Mitochondria sense changes resulting from the ischemia and subsequent reperfusion of an organ and mitochondrial reactive oxygen species (ROS) production initiates a series of events, which over time result in the development of full-fledged ischemia-reperfusion injury (IRI), severely affecting graft function and survival after transplantation. ROS activate the innate immune system, regulate cell death, impair mitochondrial and cellular performance and hence organ function. Arresting the development of IRI before the onset of ROS production is currently not feasible and clinicians are faced with limiting the consequences. Ex vivo machine perfusion has opened the possibility to ameliorate or antagonize the development of IRI and may be particularly beneficial for extended criteria donor organs. The molecular events occurring during machine perfusion remain incompletely understood. Accumulation of succinate and depletion of adenosine triphosphate (ATP) have been considered key mechanisms in the initiation; however, a plethora of molecular events contribute to the final tissue damage. Here we discuss how understanding mitochondrial dysfunction linked to IRI may help to develop novel strategies for the prevention of ROS-initiated damage in the evolving era of machine perfusion.

Reliable biomarkers for organ quality assessment during normothermic machine perfusion (NMP) are desired. ATP (adenosine triphosphate) production by oxidative phosphorylation plays a crucial role in the bioenergetic homeostasis of the liver. Thus, detailed analysis of the aerobic mitochondrial performance may serve as predictive tool towards the outcome after liver transplantation. In a prospective clinical trial, 50 livers were subjected to NMP (OrganOx Metra) for up to 24 h. Biopsy and perfusate samples were collected at the end of cold storage, at 1 h, 6 h, end of NMP, and 1 h after reperfusion. Mitochondrial function and integrity were characterized by high-resolution respirometry (HRR), AMP, ADP, ATP and glutamate dehydrogenase analysis and correlated with the clinical outcome (L-GrAFT score). Real-time confocal microscopy was performed to assess tissue viability. Structural damage was investigated by histology, immunohistochemistry and transmission electron microscopy. A considerable variability in tissue viability and mitochondrial respiration between individual livers at the end of cold storage was observed. During NMP, mitochondrial respiration with succinate and tissue viability remained stable. In the multivariate analysis of the 35 transplanted livers (15 were discarded), area under the curve (AUC) of LEAK respiration, cytochrome c control efficiency (mitochondrial outer membrane damage), and efficacy of the mitochondrial ATP production during the first 6 h of NMP correlated with L-GrAFT. Bioenergetic competence during NMP plays a pivotal role in addition to tissue injury markers. The AUC for markers of outer mitochondrial membrane damage, ATP synthesis efficiency and dissipative respiration (LEAK) predict the clinical outcome upon liver transplantation. This study was funded by a Grant from the In Memoriam Dr. Gabriel Salzner Stiftung awarded to SS and the 10.13039/501100009968 Tiroler Wissenschaftsfond granted to TH.

Biomarkers with strong predictive capacity towards transplantation outcome for livers undergoing normothermic machine perfusion (NMP) are needed. We investigated lactate clearing capacity as a basic function of liver viability during the first 6 h of NMP. A trial conducted in 6 high-volume transplant centres in Europe. All centres applied a back-to-base NMP approach with the OrganOx metra system. Perfusate lactate levels at start, 1, 2, 4 and 6 h of NMP were assessed individually and as area under the curve (AUC) and correlated with EAD (early allograft dysfunction), MEAF (model for early allograft function) and modified L-GrAFT (liver graft assessment following transplantation) scores. A total of 509 livers underwent ≥6 h of NMP before transplantation in 6 centres in the UK, Germany and Austria. The donor age was 53 (40–63) years (median, i.q.r.). The total NMP time was 10.8 (7.9–15.7) h. EAD occurred in 26%, MEAF was 4.72 (3.54–6.05) and L-GrAFT₁₀ −0.96 (−1.52–−0.32). Lactate at 1, 2 and 6 h correlated with increasing robustness with MEAF. Rather than a binary assessment with a cut-off value at 2 h, the actual 2 h lactate level correlated with the MEAF (P = 0.0306 versus P = 0.0002, Pearson r = 0.01087 versus r = 0.1734). The absolute lactate concentration at 6 h, the AUC of 0–6 h and 1–6 h (P < 0.0001, r = 0.3176) were the strongest predictors of MEAF. Lactate measured 1–6 h and lactate levels at 6 h correlate strongly with risk of liver allograft dysfunction upon transplantation. The robustness of predicting MEAF by lactate increases with perfusion duration. Monitoring lactate levels should be extended to at least 6 h of NMP routinely to improve clinical outcome.

Clostridioides difficile infection (CDI) causes severe inflammatory responses at the intestinal mucosa but the immunological mechanisms underlying CDI-related immunopathology are still incompletely characterized. Here we identified for the first time that both, non-toxigenic strains as well as the hypervirulent ribotypes RT027 and RT023 of Clostridioides difficile (formerly Clostridium difficile), induced an effector phenotype in mucosal-associated invariant T (MAIT) cells. MAIT cells can directly respond to bacterial infections by recognizing MR1-presented metabolites derived from the riboflavin synthesis pathway constituting a novel class of antigens. We confirmed functional riboflavin synthesis of C. difficile and found fixed bacteria capable of activating primary human MAIT cells in a dose-dependent manner. C. difficile-activated MAIT cells showed an increased and MR1-dependent expression of CD69, proinflammatory IFNγ, and the lytic granule components granzyme B and perforin. Effector protein expression was accompanied by the release of lytic granules, which, in contrast to other effector functions, was mainly induced by IL-12 and IL-18. Notably, this study revealed hypervirulent C. difficile strains to be most competent in provoking MAIT cell responses suggesting MAIT cell activation to be instrumental for the immunopathology observed in C. difficile-associated colitis. In conclusion, we provide first evidence for a link between C. difficile metabolism and innate T cell-mediated immunity in humans.

Transplantation represents the treatment of choice for many end-stage diseases but is limited by the shortage of healthy donor organs. Ex situ normothermic machine perfusion (NMP) has the potential to extend the donor pool by facilitating the use of marginal quality organs such as those from donors after cardiac death (DCD) and extended criteria donors (ECD). NMP provides a platform for organ quality assessment but also offers the opportunity to treat and eventually regenerate organs during the perfusion process prior to transplantation. Due to their anti-inflammatory, immunomodulatory and regenerative capacity, mesenchymal stem cells (MSCs) are considered as an interesting tool in this model system. Only a limited number of studies have reported on the use of MSCs during ex situ machine perfusion so far with a focus on feasibility and safety aspects. At this point, no clinical benefits have been conclusively demonstrated, and studies with controlled transplantation set-ups are urgently warranted to elucidate favorable effects of MSCs in order to improve organs during ex situ machine perfusion.

The liver, in combination with a functional biliary system, is responsible for maintaining a great number of vital body functions. However, acute and chronic liver diseases may lead to irreversible liver damage and, ultimately, liver failure. At the moment, the best curative option for patients suffering from end-stage liver disease is liver transplantation. However, the number of donor livers required by far surpasses the supply, leading to a significant organ shortage. Cellular therapies play an increasing role in the restoration of organ function and can be integrated into organ transplantation protocols. Different types and sources of stem cells are considered for this purpose, but highly specific immune cells are also the focus of attention when developing individualized therapies. In-depth knowledge of the underlying mechanisms governing cell differentiation and engraftment is crucial for clinical implementation. Additionally, novel technologies such as ex vivo machine perfusion and recent developments in tissue engineering may hold promising potential for the implementation of cell-based therapies to restore proper organ function.