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    A comparative study of two Lactobacillus fermentum strains that show opposing effects on intestinal barrier integrity : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Manawatu, New Zealand
    (Massey University, 2014) Sengupta, Ranjita
    The Lactobacillus species can exert health promoting effects in the gastrointestinal tract (GIT) of humans through several mechanisms, which include pathogen inhibition, maintenance of microbial balance, immunomodulation and enhancement of the GIT barrier function. However, different strains of lactobacilli can evoke different responses in the host and not all strains of the same species can be considered health promoting. Two strains identified as Lactobacillus fermentum, namely AGR1485 and AGR1487, isolated from human oral cavities, exhibit opposing effects on intestinal barrier integrity. Studies have shown that AGR1485 maintains trans-epithelial electric resistance (TEER), a measure of GIT barrier integrity, across Caco-2 cell monolayers, while AGR1487 decreases TEER by 12 hours. This work aimed to test the hypotheses that the varying effects shown by these two L. fermentum strains are related to phenotypic differences between the two strains and are mediated by the interaction of secreted and/or cell-associated bacterial components with the GIT epithelial layer. Differences in metabolic events that occur during the various phases of growth in bacteria can impact not only cellular structure and secreted molecules, but may also affect their interactions with the intestinal epithelial cells. TEER assays were conducted to investigate if variation in bacterial secreted molecules and cell wall components associated with various phases of microbial growth can affect Caco-2 cell TEER. The effect on Caco-2 cell TEER caused by both strains was independent of bacterial growth phase. To test the hypothesis that it is the bacterial structural and/or secreted components that influence Caco-2 cell TEER, assays were conducted with live versus UV-killed bacteria on Caco-2 cells. Results showed that for both strains of L. fermentum, dead bacteria have similar effects on Caco-2 cell TEER as live bacteria, implying that direct bacterial contact with Caco-2 cells is necessary for the effects. Analogous to TEER assays, live AGR1487 increased mannitol permeability while UV-killed AGR1487 did not, implying that AGR1487 uses both cell surface structures and/or metabolites through distinct mechanisms to modulate host barrier properties. Subsequent experiments conducted using secreted metabolites from bacteria, Caco-2 cells and bacteria-Caco-2 cell interactions indicated that they have no effect on Caco-2 cell TEER, strengthening the assumption that bacterial cell surface-associated components are involved in mediating these effects. The bacterial cells were subjected to ultrasonication followed by ultracentrifugation to isolate the bacterial cell wall extract. TEER assays conducted with the cell wall extracts from both strains resulted in decreasing Caco-2 cell TEER, although at high concentrations, further strengthening the role of bacterial cell surface components in influencing barrier integrity of the Caco-2 cells. To narrow down proteinaceous components of the cell wall extracts from both the strains that influence Caco-2 cell TEER, they were fractionated through size exclusion chromatography and the effects of these cell wall fractions on Caco-2 cell TEER were studied. One fraction of AGR1487 CW appeared to decrease Caco-2 cell TEER, although at a high concentration. However, the results could not be repeated when the same fraction was applied at concentrations that the proteins comprising this fraction would be found in live AGR1487. Even the high concentration tested previously did not decrease Caco-2 cell TEER and the discrepancy in results remains unexplained. The results reported in this dissertation have added to the knowledge that the two strains of L. fermentum AGR1485 and AGR1487 show differences in their genome size and in their phenotypic characteristics. In addition, these bacteria utilise both cell surface and/or secreted metabolites through multiple mechanisms to modulate host response. In the future, identification of specific bacterial effector molecules that influence host response will be a major step towards understanding strain-specific characteristics shown by Lactobacilli.
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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.
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    Epithelial development in the forestomach of pasture-fed lambs (birth to 8 weeks) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Veterinary Science at Massey University, New Zealand
    (Massey University, 1983) Ilio, Kenneth Yerro
    The histology, mitotic index, ultrastructure and Na+-K+-ATPase cytochemistry of lambs reared on pasture were studied during the period of weaning. Two embalmed mature non-pregnant Romney cross-brGd ewes and thirty Romney-cross lambs reared on Massey University sheep farms pastures were used. Five lambs {three in 1981 and two in 1982) were taken from their dams on pasture at each of the following respective ages: within 24 hours of birth, and at 12, 23, 34, 45 and 56 days. Stomach-tissue samples from 1 adult and from the lambs reared during the 1981 lambing season were prepared for histology using Haenatoxylin and Eosin, Nasson's green trichrome, Periodic-acid-Schiff and Toluidine Blue stains, and for conventional transmission electron microscopy. Tissue samples from the rumens of lambs reared during the 1982 season were used for strontium-capture technique Na+-K+-ATPase cytochemistry . Gross dissection o£ the stomach of one-day-old lambs confirmed that the largest compartment at birth is the abomasum, followed, in decreasing order of size; by the rumen, reticulum and omasum. Progressive development resulted in the forestomach compartments assuming their adult proportions by 56 days of age. Preliminary histological studies of epithelium taken from the rumen, reticulum, omasum and reticular groove of the adult sheep c0nfirmed it to be a stratified keratinizing epithelium. Five general cell layers were clearly seen: stratum basa1e, stratum spinosum, stratum granulosum, stratus transiticnale and stratum corneum. (In previous studies, the stratum granulosun and the stratum transitionale have been considered as one layer.) Mucopolysaccharides were located in the inter- cellular spaces in the stratum corneum. It is concluded that the ultrastructural features and Na+-K+-ATPase cytochemistry of the epithelium at 12 days of age appeared to be similar to those found in older animals. However, structural (and presumably functional) maturity did not appear to be complete until after 45 days of age at which stage the stratum transi- tionale had become complete and the mitotic index and the thickness of the epithelium had become stable. The increase of non-keratinocytes suggests the increasing immunocompetence of the epithelium. Tight junctions and extruded contents of membrane- coating granules in the intercellular spaces of the stratum corneum could provide a barrier to the diffusion of solutes across the epithelium. The development of gap junctions and the presence of Na+-K+-ATPase enzymatic sites in the membranes are consistent with the absorptive and transport functions of the epithelium, particularly the active transepithelial movement of sodium ions. Future studies could well show hormones, hormone-like substances and antibiotics to be important in the development of the forestomach epithelium in ruminants.