver grafts prior to transplantation. This concept has been successfully tested through nutritional supplementation in a few living donors. Utilization of deceased donor livers, however, requires defatting of explanted organs. Animal studies suggest that this can be accomplished by ex vivo warm perfusion in a time scale of a few hours. We estimate that this approach could significantly boost the size of the donor pool by increasing the utilization of steatotic livers. Here we review current knowledge on the mechanisms whereby excessive lipid storage and macrosteatosis exacerbate hepatic I/R injury, and possible approaches to address this problem, including ex vivo perfusion methods as well as metabolically induced defatting. We also discuss the challenges ahead that need to be addressed for clinical implementation.

Protective effects of hypothermic ex vivo perfusion on ischemia/reperfusion injury and transplant outcomes

Hypothermic machine preservation (HMP) has been used in renal transplantation since the late 1960s with recent robust prospective, multicenter data showing lower rates of delayed graft function and improved graft survival. Although now clearly beneficial for renal transplantation, extrarenal machine perfusion has remained predominantly in preclinical investigations. Pancreatic HMP has drawn little clinical interest because HMP has been suggested to cause graft edema and congestion, which is associated with early venous thrombosis and graft failure. Early investigation showed no benefit of HMP in whole-organ pancreas transplant. One report did show that HMP increases islet cell yield after isolation. Preclinical work in liver HMP has been promising. Short- and long-term HMP has been shown to improve graft viability and reduce preservation injury, even in animal models of steatotic and donation after cardiac death. The first clinical study of liver HMP using a centrifugal dual perfusion technique showed excellent results with lower hepatocellular injury markers and no adverse perfusion-related outcomes. In addition, a dramatic attenuation of proinflammatory cytokine expression was observed. Further studies of liver HMP are planned with focus on developing a reproducible and standard protocol that will allow the widespread availability of this technology. Future research and clinical trials of novel organ preservation techniques, solutions, and interventions are likely to bring about developments that will allow further reduction of preservation-related ischemia/reperfusion injury and improved outcomes and allow safer utilization of the precious and limited resource of donor organs.

Molecular expression of acute phase mediators is attenuated by machine preservation in human liver transplantation: Preliminary analysis of effluent, serum, and liver biopsies

BACKGROUND Hypothermic machine perfusion (HMP) mitigates the effects of ischemia/reperfusion injury (IRI) in renal transplantation and preclinical work with livers. In liver transplantation, IRI increases the likelihood of primary graft dysfunction and is associated with significant morbidity. We recently completely the first phase 1 clinical trial of liver HMP at our center, and demonstrated improved clinical parameters and shorter duration of stay for patients who received grafts stored by HMP than patients who received grafts preserved in cold storage. Biomarker analysis of venous effluent collected from the hepatic veins during HMP may yield predictive information reflecting the condition of the donor liver, such as graft injury sustained during brain death and graft preservation. The aim of this study was to characterize biomarkers released into the effluent during HMP. METHODS Effluent was collected every 30 minutes during liver HMP during our phase 1 clinical trial. Serum was extracted from blood samples obtained at incision, before explantation, and at 1, 2, and 3 hours after reperfusion. The effluent and serum samples were assayed in multiplex to determine the concentration of inflammatory cytokines and growth factors. Tissue obtained from liver biopsies was processed for either downstream reverse transcription-polymerase chain reaction or immunofluorescence. Statistical significance was determined by a two-tailed t-test. RESULTS Growth factors and most cytokines were not readily detectable in levels above baseline with this technique; however, interleukin-1 (IL-1) receptor antagonist and monocyte chemotactic protein-1 were present in significant concentrations in the effluent at all time points. This finding was confirmed with serum samples and mRNA expression obtained from liver biopsies. The concentrations of these proteins decreased from their initial values over the course of HMP, and mRNA expression levels were decreased by the use of HMP. CONCLUSION IL-1β and tumor necrosis factor (TNF)-α are key mediators of inflammation in IRI. Although difficult to measure because of short half-lives, their downstream effectors indicate their levels of activity. IL-1 receptor antagonist is secreted in response to IL-1β, and monocyte chemotactic protein in response to TNF-α. Their decreased production over the course of HMP suggests that interruption of acute-phase inflammation in the graft may attenuate reperfusion-related graft injury. Further cDNA studies and effluent analyses are required to confirm this hypothesis.

'When good kidneys pump badly': outcomes of deceased donor renal allografts with poor pulsatile perfusion characteristics.

Hypothermic machine perfusion (HMP) is a dynamic preservation technique utilizing a continuous pulsatile flow of solution, rich in metabolic substrates, to enhance viability after reperfusion. HMP has been associated with microvascular stabilization, decrease in oxidative stress as well as improved adenosine triphosphate availability upon reperfusion [1]. HMP has recently evolved into the preferred method of extended criteria donor (ECD) kidney preservation, with extensive utilization in deceased donor (DD) renal transplantation [2–6]. Strong evidence now exists that renal HMP improved early graft function [5–7] and increased the utilization of ECD kidneys [7], with recent reports suggesting a long-term graft survival benefit of HMP [3,6]. Pulsatile perfusion parameters (PPP) of flow (FL) and resistance (RES) are frequently used in the evaluation of donor kidneys and have been thought to be predictive of outcomes. Poor PPP triggered discard in many cases [2,8,9]. We sought to evaluate outcomes of DD kidneys with poor PPP that were transplanted. We identified DD grafts preserved with HMP on the Waters RM3 between 9/1/04 and 2/1/06. Kidneys were perfused with Belzer MPS at 4–6 C with settings of 50 mmHg and 60 pulses/ min. Cases with poor PPP (defined as FL <80 ml/min/ 100 g and RES > 0.4 mmHg/(ml/min/100 g) at the time of organ offer and arrival at our center were included. Donor, preservation and recipient outcomes were recorded. Eighty-nine DD kidneys underwent HMP and were transplanted during the study period. Eleven (12.4%) had PPP. Donor and Recipient age was 45.8 ± 13 and 48.6 ± 11 years respectively. Median donor terminal creatinine was 0.8 mg/dl (r, 0.7–3.7). The median cold ischemia time was 22 h (range: 14–48 h) with a median of 13 h of HMP (range: 6–30 h). Mean flow and renal resistance were 74 ± 6 cc/g/min and 0.46 ± 0.1 respectively. Case-specific donor and recipient variables are summarized in the Tables 1 and 2. All patients received thymoglobulin induction with tacrolimus, mycophenolate mofetil and steroid was discontinued within 7 days. Four patients (36.3%) required hemodialysis (HD) post-transplant, although two required only one HD session for hyperkalemia. The biopsy-proven rejection

Is extracorporeal hypothermic machine perfusion of the liver better than the 'good old icebox'?

Purpose of reviewTo underscore the advantages of hypothermic machine perfusion (HMP) in light of the recent discoveries. Recent findingsThe phase I clinical trial of liver HMP has suggested superiority over static cold storage (SCS) preservation. The liver transplant arena has gained more sophisticated insights into the molecular mechanism and biomechanical understanding behind the benefits of HMP. Its ability to attenuate ischemia-reperfusion injury translates to reduction in ischemia-reperfusion injury with improved early allograft function. HMP has the potential to predict graft function as well as enable resuscitation of grafts from extended criteria donors. Targeted therapeutic interventions via HMP will further contribute to optimal preservation and reconditioning of the allografts. SummaryHMPs advantages do not merely derive from its mechanical forte in maintaining the microvasculature patent. It is also a versatile clinical tool with the ability to deliver metabolic substrates, antioxidants and therapeutic agents to the ex-vivo graft, dilution of waste products generated by inefficient or anaerobic respiration, intraoperative ex-vivo assessment and prediction of the grafts future performance posttransplantation. With demonstrated superiority over SCS, HMP holds promise for expanding the donor pool and becoming the gold standard for liver preservation.