Browsing by Author "Kleiveland, C."
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- Insights of enteropathogenic effects of mycotoxins on the human intestinal gut mucosaPublication . Assunção, Ricardo; Alvito, Paula; Kleiveland, C.; Lea, T.Intestinal mucosa is the first biological barrier encountered by natural toxins and it could be exposed to high amounts of dietary mycotoxins1,2. Trichothecenes, ochratoxin A and patulin (PAT) are the best known enteropathogenic mycotoxins able to alter functions of the intestine3. OBJECTIVES: This study aimed to evaluate the effects of PAT, a mycotoxin produced by Penicillium spp. during fruit spoilage, on barrier properties and function of the gut mucosa. METHODOLOGIES: Viability (MTT), proliferation (3H-thymidine incorporation assay), transepithelial electrical resistance (TER), SDS-PAGE and immunoblotting and flow cytometry methodologies were applied in order to characterize the effects of PAT on intestinal cell model (Caco-2), human peripheral blood lymphocytes (PBL) and human blood monocytederived dendritic cells (DC). RESULTS: PAT exposure reduced Caco-2 cell viability at concentrations above 12μM. The integrity of the Caco-2 monolayer was affected by PAT exposure, as demonstrated by a decrease in TER values, becoming more pronounced at 50μM. No effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3 at 50μM. However, the expression of zonula occludens-1 (ZO-1) and myosin light chain (MLC) declined and levels of phospho-MLC increased, after 24h of exposure to 50μM of PAT. T cell proliferation was highly sensitive to PAT with the major effects for concentrations above 10nM of PAT. The same conditions did not affect the maturation of DC. CONCLUSIONS: PAT causes a reduction in Caco-2 barrier function mainly by perturbation of ZO-1 levels and phosphorylation of MLC. Low doses of PAT strongly inhibited T cell proliferation induced by a polyclonal activator, but had no effect on the maturation of DC. These results provide new information that strengthens the concept that the epithelium and immune cells of the intestinal mucosa are important targets for the toxic effects of food contaminants like mycotoxins.
- Interactions between dietary toxins and gut mucosa: patulin effects on the structure and function of intestinal and immune cellsPublication . Assunção, Ricardo; Alvito, Paula; Kleiveland, C.; Lea, T.The intestinal mucosa is the first biological barrier encountered by natural toxins, and consequently, it could be exposed to high amounts of dietary toxins. Mycotoxins are a wide group of fungal secondary metabolites that exert toxic effects in humans. Patulin (PAT), a mycotoxin produced by Penicillium spp. during fruit spoilage, is a major concern with regard to children's health because its ingestion could result in severe acute and chronic toxicity3,4. This study aimed to evaluate the effect of PAT on the structure and function of the gut mucosa and its interactions with intestinal and immune cells. Several methodologies were applied in order to characterize the effects of PAT on intestinal cell model (Caco-2), human peripheral blood lymphocytes (PBL) and human blood monocyte-derived dendritic cells (DCs). As evaluated by an MTT assay, PAT exposure reduced the viability of Caco-2 cells, mainly for concentrations above 12 µM. The integrity of the Caco-2 monolayer was affected after exposure to PAT, as demonstrated by a decrease in transepithelial electrical resistance values, becoming more pronounced for 50 µM of PAT. At this concentration, using SDS-PAGE and immunoblotting, no effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3. However, the expression of zonula occludens-1 and myosin light chain (MLC) declined after 24h of exposure. The phospho-MLC increased after 24h of exposure to 50 µM of PAT. T cell proliferation was evaluated by thymidine incorporation assay, incubating PBL with soluble anti-CD3 antibodies. T cell proliferation was highly sensitive to PAT with the major effects for concentrations above 10 nM of PAT. Surprisingly, the same conditions did not affect the maturation of DCs for PAT concentrations between 0.5 nM and 100 nM. These studies provide more and new information about how patulin may affect the intestinal epithelium and mucosal immune responsiveness.