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Subject: search.filters.subject.Gastrointestinal mucosa

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    Effect of Faecalibacterium prausnitzii on intestinal barrier function and immune homeostasis : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science, Massey University, Manawatū, New Zealand
    (Massey University, 2017) Maier, Eva
    Various gastrointestinal (GI) diseases, for example inflammatory bowel disease, are linked to impaired barrier function, chronic inflammation and dysbiosis of the resident microbiota. Faecalibacterium prausnitzii, an abundant obligate anaerobe of the healthy human microbiota, has reduced abundance in the GI tract of people with these diseases, and has been suggested to exert beneficial effects. Only a few studies have investigated its mechanisms of action, partly due to the difficulty of co-culturing live obligate anaerobes with oxygen-requiring human cells. The novel apical anaerobic co-culture model used in this study allows this co-culture through the separation of anaerobic and aerobic compartments. This model was used to investigate the effects of live F. prausnitzii (strains A2-165, ATCC 27768 and HTF-F) on intestinal barrier integrity, measured by transepithelial electrical resistance (TEER) of the intestinal epithelial cell line Caco-2, and on immune homeostasis, specifically on Toll-like receptor (TLR) activation. Method development was required to adapt these assays to the novel model and to optimise the growth of F. prausnitzii co-cultured with Caco-2 cells and TLR-expressing cell lines while maintaining their viabilities. Firstly, the optimised co-culture conditions were used to determine the effect of the three F. prausnitzii strains on barrier integrity of healthy and tumour necrosis factor alpha (TNF-α) treated Caco-2 cells. Live and growing F. prausnitzii did not alter the TEER across healthy Caco-2 cells. However, under TNF-α mediated inflammatory conditions, dead F. prausnitzii decreased TEER, whereas live bacteria maintained TEER. Secondly, the TLR activation assay was adapted to be carried out in the novel model. Using the adapted assay conditions it was determined that live F. prausnitzii induced greater TLR2 and TLR2/6 activation than dead F. prausnitzii. Collectively, these results indicate greater immuno-stimulatory effects of live F. prausnitzii, via TLR2 activation, and this effect is potentially linked to its barrier maintaining properties, because previous research showed enhancement of barrier integrity induced by TLR2 signalling. This new knowledge contributes to the understanding of how F. prausnitzii may maintain immune homeostasis in the GI tract. Unravelling the biological mechanisms used by prevalent species of the human microbiota, such as F. prausnitzii, will ultimately allow better comprehension of microbial regulation of GI function.
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    The aspirin augmented standardized lactulose mannitol test as a measure of the 'health' of the gastrointestinal tract : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 2015) Sequeira, Ivana Roosevelt
    In this thesis, I studied the ‘classical’ lactulose mannitol test for intestinal permeability that has been used to measure the integrity of the intestinal mucosa and thus to provide an index of recovery from inflammatory bowel disease (IBD) and from autoimmune diseases such as coeliac disease. Perusal of the literature indicates that the protocol for the test has not been standardized and a variety of different test protocols have been used. Hence there are differences in the duration of urinary sampling, the doses of the two test probes, the volumes of fluid consumed during the test and the administration of the test during the fasted or fed state. There is therefore a need for a standardized test. The bulk of the research conducted in this thesis was to develop an optimal protocol with a standardized osmolarity (720 osmol l-1) for the test solution that contained 10 g of lactulose and 5 g of mannitol dissolved in 100 ml of water. Similarly the total fluid intake was standardized to 700 ml. The volumes of fluid consumed over the experimental period were also standardized in order to control for any osmolar effects of the test drink and to hydrate the subjects sufficiently to enable them to produce half-hourly urine samples of a reasonable volume. The rates of excretion and the timings of the peaks in the excretion of mannitol and lactulose were found to vary over time in healthy subjects. Hence the rate of mannitol excretion peaked during the first two hrs whilst the rate of lactulose excretion peaked at four hrs. The correlation between urinary excretion with intestinal transit times were confirmed using a wireless motility capsule. The work with the wireless motility capsule indicated that the probe sugars were in the small intestine from 2½ - 4 hrs and in the proximal colon from 4½ - 6 hrs following dosage with the test solution. Hence a sample collected during the 2½ - 4 hr period is best for assessing permeability of the small intestinal mucosa in healthy subjects. The wireless motility capsule also confirmed that the standardized dose of the lactulose mannitol did not influence gastric transit time or that through the small intestine and large intestine. These findings confirmed that the standardized test was determining absorption during transit of the test sugars through the small and the large intestine. The effect of co-dosage with 600 mg of aspirin in the standardized test was then examined as a means of assessing the effect of a reproducible noxious stimulus on the absorption of the sugar probes. This agent augmented small intestinal permeability to lactulose and decreased its permeability to mannitol. Furthermore dosage with aspirin amplified the effect of a pre-existing adverse stimulus such as smoking. Hence the aspirin augmented test could conceivably be used to ‘unearth’ sub-clinical inflammation. Further work explored the effect of an antioxidant, ascorbic acid, on mucosal permeability. The results showed that, rather than mitigating the adverse effects of aspirin, ascorbic acid augmented intestinal permeability. In summary the work in this thesis has enabled the development of a standardized test that optimizes the ability of the lactulose mannitol test to detect clinical disorders of absorption. Further, augmenting the test with a single dose of aspirin may be useful as an index of gut health or robustness.
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    The interaction of probiotic bacteria and an oligosaccharide-enriched fraction from goat whey on in vitro intestinal barrier function and mucin production : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Manawatu, New Zealand
    (Massey University, 2014) Barnett, Alicia
    Multiple interactions occur in the human large intestine between the host, the intestinal microbiota and fermentable carbohydrates which transit relatively intact through the small intestine. A major site at which many of these interactions occur is the intestinal epithelium, which is formed from a single layer of epithelial cells. The cellular composition of the epithelial layer in the human small and large intestine varies in respect to the numbers of absorptive enterocytes and mucus-secreting goblet cells. For the human intestine the proportion of goblet cells among epithelial cell types is thought to increase from the duodenum (4%) to the distal colon (16-24%). Epithelial cell co-culture models were developed containing absorptive enterocytes (Caco-2 cells) and mucus-secreting goblet cells (HT29-MTX cells) that more closely simulate the cell proportions found in the small (90:10) and large intestine (75:25). Trans-epithelial electrical resistance (TEER) of the co-cultures was more similar to reported values of ex vivo intestinal tissue of human small and large intestine than either of the two mono-cultures. Additionally, the mucus layer thickness present at the apical surface of 75:25 co-cultures (cellular composition representative of the large intestine) was similar to the reported thickness of the inner mucus layer of human large intestine. Introduction of an oligosaccharide-enriched fraction (OEF) from goat whey to the epithelial co-culture models was shown to modulate barrier integrity as measured by TEER, in a dose-dependent manner. Oligosaccharides (1 mg/mL) increased TEER and mucin gene/protein expression of epithelial co-cultures. Finally, the interaction between probiotic bacteria and the OEF and their individual or combined effects on intestinal epithelial barrier integrity and mucin gene/protein expression was investigated. The OEF supported the growth of selected probiotic strains, and enhanced the adhesion of defined strains to the epithelial co-cultures. When in combination with the OEF, Lactobacillus plantarum 299v enhanced TEER and mucin gene/protein expression, the increase of which was greater than that for either component alone. This suggests that an interaction between Lactobacillus plantarum 299v and the OEF exists which enhances barrier integrity through increased TEER and mucin gene/protein expression, all of which are essential components of the intestinal barrier. The research presented in this dissertation has indicated that in vitro epithelial co-cultures can be used as a model to improve our understanding of the mechanisms through which probiotic bacteria/food components and intestinal epithelial cells interact, and these key findings will assist in the development of strategies to improve intestinal barrier function using novel dietary components.