As the use of probiotics and postbiotics is increasingly gaining ground in the past decade, the possibility of using bacterial strains with postbiotic activity to restore homeostasis in pathologic conditions such as IBD is extensively debated. However, clinical data as far as induction of remission is concerned has not been encouraging so far, while researchers observe that only a small number of treatments which seem promising on in vitro or mouse models translate to significant clinical benefit. This could mean that the models used so far to test potential treatments are not an accurate representation of the human intestine's rather complex micro-environment, and thus there is a need for the development of more relevant models. In this thesis, a novel organ culture model for polarized stimulation of intestinal mucosa is described. In the intestine, apical and basolateral challenge of the mucosal layer can elicit completely different results, and this is one of the issues addressed in this work. First of all, we show that it is possible to keep human intestinal mucosa in polarized culture for at least 24 hours, provided the explants are cultured in an atmosphere that is rich in oxygen. Polarized challenge is achieved by mechanical means, namely by attaching a cave plastic cylinder on the apical side of the mucosal layer, in order to confine the stimuli. We examine the impact of the cylinder and the surgical glue used to attach it on tissue morphology and survival, and show that there is no negative impact. Once optimized, this experimental set-up is used to challenge explants with pathogenic and probiotic bacteria alike and evaluate the immune response. On this model we are able to mimic a classical pathogen infection using a highly invasive Salmonella enterica serovar typhimurium strain, FB62. The response of the tissue to the pathogen was monitored by assessing the phenotype of explants after culture, while cytoine secretion profiles of the tissue were also studied. Salmonella challenge led to a damaged explant phenotype, upregulation of TNF-α and downregulation of IL-10. This response was abolished in the presence of an antiinflammatory postbiotic component, namely culture supernatant of the probiotic Lactobacillus paracasei. Moreover, as far as challenge with pathogens is concerned, we use this novel system to examine the mechanism of Shigella induced apoptosis of epithelial cells. Importantly, no animal models are available for the study of Shigella infection, as these 18 bacteria are poorly virulent in rodents. Thus, in an effort to study the involvement of Gadd45a in the apoptotic route triggered by Shigella on intestinal epitelial cells, HeLa cells, which are however a poor model for an intestinal pathogen were used. Gadd45a participates in the responses to a variety of DNA damaging agents and interacts with proteins such as Cdc2, PCNA and p21. In this work, the authors showed that after infection of HeLa cells with Shigella, Gadd45a is involved in the induction of the apoptotic process. The data was confirmed in a more relevant setting, by applying Shigella on human intestinal mucosa. Finally, we use the novel organ culture platform to test three different strains thought to exert probiotic actions. Surprisingly, we show that this is not the case, and the three strains can exert different activities even on healthy tissue. More specifically, Lactobacillus paracasei and Lactobacillus rhamnosus GG did not significantly alter the phenotype or cytokine secretion profiles of healthy explants, but challenge with Lactobacillus plantarum resulted in a detrimental effect. Of note, all three strains were detrimental for IBD explants when administered as live bacteria, even though one of the strains (L. paracasei) had previously been found to exert a prophylactic effect in a mouse model of colitis. On the contrary, we show that a potent postbiotic (L. paracasei culture supernatant) is able to ameliorate overt inflammation on both ulcerative colitis and Crohn's disease tissues as attested by cytokine secretion profiles of challenged explants. In conclusion, this work introduces a valid alternative system on which to study the interaction of various components (bacterial, pharmacological, and others) with the human intestinal mucosa.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:600108 |
| Date | January 2014 |
| Creators | Tsilingiri, Aikaterini |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=203945 |
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