BACKGROUND Primary nonfunction (PNF) is a severe complication following liver transplantation (LT), yet precise molecular biomarkers for early identification of patients at risk remain lacking, which can delay timely therapeutic intervention. MATERIAL AND METHODS Liver biopsies were collected from patients and classified into 4 groups: control, optimal graft (OG), early allograft dysfunction (EAD), and PNF. Samples were obtained at 3 time points: T0 (pre-cold perfusion), T1 (pre-reperfusion), and T2 (post-reperfusion). Isobaric tags for relative and absolute quantitation (iTRAQ) and multiple reaction monitoring (MRM) were used for proteomic analysis and biomarker verification. RESULTS Baseline characteristics of the patients showed no significant differences between groups. A total of 6505 proteins were identified in human liver samples. There were 160 differentially expressed proteins (67 upregulated and 93 downregulated) found in the PNF group compared to the control, while 54 and 36 proteins were identified in the EAD and OG groups, respectively. Ten proteins were selected for MRM verification, confirming the significant upregulation of VWF and downregulation of PRDX1, HGD, THIO, 6PGD, and HPPD, consistent with the iTRAQ results. CONCLUSIONS PRDX1, HGD, THIO, 6PGD, HPPD, and VWF were identified as candidate proteins associated with PNF and ischemia-reperfusion injury (IRI) after LT. These findings are hypothesis-generating and require validation in larger, independent cohorts to determine their potential clinical value.

The normothermic machine perfusion (NMP) technique is a promising organ preservation technology that provides an ex-vivo organ evaluation and intervention platform. Because of the limited availability of clinically discarded organs and the high cost of large animal models, the small animal NMP model has been considered a promising alternative for research. Here, we established a standardized NMP and transplantation protocol for rat kidneys, providing a step-by-step guide for kidney procurement, NMP preservation, and transplantation. The NMP details and parameters were recorded. All rat recipients successfully survived with this surgical procedure and NMP technique, and the renal graft function remained normal after transplantation. The survival rate and graft histology of NMP showed no significant difference from that of static cold storage (SCS) on postoperative day 7. This novel NMP strategy in small animal models may benefit basic studies related to organ preservation, assessment, and intervention in the future. Furthermore, this model offers a cost-effective, scalable platform for preclinical testing of therapeutics, imaging techniques, and perfusion-related interventions.

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.

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: Tumor recurrence is associated with poor post-operative survival for patients with hepatocellular carcinoma (HCC), which can be promoted by ischemia-reperfusion injury (IRI) during liver transplantation. Ischemia-free liver transplantation (IFLT) can largely abrogate IRI by avoiding graft ischemia. This study aimed to evaluate whether IFLT can improve patient survival in HCC patients by reducing the risk of cancer recurrence. Methods: This retrospective cohort study included adult patients with HCC who underwent primary liver transplantation using donation-after-brain-death (DBD) grafts between July 2017 and December 2024. Patient survival and incidence of HCC recurrence were compared between IFLT and conventional liver transplantation (CLT).Causal mediation analysis was performed to evaluate the extent to which differences in HCC recurrence contributed to the survival difference between the IFLT and CLT groups. Findings: A total of 400 patients were included, comprising 68 in the IFLT group and 332 in the CLT group. IFLT was associated with a higher 5-year survival probability than CLT (adjusted HR 0·440, 95% CI 0·229-0·847, p = 0·014). The cumulative incidence of HCC recurrence was also lower in the IFLT group than in the CLT group (SHR 0·465, 95% CI 0·236-0·916, p = 0·027). Mediation analysis showed that reduced tumor recurrence accounted for 71·8% of the 5-year survival benefit of IFLT over CLT. The oncological and survival benefits were more significant in patients beyond Milan or UCSF criteria. Interpretation: IFLT is associated with improved 5-year patient survival in patients with HCC, which is mainly mediated by reduced tumor recurrence post-transplantation. These associations might be more pronounced in patients with HCC beyond Milan or UCSF criteria. Funding: The study was supported by the National Natural Science Foundation of China (82170663, 82370664, 82300744, W2511088 and 82525012); Guangdong Provincial Key Laboratory Construction Projection on Organ Donation and Transplant Immunology (2023B1212060020); Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation) (2020A0505020003); Science and Technology Program of Guangdong (2024B1515040011, 2024A1515013030).

BACKGROUND:The liver is essential for coagulation-anticoagulation balance. Ischemia-free liver transplantation (IFLT) has been proven to prevent ischemia-reperfusion injury (IRI) and improve postoperative recovery. In this study, we explored protective effects of IFLT on the coagulation system. MATERIALS AND METHODS:Sixty-five liver transplant patients were enrolled in this post hoc analysis of the IFLT-DBD (ischemia-free transplantation of livers from donors after brain death) trial. Data of blood loss, blood product transfusion, intraoperative conventional coagulation tests, and rapid thromboelastography were evaluated. Transcriptome analysis was performed, and liver tissue specimens were collected for experimental validation. RESULTS:Total blood loss was significantly lower in the IFLT group than in the conventional liver transplantation (CLT) group [1765.0 (565.0-2965.0) vs 2600.0 (595.0-4605.0) mL, P<0.001]. The IFLT group had fewer red blood cells (RBC), fresh frozen plasma (FFP), and platelet (PLT) transfusions than the CLT group [RBC: 4 (2-6) vs 7.25 (2.25-8) units, P = 0.026; FFP: 600 (0-600) vs 600 (400-987.5) ml, P = 0.031; PLT: 6(18.8%) vs 13(39.4%), P = 0.026]. Bulk transcriptome analysis showed that hepatocyte nuclear factor 4 alpha (HNF4α) expression was higher in the IFLT group. Real-time PCR revealed higher mRNA expression of HNF4α, factor II, and V in the IFLT group. Western blotting showed higher HNF4α protein expression in the IFLT group. CONCLUSION:Adopting IFLT significantly decreased intraoperative hemorrhage and blood product transfusion. IFLT facilitates the restoration of intraoperative coagulation homeostasis by preserving the HNF4α-mediated regulation of hepatic coagulation factor synthesis.