Optimizing marginal liver grafts : development of an ex-vivo perfusion system and metabolic manipulations via an oxygenated dynamic sequence

En raison de la pénurie d'organes, des greffons hépatiques"marginaux" tels que les foies stéatosiques sont de plus en plus proposés aux équipes de transplantation, bien qu'ils tolèrent mal l’ischémie-reperfusion. La perfusion oxygénée ex-vivo des greffons hépatiques permet de minimiser l’ischémie-reperfusion, mais la variabilité des systèmes, des paramètres et l’importance des coûts des machines disponibles limitent sa généralisation. En 2014, l'équipe chirurgicale de la Pitié Salpêtrière et des ingénieurs de l'UTC ont conçu un prototype de dispositif de perfusion hépatique oxygénée. Ce projet de thèse s’est inscrit dans la continuité de ces travaux préliminaires et s’est développé autour de plusieurs axes: 1) développement d’un système de perfusion ex-vivo correspondant aux exigences métaboliques et hémodynamiques requises par la physiologie, mais aussi aux prérequis techniques définis par notre équipe. Ce développement a abouti à l’élaboration d’un prototype en partenariat avec un industriel (Livanova®).2) mise au point d’un protocole de perfusion dynamique spécifique (de l'hypo à la normothermie) avec ce système sur des foies humains « super marginaux ». Ce protocole de perfusion dynamique « cold-to-warm » sans interruption a été testé avec succès sur notre premier prototype et a permis une simplification de la séquence de perfusion par rapport aux technologies existantes. 3) évaluation du métabolisme de greffons stéatosiques perfusés et de l’efficacité d’un cocktail pharmacologique "défattant" permettant l'activation de la β-oxydation et l’export des lipides, évalué in vitro puis sur 7 foies humains ultra marginaux dont 5 stéatosiques perfusés sur notre circuit.Due to organ shortage, marginal liver grafts such as steatotic livers are increasingly proposed to transplantation teams. However, these livers have a low ischemia-reperfusion tolerance. Ex-vivo oxygenated perfusion of liver grafts represents a solution to minimize ischemia-reperfusion, but the variability of systems, parameters, and the high cost of available machines limit its expansion. In 2014, the surgical team at Pitié Salpêtrière and engineers from UTC designed a prototype of oxygenated liver perfusion device. This thesis is a continuation of this preliminary work and developed around several axes:1) development of an ex-vivo perfusion system corresponding to the metabolic and hemodynamic requirements of liver physiology, but also to the technical requirements defined by our team. This development led to the elaboration of a prototype in partnership with an industrial company (Livanova®).2) development of a specific dynamic perfusion protocol (from hypo to normothermia) with this system on "super marginal" human livers. This dynamic cold-to-warm perfusion protocol without interruption was successfully tested on our first prototype and allowed a simplification of the perfusion sequence compared to existing technologies.3) Evaluation of the metabolism of perfused steatotic grafts and of the efficacy of a defatting pharmacological cocktail. This second step, based on preliminary in vitro results showing the efficacy of a defatting cocktail on the activation of β-oxidation lipid export, led us to inject this cocktail into 7 ultra-marginal human livers, 5 of which were steatotic grafts perfused on our circuit with the "cold-to-warm" protocol

Low Levels of Microsatellite Instability at Simple Repeated Sequences Commonly Occur in Human Hepatocellular Carcinoma

International audienceBackground/Aim: The aim of this study was to assess the incidence of MSI in a large series of human hepatocellular carcinomas (HCC) with various etiologies. Materials and Methods: The MSI status was determined by polymerase chain reaction (PCR) using 5 mononucleotide and 13 CAn dinucleotide repeats. Results: None of the 122 HCC samples displayed an MSI-High phenotype, as defined by the presence of alterations at more than 30% of the microsatellite markers analyzed. Yet, limited microsatellite instability consisting in the insertion or deletion of a few repeat motifs was detected in 32 tumor samples (26.2%), regardless of the etiology of the underlying liver disease. MSI tended to be higher in patients with cirrhosis (p=0.051), possibly reflecting an impact of the inflammatory context in this process. Conclusion: Based on a large series of HCC with various etiologies, our study allowed us to definitely conclude that MSI is not a hallmark of HCC

Cold-to-warm machine perfusion of the liver: a novel circuit for an uninterrupted combined perfusion protocol

International audienceBackground: Ex-vivo perfusion of liver grafts is associated with promising results for the preservation of marginal grafts. Recent studies highlight the need for a combination of perfusion conditions, such as hypothermic followed by normothermic perfusion. While comprehensive machines dedicated to liver perfusion have been developed, these systems remain costly and poorly adaptable to perfusion condition switch, which requires a complete interruption of the perfusion process. Our team aimed at developing an adaptable and simple circuit for uninterrupted ex-vivo liver perfusion.Methods: Together with specialized bioengineers, we developed a highly adaptable circuit that can fit on already pre-existing extracorporeal oxygenation machines routinely used in cardiovascular surgery. This circuit, owing to its reservoir, allows any type of perfusion conditions without interrupting the perfusion process.Results: In a preliminary study, to assess the technical feasibility of liver perfusion using our circuit under different conditions, we performed 7 perfusions of discarded liver grafts. HOPE and DHOPE hypothermic perfusion could be performed, and a switch to normothermia was easily possible within seconds. From there, a dynamic perfusion sequence model was developed.Conclusion: This circuit may represent a simpler alternative or a new refinement to existing perfusion systems allowing uninterrupted combined perfusion protocols

Ex-Vivo Pharmacological Defatting of the Liver: A Review

International audienceThe ongoing organ shortage has forced transplant teams to develop alternate sources of liver grafts. In this setting, ex-situ machine perfusion has rapidly developed as a promising tool to assess viability and improve the function of organs from extended criteria donors, including fatty liver grafts. In particular, normothermic machine perfusion represents a powerful tool to test a liver in full 37 °C metabolism and add pharmacological corrections whenever needed. In this context, many pharmacological agents and therapeutics have been tested to induce liver defatting on normothermic machine perfusion with promising results even on human organs. This systematic review makes a comprehensive synthesis on existing pharmacological therapies for liver defatting, with special focus on normothermic liver machine perfusion as an experimental ex-vivo translational model. View Full-Tex

Complex liver resection under hepatic vascular exclusion and hypothermic perfusion with versus without veno-venous bypass: a comparative study

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Therapeutic potentials of mesenchymal stromal cells-derived extracellular vesicles in liver failure and marginal liver graft rehabilitation: a scoping review

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Liver transplantation from donors with Down Syndrome

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Consequences of Extended Spectrum Beta‐Lactamase–Producing Enterobacteriaceae and Methicillin‐Resistant Staphylococcus aureus Carriage in Awaiting Liver Transplant Patients

International audienceInfections in patients with cirrhosis are associated with liver‐related complications (LRCs), especially in patients awaiting liver transplantation (LT). The aim of this study was to evaluate the impact of methicillin‐resistant Staphylococcus aureus (MRSA) and extended spectrum beta‐lactamase colonization on infections and LRCs for patients on the wait list and on infections after LT. We retrospectively included 250 of 483 patients with cirrhosis who were placed on the wait list for LT from December 2015 to January 2018. These patients were screened for MRSA or extended spectrum beta‐lactamase–producing Enterobacteriaceae (ESBLE) at the time of wait‐list placement and after LT. Of the patients, 76% were male with a mean age of 57.5 ± 10 years, and the most frequent cause of liver disease was alcohol (39%). Median Model for End‐Stage Liver Disease (MELD) score was 19 (12‐28). Only 1 patient was positive for MRSA; 19% of patients (n = 47) had ESBLE fecal carriage at the time of wait‐list placement and 15% (n = 37) had it after LT. Infection‐free survival on the wait list and after LT, according to fecal carriage status, was not statistically different between 2 groups. LRC‐free survival at 6 and 12 months was significantly lower in ESBLE fecal carriage (HR, 1.6; P = 0.04). MELD score >19 (HR, 3.0; P = 0.01) and occurrence of infection during the first 3 months on the wait list (HR, 4.13; P < 0.001) were independent risk factors for LRC occurrence in the multivariate analysis. Our study is the first showing that in a cohort of patients with cirrhosis waiting for LT LRC‐free survival was lower in patients with ESBLE fecal carriage but that infection‐free survival was not different between the 2 groups