Figure 5 Western Blot analysis of ZO-1, Claudin-1 and Occludin (using their specific

antibodies as specified in Methods) in Caco-2 monolayers after 6 h of exposure to gliadin (1 mg/ml) alone or in combination with viable L.GG (10 8   CFU/ml), heat killed L.GG (L.GG-HK) and L.GG conditioned medium (L.GG-CM). Immunoreactive bands were quantified using Quantity One programme. The diagrams show quantification of the intensity of bands, calibrated to the intensity of the β-actin bands. All data represent the results of three different experiments (mean ± SEM). Data were analyzed by Kruskal-Wallis analysis of variance and GW-572016 nmr Dunn’s Multiple Comparison Test. (*) P < 0.05 compared www.selleckchem.com/products/pf-03084014-pf-3084014.html to gliadin treated cells. Discussion In physiological conditions, intestinal epithelium is impermeable to macromolecules, but in CD patients

the gliadin fraction of wheat gluten represents the environmental factor responsible for the alterations in the junctional structures between epithelial cells leading to compromised permeability [4]. In our in vitro conditions, administration of gliadin to Caco-2 cells caused an increase in paracellular permeability as demonstrated by the dramatic decrease in TER immediately after the exposure, with a concomitant release of zonulin. These events were followed at 90 min by a significant rising in the lactulose paracellular transport. Overall, the process was rapid. After 6 h from exposure, the release of zonulin was similar to baseline values. It is now accepted that one of the immediate consequences of gluten exposure is the increased paracellular permeability, occurring within 36 h [27] and our observations along with data in literature from in vivo studies, Selleckchem Vorinostat support that this is an early event rather than a consequence of chronic intestinal inflammation [22]. CD patients show structural alterations at TJs that are made up of transmembrane proteins such as Occludins, and Claudins with intra-cellular connections to the Zonulins, which

are members of the ZO family. These, in turn, are anchored to the cell’s actinomyosin cytoskeleton and the result is a structure that not Phloretin only provides the epithelium with a barrier function but also, by rapid assembly and disassembly, changes its permeability upon different stimuli [28]. In our study, ZO-1, Claudin-1 and Occludin expression was assessed to test their involvement in modifications of paracellular permeability of Caco-2 cells. When these cells were exposed to gliadin, a time dependent effect on TJs expression was observed. After 6 h of gliadin exposure, a slight and not significant decrease in ZO-1 and Occludin expression occurred without affecting Claudin-1. By prolonging the time of exposure up to 24 h, ZO-1 and Claudin-1 expressions decreased significantly while Occludin expression remained unchanged.