There is some evidence that zinc oxide (ZnO) protects against intestinal diseases. However, despite the suggestions that ZnO may have an antibacterial effect, the mechanisms of this protective effect have not yet been elucidated. We investigated the potential benefits of ZnO in protecting intestinal cells from damage induced by enterotoxigenic Escherichia coli (ETEC, strain K88) and the related mechanisms, using human Caco-2 enterocytes. Cell permeability, measured as transepithelial electrical resistance (TEER), was unaffected by 0.01 and 1 mmol/L ZnO treatments and moderately increased by 5 mmol/L ZnO, compared with untreated cells. Transfer of (14)C-inulin was slightly increased by 5 mmol/L ZnO compared with untreated cells; transfer was unaffected by lower concentrations. The TEER and (14)C-inulin transfer were lower in ETEC-infected cells than in uninfected cells. Treatment of ETEC exposure with 0.2 mmol/L ZnO prevented disruption of membrane integrity. The ETEC was able to adhere to enterocytes and, to some extent, invade the cells. The ZnO treatment reduced bacterial adhesion and blocked bacterial invasion. The ETEC infection upregulated the expression of the inflammatory cytokines interleukin-8, growth-related oncogene-alpha and tumor necrosis factor-alpha, and reduced that of the anti-inflammatory cytokine transforming growth factor-beta, compared with uninfected cells. The addition of 0.2 or 1 mmol/L ZnO counteracted the alteration of cytokine mRNA levels caused by ETEC. The protective effects of ZnO were not due to any antibacterial activity, because the viability of ETEC grown in a medium containing ZnO was unaffected. In conclusion, ZnO may protect intestinal cells from ETEC infection by inhibiting the adhesion and internalization of bacteria, preventing the increase of tight junction permeability and modulating cytokine gene expression.

Lactobacilli have a potential to overcome intestinal disorders; however, the exact mode of action is still largely unknown. In this study, we have used the intestinal porcine intestinal IPEC-1 epithelial cells as a model to investigate a possible protective activity of a new Lactobacillus species, the L. sobrius DSM 16698(T), against intestinal injury induced by enterotoxigenic Escherichia coli (ETEC) K88 infection and the underlying mechanisms. Treatment of infected cells with L. sobrius strongly reduced the pathogen adhesion. L. sobrius was also able to prevent the ETEC-induced membrane damage by inhibiting delocalization of zonula occludens (ZO)-1, reduction of occludin amount, rearrangement of F-actin, and dephosphorylation of occludin caused by ETEC. RT-PCR and ELISA experiments showed that L. sobrius counteracted the ETEC-induced increase of IL-8 and upregulated the IL-10 expression. The involvement of IL-8 in the deleterious effects of ETEC was proven by neutralization of IL-8 with a specific antibody. A crucial role of IL-10 was indicated by blockage of IL-10 production with neutralizing anti-IL-10 antibody that fully abrogated the L. sobrius protection. L. sobrius was also able to inhibit the internalization of ETEC, which was likely favored by the leaking barrier. The protective effects were not found with L. amylovorus DSM 20531(T) treatment, a strain derived from cattle waste but phylogenetically closely related to L. sobrius. Together, the data indicate that L. sobrius exerts protection against the harmful effects of ETEC by different mechanisms, including pathogen adhesion inhibition and maintenance of membrane barrier integrity through IL-10 regulation.

Probiotic bacteria may provide protection against intestinal damage induced by pathogens, but the underlying mechanisms are still largely unknown. We investigated whether Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG (LGG) protected intestinal Caco-2 cells from the inflammation-associated response induced by enterotoxigenic Escherichia coli (ETEC) K88, by inhibiting pathogen attachment to the cells, which is the first step of ETEC pathogenicity, and regulating neutrophil recruitment, a crucial component of inflammation. A partial reduction of ETEC adhesion was exerted by probiotics and their culture supernatant fractions either undigested or digested with proteases. ETEC viability was unaffected by the presence of B. animalis, LGG or their supernatant fractions in the culture medium, indicating an absence of probiotic bactericidal activity. Probiotics and their supernatant fractions, either undigested or digested with proteases, strongly inhibited the neutrophil transmigration caused by ETEC. Both B. animalis and LGG counteracted the pathogen-induced up regulation of IL-8, growth-related oncogene-a and epithelial neutrophil-activating peptide-78 gene expression, which are chemokines essential for neutrophil migration. Moreover, the probiotics prevented the ETEC-induced increased expression of IL-1b and TNF-a and decrease of transforming growth factor-a, which are regulators of chemokine expression. These results indicate that B. animalis MB5 and LGG protect intestinal cells from the inflammation-associated response caused by ETEC K88 by partly reducing pathogen adhesion and by counteracting neutrophil migration, probably through the regulation of chemokine and cytokine expression.

-Weaned pigs are susceptible to frequent infectious diseases. Antibiotics have been used over decades to reduce pathogen infections, but many microorganisms are becoming resistant to antibiotics. Thus, there is an urgent need to find alternatives to in-feed antibiotics. Up to now, various compounds have been used as alternatives, giving promising but sometimes contrasting results. In recent years, the in vitro models of cell culture have been proposed for the screening of various compounds and understanding their mechanisms of action. The intestine is a complex system with a continuous cross-talk among epithelial cells, the local immune system and microflora. Alternatives to in-feed antibiotics may interact with any of these constituents. This review presents data on the protective effects of probiotics, zinc salts and organic acids, used as alternatives to in-feed antibiotics, against pathogen induced damage and inflammatory processes in the intestinal cells and the underlying mechanisms, with an attempt to compare in vitro and in vivo results. All the alternatives tested resulted in effective protection against pathogens by a series of interactions with intestinal cells and pathogens, including the inhibition of adhesion and invasion of pathogens, interference in signalling pathways induced by pathogenic bacteria, maintenance of the epithelial cell cytoskeleton and junctional structure and modulation of the host immune response. microorganismes sont devenus résistants à ces substances. Aussi, est-il urgent de trouver des alternatives aux antibiotiques additifs alimentaires. Jusqu'à présent, diverses alternatives ont été envisagées, certaines donnant des résultats prometteurs, d'autres des résultats variables. Au cours des dernières années, les modèles in vitro de cultures cellulaires ont été proposés pour cribler ces substances et élucider leurs mécanismes d'action. L'intestin est un système complexe faisant intervenir des communications permanentes entre les cellules épithéliales, le système immunitaire local et la microflore. Les alternatives aux antibiotiques peuvent interagir avec n'importe lequel de ces composants. Le présent article fait la synthèse des effets protecteurs et des mécanismes d'action des probiotiques, des sels de zinc et des acides organiques, utilisés comme alternatives aux antibiotiques dans les processus inflammatoires et les altérations cellulaires épithéliales in vitro, induites par des agents pathogènes. Elle tente de comparer ces résultats avec les observations in vivo. Toutes les alternatives évaluées ont été efficaces contre les agents pathogènes, via des mécanismes d'interaction cellules épithéliales-agents pathogènes. Ces mécanismes incluaient l'inhibition de l'adhésion et de l'invasion cellulaire par ces pathogènes, la modulation des mécanismes de signalisation intracellulaire induits par les bactéries pathogènes, le maintien de la structure du cytosquelette et des jonctions serrées des cellules épithéliales, et enfin la modulation des réponses immunitaires de la cellu...

There is evidence that organic food often contains relatively high amounts of natural toxic compounds produced by fungi or plants, whereas corresponding conventional food tends to contain more synthetic toxins such as pesticide residues, but only a few studies have evaluated the impact of their consumption on health. This study proposes a novel approach to evaluate the potential health risk of organic compared to conventional food consumption, that is, the assay of sensitive markers of cell function in vulnerable conditions. The markers utilized were intestinal and splenic lymphocyte proliferative capacity and liver acute-phase reaction, both responding to the presence of toxins. The vulnerable conditions in which body defenses can be less efficient were weaning and protein-energy malnutrition. This study reports the results of a pilot experiment on one sample of eight varieties of organically and conventionally grown wheat. Weaned rats were assigned to two groups fed conventional (CV) or organic (ORG) wheat for 30 days. Each group was divided in two subgroups of well-nourished (WN) or protein-energy-malnourished (PEM) rats. For each rat, the lymphocyte proliferation was assayed by [(3)H]thymidine incorporation after stimulation of cells with a mitogen, in a culture medium containing either commercial fetal calf serum (FCS) or the corresponding rat serum (RS) to mimic the in vivo proliferative response. The acute-phase proteins (albumin, transthyretin, transferrin, ceruloplasmin, retinol-binding protein) were measured in plasma by Western blotting and immunostaining with specific antibodies. The proliferative response of lymphocytes cultured with FCS and the amount of acute-phase proteins of rats fed the ORG wheat sample, either WN or PEM, did not differ from those of rats fed the CV wheat sample. However, the proliferative response of lymphocytes cultured with RS was inhibited in PEM-CV compared with PEM-ORG. The content of mycotoxins was highest in the organic sample, and therefore the immunotoxic effect was probably due to other contaminants in the CV wheat. In conclusion, these results indicate that the conventional wheat sample tested represented a higher risk for lymphocyte function than the wheat sample organically grown, at least in vulnerable conditions.