BACKGROUND:Hypothermic machine perfusion is used to improve renal perfusion and reduce the rate of early and late graft dysfunction. It has been used in our unit since 2001. It has 2 modes of flow: continuous or pulsatile. The aim of this study is to compare the modes of perfusion in terms of perfusion-related parameters, graft survival, and estimated glomerular filtration rate.METHODS:All donation after cardiac death kidneys between 2002 and 2014 were reviewed. A total of 64 pairs of kidneys were identified of which one kidney underwent pulsatile and the other continuous perfusion. Machine parameters including resistance and perfusion flow index levels at 0, 1, 2, 3, and 4 hours were recorded and glutathione S-transferase was measured in perfusate. Estimated glomerular filtration rate from the first week of transplant until the fifth year and graft survival rates were determined.RESULTS:Machine parameters were similar at all time points. Estimated glomerular filtration rates and graft survival were the same irrespective of perfusion mode.CONCLUSION:Pulsatile perfusion may be regarded as more physiological. However, we could not identify difference in outcome following transplant of kidneys from the same donor that had been perfused under pulsatile or continuous conditions.

Introduction Kidney transplantation using donors after circulatory death can be complicated by warm ischaemic damage. The local production of carbon monoxide via the haemo-oxygenase (HMOX-1) pathway has been shown to improve outcomes. In addition, the sphingosine 1-phosphate receptor-1 (S1PR1) interacts with host immune cells and modulates immune reperfusion injury; both of these are controlled upstream by the microRNA( mir) 24-3-p. Ex situ normothermic perfusion (NMP) perfuses a kidney with oxygenated blood prior to implantation. We aimed to deliver beneficial microRNA blockers called anti-sense oligonucleotides (ASOs) during NMP and change the kidney micro-environment prior to impantation. Methods Human kidneys, deemed unsuitable for transplantation, were subjected to 6 hr of NMP using a red cell-based perfusate. Fluorescently labelled anti-miR-24-3p (n=5) or scrambled control ASO (n=5) were introduced in hypothermic perfusate and during NMP. Serial biopsies were taken at t=0, t=60 and t=360 minutes. Real time q-PCR was used to assess the expression of miR-24-3p, S1PR1 and HMOX-1. Other kidneys were perfused with ASO under hypothermic conditions. Results NMP facilitated uptake of ASOs faster than both previous in-vitro and in-vivo animal models (as early as 6 hrs, p= 0.0006), with fluorescent signal identified in both endothelium and proximal tubules. In kidneys perfused with anti-miR-24-3p (n=5) we observed a significant increase in HMOX-1 RNA(18 fold, p = 0.0033) and S1PR1 (4 fold, p =0.0217) at 6 hours. These effects were not seen when the ASO was introduced in the perfusate during hypothermic perfusion or in the scrambled control. Conclusions NMP is a highly effective platform for the pre-transplant delivery of ASO as a short acting genetic modification to human kidneys. NMP allows organ-specific targeting and reduces the risks of systemic side-effects in the transplant recipient. This is a novel and attractive strategy to prevent renal transplant ischaemia-reperfusion injury.

Background. Ex vivo perfusion is used in our unit for kidneys donated after cardiac death (DCD). Perfusion flow index (PFI), resistance, and perfusate glutathione S-transferase (GST) can be measured to assess graft viability. We assessed whether measurements taken during perfusion could predict long-term outcome after transplantation.Methods. All DCD kidney transplants performed from 2002 to 2014 were included in this study. The exclusion criteria were: incomplete data, kidneys not machine perfused, kidneys perfused in continuous mode, and dual transplantation. There were 155 kidney transplantations included in the final analysis. Demographic data, ischemia times, donor hypertension, graft function, survival and machine perfusion parameters after 3 hours were analyzed. Each perfusion parameter was divided into 3 groups as high, medium, and low. Estimated glomerular filtration rate was calculated at 12 months and then yearly after transplantation.Results. There was a significant association between graft survival and PFI and GST (P values, .020 and .022, respectively). PFI was the only independent parameter to predict graft survival.Conclusions. A low PFI during ex vivo hypothermic perfusion is associated with inferior graft survival after DCD kidney transplantation. We propose that PFI is a measure of the health of the graft vasculature and that a low PFI indicates vascular disease and therefore predicts a worse long-term outcome.

Background: Tumour transfer/development is one of the more serious risks associated with transplantation. The behaviour of a tumour can be unpredictable in immunosuppressed recipients. We report a highly sensitive method to monitor tumour behaviour in real time in a rodent tumour transplant model. This paper also explores the effect of MHC matching on tumour growth among control and immunosuppressed hosts.Methods: Luciferase expressing Wistar rat kidney tumour cells were transplanted into either Wistar or Lewis recipients which mimic a well and poorly matched combination to assess the effects of MHC matching on transplanted tumour cells. Experimental groups included controls with no immunosuppression and animals immunosuppressed with cyclosporine. The latter group was further divided into a continuous treatment group which received four weeks of immunosuppression and a treatment withdrawal group where immunosuppression was stopped after two weeks to assess the effects of rejection on tumour growth.Results: All the tumour cells were rejected in the control animals that received no immunosuppression, within 2 weeks among well-matched combination and within one week in the poorly matched combination (p 0.001). The transplanted tumour cells continued to grow in both well-matched and poorly matched groups who were treated with cyclosporine, but growth was significantly faster in the well-matched combination (p 0.033). After treatment withdrawal the tumour cells were rejected in all the animals of the poorly matched group compared to 50% in well matched animals within the four-week study period (p 0.039).Conclusion: In the absence of immunosuppression the hosts reject the transplanted tumour cells, and the antitumour response is stronger when there is a greater mismatch in MHC with the recipient. In the presence of cyclosporine immunosuppression the tumour continues to grow, however, after withdrawal of the immunosuppression, tumour clearance is quicker in the poorly matched background. This data supports the idea of expansion of the donor pool by using kidneys after ex vivo resection of small renal tumours and that these organs should be transplanted into a less well-matched HLA recipient. We hypothesise that should a tumour recurrence occur a poorly matched recipient could clear the tumour through withdrawal of immunosuppression. (C) 2015 Elsevier B.V. All rights reserved.

Introduction: Increasing the usage of hearts from marginal donors and from donors after circulatory death (DCD) has the potential to expand the donor pool. Such hearts may be resuscitated when subjected to ex vivo perfusion. In addition, this may provide an opportunity for viability testing prior to transplant. We describe a novel ex vivo perfusion system designed to reanimate porcine DCD hearts. The same system was subsequently tested using a human heart from a marginal brainstem-dead donor. Method: In the first phase of the study 23 porcine hearts were procured following circulatory death. All hearts were subjected to a period of primary warm ischemia followed by 120 minutes of hypothermic preservation. The period of hypothermic preservation was initially static cold storage (SCS); then oxygenated machine perfusion and finally a combination of static cold storage and oxygen persufflation via the coronary sinus. Ex vivo perfusion of the hearts was performed with a normothermic, oxygenated blood-based solution in our Langendorff system. In the second phase of the study the same system was used to perfuse a human heart from a marginal brainstem-dead donor. This donor had not met criteria for consideration of heart donation. Results: 15 of the 23 (65.2%) DCD porcine hearts reanimated following reperfusion on the ex vivo system. Reanimation was achieved with 63.6% (7/11) in the SCS group; 33.3% (2/6) in the machine perfusion group and 100% (6/6) in the persufflation group. The human heart was placed in the system after a cold ischemic period of 7 hours 4 minutes and perfused with a mid-thermic temperature solution for a further 2 hours 40 minutes to allow for correction of hyperkalemia before warming. The heart started to work after a further hour and then maintained until the experiment was terminated after a further two hours. Conclusion: The ex vivo perfusion system described can potentially resuscitate marginal hearts including those from DCD. The system devised in this study could also be used as a platform to functionally assess marginal human hearts. This mode of viability testing would be an essential step to determine suitability for transplant.

Background: This study reports on the development of a novel method for achieving ex vivo reanimation of hearts from a porcine donation after circulatory death (DCD) model without the use of donor pretreatment.Methods: Porcine hearts (n = 23) were procured 10-29 min after confirmation of asystole. All hearts underwent initial flush with AQIX RS-I solution (London, UK). A 2-h preservation period followed: group 1 hearts (n1-n11) were preserved using static cold storage, group 2 hearts (n12-n17) were preserved using oxygenated, hypothermic machine perfusion (MP), and group 3 hearts (n18-n23) were subjected to retrograde oxygen persufflation. Reperfusion was performed on a Langendorff modification of a Model 33 Functional Circulation circuit. In hearts n16-n23, a dialysis circuit was incorporated into the circuit to facilitate removal of metabolites. The experimental protocol was allowed to follow an evolutionary course, with the aim of achieving greater success with reanimation.Results: In group 1 (static cold storage), 7 of the 11 hearts (63.6%) achieved reanimation on the ex vivo circuit. Two of the six hearts (33.3%) in group 2 (MP) were successfully reanimated. All the six hearts (100%) in group 3 (persufflation) were successfully reanimated. The period of sustained reanimation increased when dialysis was incorporated into the circuit with a maximum of 300 min.Conclusions: Porcine DCD hearts after 29 min of warm ischemia can be reanimated using the method described. A mechanism of reoxygenation (oxygenated MP or coronary sinus oxygen persufflation) during preservation appears mandatory for hearts from DCDs. Persufflation was associated with a higher probability of successful reanimation. Dialysis in the warm phase was useful in removing metabolites that could interfere with reanimation.

Background: Deceased cardiac donors (DCDs) have become a useful source of organs for liver transplantation; nevertheless, there are concerns about the longevity of these grafts. The aim of this study was to evaluate the use of extracorporeal membrane oxygenation (ECMO) to resuscitate DCD porcine livers as a preclinical model using hepatocyte isolation and viability as a marker to assess whole-graft preservation.Materials and methods: We randomized Landrace pigs into three groups after cardiac death and 30 min of warm ischemia: group 1, peritoneal cooling with intravascular cooling for 2 h; group 2, ECMO for 2 h; and group 3, control (conventional intravascular cooling and retrieval). We then reperfused group 1 and 2 livers for 2 h on an ex vivo reperfusion circuit and isolated hepatocytes.Results: After reperfusion, hepatocyte viability was significantly improved in the ECMO group compared to the cooling groups, as measured by trypan blue, methylthiazolyldiphenyl-tetrazolium bromide, and seeding efficiency. Glycogen and reduced glutathione content were significantly used in the ECMO group both before and after reperfusion compared with group 2. The adenosine diphosphate: adenosine triphosphate ratio showed an improved trend (lower) in the ECMO group compared with the cooling group but did not reach statistical significance either before or after reperfusion.Conclusions: This preclinical study suggests that ECMO is a viable technique for liver preservation that gives an improved yield of hepatocytes when isolated from a DCD liver, suggesting improved liver preservation. (C) 2013 Elsevier Inc. All rights reserved.

Introduction: Evasion from immune system is one of the many steps required for tumour development and progression. In allografts tumour can either be transplanted inadvertently or develop de novo. Transplanted tumour being “nonself” to host is expected to be rejected by the immune system in the absence of any immunosuppression. Thus acute rejection could be employed as a therapeutic strategy should a tumour recur by stopping the immunosuppression. This study looks at the influence of matching and role of rejection on transplanted tumours. Methods: Luciferase labelled kidney tumour cells of Wistar origin were injected subcutaneously into either Wistar or Lewis strains mimicking a well and a poorly matched combination respectively. Animals were divided into controls with no immunosuppression and experimental group receiving one of the 3 agents (Cyclosporine, Sirolimus, leflunomide) for 2 weeks followed by treatment withdrawal to mimic acute rejection. Weekly IVIS spectrum imaging was performed to monitor the tumour behaviour.Figure: [Control animals]Results: With no immunosuppression, the transplanted tumour cells were rejected in two weeks in well matched and within one week in poorly matched animals (p < 0.001). In Cyclosporine group, only 4/8 Wistar animals rejected the tumour, while all the Lewis animals rejected the tumour within 2 weeks of treatment withdrawal (p < 0.001). In Sirolimus group, both the Wistars and Lewis rats rejected the tumour within one week of treatment withdrawal as opposed to the treatment continue group which took a total of 4 weeks of treatment to destroy the tumour (p < 0.05). In Leflunomide group, 5/7 animals rejected tumour after treatment withdrawal (Only Wistar rats were studied in this group). Control animals Conclusions: In both the controls and treatment withdrawal groups the tumour rejection was significantly stronger among poorly matched animals. Clinically, patients receiving high risk organs (eg; kidneys from patients with small RCC after ex vivo resection) could benefit from a less well HLA matching, to enable rejection “therapy” should a tumour recur in allograft.

Introduction: Transplantation has immense benefits for the recipients, but there can be some serious risks associated both in short and long terms. One such risk factor is either the development of a de-novo tumour or transmission of a tumour from the donor inadvertently. The use of immunosuppression in transplant recipients can remove the natural checks against cancerous cells by the immune system and thus the behaviour of tumours can be unpredictable. We describe a tumour transplant model to assess the behaviour of transplanted tumour in hosts in the presence of different immunosuppressive agents. Methods: Rat kidney tumour cells (BP36B) were transfected with a lentivirus based construct designed to produce stable transfects that would express both green fluorescence protein (GFP) and Luciferase. The presence of Luciferase can be detected by supplying a substrate, luciferin, which produces a bioluminescent product. The substrate can be injected into live animals and the cells expressing the luciferase be detected in real time. Fixed number of tumour cells of Wistar origin (1.8* 107) were injected subcutaneously into two different strains of rats - Wistar and Lewis; to mimic well-matched (Wistar) and poorly-matched (Lewis) groups. Animals were further divided into treatment group receiving cyclosporine and a control group with no immunosuppression. Tumour behaviour was monitored in real time with IVIS spectrum in vivo imaging system. 10-15 minutes before imaging, the animals were injected with Luciferin (substrate for Luciferase) and the anaesthetised animals were placed in a heated dark chamber and photons emitted from the labelled tumour cells were detected on an ultra sensitive CCD camera. Animals were scanned once weekly for 4-6 weeks and tumour behaviour was monitored in different experimental conditions. Cyclosporine treatment in half of the animals from both strains was stopped midway to further determine the role of rejection on transplanted tumour cells. Tumour depth was determined in some animals with 3D reconstruction and was reconfirmed after post mortem examination in palpable tumours. Results: All the animals displayed good bioluminescent signals. Due to the high sensitivity of the imaging system, we were able to monitor the tumour cells even in the absence of palpable tumour lumps. Significantly varied tumour behaviour under different experimental conditions was observed using this model. Conclusions: This model provides a highly sensitive approach of monitoring tumour behaviour under different experimental conditions even in the absence of palpable tumours. Accurate measurements for local and distant spread can be made in live animals. Furthermore post mortem analysis of the tumour cells can be done with florescent microscopy for tissue invasion and flow cytometry for hematopoietic metastasis.Figure: [Rat image]

Introduction: Recently, kidneys from patients with small renal cell carcinoma (RCC) have been used for transplantation after ex vivo resection of tumour with excellent results. Such an approach can have great potential to increase the donor pool. Concerns regarding the behaviour of the tumour (inadvertently transplanted/recurrence) under the influence of standard immunosuppression prevents this potential source from being popularised. Aim of our study was to compare the tumour behaviour with standard immunosuppression (Cyclosporine) and immunosuppressives with anti proliferative properties (Sirolimus and Leflunomide). Methods: This was a rodent tumour transplant model. Wistar rat kidney tumour cells were labelled with Green Florescent Protein (GFP) and Luciferase to enable real time in vivo monitoring of tumour cells injected into the subcutaneous tissue of Wistar/Lewis rats to mimic well matched and poorly matched groups respectively. Both strains of rats were divided into groups of 12, receiving Cyclosporine (25mg/kg), Sirolimus (2mg/kg) and Sirolimus (0.5mg/kg). Leflunomide (20mg/kg) group comprised of 14 wistar rats. After two weeks of continuous immunosuppression, these groups were further segregated by stopping immunosuppression in half of the animals to monitor the role of rejection. Tumour progression was monitored with IVIS spectrum imaging system once a week. After 4 weeks of study period the animals were humanely euthanased and isolation of splenocytes followed by Flwo cytometric analysis of CD4, CD8, NK and T regulatory cells supplemented gross post mortem findings. Results: With Cyclosporine immunosuppression, tumour continued to grow to palpable sizes among both strains, more in Wistar than in Lewis (p 0.033) for the study period. With high dose Sirolimus, the tumour was eradicated within 2 weeks in all Wistars and 3 weeks in all Lewis rats (p < 0.001 between Cyclosporine and SIrolimus groups). Both Wistar and Lewis rats receiving low dose Sirolimus also eradicated the tumour within four weeks of continuous treatment (p < 0.01 between Cyclosporine and Low dose Sirolimus). In Leflunomide group, 5/7 animals rejected the tumour within the 4 weeks of study period (p < 0.001 between Cyclosporine and Leflunomide).Figure: [Cyclosporine v Sirolimus]Conclusions: 1) Transplanted tumour continues to grow under Cyclosporine immunosuppression 2) Sirolimus (low and high dose) immunosuppression was significantly superior to Cyclosporine and Leflunomide for tumour destruction. 3) Leflunomide immunosuppression was significantly better than cyclosporine. 4) Clinically, recipients of such kidneys should perhaps be immunosuppressed with agents with anti proliferative properties eg Sirolimus to prevent/counter tumour recurrence.

Introduction: Utilisation of hearts from Donors after Circulatory Death (DCD) could increase the donor pool and stem the decline in heart transplantation. Persufflation is a method of introducing oxygen into the organ during the cold preservation phase. This may offset the effects of warm ischaemia inflicted on the DCD heart, enabling a greater chance of functional recovery. Methods: Seventeen cross-Yorkshire Landrace pigs (divided into three groups) were euthanased humanely by Schedule-1 (intravenous administration of phenobarbitone) or exsanguination. The non-beating hearts were procured after being subjected to 10 - 29 minutes of warm ischaemia. All hearts underwent initial antegrade flush with AQIX® RS-I solution (a novel non-phosphate pH buffered preservation solution). Group I and II hearts were subjected to static cold storage (SCS). The Group III hearts were subjected to retrograde oxygen persufflation via the coronary sinus, at a pressure of 12mmHg, whilst the heart was immersed in cold AQIX® RS-I solution. 10-12 small holes were made in the myocardium, using a 21G needle, to create an outlet for excess oxygen. Reperfusion was performed on a Langendorff modification of Model 30 Functional Circulation circuit, using a mixture of heparinised, leukocyte-depleted blood and AQIX® RS-I solution. Drugs (adrenaline, calcium gluconate, dopamine) and DC cardioversion were used to initiate left ventricular activity. Results:[Figure 1Discussion: DCD hearts subjected to preservation using coronary sinus oxygen persufflation can be successfully reanimated. This study compared persufflation with a retrospective control group (SCS). However, persufflation has demonstrated promise as a potential method for introducing oxygen into the heart during the cold preservation phase, with subsequent ability to achieve sustained reanimation.