Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.

Novel insights into the inflammatory basis of gastrointestinal disease

It has become clear that inflammation is beneficial to man, there are situations though that the inflammatory response causes damage to the host that is harmful to health. When the inflammatory response fails or is too strong, the health of the host is damaged and disease can occur. The implication of intestinal disease caused by an ineffective immune response is of great social and economic burden to society. The overarching purpose of this thesis is to assess inflammatory signalling targets associated with immune mediated disorders such as IBD, IBS and inflammatory liver disease. By assessing these targets and modifying their function I hope to contribute and expand further the pre-existing information on these disorders and improve the therapeutic interventions available in these debilitating conditions. I will assess the role of inflammation in disorders of the GI tract and liver IBD, IBS, hepatic inflammatory injury and furthermore, I will use pharmaceutical agents to activate and suppress components of the immune system. I will examine the inflammatory response in experimental models of disease for IBD and liver injury, I will attempt to alter these pathways using pharmaceutical intervention to delineate the disease causing mechanism that may lead to clinically relevant therapeutic interventions. In regards to IBS, I will attempt to improve the existing knowledge that exists in relation to the pathogenesis of this functional bowel disorder. I will attempt to define a mechanism by which the low grade mucosal inflammation that has been demonstrated by others arises and what this inflammation is induced by. The overall aim of this thesis is to attempt to further understand the mechanisms behind GI and liver disease. Looking at the inflammatory response in these specific conditions and how they can be altered may lead to exciting new therapies for inflammatory conditions in the gastrointestinal tract.

CALR mutation profile in Irish patients with myeloproliferative neoplasms

Insertion and/or deletion mutations of the CALR gene have recently been demonstrated to be the second most common driver mutations in the myeloproliferative neoplasms (MPNs) of essential thrombocythemia (ET) and primary myelofibrosis (PMF). Given the diagnostic and emerging prognostic significance of these mutations, in addition to the geographical heterogeneity reported, the incidence of CALR mutations was determined in an Irish cohort of patients with MPNs with a view to incorporate this analysis into a prospective screening program. A series of 202 patients with known or suspected ET and PMF were screened for the presence of CALR mutations. CALR mutations were detected in 58 patients. Type 1 and Type 1-like deletion mutations were the most common (n = 40) followed by Type 2 and Type 2-like insertion mutations (n = 17). The CALR mutation profile in Irish ET and PMF patients appears similar to that in other European populations. Establishment of this mutational profile allows the introduction of a rational, molecular diagnostic algorithm in cases of suspected ET and PMF that will improve clinical management.

Tryptophan degradation in irritable bowel syndrome: evidence of indoleamine 2,3-dioxygenase activation in a male cohort

Background: Irritable bowel syndrome (IBS) is a common disorder that affects 10–15% of the population. Although characterised by a lack of reliable biological markers, the disease state is increasingly viewed as a disorder of the brain-gut axis. In particular, accumulating evidence points to the involvement of both the central and peripheral serotonergic systems in disease symptomatology. Furthermore, altered tryptophan metabolism and indoleamine 2,3-dioxygenase (IDO) activity are hallmarks of many stress-related disorders. The kynurenine pathway of tryptophan degradation may serve to link these findings to the low level immune activation recently described in IBS. In this study, we investigated tryptophan degradation in a male IBS cohort (n = 10) and control subjects (n = 26). Methods: Plasma samples were obtained from patients and healthy controls. Tryptophan and its metabolites were measured by high performance liquid chromatography (HPLC) and neopterin, a sensitive marker of immune activation, was measured using a commercially available ELISA assay. Results: Both kynurenine levels and the kynurenine:tryptophan ratio were significantly increased in the IBS cohort compared with healthy controls. Neopterin was also increased in the IBS subjects and the concentration of the neuroprotective metabolite kynurenic acid was decreased, as was the kynurenic acid:kynurenine ratio. Conclusion: These findings suggest that the activity of IDO, the immunoresponsive enzyme which is responsible for the degradation of tryptophan along this pathway, is enhanced in IBS patients relative to controls. This study provides novel evidence for an immune-mediated degradation of tryptophan in a male IBS population and identifies the kynurenine pathway as a potential source of biomarkers in this debilitating condition.

Genome-scale analyses of health-promoting bacteria: probiogenomics

The human body is colonized by an enormous population of bacteria (microbiota) that provides the host with coding capacity and metabolic activities. Among the human gut microbiota are health-promoting indigenous species (probiotic bacteria) that are commonly consumed as live dietary supplements. Recent genomics-based studies (probiogenomics) are starting to provide insights into how probiotic bacteria sense and adapt to the gastrointestinal tract environment. In this Review, we discuss the application of probiogenomics in the elucidation of the molecular basis of probiosis using the well-recognized model probiotic bacteria genera Bifidobacterium and Lactobacillus as examples.

Molecular analysis of virulence mechanisms associated with adherent-invasive Escherichia coli (AIEC)

Crohn's Disease (CD) is a chronic inflammatory bowel disease of unknown etiology. Recent work has shown that a new pathotype of Escherichia coli, Adherent Invasive E. coli (AIEC) may be associated with CD. AIEC has been shown to adhere to and invade epithelial cells and to replicate within macrophages (together this is called the AIEC phenotype). In this thesis, the AIEC phenotype of 84 E. coli strains were determined in order to identify the prevalence of this phenotype within the E. coli genus. This study showed that a significant proportion of E. coli strains (approx. 5%) are capable of adhering to and invading epithelial cells and undergoing intramacrophage replication. Moreover, the results presented in this study indicate a correlation between survival in macrophage and resistance to grazing by amoeba supporting the coincidental evolution hypothesis that resistance to amoebae could be a driving force in the evolution of pathogenicity in some bacteria, such as AIEC. In addition, this study has identified an important regulatory role for the CpxA/R two component system (TCS) in the invasive abilities of AIEC HM605, a colonic mucosa-associated CD isolate. A mutation in cpxR was shown to be defective in the invasion of epithelial cells and this defect was shown to be independent of motility or the expression of Type 1 fimbriae, factors that have been shown to be involved in the invasion of another strain of AIEC, isolated from a patient with ileal CD, called LF82. The CpxA/R TCS responds to disturbances in the cell envelope and has been implicated in the virulence of a number of Gram negative pathogens. In this study it is shown that the CpxA/R TCS regulates the expression of a potentially novel invasin called SinH. SinH is found in a number of invasive strains of E. coli and Salmonella. Moreover work presented here shows that a critical mechanism underpinning AIEC persistence in macrophages is the repair of DNA bases damaged by macrophage oxidants. Together these findings provide evidence to suggest that AIEC are a diverse group of E. coli and possess diverse molecular mechanisms and virulence factors that contribute to the AIEC phenotype. In addition, AIEC may have gone through different evolutionary histories acquiring various molecular mechanisms ultimately culminating in the AIEC phenotype. The gastrointestinal (GI) tract harbors a diverse microbiota; most are symbiotic or commensal however some bacteria have the potential to cause disease (pathobiont). The work presented here provides evidence to support the model that AIEC are pathobionts. AIEC strains can be carried as commensals in healthy guts however, when the intestinal homeostasis is disrupted, such as in the compromised gut of CD patients, AIEC may behave as opportunistic pathogens and cause and/or contribute to disease by driving intestinal inflammation.

Investigation of growth and stress tolerance characteristics of Cronobacter spp.

Cronobacter spp. are opportunistic pathogens which can be isolated from a wide variety of foods and environments. They are Gram negative, motile, non-spore forming, peritrichous rods of the Enterobacteriaceae family. This food-borne pathogen is associated with the ingestion of contaminated infant milk formula (IMF), causing necrotizing enterocolitis, sepsis and meningitis in neonatal infants. The work presented in this thesis involved the investigation and characterisation of a bank of Cronobacter strains for their ability to tolerate physiologically relevant stress conditions that are commonly encountered in the gastrointestinal tract. While all strains were able to endure the suboptimal conditions tested, noteworthy variations were observed between strains. A collection of these strains were Lux-tagged to determine if their growth could be tracked in IMF by measuring bioluminescence. The resulting strains could be easily and reproducibly monitored in real time by measuring light emission. Following this a transposon mutagenesis library was created in one of the Lux-tagged strains of Cronobacter sakazakii. This library was screened for mutants with affected growth in milk. The majority of mutants identified were associated with amino acid metabolism. The final section of this thesis identified genes involved in the tolerance of C. sakazakii to the milk derived antimicrobial peptide, Lactoferricin B (Lfcin B). This was achieved by creating a transposon mutagenesis library in C. sakazakii and screening for mutants with increased susceptibility to Lfcin B. Overall this thesis demonstrates the variation between Cronobacter strains. It also identifies genes required for growth of the bacteria in milk, as well as genes needed for antimicrobial peptide tolerance.

Effects of synbiotics on cancer risk biomarkers

Colorectal cancer (CRC) is the fourth most common cause of death from cancer in the world and second most common (behind lung cancer) in developed countries. In recent years there has been much interest in the potential use of prebiotics, probiotics and synbiotics in the prevention and treatment of CRC. We have previously shown that synbiotic consumption in Azoxymethane treated rats modulates the immune system, influences the genotoxic potential of caecal contents and reduces the number of colonic tumours compared to control rats who did not receive the synbiotic. The aim of the current study was to identify biomarkers suitable for use as cancer risk markers and as intervention markers. A second aim was to determine the influence of synbiotic consumption on cancer risk biomarkers such as in vivo colonic mucosal proliferation and genotoxic damage along with examining the genotoxic, cytotoxic and tumour promoting potential of faecal water (FW). Synbiotic consumption altered the composition of the gastrointestinal flora and reduced in vivo genotoxic damage and the genotoxic potential of FW in cancer and polyp subjects. Synbiotic consumption also reduced the proliferative activity in the colonic mucosa in polyp subjects. In both cancer and polyp subjects gene expression in the colonic mucosa was modulated in synbiotic consuming subjects. In this and other studies the activity of natural killer cells, the level of PGE2 in FW, IL-12 production by PBMCs, genotoxic damage in the colonic mucosa and the tumour promoting activities of FW have been identified as possible biomarkers of cancer risk. Future large scale studies investigating these parameters in healthy and diseased individuals are needed to confirm the suitability of these markers in assessing cancer risk and the role of synbiotics in modulating them.

Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer

Despite studies demonstrating that inhibition of cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) has significant chemotherapeutic benefits in vitro and in vivo, inhibition of COX enzymes is associated with serious gastrointestinal and cardiovascular side effects, limiting the clinical utility of these drugs. PGE2 signals through four different receptors (EP1–EP4) and targeting individual receptor(s) may avoid these side effects, while retaining significant anticancer benefits. Here, we show that targeted inhibition of the EP1 receptor in the tumor cells and the tumor microenvironment resulted in the significant inhibition of tumor growth in vivo. Both dietary administration and direct injection of the EP1 receptor-specific antagonist, ONO-8713, effectively reduced the growth of established CT26 tumors in BALB/c mice, with suppression of the EP1 receptor in the tumor cells alone less effective in reducing tumor growth. This antitumor effect was associated with reduced Fas ligand expression and attenuated tumor-induced immune suppression. In particular, tumor infiltration by CD4+CD25+Foxp3+ regulatory T cells was decreased, whereas the cytotoxic activity of isolated splenocytes against CT26 cells was increased. F4/80+ macrophage infiltration was also decreased; however, there was no change in macrophage phenotype. These findings suggest that the EP1 receptor represents a potential target for the treatment of colon cancer.

An evaluation of reovirus as an effective therapeutic for cancer

Cancer is a global problem. Despite the significant advances made in recent years, a definitively effective therapeutic has yet to be developed. Oncolytic virology has fallen back into favour for the treatment of cancer with several viruses and viral vectors currently under investigation including vesicular stomatitis virus (VSV), adenovirus vectors and herpes simplex virus (HSV) vectors. Reovirus has an advantage over many viral vectors in that its wild-type form is non-pathogenic and will selectively infect transformed cells, particularly those mutated in the Ras pathway. These advantages make Reovirus an ideal candidate as a safe and non-toxic therapeutic. The aim of the first part of this study was to determine the effect, if any, of Reovirus on cell lines derived from cancers of the gastrointestinal tract. These cancers, particularly those of the oesophagus and stomach, have extremely poor prognoses and little improvement has been seen in survival of these patients in recent years. Reovirus as a single therapy showed promising results in cell lines of oesophageal, gastric and colorectal origin. Further study of partially resistant cell lines using a combination of Reovirus and conventional therapies, either chemotherapy or radiation, showed that a multi-modal approach to therapy is possible with Reovirus and no antagonism between Reovirus and other treatments was observed. The second part of this study focused on investigating a novel use of Reovirus in an in vivo setting. Cancer vaccination or the use of vaccines in cancer therapy is gaining momentum and success has been seen both in a prophylactic approach and a therapeutic approach. A cell-based Reovirus vaccine was used in both these approaches with encouraging success. When used as a prophylactic vaccine tumour development was subsequently inhibited even upon exposure to a tumorigenic dose of cells. The use of the cell-based Reovirus vaccine as a therapeutic for established tumours showed significant delay in tumour growth and a prolongation of survival in all models. This study has proven that Reovirus is an effective therapeutic in a range of cancers and the successful use of a cell-based Reovirus vaccine leads the way for new advancements in cancer immunotherapy.

The role of metabolism in the pathogenesis of adherent invasive Escherichia coli (AIEC)

Crohn’s disease (CD) is a chronic, relapsing inflammatory condition affecting the gastrointestinal tract of humans, of which there is currently no cure. The precise etiology of CD is unknown, although it has become widely accepted that it is a multifactorial disease which occurs as a result of an abnormal immune response to commensal enteric bacteria in a genetically susceptible host. Recent studies have shown that a new group of Escherichia coli, called Adherent Invasive Escherichia coli (AIEC) are present in the guts of CD patients at a higher frequency than in healthy subjects, suggesting that they may play a role in the initiation and/or maintenance of the inflammation associated with CD. Two phenotypes define an AIEC and differentiate them from other groups of E. coli. Firstly, AIEC can adhere to and invade epithelial cells; and secondly, they can replicate in macrophages. In this study, we use a strain of AIEC which has been isolated from the colonic mucosa of a CD patient, called HM605, to examine the relationship between AIEC and the macrophage. We show, using a systematic mutational approach, that while the Tricarboxylic acid (TCA) cycle, the glyoxylate pathway, the Entner-Doudoroff (ED) pathway, the Pentose Phosphate (PP) pathway and gluconeogenesis are dispensable for the intramacrophagic growth of HM605, glycolysis is an absolute requirement for the ability of this organism to replicate intracellularly. We also show that HM605 activates the inflammasome, a major driver of inflammation in mammals. Specifically, we show that macrophages infected with HM605 produce significantly higher levels of the pro-inflammatory cytokine IL-1β than macrophages infected with a non-AIEC strain, and we show by immunoblotting that this is due to cleavage of caspase-1. We also show that macrophages infected with HM605 exhibit significantly higher levels of pyroptosis, a form of inflammatory cell death, than macrophages infected with a non-AIEC strain. Therefore, AIEC strains are more pro-inflammatory than non-AIEC strains and this may have important consequences in terms of CD pathology. Moreover, we show that while inflammasome activation by HM605 is independent of intracellular bacterial replication, it is dependent on an active bacterial metabolism. Through the establishment of a genetic screen aimed at identifying mutants which activate the inflammasome to lower levels than the wild-type, we confirm our observation that bacterial metabolism is essential for successful inflammasome activation by HM605 and we also uncover new systems/structures that may be important for inflammasome activation, such as the BasS/BasR two-component system, a type VI secretion system and a K1 capsule. Finally, in this study, we also identify a putative small RNA in AIEC strain LF82, which may be involved in modulating the motility of this strain. Overall this works shows that, in the absence of specialised virulence factors, the ability of AIEC to metabolise within the host cell may be a key determinant of its pathogenesis.

The gut microbiota as a contributing factor to antipsychotic-induced weight gain and metabolic dysfunction

Schizophrenia represents one of the world’s most devastating illnesses due to its often lifelong course and debilitating nature. The treatment of schizophrenia has vastly improved over recent decades with the discovery of several antipsychotic compounds; however these drugs are not without adverse effects that must be addressed to maximize their therapeutic value. Newer, atypical, antipsychotics are associated with a compilation of serious metabolic side effects including weight gain, insulin resistance, fat deposition, glucose dysregulation and ensuing co-morbidities such as type II diabetes mellitus. The mechanisms underlying these side effects remain to be fully elucidated and adequate interventions are lacking. Further understanding of the factors that contribute these side effects is therefore required in order to develop effective adjunctive therapies and to potentially design antipsychotic drugs in the future with reduced impact on the metabolic health of patients. We investigated if the gut microbiota represented a novel mechanism contributing to the metabolic dysfunction associated with atypical antipsychotics. The gut microbiota comprises the bacteria that exist symbiotically within the gastrointestinal tract, and has been shown in recent years to be involved in several aspects of energy balance and metabolism. We have demonstrated that administration of certain antipsychotics in the rat results in an altered microbiota profile and, moreover, that the microbiota is required for the full scale of metabolic dysfunction to occur. We have further shown that specific antibiotics can attenuate certain aspects of olanzapine and risperidone–induced metabolic dysfunction, in particular fat deposition and adipose tissue inflammation. Mechanisms underlying this novel link appear to involve energy utilization via expression of lipogenic genes as well as reduced inflammatory tone. Taken together, these data indicate that the gut microbiota is an important factor involved in the myriad of metabolic complications associated with antipsychotic therapy. Furthermore, these data support the future investigation of microbial-based therapeutics for not only antipsychotic-induced weight gain but also for tackling the global obesity epidemic.

An exploration of the positive and negative relationships associated with the development of asthma and atopic disorders in primary school children in Cork

Childhood asthma, allergic rhinitis and eczema are complex heterogenic chronic inflammatory allergic disorders which constitute a major burden to children, their families. The prevalence of childhood allergic disorders is increasing worldwide and merely rudimentary understanding exists regarding causality, or the influence of the environment on disease expression. Phase Three of the International Study of Asthma and Allergy in Childhood (ISAAC) reported that Irish adolescents had the 4th highest eczema and rhinoconjunctivitis prevalence and 3rd highest asthma prevalence in the world. There are no ISAAC data pertaining to young Irish children. In 2002, Sturley reported a high prevalence of current asthma in Cork primary school children aged 6-9 years. This thesis comprises of three cross-sectional studies which examined the prevalence of and associations with childhood allergy and a quasi-retrospective cohort study which observed the natural history of allergy from 6-9 until 11-13 years. Although not part of ISAAC, data was attained by parentally completed ISAAC-based questionnaires, using the ISAAC protocol. The prevalence, natural history and risk factors of childhood allergy in Ireland, as described in this thesis, echo those in worldwide allergy research. The variations of prevalence in different populations worldwide and the recurring themes of associations between childhood allergy and microbial exposures, from farming environments and/or gastrointestinal infections, as shown in this thesis, strengthen the mounting evidence that microbial exposure on GALT may hold the key to the mechanisms of allergy development. In this regard, probiotics may be an area of particular interest in allergy modification. Although their effects in relation to allergy, have been investigated now for several years, our knowledge of their diversity, complex functions and interactions with gut microflora, remain rudimentary. Birth cohort studies which include genomic and microbiomic research are recommended in order to examine the underlying mechanisms and the natural course of allergic diseases.

Novel ingredients from brewers' spent grain - bioactivity in cell culture model systems and bioactivity retention in fortified food products

Functional food ingredients, with scientifically proven and validated bioactive effects, present an effective means of inferring physiological health benefits to consumers to reduce the risk of certain diseases. The search for novel bioactive compounds for incorporation into functional foods is particularly active, with brewers’ spent grain (BSG, a brewing industry co-product) representing a unique source of potentially bioactive compounds. The DNA protective, antioxidant and immunomodulatory effects of phenolic extracts from both pale (P1 - P4) and black (B1 – B4) BSG were examined. Black BSG extracts significantly (P < 0.05) protected against DNA damage induced by hydrogen peroxide (H2O2) and extracts with the highest total phenolic content (TPC) protected against 3-morpholinosydnonimine hydrochloride (SIN-1)-induced oxidative DNA damage, measured by the comet assay. Cellular antioxidant activity assays were used to measured antioxidant potential in the U937 cell line. Extracts P1 – P3 and B2 - B4 demonstrated significant (P < 0.05) antioxidant activity, measured by the superoxide dismutase (SOD) activity, catalase (CAT) activity and gluatathione (GSH) content assays. Phenolic extracts P2 and P3 from pale BSG possess anti-inflammatory activity measured in concanavalin-A (conA) stimulated Jurkat T cells by an enzyme-linked immunosorbent assay (ELISA); significantly (P < 0.05) reducing production of interleukin-2 (IL-2), interleukin-4 (IL-4, P2 only), interleukin-10 (IL-10) and interferon-γ (IFN-γ). Black BSG phenolic extracts did not exhibit anti-inflammatory effects in vitro. Hydroxycinnamic acids (HA) have previously been shown to be the phenolic acids present at highest concentration in BSG; therefore the HA profile of the phenolic extracts used in this research, the original barley (before brewing) and whole BSG was characterised and quantified using high performance liquid chromatography (HPLC). The concentration of HA present in the samples was in the order of ferulic acid (FA) > p-coumaric acid (p-CA) derivatives > FA derivatives > p-CA > caffeic acid (CA) > CA derivatives. Results suggested that brewing and roasting decreased the HA content. Protein hydrolysates from BSG were also screened for their antioxidant and anti-inflammatory potential. A total of 34 BSG protein samples were tested. Initial analyses of samples A – J found the protein samples did not exert DNA protective effects (except hydrolysate H) or antioxidant effects by the comet and SOD assays, respectively. Samples D, E, F and J selectively reduced IFN-γ production (P < 0.05) in Jurkat T cells, measured using enzyme linked immunosorbent assay (ELISA). Further testing of hydrolysates K – W, including fractionated hydrolysates with molecular weight < 3, < 5 and > 5 kDa, found that higher molecular weight (> 5 kDa) and unfractionated hydrolysates demonstrate greatest anti-inflammatory effects, while fractionated hydrolysates were also shown to have antioxidant activity, by the SOD activity assay. A commercially available yogurt drink (Actimel) and snack-bar and chocolate-drink formulations were fortified with the most bioactive phenolic and protein samples – P2, B2, W, W < 3 kDa, W < 5 kDa, W > 5 kDa. All fortified foods were subjected to a simulated gastrointestinal in vitro digestion procedure and bioactivity retention in the digestates was determined using the comet and ELISA assays. Yogurt fortified with B2 digestate significantly (P < 0.05) protected against H2O2-induced DNA damage in Caco-2 cells. Greatest immunomodulatory activity was demonstrated by the snack-bar formulation, significantly (P < 0.05) reducing IFN-γ production in con-A stimulated Jurkat T cells. Hydrolysate W significantly (P < 0.05) increased the IFN-γ reducing capacity of the snack-bar. Addition of fractionated hydrolysate W < 3 kDa and W < 5 kDa to yogurt also reduced IL-2 production to a greater extent than the unfortified yogurt (P < 0.05).

Investigations into selective metabolic aspects of bifidobacteria: carbohydrate metabolism, fatty acid biosynthesis and plasmid biology

The gastrointestinal tract (GIT) is a diverse ecosystem, and is colonised by a diverse array of bacteria, of which bifidobacteria are a significant component. Bifidobacteria are Gram-positive, saccharolytic, non-motile, non-sporulating, anaerobic, Y-shaped bacteria, which possess a high GC genome content. Certain bifidobacteria possess the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA) by a biochemical pathway that is hypothesised to be achieved via a linoleic isomerase. In Chapter two of this thesis it was found that the MCRA-specifying gene is not involved in CLA production in B. breve NCFB 2258, and that this gene specifies an oleate hydratase involved in the conversion of oleic acid into 10-hydroxystearic acid. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the colon. Key to the development of such novel prebiotics is to understand which carbohydrates support growth of bifidobacteria and how such carbohydrates are metabolised. In Chapter 3 of this thesis we describe the identification and characterisation of two neighbouring gene clusters involved in the metabolism of raffinose-containing carbohydrates (plus related carbohydrate melibiose) and melezitose by Bifidobacterium breve UCC2003. The fourth chapter of this thesis describes the analysis of transcriptional regulation of the raf and mel clusters. In the final experimental chapter two putative rep genes, designated repA7017 and repB7017, are identified on the megaplasmid pBb7017 of B. breve JCM 7017, the first bifidobacterial megaplasmid to be reported. One of these, repA7017, was subjected to an in-depth characterisation. The work described in this thesis has resulted in an improved understanding of bifidobacterial fatty acid and carbohydrate metabolism, Furthermore, attempts were made to develop novel genetic tools.