Comment motiver des élèves de 1ère ES pour un investissement en SVT ?:Un travail commun SES-SVT devrait permettre de susciter l’intérêt des élèves pour cet enseignement scientifique.

Professorat des lycées et collègesNous avons tenté une approche interdisciplinaire SES-SVT des techniques médicales liées à la procréation et leurs répercussions dans notre société. Ce projet a été réalisé en parallèle dans deux classes de première ES, au lycée M. CURIE et au lycée M. RUDLOFF. Nous avons cherché, au travers d’un travail en groupe autour de la réalisation d’une affiche, à motiver nos élèves. L’aspect interdisciplinaire de notre projet n’a pas été pleinement perçu par nos élèves. Mais ils ont, par contre, montré un certain enthousiasme pour la création des affiches et leur exposition à l’ensemble de la classe. Les résultats sont certes mitigés, mais le bilan nous apparaît positif, car nous avons réussi à susciter l’intérêt d’élèves non scientifiques avec ce projet

Characterization of the Peri-Membrane Fluorescence Phenomenon Allowing the Detection of Urothelial Tumor Cells in Urine

International audienceSimple Summary To detect bladder cancer (BC), urinary cytology and cystoscopy are the primary diagnostic tests used. Urine cytology is non-invasive, easy to collect, with medium sensitivity and high specificity. It is an effective way to detect high-grade BC, but it is less effective on low-grade BC because the rate of equivocal results is much higher, making them difficult to detect. Despite the implementation of new diagnostics, urinary cytology and cystoscopy remain the gold standard. Instead of looking for new diagnostics, one of the new research areas is the improvement of urinary cytology. Recently, the fluorescent properties of plasma membranes of urothelial tumor cells, called peri-membrane fluorescence, found in urinary cytology have been shown to be useful in improving the early detection of BC. The main objective of this study was to characterize the peri-membrane fluorescence allowing the detection of urothelial tumor cells in urine. Urine cytology is non-invasive, easy to collect, with medium sensitivity and a high specificity. It is an effective way to detect high-grade bladder cancer (BC), but it is less effective on low-grade BC because the rate of equivocal results is much higher. Recently, the fluorescent properties of plasma membranes of urothelial tumor cells (UTC) found in urine cytology have been shown to be useful in improving the early detection of BC. This phenomenon is called peri-membrane fluorescence (PMF). Based on previous studies that have identified the PMF on UTCs, the main objective was to characterize this phenomenon. For this study, a software was specially created to quantify the PMF of all tested cells and different treatments performed. PMF was not found to be a morphological and discriminating feature of UTCs, all cells in shape and not from urine show PMF. We were able to highlight the crucial role of plasma membrane integrity in the maintenance of PMF. Finally, it was found that the induction of a strong cellular stress induced a decrease in PMF, mimicking what was observed in non-tumor cells collected from urine. These results suggest that PMF is found in cells able to resist this stress, such as tumor cells

Inhibition of the differentiation of monocyte-derived dendritic cells by human gingival fibroblasts.

We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism

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 Transwell® 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 µm, original magnification ×400.</p

Antibodies against VEGF and IL-6 reduce the inhibitory effect of conditioned medium (CM) from GFs.

<p>A. Neutralizing antibodies against VEGF (10 µg/ml) and IL-6 (10 µg/ml) significantly reduced (P<0.05) the inhibition of CD1a+ monocytes-derived cells by CM from GFs. In contrast, neutralizing antibodies against TGFβ1 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

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 (P = 0.002) and **Significant difference (P<0.001).</p

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

Detection of IL-6, VEGF and TGFβ1 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⁺ gingival fibroblasts (GFs) and CD14⁺ monocytes in superficial gingival connective tissue.

<p>A. Numerous GFs are revealed as red fluorescence by PE-conjugated secondary antibody (magnification ×40). B. CD14⁺ monocytes are revealed as green fluorescence by FITC-conjugated secondary antibody (magnification ×40). 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, ×40). D. Isotype-matched antibodies as negative controls (magnification ×40), scale bar: 50 µm.</p

Soluble factors from gingival fibroblasts (GFs) inhibit the differentiation of CD1a⁺ 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 Transwell® chamber system. The effect of several GF/monocyte ratios (1∶25 to 1∶5) on CD1a and DC-SIGN expression was analyzed by flow cytometry. Results are expressed as percentages ± SD of CD1a⁺ DC-SIGN⁺ positive cells in the upper right part of each figure. Percentages of CD1a⁺ DC-SIGN⁺ 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⁺ DC-SIGN⁺ 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⁺ dendritic cells was significantly lower (P<0.0001) and that of CD14⁺ 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