Successful Transplantation of Human Kidneys Deemed Untransplantable but Resuscitated by Ex Vivo Normothermic Machine Perfusion

We report the successful transplantation of a pair of human kidneys that were declined for transplantation due to inadequate in situ perfusion but subsequently transplanted after perfusion and assessment using ex vivo normothermic perfusion (EVNP). The kidneys were from a 35-year-old man, a donation after circulatory death donor. Both kidneys were declined by all UK transplant centers. On arrival, the kidneys had significant areas of incomplete clearance of blood from the microcirculation that did not clear after a further attempt to flush them. Kidneys underwent 60 min of EVNP with an oxygenated packed red blood cell–based solution warmed to 35.2°C. During EVNP, the patchy areas cleared in both kidneys. The mean renal blood flow and total urine output were 68.0 mL/min/100 g and 560 mL in the left kidney and 59.9 mL/min/100 g, 430 mL in the right, respectively. Based on the EVNP perfusion parameters, both kidneys were deemed suitable for transplantation. They were transplanted without any complications, and both recipients had initial graft function. The serum creatinine levels at 3 months were 1.2 mg/dl in the recipient of the left kidney and 1.62 mg/dl in the recipient of the right kidney. EVNP technology can be used to assess and rescue kidneys previously deemed unsuitable for transplantation.This study was supported by Kidney Research UK. The research was also funded by the National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Organ Donation and Transplantation at the University of Cambridge in collaboration with Newcastle University and in partnership with NHS Blood and Transplant (NHSBT). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health, or NHSBT

Engaging Cold to Upregulate Cell Proliferation in Alginate-Encapsulated Liver Spheroids.

For many years, the impact of hyper- and hypothermia on mammalian cells has been examined. With the exception of short, low temperature storage, which has uses in areas such as preservation for transplantation or regenerative medicine, advantages for the use of low temperature treatment in hepatocytes have not been previously reported. We have observed that alginate-encapsulated HepG2 liver spheroids that are cryopreserved or experience a cold reduction in temperature (≤10°C) for periods between 1 and 90 min display an enhanced cell proliferation during culture 7-16 days post-treatment compared with untreated samples. Following 8-12 days post-treatment, alginate-encapsulated liver spheroids experienced a cell density of 1.71 ± 0.35 times that of control samples (p < 0.001). This effect occurred in samples with a variety of cold treatments. This low temperature treatment offers a simple method to rapidly increase cell proliferation rates for extended culture systems, such as bioartificial liver devices. This would allow the manufacture of required biomass more rapidly, and to a higher cell density, reducing final required biomass volume. This could enable bioartificial liver devices to be prepared more cheaply, making them a more cost effective treatment.Funding for this work was provided through a Medical Research Council (United Kingdom) Industrial Case Studentship (9203) and by Innovate UK (101103) between University College London and Asymptote Ltd. and a Medical Research Council Proximity to Discovery Grant (RG79366) between University of Cambridge and Asymptote Ltd

Pancreatic Allograft Thrombosis: Suggestion for a CT grading system and management algorithm

Pancreatic allograft thrombosis (PAT) remains the leading cause of non-immunological graft failure. Herein we propose a new CT grading system of PAT to identify risk factors for allograft loss and outline a management algorithm by retrospective review of consecutive pancreatic transplants between 2009-2014. Triple-phase CT scans were graded independently by two radiologists as; Grade 0 – no thrombosis, Grade 1 – peripheral thrombosis, Grade 2 – intermediate non-occlusive thrombosis and Grade 3 – central occlusive thrombosis. Twenty-four of 103 (23.3%) recipients were diagnosed with PAT (including grade 1). Three grafts (2.9%) were lost due to portal vein thrombosis. On multivariate analysis, pancreas after SPK/PAK transplant, acute rejection and CT finding peri-pancreatic oedema and/or inflammatory change were significant risk factors of PAT. Retrospective review of CT images revealed more grade 1 and 2 thromboses than were initially reported. There was no significant difference in graft or patient survival, post-operative stay or morbidity of recipients with grade 1 or 2 thrombosis who were or were not anticoagulated. Our data suggest that therapeutic anticoagulation is not necessary for grade 1 and 2 arterial and grade 1 venous thrombosis. The proposed grading system can assist clinicians in decision making and provide standardised reporting for future studies

Protocol of a randomised controlled, open-label trial of ex vivo normothermic perfusion versus static cold storage in donation after circulatory death renal transplantation.

Introduction: Ex vivo normothermic perfusion (EVNP) is a novel technique that reconditions the kidney and restores renal function prior to transplantation. Phase I data from a series of EVNP in extended criteria donor kidneys have established the safety and feasibility of the technique in clinical practice. Methods and analysis: This is a UK-based phase II multicentre randomised controlled trial to assess the efficacy of EVNP compared with the conventional static cold storage technique in donation after circulatory death (DCD) kidney transplantation. 400 patients receiving a kidney from a DCD donor (categories III and IV, controlled) will be recruited into the study. On arrival at the transplant centre, kidneys will be randomised to receive either EVNP (n=200) or remain in static cold storage (n=200). Kidneys undergoing EVNP will be perfused with an oxygenated packed red cell solution at near body temperature for 60 min prior to transplantation. The primary outcome measure will be determined by rates of delayed graft function (DGF) defined as the need for dialysis in the first week post-transplant. Secondary outcome measures include incidences of primary non-function, the duration of DGF, functional DGF defined as <10% fall in serum creatinine for 3 consecutive days in the first week post-transplant, creatinine reduction ratio days 2 and 5, length of hospital stay, rates of biopsy-proven acute rejection, serum creatinine and estimated glomerular filtration rate at 1, 3, 6 and 12 months post-transplant and patient and allograft survival. The EVNP assessment score will be recorded and the level of fibrosis and inflammation will also be measured using tissue, blood and urine samples. Ethics and dissemination. The study has been approved by the National Health Service (NHS) Health Research Authority Research Ethics Committee. The results are expected to be published in 2020. Trial registration number: ISRCTN15821205; Pre-results.Kidney Research UK (SP/MEKC/1/2014); University of Cambridge and University Hospitals of Cambridge Foundation Trust, Cambridge CB2 OQQ

Ex vivo normothermic perfusion of isolated segmental porcine bowel: a novel functional model of the small intestine.

BACKGROUND: There is an unmet need for suitable ex vivo large animal models in experimental gastroenterology and intestinal transplantation. This study details a reliable and effective technique for ex vivo normothermic perfusion (EVNP) of segmental porcine small intestine. METHODS: Segments of small intestine, 1.5-3.0 m in length, were retrieved from terminally anaesthetized pigs. After a period of cold ischaemia, EVNP was performed for 2 h at 37°C with a mean pressure of 80 mmHg using oxygenated autologous blood diluted with Ringer's solution. The duration of EVNP was extended to 4 h for a second set of experiments in which two segments of proximal to mid-ileum (1.5-3.0 m) were retrieved from each animal and reperfused with whole blood (control) or leucocyte-depleted blood to examine the impact of leucocyte depletion on reperfusion injury. RESULTS: After a mean cold ischaemia time of 5 h and 20 min, EVNP was performed in an initial group of four pigs. In the second set of experiments, five pigs were used in each group. In all experiments bowel segments were well perfused and exhibited peristalsis during EVNP. Venous glucose levels significantly increased following luminal glucose stimulation (mean(s.e.m.) basal level 1.8(0.6) mmol/l versus peak 15.5(5.8) mmol/l; P < 0.001) and glucagon-like peptide 1 (GLP-1) levels increased in all experiments, demonstrating intact absorptive and secretory intestinal functions. There were no significant differences between control and leucocyte-depleted animals regarding blood flow, venous glucose, GLP-1 levels or histopathology at the end of 4 h of EVNP. CONCLUSIONS: This novel model is suitable for the investigation of gastrointestinal physiology, pathology and ischaemia reperfusion injury, along with evaluation of potential therapeutic interventions

Use of Ex Vivo Normothermic Perfusion for Quality Assessment of Discarded Human Donor Pancreases.

A significant number of pancreases procured for transplantation are deemed unsuitable due to concerns about graft quality and the associated risk of complications. However, this decision is subjective and some declined grafts may be suitable for transplantation. Ex vivo normothermic perfusion (EVNP) prior to transplantation may allow a more objective assessment of graft quality and reduce discard rates. We report ex vivo normothermic perfusion of human pancreases procured but declined for transplantation, with ABO-compatible warm oxygenated packed red blood cells for 1-2 h. Five declined human pancreases were assessed using this technique after a median cold ischemia time of 13 h 19 min. One pancreas, with cold ischemia over 30 h, did not appear viable and was excluded. In the remaining pancreases, blood flow and pH were maintained throughout perfusion. Insulin secretion was observed in all four pancreases, but was lowest in an older donation after cardiac death pancreas. Amylase levels were highest in a gland with significant fat infiltration. This is the first study to assess the perfusion, injury, as measured by amylase, and exocrine function of human pancreases using EVNP and demonstrates the feasibility of the approach, although further refinements are required.This study was financially supported by a grant from the Mason Medical Research Foundation.This is the author accepted manuscript. The final version is available via Wiley at http://onlinelibrary.wiley.com/doi/10.1111/ajt.13303/abstract

Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue.

Pediatric liver transplantation is often required as a consequence of biliary disorders because of the lack of alternative treatments for repairing or replacing damaged bile ducts. To address the lack of availability of pediatric livers suitable for transplantation, we developed a protocol for generating bioengineered biliary tissue suitable for biliary reconstruction. Our platform allows the derivation of cholangiocyte organoids (COs) expressing key biliary markers and retaining functions of primary extra- or intrahepatic duct cholangiocytes within 2 weeks of isolation. COs are subsequently seeded on polyglycolic acid (PGA) scaffolds or densified collagen constructs for 4 weeks to generate bioengineered tissue retaining biliary characteristics. Expertise in organoid culture and tissue engineering is desirable for optimal results. COs correspond to mature functional cholangiocytes, differentiating our method from alternative organoid systems currently available that propagate adult stem cells. Consequently, COs provide a unique platform for studies in biliary physiology and pathophysiology, and the resulting bioengineered tissue has broad applications for regenerative medicine and cholangiopathies

RNF43/ZNRF3 loss predisposes to hepatocellular-carcinoma by impairing liver regeneration and altering the liver lipid metabolic ground-state

RNF43/ZNRF3 negatively regulate WNT signalling. Both genes are mutated in several types of cancers, however, their contribution to liver disease is unknown. Here we describe that hepatocyte-specific loss of Rnf43/Znrf3 results in steatohepatitis and in increase in unsaturated lipids, in the absence of dietary fat supplementation. Upon injury, Rnf43/Znrf3 deletion results in defective hepatocyte regeneration and liver cancer, caused by an imbalance between differentiation/proliferation. Using hepatocyte-, hepatoblast- and ductal cell-derived organoids we demonstrate that the differentiation defects and lipid alterations are, in part, cell-autonomous. Interestingly, ZNRF3 mutant liver cancer patients present poorer prognosis, altered hepatic lipid metabolism and steatohepatitis/NASH signatures. Our results imply that RNF43/ZNRF3 predispose to liver cancer by controlling the proliferative/differentiation and lipid metabolic state of hepatocytes. Both mechanisms combined facilitate the progression towards malignancy. Our findings might aid on the management of those RNF43/ZNRF3 mutated individuals at risk of developing fatty liver and/or liver cancer

Assessment of H2_{2}S in vivo using the newly developed mitochondria-targeted mass spectrometry probe MitoA

Hydrogen sulfide (H$_{2}$S) is produced endogenously in vivo and has multiple effects on signaling pathways and cell function. Mitochondria can be both an H$_{2}$S source and sink, and many of the biological effects of H$_{2}$S relate to its interactions with mitochondria. However, the significance of mitochondrial H$_{2}$S is uncertain, in part due to the difficulty of assessing changes in its concentration in vivo Although a number of fluorescent H$_{2}$S probes have been developed these are best suited to cells in culture and cannot be used in vivo To address this unmet need we have developed a mitochondria-targeted H$_{2}$S probe, MitoA, which can be used to assess relative changes in mitochondrial H$_{2}$S levels in vivo MitoA comprises a lipophilic triphenylphosphonium (TPP) cation coupled to an aryl azide. The TPP cation leads to the accumulation of MitoA inside mitochondria within tissues in vivo There, the aryl azido group reacts with H$_{2}$S to form an aryl amine (MitoN). The extent of conversion of MitoA to MitoN thus gives an indication of the levels of mitochondrial H$_{2}$S in vivo Both compounds can be detected sensitively by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tissues, and quantified relative to deuterated internal standards. Here we describe the synthesis and characterization of MitoA and show that it can be used to assess changes in mitochondrial H$_{2}$S levels in vivo As a proof of principle we used MitoA to show that H$_{2}$S levels increase in vivo during myocardial ischemia.This work was supported in part by Medical Research Council UK Grant MC_U105663142, Wellcome Trust Investigator award 110159/Z/15/Z (to M. P. M.), Biotechnology and Biological Sciences Research Council Grant BB/I012826/1, Wellcome Trust Investigator award 110158/Z/15/Z (to R. C. H.), and a Consejo Nacional de Ciencia y Technología studentship (to C. B.-G.)

Selection of Oncogenic Mutant Clones in Normal Human Skin Varies with Body Site

Skin cancer risk varies substantially across the body, yet how this relates to the mutations found in normal skin is unknown. Here we mapped mutant clones in skin from high- and low-risk sites. The density of mutations varied by location. The prevalence of NOTCH1 and FAT1 mutations in forearm, trunk, and leg skin was similar to that in keratinocyte cancers. Most mutations were caused by ultraviolet light, but mutational signature analysis suggested differences in DNA-repair processes between sites. Eleven mutant genes were under positive selection, with TP53 preferentially selected in the head and FAT1 in the leg. Fine-scale mapping revealed 10% of clones had copy-number alterations. Analysis of hair follicles showed mutations in the upper follicle resembled adjacent skin, but the lower follicle was sparsely mutated. Normal skin is a dense patchwork of mutant clones arising from competitive selection that varies by location. / Significance: Mapping mutant clones across the body reveals normal skin is a dense patchwork of mutant cells. The variation in cancer risk between sites substantially exceeds that in mutant clone density. More generally, mutant genes cannot be assigned as cancer drivers until their prevalence in normal tissue is known