Introduction: Deaths due to chronic liver disease have increased significantly in recent decades. This is due to increases in alcohol consumption and obesity during this time period, and insensitive screening tests (liver function blood tests) utilised in primary care. This thesis describes a new liver disease community diagnostic pathway which focussed upon defined risk factors for chronic liver disease and uses Transient Elastography (TE) as the primary investigation modality. The aims of the thesis are to assess the feasibility of this pathway for detecting liver disease due to alcohol or non-alcoholic fatty liver disease within the United Kingdom healthcare system, to quantify the number of new cases detected with this approach and to evaluate patient experience of these investigations. Methods: Following a systematic review of the literature, an investigation pathway was derived and piloted in 4 general practice sites in Nottinghamshire in two phases between February 2012 and September 2014. Patients with hazardous alcohol use, type 2 diabetes or persistently raised alanine aminotransferase (ALT) level and negative liver serology were eligible for study. TE was performed in the community; a liver stiffness reading of ≥8 kilopascals defined clinically significant liver disease and subsequent review in a consultant led community clinic. Risk factors for new diagnoses of liver disease and cirrhosis were identified and the association with obesity investigated. A qualitative interview substudy was conducted to explore the experiences of 20 patients undergoing investigation. Results: In a total adult population of 20,868 patients, 2,022 patients were eligible for study of whom 909 (45%) underwent TE. Valid liver stiffness measurements were possible in 98% of patients. Overall, 230 cases of elevated liver stiffness and 27 new cases of cirrhosis were identified. Minimum cirrhosis prevalence in patients with type 2 diabetes was 2%. Obesity was significantly associated with diagnosis of cirrhosis in type 2 diabetics (odds ratio 9.4 (95% CI 2.2-40.9)) and hazardous alcohol users (OR 5.6 (95% CI 1.6-19.7)). The majority of new cases of liver disease had normal ALT levels. From the initial pilot phase in two general practices in Rushcliffe (Nottingham), in which liver function test data from 378 patients undergoing TE was analysed, 72.4% with elevated liver stiffness measurement, 60% with biopsy proven cirrhosis and 90% with cirrhosis diagnosis had normal ALT. Patients felt that TE was a useful adjunct to lifestyle change and described a positive experience of liver disease investigation. Conclusion: A new non-invasive diagnostic pathway for liver disease was feasible to implement in Nottinghamshire primary care and resulted in significantly increased diagnosis of chronic liver disease and cirrhosis. These findings warrant exploration of the pathway in a larger primary care population.

The majority of complications in patients with cirrhosis result from the development and progression of portal hypertension characterised by increased intrahepatic resistance and progressive splanchnic vasodilation. Hepatic venous pressure gradient (HVPG) measurement is the only validated technique to accurately evaluate changes in portal pressure. However, HVPG measurements are invasive and available only in specialised hepatology units, precluding its use in routine clinical practice. In the first study, we evaluated the use of non-contrast quantitative magnetic resonance imaging (MRI) as a surrogate measure of portal pressure. 30 patients undergoing HVPG measurement were prospectively recruited. MR parameters of longitudinal relaxation time (T1), perfusion of the liver and spleen (by arterial spin labelling), and blood flow in the portal, splanchnic and collateral circulation (by phase-contrast MRI) were assessed. We estimated the liver stiffness measurement (LSM) and Enhanced Liver Fibrosis (ELF) score. The correlation of all non-invasive parameters with HVPG was evaluated. The mean (range) HVPG of the patients was 9.8 (1-22) mmHg, and 14 patients (48%) had clinically significant portal hypertension (CSPH, HVPG ≥10 mmHg). Liver T1 relaxation time, splenic artery and superior mesenteric artery velocity correlated significantly with HVPG. Using multiple linear regression, liver T1 and splenic artery velocity remained as the two parameters in the multivariate model significantly associated with HVPG (R=0.90, p < 0.001). This correlation was maintained in patients with CSPH (R=0.85, p < 0.001). A validation cohort (n=10) showed this linear model provided a good prediction of HVPG. LSM and ELF score correlated significantly with HVPG in the whole population but the correlation was absent in CSPH. In conclusion, MR parameters related to both hepatic architecture and splanchnic haemodynamics correlate significantly with HVPG. This proposed model, confirmed in a validation cohort, could replace the invasive HVPG measurement. In the second part, we studied the use non-invasive MRI to dynamically assess changes in hepatic and collateral blood flow, liver perfusion and oxygenation in response to ingestion of a test meal (meal challenge), and hyperoxia and hypercapnia (gas challenge). These changes were compared between healthy subjects and patients with compensated cirrhosis (CC). In the meal challenge study, we evaluated the blood flow in the portal vein, hepatic artery and azygos vein, liver perfusion and blood oxygenation (using transverse relaxation time (T2*) mapping). Measures were collected at baseline and at 6-7 minute intervals from 20 to 65 minutes following a test meal (440 ml; 660 kcal) in 10 healthy participants and 10 CC patients. In healthy participants, we observed a postprandial increase in portal vein flow from baseline coupled with a reduction in hepatic artery flow from baseline, reflecting the hepatic artery buffer response (HABR). In CC patients, changes in portal vein and hepatic artery flow were smaller, with no clear HABR. In healthy participants, postprandial liver perfusion increased, but not in CC patients. There was no change in liver T2* for either group. In the gas challenge study, we evaluated the blood flow in portal vein and hepatic artery, liver perfusion and liver T2* during hyperoxia and hypercapnia in 10 healthy subjects and 4 patients with compensated cirrhosis. A sequential gas delivery breathing circuit and a prospective, feed-forward gas delivery system (RespiractTM, Thornhill Research Inc., Toronto, Canada) was used to control and monitor end-tidal O2 (PETO2) and CO2 (PETCO2) partial pressures. Hyperoxia was targeted at PETO2 ~500mmHg and hypercapnia was aimed at PETCO2 ~6mmHg above resting value. The study design consisted of 5 blocks. Blocks 1, 3 and 5 were 5 min periods at resting PETCO2 and PETO2. Blocks 2 and 4 were, in a random order, 5 mins of hyperoxia (with a 2 min transition up and down) or 5 mins of hypercapnia. We observed an increase in portal vein velocity during hypercapnia among the healthy subjects and patients with cirrhosis. There was no significant changes in liver T2* but the full-width-half-maximum (FWHM) of the distribution of the liver T2* increased in response to hyperoxia and hypercapnia in both groups. Subjects with low T2* at baseline exhibited a smaller change in FWHM following the gas challenge. The within session and inter-session coefficient of variation (CoV) the blood flow measurement using phase-contrast MRI in healthy subjects was less than 15%. If our findings are confirmed in external validation studies, non-invasive MRI can be used as a surrogate measure of HVPG. Assessment of postprandial dynamic changes in hepatic, splanchnic and collateral circulation using MRI could potentially be used to stratify patients with portal hypertension and study the effects of potential novel treatments for portal hypertension.

Trendelenburg positioning is frequently used during laparoscopic surgery particularly when access to the pelvis is required. With improvements in laparoscopic skills, high risk patients and more complex procedures are now frequently being performed laparoscopically. (Improvement, 2016) The aim of this thesis is to investigate the effect of Trendelenburg positioning on intraocular pressure (IOP) and cognitive function. Chapters 2 and 4 look at the effect of Trendelenburg positioning on IOP. Perioperative vision loss occurs rarely but it is a life changing complication. A rise in IOP is a recognised risk factor for POVL. The incidence of POVL following laparoscopic colorectal surgery has been quoted as 1.24 in 10,000 cases. (Pinkney et al., 2012) Chapter 2 is an observational study during which IOP was monitored during laparoscopic colorectal surgery. This was correlated with the degree of Trendelenburg tilt used during surgery. This study revealed an increase in IOP occurred which was dependent on the degree of Trendelenburg tilt as well as the time spent in this position (Pearson’s correlation coefficient was 0.78). Patients undergoing left-sided colonic resections had a mean maximum IOP rise of 15.2mmHg. Chapter 4 is a follow-on study which looked at acetazolamide as a method of reducing the IOP rise that occurred whilst in the Trendelenburg position. This was a randomised placebo controlled cross-over healthy volunteer study. After 4 hours of Trendelenburg, the mean IOP increase was 3.17mmHg in the placebo group compared to 0.02mmHg in the acetazolamide group (P < 0.05). This suggests acetazolamide has a role in reducing the IOP rise that occurs from Trendelenburg positioning. The second half of this thesis focuses on the effect of Trendelenburg positioning on cognitive function. Post-operative cognitive decline (POCD) is defined as cognitive impairment following surgical intervention. It is associated with increased hospital stay, longer return to work/normal functioning, and in patients with existing cognitive impairment a further decline can result in loss of ability to carry out activities of daily living. (Moller et al., 1998) Chapter 5 is an observational study that explores the incidence of short- or long-term POCD following laparoscopic colorectal surgery. Post-resectional surgery, 55.4% of patients had evidence of POCD on Day 1 and 31.6% at long-term follow-up. On Day 2, 11.6% had POCD following right-sided resection compared to 16.3% in the left-sided resection group. Chapter 6 and 7 look at the effect of Trendelenburg positioning on cognitive function in healthy volunteers. Chapter 6 assessed changes in brain function using magnetoencephalography and n-back testing as well as looking at MRI structural changes after 2 hours in Trendelenburg position. Although the difference was not statistically significant, there was an increase in brain volume after 2 hours in Trendelenburg compared to pre-Trendelenburg MRI scan suggesting an element of cerebral oedema. Chapter 7 was a volunteer study designed to assess the effect of time spent in Trendelenburg position on cognitive function using cognitive tests (n back, stroop and lexical decision making tasks). This was carried out at regular intervals whilst in the Trendelenburg position and again once the volunteer was placed supine. After 3 hours in the Trendelenburg position, 40% had cognitive decline compared to 26.7% after 30 minutes.

Barrett’s oesophagus and oesophageal cancer represent two of the most important and challenging oesophageal disease processes globally, combining increasing incidences with high morbidity treatments, often with poor clinical outcomes. A major contributory factor is that disease susceptibility, progression and response to therapy are largely unpredictable, due to inherent biological complexity and variability. At present, just staging groups are used routinely as thresholds for guiding the use of therapies such as ablation, resection, and oncological therapies. However, these represent blunt tools that do not necessarily reflect patients’ experiences, or appropriately select from the range of treatments available. The aim of this thesis was to explore the potential of genomic, imaging, and pathological biomarkers in guiding more tailored and personalised therapy. The first half of this thesis explores the role of genomic markers. The first results chapter describes the identification of new loci and gene pathways associated with susceptibility to Barrett’s oesophagus, dysplasia and oesophageal adenocarcinoma, by further replication and analysis of a genome-wide association study. In addition, all reported genomic markers of these endpoints were identified and criticised by systematic review, and synthesised by meta-analysis. Validation of these was then attempted, and lessons for markers and future research drawn. The second results chapter describes a similar appraisal and synthesis of genomic markers of oesophageal cancer prognosis, response to therapy, and stage. The third describes the first next generation sequencing study performed in oesophageal adenocarcinoma (and indeed any gastrointestinal cancer as far as the author is aware), before and after neoadjuvant chemotherapy. Using whole exome sequencing a new model of genomic tumour response was developed, and the implications for biomarkers explored. The second half of this thesis follows a large cohort of patients with oesophageal cancer, from nearly 1000 undergoing staging, to more than 300 undergoing neoadjuvant chemotherapy, restaging and resection. In the fourth results chapter, the first application of decision theory to cancer staging identified the potential for routine imaging data to personalise and optimise oesophageal cancer staging. In the following chapter, positron emission tomography-computed tomography was found to be more sensitive for identifying disease progression during neoadjuvant chemotherapy than computed tomography alone. Two factors were identified that could stratify risk of progression to incurable disease, including that encountered at surgery. These included 18F-FDG avid nodes, with new concepts of metabolic nodal stage and response developed in conjunction with predictive models. Thereafter, a number of conventional and experimental metrics of metabolic tumour response were compared and refined as predictors of pathological response. Existing metrics of metabolic tumour response were found to be suboptimal, and these new concepts and classifications of metabolic nodal stage and response were found to have independent utility for clinical practice. Again, predictive models were generated. Finally, the prognostic utilities of these markers were explored. Metabolic tumour response was found to be an imperfect surrogate of pathological response. However, metabolic nodal response demonstrated independent utility in identifying patients at high risk of early recurrence and death, both when used before surgery and afterwards. Indeed, a number of analyses demonstrated the additive utility of considering the primary tumour and nodal metastases as separate entities. Finally, prognostic models were generated, and a simple risk score was generated, using the four independent prognostic markers identified to stratify prognosis.

Irritable bowel syndrome (IBS) remains a heterogeneous condition and is a common condition. The causes of IBS remain poorly understood and there is a lack in biomarkers to distinguish this condition. Recently, there have been reports on the release of immune mediators leading to symptoms of irritable bowel syndrome. Mast cells, which can be activated by allergy or stress, are thought to be important cause of symptoms in some IBS patients because they can release chemicals, which cause pain and diarrhoea. Currently, there are few effective treatments available to alleviate these symptoms. Recent small studies have shown that Mesalazine, an ‘anti-inflammatory’ drug, may be able to modify and reverse the symptoms of IBS with diarrhoea. One small study suggested Mesalazine reduced mast cell numbers. This current study is one of the largest studies looking at the use of Mesalazine as a form of treatment for IBS with diarrhoea. Unfortunately, this study did not show any beneficial effect of Mesalazine treatment in unselected patients with IBS and diarrhoea. Potentially, there is a subgroup of IBS patients who developed their symptoms following a bout of gastroenteritis who appeared to benefit from Mesalazine treatment but a larger study is needed to confirm this. In this study, the mast cell mediators released from mucosal biopsies was not a useful marker of disease since it failed to correlate with any symptoms. Magnetic resonance imaging (MRI) is a potentially useful tool to assess the physiology of the gastrointestinal tract in patients with functional gut disorders as it does not involve radiation and is not invasive. So far, there is a lack of biomarkers to assist in diagnosis and treatment of irritable bowel syndrome. The MRI marker pill used in the multiple studies in Chapter 3 to assess whole gut transit time is very promising as it is now applied, in the research setting, to patients with chronic constipation such as slow transit constipation and irritable bowel syndrome with constipation. Further use of the MRI and adding a stimulus such as laxative in patients with chronic constipation is helpful to distinguish between functional constipation and irritable bowel syndrome with constipation; thus helping with its medical management. The use of MRI as a biomarker for diagnosis of irritable bowel syndrome remains promising although it was not demonstrated in this thesis.

Human populations are at risk of exposure to constitutive androstane receptor (CAR) activators present in a range of substances, including pharmaceuticals, plasticizers and crop protection agents. What exposure to CAR activators means for human health is uncertain. Activation of CAR in rodents is associated with liver hyperplasia, increased proliferation and eventual hepatocarcinoma; however the effect in human hepatic cells is unclear. There are two methods by which a compound can achieve activation of CAR; directly or indirectly via cellular signalling pathways. Phenobarbital is a prototypical activator of CAR and does so in an indirect manner via suppression of epidermal growth factor receptor (EGFR) signalling. Direct activation of CAR in rodents also causes hepatocellular carcinoma but the human outcome is less clear. We have carried out microarray and miRNA analysis of CITCO (a potent and selective hCAR ligand) treated primary human hepatocytes. To mitigate the well documented effect of primary hepatocyte dedifferentiation primary hepatocytes were cultured in dynamic three dimensional culture in vitro. Gene expression changes indicate that direct activation of hCAR causes the promotion of a pro-proliferative and anti-apoptotic phenotype. The miRNA expression profile is crucially different to rodent data that is currently published. Despite the pro-proliferative phenotype shown there is no evidence that primary human hepatocytes proliferate in response to direct activation of CAR by CITCO. This leaves the possibility that a proliferative response may be observed in vivo or that the changes in gene expression are solely a human physiological adaptation to direct hCAR activation by CITCO and no proliferation would occur. The effect on human health and liver toxicity is unclear but this body of work has provided data that may be used to further understand the mechanistic effects of direct hCAR activation in human hepatocytes. A more complete understanding of this will help to inform the toxic potential of direct hCAR activation in vivo.

Helicobacter pylori (Hp) is a major cause of peptic ulcer disease (PUD) and gastric cancer, yet the infection remains asymptomatic in most people. One factor that influences the outcome of Hp infection is the host immune response. Expression of the immune-stimulating cytokine interleukin-17 (IL-17) is increased in the human Hp-infected gastric mucosa, but its cellular source and role in pathology are unclear. In this study dendritic cell cytokine responses to Hp stimulation were studied, and relative IL-12p70 and IL-23 concentrations compared, to assess the potential of Hp to promote differentiation of IL-17-secreting T-helper cells (Th17). The effect of Hp virulence factors on cytokine secretion was assessed and monocyte-derived DC (MoDCs) and CD1c+ myeloid DC (MyDC) responses compared. MoDCs produced high concentrations of IL-12p70 upon Hp stimulation. There was also an~ IL-23 MoDCs response, but this was >10-fold lower than the IL-12p70 response. Both IL-12p70 and IL-23 responses were significantly reduced when Hp isogenic mutants for the virulence factor dupA were used, although the effect on MoDC IL-12p70 and IL-23 secretion was less marked than previously reported for monocytes. MyDCs produced lower concentrations of IL-23 than MoDCs, and no detectable IL-12p70. It is known that Hp infection can have systemic effects, so next peripheral blood mononuclear cells (PBMCs) from 21 Hp+ and 13 uninfected patients were stimulated with Hp or control antigen and Th17 and Th1 cell frequencies analyzed by flow cytometry. A systemic Hp-specific Th17 response was identified with higher Th17 cell frequencies in the Hp+ patients compared to the uninfected controls (2.0-fold, p=0.027). A variable proportion of these cells also secreted IFNgamma (median 33%, n=21), but there was no significant correlation between Th17 and Th1 cell frequencies. Peripheral blood Th1 cells were also increased in Hp+ patients (2.1-fold, p=0.018). No significant difference was found between peripheral blood Th17 and Th1 frequencies in the Hp+ patients. The concentrations of Th17, Th1, Th2 and Treg cytokines in the gastric mucosa of Hp+ patients and uninfected controls were investigated using luminex and real-time PCR. High levels of Il-17 expression in the infected compared to uninfected gastric mucosa were confirmed at both the mRNA and protein level (mRNA: 42.6-fold, p<0.0001; protein 3.5-fold, p<0.0001). Il-17 concentrations correlated with the levels of Il-17F (p=0.80, p<0.0001) and the chemokines CCl20 (p=0.59, p<0.0001) and Il-8 (p=0.49, p=0.0004). Concentrations of the Th17-differentiating cytokines IL-1beta, IL-6, IL-21 and IL-23 were not increased in the Hp+ gastric biopsies, although IL-23 was present at high concentrations in all samples regardless of Hp infection status. IL-17 was present at higher concentrations than IFNgamma (3.9-fold, p<0.0001), IL-4 (3.0-fold, p<0.0001) and IL-10 (6.8-fold, p<0.0001) in Hp+ gastric biopsies. IFNG mRNA was also more highly expressed than IL17 mRNA (3.3-fold, p=0.016). To identify the cellular source of the IL-17 mononuclear cells were extracted from gastric biopsies, stimulated with PMA/ionomycin and analyzed by flow cytometry. Amongst biopsy CD3+ T cells from 10 Hp+ patients, IL-17 was produced mainly by CD4+ Th17 cells (68.5%), although CD8+/l-17+ (24.7%) and CD4-CD8- (18.2%) cells also made a significant contribution. High IL-17 concentrations were associated with increased inflammation (2.4-fold, p=0.024) and neutrophil infiltration (2.4-fold, p=0.031). RORC2 mRNA expression was weakly associated with PUD (p=0.046, 1.4-fold) but, surprisingly, no association was found between the IL-17 response and incidence of PUD or precancerous changes. In conclusion Hp can stimulate DCs to produce IL-23 in vitro, and high levels of this Th17-differentiating cytokine were found in gastric mucosal biopsies. Stimulation with dupA null Hp strains led to reduced IL-12p70 and IL-23 secretion, suggesting a possible mechanism of action for this recently discovered virulence factor. Culturing Hp with MoDCs and MyDCs yielded quite different results, and it remains unknown whether either model closely reflects gastric mucosal DC responses. Hp-specific Th17 responses were identified in the peripheral blood of patients with active Hp infection for the first time. Th17 cells were identified as the main cellular source of IL-17 in the Hp-infected gastric mucosa but there were also significant numbers of CDS+IL-17+ and CD4-CDS-IL-17+ T cells, which have not previously been described in this context. High mucosal concentrations of IL-17 and association of this cytokine with infiltration of immune cells indicate that it is an important component of the human immune response to Hp. However, no association between IL-17 and risk of disease was detected in this study, although RORC2 expression was weakly associated with PUD. The role of I L-17/Th 17 in Hp related disease warrants further investigation.