Liver Graft Injury Research Articles

FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

Fibroblast growth factor 21 (FGF21) is essential for modulating hepatic homeostasis, but the impact of FGF21 on liver graft injury remains uncertain. Here, we show that high FGF21 levels in liver graft and serum are associated with improved graft function and survival in liver transplantation (LT) recipients. FGF21 deficiency aggravates early graft injury and activates arachidonic acid metabolism and regional inflammation in male mouse models of hepatic ischemia/reperfusion (I/R) injury and orthotopic LT. Mechanistically, FGF21 deficiency results in abnormal activation of the arachidonate 15-lipoxygenase (ALOX15)/15-hydroxy eicosatetraenoic acid (15-HETE) pathway, which triggers a cascade of innate immunity-dominated pro-inflammatory responses in grafts. Notably, the modulating role of FGF21/ALOX15/15-HETE pathway is more significant in steatotic livers. In contrast, pharmacological administration of recombinant FGF21 effectively protects against hepatic I/R injury. Overall, our study reveals the regulatory mechanism of FGF21 and offers insights into its potential clinical application in early liver graft injury after LT.

Enhancing Liver Transplant Outcomes through Liver Precooling to Mitigate Inflammatory Response and Protect Mitochondrial Function.

Transplanted organs experience several episodes of ischemia and ischemia-reperfusion. The graft injury resulting from ischemia-reperfusion (IRI) remains a significant obstacle to the successful survival of transplanted grafts. Temperature significantly influences cellular metabolic rates because biochemical reactions are highly sensitive to temperature changes. Consequently, lowering the temperature could reduce the degradative reactions triggered by ischemia. In mitigating IRI in liver grafts, the potential protective effect of localized hypothermia on the liver prior to blood flow obstruction has yet to be explored. In this study, we applied local hypothermia to mouse donor livers for a specific duration before stopping blood flow to liver lobes, a procedure called "liver precooling". Mouse donor liver temperature in control groups was controlled at 37 °C. Subsequently, the liver donors were preserved in cold University of Wisconsin solution for various durations followed by orthotopic liver transplantation. Liver graft injury, function and inflammation were assessed at 1 and 2 days post-transplantation. Liver precooling exhibited a significant improvement in graft function, revealing more than a 47% decrease in plasma aspartate transaminase (AST) and alanine aminotransferase (ALT) levels, coupled with a remarkable reduction of approximately 50% in liver graft histological damage compared to the control group. The protective effects of liver precooling were associated with the preservation of mitochondrial function, a substantial reduction in hepatocyte cell death, and a significantly attenuated inflammatory response. Taken together, reducing the cellular metabolism and enzymatic activity to a minimum level before ischemia protects against IRI during transplantation.

Statement of Retraction: Liver X Receptors Activation Attenuates Ischemia Reperfusion Injury of Liver Graft in Rats

Statement of Retraction: Liver X Receptors Activation Attenuates Ischemia Reperfusion Injury of Liver Graft in Rats

Arachidonic acid activates NLRP3 inflammasome in MDSCs via FATP2 to promote post-transplant tumour recurrence in steatotic liver grafts

The steatotic grafts have been applied in liver transplantation frequently owing to the high incidence of non-alcoholic fatty liver disease. However, fatty livers are vulnerable to graft injury. Myeloid-derived suppressor cell (MDSC) recruitment during liver graft injury promotes tumour recurrence. Lipid metabolism exerts the immunological influence on MDSCs in tumour progression. Here, we aimed to explore the role and mechanism of inflammasome activation in MDSCs induced by lipid metabolism during fatty liver graft injury and the subsequent effects on tumour recurrence. MDSC populations and nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome levels were investigated in a clinical cohort and a rat liver transplantation model. The mechanism of NLRP3 activation by specific fatty acids was explored in mouse hepatic ischaemia/reperfusion injury (IRI) with tumour recurrence model and invitro studies. MDSC populations and NLRP3 levels were increased with higher tumour recurrent rate in patients using steatotic grafts. NLRP3 was upregulated in MDSCs with lipid accumulation post mouse fatty liver IRI. Mechanistically, arachidonic acid was discovered to activate NLRP3 inflammasome in MDSCs through fatty acid transport protein 2 (FATP2), which was identified by screening lipid uptake receptors. The mitochondrial dysfunction with enhanced reactive oxygen species bridged arachidonic acid uptake and NLRP3 activation in MDSCs, which subsequently stimulated CD4+ T cells producing more IL-17 in fatty liver IRI. Blockade of FATP2 inhibited NLRP3 activation in MDSCs, IL-17 production in CD4+ T cells, and the tumour recurrence post fatty liver IRI. During fatty liver graft injury, arachidonic acid activated NLRP3 inflammasome in MDSCs through FATP2, which subsequently stimulated CD4+ T cells producing IL-17 to promote tumour recurrence post transplantation. The high incidence of non-alcoholic fatty liver disease resulted in the frequent application of steatotic donors in liver transplantation. Our data showed that the patients who underwent liver transplantation using fatty grafts experienced higher tumour recurrence. We found that arachidonic acid activated NLRP3 inflammasome in MDSCs through FATP2 during fatty liver graft injury, which led to more IL-17 secretion of CD4+ T cells and promoted tumour recurrence post transplantation. The inflammasome activation by aberrant fatty acid metabolism in MDSCs bridged the acute-phase fatty liver graft injury and liver tumour recurrence.

Dual-regulation by Cx32 in hepatocyte to trigger and worsen liver graft injury

Liver transplantation is the ultimate treatment option for end-stage liver failure. However, liver graft injury remains a challenge. This study aimed to investigate the role of connexin32 (Cx32) in liver graft injury and elucidate its mechanism of action. Through detecting liver graft samples from 6 patients, we observed that changes in the Cx32 level coincided with liver graft injury. Therefore, we established autologous orthotopic liver transplantation (AOLT) models using Cx32-knockout and wild-type mice and hypoxia/reoxygenation (H/R) and lipopolysaccharide (LPS) pretreatment models using alpha mouse liver 12 (AML12) cells, to explore Cx32 mechanisms in liver graft injury. Following in vivo and in vitro Cx32 knockout, oxidative stress and inflammatory response were inhibited through the regulation of PKC-α/NF-κB/NLRP3 and Nrf2/NOX4/ROS signaling pathways, thereby reducing Bak/Bax-related apoptosis and ameliorating liver graft injury. When the Cx32-based gap junction (GJ) was blocked with 2-aminoethoxydiphenyl borate (2-APB), ROS transfer was attenuated between neighboring cells, exacerbated oxidative stress and inflammatory response were prevented, and aggravation of liver graft injury was mitigated. These results highlight the dual regulation mechanism of Cx32 in liver graft injury. Through interaction with PKC-α, Cx32 regulated the NF-κB/NLRP3 and Nrf2/NOX4/ROS signaling pathways, thus directly triggering oxidative stress and inflammatory response. Simultaneously, mass-produced ROS were transferred to neighboring cells through Cx32 channels, for which oxidative stress and the inflammatory response were aggravated indirectly. Finally, Bak/Bax-related apoptosis was activated, thereby worsening liver graft injury. Our findings propose Cx32 as a dual mechanistic factor for oxidative stress and inflammatory signaling pathways in regulating cell apoptosis on liver graft injury, which suggests a promising therapeutic targets for liver graft injury.

The Landscape of Aberrant Alternative Splicing Events in Steatotic Liver Graft Post Transplantation via Transcriptome-Wide Analysis.

The application of steatotic liver graft has been increased significantly due to the severe donor shortage and prevalence of non-alcoholic fatty liver disease. However, steatotic donor livers are vulnerable to acute phase inflammatory injury, which may result in cancer recurrence. Alternative splicing events (ASEs) are critical for diverse transcriptional variants in hepatocellular carcinoma (HCC). Here, we aimed to depict the landscape of ASEs, as well as to identify the differential ASEs in steatotic liver graft and their association with tumor recurrence after transplantation. The overall portrait of intragraft transcripts and ASEs were elucidated through RNA sequencing with the liver graft biopsies from patients and rat transplant models. Various differential ASEs were identified in steatotic liver grafts. CYP2E1, ADH1A, CYP2C8, ADH1C, and HGD, as corresponding genes to the common pathways involved differential ASEs in human and rats, were significantly associated with HCC patients' survival. The differential ASEs related RNA-binding proteins (RBPs) were enriched in metabolic pathways. The altered immune cell distribution, particularly macrophages and neutrophils, were perturbated by differential ASEs. The cancer hallmarks were enriched in steatotic liver grafts and closely associated with differential ASEs. Our work identified the differential ASE network with metabolic RBPs, immune cell distribution, and cancer hallmarks in steatotic liver grafts. We verified the link between steatotic liver graft injury and tumor recurrence at post-transcriptional level, offered new evidence to explore metabolism and immune responses, and provided the potential prognostic and therapeutic markers for tumor recurrence.

Interleukin-18 and High-Mobility-Group-Protein B1 are Early and Sensitive Indicators for Cell Damage During Normothermic Machine Perfusion after Prolonged Cold Ischemic Storage of Porcine Liver Grafts.

In the era of organ machine perfusion, experimental models to optimize reconditioning of (marginal) liver grafts are needed. Although the relevance of cytokine signatures in liver transplantation has been analyzed previously, the significance of molecular monitoring during normothermic machine perfusion (NMP) remains elusive. Therefore, we developed a porcine model of cold ischemic liver graft injury after prolonged static cold storage (SCS) and subsequent NMP: Livers obtained from ten minipigs underwent NMP for 6h directly after procurement (control group) or after 20h of SCS. Grafts after prolonged SCS showed significantly elevated AST, ALT, GLDH and GGT perfusate concentrations, and reduced lactate clearance. Bile analyses revealed reduced bile production, reduced bicarbonate and elevated glucose concentrations after prolonged SCS. Cytokine analyses of graft perfusate simultaneously demonstrated an increase of pro-inflammatory cytokines such as Interleukin-1α, Interleukin-2, and particularly Interleukin-18. The latter was the only significantly elevated cytokine compared to controls, peaking as early as 2h after reperfusion (11,012ng/ml vs. 1,493ng/ml; p = 0.029). Also, concentrations of High-Mobility-Group-Protein B1 were significantly elevated after 2h of reperfusion (706.00ng/ml vs. 148.20ng/ml; p < 0.001) and showed positive correlations with AST (r 2 = 0.846) and GLDH (r 2 = 0.918) levels. Molecular analyses during reconditioning of liver grafts provide insights into the degree of inflammation and cell damage and could thereby facilitate future interventions during NMP reducing acute and chronic graft injury.

Cytomegalovirus Latency Exacerbated Small-for-size Liver Graft Injury Through Activation of CCL19/CCR7 in Hepatic Stellate Cells.

The interplay between cytomegalovirus (CMV) latency and graft malfunction after living donor liver transplantation remains poorly defined because of the complexity of clinical confounding factors. Here, we aimed to investigate the effects of CMV latency on small-for-size graft injury and to get further insight into the pathogenic role of hepatic stellate cells (HSCs) in this process. Rat orthotopic liver transplantation with small-for-size grafts was performed in a CMV latent model developed in immunocompetent Sprague Dawley rats using Priscott strain. Posttransplant graft injury including hepatocyte damage, stellate cell activation, and fibrogenesis was evaluated. Differential gene expression of HSCs in response to CMV latency was screened by cDNA microarray. Clinical validation was further conducted in human biopsies. CMV latency aggravated hepatocyte apoptosis/necrosis in the early phase and enhanced HSC expansion and graft fibrosis during the middle-late phase in small-for-size liver grafts of the rat model. cDNA microarray mining revealed CCL19/CCR7 as one of the most noteworthy pathways bridging HSC activation and liver graft injury in the presence of CMV latency. Together with CCL19 upregulation, coherent overexpression of CCR7 in accumulated HSCs was confirmed in both rat and human CMV latent recipients. Moreover, addition of CCL19 in vitro promoted HSC migration by increasing the level of matrix metalloproteinase-2. Our data demonstrated that CMV latency aggravated early/late phase liver graft damage and fibrogenesis via CCL19/CCR7/HSCs axis. Blockade of CMV latency-related stellate cell activation may shed light on the strategy of graft protection clinically.

Single-cell profiling reveals distinct immune phenotypes that contribute to ischaemia-reperfusion injury after steatotic liver transplantation.

ObjectivesThe discrepancy between supply and demand of organ has led to an increased utilization of steatotic liver for liver transplantation (LT). Hepatic steatosis, however, is a major risk factor for graft failure due to increased susceptibility to ischaemia‐reperfusion (I/R) injury during transplantation.Materials and methodsTo assess the plasticity and phenotype of immune cells within the microenvironment of steatotic liver graft at single‐cell level, single‐cell RNA‐sequencing (scRNA‐Seq) was carried out on 23 675 cells from transplanted rat livers. Bioinformatic analyses and multiplex immunohistochemistry were performed to assess the functional properties, transcriptional regulation, phenotypic switching and cell‐cell interactions of different cell subtypes.ResultsWe have identified 11 different cell types in transplanted livers and found that the highly complex ecosystem was shaped by myeloid‐derived cell subsets that transit between different states and interact mutually. Notably, a pro‐inflammatory phenotype of Kupffer cells (KCs) with high expression of colony‐stimulating factor 3 (CSF3) that was enriched in transplanted steatotic livers was potentially participated in fatty graft injury. We have also detected a subset of dendritic cells (DCs) with highly expressing XCR1 that was correlated with CD8+ T cells, mediating the severer steatotic liver damage by I/R injury.ConclusionsThe findings of our study provide new insight into the mechanisms by which steatosis exacerbates liver damage from I/R injury. Interventions based on these observations create opportunities in attenuating fatty liver graft injury and expanding the donor pool.

Monocytic MDSC mobilization promotes tumor recurrence after liver transplantation via CXCL10/TLR4/MMP14 signaling

Tumor recurrence is the major obstacle for pushing the envelope of liver transplantation for hepatocellular carcinoma (HCC) patients. The inflammatory cascades activated by acute liver graft injury promote tumor recurrence. We aimed to explore the role and mechanism of myeloid-derived suppressor cell (MDSC) mobilization induced by liver graft injury on tumor recurrence. By analyzing 331 HCC patients who received liver transplantation, the patients with graft weight ratio (GWR, the weight of liver graft divided by the estimated standard liver weight of recipient) <60% had higher tumor recurrence than GWR ≥60% ones. MDSCs and CXCL10/TLR4 levels were significantly increased in patients with GWR <60% or tumor recurrence. These findings were further validated in our rat orthotopic liver transplantation model. In CXCL10−/− and TLR4−/− mice of hepatic ischemia/reperfusion injury plus major hepatectomy (IRH) model, monocytic MDSCs, instead of granulocytic MDSCs, were significantly decreased. Importantly, CXCL10 deficiency reduced the accumulation of TLR4+ monocytic MDSCs, and CXCL10 increased MDSC mobilization in the presence of TLR4. Moreover, MMP14 was identified as the key molecule bridging CXCL10/TLR4 signaling and MDSC mobilization. Knockout or inhibition of CXCL10/TLR4 signaling significantly reduced the tumor growth with decreased monocytic MDSCs and MMP14 in the mouse tumor recurrent model. Our data indicated that monocytic MDSCs were mobilized and recruited to liver graft during acute phase injury, and to promote HCC recurrence after transplantation. Targeting MDSC mobilization via CXCL10/TLR4/MMP14 signaling may represent the therapeutic potential in decreasing post-transplant liver tumor recurrence.

Inhibition of Carnitine Palmitoyltransferase 1A Aggravates Fatty Liver Graft Injury via Promoting Mitochondrial Permeability Transition.

Hepatic steatosis is a major risk factor for graft failure due to increased susceptibility of fatty liver to ischemia-reperfusion injury (IRI) during transplantation. Here, we aimed to investigate the role of carnitine palmitoyltransferase 1A (CPT1A) in fatty liver graft injury and to explore the underlying mechanism and therapeutic potential on attenuating hepatic IRI. Intragraft CPT1A expression profile and the association with fatty graft injury were investigated in human and rat liver transplantation samples. The underlying mechanism and therapeutic potential of CPT1A activator against IRI were also explored in mouse hepatic ischemia-reperfusion plus major hepatectomy model and in in vitro. CPT1A expression was significantly reduced (P = 0.0019; n = 96) in human fatty liver graft compared with normal one at early phase after transplantation. Low expression of CPT1A was significantly associated with high serum alanine aminotransferase (P = 0.0144) and aspartate aminotransferase (P = 0.0060) levels. The inhibited CPT1A and poor liver function were consistently observed in rat and mouse models with fatty livers. Furthermore, inhibition of CPT1A significantly promoted the translocation of chloride intracellular channel 1 to form chloride ion channel. The dysregulation of chloride ion channel activity subsequently triggered mitochondrial permeability transition (MPT) pore opening, exacerbated cellular oxidative stress, and energy depletion. Importantly, our intravital confocal imaging showed that CPT1A activation attenuated hepatic injury through preventing MPT after reperfusion in fatty mice. CPT1A inhibition triggered MPT contributed to severe IRI in fatty liver graft. CPT1A restoration may offer therapeutic potential on attenuating hepatic IRI.

Non-invasive screening for subclinical liver graft injury in adults via donor-specific anti-HLA antibodies

The majority of liver grafts exhibit abnormal histological findings late after transplantation, even when liver enzymes are normal. Such subclinical graft injuries were associated with rejection and fibrosis progression in recent studies. The identification of non-invasive biomarkers for subclinical graft injury might help to individualize immunosuppression. Therefore, graft injury was assessed in 133 liver biopsies with normal/near normal liver enzymes from a prospective liver biopsy program. Cytokeratin-18 cell death marker (M65) and donor specific anti-HLA antibodies (DSA) were measured as non-invasive markers in paired plasma samples in addition to routine parameters. M65 was associated with subclinical graft injury but this association was too weak for reasonable clinical application. DSA positivity was associated with more graft inflammation (OR = 5.4) and more fibrosis (OR = 4.2). Absence of DSA excluded fibrosis in 87–89%, while presence of DSA excluded histological criteria for immunosuppression minimization attempts in 92–97%. While CK18 cell death marker had no diagnostic value for the detection of subclinical liver graft injury, DSA testing can help to preselect patients for immunosuppression reduction in case of DSA negativity, while DSA positivity should prompt elastography or liver biopsy for the assessment of subclinical graft injury.

Ischemia-Reperfusion Injury in Marginal Liver Grafts and the Role of Hypothermic Machine Perfusion: Molecular Mechanisms and Clinical Implications.

Ischemia-reperfusion injury (IRI) constitutes a significant source of morbidity and mortality after orthotopic liver transplantation (OLT). The allograft is metabolically impaired during warm and cold ischemia and is further damaged by a paradox reperfusion injury after revascularization and reoxygenation. Short-term and long-term complications including post-reperfusion syndrome, delayed graft function, and immune activation have been associated with IRI. Due to the current critical organ shortage, extended criteria grafts are increasingly considered for transplantation, however, with an elevated risk to develop significant features of IRI. In recent years, ex vivo machine perfusion (MP) of the donor liver has witnessed significant advancements. Here, we describe the concept of hypothermic (oxygenated) machine perfusion (HMP/HOPE) approaches and highlight which allografts may benefit from this technology. This review also summarizes clinical applications and the main aspects of ongoing randomized controlled trials on hypothermic perfusion. The mechanistic aspects of IRI and hypothermic MP—which include tissue energy replenishment, optimization of mitochondrial function, and the reduction of oxidative and inflammatory damage following reperfusion—will be comprehensively discussed within the context of current preclinical and clinical evidence. Finally, we highlight novel trends and future perspectives in the field of hypothermic MP in the context of recent findings of basic and translational research.

Association of post-reperfusion syndrome and ischemia-reperfusion injury with acute kidney injury after liver transplantation.

Acute kidney injury (AKI) is frequently observed after orthotopic liver transplantation (OLT) even in patients with previously normal renal function. In this study, we investigated the impact of factors such graft steatosis, post-reperfusion syndrome (PRS), and hepatic ischemia reperfusion injury (HIRI) on the development of AKI after OLT in adult patients. We retrospectively examined consecutive adult patients who underwent OLT at our institution between July 2011 and June 2017. AKI was diagnosed based on the criteria proposed by the International Kidney Disease Improving Global Outcomes (KDIGO) workgroup. Peak aspartate aminotransferase (AST) level within 72hours after OLT was used as a surrogate marker for HIRI. Graft steatosis was diagnosed by histopathological examination using specimens biopsied intraoperatively at the end of transplantation procedure and categorized as <10%, 10%-20%, 20%-30%, and ≥30% of hepatic steatosis. Out of 386patients, 141 (37%) developed AKI (KDIGO stage 1:71 patients; stage 2:29 patients; stage 3:41 patients). Multivariable logistic regression analysis revealed that cold ischemic time (P=.012) and HIRI (P=.007) were independent risk factors for post-OLT AKI. Multivariable analysis also revealed that graft steatosis was associated with HIRI but not directly with AKI. PRS was not associated with HIRI or AKI in the multivariable analyses. Our results indicate that greater severity of liver graft injury during transplantation negatively affects renal function after OLT. As expected, the severity of liver graft steatosis contributes to accelerated liver injury occurring during the transplantation procedure.

Successful Living-Donor Liver Transplantation With Intraoperative Endovascular Recanalization via Transsplenic Access in a Recipient With Grade III Portal Vein Thrombosis: A Case Report

Extensive portosplenomesenteric thrombosis is regarded as a relative contraindication to liver transplantation because of the complexity of the surgical procedure. This report describes a case of living-donor liver transplantation (LDLT) for a patient with extensive portosplenomesenteric thrombosis, in whom portal flow was successfully restored by intraoperative transplenic portal vein and superior mesenteric vein stenting after surgical thrombectomy. The patient’s liver function remained normal with a patent portal vein stent 6 months after LDLT, and Doppler ultrasonography demonstrated a normal wave form for portal flow. To the best of our knowledge, this is the world’s first case of endovascular management of the portal vein via percutaneous transsplenic access during LDLT, demonstrating that transsplenic access can be an alternative approach without liver graft injury when the superior mesenteric vein branch and inferior mesenteric vein cannot be used as access routes.

Auxiliary xenotransplantation as an in vivo bioreactor—Development of a transplantable liver graft from a tiny partial liver

We established a completely novel method of auxiliary xenogeneic partial liver transplantation and examined whether liver grafts procured from Syrian hamsters regenerated in nude rats, which were used as in vivo bioreactors. The hamsters and the rats were all males (n=10). Partial liver grafts from hamsters were transplanted into nude rats in an auxiliary manner. We evaluated liver graft injury, rejection, and regeneration during 7days after auxiliary xenogeneic partial liver transplantation. All rats survived until sacrifice on post-operative day (POD) 1, 3, and 7. HE-staining showed normal at POD1, mild periportal edema, and slight bile duct and venous endothelial inflammation at POD3, and moderate acute cellular rejection at POD7 without parenchymal necrosis. The liver regeneration rates at POD3 and 7 were 1.54±0.23 and 2.54±0.43, respectively. The Ki-67 labeling index was also elevated at POD3 (27.5±4.1%). Serum HGF and VEGF were elevated at POD1 and 3. ATP levels of liver grafts recovered at POD7. These results revealed that with appropriate immunosuppressive therapy, partial liver graft regeneration occurred in a xenogeneic animal, which suggests liver grafts regenerated in xenogeneic environments, such as an in vivo bioreactor, have potential to be transplantable liver grafts for humans.

Individual liver plasmacytoid dendritic cells are capable of producing IFNα and multiple additional cytokines during chronic HCV infection.

Plasmacytoid dendritic cells (pDCs) are “natural” interferon α (IFNα)-producing cells. Despite their importance to antiviral defense, autoimmunity, and ischemic liver graft injury, because DC subsets are rare and heterogeneous, basic questions about liver pDC function and capacity to make cytokines remain unanswered. Previous investigations failed to consistently detect IFNα mRNA in HCV-infected livers, suggesting that pDCs may be incapable of producing IFNα. We used a combination of molecular, biochemical, cytometric, and high-dimensional techniques to analyze DC frequencies/functions in liver and peripheral blood mononuclear cells (PBMCs) of hepatitis C virus (HCV)-infected patients, to examine correlations between DC function and gene expression of matched whole liver tissue and liver mononuclear cells (LMCs), and to determine if pDCs can produce multiple cytokines. T cells often produce multiple cytokines/chemokines but until recently technical limitations have precluded tests of polyfunctionality in individual pDCs. Mass cytometry (CyTOF) revealed that liver pDCs are the only LMC that produces detectable amounts of IFNα in response TLR-7/8 stimulation. Liver pDCs secreted large quantities of IFNα (~2 million molecules of IFNα/cell/hour) and produced more IFNα than PBMCs after stimulation, p = 0.0001. LMCs secreted >14-fold more IFNα than IFNλ in 4 hours. Liver pDC frequency positively correlated with whole liver expression of “IFNα-response” pathway (R2 = 0.58, p = 0.007) and “monocyte surface” signature (R2 = 0.54, p = 0.01). Mass cytometry revealed that IFNα-producing pDCs were highly polyfunctional; >90% also made 2–4 additional cytokines/chemokines of our test set of 10. Liver BDCA1 DCs, but not BDCA3 DCs, were similarly polyfunctional. pDCs from a healthy liver were also polyfunctional. Our data show that liver pDCs retain the ability to make abundant IFNα during chronic HCV infection and produce many other immune modulators. Polyfunctional liver pDCs are likely to be key drivers of inflammation and immune activation during chronic HCV infection.

Clinical outcomes of Laparoscopic living donor right hepatectomy without Pringle’s and Hanging maneuver

Introduction: Careful preparation and blood-saving surgery will significantly lower the postoperative morbidity in laparoscopic liver resection (LLR). To reduce bleeding during hepatectomy, it is significant to decrease central venous pressure (CVP) and apply Pringle’s & hanging maneuver (P&H). However, P&H are cumbersome and has the potential for further injury by excessive mobilization and dissection of inferior vena cava and right lobe of liver, especially in living liver donors. We would like to present the experience and outcomes of laparoscopic living donor right hepatectomy (LDRH) performed without P&H. Method: Between December 2014 and October 2018, among 97 cases of living donor right hepatectomy, 50 donors underwent LDRH. During LDRH, mean pneumoperitoneal pressure was 12 mmHg and CVP was less than 5 mmHg. The right liver was mobilized to the inferior half portion of retrohepatic IVC and large right inferior hepatic veins were preserved. The caudal approach without P&H was applied for liver parenchymal transection. The V5 and V8 for reconstruction were also preserved until just before the right hepatic duct transection. Result: Mean total operation time was 367 minutes and the warm ischemic time was 9.2 minutes. No donors required blood transfusion, conversion to open surgery, and re-operation. The postoperative course was uneventful except one donor with bile leakage from the cutting edge of the right hepatic duct stump. All donors’ liver function was recovered to normal range within 2 weeks and mean postoperative hospital stay was 8 days. Conclusion: Conclusively, although P&H is not used in LDRH, LLR under low CVP and constant pneumoperitoneal pressure without P&H can help reduce blood loss and prevent further liver graft injury by excessive mobilization of liver. However, because of the donor safety which is the most important issue in living donor hepatectomy, LDRH should be performed by donor surgeon with sufficient experience in laparoscopic hepatectomy.

Delta PD-1 Expressed M2 Macrophages Promote Liver Tumor Recurrence after Transplantation

Introduction Hepatocellular Carcinoma (HCC) is one of the dominant malignancies worldwide and become the primary cause for liver transplantation. Despite its effectiveness, tumor recurrence remains to be a challenge for liver transplantation especially in patients received living donor grafts. Macrophages are key immune cells mediating the inflammatory responses during injuries. Our group previously reported that a subpopulation of macrophages (M2) enhanced tumor metastasis and contributed to poor relapse free survival in HCC patients received curative hepatectomy. Currently, the implications as well as activation mechanisms of these cells in liver transplantation are unknown. Our preliminary evidences indicated the accumulation of a subset of M2 macrophages, expressed with Δ42PD-1, a natural isoform of a immune regulator PD-1, in the post-transplant tissues. We hypothesized that liver graft injury during transplantation activate M2 macrophages, via Δ42PD-1 mechanism, leads to tumor recurrence and poor outcome in HCC patients. Materials and Methods A clinical cohort of 100 HCC patients who underwent deceased (DDLT) and living (LDLT) donor liver transplantation were included. To understand its clinical implications, the expression level of tissue M2 macrophages with and without Δ42PD-1+ were examined followed by the association analyses with patients post-transplant outcomes. To investigate its roles in recurrence, Δ42PD-1+ populations isolated from patients were studied in rat tumor transplant model and in vitro co-cultivation system with HCC cells (Figure 1). Results and Discussion Clinically, 2.1 fold elevation of intra-hepatic M2 macrophages were found in living donor graft compared to deceased graft during the early phase of post-transplantation (P<0.01)(Figure 2). In addition, we identified the increased of Δ42PD-1 expressed in the elevated M2 sub-population (Figure 3). Further analysis indicated the M2 macrophages contributed to the higher recurrence incidences and shorter relapse free survival in patients (P=0.036)(Figure 4A and 4B). In vivo study showed that liver graft injuries significantly induced the accumulation of M2 macrophages after transplantation and resulted in larger tumor volume in the rat transplant tumor model. We showed that that Δ42PD-1[high] macrophages expressed characteristic M2 phenotypes including pro-tumor cytokine secretory profiles compared to Δ42PD-1[low] cells. Co-cultivation of Δ42PD-1[high] population with HCC cells significant increased tumor cells proliferation. Conclusion Here we revealed a novel cellular mechanism in promoting the post-transplant recurrence in liver transplantation. Liver graft injury induced the accumulation and activation of tumor promoting Δ42PD-1 M2 macrophages. Targeting the specific immune population and the regulatory mechanism represent a potential therapeutic strategy in attenuating tumor recurrence in HCC.

Intestinal Mucosal Barrier Disruption and Microfold Cells Impairment were Required for Invasive Fungal Infection after Living Donor Liver Transplantation.

Introduction and Objective Post-transplant invasive fungal infection (IFI) is a critical problem with high mortality for patients receiving living donor liver transplantation (LDLT), however, the underlying mechanism stays unclear. Hence, identifying the risk factors and understanding the mechanisms of IFIs are essential to improve outcome of LDLT patients. Materials and Methods Clinical characteristics of patients diagnosed with opportunistic fungal infections compared with non-infected patients after living donor liver transplantation were analyzed in a retrospective cohort from 1994 to 2017. The correlation between marginal graft injury and incidence of IFIs was investigated by 1, 3-β-D-Glucan assay in rat orthotopic liver transplantation models using marginal grafts. And intestinal mucosal structural integrity as well as microfold cells of intestinal Peyer’s patches were also investigated in animal models. Results and Discussion 44 out of 584 patients were diagnosed with opportunistic fungal infections after LDLT, and they were presented with higher hospital mortality (15% vs 2.8%, p=0.002) as well as long term graft failure compared with patients without infections (Fig. 1A, HR=2.394, p<0.001). In addition, prolonged cold ischemic time and warm ischemic time were identified associated with post-transplant opportunistic infection occurrence (Fig. 1BC). In rat orthotopic liver transplantation models, incidence of IFIs and median serum β-D-Glucan value were higher in small-for-size graft, as well as in graft with prolonged ischemic time compared to whole-size graft and sham operation groups (Fig. 2AB). Furthermore, disruption of intestinal villi structure and loss of tight junctions were found associated with more severe liver graft injury (Fig. 2C). And damage to tight junctions of intestinal follicle associated epithelium covering Peyer’s patches as well as deletion of functional microfold cells (M cells) were also found in liver graft after transplantation, whilst the situation was even worse after ischemic time was extended (Fig. 2D). Conclusion Small-for-size and ischemic injury of liver graft rendered LDLT patients more vulnerable to gut derived invasive fungal infections by disrupting intestinal mucosal barrier and impairing intestinal innate immunity.