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Abstract

The prebiotic Bimuno is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of NCIMB 41171 using lactose as the substrate. Previous and studies demonstrating the efficacy of Bimuno in reducing serovar Typhimurium ( Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS. Here we wished to test the hypothesis that GOS, derived from Bimuno, may confer the direct anti-invasive and protective effects of Bimuno. In this study the efficacy of Bimuno, a basal solution of Bimuno without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno] and purified GOS to reduce Typhimurium adhesion and invasion was assessed using a series of and models. The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics demonstrated that the presence of ∼5 mg Bimuno ml or ∼2.5 mg GOS ml significantly reduced the invasion of Typhimurium (SL1344nal) (<0.0001). Furthermore, ∼2.5 mg GOS ml significantly reduced the adherence of Typhimurium (SL1344nal) (<0.0001). It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions. In the murine ligated ileal gut loops, the presence of Bimuno or GOS prevented the adherence or invasion of Typhimurium to enterocytes, and thus reduced its associated pathology. This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen. In all assays, Bimuno without GOS conferred no such protection, indicating that the basal solution confers no protective effects against Typhimurium. Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno can be attributed to GOS.

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2010-12-01
2019-10-23
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vol. , part 12, pp. 1428 - 1439

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SEM of HT-29-16E (mucus-secreting) cells cultured using standard procedures as monolayers (A) or as 3D aggregates (B) illustrating superior brush border formation and cellular differentiation in the RWV cultured 3D cells.

TEM of HT-29-16E (mucus-secreting) cells cultured using standard procedures as monolayers (A) or as 3D aggregates (B), illustrating the formation of multilayered aggregates of cells in the 3D cell culture system as compared to single layered cells when cultured as conventional monolayers.

Confocal microscopy of HT-29-16E (mucus-secreting) cells cultured using standard procedures as monolayers (A, C, E, G, I, K and M) or 3D aggregates (B, D, F, H, J, L and N) incubated with phalloidin-TRITC (A and B), villin (C, D), β-catenin (E, F), E-cadherin (G, H), ZO-1 (I, J), collagen IV (K, L) and cytokeratin 18 (M, N).

SEM of 3D cultured HT-29-16E cells incubated with PBS (A), Bimuno® (B), filtered Bimuno® (C), filtered Bimuno® without GOS (D), filtered GOS (E), . Typhimurium SL1344nalr (F), Bimuno® before SL1344nalr (G), Bimuno®+SL13444nalr (H), filtered Bimuno®+SL1344nalr (I), Bimuno® without GOS+SL1344nalr (J), filtered Bimuno® without GOS+SL1344nalr (K), GOS+SL1344nalr (L) and filtered GOS+SL1344nalr (M).

Confocal microscopy of 3D cultured HT-29-16E cells incubated with . Typhimurium SL1344nalr (A), Bimuno® before SL1344nalr (B), Bimuno®+SL13444nalr (C), filtered Bimuno®+SL1344nalr (D), Bimuno® without GOS+SL1344nalr (E), filtered Bimuno® without GOS+SL1344nalr (F), GOS+SL1344nalr (G) and filtered GOS+SL1344nalr (H).

TEM of murine ligated ileal gut loops inoculated with PBS (A), Bimuno® (B), filtered Bimuno® (C), filtered Bimuno® without GOS (D), filtered GOS (E).

Immunohistochemical detection of CSA-1 in murine ligated ileal gut loops inoculated with PBS (A) or . Typhimurium SL1344nalr (B).



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