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 .
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  and our observations along with data in literature from in vivo studies, support that this is an early event rather than a consequence of chronic intestinal inflammation .
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 only provides the epithelium with a barrier function but also, by rapid assembly and disassembly, changes its permeability upon different stimuli . 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. This evidence let us hypothesize that the continuous exposure to a toxic agent such as gliadin could bring cells to a rearrangement in TJ architecture, beyond the immediate disruption of the epithelial barrier. Few data are available on this item. Previously, Sander et al.  reported a fast disruption of intestinal barrier function in Caco-2 cells (after 4 h of exposure to gliadin peptic-tryptic digest) that markedly involved Occludin, ZO-1 and E-cadherin. In our study, the events were not so rapid even if, in agreement with these authors, we also found that permeability, as measured by TER, increased immediately after gliadin addition reaching its maximum after 60 minutes. The differences in TJ expression between the two studies probably rely on the toxic agent administered. In fact, we used wheat gliadin instead of the peptic-tryptic (PT) digests that are known to have different modes of action in regard to their toxicity. PT treatment induces the production of alkenals that in turn can modify the activity of membrane-associated proteins and enzymes .
The modifications in paracellular permeability went together with a rising in the single and total polyamine content that was evident and significant after 6 h of exposure. A clear role for polyamines at cellular and molecular levels in the gliadin-triggered damage of intestinal epithelia is still under debate. Regulation of brush border functions by spermidine and spermine has been suggested to be mediated by a transglutaminase-induced incorporation of polyamines into membrane proteins . Besides, it has been hypothesized that epithelial binding of gliadin peptides may occur in the form of IgA immune complexes which then translocate across the epithelium . This binding could represent powerful extraneous growth factors for the gut and, as a result, induce extensive proliferation and changes in the metabolism of epithelial cells via activation of second messenger pathways. These metabolic changes may release huge amounts of polyamines, mostly spermidine . On the other hand, the increase in polyamine content probably results from increased cell proliferation during the repair phase of mucosal injury. In this context, polyamine levels could be regarded as markers of a hyperproliferative state in response to toxic effects of gliadin. This behavior by polyamines has already been reported during inflammation of intestine leading to derangement of the mucosa .
The second aim of the study was to investigate the possible effects on paracellular permeability and polyamine content following co-administration of viable L.GG, LGG-HK or its conditioned medium with gliadin. In previous experiments by our group, L.GG was proven to be effective in modulating cell proliferation and polyamine metabolism and biosynthesis also when its components (namely cytoplasm extracts and cell wall extracts) were tested, supporting the hypothesis that intact cells is not a pre-requisite for the L.GG protective effects [19, 20].
Although members of the Lactobacillus genera are a subdominant part of the normal colonic microbiota, they represent a much greater component higher up in the gut . Animal models and cell culture systems have provided indications that lactobacilli are able to counteract alterations in paracellular permeability evoked by cytokines, chemicals, peptides, infections or stress . A paper by Seth et al.  reported that the administration of live and heat inactivated L.GG, bacterial supernatants and peculiar L.GG purified soluble proteins to Caco-2 cells treated by hydrogen peroxide that destroys TER and increases permeability, caused the secretion of proteins of this strain effective against the insult.
In our study, the administration of viable and heat killed L.GG as well as its conditioned medium, caused only a slight and not significant increase in TER after 90 min from exposure without any effects on lactulose flux and zonulin release. By opposite, in Caco-2 cells treated with gliadin, the addition of viable L.GG, but also L.GG-HK and L.GG-CM, significantly restored cell barrier function. Also the single and total polyamine levels diminished significantly when Caco-2 cells were exposed to gliadin in combination with viable and heat killed L.GG. Recently, our group reported that the administration of viable, heat killed L.GG and L.GG homogenate to DLD-1 and HGC-27 cell lines significantly reduced neoplastic proliferation as well as polyamine content and biosynthesis [19, 20, 38].
As regards the protective effects of some probiotics against gliadin, our findings are in line with data in literature  and different mechanisms could be evoked to explain the effects exerted by L.GG, not only as viable bacteria, but also when they were heat inactivated or their conditioned medium was used. Firstly, L.GG might inhibit gliadin-induced damage in Caco-2 cells by hydrolyzing gliadin similarly to other live probiotic bacteria as in the VSL3# probiotic preparation . These strains showed the ability to colonize the human stomach and duodenum, where the hydrolysis of gliadin epitopes may be relevant for decreasing the abnormal secretion of zonulin and the initial step of immune response to gliadin [41, 42]. Secondly, the peculiar set of peptidases shown by L.GG was probably able to inhibit the gliadin-induced damage to Caco-2 cells breaking up wheat gliadin into small harmless peptide products . Thirdly, L.GG might modulate directly the function of epithelial cells. It has already been reported that different probiotic strains, probiotic bacterial lysates or conditioned medium increase epithelial barrier function as measured by TER . In addition, L.GG might protect the epithelium from the gliadin insult by direct action on the cells.
One interesting finding of the present study is that viable L.GG per se was able to significantly increase ZO-1, Claudin-1 and Occludin expression after 6 h of exposure. Even if the gliadin effects on TJ expression were significant only after 24 h, the co-administration of viable L.GG with gliadin caused an early and significant increase in the expression of the tested proteins compared to gliadin treated cells. Besides, after 24 h, viable L.GG with gliadin continued to significantly increase the expression of Claudin-1 and Occludin, but exerted only a slight and not significant decrease on ZO-1 levels. Available data support the capability of peculiar probiotic strains in modulating TJ protein expression. Pretreatment of Caco-2 monolayers with L. plantarum significantly attenuated the effects of phorbol ester-induced dislocation of ZO-1 and Occludin and the associated increase in epithelial permeability . Additionally, treatment of Caco-2 cells with the probiotic L. plantarum MB452 resulted in augmented transcription of Occludin and Cingulin genes, suggesting that bacteria-induced improvements to intestinal barrier integrity may also be regulated at the gene expression level .
Of note, the presence of polyamines was required for viable L.GG to exert its effects on TJ expression. As a matter of fact, when Caco-2 monolayers were deprived in the polyamine content by DFMO, the expression of TJ proteins was not significantly different from that in controls or cells treated with gliadin alone. Cellular polyamines spermidine, spermine and their precursor putrescine, have been indicated as playing a role in the maintenance of the intestinal epithelial integrity by their ability to modulate expression and functions of various genes, such as intercellular junction proteins . Present findings let us hypothesize that the action of viable L.GG in modulating the expression of TJ proteins could be mediated also by the presence of cellular polyamines, although the exact mechanisms are still not completely elucidated. Possibly, they may be related to the specific molecular structure of these compounds. At physiological pH, putrescine, spermidine, and spermine possess two, three, and four positive charges, respectively . These compounds can bind to negatively charged macromolecules such as DNA, RNA, and proteins to influence the sequence-specific DNA-, RNA- or protein-protein interactions, which alter gene transcription and translation and the stability of mRNAs and proteins.