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Colostrum is the first milk produced after birth, is particularly rich in antibodies, antibacterial proteins and bioactive substances. Recent studies have suggested that colostrum might be useful in treating gut damage. The value of colostral preparations in patients taking long-term non -steroidal anti- inflammatory drugs (NSAIDs) was examined by studying the influence of colostrum on dyspepsia symptoms, intestinal permeability and faecal calprotectin as markers of gut injury. ANOVA of symptom scores showed that both colostrum and milk groups had a small but significant (p<O.Ol) reduction over time. However, there was no significant effect of treatment (p = 0.28). Initial intestinal permeability values were normal or only mildly elevated (0.07-0.80). ANOVA showed no significant effect of time (p = 0.66). Also initial faecal calprotectin levels of only 4 out of 12 subjects were elevated. Further analysis as to the effect of colostrum was not carried out because of incomplete stool collection by some of the subjects. Colostrum contains many factors that may have relevance for gut repair. The effect of nucleotides known to be present in colostrum and lipocalin 24p3, also present in colostrum, were studied using cell migration, proliferation assays and indomethacin gastric damage model. Nucleotide mixture caused a dose dependent increase in cell migration. Addition of individual nucleotides caused a significant increase in the rate of migration. Nucleotides had either no effect on cell proliferation or caused a growth inhibition. Subcutaneous administration of nucleotide mixture increased gastric damage. In contrast, oral administration of nu cl eo tides caused a dose related reduction in gastric injury. 24p3 caused cell migration in a dose dependent fashion but did not have an affect on cell proliferation. Administration of wild type 24p3 and mutant 24p3 (C98A) were both able to reduce the amount of gastric injury that occurred (both p < 0.01 vs. control).

Infectious intestinal disease (IID) causes significant morbidity and mortality worldwide. Detection and identification of the causative microorganism is important to allow for effective patient care, outbreak detection and timely public health interventions. which may reduce the number of cases and deaths. Routine detection of bacterial pathogens in foods and faeces relies on culture-based methods and phenotypic identification. However, these methods arc laborious and take several days to achieve results. Also separate methods must be performed for each target pathogen, increasing labour further. PCR-based approaches offer a rapid altemative method. which maintains specific and sensitive detection.

Gastrointestinal parasites are one of the most prevalent infections known to infect mammals, estimated to affect over one billion people globally. It is well established that the generation of a type two response, characterised by CD4+ T cell secretion of interleukin (IL)-13, IL-4 and IL-9 will expel most gastrointestinal helminths. Expulsion is mediated by a variety of effector mechanisms including altered mucin production, intestinal mast cell activation, increased epithelial cell turnover and increased muscle contraction. Protective immunity can be generated over time; however, infections can persist to chronicity following the modulation of a protective type two response, such as the generation of a type one response. This response, characterised by IFN-γ and IL 12 secretion, can lead to long lived infections, placing huge morbidity upon infected individuals. The mechanisms leading to the generation of a type one or type two response are not fully understood and are of paramount importance if a vaccine to helminth parasites is to be developed. In addition, the dysregulation of these responses is thought to be the cause of allergy and autoimmunity and understanding the regulation of type two responses will also add to our understanding of these diseases.Numerous labs have recently reported roles for new innate cells, such as nuocytes, as sources of IL 13, a cytokine critical to worm expulsion. In addition, data from this lab have demonstrated that NK cell derived IL-13 is the cause of pathology during infection with Trichinella spiralis. It has also been shown that NK cells provide a supplementary source of IL-13 in Trichuris muris infected IL-4-/- mice. This thesis has used 2 strains of mice; the E4bp4-/- mouse and the Il13eGFP reporter mouse, in order to dissect the role of innate cells in the immune response to the related helminth parasites T. muris and T. spiralis. The transcription factor E4BP4 has been shown to control CD4+ T cell IL-5 and 13 secretion and the development of NK cells. Data presented in this thesis show that following a low dose infection, which usually persists to chronicity in WT mice, E4bp4-/- mice were able to expel the parasite following the generation of an elevated type two response. This effect was abrogated following CD4+ T cell depletion, indicative of an essential role for these cells in this response. It was also shown that E4BP4 ablation was sufficient to delay expulsion of a high dose of T. muris, indicative of a key role for E4BP4 in regulating type two responses to T. muris. Contrary to the hypothesis, it was demonstrated that NK cells did not play a major role in protective immunity to T. muris.In addition, the data presented here show, for the first time, the induction of nuocytes in the MLN following both T. muris and T. spiralis infection. Nuocytes made an early contribution to IL-13 secretion following T. spiralis infection; whereas low levels of Il13eGFP+ cells were observed only at day 18-21 post T. muris infection, with nuocytes making a less significant contribution to IL-13 levels. The levels of different cell lineages in the MLN post infection and their contribution to IL-13 expression was also mapped in detail.Finally, an analysis of the haematopoietic response in the bone marrow following T. muris infection was undertaken. The percentage and number of early progenitor Lineage-Sca-1+c-kit+ (LSK) cells in the bone marrow increased at day 21 post T. muris infection. LSK levels returned to normal after this time, indicative of an inflammation dependent response. Interestingly, E4bp4 ablation abrogated this effect, in line with the hypothesised role of IFN-γ in this expansion. Taken together, these data have added to our knowledge of the orchestration of immune responses to two intestinal helminth parasites; T. muris and T. spiralis.

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.

Background There have been many conflicting changes in the prevalence of the risk factors for upper gastrointestinal bleeding and therefore it is not clear what the current trends in mortality or incidence are, nor which factors are important in driving these trends. As populations in many countries are ageing with an increasing burden of co-morbidity, this thesis investigates whether the relationship between non gastrointestinal co-morbidity and upper gastrointestinal bleeding might be an explanation for current trends. I hypothesised that non gastrointestinal co-morbidity was responsible for a large proportion of bleeds in the population and the deaths that occur following a bleed. Methodology Large scale routine population based data records were used to assess the current incidence and mortality trends of upper gastrointestinal bleeding in England, as well as more in depth studies of predictors of its occurrence and subsequent mortality. The databases were examined and compared to external sources to assess their representativeness, and methods for defining cases in linked primary and secondary care were developed. The specific questions addressed in the studies were: 1. What are the current trends and variations in occurrence of upper gastrointestinal bleeding? Incidence rates and adjusted incidence rate ratios were calculated by quintiles of socioeconomic status, age group, sex, region, and calendar year. 2. Has there been an improvement in 30 day mortality following upper gastrointestinal bleeding? A nested case control study using Hospital Episodes Statistics from England 1999-2007 examined mortality trends by age, sex, co-morbidity and type of bleed. 3. Does non gastrointestinal co-morbidity predict upper gastrointestinal bleeding? A matched nested case control study used the linked Hospital Episodes Statistics and General Practice Research Database to examine non gastrointestinal co-morbidity as a risk factor adjusted for other known risk factors for bleeding. Sequential population attributable fractions were calculated to estimate what each risk factor contributed to the disease burden. 4. What are the excess causes of death following upper gastrointestinal bleeding? Causes of death by ICD 10 category were extracted following a bleed from the linked Office for National Statistics death register. Crude mortality rates and excess cumulative incidence functions were calculated; the latter adjusted for the competing risks between different causes of death. Results 1. A higher incidence of upper gastrointestinal bleeding was observed in the north of England, but this variation was dwarfed by the variation associated with deprivation. Areas of greater deprivation had 2-3 fold higher rates of hospitalisation for upper gastrointestinal bleeding than areas of less deprivation suggesting that strong modifiable risk factors exist. 2. Over the last decade there was a 20% improvement in 28 day mortality following upper gastrointestinal bleeding, and those admitted with bleeding were increasingly older and had more co-morbidity. 3. A combined measure of non gastrointestinal co-morbidity was a significant independent predictor of upper gastrointestinal bleeding and explained a greater proportion of the burden of bleeding (19%) than any other risk factor in the population, including medications such as aspirin and NSAIDs. 4. More than half the absolute excess risk of death was due to co-morbidity not related to the upper gastrointestinal tract. Conclusions Non gastrointestinal co-morbidity both strongly predicts an event of upper gastrointestinal bleeding, and is responsible for a large proportion of the subsequent long term mortality. The magnitude of the association in the population explains both why its incidence had not decreased, and why the improvements in mortality were observed irrespective of endoscopic management or bleed type. Furthermore a bleed can be an indicator for a re-assessment of the severity of co-existing non gastrointestinal morbidity.

Vibrio parahaemolyticus is a seafood-borne pathogenic bacterium which is a major cause of gastroenteritis worldwide. In the present study, the genetic relationships and population structure of isolates originating from clinical and seafood production sources in Thailand were investigated by multilocus sequence typing (MLST). Nucleotide sequence variation of virulence-related genes including haemolysin and TTSS1 genes among Thai and worldwide isolates was also analyzed. The outer membrane proteome of V. parahaemolyticus isolate RIMD2210633 was predicted using bioinformatic approaches, and the outer membrane proteomes of eight isolates from different sources and representing different MLST sequence type characterized using proteomics. The 101 Thai V. parahaemolyticus isolates examined were recovered from clinical samples (n=15), healthy human carriers (n=18), various fresh seafood (n=18), frozen shrimps (n=16), fresh-farmed shrimp tissue (n=18) and shrimp-farm water (n=16). Phylogenetic analysis revealed a high degree of genetic diversity within the V. parahaemolyticus population, although isolates recovered from clinical samples, farmed shrimp and water samples represented five distinct clusters. The majority of clinical isolates were resolved into two genetic clusters and none of these isolates were found to share sequence types (STs) with strains isolated from human carriers, seafood, or water. Similarly, STs representing human carrier isolates differed from those of clinical, seafood and water isolates. The limited genetic diversity of the clinical isolates suggested non-random selection for pathogenic strains, but the absence of such strains in local seafood raises questions about the likely source of infection. Extensive serotypic diversity occurred among isolates representing the same STs and recovered from the same source at the same time point. Furthermore, evidence of interspecies horizontal gene transfer and intragenic recombination was observed at the recA locus in a large proportion of isolates; this has a substantial effect on the apparent phylogenetic relationships of the isolates. Notably, the majority of these recombinational exchanges occurred among clinical and carrier isolates, suggesting that the human intestinal tract is serving as a reservoir that is driving evolutionary change and leading to the emergence of new, potentially pathogenic strains. MLST was also applied to study genetic relationships between V. parahaemolyticus isolates from Thailand (n=101) and those from European countries (n=9). With the exception of the pandemic ST3 which was resolved from two isolates from Thai human carriers, two clinical isolates from England and a clinical isolate from Norway, none of the other European isolates examined in this study shared the same ST with the Thai isolates. This study demonstrated that Thai human carrier isolates are capable of harbouring virulence-related genes including the haemolysin-encoding genes tdhA, tdhS, trh1 and trh2, and the TTSS1-related genes vcrD1, vscC2 and VP1680, that are present in clinical isolates. In particular, the Thai human carrier isolate VP132 shared identical TTSS1-related gene fragments with the pandemic V. parahaemolyticus serotype O3:K6 (RIMD2210633) and related strains (AQ3810, AQ4037, Peru466, AN5034 and K5030) of worldwide distribution. A total of 117 outer membrane proteins (OMPs) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633. A total 73 OMPs proteins were identified from eight V. parahaemolyticus isolates recovered from clinical samples, human carriers, oyster, shrimp tissue and water in Thailand. Of the 117 predicted OMPS, 32 were identified in eight strains by proteomic analysis. OmpU, a non-specific porin protein, represents the most abundantly expressed protein in all eight isolates. OMPs involved in TTSSs (YscW, YscJ, YscC, PopN and VscC2) and iron uptake (IrgA, putative 83 Da decaheme outer membrane cytochrome C, PvuA1, PvuA2, LutA, FhuE, HutA and putative-regulated protein B) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633, but were not recovered from any of the eight Thai isolates. The absence of TTSS and iron uptake related OMPs in the eight representative strains that were grown under in vitro conditions may suggest an important requirement for in vivo growth conditions to induce expression of important virulence factor-related OMPs in V. parahaemolyticus. There was no clear association between OMP profile and the source of isolation, ST or serotype. However, a high degree of variation of OMP profiles was observed in isolates from different sources as well as in the isolates representing the same ST. This study demonstrated the usefulness of a multidisciplinary approach that includes MLST, virulence-related gene DNA sequence analysis, bioinformatic prediction and gel-based proteomic analyses for the study of molecular evolutionary relationships and the epidemiology of V. parahaemolyticus isolates from clinical and seafood production sources. The outcomes of this study highlight the role of human carriers as a reservoir of potentially pathogenic V. parahaemolyticus and this should be considered as one of the possible contamination sources in the surveillance of seafood safety.

The utility of breath trace compounds as bio-markers for various physiological conditions has long been exploited for the diagnosis of various diseases. Urea breath tests have been adopted as the gold standard for the detection of Helicobacter pylori which is a primary cause for acute gastritis and peptic ulcers. In these tests, small changes in the ratio of stable CO2 isotopomers, 13CO2 and 12CO2, present in exhaled breath are measured precisely and this is conventionally done by using an Isotope Ratio Mass Spectrometer. However, the huge cost and complexity involved in operating these instruments has restricted their widespread use. A viable and low cost alternative is offered by instruments employing non-dispersive infrared absorption techniques. The feasibility of such an instrument has been explored in this work. The instrument presented here is a two channel isotope ratiometer that performs whole band integrated absorption measurements. Detection is based on a novel feedback mech- anism whereby an imbalance in the channel absorptions causes the pathlength along one of the channels to be altered in order to bring the system back to balance. This change in ratio of pathlengths is directly related to the change in the 13CO2/12CO2 concentration. Signffcant amount of work has already been done to investigate the effects of interferences from coincident absorption bands and other spectral effects that can lead to spurious results. A comprehensive description of the overall system design, development and performance evaluation of the first prototype instrument has been presented here. This involved significant computer modeling and simulations and the results were verified experimentally. These results provided sufficient evidence to suggest the feasibility of such an instrument as a diagnostic tool. It was also concluded that some design improvements were required to circumvent issues related to pathlength variation and a list of recommendations has been provided for this purpose. On the basis of the results obtained as part of this research endeavour, it was concluded that the non-dispersive instrument design presented here can form the basis for a low cost commercial alternative for performing carbon isotope ratio breath tests.

Les principaux virus entériques à l’origine de toxi-infections alimentaires collectives sont les norovirus génogroupes I et II (NoV GI, NoV GII) et le virus de l’hépatite A (VHA) responsables respectivement de gastro-entérites et d’hépatites. Ces virus sont transmissibles par la voie féco-orale directe ou via l’ingestion d’eaux ou d’aliments consommés crus ou peu cuits (coquillages, fruits et légumes). Le niveau de contamination virale des aliments souvent faible nécessite d’utiliser des méthodes de détection très sensibles. La plupart des virus entériques étant non cultivable, ces méthodes reposent sur la détection / quantification des génomes viraux par RT-qPCR ce qui ne permet pas de déterminer l’infectiosité des virus et limite l’appréciation du risque viral en santé publique. Les travaux de thèse visaient à proposer des méthodes moléculaires pour la détection, la quantification et le typage des virus entériques, à évaluer l’apport de nouvelles technologies moléculaires (comme la Digital RT-PCR (RT-dPCR) et la RT-PCR à haut débit) dans le cadre du diagnostic viral dans les aliments et enfin à développer des traitements précédant les réactions de RT-qPCR pour détecter des génomes issus de particules virales infectieuses. Une nouvelle technique d’extraction du VHA à partir de la laitue a été développée et évaluée équivalente à la technique de référence décrite dans les spécifications techniques publiées en 2013 (ISO/TS 15216-1 et 15216-2). Pour favoriser les études phylogénétiques dans le domaine alimentaire, 6 modèles moléculaires de RT-qPCR spécifiques des 6 sous-types humains du VHA (IA, IB, IIA, IIB, IIIA, IIIB) ont été développés et évalués pour le génotypage d’échantillons cliniques contaminés par le VHA. Ils peuvent être utiles pour tracer les sous-types du VHA dans des échantillons faiblement contaminés comme des matrices alimentaires, mais aussi permettre l'identification de co-infection de l'homme ou de souches de VHA recombinantes. La RT-dPCR en nanofluidique a été comparée à la RT-qPCR pour la quantification des génomes de NoV GI, NoV GII et VHA en présence de 2 contrôles de process (mengovirus et norovirus murin) dans des échantillons de laitues et d’eau embouteillée artificiellement contaminés. Un contrôle externe d’amplification a permis d’évaluer et de comparer l’inhibition des réactions de RT-qPCR et RT-dPCR. Les rendements d’extraction viraux se sont révélés significativement plus élevés après RT-dPCR qu’après RT-qPCR pour les NoV GI et mengovirus dans l'eau et pour les NoV GII et VHA dans les échantillons de laitue. De plus, les essais de RT-dPCR se sont avérés être plus tolérants à la présence de substances inhibitrices issues de laitues. La technologie qPCR en nanofluidique a également été utilisée afin de proposer une « puce » capable de détecter 20 virus entériques. Des limites de détection similaires ont été obtenues avec la qPCR et la dPCR. La qPCR nanofluidique a été trouvé moins sensible d’environ 1 à 3 log10 (du fait des faibles volumes (~nanolitre) d’échantillons analysés). Des prétraitements à base de monoazide +/- détergent à réaliser avant la RT-qPCR pour la détection de virus infectieux (VHA, rotavirus) ont été développés et évalués en réalisant des cinétiques d’inactivations thermiques. [...] Suite et fin du résumé dans la thèse. / The main enteric viruses that cause foodborne outbreaks are noroviruses genogroupe I and II (NoV GI and NoV GII) and hepatitis A virus (HAV), respectively responsible for gastroenteritis and hepatitis. They are mainly transmitted via the faecal-oral route either by person-to-person contact or by ingestion of contaminated water, raw and undercooked food, particularly shellfish, soft fruits and vegetables. Viral contamination level is often low and requires sensitive methods of detection. As most enteric viruses are not cultivable, these methods are based on viral genome detection and quantification by real time RT-PCR. Such an approach provides no information regarding virus infectivity and therefore limits viral risk assessment in public health. These thesis works aim to propose molecular methods for enteric viruses detection, quantification and typing, also to evaluate new molecular technologies contribution (as Digital PCR and PCR high throughput) for food viral diagnosis and finally to develop treatments combined with RT-qPCR to only detect genomes from infectious viral particles. A new HAV extraction from lettuce method was developed and assessed as similar to the reference method which is described in the technical specifications published in 2013 (ISO/TS 15216-1; ISO/TS 15216-2). In order to facilitate phylogenetic analysis in food microbiology, six subtype-specific RT-qPCR assays for human HAV (HAV IA, IB, IIA, IIB, IIIA, IIIB) were developed and evaluated by testing HAV contaminated clinical samples genotyping. These assays may be particularly useful for accurately tracing HAV in low-level contaminated samples such as food matrices and moreover, to allow co-infection identification in human samples and/or HAV recombinant strains. Nanofluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for NoV GI, NoV GII, and HAV genomes quantification, in presence of two process controls (mengovirus and murine norovirus) in artificially contaminated bottled water and lettuce samples. External amplification control allowed evaluating and comparing RT-qPCR and RT-dPCR assays inhibitions. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. Nanofluidic PCR Array (PCR Array) has also been used in order to propose an array able to simultaneously detect 20 enteric viruses. Similar detection limits were obtained with qPCR and dPCR but PCR Array was found less sensitive of 1 to 3 log10 (due to the weak volumes (nanolitre) of analyzed samples). Pretreatments based on the use of monoazides +/- surfactant and to do before RT-qPCR were developed for discriminating between infectious and non-infectious particles of HAV and rotavirus. They have been evaluated with thermal inactivation kinetic curves. Last and final summary in the thesis.