Effect of PGs on the proliferation of stimulated PBMC in presence or absence of CMs obtained from hAMTC or hAM treated or not with indometahcin.

<p>(<b>A</b>) Lymphocyte proliferation, stimulated with anti-CD3, in presence of different amount of prostaglandin (PG) PGD2 (⧫), PGE2 (□), PGF2α (<b>Δ</b>) or PGI2 (X). (<b>B</b>) Lymphocyte proliferation, stimulated with anti-CD3, in presence of 100 μl of control medium (UltraCulture) or CM derived from untreated (CM-hAMTC) or indomethacin treated (CM-hAMTC Indo) hAMTC with (white bars) or without (black bars) the addition of a mix of PGs (PGE2 500 ng/ml + PGD2 100 ng/ml + PGF2α 25 ng/ml). (<b>C</b>) Lymphocyte proliferation, stimulated with anti-CD3, in presence of 100 μl of control medium (UltraCulture) or CM derived from untreated (CM-hAM) or indomethacin treated (CM-hAM Indo) hAM with (white bars) or without (black bars) the addition of a mix of PGs (PGE2 25 ng/ml + PGD2 6 ng/ml + PGF2α 10 ng/ml). * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Effects of proteinase K treatment on the inhibitory activity of CM-hAMTC and CM-hAM.

<p>(<b>A</b>) Representative SDS-PAGE of untreated and proteinase K-treated (for 2 and 6 hrs) UltraCulture medium, CM-hAMTC and CM-hAM. Digestion level was calculated as the reduction of the density of the BSA band (arrow). (<b>B</b>) Inhibitory effect on lymphocyte proliferation, induced by anti-CD3 stimulation, of 100 μl of untreated (NT) and proteinase K-treated UltraCulture medium, CM-hAMTC and CM-hAM. Similar results were also obtained using 50 μl of CMs (data not shown). Data represent the mean and SD of at least five independent experiments. * = p<0.05, ** = p<0.01 versus UltraCulture.</p

Inhibition of production of NO, kynurenine, and prostaglandins (PGs) in PBMC culture.

<p>Lymphocyte proliferation, stimulated with anti-CD3, in presence of 100 μl of control medium or CM-hAMTC and CM-hAM with (white bars) or without (black bars) the addition of indomethacin (<b>A</b>), L-NAME (<b>B</b>) or DL-methyl-tryptophan (<b>C</b>). Data represent the mean and SD of at least three independent experiments. * = p<0.05, ** = p<0.01, *** = p<0.001 versus PBMC + αCD3.</p

Inhibitory effect of CM-hAMTC and CM-hAM in presence of neutralizing antibodies.

<p>Lymphocyte proliferation, stimulated with anti-CD3 (PBMC + αCD3), in presence of 100 μl of control medium or CM with (white bars) or without (black bars) the addition of neutralizing antibodies against IL-10 (<b>A</b>), TGF-β (<b>B</b>), HGF (<b>C</b>), IL-6 (<b>D</b>), mix of antibodies anti-IL-10, -TGF-β, -HGF and -IL-6 (<b>E</b>). Data represent the mean and SD of at least four independent experiments. * = p<0.05, ** = p<0.01 versus PBMC + αCD3.</p

Cytokine quantification in UltraCulture medium, CM-hAMTC and CM-hAM.

<p>MAD: Median Absolute Deviation.</p><p>Values are expressed in pg/ml.</p>*<p>quantified by ELISA.</p

Prostanoids quantification in UltraCulture medium and CM derived from untreated or indomethacin treated hAMTC and hAM.

<p>Values are expressed in ng/ml.</p

Analysis of inhibitory effect of hAMTC, BM-MSC and of the respective CMs.

<p>Effects of the following on the proliferation of anti-CD3-stimulated lymphocytes (PBMC + αCD3): (<b>A</b>) hAMTC at passage 0 (white-grey striped bar), different amounts (50–75–100 μl) of CM obtained from the culture of hAMTC at p0 when plated at density of 1×10⁶ cells/ml (CM-hAMTC p0 1*×10⁶/ml) or 0.25×10⁶ cells/0.5 ml (CM-hAMTC p0 0.25*×10⁶/0.5 ml) and different amounts (50–75–100 μl) of CM-hAM; (<b>B</b>) hAMTC at passage 3 (vertical black line bar), BM-MSC at passage 3 (horizontal black line bar) and different amounts (50–100 μl) of CM derived from the culture of these cells. Data represent the mean and standard deviation (SD) of at least four independent experiments. * = p<0.05, ** = p<0.01, *** = p<0.001 versus PBMC + αCD3; ## = p<0.01.</p

Study of the potential molecular weight of the inhibitory factor(s).

<p>(<b>A</b>) Lymphocyte proliferation, stimulated by anti-CD3, in presence of 100 μl of CM-hAMTC (white bar), CM-hAMTC-fraction-mix (grey bars) or mix of fractions with different molecular weights (white-grey- and black-grey-striped bars). (<b>B</b>) Lymphocyte proliferation, stimulated by anti-CD3, in presence of 100 μl of complete UltraCulture medium (black bar) or its fractions (grey and black-grey-striped bars). Data represent the mean and SD from at least six independent experiments. ** = p<0.01, *** = p<0.001 versus PBMC + αCD3; # = p<0.05.</p

NO, kynurenine, and prostaglandins (PGs) in CM-hAMTC and CM-hAM.

<p>(<b>A</b>) NO quantification (total nitrate quantification) in UltraCulture, CM-hAMTC, and CM-hAM performed by means of the Griess test. (<b>B</b>) Lymphocyte proliferation (expressed in cpm) after stimulation with anti-CD3 and in presence of 100 μl of complete UltraCulture medium, CM derived from untreated (black bar), or L-NAME-treated (white bar) hAMTC and hAM cultures. (<b>C</b>) IDO activity, shown as the ratio between the concentration of kynurenine and tryptophan, in the control medium, CM-hAMTC, CM-MLR or CM-MLR-hAMTC, and in the presence of 0.5 mM DL-methyl-tryptophan (CM-MLR-hAMTC+MT). (<b>D</b>) Lymphocyte proliferation (expressed in cpm), induced by MLR, in the presence of hAMTC cultured with or without the addition of DL-methyl-tryptophan. (<b>E</b>) Lymphocyte proliferation (expressed in cpm), stimulated with anti-CD3, in presence of 100 μl of control medium or CM derived from untreated (black bar) or 0.5 mM DL-methyl-tryptophan-treated (white bar) hAMTC and hAM cultures. (<b>F–I</b>) Lymphocyte proliferation (expressed in cpm) after stimulation with anti-CD3, in presence of 100 μl of control medium or CM derived from untreated (black bar) or indomethacin- (<b>F</b>), ketoprofen- (<b>G</b>), niflumic acid- (<b>H</b>) or inhibitors-mix- (<b>I</b>) treated (white bars) hAMTC and hAM cultures. Data represent the mean and the SD of at least three independent experiments. * = p<0.05, ** = p<0.01, *** = p<0.001.</p

sj-png-1-cll-10.1177_09636897231166209 – Supplemental material for Mapping of the Human Amniotic Membrane: In Situ Detection of Microvesicles Secreted by Amniotic Epithelial Cells

Supplemental material, sj-png-1-cll-10.1177_09636897231166209 for Mapping of the Human Amniotic Membrane: In Situ Detection of Microvesicles Secreted by Amniotic Epithelial Cells by Mariangela Basile, Lucia Centurione, Francesca Passaretta, Gianmarco Stati, Olga Soritau, Sergiu Susman, Florelle Gindraux, Antonietta Silini, Ornella Parolini and Roberta Di Pietro in Cell Transplantation</p