Abstract Background Up to 85% of patients with liver metastases have inoperable disease. Isolated liver chemoperfusion involves vascular isolation of the liver with regional delivery of chemotherapy, and aims to maximise the therapeutic response and limit extra-hepatic toxicity. Historically, the morbidity associated with drug leak from the liver circuit has limited its success. To improve the safety of this approach, we developed a surgical protocol for Isolated Normothermic Liver Chemoperfusion (INLiC) and assessed its short-term safety, and feasibility for enhancing drug delivery. Methods Laparotomy and complete, vascular isolation of the liver was performed on (n=9) 55–65 Kg pigs. The gastroduodenal artery, portal vein and inferior vena cava (IVC) were cannulated and normothermic machine perfusion of the liver established in situ. Systemic circulation was maintained with veno-venous bypass. High-dose, intra-arterial doxorubicin was delivered to the isolated liver, circulated for 1 hour and vascular reconnection performed. Biochemical and physiological parameters were assessed and doxorubicin quantified in blood, bile and tissue by high performance liquid chromatography. Results A standardised, surgical protocol was developed to enable INLiC. Physiological median liver outflows (1.1 L/min (0.9–1.1)), pH (7.35 (7.26–7.42)), lactate concentration (1.1 mM (0.33–2.3)) and an acceptable peak AST (805 U/L (308–1667)) were achieved. Population two-compartmental analysis of plasma doxorubicin decay demonstrated a distribution half-life of 1.3 minutes and a Cmax 8-times higher than tolerable by systemic delivery, due to cardiotoxicity. The resulting hepatic tissue concentration of doxorubicin was significantly higher than could be safely achieved with systemic delivery (p=0.019). Notably, there was no extra-hepatic leak of doxorubicin and it did not accumulate in cardiac or renal tissue. Conclusions INLiC is feasible, safe and enables high-dose drug delivery to the liver without extra-hepatic tissue accumulation.

BACKGROUND:Advances in organ preservation, reconditioning and assessment have been driven by the increasing necessity to utilise organs from extended criteria donors, particularly donors after circulatory death. Research efforts in this area have aided translation of machine perfusion technology into clinical practice. Pigs are anatomically and physiologically similar to humans and are an excellent model. However, conducting large animal experimental research is challenging and typically limited by ethical and economic constraints. Here we describe a reproducible, cost-effective multi-organ abdominal procurement model of porcine organs from the slaughterhouse.METHODS:Domestic pigs are electrically stunned and exsanguinated following the standard abattoir process. Via a longitudinal midline incision, the thoracoabdominal viscera are removed en bloc by incising along the anterior vertebral plane. The abdominal organs are isolated, perfused and separated preserving their respective vasculature, allowing individual organ use for specific experiments.RESULTS:The warm ischaemic time is kept between 15-30 minutes. Using this highly protocolized procurement technique we have procured 12 livers, 162 kidneys and 12 pancreata for research, the majority of which have been utilized for ex situ perfusion experiments.CONCLUSIONS:We have described a reliable and reproducible procedure for abdominal multi-organ procurement from slaughterhouse pigs.

PURPOSE:Postoperative pancreatic fistula (POPF) with reported incidence rates up to 45% contributes substantially to overall morbidity. In this study, we conducted a retrospective evaluation of POPF along with its potential perioperative clinical risk factors and its effect on tumor recurrence. METHODS:Clinical data on patients who had received pancreatoduodenectomy (PD), distal pancreatectomy (DP), or duodenum-preserving pancreatic head resection (DPPHR) were prospectively collected between 2007 and 2016. A Picrosirius red staining score was developed to enable morphological classification of the resection margin of the pancreatic stump. The primary end point was the development of major complications. The secondary end points were overall and recurrence-free survival. RESULTS:340 patients underwent pancreatic resection including 222 (65.3%) PD, 87 (25.6%) DP, and 31 (9.1%) DPPHR. Postoperative major complications were observed in 74 patients (21.8%). In multivariable logistic regression analysis, POPF correlated with body mass index (BMI) (p = 0.025), prolonged stay in hospital (p<0.001), high Picrosirius red staining score (p = 0.049), and elevated postoperative levels of amylase or lipase in drain fluid (p≤0.001). Multivariable Cox regression analysis identified UICC stage (p<0.001), tumor differentiation (p<0.001), depth of invasion (p = 0.001), nodal invasion (p = 0.001), and the incidence of POPF grades B and C (p = 0.006) as independent prognostic markers of recurrence-free survival. CONCLUSION:Besides the known clinicopathological risk factors BMI and amylase in the drain fluid, the incidence of POPF correlates with high Picrosirius red staining score in the resection margins of the pancreatic stumps of curatively resected pancreatic ductal adenocarcinoma (PDAC). Furthermore, clinically relevant POPF seems to be a prognostic factor for tumor recurrence in PDAC.

Abstract Aims Up to 85% of patients with liver metastases have inoperable hepatic tumour burden. Isolated liver perfusion involves vascular isolation of the liver in situ and regional delivery of chemotherapy, avoiding dose-limiting extra-hepatic toxicity. In this series, we develop a surgical protocol to demonstrate the feasibility of isolated normothermic liver perfusion (INLP) and investigate short-term safety and feasibility of delivering high-dose chemotherapy. Methods Laparotomy and complete, vascular isolation of the liver was performed on 55-65Kg pigs (n = 6). The hepatic artery (HA), portal vein (PV) and inferior vena cava were cannulated and liver NMP established. Veno-venous bypass maintained systemic circulation. High-dose doxorubicin was administered to the isolated liver, circulated for 1 hour and vascular reconnection performed. Physiological parameters were measured and doxorubicin quantified in blood, bile and tissue by high-performance liquid chromatography. Results INLP with doxorubicin delivery achieved physiological flow rates (PV 0.7L/min (0.6-0.9L/min); HA 0.3L/min (0.2-0.4L/min)) and pH (median 7.3 (7.24-7.38)), with a median lactate of 0.42mmol/L. Median peak AST and ALT were 1045 U/L and 47 U/L respectively. Doxorubicin decay was fitted with a 2-compartmental model; distribution half-life was 1.9 minutes and plasma Cmax was higher than if given systemically resulting in mean hepatic tissue levels of 26+/-11.6 µg/g. There was no leak during INLP and doxorubicin was undetectable in kidney or heart. Conclusions Surgical isolation and NMP of the liver in situ, with concurrent veno-venous bypass is feasible and enables high-dose drug delivery resulting in therapeutic tissue levels with no off-target toxicity. Further safety studies are required.

La conservation statique hypothermique (CSH) est actuellement l’unique méthode de préservation des transplants pancréatiques. Le développement récent de la perfusion normothermique ex situ sur machine (NMP) des greffons hépatiques et rénaux permet une évaluation et une optimisation des transplants issus de donneurs non optimaux. L’objectif de cette étude est de mettre au point un modèle de NMP adapté au pancréas. Cette étude a été réalisée sur un modèle porcin de donneur décédé après arrêt circulatoire avec ischémie chaude de 30 minutes. Trois groupes perfusion NMP ont été définis : après 2,5 heures de CSH (G1), après 6 heures de CSH (G2), et après 6 h de perfusion hypothermique sur machine (HMP) (G3). La NMP a été réalisée sur une machine KidneyAssist (Organ Assist). Le perfusât a été composé de plasma et de globules rouges autologues. L’oxygénation a été de 21 %. Les paramètres de perfusion ont été une pression de 40 mmHg et une température de 37 ° C. Les paramètres de perfusion analysés ont été le flux (F) et l’index de résistance (IR). Sept perfusions NMP ont été réalisées. Toutes les NMP ont été techniquement réalisables avec une perfusion macroscopique efficace sur le pancréas et le duodénum (Fig. 1). Les pancréas G1 et G3 ont eu une évolution favorable des paramètres de perfusion à 60 min G1 (F = 105 mL/min5 et RI = 0,50,2) G3 (F = 45 mL/min5 et RI = 0,60,3). Les pancréas G2 ont présenté une évaluation rapidement défavorable des paramètres de perfusion imposant un arrêt de la NMP à 45 min. Le taux de lactate est resté stable pour toutes les groupes (62 mmol/L à 45 min). Des tests de glucostimulation des TP avec 27,5 mmol/L de glucose ont permis des évaluations de la sécrétion insulinique en réponse. La perfusion NMP de pancréas est réalisable et permet une évaluation fonctionnelle des transplants. Cette évaluation pourrait être particulièrement intéressante pour des transplants provenant de donneurs décédés après arrêt circulatoire ou encore d’autres donneurs non optimaux. Cette technique innovante est une voie de recherche permettant d’envisager de futures potentialités d’optimisation des transplants.

Liver transplantation is increasingly dependent on the use of extended criteria donors (ECD) to increase the organ donor pool and address rising demand. This has necessitated the adoption of innovative technologies and strategies to protect these higher-risk grafts from the deleterious effects of traditional preservation and ischaemia reperfusion injury (IRI). The advent of normothermic machine perfusion (NMP) and rapid growth in the clinical adoption of this technology has accelerated efforts to utilise NMP as a platform for therapeutic intervention to optimise donor livers. In this review we will explore the emerging preclinical data related to ameliorating the effects of IRI, protecting the microcirculation and reducing the immunogenicity of donor organs during NMP. Exploiting the window of opportunity afforded by NMP, whereby the liver can be continuously supported and functionally assessed while therapies are directly delivered during the preservation period, has clear logistical and theoretical advantages over current preservation methods. The clinical translation of many of the therapeutic agents and strategies we will describe is becoming more feasible with widespread adaptation of NMP devices and rapid advances in molecular biology and gene therapy, which have substantially improved the performance of these agents. The delivery of novel therapeutics during NMP represents one of the new frontiers in transplantation research and offers real potential for successfully tackling fundamental challenges in transplantation such as IRI.

Die Pankreatoduodenektomie (PD) ist die chirurgische Therapie bei der Behandlung von Erkrankungen des Pankreaskopfes. Postop Pankreasfisteln (POPF) mit Inzidenzraten von 3 – 45% tragen substantiell zur Morbidität nach PD bei.

P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis.

Caveolae and caveolins, structural components of caveolae, are associated with specific ion channels in cardiac myocytes. We have previously shown that P2X purinoceptor 7 (P2X7R), a ligand-gated ion channel, is increased in atrial cardiomyocytes of caveolin-1 knockout mice; however, the specific biochemical relationship of P2X7R with caveolins in the heart is not clear. The aim of this work was to study the presence of the P2X7R in atrial cardiomyocytes and its biochemical relationship to caveolin-1 and caveolin-3. Caveolin isoforms and P2X7R were predominantly localized in buoyant membrane fractions (lipid rafts/caveolae) prepared from hearts using detergent-free sucrose gradient centrifugation. Caveolin-1 knockout mice showed normal distribution of caveolin-3 and P2X7R to buoyant membranes indicating the importance of caveolin-3 to formation of caveolae. Using clear native-PAGE, we showed that caveolin-1, -3 and P2X7R contribute to the same protein complex in the membranes of murine cardiomyocytes and in the immortal cardiomyocyte cell line HL-1. Western blot analysis revealed increased caveolin-1 and -3 proteins in tissue homogenates of P2X7R knockout mice. Finally, tissue homogenates of atrial tissues from caveolin-3 knockout mice showed elevated mRNA for P2X7R in atria. The colocalization of caveolins with P2X7R in a biochemical complex and compensated upregulation of P2X7R or caveolins in the absence of any component of the complex suggests P2X7R and caveolins may serve an important regulatory control point for disease pathology in the heart.

Changes in intracellular calcium concentration [Ca(2+)](i) are believed to influence the proliferation and differentiation of airway epithelial cells both in vivo and in vitro. In the present study, using mouse alveolar epithelial E10 cells, we demonstrated that the treatment of lung epithelial cells with BLM resulted in elevated intracellular Ca(2+) levels. BLM further increased P2rx7 mRNA expression and P2X7R protein levels, paralleled by increased PKC-β1 levels. BLM treatment or stimulation of the P2X7R with the P2X7R agonist BzATP induced translocation of PKC-β1 from the cytoplasm to the membrane. The expression of PKC-β1 was repressed by the P2X7R inhibitor oxATP, suggesting that PKC-β1 is downstream of P2X7R activation. Furthermore, cells exposed to BLM contained increased amounts of P2X7R and PKC-β1 in Cav-1 containing lipid raft fractions. The comparison of lung tissues from wild-type and P2rx7(-/-) mice revealed decreased protein and mRNA levels of PKC-β1 and CaM as well as decreased immunoreactivity for PKC-β1. The knockdown of P2X7R in alveolar epithelial cells resulted also in a loss of PKC-β1. These data suggest that the effect of P2X7R on expression of PKC-β1 detected in alveolar epithelial cells is also functioning in the animal model. Immunohistochemical evaluation of fibrotic lungs derived from a BLM-induced mouse model revealed a strong increase in PKC-β1 immunoreactivity. The present experiments demonstrated that the increased expression of P2X7R influences PKC-β1. We predict that increased Ca(2+) concentration stimulates PKC-β1, whereas the prerequisite for activating PKC-β1 after P2X7R increase remained to be determined. Our findings suggest that PKC-β1 is important in the pathogenesis of pulmonary fibrosis.