Liver Perfusion Research Articles

Multiparametric Flow Cytometry-Based Immunophenotyping of Mouse Liver Immune Cells.

The liver is a complex organ that governs many types of metabolisms, including energy metabolism and other cellular processes. The liver also plays a crucial role in important functions in immunity, and the activity of liver tissue-associated immunity affects the outcome of many liver pathologies. A thorough characterization of the liver immune microenvironment may contribute to a better understanding of immune signaling, the mechanisms of specific immune responses, and even to improved predictions about therapy outcomes. In this paper, we present an optimized, simple, and rapid protocol to characterize the liver-associated immune cell milieu. We believe that the most suitable technique for obtaining a complex immune cell suspension and for removing contaminating blood cells is to perform mouse liver perfusion, using only phosphate buffer saline. Combining an enzymatic digestion and a mechanical dissociation of liver tissue, followed by cell purification, improves downstream applications. This combination is an essential prerequisite for immune cell determination and characterization. We then demonstrate a flow cytometry-based multiparametric immunophenotyping along with a gating strategy to detect and quantify liver endothelial cells, T cells (helper and cytotoxic), B cells, NK cells, NKT cells, neutrophils, monocytes (subsets included), dendritic cells (subsets included), macrophages and Kupffer cells.

Normothermic liver machine perfusion as a dynamic platform for regenerative purposes: What does the future have in store for us?

Liver transplantation has become an immense success; nevertheless, far more recipients are registered on waiting lists than there are available donor livers for transplantation. High-risk, extended criteria donor livers are increasingly used to reduce the discrepancy between organ demand and supply. Especially for high-risk livers, dynamic preservation using machine perfusion can decrease post-transplantation complications and may increase donor liver utilisation by improving graft quality and enabling viability testing before transplantation. To further increase the availability of donor livers suitable for transplantation, new strategies are required that make it possible to use organs that are initially too damaged to be transplanted. With the current progress in experimental liver transplantation research, (long-term) normothermic machine perfusion may be used in the future as a dynamic platform for regenerative medicine approaches, enabling repair and regeneration of injured donor livers. Currently explored therapeutics such as defatting cocktails, RNA interference, senolytics, and stem cell therapy may assist in the repair and/or regeneration of injured livers before transplantation. This review will provide a forecast of the future utility of normothermic machine perfusion in decreasing the imbalance between donor liver demand and supply by enabling the repair and regeneration of damaged donor livers.

P11.11: A New Designed Machine Perfusion System to Mimic the Cardiac Cycle’s Heart Function for a Better Hepatic Microcirculation Perfusion

Background: Machine perfusion is a relatively new concept developed for protecting marginal liver transplantation. However, so far, limited studies have reported on liver transplantation donated after cardiac death (DCD). Objective: To establish and verify a novel machine perfusion concept to eliminate these risk factors and recover DCD livers. Methods: To explore a novel hypothermic oxygenated perfusion (HOPE) system, two pumps and an elastic water sac were integrated into the perfusion system to mimic the cardiac cycle’s heart function. Compared to the other two traditional systems (HOPE S1 and S2), the novel HOPE system (HOPE S3) was verified in rats by no injured livers (perfused with methylene blue diluted by UW-G) or DCD livers subjected to 60 min in situ warm ischemia, without application of heparin. Liver perfusion outcomes were compared using macroscopy, microscopy, molecular tests, and orthotopic liver transplantation (OLT). Results: DCD livers, subjected to HOPE systems’ perfusion, disclosed reduced injury and improved survival compared to static cold storage after warm ischemia of 60 min (DCD+SCS). The post-transplantation survival rates of 4 weeks were 0%, 20 and 30% in DCD+SCS, DCD+SCS+HOPE S1, and DCD+SCS+HOPE S2 group, respectively. In contrast, HOPE S3 protected from hepatocyte and non-parenchymal cell injury and led to 60% (6/10) animal survival after 60 min of warm donor ischemia (DCD+SCS+HOPE S3). These data were further demonstrated by monitoring the hepatic sinusoid microcirculation, function morphological, and molecular changes of preserved livers. Conclusion: The newly designed novel machine perfusion system achieved complete and homogeneous liver perfusion and is regarded as a useful tool for resuscitating DCD liver grafts with severe warm ischemic injuries. Key words：Liver transplantation; Hypothermic oxyenated perfusion; Donation after cardiac death; Organ recovery; Perservayion manner; Ischemic reperfusion injury; Static cold storage; Hepatic sinusoidal; Hepatic microcirculation; Inferior vena cava. National Natural Science Foundation of China (Grant No. 81970548). Medical Science Advancement Program (Youth Scholars) of Wuhan University (Grant No. TFZZ2018035). Science and Technology Innovation Cultivation Fund of Zhongnan Hospital of Wuhan University(No.znpy2019041).

313.5: Meeting Packed Red Blood Cell Requirement for the Transmedics Organ Care System (OCS) Liver Transplantation Trial (PROTECT Trial) At Henry Ford Hospital

Introduction: In January 2016, HFHS Transplant Institute began the PROSPECT trial, an interventional trial studying normothermic preservation of the donated liver before transplant using the Liver Organ Care System (OCSTM). The Henry Ford Hospital Transfusion Service (TS) provided the required packed red blood cells (PRBC). Background: The TransMedics (OCSTM) liver trial was an international, randomized trial studying the intervention of a portable liver system to preserve donor livers for transplant. The system utilizes an oxygenated blood-based perfusate mixed with nutrient solution in normothermic perfusion for organ preservation. The TS provided PRBC needed for the study, final PRBC used dictated by the condition and size of the retrieved liver. TS study goal was to secure proper transfusion needs for liver perfusion while also adhering to blood inventory management. Methods: Organ procurement candidates for the PROTECT trial were selected based on pre-determined conditions and consented according to that protocol. Once the patient was consented and the liver transplantation was scheduled, the PROTECT trial team notified the TS,and 5 units of group O Positive, leukoreduced PRBC less than 10 days old were selected, and placed inside a temperature-controlled packaging container along with a remote temperature monitoring device. The container was then transported with the procurement team to the harvesting location. Once the insulated container was returned to the TS, the temperature-monitoring device information was uploaded to verify that the returned PRBC have been stored within their proper temperature limits. Results: The TS support for the PROTECT trial at HFHS occurred from January 2016 until September 2021. The table below shows the activity related to this trial. - Issued Utilized Returned Wasted PRBC units 240 132 104 4 In summary, 29/48 study events used 4 units; 3/48 study events used 5 units; on 15/48 events the organ harvesting was cancelled; all units returned had an acceptable temperature range and returned to the available inventory. For one study event, 4 PRBC were spiked in preparation for use in the perfusate prior to the event being cancelled. These PRBC were discarded and deemed wasted. Conclusions: During the PROTECT trial no PRBCs were wasted due to inappropriate storage conditions. The joint use of a temperature-controlled container and remote temperature monitoring device allowed for the appropriate return of unused PRBCs to the TS blood inventory, thus avoiding any wastage. Only 3/48 events used 5 units, the\us issuing 4 PRBC to meet the OCS™ support may suffice, further decreasing the risk for wastage of any PRBC units. After receiving FDA in September 2021, alternatives to maintain the required PRBC temperature should be addressed to minimize wastage at this time of limited blood availability for the support of the TransMedics Organ Care System (OCSTM) for liver organ perfusion.

P11.07: BMSCs Combined With Normothermic Machine Perfusion Resuscitate Aged Donor Liver With Long Warm Ischemia Time in Rats

Background: Both aged and donation after cardiac death (DCD) donor liver, considered as marginal organs, were used for transplantation due to huge organ shortage in the world. Organs combined with both danger factors would impair the donor liver viability. Using normothermic machine perfusion (NMP), we investigated the possibility of mesenchymal stem cells (MSCs) to revive the aged DCD donor liver in a rat model. Methods: The SD rats used for NMP were all over 14 months of age. BMSCs were isolated from the bone marrow of SD rats. Rats were anesthetized and cardiac arrest was induced by compression of the heart for 30 minutes as warm ischemia prior to donor liver harvesting. The liver was connected to a rat liver perfusion machine for NMP with or without 10^6 BMSCs infusion. Results: The weight of the aged liver in the experimental and control groups was 23.03 ± 0.25 g and 21.40 ± 0.87 g. the donor liver appeared to multiple bruised foci in both prior- and post- perfusion, while liver with BMSCs perfusion had fewer bruised foci (Figure 1A, B). The pH was maintained between 7.35 and 7.45 in both groups during perfusion. Lactate in the BMSCs group decreased after 1 hour during perfusion and fell below 1 gradually within 3 hours, while in the control group it decreased more slowly and remained around 4 even after 4 hours of perfusion (Figure 1C). Pathological appearance showed that the pathological Suzuki‘s sores of liver in the control group were higher than MSCs group, with bruised liver sinusoids, apoptosis and focal necrosis of hepatocytes. In contrast, pathological change in the liver of the BMSC group showed significant improvement with no obvious necrosis and structural integrity of the liver lobules (Figure 1D). The climbing rate and peak value of AST and ALT in the BMSCs group were lower than that in the control group, indicating that BMSCs protected liver from damage during perfusion (Figure 1E, 1F). Conclusion: Our results demonstrate that the use of MSCs during normothermic machine perfusion is a feasible strategy to resuscitate the aged DCD donor liver.National Natural Science Foundation of China (81401324 and 81770410).

P11.27: Changes in Markers of Coagulation and Fibrinolysis Offer Insights Into Pathophysiological Aspects of Preservation Injury During Normothermic Liver Perfusion

Background: The perfusate used in normothermic machine perfusion (NMP) is mainly composed of red packed blood cells and colloid solution and is therefore free from thrombocytes and coagulation factors. Our aim was to monitor changes in composition of coagulation factors during NMP and to investigate their association with liver function during NMP and after transplantation. Methods: NMP has been performed on a heterogenous study group of 16 grafts that included 12 extended criteria donor livers. Factor V activity (FV), factor XIII activity (FXIII), van Willebrand factor activity (vWF), D-dimer, plasminogen-activator-inhibitor (PAI-1) and thrombocyte count in perfusate were assessed. Liver and bile duct biopsies were taken before and after perfusion and after reperfusion. Median follow-up after transplantation was 11 months (IQR: 6.4-12). Results: NMP and assessment of 16 livers resulted in 10 livers that were successfully transplanted. Main reasons for decline included inability to clear lactate, low bile quality, or signs of fibrosis in the frozen section. Two livers presented with increased D-dimer levels during perfusion (8.7 µg/mL and 12.3 µg/mL vs. mean peak D-dimer of 3.7 µg/mL SD: 3.1). One liver was transplanted, the histological analysis of the bile duct biopsies taken before implantation showed sclerosing cholangitis. Additionally, vWF activity during perfusion of this liver was increased (13 % vs. mean peak vWF activity of 6.4% SD: 2.5). The recipient had to be treated with a stent for an anastomotic stricture. The second liver with elevated D-dimer was severely steatotic and the only liver out of the study group that did not produce bile and was therefore declined for transplantation. The graft additionally presented with high levels of PAI-1 (299 IU/mL vs. mean peak PAI-1 of 186 IU/mL SD: 155) and increased FXIII activity in perfusate (158 % vs. mean peak FXIII activity 45% SD: 49). PAI-1 was also elevated in another three livers during perfusion, of which one was discarded due to increasing lactate after initial clearing and high grade of steatosis (peak PAI-1 605 IU/mL). Both other livers were successfully transplanted, one of the two recipients later developed an anastomotic stricture. Each of the three cases with increased PAI-1 levels coincided with peaks in either thrombocyte count, bilirubin or FV activity. Conclusion: Changes in perfusate composition of coagulation and fibrinolysis factors offer interesting new insights in pathophysiological aspects of preservation injury and might allow for additional graft assessment during longer courses of NMP. Prospective studies are needed to validate these initial findings.

313.16: Glycocalyx Damage Marker Syndecan-1 Correlates With Early Allograft Dysfunction During Hypothermic Liver Machine Perfusion

Introduction: The endothelial glycocalyx is mainly consisting of Syndecan-1 (Sdc-1) which is released into human serum upon glycocalyx degradation. Glycocalyx alterations are connected to different aspects of organ damage and have been recently investigated in the context of liver transplantation. Due to shortage of donor organs, new preservation methods as hypothermic oxygenated machine perfusion (HOPE) have been introduced to enable transplantation of organs with a higher risk profile. For safe expansion of the donor pool, reliable organ assessment markers for evaluation during machine perfusion are missing. We aimed to measure glycocalyx damage during hypothermic liver perfusion. Methods: HOPE was performed with the Organ Assist® perfusion system on 40 livers, prior to organ transplantation. Samples were collected during (perfusate at 0 and 60 min) and after HOPE (effluent). Sdc-1 concentration in samples was measured by ELISA as indicator for glycocalyx destruction. We compared clinical parameters with Sdc-1 levels in patients regarding the development of early allograft dysfunction (EAD) using Mann-Whitney U test, Pearson correlation and receiver operating characteristics (ROC). Results: The 13 patients which developed EAD, showed an elevation in Sdc-1 concentration compared to non-EAD patients during HOPE at 0 min: [598 (±526) vs. 276 (±150) ng/ml; p=0.076] and 60 min: [1099 (±739) vs. 521 (±382) ng/ml; p=0.016], as well as afterwards in the effluent: [2074 (±1273) vs. 443 (±226) ng/ml; p=0.001 (n=15, 4 with EAD)]. Concentration of Sdc-1 during HOPE correlated with EAD at 0 min: (R=0.433, p=0.006), at 60 min: (R=0.471, p=0.003) and in the effluent: (R=0.769, p<0.001, n=15, 4 with EAD). Furthermore, an association between EAD and Sdc-1 concentrations was indicated by ROCs: during HOPE at 60 minutes: (AUC=0.704 and p=0.018) and in the effluent: (AUC=1 and p=0.004 (n=15, 4 with EAD)). No association of graft survival with Sdc-1 (p=0.339) was detected in cox regression analysis however, reduced graft survival (log-rank=0.009) of EAD patients compared to those without EAD could be shown. Conclusion: We found that measuring glycocalyx degradation during HOPE could indicate transplantation outcome regarding EAD. Consequently, we argue that Sdc-1 could be a useful biomarker for organ assessment during HOPE.

Machine Perfusion as "Comfort Zone": What Are Key Challenges of Liver Viability Assessment Today?

Machine Perfusion as "Comfort Zone": What Are Key Challenges of Liver Viability Assessment Today?

Predicting Early Allograft Function After Normothermic Machine Perfusion.

Normothermic ex situ liver perfusion is increasingly used to assess donor livers, but there remains a paucity of evidence regarding criteria upon which to base a viability assessment or criteria predicting early allograft function. Perfusate variables from livers undergoing normothermic ex situ liver perfusion were analyzed to see which best predicted the Model for Early Allograft Function score. One hundred fifty-four of 203 perfused livers were transplanted following our previously defined criteria. These comprised 84/123 donation after circulatory death livers and 70/80 donation after brain death livers. Multivariable analysis suggested that 2-h alanine transaminase, 2-h lactate, 11 to 29 mmol supplementary bicarbonate in the first 4 h, and peak bile pH were associated with early allograft function as defined by the Model for Early Allograft Function score. Nonanastomotic biliary strictures occurred in 11% of transplants, predominantly affected first- and second-order ducts, despite selection based on bile glucose and pH. This work confirms the importance of perfusate alanine transaminase and lactate at 2-h, as well as the amount of supplementary bicarbonate required to keep the perfusate pH > 7.2, in the assessment of livers undergoing perfusion. It cautions against the use of lactate as a sole indicator of viability and also suggests a role for cholangiocyte function markers in predicting early allograft function.

Possible Role of Extracellular Vesicles in Hepatotoxicity of Acetaminophen.

We examined proteomic profiles of rat liver extracellular vesicles (EVs) shed following treatment with a sub-toxic dose (500 mg/kg) of the pain reliever drug, acetaminophen (APAP). EVs representing the entire complement of hepatic cells were isolated after perfusion of the intact liver and analyzed with LC-MS/MS. The investigation was focused on revealing the function and cellular origin of identified EVs proteins shed by different parenchymal and non-parenchymal liver cells and their possible role in an early response of this organ to a toxic environment. Comparison of EV proteomic profiles from control and APAP-treated animals revealed significant differences. Alpha-1-macroglobulin and members of the cytochrome P450 superfamily were highly abundant proteins in EVs shed by the normal liver. In contrast, proteins like aminopeptidase N, metalloreductase STEAP4, different surface antigens like CD14 and CD45, and most members of the annexin family were detected only in EVs that were shed by livers of APAP-treated animals. In EVs from treated livers, there was almost a complete disappearance of members of the cytochrome P450 superfamily and a major decrease in other enzymes involved in the detoxification of xenobiotics. Additionally, there were proteins that predominated in non-parenchymal liver cells and in the extracellular matrix, like fibronectin, receptor-type tyrosine-protein phosphatase C, and endothelial type gp91. These differences indicate that even treatment with a sub-toxic concentration of APAP initiates dramatic perturbation in the function of this vital organ.

Non-hepatotropic viral hepatitis and its causative pathogens: The ongoing need for monitoring in children with severe acute hepatitis of unknown etiology.

Non-hepatotropic viral hepatitis and its causative pathogens: The ongoing need for monitoring in children with severe acute hepatitis of unknown etiology.

The metabolic and toxic acute effects of phloretin in the rat liver

The current study sought to evaluate the acute effects of phloretin (PH) on metabolic pathways involved in the maintenance of glycemia, specifically gluconeogenesis and glycogenolysis, in the perfused rat liver. The acute effects of PH on energy metabolism and toxicity parameters in isolated hepatocytes and mitochondria, as well as its effects on the activity of a few key enzymes, were also evaluated. PH inhibited gluconeogenesis from different substrates, stimulated glycogenolysis and glycolysis, and altered oxygen consumption. The citric acid cycle activity was inhibited by PH under gluconeogenic conditions. Similarly, PH reduced the cellular ATP/ADP and ATP/AMP ratios under gluconeogenic and glycogenolytic conditions. In isolated mitochondria, PH inhibited the electron transport chain and the FoF1–ATP synthase complex as well as acted as an uncoupler of oxidative phosphorylation, inhibiting the synthesis of ATP. PH also decreased the activities of malate dehydrogenase, glutamate dehydrogenase, glucose 6-phosphatase, and glucose 6-phosphate dehydrogenase. Part of the bioenergetic effects observed in isolated mitochondria was shown in isolated hepatocytes, in which PH inhibited mitochondrial respiration and decreased ATP levels. An aggravating aspect might be the finding that PH promotes the net oxidation of NADH, which contradicts the conventional belief that the compound operates as an antioxidant. Although trypan blue hepatocyte viability tests revealed substantial losses in cell viability over 120 min of incubation, PH did not promote extensive enzyme leakage from injured cells. In line with this effect, only after a lengthy period of infusion did PH considerably stimulate the release of enzymes into the effluent perfusate of livers. In conclusion, the increased glucose release caused by enhanced glycogenolysis, along with suppression of gluconeogenesis, is the opposite of what is predicted for antihyperglycemic agents. These effects were caused in part by disruption of mitochondrial bioenergetics, a result that should be considered when using PH for therapeutic purposes, particularly over long periods and in large doses.

O023 Artificial intelligence in photography of normothermic machine perfusion of livers

Abstract Introduction Livers donated for use in transplantation are underutilised. A lack of clear guidelines regarding the visual assessment of their quality means many borderline grafts are needlessly discarded; macroscopic visual assessment by transplanting clinicians is subjective and inconsistent. Deep learning (DL), a form of artificial intelligence, has previously been used to aid medical image analysis. Normothermic machine perfusion (NMP) is often used to evaluate borderline livers for transplantation. This study aimed to develop an objective DL model to visually assess organ transplantability during NMP. Methods Five DL models were trained and tested on 100 images of donor livers, each labelled with scores from three transplant clinicians on steatosis, perfusion, and transplantability. Models were trained to classify liver transplantability through either image data and transplantability scoring data, or image data and all clinician scoring data. Model accuracy, specificity, and sensitivity were calculated. Evaluations of clinician scoring agreement were carried out using intraclass correlation coefficient (ICC) and Fleiss kappa. Results In the classification of transplantability, the highest performing models achieved training and testing accuracies of 64.3% and 76%, respectively. Sensitivity and specificity ranged between 44.1%–94.1% and 6.3%–75%, respectively. ICC and Fleiss kappa values indicated a fair-to moderate scoring agreement between clinicians. Conclusion The performance of DL image analysis in assessing liver quality during NMP has been modest. Visual assessment during NMP is more challenging than on the back table at organ retrieval. Broader, more varied data sets are required to maximise model performance. Take-home message Livers donated for use in transplantation are underutilised due to a lack of clear guidelines and the subjective nature of their visual assessment. Artificial intelligence can be used to consistently and objectively assess donated livers in order to improve their utilisation.

Acute ischaemic hepatitis with secondary coagulopathy as an incidental finding on 99m Tc-3,3-diphosphono-1,2-propanedicarboxylic acid (99m Tc-DPD) scintigraphy

Acute ischaemic hepatitis with secondary coagulopathy as an incidental finding on 99m Tc-3,3-diphosphono-1,2-propanedicarboxylic acid (99m Tc-DPD) scintigraphy

Series of Intravoxel Incoherent Motion and T2* Magnetic Resonance Imaging Mapping in Detection of Liver Perfusion Changes and Regeneration Among Partial Hepatectomy in Sprague-Dawley Rats

To evaluate liver perfusion changes and their effect on liver regeneration (LR) after partial hepatectomy (PH) using intravoxel incoherent motion (IVIM) and T2* mapping in a rat model. One hundred and two rats underwent 30%, 50%, or 70% PH. Within each group (n=34), rats in MR imaging subgroup (n=10) underwent liver IVIM and T2* mapping before and within 2 h, 1, 2, 3, 5, 7, 14, and 21 days post-PH to measure D*, perfusion fraction (PF), and T2* values. Three rats from histologic subgroup (n=24) sacrificed at each time point for hepatocyte Ki-67 indices and diameters measurement. Liver D* and PF values decreased immediately post-PH, then returned to original level as LR progressed in all groups. PF values in 70% PH group were significantly lower than in the other two groups (p < .05). D* and PF values correlated significantly with hepatocyte Ki-67 indices (r = -0.588 to -0.915; p < .05) and hepatocyte diameter (r = -0.555 to -0.792; p < .05). Liver T2* values decreased immediately within 2 h post-PH, then increased to a high level and followed with returning to original level gradually. The duration of the high T2* levels was consistent with Ki-67 indices. Liver perfusion decreased immediately followed with increasing gradually after PH. IVIM and T2* mapping are promising methods for monitoring changes of liver perfusion. IVIM-derived D* value is the best indicator in reflecting the process of LR noninvasively.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Hepatoprotective Role of Carvedilol against Ischemic Hepatitis Associated with Acute Heart Failure via Targeting miRNA-17 and Mitochondrial Dynamics-Related Proteins: An In Vivo and In Silico Study.

Acute heart failure (AHF) is one of the most common diseases in old age that can lead to mortality. Systemic hypoperfusion is associated with hepatic ischemia–reperfusion injury, which may be irreversible. Ischemic hepatitis due to AHF has been linked to the pathogenesis of liver damage. In the present study, we extensively investigated the role of mitochondrial dynamics-related proteins and their epigenetic regulation in ischemic liver injury following AHF and explored the possible hepatoprotective role of carvedilol. The biochemical analysis revealed that the ischemic liver injury following AHF significantly elevated the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) enzymes, the level of total and direct bilirubin, and the expression of hepatic mitogen-activated protein kinase (MAPK), dynamin-1-like protein (DNM1L), and hepatic miRNA-17. At the same time, it significantly reduced the serum albumin level, the activity of hepatic superoxide dismutase (SOD), and the expression of mitochondrial peroxisome proliferator-activated receptor-1α (PGC-1α), and mitofusin 2 (Mtf2). The histological examination of the liver tissue revealed degenerated hepatocytes. Interestingly, administration of carvedilol either prior to or after isoprenaline-induced AHF significantly improved the liver function and reversed the deterioration effect of AHF-induced ischemic hepatitis, as demonstrated by biochemical, immunohistochemical, and histological analysis. Our results indicated that the hepatoprotective effect of carvedilol in ameliorating hepatic ischemic damage could be attributed to its ability to target the mitochondrial dynamics-related proteins (Mtf2, DNM1L and PGC-1α), but also their epigenetic regulator miRNA-17. To further explore the mode of action of carvedilol, we have investigated, in silico, the ability of carvedilol to target dynamin-1-like protein and mitochondrial dynamics protein (MID51). Our results revealed that carvedilol has a high binding affinity (−14.83 kcal/mol) toward the binding pocket of DNM1L protein. In conclusion, our study highlights the hepatoprotective pharmacological application of carvedilol to attenuate ischemic hepatitis associated with AHF.

Human Hepatic CD56bright NK Cells Display a Tissue-Resident Transcriptional Profile and Enhanced Ability to Kill Allogenic CD8+ T Cells.

Liver-resident CD56brightCD16- natural killer (NK) cells are enriched in the human liver and are phenotypically distinct from their blood counterparts. Although these cells are capable of rapid cytotoxic effector activity, their functional role remains unclear. We hypothesise that they may contribute to immune tolerance in the liver during transplantation. RNA sequencing was carried out on FACS sorted NK cell subpopulations from liver perfusates (n=5) and healthy blood controls (n=5). Liver-resident CD56brightCD16+/- NK cells upregulate genes associated with tissue residency. They also upregulate expression of CD160 and LY9, both of which encode immune receptors capable of activating NK cells. Co-expression of CD160 and Ly9 on liver-resident NK cells was validated using flow cytometry. Hepatic NK cell cytotoxicity against allogenic T cells was tested using an in vitro co-culture system of liver perfusate-derived NK cells and blood T cells (n=10-13). In co-culture experiments, hepatic NK cells but not blood NK cells induced significant allogenic T cell death (p=0.0306). Allogenic CD8+ T cells were more susceptible to hepatic NK cytotoxicity than CD4+ T cells (p<0.0001). Stimulation of hepatic CD56bright NK cells with an anti-CD160 agonist mAb enhanced this cytotoxic response (p=0.0382). Our results highlight a role for donor liver NK cells in regulating allogenic CD8+ T cell activation, which may be important in controlling recipient CD8+ T cell-mediated rejection post liver-transplant.

Bacterial and Viral Infections in Liver Transplantation: New Insights from Clinical and Surgical Perspectives.

End-stage liver disease patients undergoing liver transplantation are prone to develop numerous infectious complications because of immunosuppression, surgical interventions, and malnutrition. Infections in transplant recipients account for the main cause of mortality and morbidity with rates of up to 80%. The challenges faced in the early post-transplant period tend to be linked to transplant procedures and nosocomial infections commonly in bloodstream, surgical, and intra-abdominal sites. Viral infections represent an additional complication of immunosuppression; they can be donor-derived, reactivated from a latent virus, nosocomial or community-acquired. Bacterial and viral infections in solid organ transplantation are managed by prophylaxis, multi-drug resistant screening, risk assessment, vaccination, infection control and antimicrobial stewardship. The aim of this review was to discuss the epidemiology of bacterial and viral infections in liver transplants, infection control issues, as well as surgical frontiers of ex situ liver perfusion.

The Immunological Effect of Oxygen Carriers on Normothermic Ex Vivo Liver Perfusion.

IntroductionNormothermic ex vivo liver perfusion (NEVLP) is an organ preservation method that allows liver graft functional assessment prior to transplantation. One key component of normothermic perfusion solution is an oxygen carrier to provide oxygen to the liver to sustain metabolic activities. Oxygen carriers such as red blood cells (RBCs) or hemoglobin-based oxygen carriers have an unknown effect on the liver-resident immune cells during NEVLP. In this study, we assessed the effects of different oxygen carriers on the phenotype and function of liver-resident immune cells.MethodsAdult Lewis rat livers underwent NEVLP using three different oxygen carriers: human packed RBCs (pRBCs), rat pRBCs, or Oxyglobin (a synthetic hemoglobin-based oxygen carrier). Hourly perfusate samples were collected for downstream analysis, and livers were digested to isolate immune cells. The concentration of common cytokines was measured in the perfusate, and the immune cells underwent phenotypic characterization with flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The stimulatory function of the liver-resident immune cells was assessed using mixed lymphocyte reactions.ResultsThere were no differences in liver function, liver damage, or histology between the three oxygen carriers. qRT-PCR revealed that the gene expression of nuclear factor κ light chain enhancer of activated B cells (NF-kB), Interleukin (IL-1β), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 7 (CCL7), and CD14 was significantly upregulated in the human pRBC group compared with that in the naive, whereas the rat pRBC and Oxyglobin groups were not different from that of naive. Flow cytometry demonstrated that the cell surface expression of the immune co-stimulatory protein, CD86, was significantly higher on liver-resident macrophages and plasmacytoid dendritic cells perfused with human pRBC compared to Oxyglobin. Mixed lymphocyte reactions revealed increased allogeneic T-cell proliferation in the human and rat pRBC groups compared to that in the Oxyglobin group.ConclusionsLiver-resident immune cells are important mediators of rejection after transplantation. In this study, we show that the oxygen carrier used in NEVLP solutions can affect the phenotype of these liver-resident immune cells. The synthetic hemoglobin-based oxygen carrier, Oxyglobin, showed the least amount of liver-resident immune cell activation and the least amount of allogeneic proliferation when compared to human or rat pRBCs. To mitigate liver-resident immune cell activation during NEVLP (and subsequent transplantation), Oxyglobin may be an optimal oxygen carrier.