No description available.

published_or_final_version / abstract / Psychiatry / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

This thesis sought to investigate novel methods for protecting the liver from ischaemia reperfusion injury in the context of liver transplantation. Research in the heart, brain and kidneys has suggested that hypoxia inducible factor (HIF) may play a key role in the delayed phase of ischaemic preconditioning and can protect organs for up to 3 days. However, although there is good evidence for the potential of HIF to protect organs from ischaemia, the HIF pathway still presents some what of a paradox because it targets both pro-death (e.g. BNIP3,NIX) as well as pro-survival genes (e.g. HO-I, EPO). HIF is primarily controlled by 3 oxygen dependent prolyl hydroxylases (PHD 1 , PHD2, PHD3), and inhibition of these prolyl hydroxylases leads to HIF activation. It was hypothesised that differential inhibition of PHD 1,2 or 3 may result in selective gene regulation and may confer greater or less protection against ischaemia reperfusion injury. To investigate this hypothesis mouse embryonic fibroblasts (MEFs) were isolated from PHDl, 2, and 3 knock-out (KO) embryos and compared to MEFs derived from WT littermate controls. In these MEFs, cell growth and proliferation, as well as cell survival following exposure to anoxia and inducers of apoptosis was studied. The principal findings were that PHD2 is the dominant regulator of HIF in normoxia. PHD2 knock-out MEFs exhibited glycolytic metabolism and had a lower oxygen consumption compared to wild-type MEFs. Gene array studies confirmed the dominant role of PHD2 but also demonstrated that PHD 1 upregulates a number of HIF target genes, albeit to a lesser extent than PHD2. There were no differences, however, in susceptibility to hypoxic injury in the PHDl, 2, and 3 knock-out MEFs compared to wild-type controls. A further aim of the study was to investigate whether prolyl hydroxylase inhibition using dimethyloxalyglycerine (DMOG) may protect the liver in a rodent model of ischaemia reperfusion injury. DMOG effectively upregulated HIF and IllF target genes. Serum transaminases (AST and AL T) were significantly lower in the DMOG treated animals compared to the normal saline treated controls 24 hours following ischaemia. This protection was similar to the protection conferred by surgically induced ischaemic preconditioning. This thesis provides important insights into the individual function of the prolyl hydroxylases and provides preliminary evidence that prolyl hydroxylase inhibitors may be useful in the treatment of ischaemia reperfusion injury in liver transplantation.

The use of organs from donors after circulatory death (DCD) has been recommended as one strategy to enlarge the donor pool and raise the transplant rate. However, DCD allografts had higher incidence of early post-transplant dysfunction. The general aim of this research project was to develop clinical and experimental strategies to reduce the incidence of early post-transplant dysfunction of kidney and liver allografts from DCD. First the ability of a clinical scoring system based on donor data for identifying DCD kidneys with high-risk of post-transplant dysfunction was evaluated using the Oxford and the UK National DCD kidney transplant cohorts. This works suggest that stratification of DCD kidneys before transplantation might allow early identification of kidneys in which lower graft function and survival could be expected if any additional therapeutic intervention is implemented. Second, as it has been suggested that hypothermic machine perfusion (HMP) may protect DCD kidneys from additional preservation injury and improve their outcome after transplantation, this work explored the benefit of HMP as preservation technique fo DCD kidneys in Oxford and discusses the potential of this technique for reducing the incidence of post-transplant dysfunction in DCD kidneys. The Oxford. Liver Group has provided evidence of the benefit of preservation with normothermic machine perfusion (NMP) on post-transplant function and survival of DCD liver allografts. In this work, the molecular mechanisms associated with this benefit were characterized using micro array technology. This analysis suggests that the beneficial effect ofNMP may be associated with the induction of the ischaemic preconditioning phenomenon and highlights a group of genes with potential for gene therapy. Finally, this works provides the "proof-of-concept" that the use of a non-mammalian viral vector for gene transfer of kidneys and livers during conventional cold preservation is feasible and is not associated with additional tissue injury.

Cellular immune responses to Hepatitis C (HCV) epitopes are crucial for successful host response to HCV infection. We investigated a platform to assess specific and global immune responses in HCV infection. We identified 57 HCV peptides from literature (24 of CD4+, 33 of CD8+ specificity) and tested them in two peptide pools to assess specific response in non-transplanted and post-liver transplant (LT) patients. Robust interferon-gamma (IFN) response to CD4+ peptide and mitogen stimulation was seen in sustained virological clearance. IFN response to the CD4+ peptide pool could differentiate between SVR and NR with 82% accuracy. In patients with recurrent HCV post-LT, HCV-specific responses were attenuated, but global immune responses were preserved. Significantly lower specific (CD4+) and global immune responses (mitogen response) were observed in patients with advanced allograft disease (fibrosis score>2). Quantiferon-HCV may identify patients likely to respond to anti-HCV treatment, as well as post-LT patients with aggressive HCV recurrence. / Experimental Surgery

Die orthotope Lebertransplantation (OLT) ist ein etabliertes Standardtherapieverfahren von Endzuständen verschiedenster Lebererkrankungen. Bei Patienten mit Lebererkrankungen kommt es zu einer deutlichen Reduktion der hepatischen Metabolisierung und Elimination von verschiedensten Medikamenten. Fentanyl ist das Opioid der Wahl im Rahmen der Anästhesie bei Patienten mit Lebererkrankungen. Die Pharmakokinetik von Fentanyl ist besonders in der anhepatischen Phase durch einen Ausfall der hepatischen Elimination gekennzeichnet, sodass es zu hohen Plasmakonzentrationen von Fentanyl kommen kann. Besonders bei der Fentanylinfusion bis zur Reperfusion kann dies zu einer verzögerten Extubation führen, welche im Rahmen des Fast-Track-Verfahrens vermieden werden sollte. Hauptanliegen unserer Studie ist der Vergleich zweier Infusionsregimes für das Opioid Fentanyl, nämlich der Beendigung der Fentanylapplikation mit Beginn der anhepatischen Phase (Studiengruppe) und zum Zeitpunkt der Reperfusion (Kontrollgruppe). Dazu wurden von 22 Patienten (Studiengruppe: n=10; Kontrollgruppe: n=12) intraoperativ zu neun definierten Messzeitpunkten (MZP) die Verläufe der arteriellen Fentanylplasmakonzentration, sowie von Parametern des Säure-Basen-Haushaltes und der hämodynamische Messwerte erfasst. Die Bestimmung der Fentanylkonzentration erfolgte mit Hilfe der Flüssigchromatographie mit Massenspektrometrie (SSQ 7000, Finnigan), der Säure-Basen-Haushalt mittels Blutgasanalyse (ABL 700, Radiometer Medical A/S, Kopenhagen). Hinsichtlich der Daten für MELD-Score, Diagnose und Gesamtoperationsdauer unterschieden sich die beiden Gruppen nicht signifikant voneinander. Im Verlauf der anhepatischen Phase lag die Fentanylkonzentration im Plasma in der Kontrollgruppe signifikant höher als in der Studiengruppe. Die gefundenen höheren Fentanylspiegel in der Kontrollgruppe sind Ausdruck einer nicht vorhandenen hepatischen Fentanylclearance in der anhepatischen Phase. Basierend auf unseren Daten wollen wir zu einer Überprüfung des Infusionskonzeptes von Fentanyl bei OLT anregen. Während einer OLT sollte im Hinblick auf das Fast-Track-Konzept über eine Beendigung der Fentanylzufuhr zum Beginn der anhepatischen Phase nachgedacht werden.

published_or_final_version / Surgery / Master / Master of Research in Medicine

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Background: Hepatocellular carcinoma is one of the most fatal diseases worldwide. Liver transplantation dramatically improved the survival rate of HCC patients. However, tumor recurrence remains a huge threat to HCC patients without any promising curative treatment. Chemotherapy, as one of the potential treatments to recurrent HCC, did not show any significant effect either. Objective: We aim to investigate the role of interferon-gamma inducible protein 10 (IP10) in acute-phase liver graft injury induced late-phase cisplatin resistance after liver transplantation and to explore the underlying mechanism. Furthermore, a potential adjuvant therapy was expected to be identified to sensitize cisplatin treatment in HCC. Materials and methods: A rat orthotopic liver transplantation model was established with applying whole or small-for-size (50%) graft. Afterwards, a rat hepatoma cell (MH7777) was injected via portal vein to generate recurrent tumor. The expressions of genes linked to multi-drug resistance and graft injury were compared between tumors developed after liver transplantation using small and whole grafts. IP10 expression was further validated in clinical samples from two cohorts of patients including HCC patients with hepatectomy and HCC patients with liver transplantation. The extracellular and intracellular roles of IP10 were examined in vitro by using IP10 recombinant protein and IP10 stable transfectants in HCC cell lines. The correlation between IP10 expression and tumor growth was investigated in three in vivo nude mice models including a subcutaneous model, an orthotopic model and ischemia reperfusion injury model. The underlying mechanism was further explored in vitro, in vivo and in clinical samples. IP10 neutralizing antibody was employed as an adjuvant therapy to identify its effect on sensitizing cisplatin treatment in HCC. Results: The expressions of multidrug resistant genes were significantly up-regulated in liver and tumor from small-for-size group in rat liver transplantation model. IP10 was selected as the potential target for its constantly higher expression in liver and tumor tissues in small-for-size group. In clinical studies, IP10 was overexpressed in around 45% HCC patients with hepatectomy. The expression of circulating IP10 well correlated with tumor recurrence and small graft ratio in HCC patients after liver transplantation. In in vitro studies, it was demonstrated that overexpression of IP10 could significantly promote HCC cell proliferation either in short term or in long term cisplatin administration. In in vivo studies, subcutaneous and orthotopic nude mice models showed that the overexpression of IP10 have significant correlations with larger tumor volume and less tumor necrosis after cisplatin treatment. In mechanism studies, IP10 overexpression was found to be well correlated with the activation of endoplasmic reticulum (ER) stress signaling pathways in vitro and further validated in vivo models and in clinical specimens. IP10 neutralizing antibody was identified as a potential therapy which could sensitize cisplatin treatment in vitro and in vivo. Conclusions: The high expression of IP10 was identified in two cohorts of clinical samples and showed significant correlations with tumor recurrence. Graft injury induced IP10 overexpression could significantly increase cisplatin resistance after liver transplantation via ER stress signaling pathways. IP10 neutralizing antibody may be applied as an alternative treatment for recurrent HCC after liver transplantation. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy