No description available.

No description available.

No description available.

No description available.

Intestinal ischaemia reperfusion (IR) injury is associated with various clinical conditions such as hypovolemic shock, strangulation-obstruction, cardiovascular surgery, abdominal aortic surgery and small bowel transplantation. IR injury of the intestine is a systemic phenomenon resulting in bacterial translocation, endotoxaemia, acute respiratory distress syndrome and acute hepatic injury culminating in multiple organ failure. It has been hypothesised that ischaemic preconditioning may protect against IR injury of the intestine through haem oxygenase formation. This thesis (study) investigated the relationship of haem oxygenase metabolism with intestinal microvascular perfusion, tissue oxygenation and function with ischaemic preconditioning of the intestine in an experimental model of ischaemia reperfusion injury of the small intestine. Male Sprague Dawley rats (250-300 grams) were subjected to 30 mins of ischaemia by clamping of superior mesenteric artery followed by 2 hrs (early phase) or 24 hrs (late phase) of reperfusion. Ischaemic preconditioning was performed with 10 min ischaemia and 10 min reperfusion before the sustained ischaemia. Pyrrolidine dithiocarbamate (PDTC) or Zinc Protoporrphyrrin (ZnPP) were administered to stimulate or block heme oxygenase synthesis. The study demonstrated that ischaemic preconditioning resulted in significantly improvement in intestinal microvascular perfusion, tissue oxygenation as well as decreased leukocyte-endothelial interactions and intestinal and pulmonary injury following both early and late phases of IR injury. The preconditioning effect was associated with significantly increased haem oxygenase production suggested by intestinal tissue haeme oxygenase levels as demonstrated by haem oxygenase assays and western blotting. PDTC treatment reproduced the protective effect of ischaemic preconditioning. Haem oxygenase inhibition with ZnPP antagonized the protective effect of ischaemic preconditioning. This thesis has shown that the protective effect of intestinal ischaemic preconditioning against both early and late phases of IR injury is associated with increased haem oxygenase production. These data may have important implications in intestinal surgery and transplantation and may lead to the development of pharmacological strategies for protecting the intestine from ischaemic injury.

Oesophageal atresia (OA) and tracheo-oesophageal fistula (TOF) are important human malformations of the foregut, the development of which is poorly understood. In this thesis, the mechanisms that underlie the development of OA/TOF, as well as normal tracheo-oesophageal development, were investigated. The rat model of OA/TOF, based on exposure of embryos to the anticancer agent Adriamycin, was adapted to the mouse in order to allow more in depth study of the cellular and molecular events that underlie the malformations. The Adriamycin-treated mouse was shown to have persistence of an undivided foregut at El 1.5, in contrast to saline controls in which the foregut was in the process of separating into a ventral structure (trachea) and a dorsal structure (oesophagus). This failure of tracheo-oesophageal separation was also a feature of the Sonic hedgehog (Shh) null mutant mouse. The study of the respiratory marker Nkx2.1 confirmed that the foregut normally divides along the dorsoventral boundary of respiratory/ gastrointestinal specification and that the fundamental defect in both the Adriamycin-treated and Shh null mutant mice is persistence of an undivided foregut in which both the respiratory and gastrointestinal lineages are represented, with a preservation of the dorsoventral pattern of expression of that marker. The study of expression of Shh showed this to have a dorsoventral pattern that was closely related to the separation boundary and which changed as separation progressed. Moreover, this dorsoventral expression pattern was disturbed in those Adriamycin-treated embryos that had failed to separate the trachea and oesophagus. The process of tracheo-oesophageal separation was also found to be associated with a distinct pattern of programmed cell death (PCD) in the dorsal foregut and at the dorsoventral boundary. PCD cells were present before any morphological evidence of separation, suggesting a possible role for PCD in controlling the separation process. When a PCD inhibitor was applied to an in vitro, whole embryo, culture system, the process of tracheo-oesophageal separation was arrested, suggesting that PCD is a requirement and not just a consequence of separation.

Σε αυτή τη μελέτη εξατάσαμε κατά πόσον οι φλεγμονώδεις κυτοκίνες ρυθμίζουν την έκφραση της δισακχαριδάσης της ψηκτροειδούς παρυφής των εντεροκυττάρων, σουκράσης-ισομαλτάσης(ΣΙ), στην κυτταρική σειρά Caco2. Επίσης εξετάσαμε την πιθανότητα οι κυτοκίνες να ρυθμίζουν την έκφραση και άλλων ειδικών πρωτεϊνών των εντεροκυττάρων, χρησιμοποιώντας την λακτάση σαν πρότυπο. Τέλος εξετάσαμε την πιθανότητα καταστολής του γονιδίου της ΣΙ στα εντεροκύτταρα των λαχνών in vivo, κατά την τοπική φλεγμονώδη αντίδραση της νόσου του Crohn. Για τις μελέτες μας χρησιμοποιήσαμε τεχνικές κυτταροκαλλέργειας, βιοσυνθετικής σήμανσης-ανοσοκαθίζησης-ηλεκτροφόρησης, ανάλυση προστασίας ριβονουκλεάσης, ανοσοϊστοχημεία και ανάλυση υβριδισμού in situ. Τα αποτελέσματα δείχνουν ότι η IL-6 και η IFN-γ προκαλούν ελάττωση, και ο TNFα αύξηση της ΣΙ στα Caco2 κύτταρα. Η σύνθεση τηε λακτάσης δεν επηρεάζεται. Υπάρχει μια εκσεσημασμένη και ειδική ελάττωση της γονιδιακής έκφρασης της ΣΙ στα εντεροκύτταρα των λαχνών ειλεού νόσου του Crohn με οξεία φλεγμονή, σε σύγκριση με παρακείμενο μη φλεγμαίνοντα ειλεό και με φυσιολογικό ειλεό. Τα αποτελέσματα παρέχουν απόδειξη για ένα μέχρι τώρα άγνωστο μηχανισμό ανεπάρκειας δισακχαριδάσης στην εντερική φλεγμονή. / In the present study we examined whether inflammatory cytokines regulate the expression of the enterocyte brush border disaccharidase sucrase-isomaltase(SI), in the Caco2 cell line. We also examined the possibility that inflammatory cytokines regulate the expression of other enterocyte-specific proteins, using lactase as a prototype. Last we examined the possibility that SI gene expression is down-regulated in villous enterocytes in vivo during the local inflammatory response of Crohn´s disease. For our studies we used techniques including cell culture, biosynthetic labeling-immunoprecipitation-electrophoresis, ribonuclease protection assay, immunocytochemistry and in situ hybridization anlysis. The results show that IL-6 and IFN-γ mediate a decrease, whereas TNFα mediates an incrase in SI synthesis in Caco2 cells. Synthesis of lactase is not affected. There is a marked and specific decrease in SI gene expression in villous enterocytes in acutely inflammed Crohn´s ileum, as compared to adjacent uninflammed ileum and normal ileum. The results provide evidence for a previously unrecognized mechanism for disaccharidase deficiency in intestinal inflammation.

- / -

- / -

This study was designed to identify the processes which underlie pain in symptomatic diverticular disease (SDD). Our hypothesis was that a spectrum of both peripheral and central pathologies were involved, with those that had a more peripheral problem having abdominal symptoms only while those with multiple symptoms throughout the body, having an altered central pain processing. The first study examining the brain response to cutaneous pain using functional magnetic resonance imaging (fMRI) has supported this hypothesis. Although a statistically significant difference in sensory pain threshold was not demonstrated between the groups, fMRI imaging has shown greater emotional processing during pain and reduced anticipatory inhibitory responses in the high somatising symptomatic diverticular disease (HSDD) groups. However this is not as clear cut as we had anticipated which may be due to subject selection and demonstrate a spectrum of mixed peripheral and central changes as well as those with only peripheral or central components. In the second part we performed a randomized placebo controlled study of mesalazine 3gm versus placebo. Mesalazine significantly reduced expression of many genes associated with inflammation in SDD patients. A reduction in the median number of hours of pain per week was seen. The study was not designed to allow intention to treat analysis but has shown promising results which will need to be consolidated with future large scale studies. Both these studies support a tailored approach to SDD patient treatment based on the underlying pain process which can be both central and peripheral. The Patient health questionnaire 12(PHQ12) may be one simple measure of doing this, but again needs to be confirmed with further larger studies.