Figure 8.

Epithelial cells transport Stx from the lumen to the basolateral surface. (A) TEER measurements in enteroid monolayers formed in Transwells. Stx2a (30 ng) was added to the apical media (upper chamber) of the Transwell on day 25. Means ± SD of experiments performed in triplicate is plotted. (B) Epithelial barrier function was verified. FITC-dextran (0.5 μmol/L) was added to the upper chamber on day 24 and Stx2a (30 ng) was added on day 25. Fluorescence in the lower chamber was assessed 24 hours later (day 26). Fluorescence was quantified using ImageJ software, and plotted as means ± SD. Statistical significance compared with saline control was assessed using 1-way analysis of variance, Dunnett’s posttest. ∗∗∗P < .0001, n = 3. (C) Schematic representation of the Stx2a toxicity assay to detect transcytosis. Stx2a (30 ng) was added to the luminal side of the enteroid monolayer (upper chamber) on day 25. The basolateral medium was isolated from the lower chamber 24 hours later and added to mRuby2-expressing mesenchymal cells isolated from HIOs. (D) Stx2a added to the apical surface was recovered in the basolateral medium. Mesenchymal cells were incubated for 27 hours with basolateral medium (lower chamber) from the Stx2a-treated epithelial monolayers and stained with Sytox green, with or without neutralizing monoclonal antibodies to the Stx2a A and B subunits. Statistical significance compared with saline control was assessed using 1-way analysis of variance, Bonferroni multiple comparison posttest. ∗∗∗P < 0.0001, n = 3.