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Figure 1: Confocal analysis of FITC-labelled L. casei uptake by adherent DC. (a) Schematic view of the DC/epithelial cell culture model. The dotted arrow indicates the section where the analysis was done. (b) The pictures represent different sections of the same DC and show the colocalization of FITC-bacteria into the cytoplasm of DC.
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Figure 2: DC take up FITC-labelled L. casei through an intestinal epithelial layer. Monocytes from allergic patients and healthy donors were differentiated inDC in the presence of GM-CSF and IL-4 for 5- 6 days. Cells were then pulsed with unlabelled or with FITC-labelled L.casei (ratio bacteria/DC: 100/1) for 24 or 48 hours in the presence of an intestinal epithelial layer. DC, adherent and nonadherent to the filter, were harvested and analyzed by flow cytometry. (a) The percentage of FITC-positive cells is evaluated into the gate represented on the dot plots. (b) Open histograms represent the fluorescence of DC incubated with unlabelled L. casei, and closed histograms represent the capture of FITC-labelled L. casei by DC. The percentages of FITC-positive cells are indicated. One representative experiment is shown for both donor types.e, winFeature
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Figure 3: Effect of L. casei stimulation on DC phenotype. Monocyte-derived dendritic cells from house dust mite sensitive-patients (a) and (b) and from healthy donors (c) and (d) were exposed (dark grey line) or not (grey line) to L. casei for 48 hours in the Transwell model. (a) and (c) Adherent DC were analyzed by flow cytometry for CD80, CD86, HLA-DR, and CD54 expression. the black line represents the reactivity of fluorochrome-matched isotype control mAbs. One representative experiment (out of 6 and 7, for allergic and healthy donors, resp.) is shown. (b), (d) The mean fluorescence intensity (.MFI) of the expression of each marker by adherent and nonadherent DC after stimulation with medium, and L.casei is represented for both allergic ((b), n = 6) and healthy donors ((d), n = 7). *P
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Figure 4: L. casei-stimulated DC produce IL-12 and IL-10. Monocytes-derived DC from healthy (a) and allergic (b) donors were pulsed or not for 48 hours with L. casei through an intestinal epithelial layer or not. Supernatants were collected and the amounts of IL-10 and IL-12 were measured by specific ELISA. Results are expressed as mean ± SEM (n = 5 for allergic donors and n = 5 healthy donors). *P
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Figure 5: Production of cytokine by native CD4+ T cells.Monocyte-derived DC from allergic patients and healthy donors were incubated with L. casei or medium only for 24 hours, either directly in contact with cells (without epithelium) or through an intestinal epithelial layer (with epithelium). Adherent DC were harvested from the lower chamber of the Transwell, cultured with na¨ive autologous CD4+ T cells for 5 days. Supernatants were collected and the amounts of IFN-., IL-10, and IL-5 were measured by specific ELISA. Results are expressed as mean ± SEM of cytokine percentage increase above L. casei stimulation (n = 5 for both allergic and healthy donors).
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