BACKGROUND:Dendritic cell (DC) migration-triggered T cell activation is a pivotal event in transplant rejection. Previous studies have established a critical role of the C-type lectin receptor 2（CLEC-2）and podoplanin (PDPN) axis in regulating DC migration. We therefore hypothesized that targeting CLEC-2-PDPN axis could prolong allograft survival in mice by impairing DC migration. MATERIALS AND METHODS:Bioinformatics analysis was performed to assess differential CLEC-2 expression between allograft and isograft tissues and to identify potential associated biological processes. Bone marrow-derived dendritic cells (BMDCs) were generated from donor mice and treated with anti-CLEC-2 to inhibit CLEC-2 function. After LPS stimulation, surface expression of costimulatory molecules and MHC class II, as well as phagocytic capacity, were analyzed. Migration in response to PDPN and CCL19 gradients and the ability to stimulate T cell proliferation were evaluated using a Transwell assay. A murine skin allograft model (BALB/c to C57BL/6) was established. Then anti-PDPN was injected into recipient mice during transplantation. Graft survival was monitored, and pathological examination of grafts, flow cytometric analysis of draining lymph nodes (assessing DC migration and T cell activation/proliferation), and serum cytokine detection by ELISA were performed. RESULTS:Bioinformatics analysis revealed that CLEC-2 was significantly upregulated in the allograft group. In vitro, CLEC-2 suppression had no significant effect on the expression of costimulatory molecules or phagocytosis in dendritic cells. However, it specifically inhibited PDPN-mediated migration and suppressed T cell proliferation in the Transwell assay. Anti-PDPN treatment significantly prolonged skin allograft survival and attenuated rejection in mice. Flow cytometry revealed a reduced proportion of migratory DCs, particularly of the cDC2 subset in the draining lymph nodes of treated mice. Furthermore, both CD4 + and CD8 +T cell proliferation and activation were markedly suppressed. ELISA results showed decreased serum levels of IL-18 and IL-1β, while elevated IL-10. CONCLUSION:Targeting the CLEC-2-PDPN axis significantly prolongs murine allograft survival via the inhibiting DC migration and subsequent T-cell proliferative responses.

Ischemia-free liver transplant (IFLT) has been developed to reduce ischemia-reperfusion injury (IRI). This study aims to investigate how this procedure impacts local and systemic immunity compared to conventional liver transplantation (CLT). Immunohistochemistry, immunofluorescence staining, single-cell RNA sequencing (scRNA-seq), and multiplex cytokine are used to illustrate distinct local and systemic immunity. In contrast to CLT, IFLT reduces neutrophil infiltration and neutrophil extracellular trap formation in grafts. By constructing an immune cell chimerism atlas, we reveal that IFLT reduces recipient-derived monocyte infiltration by suppressing ANXA1-FPR1 signaling through the STAT3-HIF-1α pathway, thereby attenuating inflammatory responses in graft monocytes. Additionally, IFLT confers graft protection by upregulating HMOX1 expression in monocytes and macrophages. Peripherally, IFLT significantly reduces the expression of MHC II molecules in circulating monocytes. Accordingly, CD8+ effector T cell composition, T helper 1 (Th1) and Th17 cytokine levels are reduced, while regulatory T cell (Treg) composition and Th2 cytokine levels are increased in IFLT versus CLT recipients. These results show that IFLT profoundly affects local and systemic immunity in liver transplantation. Recipient-circulating monocytes might play a key role in the interaction between graft IRI and allograft rejection.

INTRODUCTION:To explore the relationship among portal vein velocity (PVV) on the first postoperative day after liver transplantation, the occurrence of hepatic artery thrombosis (HAT), and 90-day mortality in hepatocellular carcinoma (HCC). METHODS:In total, 84 patients' clinical records were retrospectively analyzed. The receiver operating characteristic curve was employed. According to the optimal cutoff value, 84 patients were separated into the high PVV group and the normal PVV group. Fisher's exact test compared the occurrence of HAT and the 90-day mortality. The risk factors of HAT were analyzed by logistic regression. RESULTS:HAT was documented in 8 (9.5%) patients of the high PVV group and no patient in the normal PVV group, p < 0.001. During the 90 days after surgery, 6(7.1%) deaths occurred in the high PVV group, and 1(1.2%) death occurred in the normal PVV group, p = 0.019. PVV ≥51.2 cm/s on the first postoperative day constitutes an independent risk factor for HAT. CONCLUSION:PVV ≥51.2 cm/s on the first postoperative day in HCC constitutes an independent risk factor for HAT, associated with high 90-day mortality.

The persistent disparity between organ donation rates and clinical demand has driven the increasing use of extended-criteria donor livers. However, conventional static cold storage inadequately preserves extended-criteria donor with severe ischemia-reperfusion injury (IRI), contributing to high rates of mortality and morbidity. Although different machine perfusion technologies have been used to reduce IRI in clinical practice, organ ischemia remains unavoidable throughout the entire transplantation procedure. To minimize IRI to the greatest extent possible, we developed a novel ischemia-free liver transplantation (IFLT) method based on surgical innovation and continuous normothermic machine perfusion. IFLT not only effectively preserves graft quality but also expands the donor pool, making it possible to utilize high-risk livers. Classic IFLT increases the complexity of donor liver procurement and prolongs the anhepatic phase during implantation. Here we develop a simplified IFLT (SIFLT) technique. By streamlining the donor liver retrieval procedure and optimizing the sequence of vascular anastomosis during implantation, the efficacy and safety data for SIFLT are comparable to those of classic IFLT, with similar rates of postoperative complications, graft survival and patient survival. Thus, SIFLT represents a more efficient, safer and widely applicable approach to minimize organ ischemia, offering a robust strategy to improve outcomes and maximize organ utilization.

Background:Patients with BCLC stage B or C hepatocellular carcinoma (HCC) often face long waiting times. The effectiveness of different tumor control strategies remains unclear. This study evaluates preoperative conversion therapies for liver transplantation (LT). Methods:This study retrospectively analyzed 269 patients with HCC at BCLC stage B or C from a single center from January 2015 to August 2024. All these patients underwent liver transplantation. They were divided into three groups by treatment: transcatheter arterial chemoembolization (TACE) (n = 150), TACE + tyrosine kinase inhibitor (TKI) (n = 74), and TACE + TKI+immune checkpoint inhibitors (ICI) (n = 45). Preoperative assessments included blood tests, liver and kidney function, coagulation, tumor markers, and imaging to evaluate tumor response and monitor adverse events. Data collected included baseline characteristics, lab results, surgical details, preoperative treatment response, adverse events, 1-year survival, tumor-free survival, and donor information. Results:Baseline characteristics were well-balanced across the three groups. The TACE + TKI + ICI group showed higher DCR, ORR, and CRR than both the TACE-alone and TACE + TKI groups (all P < 0.05; χ2 = 11.592, 18.111, 17.558). It also had longer RFS versus both control groups (vs. TACE alone: P = 0.008; vs. TACE + TKI: P = 0.040). One-year OS did not differ significantly among groups (P = 0.707). Safety analyses revealed no significant differences in preoperative lab values, adverse event rates, or intraoperative liver transplant parameters (all P > 0.05), confirming prior treatments did not compromise surgical feasibility or safety. Multivariate logistic regression identified treatment regimen as an independent predictor of DCR (P=0.002), ORR (P<0.001), and CRRP (P<0.001). Multivariate Cox regression confirmed it as an independent predictor of RFS (P<0.01). After a 1-month washout, rejection rates did not differ significantly among the three patient groups, and treatment method was not associated with rejection risk (P=0.549). Conclusion:In the conversion therapy of HCC, the combination of TACE + TKI + ICI has demonstrated higher efficacy, longer recurrence-free survival, manageable safety, and does not interfere with subsequent LT.

The objective of this study was to examine the effect of PRDM1 inhibition or overexpression on CD4 + T cells, and islet and skin transplantation in mice. Bioinformatics analysis revealed PRDM1 showed a significant downregulation in the Allograft group in CD4 + T cells. Mice CD4 + T cells were cultured, purified and enriched, and then transfected with shRNA lentiviral vector PRDM1-RNAi-GFP and overexpression lentiviral vector LV-PRDM1-puro. Murine islet transplantation and skin transplantation model were established, and the mice were injected with CD4 + T cells transfected with lentivirus. Levels of cytokines were measured, and the survival times of the grafts were measured and compared. T cell subsets were analyzed by flow cytometry. The expressions of IL-4 and IL-10 were up-regulated after overexpression of PRDM1, and the expression of IFN-γ was up-regulated in CD4 + T cells after inhibition of PRDM1 (P < 0.05). Overexpression of PRDM1 resulted in longer islet and skin graft survival and better graft function, while inhibition of PRDM1 shortened islet and skin graft survival. Microscopic examination showed evidence of mild rejection of islet and skin grafts in the LV-PRDM1-puro, but severe rejection in the PRDM1-RNAi group. Taken together, the results indicate that overexpression of PRDM1 can prolong graft survival and induce the formation of transplant immune tolerance via secretion of Th2 cytokines by CD4 + T cells. Inhibition of PRDM1 can promote transplant rejection and shorten graft survival via secretion of Th1 cytokines.

IFI16 (the murine homologue is IFI204) is an intracellular double-stranded DNA (dsDNA) pattern recognition receptor (PRR) that plays a crucial role in bridging innate and adaptive immunity. However, its function in dendritic cell (DC) activation and anti-hepatocellular carcinoma (HCC) efficacy remains poorly characterised. This study demonstrates that IFI16 promotes DC maturation, functional activation and antitumor immunity. This effect occurs through activation of the STING-TBK1-IRF3 signalling pathway. Our findings establish IFI16 as a key molecule enabling DCs to sense dsDNA and initiate antitumor responses. Consequently, targeting IFI16 and its downstream STING-TBK1-IRF3 signalling pathway represents a potential therapeutic strategy for hepatocellular carcinoma.

Cyclic GMP-AMP synthase (cGAS) is critical for dendritic cell (DC) maturation. This study investigates how cGAS suppression in DCs influences graft immune tolerance. Bioinformatics analysis assessed cGAS involvement in transplant immunity. Immature DCs were transduced with an adenoviral vector to knockdown cGAS and divided into three groups: cGAS-shRNA-DCs, EGFP-DCs and PBS (control). These were administered intravenously before transplantation to establish a mouse model. Graft survival and histopathology were evaluated. Splenic T cell subsets were analyzed by flow cytometry. After lipopolysaccharide stimulation, MHC-II and co-stimulatory molecule expression, antigen uptake, and T cell proliferation were measured in three groups. Cytokine levels in supernatants were quantified. Western blotting was used to explore the mechanism of cGAS in DC maturation. Bioinformatics revealed elevated cGAS expression in allografts. cGAS-shRNA-DCs showed reduced MHC-II and co-stimulatory molecule expression, enhanced phagocytosis, and decreased T cell activation. IFN-γ, IL-1β, TNF-α, and IL-6 levels were lower, while IL-10 was higher. Mice receiving cGAS-shRNA-DCs exhibited prolonged graft survival and improved function. Flow cytometry showed increased regulatory T cells and reduced Th1 and Th17 cells. WB indicated that cGAS regulates DC maturation via NF-κB signaling. In conclusion, suppressing cGAS in DCs inhibits their maturation, thereby enhancing graft survival.

Purpose:This pilot study aimed to explore the necessity for 47,XYY syndrome males (couples) to perform PGT rather than conventional In Vitro Fertilization (IVF)/Intracytoplasmic Sperm Injection (ICSI) cycles. Methods:A retrospective cohort study was conducted with 36 nonmosaic and mosaic 47,XYY syndrome patients (couples) undergoing 43 oocyte retrieval cycles (37 planned for PGT and 6 for IVF/ICSI) between December 2017 and December 2023. The couples were given either next-generation sequencing-based PGT or conventional IVF/ICSI followed by 45 embryo transfer (ET) cycles (38 from PGT and 7 from IVF/ICSI). The detailed cytogenetic results of the 129 embryos from PGT were analyzed, and the pregnancy and neonatal outcomes between PGT-ET and conventional IVF/ICSI-ET cycles were compared. Results:The PGT results showed that the chance of sex chromosome abnormalities was low (1.55%), with chromosomal errors being observed more often in autosomes. Importantly, no differences were observed in the rates of biochemical pregnancy, implantation, clinical pregnancy, ongoing pregnancy, pregnancy loss, live birth, and preterm delivery between PGT-ET cycles and conventional IVF/ICSI-ET cycles. Comparable results regarding gestational age, birthweight, low birthweight rate, macrosomia rate, male rate, as well as the rate of congenital anomalies were also observed between the two groups. Conclusions:Preimplantation genetic testing might not be necessary to conduct for 47,XYY syndrome males unless there are other indications. Studies with large populations are in demand to confirm the present results.

Background & Aims: Ischemia-free liver transplantation (IFLT) is a novel technique designed to avoid ischemia–reperfusion injury (IRI). Here, we report the first detailed 5-year follow-up outcomes. Methods: We conducted a cohort study comparing long-term outcomes between IFLT and conventional liver transplantation (CLT) recipients of livers donated after brain death (DBD). The primary objective was to evaluate 5-year patient and graft survival. Additional endpoints included graft loss, biliary complications, rejection, infections, and liver-related laboratory tests. Subgroup analysis was performed to validate the generalizability of the results in patients with pre-transplant hepatocellular carcinoma (HCC). Results: A total of 168 patients were enrolled, with 38 patients in the IFLT group and 130 patients in the CLT group. Five-year patient survival (86.84% vs. 56.92%; hazard ratio [HR] 0.246, 95% confidence interval [CI] 0.098–0.620; p <0.01) and graft survival (84.61% vs. 56.92%; HR 0.307, 95% CI 0.131–0.719; p <0.01) rates were significantly improved in the IFLT group compared with the CLT group. In the multivariate analysis, IFLT emerged as an independent protective factor for 5-year patient survival (HR 0.246, 95% CI 0.098–0.620; p <0.01). Conversely, HCC before transplantation (HR 2.039, 95% CI 1.159–3.590; p <0.05), donor age (HR 1.022, 95% CI 1.001–1.040; p <0.05), and extended criteria donor (HR 2.088, 95% CI 1.215–3.590; p <0.01) were identified as independent risk factors for impaired 5-year patient survival. In patients with pre-transplant HCC, the 5-year overall survival rate of the IFLT group was also significantly higher than that of the CLT group after adjustment for HCC risk factors (82.35% vs. 42.03%; HR 0.249, 95% CI 0.074–0.831; p <0.05). Conclusions: Long-term (5-year) follow-up data demonstrate that the use of IFLT potentially improves both patient and graft survival, when compared with CLT, in transplantation of brain-dead donor livers. Impact and implications: Ischemia-free liver transplantation (IFLT) has emerged as a new approach designed to avoid IRI throughout all episodes of the transplant procedure. It has been confirmed that the use of IFLT can substantially reduce early-onset graft IRI-related complications. In this first 5-year follow-up study on the IFLT technique, we demonstrate that, compared with conventional liver transplantation, IFLT can potentially improve long-term patient and graft survival by reducing cancer recurrence. This new technique has the potential to change current clinical practice, particularly in the use of marginal grafts and in patients with HCC. Clinical Trials registration: chictr.org (ChiCTR-OPN-17012090).

Dendritic cells (DCs) serve a pivotal and diverse role in the modulation of transplant immunity, wherein alterations in their maturation state influence the balance between promoting immunotolerance and exacerbating inflammatory responses. Recent studies have revealed a spectrum of novel mechanisms through which absent in melanoma 2 (AIM2) regulates the functions of immune cells. However, the impact of AIM2 on the regulatory functions of dendritic cells in alloimmunity has not been thoroughly investigated. In this study, we constructed a recombinant adenovirus vector containing AIM2 interference sequences for the transduction of DCs, aiming to suppress AIM2 expression within these cells. Our findings indicate that silencing AIM2 preserved a semi-mature status of DCs upon lipopolysaccharide (LPS) exposure, alongside maintaining reduced levels of pro-inflammatory cytokine secretion and enhanced phagocytic activity. When co-cultured with allogeneic naive T cells, AIM2-silenced DCs demonstrated reduced activation potential of CD4+ and CD8+ T cells in response to LPS stimulation. The transfusion of AIM2-silenced DCs into recipient mice led to a notable prolongation of graft survival and a decrease in inflammatory cell infiltration within the graft. Furthermore, mice treated with AIM2-silenced DCs exhibited a higher percentage of regulatory T cells in their spleen and abdominal lymph nodes, concomitant with a decrease in the proportions of Th1 and Th17 cell subpopulations. AIM2-silenced DCs exhibit stable immunosuppressive capabilities, offering an effective strategy for alleviating allograft rejection.

BACKGROUND:Aberrant DNA methylation plays a pivotal role in cancer progression by enhancing oncogene activation or silencing tumor suppressor genes, contributing to malignant phenotypes. Methylation driver genes (MDGs) are characterized by an inverse correlation between DNA methylation levels and mRNA expression, making them critical targets for cancer research. METHODS:We analyzed the liver hepatocellular carcinoma (LIHC) dataset from The Cancer Genome Atlas (TCGA) using the R package MethylMix to identify MDGs. Prognostic models were developed through univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and multivariate Cox regression to identify core genes. We further evaluated the associations of these genes with the tumor immune microenvironment, immune checkpoint inhibitors (ICIs), and chemotherapeutic sensitivity. Finally, liver cancer tissue organoid culture experiments combined with DNA methylation sequencing were conducted to validate predictions of drug sensitivity. RESULTS:A total of 21 MDGs were identified, among which GNA14, glutaminase (GLS), and GNG4 were selected to construct a prognostic risk score model. The model demonstrated robust predictive performance, with Receiver Operating Characteristic (ROC) values of 0.723, 0.764, and 0.716 for 1-, 3-, and 5-year survival, respectively. Among these, GLS emerged as a key gene, showing low methylation levels and high mRNA expression, which were associated with poor prognosis, significant alterations in the tumor immune microenvironment, and differential sensitivity to ICIs and chemotherapeutic agents. CONCLUSION:The three-gene MDG-based prognostic model effectively predicts survival outcomes in LIHC patients. Moreover, the methylation status of GLS serves as a biomarker for assessing immune microenvironment characteristics, responsiveness to immunotherapy, and chemotherapy sensitivity, highlighting its potential as a therapeutic target in liver cancer.

Bile leakage is a common complication after liver transplantation that may usually be cured with endoscopic retrograde cholangiopancreatography (ERCP), percutaneous transhepatic cholangial drainage (PTCD), percutaneous transhepatic cholangioscopy (PTCS)and surgery. We report a novel treatment for biliary leakage lasting 9 months after liver transplantation, during which ERCP, PTCD, PTCS, and surgical therapy were in vain. We have used a new method of multi-endoscopic treatment. First, ERCP and peroral single operator cholangioscopy are used to place a plastic stent as a marker at the distal end of the bile leakage site. Second, PTCS is performed through the PTCD tube. During the operation, the proximal end of the bile leakage site is located by the plastic stent and B ultrasound, and the continuity of the biliary tract is temporarily reconstructed through the guide wire and urinary catheter. Third, a double-headed guidewire is placed, the urinary catheter is removed, the stents are placed, and biliary continuity is reconstructed. In conclusion, we have found a new method combining ERCP, peroral single operator cholangioscopy, and PTCS to treat complex bile leakage after liver transplantation.

IntroductionDespite years of research, knowledge about the microbial populations of human physiological bile has remained limited. Bile sampling techniques, such as Endoscopic Retrograde Cholangiopancreatography (ERCP), percutaneous biliary drainage, and intra-operative sampling, are invasive procedures typically performed only in the presence or suspicion of biliary tract disease. Furthermore, the increased incidence of bacterial infections following biliary drainage poses a significant clinical concern; however, the relationship between biliary drainage and biliary flora remains poorly understood. In this study, we present a distinct taxonomic composition of bacterial communities identified in bile samples from disease-free individuals, as well as from obstructive and post-drainage biliary tracts.MethodsA metagenomic sequence analysis of bile samples from patients with MBO who underwent percutaneous biliary drainage (PTBD) at our center from 1st May 2021 to 1st March 2022, which were divided into 2 groups, as the MBO group (n = 29) and BD group (n = 27). Eight liver donors were included as a control group.ResultsAbundant bacterial populations were detected in the bile of liver donors, revealing a highly similar microbial composition in both disease-free and malignant obstructive biliary trees. Notably, biliary drainage was found to alter the composition of bile microbiota, resulting in decreased microbial diversity and an association with an increase in antibiotic resistance genes.DiscussionThese findings provide fundamental knowledge on the composition of the human bile microbiota and present new evidence to support that biliary drainage induces a shift in bile microbiota, rendering it more aggressive and resistant to antibiotics.

INTRODUCTION:Hepatitis B virus (HBV)-related liver diseases, including hepatitis, cirrhosis, and liver failure, seriously threaten human lives and health worldwide. Innate and adaptive immune cells are all thought to participate in HBV-related diseases. However, there is a lack of information on the comprehensive landscape of the immune microenvironment. METHODS:In this study, single-cell ribonucleic acid sequencing was performed on liver samples obtained from patients diagnosed with hepatitis, cirrhosis, and acute-on-chronic liver failure, which were caused by HBV. Trajectory analysis was performed to analyze the evolution of cell subsets, and branch expression analysis modeling was applied to visualize the changes in gene expression during evolution. RESULTS:Finally, there was a significant increase in adaptive immune cells in the hepatitis and cirrhosis groups, whereas more innate immune cells were observed in the liver failure group. Furthermore, we found that monocytes underwent remarkable transcriptomic changes into FABP5+ macrophages, promoting the degranulation and chemotaxis of neutrophils through RESISTIN signaling, and LGMN+ macrophages, with the sequential activation of antigen presentation and defense to pathogens through SPP1 signaling. CONCLUSION:Macrophages were revealed as central to the progression of acute-on-chronic liver failure as they regulated the activation or inhibition of other immune cells, which could help in developing an effective novel therapy.

Donation after circulatory death (DCD) organs are associated with compromised transplant outcomes and limited utilization. Normothermic regional perfusion (NRP) has been shown to significantly improve organ quality and transplant outcomes. Our group previously demonstrated the feasibility and efficacy of ischemia‐free liver implantation (IFLI) using grafts from donation after brain death. We hypothesized that NRP sequential IFLI would be feasible and effective in DCD liver transplantation. This study presents clinical case attempts that validate the viability of this hypothesis. We report the first case of IFLI of a DCD liver following NRP. The graft was donated by a 75‐year‐old man with a total warm ischemia time of 24 min. The abdominal organs underwent 67 min of NRP. The liver was then cold‐stored in University of Wisconsin solution and transported, with a total cold ischemia time of 548 min. After back‐table preparation, the liver was subjected to 373 min of normothermic machine perfusion (NMP) before being implanted using an ischemia‐free implant technique. Perfusion parameters, lactate clearance, and liver enzyme levels during in situ NRP indicated that the liver was transplantable. In addition, perfusion parameters, lactate clearance, and bile production during ex situ NMP fulfilled the VITTAL criteria for transplantation. The recipient was a 65‐year‐old woman with primary biliary cirrhosis. The peak post‐transplant alanine aminotransferase level was 77 U/L, and liver function normalized within 9 days after surgery. The patient recovered uneventfully, with no evidence of early allograft dysfunction or other major complications, and was discharged on day 16 post‐transplantation. This first report demonstrates the feasibility of NRP sequential IFLI in DCD liver transplantation.

BACKGROUND:The mature state of dendritic cells (DCs) determines their ability to regulate immune responses. Retinoic acid-inducible gene-1 (RIG-1) plays a critical role in DC activation and maturation. RIG-1 activation triggers mitogen-activated protein kinase and nuclear factor-kappa B signal transduction. In this study, we aimed to investigate the effects of inhibiting RIG-1 expression in DCs and its potential in inducing immune tolerance.METHODS:DCs were transduced with the recombinant lentiviral vector (Lv) to inhibit RIG-1 expression. A murine islet and skin transplantation model were constructed to find out whether DC-DDX58-RNAi could prolong allograft survival. The phenotypes of DCs and T-cells were analyzed using flow cytometry. Cytokines in serum were detected by the enzyme-linked immunosorbent assay. Protein levels were determined by Western blot.RESULTS:RIG-1-deficient DCs had low expression of costimulatory molecules and major histocompatibility complex and a strong phagocytic ability. DC-DDX58-RNAi induced regulatory T cell differentiation in the transplant recipient spleens. The DC-DDX58-RNAi-treated recipients showed satisfactory islet allograft function and longer survival time.CONCLUSION:Inhibition of RIG-1 with DDX58-RNAi prevented the activation and maturation of the DCs, affected T cell differentiation, protected the biological function of the allograft, and prolonged graft survival. These findings may have important therapeutic implications for new immunomodulatory regimens.