Prolonged xenokidney graft survival in sensitized NHP recipients by expression of multiple human transgenes in a triple knockout pig.

Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (ß-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.

Subnormothermic Oxygenated Machine Perfusion (24 h) in DCD Kidney Transplantation.

Background: Ex vivo kidney perfusion is an evolving platform that demonstrates promise in preserving and rehabilitating the kidney grafts. Despite this, there is little consensus on the optimal perfusion conditions. Hypothermic perfusion offers limited functional assessment, whereas normothermic perfusion requires a more complex mechanical system and perfusate. Subnormothermic machine perfusion (SNMP) has the potential to combine the advantages of both approaches but has undergone limited investigation. Therefore, the present study sought to determine the suitability of SNMP for extended kidney preservation. Methods: SNMP at 22-25 °C was performed on a portable device for 24 h with porcine kidneys. Graft assessment included measurement of mechanical parameters and biochemical analysis of the perfusate using point-of-care tests. To investigate the viability of kidneys preserved by SNMP, porcine kidney autotransplants were performed in a donation after circulatory death (DCD) model. SNMP was also compared with static cold storage (SCS). Finally, follow-up experiments were conducted in a subset of human kidneys to test the translational significance of findings in porcine kidneys. Results: In the perfusion-only cohort, porcine kidneys all displayed successful perfusion for 24 h by SNMP, evidenced by stable mechanical parameters and biological markers of graft function. Furthermore, in the transplant cohort, DCD grafts with 30 min of warm ischemic injury demonstrated superior posttransplant graft function when preserved by SNMP in comparison with SCS. Finally, human kidneys that underwent 24-h perfusion exhibited stable functional and biological parameters consistent with observations in porcine organs. Conclusions: These observations demonstrate the suitability and cross-species generalizability of subnormothermic machine perfusion to maintain stable kidney perfusion and provide foundational evidence for improved posttransplant graft function of DCD kidneys after SNMP compared with SCS.

Contemporary trends and outcomes after liver transplantation and resection for intrahepatic cholangiocarcinoma.

BACKGROUND: Liver transplantation (LT) has been shown to be superior to resection in highly selected patients with perihilar cholangiocarcinoma (CCA), yet has traditionally been contraindicated for intrahepatic CCA (iCCA). Herein, we aimed to examine contemporary trends and outcomes for surgical resection and LT for iCCA. METHODS: The National Cancer Database was queried for patients presenting with stage I-III iCCA between 2010 and 2018 who underwent resection or LT. Overall survival (OS) was compared with Kaplan-Meier and multivariable Cox proportional hazards methods stratified by management. Secondary analysis of patients undergoing transplant for CCA was performed with the United Network for Organ Sharing database. RESULTS: Of 2565 patients, 2412 (94.0%) underwent resection and 153 (5.96%) LT of whom 84 (54.9%) received neoadjuvant therapy. Utilization of LT remained between 3.9% and 7.8% annually. Unadjusted 5-year OS was higher for LT than resection (59.8% vs 39.9%, P = .0067), yet adjusted analysis revealed no significant difference in mortality (hazard ratio, 0.91; 95% CI, 0.66-1.27; P = .58). On secondary analysis including 437 patients with all subtypes of CCA, unadjusted 5-year OS was higher for non-CCA indications (79% vs 52%-54%, P < .001). CONCLUSION: Utilization of LT for iCCA remains low and many cases are likely incidental. Although partial hepatectomy remains the standard of care for patients with resectable disease, our findings suggest that highly selected patients with unresectable iCCA may achieve favorable outcomes after LT. Granular, prospective data are needed to identify patients most likely to benefit from transplant and allocate scarce liver grafts.

What Is Hot and New in Basic and Translational Science in Liver Transplantation in 2023? Report of the Basic and Translational Research Committee of the International Liver Transplantation Society.

The 2023 Joint Annual Congress of the International Liver Transplantation Society, European Liver and Intestine Transplant Association, and Liver Intensive Care Group of Europe were held in Rotterdam, the Netherlands, from May 3 to 6, 2023. This year, all speakers were invited to attend the Congress in person for the first time since the COVID-19 pandemic. The congress was attended by 1159 registered delegates from 54 countries representing 5 continents, with the 10 countries comprising the bulk of the delegates. Of the 647 abstracts initially submitted, 542 were eventually presented at the meeting, coming from 38 countries (mainly North America, Europe, and Asia) and 85% of them (462 abstracts) came from only 10 countries. Fifty-three (9.8%) abstracts, originated from 17 countries, were submitted under the Basic/Translational Scientific Research category, a similar percentage as in 2022. Abstracts presented at the meeting were classified as (1) ischemia and reperfusion injury, (2) machine perfusion, (3) bioengineering and liver regeneration, (4) transplant oncology, (5) novel biomarkers in liver transplantation, (6) liver immunology (rejection and tolerance), and (7) artificial intelligence and machine learning. Finally, we evaluated the number of abstracts commented in the Basic and Translational Research Committee-International Liver Transplantation Society annual reports over the past 5 y that resulted in publications in peer-reviewed journals to measure their scientific impact in the field of liver transplantation.

Gene delivery followed by ex vivo lung perfusion using an adeno-associated viral vector in a rodent lung transplant model.

OBJECTIVE: Ex vivo lung perfusion has emerged as a platform for organ preservation, evaluation, and restoration. Gene delivery using a clinically relevant adeno-associated vector during ex vivo lung perfusion may be useful in optimizing donor allografts while the graft is maintained physiologically active. We evaluated the feasibility of adeno-associated vector-mediated gene delivery during ex vivo lung perfusion in a rat transplant model. Additionally, we assessed off-target effects and explored different routes of delivery. METHODS: Rat heart-lung blocks were procured and underwent 1-hour ex vivo lung perfusion. Before ex vivo lung perfusion, 4e11 viral genome luciferase encoding adeno-associated vector 9 was administered via the left bronchus (Br group, n = 4), via the left pulmonary artery (PA group, n = 3), or directly into the circuit (Circuit group, n = 3). Donor lungs in the Control group (n = 3) underwent ex vivo lung perfusion without adeno-associated vector 9. Only the left lung was transplanted. Animals underwent bioluminescence imaging weekly before being killed at 2 weeks. Tissues were collected for luciferase activity measurement. RESULTS: All recipients tolerated the transplant well. At 2 weeks post-transplant, luciferase activity in the transplanted lung was significantly higher among animals in the Br group compared with the other 3 groups (Br: 1.1 × 106 RLU/g, PA: 8.3 × 104 RLU/g, Circuit: 3.8 × 103 RLU/g, Control: 2.5 × 103 RLU/g, P = .0003). No off-target transgene expression was observed. CONCLUSIONS: In this work, we demonstrate that a clinically relevant adeno-associated vector 9 vector mediates gene transduction during ex vivo lung perfusion in rat lung grafts when administered via the airway and potentially the pulmonary artery. Our preliminary results suggest a higher transduction efficiency when adeno-associated vector 9 was delivered via the airway, and delivery during ex vivo lung perfusion reduces off-target effects after graft implant.

Comparison of the effects of normothermic machine perfusion and cold storage preservation on porcine intestinal allograft regenerative potential and viability.

Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal allografts. However, CS and subsequent transplantation induce ischemia-reperfusion injury (IRI). Severe IRI impairs epithelial barrier function, including loss of intestinal stem cells (ISC), critical to epithelial regeneration. Normothermic machine perfusion (NMP) preservation of kidney and liver allografts minimizes CS-associated IRI; however, it has not been used clinically for IT. We hypothesized that intestine NMP would induce less epithelial injury and better protect the intestine's regenerative ability when compared with CS. Full-length porcine jejunum and ileum were procured, stored at 4 °C, or perfused at 34 °C for 6 hours (T6), and transplanted. Histology was assessed following procurement (T0), T6, and 1 hour after reperfusion. Real-time quantitative reverse transcription polymerase chain reaction, immunofluorescence, and crypt culture measured ISC viability and proliferative potential. A greater number of NMP-preserved intestine recipients survived posttransplant, which correlated with significantly decreased tissue injury following 1-hour reperfusion in NMP compared with CS samples. Additionally, ISC gene expression, spheroid area, and cellular proliferation were significantly increased in NMP-T6 compared with CS-T6 intestine. NMP appears to reduce IRI and improve graft regeneration with improved ISC viability and proliferation.

Sterile inflammation in liver transplantation.

Sterile inflammation is the immune response to damage-associated molecular patterns (DAMPs) released during cell death in the absence of foreign pathogens. In the setting of solid organ transplantation, ischemia-reperfusion injury results in mitochondria-mediated production of reactive oxygen and nitrogen species that are a major cause of uncontrolled cell death and release of various DAMPs from the graft tissue. When properly regulated, the immune response initiated by DAMP-sensing serves as means of damage control and is necessary for initiation of recovery pathways and re-establishment of homeostasis. In contrast, a dysregulated or overt sterile inflammatory response can inadvertently lead to further injury through recruitment of immune cells, innate immune cell activation, and sensitization of the adaptive immune system. In liver transplantation, sterile inflammation may manifest as early graft dysfunction, acute graft failure, or increased risk of immunosuppression-resistant rejection. Understanding the mechanisms of the development of sterile inflammation in the setting of liver transplantation is crucial for finding reliable biomarkers that predict graft function, and for development of therapeutic approaches to improve long-term transplant outcomes. Here, we discuss the recent advances that have been made to elucidate the early signs of sterile inflammation and extent of damage from it. We also discuss new therapeutics that may be effective in quelling the detrimental effects of sterile inflammation.

Normothermic Machine Perfusion of Donor Livers for Transplantation in the United States: A Randomized Controlled Trial.

OBJECTIVE: To compare conventional low-temperature storage of transplant donor livers [static cold storage (SCS)] with storage of the organs at physiological body temperature [normothermic machine perfusion (NMP)]. BACKGROUND: The high success rate of liver transplantation is constrained by the shortage of transplantable organs (eg, waiting list mortality >20% in many centers). NMP maintains the liver in a functioning state to improve preservation quality and enable testing of the organ before transplantation. This is of greatest potential value with organs from brain-dead donor organs (DBD) with risk factors (age and comorbidities), and those from donors declared dead by cardiovascular criteria (donation after circulatory death). METHODS: Three hundred eighty-three donor organs were randomized by 15 US liver transplant centers to undergo NMP (n = 192) or SCS (n = 191). Two hundred sixty-six donor livers proceeded to transplantation (NMP: n = 136; SCS: n = 130). The primary endpoint of the study was "early allograft dysfunction" (EAD), a marker of early posttransplant liver injury and function. RESULTS: The difference in the incidence of EAD did not achieve significance, with 20.6% (NMP) versus 23.7% (SCS). Using exploratory, "as-treated" rather than "intent-to-treat," subgroup analyses, there was a greater effect size in donation after circulatory death donor livers (22.8% NMP vs 44.6% SCS) and in organs in the highest risk quartile by donor risk (19.2% NMP vs 33.3% SCS). The incidence of acute cardiovascular decompensation at organ reperfusion, "postreperfusion syndrome," as a secondary outcome was reduced in the NMP arm (5.9% vs 14.6%). CONCLUSIONS: NMP did not lower EAD, perhaps related to the inclusion of lower-risk liver donors, as higher-risk donor livers seemed to benefit more. The technology is safe in standard organ recovery and seems to have the greatest benefit for marginal donors.

Development and validation of a REcurrent Liver cAncer Prediction ScorE (RELAPSE) following liver transplantation in patients with hepatocellular carcinoma: Analysis of the US Multicenter HCC Transplant Consortium.

HCC recurrence following liver transplantation (LT) is highly morbid and occurs despite strict patient selection criteria. Individualized prediction of post-LT HCC recurrence risk remains an important need. Clinico-radiologic and pathologic data of 4981 patients with HCC undergoing LT from the US Multicenter HCC Transplant Consortium (UMHTC) were analyzed to develop a REcurrent Liver cAncer Prediction ScorE (RELAPSE). Multivariable Fine and Gray competing risk analysis and machine learning algorithms (Random Survival Forest and Classification and Regression Tree models) identified variables to model HCC recurrence. RELAPSE was externally validated in 1160 HCC LT recipients from the European Hepatocellular Cancer Liver Transplant study group. Of 4981 UMHTC patients with HCC undergoing LT, 71.9% were within Milan criteria, 16.1% were initially beyond Milan criteria with 9.4% downstaged before LT, and 12.0% had incidental HCC on explant pathology. Overall and recurrence-free survival at 1, 3, and 5 years was 89.7%, 78.6%, and 69.8% and 86.8%, 74.9%, and 66.7%, respectively, with a 5-year incidence of HCC recurrence of 12.5% (median 16 months) and non-HCC mortality of 20.8%. A multivariable model identified maximum alpha-fetoprotein (HR = 1.35 per-log SD, 95% CI,1.22-1.50, p < 0.001), neutrophil-lymphocyte ratio (HR = 1.16 per-log SD, 95% CI,1.04-1.28, p < 0.006), pathologic maximum tumor diameter (HR = 1.53 per-log SD, 95% CI, 1.35-1.73, p < 0.001), microvascular (HR = 2.37, 95%-CI, 1.87-2.99, p < 0.001) and macrovascular (HR = 3.38, 95% CI, 2.41-4.75, p < 0.001) invasion, and tumor differentiation (moderate HR = 1.75, 95% CI, 1.29-2.37, p < 0.001; poor HR = 2.62, 95% CI, 1.54-3.32, p < 0.001) as independent variables predicting post-LT HCC recurrence (C-statistic = 0.78). Machine learning algorithms incorporating additional covariates improved prediction of recurrence (Random Survival Forest C-statistic = 0.81). Despite significant differences in European Hepatocellular Cancer Liver Transplant recipient radiologic, treatment, and pathologic characteristics, external validation of RELAPSE demonstrated consistent 2- and 5-year recurrence risk discrimination (AUCs 0.77 and 0.75, respectively). We developed and externally validated a RELAPSE score that accurately discriminates post-LT HCC recurrence risk and may allow for individualized post-LT surveillance, immunosuppression modification, and selection of high-risk patients for adjuvant therapies.

Incidence of Postreperfusion Hyperfibrinolysis in Liver Transplantation by Donor Type and Observed Treatment Strategies.

BACKGROUND: Hyperfibrinolysis is a possible complication during liver transplantation, particularly immediately after reperfusion. METHODS: We performed a retrospective study to examine the incidence, treatment, and resolution of postreperfusion hyperfibrinolysis in patients undergoing liver transplantation at Duke University Hospital from 2015 to 2020. RESULTS: Out of 535 patients undergoing liver transplantation, 21 or 3.9%, 95% CI (2.5-5.9), had hyperfibrinolysis after reperfusion. Hyperfibrinolysis occurred in 16 of 511 (3.1%) patients receiving livers from DBD donors, 5 of 18 (27.8%) patients receiving livers from donation after circulatory death (DCD) donors, and 0 of 6 (0.0%) patients receiving livers from living donors. Fibrinolysis was treated with cryoprecipitate (12/21), a combination of cryoprecipitate and tranexamic acid (3/21), or neither (6/21) and resolved within several hours in all cases. CONCLUSIONS: Anesthesiologists should be aware of the possibility of postreperfusion hyperfibrinolysis in liver transplantation, particularly with DCD donors, and may consider treatment with cryoprecipitate or tranexamic acid. Further work is needed to identify any potential differences, such as faster resolution of fibrinolysis, between different treatment modalities.

American Society of Transplant Surgeons recommendations on best practices in donation after circulatory death organ procurement.

The American Society of Transplant Surgeons supports efforts to increase the number of organs that are critically needed for patients desperately awaiting transplantation. In the United States, transplantation using organs procured from donation after circulatory death (DCD) donors has continued to increase in number. Despite these increases, substantial variability in the utilization and practices of DCD transplantation still exists. To improve DCD organ utilization, it is important to create a set of best practices for DCD recovery. The following recommendations aim to provide guidance on contemporary issues surrounding DCD organ procurement in the United States. A work group was composed of members of the American Society of Transplant Surgeon Scientific Studies Committee and the Thoracic Organ Transplantation Committee. The following topics were identified by the group either as controversial or lacking standardization: prewithdrawal preparation, definition of donor warm ischemia time, DCD surgical technique, combined thoracic and abdominal procurements, and normothermic regional perfusion. The proposed recommendations were classified on the basis of the grade of available evidence and the strength of the recommendation. This information should be valuable for transplant programs as well as for organ procurement organizations and donor hospitals as they develop robust DCD donor procurement protocols.

Textbook Outcome: Definition and Analysis of a Novel Quality Measure in Lung Transplantation.

OBJECTIVE: To define textbook outcome (TO) for lung transplantation (LTx) using a contemporary cohort from a high-volume institution. SUMMARY BACKGROUND DATA: TO is a standardized, composite quality measure based on multiple postoperative endpoints representing the ideal "textbook" hospitalization. METHODS: Adult patients who underwent LTx at our institution between 2016 and 2019 were included. TO was defined as freedom from intraoperative complication, postoperative reintervention, 30-day intensive care unit or hospital readmission, length of stay >75th percentile of LTx patients, 90 day mortality, 30-day acute rejection, grade 3 primary graft dysfunction at 48 or 72 hours, postoperative extracorporeal membrane oxygenation, tracheostomy within 7 days, inpatient dialysis, reintubation, and extubation >48 hours post-transplant. Recipient, operative, financial characteristics, and post-transplant outcomes were recorded from institutional data and compared between TO and non-TO groups. RESULTS: Of 401 LTx recipients, 97 (24.2%) achieved TO. The most common reason for TO failure was extubation >48 hours post-transplant (N = 119, 39.1%); the least common was mortality (N = 15, 4.9%). Patient and graft survival were improved among patients who achieved versus failed TO (patient survival: log-rank P < 0.01; graft survival: log-rank P < 0.01). Rejection-free and chronic lung allograft dysfunction-free survival were similar between TO and non-TO groups (rejection-free survival: log-rank P = 0.07; chronic lung allograft dysfunction-free survival: log-rank P = 0.3). On average, patients who achieved TO incurred approximately $638,000 less in total inpatient charges compared to those who failed TO. CONCLUSIONS: TO in LTx was associated with favorable post-transplant outcomes and significant cost-savings. TO may offer providers and patients new insight into transplant center quality of care and highlight areas for improvement.

Adeno-associated virus mediates gene transduction after static cold storage treatment in rodent lung transplantation.

OBJECTIVE: Adeno-associated virus is a clinically used gene therapy vector but has not been studied in lung transplantation. We sought to determine the efficacy of adeno-associated virus delivery during static cold storage via the airway versus the pulmonary artery before lung transplantation in a rodent model. METHODS: Lewis rat lung grafts were treated with a dose of 8e8 or 4e9 viral genome/µL recombinant adeno-associated virus subtype-9 vectors containing firefly luciferase genomes administered via the pulmonary artery or airway during cold storage. A control group did not receive adeno-associated virus. Recipient syngeneic rats then underwent single left lung transplantation. Animals underwent bioluminescence imaging on postoperative days 7, 14, 28, and 56. Explanted tissues were prepared as lysates to quantify luciferase activity. Immunohistochemistry was performed to evaluate cellular transgene expression patterns. RESULTS: Control animals with no luminescent signal produced a background radiance of 6.1e4 p/s/cm2/sr. In the airway delivery group, mean radiance was greater than the control at 4e9 viral genome/µL postoperative day 7 radiance 6.9e4 p/s/cm2/sr (P = .04). In the pulmonary artery delivery group, we observed greater in vivo luminescence in animals receiving 4e9 viral genome/µL compared with all other groups. However, analysis of tissue lysate revealed greater luminescence in the airway delivery group and suggested off-target expression in heart and liver tissue in the pulmonary artery delivery group. Immunohistochemistry demonstrated transgene staining in distal airway epithelium and alveoli but sparing of the vasculature in the airway delivery group. CONCLUSIONS: Adeno-associated virus mediates gene transduction during static cold storage in rat lung isografts when administered via the airway and pulmonary artery. Airway administration leads to robust transgene expression in respiratory epithelial cells, whereas pulmonary artery administration targets alternative cell types and increases extrapulmonary transgene expression.

Complement-targeting therapeutics for ischemia-reperfusion injury in transplantation and the potential for ex vivo delivery.

Organ shortages and an expanding waitlist have led to increased utilization of marginal organs. All donor organs are subject to varying degrees of IRI during the transplant process. Extended criteria organs, including those from older donors and organs donated after circulatory death are especially vulnerable to ischemia-reperfusion injury (IRI). Involvement of the complement cascade in mediating IRI has been studied extensively. Complement plays a vital role in the propagation of IRI and subsequent recruitment of the adaptive immune elements. Complement inhibition at various points of the pathway has been shown to mitigate IRI and minimize future immune-mediated injury in preclinical models. The recent introduction of ex vivo machine perfusion platforms provides an ideal window for therapeutic interventions. Here we review the role of complement in IRI by organ system and highlight potential therapeutic targets for intervention during ex vivo machine preservation of donor organs.

Orthotopic Transplantation of the Full-length Porcine Intestine After Normothermic Machine Perfusion.

Successful intestinal transplantation is currently hindered by graft injury that occurs during procurement and storage, which contributes to postoperative sepsis and allograft rejection. Improved graft preservation may expand transplantable graft numbers and enhance posttransplant outcomes. Superior transplant outcomes have recently been demonstrated in clinical trials using machine perfusion to preserve the liver. We hypothesized that machine perfusion preservation of intestinal allografts could be achieved and allow for transplantation in a porcine model. Methods: Using a translational porcine model, we developed a device for intestinal perfusion. Intestinal samples were collected at the time of organ procurement, and after 6 h of machine perfusion for gross and histologic evaluation, hourly chemistry panels were performed on the perfusate and were used for protocol optimization. Following transplantation, porcine recipient physical activity, systemic blood parameters, and vital signs were monitored for 2 d before sacrifice. Results: In initial protocol development (generation 1, n = 8 grafts), multiple metabolic, electrolyte, and acid-base derangements were measured. These factors coincided with graft and mesenteric edema and luminal hemorrhage and were addressed with the addition of dialysis. In the subsequent protocol (generation 2, n = 9 grafts), differential jejunum and ileum perfusion were observed resulting in gross evidence of ileal ischemia. Modifications in vasodilating medications enhanced ileal perfusion (generation 3, n = 4 grafts). We report successful transplantation of 2 porcine intestinal allografts after machine perfusion with postoperative clinical and gross evidence of normal gut function. Conclusions: This study reports development and optimization of machine perfusion preservation of small intestine and successful transplantation of intestinal allografts in a porcine model.