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Title: The regulation of tight junctions in colonic epithelial cells by inflammation and related cytokines
Author: Prasad, Shyam
ISNI:       0000 0001 3498 507X
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2005
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This thesis examines the hypothesis that cytokines important in the pathogenesis of IBD impair the barrier function of the intestinal epithelium by inducing alterations in the regulation of TJ proteins. The expression of TJ proteins in IBD and normal colonic epithelia was examined by immunohistochemistry, western blotting and quantitative reverse transcriptase-mediated polymerase chain reaction. Claudin 2 was strongly expressed in inflamed epithelia, whilst being barely detectable in normal colon. In contrast, claudins 3 and 4, occluding and ZO-1 were highly expressed in normal colon, but were reduced or redistributed in the surface epithelium of many IBD cases. To functionally test the effects of inflammatory cytokines implicated in the pathogenesis of IBD, the T84 cell line was cultured on collagen S-coated, semi-permeable supports to model the epithelial barrier. Measurements of transepithelial electrical resistance (TER) and the passage of uncharged dextran were utilised to monitor permeability. The expression of claudins 2, 3 and 4 was studied by immunfluorescence and western blotting. TER increased over 15 days, correlating positively with the incorporation of claudins 3 and 4 into the TJ, but negatively with the amount of claudin 2 detected. Claudin 2 expression did not correlate with either proliferation or junctional reorganisation after calcium ion depletion and replenishment. Interleukin-(IL-)13 and combined interferon-gamma and tumour necrosis factor-alpha (IFNγ/TNFα) increased permeability to dextran and reduced TER.  IFNγ/TNFα led to decreases in claudins 2 and 3 and the redistribution of claudin 4, whereas IL-13 dramatically increased claudin 2 mRNA and protein expression, with little effect on claudins 3 and 4. The effects of IL-13 on TER and claudin 2 mRNA and protein levels could be inhibited by co-incubation with Ly294002, an inhibitor of phosphatidylinositol-3-kinase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available