Outcomes of normothermic machine perfusion of liver grafts in repeat liver transplantation (NAPLES initiative).

BACKGROUND: Retransplantation candidates are disadvantaged owing to lack of good-quality liver grafts. Strategies that can facilitate transplantation of suboptimal grafts into retransplant candidates require investigation. The aim was to determine whether late liver retransplantation can be performed safely with suboptimal grafts, following normothermic machine perfusion. METHODS: A prospectively enrolled group of patients who required liver retransplantation received a suboptimal graft preserved via normothermic machine perfusion. This group was compared with both historical and contemporaneous cohorts of patient who received grafts preserved by cold storage. The primary outcome was 6-month graft and patient survival. RESULTS: The normothermic machine perfusion group comprised 26 patients. The historical (cold storage 1) and contemporaneous (cold storage 2) groups comprised 31 and 25 patients respectively. The 6-month graft survival rate did not differ between groups (cold storage 1, 27 of 31, cold storage 2, 22 of 25; normothermic machine perfusion, 22 of 26; P = 0.934). This was despite the normothermic machine perfusion group having significantly more steatotic grafts (8 of 31, 7 of 25, and 14 of 26 respectively; P = 0.006) and grafts previously declined by at least one other transplant centre (5 of 31, 9 of 25, and 21 of 26; P < 0.001). CONCLUSION: In liver retransplantation, normothermic machine perfusion can safely expand graft options without compromising short-term outcomes.

Liver transplantation is a life-saving procedure for many different diseases. In the UK, one in 10 patients awaiting transplant have had a previous liver transplant. These retransplant operations are complex, and the general belief is that a good-quality donor liver graft is required for best outcomes. However, there is a significant shortage of good-quality organs for liver transplantation, so many patients awaiting retransplantation spend longer on the waiting list. This study investigated whether a new technology, called normothermic machine perfusion, could be used to preserve lower-quality donor livers and have successful outcomes for patients undergoing retransplantation. Traditionally, good-quality livers are preserved in an ice box and the study compared the outcomes of these two different approaches. The aim was to prove that normothermic machine perfusion improves access to transplantation for this group of patients, without compromising outcomes. A group of patients who underwent retransplantation and received a lesser-quality liver preserved with normothermic machine perfusion was compared with two groups of patients who had received a transplant with traditional ice-box preservation. The complications, graft, and patient survival of the former group was compared with those in the latter two groups who underwent liver retransplantation with better-quality liver grafts. The rate of survival and adverse surgical outcomes were comparable between the groups of patients who received a liver preserved via traditional ice-box preservation, and those who received a lesser-quality liver preserved via normothermic machine perfusion. Normothermic machine perfusion can potentially expand the number of suitable donor livers available for retransplant candidates.

The effect of end-ischaemic normothermic machine perfusion on donor hepatic artery endothelial integrity.

BACKGROUND: Ex vivo normothermic machine liver perfusion (NMLP) involves artificial cannulation of vessels and generation of flow pressures. This could lead to shear stress-induced endothelial damage, predisposing to vascular complications, or improved preservation of donor artery quality. This study aims to assess the spatial donor hepatic artery (HA) endothelial quality downstream of the cannulation site after end-ischaemic NMLP. METHODS: Remnant HA segments from the coeliac trunk up to the gastroduodenal artery branching were obtained after NMLP (n = 15) and after static cold storage (SCS) preservation (n = 15). Specimens were fixed in 10% neutral buffered formalin and sectioned at pre-determined anatomical sites downstream of the coeliac trunk. CD31 immunohistostaining was used to assess endothelial integrity by a 5-point ordinal scale (grade 0: intact endothelial lining, grade 5: complete denudation). Endothelial integrity after SCS was used as a control for the state of the endothelium at commencement of NMP. RESULTS: In the SCS specimens, regardless of the anatomical site, near complete endothelial denudation was present throughout the HA (median scores 4.5-5). After NMLP, significantly less endothelial loss in the distal HA was present compared to SCS grafts (NMLP vs. SCS: median grade 3 vs. 4.5; p = 0.042). In NMLP specimens, near complete endothelial denudation was present at the cannulation site in all cases (median grade: 5), with significantly less loss of the endothelial lining the further from the cannulation site (proximal vs. distal, median grade 5 vs. 3; p = 0.005). CONCLUSION: Loss of endothelial lining throughout the HA after SCS and at the cannulation site after NMLP suggests extensive damage related to surgical handling and preservation injury. Gradual improved endothelial lining along more distal sites of the HA after NMLP indicates potential for re-endothelialisation. The regenerative effect of NMLP on artery quality seems to occur to a greater extent further from the cannulation site. Therefore, arterial cannulation for machine perfusion of liver grafts should ideally be as proximal as possible on the coeliac trunk or aortic patch, while the site of anastomosis should preferentially be attempted distal on the common HA.

Banff Lung Report: Current knowledge and future research perspectives for diagnosis and treatment of pulmonary antibody-mediated rejection (AMR).

The Lung session of the 2017 14th Banff Foundation for Allograft Pathology Conference, Barcelona focused on the multiple aspects of antibody-mediated rejection (AMR) in lung transplantation. Multidimensional approaches for AMR diagnosis, including classification, histological and immunohistochemical analysis, and donor- specific antibody (DSA) characterization with their current strengths and limitations were reviewed in view of recent research. The group also discussed the role of tissue gene expression analysis in the context of unmet needs in lung transplantation. The current best practice for monitoring of AMR and the therapeutic approach are summarized and highlighted in this report. The working group reached consensus of the major gaps in current knowledge and focused on the unanswered questions regarding pulmonary AMR. An important outcome of the meeting was agreement on the need for future collaborative research projects to address these gaps in the field of lung transplantation.

Transplantation of Declined Liver Allografts Following Normothermic Ex-Situ Evaluation.

The demand for liver transplantation (LT) exceeds supply, with rising waiting list mortality. Utilization of high-risk organs is low and a substantial number of procured livers are discarded. We report the first series of five transplants with rejected livers following viability assessment by normothermic machine perfusion of the liver (NMP-L). The evaluation protocol consisted of perfusate lactate, bile production, vascular flows, and liver appearance. All livers were exposed to a variable period of static cold storage prior to commencing NMP-L. Four organs were recovered from donors after circulatory death and rejected due to prolonged donor warm ischemic times; one liver from a brain-death donor was declined for high liver function tests (LFTs). The median (range) total graft preservation time was 798 (range 724-951) min. The transplant procedure was uneventful in every recipient, with immediate function in all grafts. The median in-hospital stay was 10 (range 6-14) days. At present, all recipients are well, with normalized LFTs at median follow-up of 7 (range 6-19) months. Viability assessment of high-risk grafts using NMP-L provides specific information on liver function and can permit their transplantation while minimizing the recipient risk of primary graft nonfunction. This novel approach may increase organ availability for LT.

Comparison of energy metabolism in liver grafts from donors after circulatory death and donors after brain death during cold storage and reperfusion.

BACKGROUND: Donation after circulatory death (DCD) liver grafts have supplemented the donor organ pool, but certain adverse outcomes have prevented exploration of the full potential of such organs. The aim of this study was to determine key differences in basic energy metabolism between DCD and donation after brainstem death (DBD) grafts. METHODS: Microdialysis samples from DCD and DBD allograft parenchyma from cold storage to 48 h after reperfusion were analysed by colorimetric methods. Interstitial lactate, pyruvate and glycerol levels were measured and the lactate/pyruvate ratio was calculated to estimate energy depletion of the grafts. Histological features of ischaemia and reperfusion injury were assessed. RESULTS: Donor age, extent of steatosis and cold ischaemia time were comparable between ten DCD and 20 DBD organs. DCD grafts had higher levels of interstitial lactate (median 11.6 versus 1.2 mmol/l; P = 0.015) and increased lactate/pyruvate ratio (792 versus 38; P = 0.001) during cold storage. There was no significant difference in glycerol levels between DCD and DBD grafts (225.1 versus 127.5 µmol/l respectively; P = 0.700). Rapid restoration of energy levels with lactate clearance, increased pyruvate levels and reduced lactate/pyruvate ratio was seen following reperfusion of functioning DCD grafts, parallel with levels in DBD grafts. Histology revealed more pronounced glycogen depletion in DCD grafts. Three allografts that failed owing to primary non-function showed energy exhaustion with severe glycogen depletion. CONCLUSION: Liver grafts from DCD donors exhibited depletion of intracellular energy reserves during cold storage. Failed allografts showed severe energy depletion. Modified organ preservation techniques to minimize organ injury related to altered energy metabolism may enable better utilization of donor organs after circulatory death.

Influence of preformed donor-specific antibodies and C4d on early liver allograft function.

INTRODUCTION. The impact of preformed donor-specific antibodies (DSA) is incompletely understood in liver transplantation. The incidence and impact of preformed DSA on early post liver transplant were assessed and these were correlated with compliment fragment C4d on allograft biopsy. METHODS. Pretransplant serum from 41 consecutive liver transplant recipients (brain dead donors; DBD = 27 and cardiac death donors; DCD = 14) were tested for class-specific anti-human leukocyte antigen (HLA) and compared against donor HLA types. Liver biopsies were taken during cold storage (t-1) and post-reperfusion (t0) stained with C4d and graded for preservation-reperfusion injury (PRI). RESULTS. Of the 41 recipients, 8 (20%) had anti-HLA class I/II antibodies pretransplant, 3 (7%) were confirmed preformed DSA; classes I and II (n=1) and class I only (n=2). No biopsies showed definite evidence of antibody-mediated rejection. Graft biopsies in overall showed only mild PRI with ischemic hepatocyte C4d pattern similar in both positive and negative DSA patients. One DSA-positive (33%) compared with four DSA-negative patients (10%) had significant early graft dysfunction; severe PRI causing graft loss from primary nonfunction was seen only in DSA-negative group. Allograft biopsy of preformed DSA-positive patient demonstrated only minimal PRI; however, no identifiable cause could be attributed to graft dysfunction other than preformed DSA. CONCLUSION. Preformed DSA are present in 5-10% liver transplant recipients. There is no association between anti-HLA DSA and PRI and C4d, but preformed DSA may cause early morbidity. Larger studies on the impact of DSA with optimization of C4d techniques are required.

Use of bioelectrical impedance analysis to assess liver steatosis.

BACKGROUND: The shortage of donor livers has led to increased utilization of steatotic marginal livers. Bioelectrical impedance analysis (BIA) uses the principles of electric current flows through tissue, with less resistance offered if the water content is high and the opposite in the presence of fat. Our hypothesis was that liver steatosis would result in an increased resistance to current flow, and correlate with the degree of liver steatosis. METHODS: Before studying cadaveric donor livers for transplantation, this study was performed in patients undergoing liver resection. A total of 37 patients undergoing liver resection for cancer were analysed with BIA, using a handheld, specially calibrated Maltron BIA analyser (BioScan 915) with modified tertrapolar electrodes. These electrodes were applied to the liver surface and resistance was recorded. To validate the results of BIA, a liver biopsy was performed. Histopathology was graded quantitatively as no steatosis, mild, moderate, or severe steatosis according the percentage of fat as well as qualitatively by type of fat (micro and macrovesicular). RESULTS: Bioelectric resistance showed a correlation with macroveiscular steatosis (P = .03). CONCLUSION: BIA is a simple, noninvasive technique and its use should be explored in donor livers to assess steatosis.

End-of-life decisions in medical practice: a survey of doctors in Victoria (Australia).

OBJECTIVES: To discover the current state of opinion and practice among doctors in Victoria, Australia, regarding end-of-life decisions and the legalisation of voluntary euthanasia. Longitudinal comparison with similar 1987 and 1993 studies. DESIGN AND PARTICIPANTS: Cross-sectional postal survey of doctors in Victoria. RESULTS: 53% of doctors in Victoria support the legalisation of voluntary euthanasia. Of doctors who have experienced requests from patients to hasten death, 35% have administered drugs with the intention of hastening death. There is substantial disagreement among doctors concerning the definition of euthanasia. CONCLUSIONS: Disagreement among doctors concerning the meaning of the term euthanasia may contribute to misunderstanding in the debate over voluntary euthanasia. Among doctors in Victoria, support for the legalisation of voluntary euthanasia appears to have weakened slightly over the past 17 years. Opinion on this issue is sharply polarised.

Synbiotic therapy (Bifidobacterium longum/Synergy 1) initiates resolution of inflammation in patients with active ulcerative colitis: a randomised controlled pilot trial.

BACKGROUND AND AIMS: Ulcerative colitis (UC) is an acute and chronic inflammatory disease of the large bowel with unknown aetiology. The immune response against normal commensal microorganisms is believed to drive inflammatory processes associated with UC. Therefore, modulation of bacterial communities on the gut mucosa, through the use of probiotics and prebiotics, may be used to modify the disease state. METHODS: A synbiotic was developed for use in UC patients combining a probiotic, Bifidobacterium longum, isolated from healthy rectal epithelium, and a prebiotic (Synergy 1), a preferential inulin-oligofructose growth substrate for the probiotic strain. Treatment was employed in a double blinded randomised controlled trial using 18 patients with active UC for a period of one month. Clinical status was scored and rectal biopsies were collected before and after treatment, and transcription levels of epithelium related immune markers were measured. RESULTS: Sigmoidoscopy scores (scale 0-6) were reduced in the test group (start 4.5 (1.4), end 3.1 (2.5)) compared with placebo (start 2.6 (2.1), end 3.2 (2.2)) (p=0.06). mRNA levels for human beta defensins 2, 3, and 4, which are strongly upregulated in active UC, were significantly reduced in the test group after treatment (p=0.016, 0.038, and 0.008, respectively). Tumour necrosis factor alpha and interleukin 1alpha, which are inflammatory cytokines that drive inflammation and induce defensin expression, were also significantly reduced after treatment (p=0.018 and 0.023, respectively). Biopsies in the test group had reduced inflammation and regeneration of epithelial tissue. CONCLUSIONS: Short term synbiotic treatment of active UC resulted in improvement of the full clinical appearance of chronic inflammation in patients receiving this therapy.

HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia.

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.

Are parenchymal changes in early post-transplant biopsies related to preservation-reperfusion injury or rejection?

BACKGROUND: The progression of parenchymal changes in liver allograft biopsies due to preservation-reperfusion injury (PRI) and their differentiation from rejection related changes is poorly understood. The aim of this study was to determine which changes in a 1-week posttransplant biopsy could be attributed to PRI and which to acute rejection. METHODS: One week protocol liver transplant biopsies from patients with mild PRI (day 1 AST<400 IU/L) were compared with those from patients with severe PRI (day 1 AST>2000 IU/L). Parenchymal changes (cholestasis, ballooning, steatosis, necrosis) and rejection-related inflammatory features (portal tract inflammation, bile duct inflammation, portal vein endothelial inflammation, hepatic vein endothelial inflammation, and centrilobular inflammation) were blindly assessed semiquantitatively. RESULTS: Fat, cholestasis, and hepatocyte ballooning were significantly worse in the severe PRI group, and these features showed no correlation with histological features related to acute rejection. Centrilobular hepatocyte necrosis correlated with hepatic venular endothelial inflammation and centrilobular inflammation but not with rejection related features in portal tracts or with PRI. These findings suggest that centrilobular necrosis is a manifestation of a rejection-related parenchymal injury and may involve different pathogenetic mechanisms to rejection-related features in portal tracts. CONCLUSIONS: This study indicates that in early posttransplant biopsies, fat, cholestasis, and ballooning can largely be attributed to PRI. By contrast, centrilobular hepatocyte loss should be suspected as a rejection related phenomenon, even if typical portal tract changes are not prominent, and augmentation of immunosuppression should be considered.

Regulation of human beta-defensins by gastric epithelial cells in response to infection with Helicobacter pylori or stimulation with interleukin-1.

Gastric epithelial cells in vitro and in vivo are shown to constitutively express the peptide antibiotic human beta-defensin type 1 (hBD-1). In contrast, hBD-2 expression is regulated in gastric epithelial cells and increases in response to infection with Helicobacter pylori or stimulation with the proinflammatory cytokine interleukin-1. These data suggest that hBD-2 is a component of the regulated host gastric epithelial cell response to H. pylori infection and proinflammatory mediators.

Endothelium during microarterial graft procurement and transplantation.

Microarterial grafts are prone to mechanical endothelial injury that can have profound effects on the transplanted vessel, a factor neglected in studies on vascular changes post-transplantation. The aim of this study was to document the integrity of the endothelial lining after both procurement and transplantation of rat aortic grafts, using a minimal touch technique with and without the use of topical papaverine. It was found that procurement using a simple minimal touch technique preserved the endothelium and transplantation could be performed without endothelial injury. The appearance of "normal" endothelium varied with the degree of distension of the artery, suggesting a dynamic endothelial architecture to accommodate changes in the surface area of the artery during pulsation. These findings indicate that transplantation of an arterial segment without injury to the intima is possible and stress the importance of technical controls after both procurement and transplantation to prevent the use of injured grafts and misleading results.

Coronary arteries from a single coronary ostium in the right coronary sinus: a previously unreported anatomy.

The coronary circulation originating from a single coronary ostium is rare. All possible anatomical variations were the basis of a recent classification. This case report describes a previously unreported IID(1) pattern, comprising a solitary coronary ostium in the right coronary sinus with an anatomical course of the right coronary artery. The left circumflex coronary artery arises from the proximal right coronary artery coursing behind the aorta to the left. The left anterior descending coronary artery arises from the proximal right coronary artery coursing to the left side anterior to the right ventricle.

Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium.

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.

Serum concentrations and peripheral secretion of the beta chemokines monocyte chemoattractant protein 1 and macrophage inflammatory protein 1alpha in alcoholic liver disease.

BACKGROUND: Alcoholic liver disease is associated with increased hepatic expression of monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1alpha (MIP-1alpha). AIMS: To determine whether concentrations of chemokines in the peripheral circulation reflect disease activity, and whether chemokine secretion is restricted to the liver or is part of a systemic inflammatory response in alcoholic liver disease. PATIENTS: Fifty one patients with alcoholic liver disease and 12 healthy controls. METHODS: Peripheral vein (and hepatic vein in patients undergoing transjugular liver biopsy) chemokine concentrations were measured by ELISA. Chemokine secretion and transcription in isolated peripheral mononuclear cells were assessed using ELISA and in situ hybridisation in patients with severe alcoholic hepatitis. RESULTS: Serum MCP-1 concentrations were higher in alcoholic hepatitis compared with cirrhosis or healthy controls. MIP-1alpha concentrations were below the assay sensitivity in most patients. Serum MCP-1 concentrations correlated significantly with serum aspartate aminotransferase and creatinine. In severe alcoholic hepatitis, MCP-1 concentrations were higher in hepatic compared with peripheral veins; in mild alcoholic hepatitis there was no difference. Mononuclear cell secretion of both MCP-1 and MIP-1alpha was higher in severe alcoholic hepatitis compared with healthy controls, and chemokine mRNA was identified in monocytes. CONCLUSIONS: Serum MCP-1 concentrations are raised in alcoholic liver disease and reflect severity of hepatic inflammation. Monocyte secretion of both MCP-1 and MIP-1alpha is increased in severe alcoholic hepatitis. Both intrahepatic sources and peripheral mononuclear cells contribute to the raised serum MCP-1 concentrations.