7 research outputs found
Percentage of the differentiation of CD1a+ monocyte-derived dendritic cells using conditioned media from 4 independent gingival fibroblasts donors.
<p>MD: medium for differentiation (rGM-CSF+rIL-4).</p><p>CM1, CM2, CM3 and CM4: conditioned media from independent gingival fibroblasts donors 1 to 4.</p><p>For each CM, results are shown as mean ± SD of at least 3 independent experiments in duplicate.</p>*<p>P<0.0001.</p
Gingival fibroblasts (GFs) or conditioned medium from GFs modify the morphology of monocyte-derived dendritic cells.
<p>Giemsa staining on cytospin slides of cells prepared from cultures of A. Monocytes (day 0). B. Monocyte-derived dendritic cells after 7 days in culture with medium for differentiation (MD, containing rGM-CSF and rIL-4); these cells present the typical characteristics of DC with clearly visible dendrites. C. Monocyte-derived cells co-cultured for 7 days with GFs in a TranswellÂź system with MD. D. Monocyte-derived cells cultured for 7 days in the presence of conditioned medium from GFs with MD. Monocyte-derived cells (C and D) and monocyte-dendritic cells (B) show morphological differences, in particular the absence of typical dendritesfrom monocyte-derived cells. The results are representative of three independent experiments. Scale bar: 20 ”m, original magnification Ă400.</p
Mature monocyte-derived DCs obtained with (CM+) or without (CM-) conditioned medium from GFs were used to stimulate allogeneic CD4+ T cells purified from the PBMCs from two donors (PBMC1 and PBMC2); thymidine incorporation is expressed in counts per minute (cpm).
<p>Mature monocyte-derived DCs obtained in the presence of CM from GFs displayed a significantly capacity than controls to stimulate the proliferation of allogeneic CD4+T cells. Mean ± SD of triplicates are shown and results are representative of two independent experiments. *Significant difference (Pâ=â0.002) and **Significant difference (P<0.001).</p
Antibodies against VEGF and IL-6 reduce the inhibitory effect of conditioned medium (CM) from GFs.
<p>A. Neutralizing antibodies against VEGF (10 ”g/ml) and IL-6 (10 ”g/ml) significantly reduced (P<0.05) the inhibition of CD1a+ monocytes-derived cells by CM from GFs. In contrast, neutralizing antibodies against TGFÎČ1 significantly increased (P<0.05) this inhibitory effect. B. The effect of the neutralizing antibodies against VEGF and IL-6 was dose-dependent. The results reported are means ±SD for four independent experiments in duplicate. *Significant difference to the value for MD+CM (P<0.05).</p
Soluble factors from gingival fibroblasts (GFs) inhibit the differentiation of CD1a<sup>+</sup> dendritic cells.
<p>A. Analysis of the induction of the differentiation of monocyte-derived DCs by MD (rGM-CSF and rIL-4) after 7 days co co-culture of GFs (lower compartment) with monocytes (upper compartment) in a TranswellÂź chamber system. The effect of several GF/monocyte ratios (1â¶25 to 1â¶5) on CD1a and DC-SIGN expression was analyzed by flow cytometry. Results are expressed as percentages ± SD of CD1a<sup>+</sup> DC-SIGN<sup>+</sup> positive cells in the upper right part of each figure. Percentages of CD1a<sup>+</sup> DC-SIGN<sup>+</sup> dendritic cells decreased with increasing GF/monocyte ratio. Percentages are means ± SD of three independent experiments. B. Monocytes cultured with conditioned medium (CM) from GFs in the presence of MD for 7 days. Results are representative of four separate experiments and results are expressed as percentages ± SD of CD1a<sup>+</sup> DC-SIGN<sup>+</sup> positive cells in the upper right part of each figure. C. The differentiation of monocyte-derived DCs was also monitored by assessing the expression of CD1a and CD14 on day 7. The percentage of CD1a<sup>+</sup> dendritic cells was significantly lower (P<0.0001) and that of CD14<sup>+</sup> cells significantly higher (P<0.0001) in the presence of CM than in controls. Results are expressed as means ± SD and the histogram reports the results of 12 independent experiments using monocytes from 12 different donors. CM: conditioned medium. * Significant difference (P<0.0001).</p
Detection of IL-6, VEGF and TGFÎČ1 by ELISA in CM and supernatant from cell culture (pg/mL).
<p>Results are representative of 4 independent experiments realized in duplicate.</p><p>IL-13 and IL-10 were not detected in CM and supernatant from cell culture.</p><p>MD: medium for differentiation (rGM-CSF+rIL-4).</p><p>CM: conditioned medium from gingival fibroblasts.</p><p>D7: after 7 days in culture.</p><p>ND: not detected.</p
Co-localization of CD90<sup>+</sup> gingival fibroblasts (GFs) and CD14<sup>+</sup> monocytes in superficial gingival connective tissue.
<p>A. Numerous GFs are revealed as red fluorescence by PE-conjugated secondary antibody (magnification Ă40). B. CD14<sup>+</sup> monocytes are revealed as green fluorescence by FITC-conjugated secondary antibody (magnification Ă40). C. DAPI staining with superposition of red and green fluorescence showing the close proximity of the two cell populations in the gingival tissue, consistent with communication between these cell types (magnification, Ă40). D. Isotype-matched antibodies as negative controls (magnification Ă40), scale bar: 50 ”m.</p