Cell cytotoxicity during interaction between human colonic explants and <i>E. histolytica</i> or <i>E. dispar</i>.

<p>Mean LDH concentrations (IU/L) released into the supernatant of the organotypic culture after incubation with <i>E. histolytica</i> WT or <i>E. dispar</i> or in the absence of amoeba (control) from 1 to 7 hours. Data are from 8 individual experiments. <b>*</b> indicates a significant difference between WT and control (<i>p</i><0.03) and between WT and <i>E. dispar</i> (<i>p</i><0.05). <b>#</b> indicates a significant difference between WT and control (<i>p</i><0.001) and between WT and <i>E. dispar</i> (<i>p</i><0.02)</p

<i>E. histolytica</i>-induced secretion of pro-inflammatory cytokines.

<p>Histogram showing mean ± SD concentrations (pg/ml) of individual analytes (IL-1β, IL-6, IL-8, IFN-γ and TNF) secreted from 8 human colonic explants incubated with WT, <i>E. dispar</i> or without amoeba (control) after 4 and 7 hours of incubation, as measured on a Luminex100 system. Levels of secreted pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IFN-γ and TNF) were significantly higher at 4 hours (<b>*</b><i>p</i><0.05) and 7 hours (<b># </b><i>p</i><0.03) in the explants incubated with WT, in comparison with both those secreted by explants incubated with <i>E. dispar</i> and the amoeba-free control.</p

Pro-inflammatory cytokines secretion induced in the <i>ex-vivo</i> human colonic model by sub-strains of <i>E. histolytica</i>.

<p>Mean concentrations (pg/ml) of IL-1β, IL-8, IFN-γ and TNF secreted after 4 hours (grey bars) and 7 hours (black bars) of incubation of HGL2, NEO, RBV, G3, WT and RB8 trophozoites with 3 individual human colonic explants. NEO and HGL2 parasites induced significantly higher levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IFN-γ and TNF in the explants incubated for 4 hours (<i>p</i><0.05) and 7 hours (<i>p</i><0.02), compared with the control in the absence of amoeba. RBV, G3 and WT strains induced significantly higher levels of pro-inflammatory cytokines [IL-1β (<i>p</i><0.05,<0.01 and <0.01 respectively) IL-8 (<0.04,<0.01 and <0.01 respectively), IFN-γ(<0.009,<0.003 and <0,01 respectively) and TNF (<0.02,<0.02 and <0.02 respectively)] in the explants incubated for 4 hours and IL-8 (<0.009, <0.001 and <0.01 respectively), IFN-γ (<0.002,<0.0004 and <0.01 respectively) and TNF (<0.008,<0.008 and <0.02 respectively)] at 7 hours, compared with the control in the absence of amoeba. WT secreted IL1β (0.04/0.03), IL-8 (0.001/0.001), IFN-γ (0.001/0.002) and TNF (0.008/0.01) at 4 and 7 hours respectively, compared with RB8.</p

Cell cytotoxicity during interplay between <i>E. histolytica</i> trophozoites affected for virulent factors and human colonic explants.

<p>Mean LDH concentrations (IU/L, from 3 individual experiments) released after incubation of human colonic explants with HGL2, NEO, RBV, G3, WT and RB8 trophozoites and in the absence of amoeba (control) for 4 hours (grey bars) and 7 hours (black bars). <b>*</b> indicates a significant difference between HGL2 and control (<i>P</i><0.01), NEO and control (<i>P</i><0.03) and WT and control (<i>P</i><0.05). <b>#</b> indicates a significant difference between HGL2 and control (<i>p</i><0.008), NEO and control (<i>p</i><0.01), RBV and control (<i>p</i><0.01), G3 and control (<i>p</i><0.009), WT and control (<i>p</i><0.007), RBV and RB8 (<i>p</i><0.03), G3 and RB8 (<i>p</i><0.01) and WT and RB8 (<i>p</i><0.01)</p

Migration through the lamina propria of <i>E. histolytica</i> sub-strains impaired in virulent functions.

<p>Representative images from three individual experiments are shown. Histological examination of colonic tissue sections after seven hours of incubation, with HGL2, G3, RBV and RB8. Transversal tissue slices were stained with haematoxylin-eosin. Trophozoites were immunostained with antibodies against the Gal/GalNAc lectin. Experiments with HGL2, G3 and RBV revealed that trophozoites were able to invade the mucosa, as described for the WT. In contrast, RB8 parasites were unable to penetrate deeper into the lamina propria and were blocked at the surface of the mucosa, although they were still able to disorganize and detach cells from the upper side of the mucosa.</p

Interaction between Entamoeba and the lumen surface of the human colonic explants.

<p><b>A</b>. Analyse by histology of the mucus layer at the surface of Human colonic fragments incubated for seven hours without <i>Entamoeba</i> (left panel) and with <i>E. histolytica</i> (right panel). The mucus layer covering the epithelium at the surface was observable after seven hours of organotypic culture but not in the presence of <i>E. histolytica</i>. <b>B</b>. Scanning electron micrographs of the luminal surface of the human colonic explants incubated with <i>E. histolytica</i> or <i>E. dispar</i>. Representative images from three individual experiments are shown. (a) <i>E. histolytica</i> trophozoites adhering to the mucus layer at time 0; (b) 2 hours after incubation, the mucus layer had been degraded by <i>E. histolytica</i> and the regular mucosal architecture of the colonic epithelium was visible. Holes corresponded to the crypts of Lieberkühn and abundant aggregates were seen in the interglandular regions. (c) The aggregates were composed of human cells and trophozoites, as seen in an enlargement of this region (d) After 4 hours, the epithelium was damaged and (e) <i>E. histolytica</i> trophozoites began to penetrate into the tissue (f) After 4 hours, <i>E. dispar</i> trophozoites were still adhering to the mucus but had not degraded it and (g) had not evoked the recruitment of cells to the interglandular region, as shown after manually scraping the mucus after SEM fixation procedure of the sample.</p