Background: VSL#3 has been extensively investigated and is currently recommended for
the prevention and treatment of chronic pouchitis and ulcerative colitis. Nonetheless, in vitro and in
vivo studies have recently shown variability in the VSL#3 efficacy often attributed to the manufacturing
Objective: The aim was to comparatively study the in vitro effects of two VSL#3 preparations produced
in different sites (named US- and Italy-made VSL#3) on CaCo-2 epithelial barrier model in
terms of trans-epithelial electrical resistance (TEER), dextran flux and expression of Tight Junctions
(TJ) proteins i.e. zonulin-1 (ZO-1) and occludin, in the absence or presence of a heat stress-related
damage of monolayer.
Methods: TEER was evaluated on CaCo-2 differentiated monolayers. Epithelial permeability of polarized
monolayers was assessed by measuring the FITC-labeled dextran flux from the apical to basolateral
chambers. ZO-1/occludin levels were analyzed by western blot analysis. A set of experiments
was performed to compare the effects of both VSL#3 on TEER values, dextran flux and ZO-1/occludin
expression in CaCo-2 monolayers after heat stress exposure.
Results: US- and Italy-made VSL#3 have opposing effects on TEER values, dextran flux, and ZO-
1/occludin expression, being all these parameters negatively influenced just by Italy-made product.
US-made probiotic did not affect baseline TEER, dextran flux and ZO-1 expression and strongly increased
occludin levels. Of note, pre-treatment of monolayer with US-made VSL#3, but not Italy-made
product, totally prevented the heat-induced epithelial barrier integrity loss.
Conclusion: Our data trigger the need for reassessing efficacy or safety of the Italy-made VSL#3 considering
intestinal epithelial barrier plays an important role in maintaining host health.