Activation of farnesoid X receptor enhances the efficacy of normothermic machine perfusion in ameliorating liver ischemia-reperfusion injury.

The shortage of transplant organs remains a severe global issue. Normothermic machine perfusion (NMP) has the potential to increase organ availability, yet its efficacy is hampered by the inflammatory response during machine perfusion. Mouse liver ischemia-reperfusion injury (IRI) models, discarded human liver models, and porcine marginal liver transplantation models were utilized to investigate whether farnesoid X receptor (FXR) activation could mitigate inflammation-induced liver damage. FXR expression levels before and after reperfusion were measured. Gene editing and coimmunoprecipitation techniques were employed to explore the regulatory mechanism of FXR in inflammation inhibition. The expression of FXR correlates with the extent of liver damage after reperfusion. Activation of FXR significantly suppressed the inflammatory response triggered by IRI, diminished the release of proinflammatory cytokines, and improved liver function recovery during NMP, assisting discarded human livers to reach transplant standards. Mechanistically, FXR disrupts the interaction between p65 and p300, thus inhibiting modulating the nuclear factor kappa-B signaling pathway, a key instigator of inflammation. Our research across multiple species confirms that activating FXR can optimize NMP by attenuating IRI-related liver damage, thereby improving the utilization of marginal livers for transplantation.

Development of a Large Animal Model of Ischemia-free Liver Transplantation in Pigs.

Background: In organ transplantation, ischemia, and reperfusion injury (IRI) is considered as an inevitable event and the major contributor to graft failure. Ischemia-free liver transplantation (IFLT) is a novel transplant procedure that can prevent IRI and provide better transplant outcomes. However, a large animal model of IFLT has not been reported. Therefore, we develop a new, reproducible, and stable model of IFLT in pigs for investigating mechanisms of IFLT in IRI. Methods: Ten pigs were subjected to IFLT or conventional liver transplantation (CLT). Donor livers in IFLT underwent 6-h continuous normothermic machine perfusion (NMP) throughout graft procurement, preservation, and implantation, whereas livers in CLT were subjected to 6-h cold storage before implantation. The early reperfusion injury was compared between the 2 groups. Results: Continuous bile production, low lactate, and liver enzyme levels were observed during NMP in IFLT. All animals survived after liver transplantation. The posttransplant graft function was improved with IFLT when compared with CLT. Minimal histologic changes, fewer apoptotic hepatocytes, less sinusoidal endothelial cell injury, and proinflammatory cytokine (interleukin [IL]-1ß, IL-6, and tumor necrosis factor-α) release after graft revascularization were documented in the IFLT group versus the CLT group. Conclusions: We report that the concept of IFLT is achievable in pigs. This innovation provides a potential strategy to investigate the mechanisms of IRI and provide better transplant outcomes for clinical practice.

Ischemia-free liver transplantation improves the prognosis of recipients using Functioned Marginal Liver grafts.

Background & Aims: The shortage of donor livers hinders the development of liver transplantations. This study aimed to clarify the poor outcomes of functioned marginal liver grafts (FMLs) and provide evidence for the improvement of ischemia-free liver transplantation (IFLT) on transplantation with FMLs. Methods: Propensity score matching was used to control for confounding factors. The outcomes of the control group and FMLs were compared to demonstrate the negative impact of FMLs in liver transplantation patients. We compared the clinical improvements of the different surgical types. To elucidate the underlying mechanism, we conducted bioinformatic analysis based on transcriptome and single-cell profiles. Results: FMLs showed a significantly higher Hazard Ratio (HR: 1.969, P = 0.018) than other marginal livers. A worse 90-days survival (12.3% vs. 5.0%, P = 0.007) was observed in patients who underwent FMLs. Patients receiving FMLs had a significant overall survival benefit after IFLT (10.4% vs. 31.3%, P = 0.006). Pyroptosis and inflammation are inhibited in patients who undergo IFLT. The infiltration of Natural Killer cells was lower in liver grafts from these patients. A positive relationship was observed between IL32 and Caspase 1 (R = 0.73, P = 0.01) and Gasdermin D (R = 0.84, P = 0.0012) in the bulk transcriptome profiles. Conclusion: FMLs function as a more important negative prognostic parameter than other marginal livers do. IFLT might ameliorate liver injury in FMLs by inhibiting the infiltration of NK cells, consequently leading to the abortion of IL-32, which drives pyroptosis in monocytes and macrophages.

The establishment of diseased human whole organ model by normothermic machine perfusion technique: Principle of concept.

BACKGROUND: The global incidence of liver diseases is rising, yet there remains a dearth of precise research models to mimic these diseases. The use of normothermic machine perfusion (NMP) to study diseased livers recovered from liver transplantation (LT) recipients presents a promising avenue. Accordingly, we have developed a machine perfusion system tailored specifically for the human whole diseased livers and present our experience from the NMP of diseased livers. METHODS: Six diseased livers recovered from LT recipients with different diagnoses were collected. The diseased livers were connected to the machine perfusion system that circulated tailored perfusate, providing oxygen and nutrients. The pressure and flow of the system were recorded, and blood gas analysis and laboratory tests of perfusate and bile were examined to analyze the function of the diseased livers. Liver tissues before and after NMP were collected for histological analysis. RESULTS: Experiments showed that the system maintained the diseased livers in a physiological state, ensuring stable hemodynamics and a suitable partial pressure of oxygen and carbon dioxide. The results of blood gas analysis and laboratory tests demonstrated a restoration and sustenance of metabolism with minimal damage. Notably, a majority of the diseased livers exhibited bile production continuously, signifying their vivid functional integrity. The pathological characteristics remained stable before and after NMP. CONCLUSION: We successfully established the machine perfusion system tailored specifically for diseased human whole livers. Through the application of this system, we have developed a novel in vitro model that faithfully recapitulates the main features of human liver disease. This model holds immense promise as an advanced disease modeling platform, offering profound insights into liver diseases and potential implications for research and therapeutic development.

Application of extended criteria donor grafts in liver transplantation for acute-on-chronic liver failure: A retrospective cohort study.

BACKGROUND: There is no consensus on the usage of extended criteria donor (ECD) grafts in liver transplantation (LT) for acute-on-chronic liver failure (ACLF) patients. AIM: To summarize the experience of using ECD livers in ACLF-LT. METHODS: A retrospective cohort study was conducted, enrolling patients who underwent LT at the First Affiliated Hospital of Sun Yat-Sen University from January 2015 to November 2021. The patients were divided into ECD and non-ECD groups for analysis. RESULTS: A total of 145 recipients were enrolled in this study, of which ECD and non-ECD recipients accounted for 53.8% and 46.2%, respectively. Donation after cardiac death (DCD) recipients accounted for the minority compared with donation after brain death (DBD) recipients (16.6% vs 83.4%). Neither overall survival nor graft survival significantly differed between ECD and non-ECD and DCD and DBD recipients. ECD grafts were associated with a significantly higher incidence of early allograft dysfunction (EAD) than non-ECD grafts (67.9% vs 41.8%, P = 0.002). Postoperative outcomes between DCD and DBD recipients were comparable (P > 0.05). ECD graft (P = 0.009), anhepatic phase (P = 0.034) and recipient gamma glutamyltransferase (P = 0.016) were independent risk factors for EAD. Recipient preoperative number of extrahepatic organ failures > 2 (P = 0.015) and intraoperative blood loss (P = 0.000) were independent predictors of poor post-LT survival. CONCLUSION: Although related to a higher risk of EAD, ECD grafts can be safely used in ACLF-LT. The main factors affecting post-LT survival in ACLF patients are their own severe preoperative disease and intraoperative blood loss.

Clinical utilization of multiple antibodies of Mycobacterium tuberculosis for serodiagnosis evaluation of tuberculosis: a retrospective observational cohort study.

OBJECTIVES: We aimed to investigate clinical uncertainties by characterizing the accuracy and utility of commercially available antibodies of Mycobacterium tuberculosis in the diagnostic assessment of suspected tuberculosis in high-burden countries. METHODS: We conducted a retrospective, descriptive, cohort study among participants aged ≥ 18 years with suspected tuberculosis in Nanning, Guangxi, and China. Participants were tested for M. tuberculosis infection using commercially available antibodies against Mycobacterum tuberculosis. Specificity, sensitivity, negative and positive predictive values, and negative and positive likelihood ratios of the tests were determined. Sputum specimens and bronchoalveolar lavage fluid were sent for mycobacterial culture, Xpert MTB/RIF assay, and cell-free M. tuberculosis DNA or RNA assay. Blood samples were used for IGRAs, T-cell counts (CD3 + CD4+ and CD3 + CD8+), and antibodies to tuberculosis test. RESULTS: Of the 1857 participants enrolled in this study, 1772 were included in the analyses, among which, 1311 were diagnosed with active tuberculosis. The specificity of antibody against 16kD for active tuberculosis was 92.7% (95% confidence interval [CI]: 89.3-95.4) with a positive likelihood ratio for active tuberculosis cases of 3.1 (95% CI: 2.1-4.7), which was higher than that of antibody to Rv1636 (90.5% [95% CI: 86.6-93.5]), antibody to 38kD (89.5% [95% CI: 85.5-92.7]), antibody against CFP-10 (82.6% [95% CI: 77.9-86.7]), and antibody against LAM (79.3% [95% CI: 74.3-83.7]). Sensitivity ranged from 15.8% (95% CI: 13.9-17.9) for antibody against Rv1636 to 32.9% (95% CI: 30.4-35.6) for antibody to LAM. CONCLUSIONS: Commercially available antibodies against to Mycobacterium tuberculosis do not have sufficient sensitivity for the diagnostic evaluation of active tuberculosis. However, antibody against Rv1636 and 16kD may have sufficiently high specificities, high positive likelihood ratios, and correspondingly high positive predictive values to facilitate the rule-in of active tuberculosis.

Existing M. tuberculosis antigens do achieve a limited sensitivity and negative predictive value to rule out a diagnosis of tuberculosis.M. tuberculosis antigens may help to rule in a diagnosis of active or latent tuberculosis in clinical setting among the high burden tuberculosis countries.This study is the largest retrospective, descriptive, cohort study to evaluate the clinical utilization of existing M. tuberculosis antigens integrating M. tuberculosis immunogens in patients with suspected active tuberculosis in high-burden country.

A randomized-controlled trial of ischemia-free liver transplantation for end-stage liver disease.

BACKGROUND & AIMS: Ischemia-reperfusion injury (IRI) has thus far been considered as an inevitable component of organ transplantation, compromising outcomes, and limiting organ availability. Ischemia-free organ transplantation is a novel approach designed to avoid IRI, with the potential to improve outcomes. METHODS: In this randomized-controlled clinical trial, recipients of livers from donors after brain death were randomly assigned to receive either an ischemia-free or a 'conventional' transplant. The primary endpoint was the incidence of early allograft dysfunction. Secondary endpoints included complications related to graft IRI. RESULTS: Out of 68 randomized patients, 65 underwent transplants and were included in the analysis. 32 patients received ischemia-free liver transplantation (IFLT), and 33 received conventional liver transplantation (CLT). Early allograft dysfunction occurred in two recipients (6%) randomized to IFLT and in eight (24%) randomized to CLT (difference -18%; 95% CI -35% to -1%; p = 0.044). Post-reperfusion syndrome occurred in three recipients (9%) randomized to IFLT and in 21 (64%) randomized to CLT (difference -54%; 95% CI -74% to -35%; p <0.001). Non-anastomotic biliary strictures diagnosed with protocol magnetic resonance cholangiopancreatography at 12 months were observed in two recipients (8%) randomized to IFLT and in nine (36%) randomized to CLT (difference, -28%; 95% CI -50% to -7%; p = 0.014). The comprehensive complication index at 1 year after transplantation was 30.48 (95% CI 23.25-37.71) in the IFLT group vs. 42.14 (95% CI 35.01-49.26) in the CLT group (difference -11.66; 95% CI -21.81 to -1.51; p = 0.025). CONCLUSIONS: Among patients with end-stage liver disease, IFLT significantly reduced complications related to IRI compared to a conventional approach. CLINICAL TRIAL REGISTRATION: chictr.org. ChiCTR1900021158. IMPACT AND IMPLICATIONS: Ischemia-reperfusion injury has thus far been considered as an inevitable event in organ transplantation, compromising outcomes and limiting organ availability. Ischemia-free liver transplantation is a novel approach of transplanting donor livers without interruption of blood supply. We showed that in patients with end-stage liver disease, ischemia-free liver transplantation, compared with a conventional approach, led to reduced complications related to ischemia-reperfusion injury in this randomized trial. This new approach is expected to change the current practice in organ transplantation, improving transplant outcomes, increasing organ utilization, while providing a clinical model to delineate the impact of organ injury on alloimmunity.

Metabolomics Differences of the Donor Livers Between In Situ and Ex Situ Conditions During Ischemia-free Liver Transplantation.

BACKGROUND: Ischemia-free liver transplantation (IFLT) has been innovated to avoid graft ischemia during organ procurement, preservation, and implantation. However, the metabolism activity of the donor livers between in the in situ and ex situ normothermic machine perfusion (NMP) conditions, and between standard criteria donor and extend criteria donor remains unknown. METHODS: During IFLT, plasma samples were collected both at the portal vein and hepatic vein of the donor livers in situ during procurement and ex situ during NMP. An ultra-high performance liquid chromatography-mass spectrometry was conducted to investigate the common and distinct intraliver metabolite exchange. RESULTS: Profound cysteine and methionine metabolism, and aminoacyl-tRNA biosynthesis were found in both in situ and ex situ conditions. However, obvious D-arginine and D-ornithine metabolism, arginine and proline metabolism were only found in the in situ condition. The suppressed activities of the urea cycle pathway during ex situ condition were confirmed in an RNA expression level. In addition, compared with extend criteria donor group, standard criteria donor group had more active intraliver metabolite exchange in metabonomics level. Furthermore, we found that the relative concentration of p-cresol, allocystathionine, L-prolyl-L-proline in the ex situ group was strongly correlated with peak alanine aminotransferase and aspartate aminotransferase at postoperative days 1-7. CONCLUSIONS: In the current study, we show the common and distinct metabolism activities during IFLT. These findings might provide insights on how to modify the design of NMP device, improve the perfusate components, and redefine the criteria of graft viability.

TUBB2B facilitates progression of hepatocellular carcinoma by regulating cholesterol metabolism through targeting HNF4A/CYP27A1.

Cholesterol metabolism plays a critical role in the progression of hepatocellular carcinoma (HCC), but it is not clear how cholesterol metabolism is regulated. The tubulin beta class I genes (TUBBs) are associated with the prognosis of many different cancers. To confirm the function of TUBBs in HCC, the Kaplan-Meier method and Cox analyses were performed using TCGA and GSE14520 datasets. A higher expression of TUBB2B is an independent prognostic factor for shorter over survival in HCC patients. Deletion of TUBB2B in hepatocytes inhibits proliferation and promotes tumor cell apoptosis, while over-expression of TUBB2B has the opposite function. This result was confirmed in a mouse xenograft tumor model. Mechanistically, TUBB2B induces the expression of CYP27A1, an enzyme responsible for the conversion of cholesterol to 27-hydroxycholesterol, which leads to the up-regulation of cholesterol and the progression of HCC. In addition, TUBB2B regulates CYP27A1 via human hepatocyte nuclear factor 4alpha (HNF4A). These findings indicated that TUBB2B functions as an oncogene in HCC, and plays a role in promoting cell proliferation and anti-apoptosis through targeting HNF4A/CYP27A1/cholesterol.

Neoadjuvant programmed cell death 1 inhibitor before liver transplantation for HCC is not associated with increased graft loss.

Immune checkpoint inhibitors (ICIs) may lead to rejection and even graft loss of solid organ transplant recipients, making them not widely used in transplant patients. There is insufficient clinical experience in using ICIs as a bridging or downstaging therapy before transplantation. We performed a retrospective review of patients receiving programmed cell death 1 inhibitor (PD1) before liver transplantation for HCC in our center and analyzed the data of these patients with the purpose of investigating the safety and feasibility of preoperative PD1 inhibitor among liver transplant recipients and exploring the preoperative correlation ICIs and the postoperative risk of rejection and immune-related graft loss. A total of 16 patients enrolled in this study. Acute rejection occurred in 9 patients, with an incidence of 56.3%. The median time of rejection was 7 days after surgery. The median FK506 concentration at the time of rejection was 7.1 µg/L. All rejection reactions were reversed after adjusting the immunosuppression regimen. The interval between the last PD1 inhibitor and transplantation in the rejection group was shorter than that in the nonrejection group, and there was a statistical difference [21.0 (15.5-27.5) days vs. 60.0 (34.0-167.0) days, p =0.01]. In conclusion, PD1 inhibitor is a safe and feasible method for bridging or downstaging treatment before liver transplantation. Although preoperative PD1 inhibitor may increase the incidence of postoperative rejection, it is not associated with increased immune-related graft loss and patient death.

Abrogation of graft ischemia-reperfusion injury in ischemia-free liver transplantation.

BACKGROUND: Ischemia-reperfusion injury (IRI) is considered an inherent component of organ transplantation that compromises transplant outcomes and organ availability. The ischemia-free liver transplantation (IFLT) procedure has been developed to avoid interruption of blood supply to liver grafts. It is unknown how IFLT might change the characteristics of graft IRI. METHODS: Serum and liver biopsy samples were collected from IFLT and conventional liver transplantation (CLT) recipients. Pathological, metabolomics, transcriptomics, and proteomics analyses were performed to identify the characteristic changes in graft IRI in IFLT. RESULTS: Peak aspartate aminotransferase (539.59 ± 661.76 U/L versus 2622.28 ± 3291.57 U/L) and alanine aminotransferase (297.64 ± 549.50 U/L versus 1184.16 ± 1502.76 U/L) levels within the first 7 days and total bilirubin levels by day 7 (3.27 ± 2.82 mg/dl versus 8.33 ± 8.76 mg/dl) were lower in the IFLT versus CLT group (all p values < 0.001). The pathological characteristics of IRI were more obvious in CLT grafts. The antioxidant pentose phosphate pathway remained active throughout the procedure in IFLT grafts and was suppressed during preservation and overactivated postrevascularization in CLT grafts. Gene transcriptional reprogramming was almost absent during IFLT but was profound during CLT. Proteomics analysis showed that "metabolism of RNA" was the major differentially expressed process between the two groups. Several proinflammatory pathways were not activated post-IFLT as they were post-CLT. The activities of natural killer cells, macrophages, and neutrophils were lower in IFLT grafts than in CLT grafts. The serum levels of 14 cytokines were increased in CLT versus IFLT recipients. CONCLUSIONS: IFLT can largely avoid the biological consequences of graft IRI, thus has the potential to improve transplant outcome while increasing organ utilization.

Single-Cell RNA Sequencing Identifies Intra-Graft Population Heterogeneity in Acute Heart Allograft Rejection in Mouse.

Transplant rejection remains a major barrier to graft survival and involves a diversity of cell types. However, the heterogeneity of each cell type in the allograft remains poorly defined. In the present study, we used single-cell RNA sequencing technology to analyze graft-infiltrating cells to describe cell types and states associated with acute rejection in a mouse heart transplant model. Unsupervised clustering analysis revealed 21 distinct cell populations. Macrophages formed five cell clusters: two resident macrophage groups, two infiltrating macrophage groups and one dendritic cell-like monocyte group. Infiltrating macrophages were predominantly from allogeneic grafts. Nevertheless, only one infiltrating macrophage cluster was in an active state with the upregulation of CD40, Fam26f and Pira2, while the other was metabolically silent. Re-clustering of endothelial cells identified five subclusters. Interestingly, one of the endothelial cell populations was almost exclusively from allogeneic grafts. Further analysis of this population showed activation of antigen processing and presentation pathway and upregulation of MHC class II molecules. In addition, Ubiquitin D was specifically expressed in such endothelial cell population. The upregulation of Ubiquitin D in rejection was validated by staining of mouse heart grafts and human kidney biopsy specimens. Our findings present a comprehensive analysis of intra-graft cell heterogeneity, describe specific macrophage and endothelial cell populations which mediate rejection, and provide a potential predictive biomarker for rejection in the clinic.

Ischemic-Free Liver Transplantation Reduces the Recurrence of Hepatocellular Carcinoma After Liver Transplantation.

Ischemia reperfusion injury (IRI) is an adverse factor for hepatocellular carcinoma (HCC) recurrence after liver transplantation. Ischemic-free liver transplantation (IFLT) is a novel transplant procedure that can largely reduce or even prevent IRI, but the clinical relevance of IFLT and the recurrence of HCC after liver transplantation are still unknown. This retrospective study compared survival outcomes, HCC recurrence, perioperative data and IRI severity following liver transplantation (LT). 30 patients received IFLT and 196 patients received conventional liver transplantation (CLT) were chosen for the entire cohort between June 2017 and August 2020. A 1:3 propensity score matching was performed, 30 IFLT recipients and 85 matched CLT patients were enrolled in propensity-matched cohorts. An univariate and multivariate Cox regression analysis was performed, and showed surgical procedure (CLT vs IFLT) was an independent prognostic factor (HR 3.728, 95% CI 1.172-11.861, P=0.026) for recurrence free survival (RFS) in HCC patients following liver transplantation. In the Kaplan-Meier analysis, the RFS rates at 1 and 3 years after LT in recipients with HCC in the IFLT group were significantly higher than those in the CLT group both in the entire cohort and propensity-matched cohort (P=0.006 and P=0.048, respectively). In addition, patients in the IFLT group had a lower serum lactate level, lower serum ALT level and serum AST level on postoperative Day 1. LT recipients with HCC in the IFLT group had a lower incidence of early allograft dysfunction than LT recipients with HCC in the CLT group. Histological analysis showed no obvious hepatocyte necrosis or apoptosis in IFLT group. In conclusion, IFLT can significantly reduce IRI damage and has the potential to be a useful strategy to reduce HCC recurrence after liver transplantation.

Ischaemia-free liver transplantation in humans: a first-in-human trial.

Background Ischaemia-reperfusion injury is considered an inevitable component of organ transplantation, compromising organ quality and outcomes. Although several treatments have been proposed, none has avoided graft ischaemia and its detrimental consequences. Methods Ischaemia-free liver transplantation (IFLT) comprises surgical techniques enabling continuous oxygenated blood supply to the liver of brain-dead donor during procurement, preservation, and implantation using normothermic machine perfusion technology. In this non-randomised study, 38 donor livers were transplanted using IFLT and compared to 130 conventional liver transplants (CLT). Findings Two recipients (5•3%) in the IFLT group experienced early allograft dysfunction, compared to 50•0% in patients receiving conventional transplants (absolute risk difference, 44•8%; 95% confidence interval, 33•6-55•9%). Recipients of IFLT had significantly reduced median (IQR) peak aspartate aminotransferase levels within the first week compared to CLT recipients (365, 238-697 vs 1445, 791-3244 U/L, p<0•001); likewise, median total bilirubin levels on day 7 were significantly lower (2•34, 1•39-4•09 mg/dL) in the IFLT group than in the CLT group (5•10, 1•90-11•65 mg/dL) (p<0•001). Moreover, IFLT recipients had a shorter median intensive care unit stay (1•48, 0•75-2•00 vs 1•81, 1•00-4•58 days, p=0•006). Both one-month recipient (97•4% vs 90•8%, p=0•302) and graft survival (97.4% vs 90•0%, p=0•195) were better for IFLT than CLT, albeit differences were not statistically significant. Subgroup analysis showed that the extended criteria donor livers transplanted using the IFLT technique yielded faster post-transplant recovery than did the standard criteria donor livers transplanted using the conventional approach. Interpretation IFLT provides a novel approach that may improve outcomes, and allow the successful utilisation of extended criteria livers. Funding This study was funded by National Natural Science Foundation of China, Guangdong Provincial Key Laboratory Construction Projection on Organ Donation and Transplant Immunology, and Guangdong Provincial international Cooperation Base of Science and Technology. Panel: Research in context.

lncRNA NEAT1 regulates the proliferation and migration of hepatocellular carcinoma cells by acting as a miR­320a molecular sponge and targeting L antigen family member 3.

Long non­coding RNAs (lncRNAs) serve a pivotal role in hepatocellular carcinoma (HCC) progression and have been confirmed to participate in the carcinogenesis and development of HCC. Certain studies have focused on lncRNA nuclear enriched abundant transcript 1 (NEAT1) in HCC. However, the relationship between lncRNA NEAT1 and HCC remains unclear. The present study found that NEAT1 was significantly overexpressed in HCC cell lines compared with LX­2 hepatic stellate cells. NEAT1 expression in Huh7 and MHCC­97H cells was increased following transfection with lentivirus (LV)­NEAT1 but inhibited by LV­short hairpin NEAT1. Knockdown of NEAT1 significantly repressed HCC cell viability, increased cell apoptosis, and inhibited cell migration and invasion capacity. By contrast, upregulation of NEAT1 demonstrated the reverse effects. Furthermore, microRNA­320a (miR­230a) was predicted to be a direct target of NEAT1 and was significantly reduced in HCC cells. Additionally, a luciferase activity reporter assay and RNA immunoprecipitation assay were performed to confirm the interaction between miR­320a and NEAT1. Using a dual­luciferase activity assay, L antigen family member 3 (LAGE3) was found to be a target of miR­320a. Finally, in vivo nude mouse models were established, and the results indicated that NEAT1 suppressed HCC progression by targeting miR­320a. In conclusion, the present findings revealed that the NEAT1/miR­320a/LAGE3 axis participates in HCC development and that NEAT1 could be used as a biomarker for HCC.

Deceased Donor Predictors for Pediatric Liver Allograft Utilization.

BACKGROUND: The number of pediatric deceased organ donors has recently declined, and the nonutilization of pediatric liver allografts has limited the development of liver transplantation. We determined the utilization rate of pediatric livers and identified risk factors for graft discard. METHODS: We used data from the Scientific Registry of Transplant Recipients database from January 1, 2000, to December 31, 2012. The trends of pediatric liver donors and utilization rates were analyzed. Donor risk factors that impacted the graft use of pediatric livers were measured. Logistic regression modelling was performed to evaluate graft utilization and risk factors. RESULTS: A total of 11,934 eligible pediatric liver donors were identified during this period. A total of 1191 authorized liver grafts did not recover or recovered without transplantation. Factors including pediatric donors >1 year of age (odds ratio [OR] = 2.956, 95% confidence interval [CI] 2.494-3.503, P < .001), nonhead trauma (OR = 2.243, 95% CI 1.903-2.642, P < .001), lack of heartbeat (OR = 7.534, 95% CI 5.899-9.623, P < .001), hepatitis B surface antigen positivity (OR = 4.588, 95% CI 1.021-20.625, P = .047), anti-hepatitis C virus positivity (OR = 4.691, 95% CI 1.352-16.280, P = .015), total bilirubin >1 mg/dL (OR = 1.743, 95% CI 1.469-2.068, P < .001), and blood urea nitrogen >21 mg/dL (OR = 1.941, 95% CI 1.546-2.436, P < .001) were significantly related to graft nonutilization. Steroids or diuretics administered prerecovery were significantly related to graft utilization (OR = 0.684, 95% CI 0.581-0.806, P < .001; OR = 0.744, 95% CI 0.634-0.874, P < .001; respectively). CONCLUSIONS: The pediatric liver allograft utilization rate and risk factors for nonutilization of grafts were determined.

Association of Perfusion Characteristics and Posttransplant Liver Function in Ischemia-Free Liver Transplantation.

It has been shown that normothermic machine perfusion (NMP), a novel preservation method, is able to assess and resuscitate liver grafts with risk factors. However, there is no consistent criteria for the assessment of liver grafts with NMP. Ischemia-free liver transplantation (IFLT) includes innovative surgical techniques and NMP, which can protect liver grafts from ischemia throughout organ procurement, preservation, and implantation. In our center, 28 human livers from donation after brain death donors were subjected to IFLT between July 2017 and October 2018. The correlation between posttransplant liver function tests with the perfusion parameters, blood gas analysis of perfusate, and bile biochemistry were analyzed. During the preservation phase, the vascular flow was stable, and the lactate level decreased rapidly. The transaminase release in the perfusate was low but stable, whereas the glucose level remained high. The perfusate lactate and aspartate aminotransferase (AST) levels at 1 hour of perfusion were correlated with the posttransplant peak AST level. There were negative correlations between the portal vein and hepatic artery flows at the end of perfusion and the peak transaminase levels within 7 days after transplantation. In conclusion, during IFLT, NMP is able to bridge the liver grafts from donors to recipients and can allow the assessment of liver function by perfusion characteristics.

Prospective, single-centre, randomised controlled trial to evaluate the efficacy and safety of ischaemia-free liver transplantation (IFLT) in the treatment of end-stage liver disease.

INTRODUCTION: During conventional liver transplantation (CLT), ischaemia-reperfusion injury (IRI) is inevitable and is associated with complications such as early allograft dysfunction (EAD), primary non-function and ischaemic-type biliary lesions. We have established a novel procedure called ischaemia-free liver transplantation (IFLT). The results from a pilot study suggest that IFLT might prevent IRI and yield better transplant outcomes than CLT. The purpose of this study was to further assess the efficacy and safety of IFLT versus CLT in patients with end-stage liver disease. METHODS AND ANALYSIS: This is an investigator-initiated, open-label, phase III, prospective, single-centre randomised controlled trial on the effects of IFLT in patients with end-stage liver disease. Adult patients (aged 18-75 years) eligible for liver transplantation will be screened for participation in this trial and will be randomised between the IFLT group (n=34) and the CLT group (n=34). In the IFLT group, the donor liver will be procured, preserved and implanted with continuous normothermic machine perfusion (NMP). In the CLT group, the donor liver will be procured after a fast cold flush, preserved in 0°C-4°C solution and implanted under hypothermic and hypoxic conditions. Patients in both groups will be managed according to the standard protocol of our centre. The primary end point is the incidence of EAD after liver transplantation. Intraoperative and postoperative parameters of donor livers and recipients will be observed and recorded, and postoperative liver graft function, complications and recipient and graft survival will be evaluated. After a 12-month follow-up of the last enrolled recipient, the outcomes will be analysed to evaluate the safety and efficacy of IFLT versus CLT in patients with end-stage liver disease. ETHICS AND DISSEMINATION: The protocol was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Sun Yat-sen University. The findings will be disseminated to the public through conference presentations and peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: ChiCTR1900021158.

First Preliminary Experience with Preservation of Liver Grafts from Extended-Criteria Donors by Normothermic Machine Perfusion in Asia.

BACKGROUND Normothermic machine perfusion (NMP) can provide access to evaluate and resuscitate high-risk donor livers before transplantation. The purpose of this study was to determine the efficacy of NMP in preservation and assessment of extended-criteria donor (ECD) livers in China. CASE REPORT From September 2018 to March 2019, 4 liver grafts from 3 transplant center defined as ECD were subjected to NMP, and then were transplanted successfully. During perfusion, perfusion parameters such as vascular flow, glucose level, lactate clearance, and bile production/composition were recorded to assess graft viability. All recipients were followed up 6 months after transplantation. CONCLUSIONS NMP provides a potential tool for preservation and assessment of ECD livers in China.