Chronic hepatitis B (CHB) results from a complex interaction between a replicating non-cytopathic virus and an impaired antiviral host immune response. The Programmed Cell Death (PD-1) pathway is an immunoinhibitory T-cell pathway implicated in virus-specific T-cell dysfunction in several chronic viral infections. The role of the PD-1 pathway in the immunopathogenesis of chronic hepatitis B was investigated through several different approaches. Firstly, longitudinal changes in PD-1 expression in patients with CHB undergoing oral antiviral therapy was investigated. A direct correlation between viral load and PD-1 expression on virus-specific CD8+T-cells was observed in this patient cohort, and treatment induced suppression of viraemia resulted in a significant decrease in PD-1 expression on virus-specific CD8+ T-cells with a decrease in HBV-DNA and improvement in virus specific T-cell reactivity. Secondly, through the employment of a purposely-designed in vitro cell co-culture model of Hepatitis B virus infection the interactions between HBV-producing hepatoma cells (target cells) and HBV-specific CD8+ T-cells (effector cells) was investigated. This model provided evidence that both cytolytic and non-cytolytic CD8+ T-cell effector functions are important in effective control of viral replication, and blockade of the PD-1 pathway distorts the balance between these differential effector functions in vitro. Finally through the transfection of a human hepatoma cell line with hepatitis B virus (HBV) and the analysis of hepatoma cell lines that differentially express HBV these studies demonstrate that the Hepatitis B virus itself upregulates PDL1 expression on infected hepatocytes in vitro and in doing so, are able to alter the balance between cytolytic and non-cytolytic CD8+ T-cell effector functions favouring chronicity of infection. Manipulation of the PD-1 pathway may be a possible mechanism to improve virus-specific host immune responses and allow control of HBV infection. However, these immunotherapeutic strategies require careful application as there is a potential risk of immune-mediated host tissue damage.

Malnutrition can be defined as a state of deficiency, excess or imbalance of nutrition which has an adverse effect on bodily function, composition or clinical outcome. Despite justifiable concerns regarding the increasing prevalence of over-nutrition in children, under-nutrition remains an underlying issue in paediatric hospitals. Under-nutrition is commonly seen in hospitalised patients as a result of decreased dietary intake, an inability to absorb nutrients or increased metabolic requirements. Apart from the well-recognised detrimental metabolic and clinical consequences, the long term effects of under-nutrition on foetal, infant and childhood growth have been acknowledged to predispose a child to an increased risk of chronic disease in adulthood. General estimates suggest that 9-47% of hospitalised children are at risk of under nutrition. Apart from certain disease states known to predispose to under-nutrition, such as cystic fibrosis, cardiology and oncology conditions, prevalence estimates are highly dependent on the indices used to define under-nutrition. Suggested indices for the assessment of acute under-nutrition include weight-for-height, weight-for-age and BMI-forage, while height-for-age is suggested for the assessment of chronic under-nutrition. At present, there is a lack of universally acceptable criteria by which to define under-nutrition. The recent emergence of paediatric specific nutrition screening tools for use in hospitals, including STAMp© and PYMS in the UK and Strongkids in the Netherlands, has increased interest in under-nutrition in hospitalised children. These tools aim to identify under nutrition in order to initiate effective referral for treatment and thus potentially reduce the detrimental effects on growth and clinical outcomes. Their use in infants, however, has been largely overlooked. This thesis considers the identification and prevalence of malnutrition in hospitalised children.

Early age at hepatitis B virus (HBV) infection is known to increase the risk of chronic HBV (CHB) infection. This thesis investigated whether, in addition to increasing the risk of chronicity, early age at HBV infection further increases the risk of hepatocellular carcinoma (HCC) by maintaining high viral replication. A systematic review of observational studies suggested that early age at HBV infection might increase the risk of sequelae of CHB infection. However, there was no data from Africa. A project was therefore designed to explore the effect of age at HBV infection on HCC and its predictors in The Gambia, West Africa, using two proxy variables for the age at infection: birth order and maternal HBV sero-status. A historical cross-sectional study of children born to HBeAg-positive mothers and HBeAg-negative mothers found that having an HBeAg-positive mother is associated with higher risk of positive HBeAg in children. The distribution of birth order was compared between HBV-related HCC cases and HBsAg-positive controls using a historical case-control study and data from the PROLIFICA (Prevention of Liver Fibrosis and Cancer in Africa) project. The former study did not find statistically significant association whilst the latter found an inverse association between birth order and HCC, suggesting that chronic carriers with low birth order might have an increased risk of HCC. Finally, an open community cohort study of HBsAg-positive people in three Gambian villages found that having an HBsAg-positive mother is associated with a number of factors predictive of disease (delayed HBeAg seroclearance, high HBV DNA and alanine transaminase levels over time, active CHB disease, significant liver fibrosis and condition requiring antiviral treatment). These findings suggest that interrupting 6 perinatal mother-to-infant transmission might significantly reduce the burden of liver disease associated with CHB infection in The Gambia.

A major hurdle facing current hepatocyte transplantation practice is the marginal quality of isolated hepatocytes. Previous studies showed that mesenchymal stem cells (MSCs) could maintain morphology and improve liver-specific metabolism of co-cultured hepatocytes. The present work aimed to optimise the MSCs co-culture system by testing adipose tissue (AT), bone marrow, and umbilical cord-derived MSCs at predefined seeding ratios. Liver-specific metabolism and apoptosis assays were performed to investigate hepatotrophic and antiapoptotic effects of MSCs co-culture. Indirect co-culture was established to investigate the role of paracrine factors in hepatotrophic effect of MSCs co-culture. Hypoxia-preconditioned (HPc) MSCs were co-cultured with hepatocytes to investigate potentiative effect of HPc induction. Intracellular reactive oxygen species (ROS) activity quantitation and antagonisation experiments were performed to investigate whether HPc potentiated MSCs co-culture by an intracellular ROS-dependent mechanism. Tumour necrosis factor alpha (TNF-α), transforming growth factor beta1 (TGF-β1), extracellular collagen, and apoptosis- associated caspase and BAX/BCL-2 signalling pathways were analysed to investigate the contribution of soluble factors, extracellular collagen, and gene signalling to the hepatotrophic effects of MSCs co-culture and potentiative effect of HPc induction. All the three types of MSCs exhibited a similar hepatotrophic effect, with a comparable effect even in low-density AT-MSCs co- culture. Hepatotrophic and antiapoptotic effects of MSCs showed a cell contact dependent manner, and HPc potentiated MSCs co-culture by a cell-contact intracellular ROS-dependent mechanism. Decreased hepatocyte autocrine TNF-α, increased MSC autocrine TGF-β1, and enhanced MSCs deposition of extracellular collagen contributed to the hepatotrophic effects of MSCs co-culture and potentiative effect of HPc induction, with downregulated expression of proapoptotic CASP9, BAX, and BID and upregulated expression of antiapoptotic BCL-2. It is concluded that synergistic effects of cell contact, intracellular ROS-dependent soluble factors, extracellular matrix, and apoptosis- associated signalling in MSCs co-culture contribute to hepatotrophic effect and HPc-induced potentiative effect. Co-transplantation with MSCs should improve therapeutic effects of HCT by enhancing survival and metabolism of co-transplanted hepatocytes.

The intestinal IgA response has features that are different to those of the systemic humoral response, which is dominated by IgG. Although the IgA response, like the IgG response, includes an antigen specific component, it is also associated with polyspecificity and autoimmunity. The profile of intestinal immunoglobulins changes in inflammatory bowel disease (IBD) where there is a disproportionate increase in IgG production and in ulcerative colitis (UC), this includes the production of autoantibodies. In this thesis, two immunoregulatory T cell subsets that could influence the intestinal B cell response have been studied; T follicular helper cells (TFH) and regulatory T cells (Treg). Results in chapter 3 show that there is a higher density of TFH in gut associated lymphoid tissue (GALT) compared to peripheral lymphoid tissue due to a higher density of CD57- TFH- The expression of cytokines and CD40L was almost comparable between CD57+ and CD57- TFH- However, culturing experiments suggest that CD57-TFH may develop into CD57+ TFH and there is a constant turnover of TFH in the gut. Experiments in chapter 4 attempted to seek evidence for a developmental relationship between TFH and Treg by analysis of T cell receptor sequences. No evidence of plasticity between these subsets was observed. Experiments in chapter 5 set out to characterise TFH in IBD. In the appendix of UC patients, nearly all TFH were CD57+ and at a high density within germinal centres (GC). This thesis concludes that TFH are more phenotypically diverse and denser in GALT compared to peripheral lymphoid tissue. This may reduce the threshold for GC B cell survival in the gut permitting the propagation of plasma cells that secrete polyspecific and autoreactive IgA. TFH are denser still in the small GC in UC appendix. This may be relevant to the production of autoantibodies and disease pathogenesis.

The following review discusses some of the cognitive and functional problems in liver disease. Some medical literature is included which is consistent with difficulties reported by patients. Prevalence, possible causes, and types of liver disease are reviewed, including an outline of various complications associated with the disease. Hepatic encephalopathy (HE) is one such complication and a general background to this is given. It has been suggested that subgroups of patients with liver disease have mild cognitive deficits and demonstrate poorer performances on neuropsychological tests compared with matched controls. This has been termed minimal hepatic encephalopathy (MHE), a syndrome that occurs in patients with liver disease without overt symptoms of hepatic encephalopathy. The full spectrum of cognitive impairment in MHE is unknown (Collie, 2005). Research has attempted to understand the profile of cognitive deficits in patients with liver disease. Studies have investigated various areas of functioning (e.g. psychomotor skills, attention and memory) by neuropsychological testing. The main studies are presented in the review. Some of the limitations of the minimal hepatic encephalopathy hypothesis are discussed. There is some debate about possible causes of observed cognitive deficits and various psychological models including health (coping and quality of life) and clinical (mood issues) are proposed. Further research and clinical implications are also discussed.

FTO encodes a nucleic acid demethylase with substrate preference towards N6-methyladenosisne (m6A). Variation in the FTO gene confers the single greatest genetic risk for common obesity amongst all obesity susceptibility loci identified to date. The mechanisms via which FTO variation promotes adiposity remain unknown. Increased hunger, increased energy intake, and enhanced preference for palatable, calorically-rich foods constitute a common denominator between ghrelin signalling and FTO perturbation. Hence, we hypothesized that altered ghrelin levels mediate the FTO-related obesogenic feeding patterns, and we undertook human, rodent and cellular studies to test our hypothesis. Here we show that Caucasian males, homozygous for the high-risk allele A of FTO rs9939609 exhibit increased BMI and adiposity compared to homozygotes for the protective variant T, even within the normal BMI-spectrum; before the development of overweight and obesity. In two independent cohorts we show that normal-BMI AA subjects exhibit attenuated hunger suppression post-meal and aberrant ghrelin profile compared to adiposity-matched TT controls; with the ghrelin phenotype of normal-BMI AA subjects reminiscing ghrelin changes seen in human obesity. Moreover, we show that global Fto deletion in mice results in alterations in circulating ghrelin levels. Utilizing reward-trait assessment questionnaires we demonstrate weight-independent increases in reward responsiveness in AA subjects. Using fMRI we show that FTO genotype weight-independently alters neuronal responsiveness to visual food cues within homeostatic and reward appetitive circuitries. Furthermore, we report that peripheral blood cells from healthy normal-weight Caucasian AA males have increased FTO and GHRL mRNA relative abundance, with decreased m6A GHRL mRNA residues compared to TT controls. Finally, we show that overexpression of FTO in HeLa cells increases abundance of pre-proghrelin, pro-ghrelin and ghrelin peptides, as well as GOAT protein in the cell lysates. Collectively, these data reveal a novel link between FTO and ghrelin, and implicate ghrelin deregulation to the adipogenic effects of FTO.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) are important causes of liver inflammation and fibrosis. More than 500 million people are persistently infected worldwide and consequently are at increased risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma. Both viruses share some similarities however, disease severity varies greatly from subject to subject and the variation in clinical course is a hallmark of viral hepatitis. The failure of the immune system to prevent persistent infection and chronic necroinflammatory change is central to understanding the disease caused by these human viruses. The Introduction to this Thesis reviews our current understanding of the immunopathogenesis and natural history of both viruses in the context of the clinical and scientific challenge that chronic viral hepatitis represents. The body of this work was to focus on immunological events which might shape and possibly dictate disease outcome in viral hepatitis. In this thesis I set out to investigate areas of cellular immunity which may contribute to pathology or protection in HBV and HCV. I have focused on the impact of cross-reactive T cells activated by HCV peptides and report that this may constitute a common occurrence in human viral infections. I explore the role of the NK cell receptor, NKG2D, in the modulation of CD8+T cell response in type B and C chronic viral hepatitis and report the impact of this novel receptor in human liver disease for the first time.

Helicobacter pylori exhibits tropism for the human stomach causing a spectrum of complications ranging from gastritis to gastric cancer in susceptible individuals. The mechanism(s) that allow the bacteria to persist and cause disease are unfolding. p-defensins are a family of endogenous, epithelial anti-microbial peptides that engage in host defense most prominently at mucosal surfaces. We and others have previously shown that human p-defensin (hBD)-2 and -3 are potent bactericidal agents against H. pylori. At present the identity of signalling pathways involved in host-bacterial cross talk leading to modulation of host antimicrobial immunity are unknown. The present study firstly investigated the potential role of bacterial virulence factors in mediating human p-defensin gene expression during H. pylori infection. AGS gastric epithelial cells were infected with cytotoxic H. pylori strains (60190, 84-183) and isogenic mutant strains (cagA-, cagE-, vacA- and CagPAl-). Human p-defensin (hBD2 & -3) gene expression quantified by RT-PCR and p-defensin transcriptional regulation was followed by transient transfection studies utilising hBD2 and -3 promoter luciferase constructs. We found hBD2 induction was dependent upon an intact cagPAI and minimal involvement was observed for the bacterial virulence factors CagA and VacA in modulating P-defensin expression. We sought to investigate the bacterial component responsible for instigating epithelial innate immune responses. Through the use of siRNA for NODI we determined a role for NODI-dependent NF-kB activation in mediating hBD2 but not hBD3 expression. Experiments utilising specific inhibitors of the MAP Kinase pathways directed us to delineate the role of each pathway in modulating p-defensin expression by the activation of stably transfected conditional MAP Kinase mutants. These studies revealed critical involvement of ERK pathway in the regulation of hBD3 but not hBD2 gene expression. Signalling upstream of ERK was explored and revealed EGFR as the host receptor responsible for detection and initiation of hBD3 gene and peptide production. Our studies demonstrated a crucial role for NODI in H. /Ty/ort-mediated hBD2 but not hBD3 expression and implicate EGFR transactivation in mediating hBD3 but not hBD2 expression, thus indicating two distinct regulatory mechanisms at play during innate immune host response to H. pylori infection.

Obesity is a fast growing medical issue worldwide and is one of the leading causes of mortality. There is evidence to suggest that various forms of dietary fibre may be used in weight management and for their general health benefits. There is evidence to suggest that the addition of alginate to a food or beverage has the potential to alter the digestion process and potentially reduce the activity of digestive enzymes. Data from this laboratory have demonstrated that alginate possesses the ability to inhibit pancreatic lipase in-vitro. Pancreatic lipase is secreted in the small intestine and hydrolyses fat so that it can be absorbed in the digestive system. If the activity of pancreatic lipase can be reduced then the amount of fat absorbed will be reduced. A modified Periodic Acid Schiffs (PAS) assay is an effective, repeatable and simple method for quantifying alginate in solution. Furthermore with any potential weight loss treatment the release rate must be determined. The model gut system developed in this laboratory is able to digest the alginate enriched bread and the modified PAS assay system is able to quantify the release rate of the alginate, with between 12-20% released in the gastric phase and 80-88% released in the small intestinal phase. The data here also confirms that the bread is protecting the alginate during the cooking process, and that the alginate retains its inhibition properties despite the cooking and digestion process. The model gut is an effective model for digesting fat substrates and foods high in fat such as butter and olive oil. Alginate enriched bread has the ability to inhibit fat substrates glyceryl trioleate, trioctanoate, tributyrate, and foods butter and olive oil 62, 39 and 32, 95 and 78% respectively after 180 minutes in the model gut. Alginate enriched bread is able to modulate fat digestion in ileostomy subjects following ingestion. This was evident by a significant increase in fat content of the effluent fluid. There was an increase in fat content of 81g and 112g when subjects consumed alginate bread when compared with control bread at 240-270-300 minutes and when total fat was combined for all time points respectively. There was also an increase of 292g in wet effluent weight for alginate bread when compared with the control bread. In addition a significant relationship between the wet effluent weight and fat content of the effluent was reported. The alginate enriched bread had no problems with palatability or adverse side effects associated with fat digestion and suggest that alginate enriched bread may be a suitable method for the delivery of alginate into a normal diet. The data in this thesis suggests that alginate enriched products are able to attenuate the digestion of fat and therefore may be used in weight management and in the treatment of obesity, however further longitudinal clinical research is required in a healthy population before any health claims can be made.