Cystic fibrosis (CF) is a fatal disease caused by a mutation of the CFTR gene and severe inflammation of the lungs. The inflammatory process is characterised by increased production of the potent neutrophil-attracting chemokine interleukin 8 (CXCL8), but the mechanism responsible is poorly understood. We tested the hypothesis that altered epigenetic regulation is responsible for the basal and cytokine-induced CXCL8 upregulation in CF airway epithelial cells. We found that CXCL8 protein levels and mRNA expression were higher in CF as compared to normal cells both basally and following cytokine stimulation. The difference in the expression was independent of increased mRNA stability or increased transcription factor activation and/or expression in CF cells. We found increased basal, but not cytokine-induced transcription factor binding to the CXCL8 promoter in a chromatin environment in CF cells in comparison with normal cells, increased histone H3 lysine 4 trimethylation, hypomethylation of CpG sites and increased binding of BRD3 and BRD4 to the CXCL8 promoter. Disruption of BRD4 association with chromatin using the selective BET bromodomain inhibitor JQ1 decreased CXCL8 protein release from CF cells to the levels observed in normal cells. Our observations suggest that epigenetic alterations are responsible for the upregulation of CXCL8 in CF and could become potential targets in the development of new therapeutic strategies.

Pancreatic cancer is a solid tumour with poor prognosis and ineffective therapeutic approaches. The role of the tumour microenvironment in supporting pancreatic tumour growth and metastasis has been demonstrated. In particular, a new concept of the stem cell niche as a mixed population of mesenchymal stem cells and niche cells (myofibroblast cells) involved in promoting these processes is emerging. Paracrine transmission of the Hedgehog (Hh) pathway in the pancreatic tumour microenvironment has previously been reported. This project aimed to identify the stromal pancreatic cancer cells able to respond to Hh pathway exogenous stimuli and to investigate their relationship to tumour progression, in order to define new targets for pancreatic cancer therapies. Gene and protein expression analysis in pancreatic primary tumours demonstrated overexpression of Shh ligand not only at the epithelial but also at the stromal level in advanced stages of pancreatic cancer. In vitro modelling of the mesenchymal stem cell niche (mSCN) using human bone marrow-derived mesenchymal stem cells (MSCs) co-cultured with cancer-associated fibroblasts (CAFs) or myofibroblast-like cells (MF-like, obtained by treating MSC with TGFβ) showed up-regulation of Shh gene and protein expression in comparison to single stromal cell populations (MSCs; CAFs and MF-like cells). The investigation of Hh paracrine signalling in pancreatic tumour microenvironment using different 2D in vitro assays (transwell and direct co-cultures, NShh treatments and tumour condition media cultures) demonstrated the inability of CAFs and MSCs, when grown in culture conditions that prevent their activation (increase of αSMA), to respond to Hh exogenous stimuli and suggested the mSCN as the stromal context in which paracrine induction of the Hh pathway takes place. Taken together, these results suggest a new concept: Shh expression is an indicator of the mSCN in the pancreatic cancer microenvironment and that its role as a possible target for chemotherapy should be explored.

Background: Patients with diarrhoea predominant irritable bowel syndrome (IBS-D) suffer from loose frequent stools with associated urgency and fear of incontinence. Relief from these symptoms is an important unmet need. The 5-HT3 receptor antagonist Alosetron has been shown to increase stool consistency, decrease urgency and reduce abdominal pain leading to a global increase in satisfaction with treatment [1]. Its use is restricted following an increased incidence of ischaemic colitis and this agent is not available in Europe. The serine proteases family of proteolytic enzymes have been identified as the source of increased faecal proteolytic activity in patients with IBS. These enzymes may be mechanistically important via their action on the Protease activated receptor (PAR) -2, inducing increases in permeability and hypersensitivity. Aims: To assess the efficacy of the commonly prescribed 5-HT3 antagonist Ondansetron, in patients with IBS-D and to identify biomarkers that might allow us to predict response defining an Ondansetron responsive endophenotype of IBS. To structurally characterise faecal serine proteases and define the impact of treatment with Ondansetron. Methods: 120 patients meeting Rome III criteria for IBS-D entered a randomised, double-blind, placebo controlled, cross-over study of 5 weeks of Ondansetron 4mg versus placebo with dose titration allowed up to two tablets thrice daily in the first 3 weeks. Patients completed daily bowel symptom diaries documenting stool consistency using the Bristol Stool Form Score (BSFS). Gut transit and small bowel water content were measured in the last week of each treatment. The primary endpoint was average stool consistency in the last 2 weeks of treatment. Faecal samples were obtained from 30 healthy volunteers (HV) and 79 IBS-D patients participating in a trial of Ondansetron versus placebo. Colonic transit was measured using radio-opaque markers. Faecal serine proteases (FSP) were purified from faecal extracts using Benzamidine-Sepharose affinity chromatography. SDS-PAGE profiled components were identified using trypsinolysis and tandem-mass-spectrometry. Functional protease activity in faecal extracts was measured using a colourimetric assay based upon the proteolysis of azo-casein. Results: Ondansetron significantly improved stool consistency In the intention to treat analysis n= 101, with a 1.39 (95% CI1.20-1.58)point decrease on the Bristol stool form scale whilst taking Ondansetron compared to a 0.51 (95% CI 0.32-0.72) point reduction whilst taking placebo p=<0.0001. Compared to placebo patients on Ondansetron experienced fewer days with urgency (p=0.01), lower urgency scores (P<0.001), reduced frequency of defecation (p=0.001) and less bloating (p=0. 25) although pain scores did not change significantly. Protein analysis identified the most abundant FSPs as being of human origin and likely pancreatic juice derived. Functional assays showed increased FSP and faecal amylase in IBS-D compared to HV. Those with higher amylase had significantly higher FSP and greater anxiety. FSP activity correlated negatively with whole gut transit in IBS-D (Spearman r=-0.32, p=0.005) and HV (r=-0.55, p=0.014), but was not affected by treatment with Ondansetron. Conclusions: Ondansetron is an effective and well tolerated treatment in patients with IBS-D with a low number of side effects. It slows whole gut transit, but without a demonstrable difference in small bowel water content. Clinical rather than biochemical indicators predicted responsiveness to Ondansetron best. Patients with less severe symptoms are more likely to respond well to Ondansetron which should prove a useful addition to the current rather limited therapies available for this important group of patients. Previous reports that FSP activity is elevated in some patients with IBS-D has been confirmed. We have increased our understanding of this phenomenon by characterising the proteins responsible for the serine protease activity, showing that most of this activity is likely due to human pancreatic enzymes.

This thesis presents the development and evaluation of two applications for scaffolds in the field of liver tissue engineering. In the first study a poly (D,L lactic acid) (PDLLA) scaffold is used as a three-dimensional template for hepatocyte–hepatic stellate cell (HSC) co-culture. To enhance PDLLA ligand binding capacity scaffolds are surface modified using allylamine plasma deposition and treatment with NaOH. Primary adult rat hepatocytes and HSC are then seeded onto these scaffolds and cultured in static conditions. Scanning electron microscopy (SEM) is used to assess mono-culture and co-culture morphology whilst synthetic and cytochrome P450 function are measured using albumin and testosterone assays. The second study explores the potential for intrahepatic growth factor and extracellular matrix (ECM) delivery from a biodegradable polymer scaffold to promote liver growth and to enhance regeneration. The study is undertaken in rats. The scaffold design and implantation technique are first piloted in a short survival study. Hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF)1, FGF2 and liver derived ECM (L-ECM) are then loaded into poly(lactic-co-glycolic acid) (PLGA) + 5% poly(ethylene glycol) (PEG) scaffolds and implanted into normal and partially hepatectomised liver. Implant morphology is assessed by micro-CT reconstruction. Growth factor bioactivity and release are confirmed by in vitro profiling. Liver growth and volume redistribution are assessed by liver weight analysis. Parenchymal injury and function are quantified by measuring serum aspartate aminotransferase (AST) & bilirubin. 5-bromodeoxyuridine (BrdU) inclusion & MIB-5 immunohistochemistry (IHC) are used to identify hepatocyte and non-parenchymal cell proliferation. Liver-scaffold interaction is characterised by H&E and Masson’s trichrome staining. Non-parenchymal cell migration is assessed by ED-1 and desmin IHC. All histology is then subjected to image analysis.

The inflammatory bowel diseases, ulcerative colitis and Crohn's disease are characterised by spontaneously relapsing and remitting inflammation, associated with increased mucosal levels of the inflammatory cytokine, interleukin-1 (IL-1)β. IL-1β processing and release is mediated by ATP stimulation of the purine receptor, P2X7. P2X7 is a membrane ion channel highly expressed in immune cells. Signal transduction occurs via rapid cation exchange, plasma membrane depolarisation and increased intracellular calcium. Additionally, prolonged or repeated P2X7 stimulation leads to formation of a non-selective membrane pore permeable to small molecules, and ultimately to cell death. The aim of this project was to investigate the properties of the P2X7 receptor in mononuclear cells, to show that it is associated with IL-1β release in the colon, and that this release can be modified by P2X7 antagonists. Studies of ethidium bromide uptake, a functional assay, showed that P2X7 receptors are present on LPMCs and displayed properties similar to those of PBMCs and THP-1 cells. P2X7 receptor-stimulation released mature IL-1β from LPMCs in a dose-dependent manner that, in IBD patients, matched the severity of their inflammation, and could be markedly reduced by P2X7 antagonists. P2X7 stimulation also results in increased exposure of phosphatidylserine on the outer cell membrane (PS flip), often considered to be a marker of apoptotic cell death. P2X7-stimulated PS flip however is reversible and is not associated with cell death following brief stimulation times. Cell death caused by longer stimulation did not have features of apoptosis, was more evident in monocytes than lymphocytes, with LPMCs being less susceptible than PBMCs and THP-1 cells. These studies have shown that the P2X7 receptor is intimately involved in the release of IL-1β from human colonic mononuclear cells, that the release is greater in cells from IBD tissue and can be markedly inhibited by P2X7 antagonists.

Chronic infections with Pseudomonas aeruginosa are primarily responsible for the decline in lung function and ultimate mortality in cystic fibrosis patients. The overall aim of this project was to elucidate some of the molecular mechanisms governing the pathogenesis of P. aeruginosa in the cystic fibrosis lung. This was with particular reference to (a) quorum sensing, a cell-to-cell communication system controlling the production of virulence determinants in a population density dependent manner using diffusible signal molecules and (b) the pseudomonas lectins, LecA and LecB, which are known to contribute to biofilm formation. Serial sputum samples were collected from adult and paediatric patients with cystic fibrosis and a cohort of clinical P. aeruginosa isolates was assembled. Using bioreporters, these isolates were shown to synthesise a range of quorum sensing signal molecules. Furthermore, the direct detection of these P. aeruginosa products from infected sputum samples in conjunction with patient clinical data implied an association between sputum quorum sensing signal molecule level and cystic fibrosis disease status, response to intravenous antibiotics and the presence of non-culturable P. aeruginosa. Quorum sensing also makes an important contribution to P. aeruginosa biofilm maturation, antibiotic tolerance and resistance to host defences. There is evidence that the quorum sensing regulated lectins LecA and LecB contribute to biofilm development and this was investigated using different biofilm assays, including the flowchamber biofilm system. This work demonstrated that LecA contributed to biofilm maturation in both laboratory and clinical strains and hydrophobic galactosides were shown to be able to inhibit biofilm development. The putative biofilm target ligand for LecA was tentatively identified as the Psl exopolysaccharide. Mutants deficient in either lecA or lecB produced defective biofilms, which could be inhibited and/or dispersed by galactosides or furanosides respectively, including novel synthetic furanoside dendrimers. The latter proved inhibitory to both laboratory and clinical P. aeruginosa isolates and constitute a potential novel therapeutic.

Tumour hypoxia has been linked to increased resistance to both radiotherapy and chemotherapy, especially in solid metastatic GI tumours. Under hypoxic conditions, genes that promote tumour growth and survival are up-regulated, via the transcription factor hypoxia-inducible factor-1 (HIF-1). The digestive hormone gastrin, which is often over-expressed in GI cancers, has also been shown to act as a pro-survival factor, up-regulating processes such as tumour proliferation, angiogenesis and migration, and down-regulating apoptosis. Due to the high level of similarity between the downstream events mediated by the two proteins, the relationship between gastrin and HIF-1 was investigated. HIF-1α nuclear protein expression was inducible under hypoxic conditions, which led to an expected increase in VEGF gene expression, followed by a 12-50 fold increase in hypoxic gastrin mRNA expression. HIF-1α expression and transcriptional activity were not consistently affected by exogenous gastrin. RNA-interference-mediated knockdown of HIF-1α resulted in a 40-60% down-regulation of gastrin gene expression under hypoxic conditions suggesting that HIF-1α is partially responsible for gastrin up-regulation in hypoxia. Potential hypoxia-response elements (HREs) were identified within the gastrin promoter, but were only partially responsive to hypoxic incubation in GI carcinoma cells in luciferase-reporter assays. Other possible mechanisms that may account for the increased gastrin gene expression induced under hypoxic conditions include interactions of gastrin with other transcriptional regulators, either in synergy with or independent from HIF-1, or the sequestration of gastrin within the cell by ‘P’-bodies or RNA-binding proteins. These findings may indicate that the addition of anti-gastrin agents such as CCK-2 receptor antagonists or gastrin immunogens to the treatment regime of patients with solid GI tumours may be clinically beneficial, especially if combined with agents used to reduce radiotherapy and chemotherapy resistance.

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