Effects of combined IL-12 and IL-27 given as a single shot or continuously in culture.

<p><i>(A)</i>. CD4⁺ T cells were cultured with the relevant peptide and either left untreated (peptide) or treated for 2 days with combined IL-12 (5 ng/ml) and IL-27 (100 ng/ml) and then washed and left untreated (IL-12+IL-27 (2-days)) or treated continuously with IL-12 and IL-27 at the same doses for 2 weeks (IL-12+IL-27 (14 days)). At day 14, cells were tested in the presence or the absence of the relevant peptide and the specific IL-5, IL-13 and IFN-γ release in the supernatant tested by ELISA. The data are means of duplicate determination±SD. The basal level of cytokines secretion of CD4⁺ T cells in the presence of LCL only was subtracted from the sample values and was as follows: IL-5 (0,093±0,006 ng/ml), IL-13 (0,468±0,028 ng/ml) and IFN-γ (0 ng/ml). The data are representative of four experiments. (<i>B</i>). Surface expression of CRTH2, CCR4 CCR5 by CD4⁺ T cells after treatment with combined IL-12+IL-27. Analysis was performed on cells treated for 2 days and then left untreated for a further week. Filled histograms represent isotype controls; open histograms samples stained with the indicated markers.</p

Combined treatment with IL-12 and IL-27 modulates polarization of Th2 and enhances IFN-γ production by pre-existing Th1 anti-CEA CD4⁺ T cells.

<p>CD4⁺ T cells from pt#43 and ND#11 were cultured in five replicates, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007234#s2" target="_blank">Materials and Methods</a>, with the responsive CEA peptides, in the absence (grey bars) or in the presence (black bars) of the combined treatment with IL-12 (5 ng/ml) plus IL-27 (100 ng/ml). After 14 days, IL-5, IL-13, GM-CSF and IFN-γ release was tested by ELISA. Data reported are means of duplicate determination±SD. Dashed lines identify the basal level of cytokine secretion in the presence of APC only. n.d. = not determined.</p

Effect of titrated doses of IL-12 or IL-27 as single agent on the repertoire of cytokine secreted by CEA_{177–189/355–367} specific CD4⁺ T cells.

<p><i>(A).</i> CD4⁺ T cells were cultured with the relevant peptide and irradiated LCL as APC in the presence of increasing concentrations of IL-27 (0-0,1-1-10-100 ng/ml); after 2 days the cytokine release was evaluated by CBA (IL-5 IL-4, and IFN-γ) or ELISA (IL-13). The data for IL-13 are means of duplicate determination±SD. <i>(B).</i> CD4⁺ T cells were cultured and tested, as described above, in the presence of increasing concentrations of IL-12 (0-5-20 ng/ml). The basal level of cytokines secretion of CD4⁺ T cells in the presence of LCL only was subtracted from the sample values and was comprised between 0 and 0,018 ng/ml. The data are representative of at least three experiments.</p

Modulation of Th2 polarization of CEA_{177–189/355–367} specific CD4⁺ T cells by combined IL-12 and IL-27 treatment.

<p>CD4⁺ T cells were cultured with the relevant peptide in the absence (basal) or in the presence of IL-12 (5 ng/ml), as single agent; or IL-27 (100 ng/ml), as single agent; or combined IL-12 and IL-27 in a 2-day stimulation assay and then tested for cytokine release by CBA (IL-5 and IFN-γ) or ELISA (IL-13 and GM-CSF). The data for IL-13 and GM-CSF are means of duplicate determination±SD. The basal level of cytokines secretion of CD4⁺ T cells in the presence of LCL only was subtracted from the sample values and was comprised between 0 and 0,006 ng/ml. The data are representative of five experiments.</p

Characterization of CEA_{177–189/355–367} specific CD4⁺ T cell clones from pt#15.

<p><i>Profile of cytokine secreted (A)</i>. CD4⁺ T cells were cultured with irradiated APC in the presence or the absence of the relevant peptide in a 2-day stimulation assay and the concentration of the indicated cytokines in the supernatants was determined either by CBA (upper panel) or ELISA (lower panel). The data are representative of at least six experiments; for ELISA assays, the data are means of duplicate determination±SD. <i>HLA restriction (B)</i>. CD4⁺ T cells were cultured with irradiated autologous PBMC or HLA-DR matched LCL, as indicated, in the presence or the absence of the relevant peptide (10 µg/ml) and in the absence or the presence of anti-HLA-DR, anti-HLA-DP and anti-HLA-DQ mAbs: after 2 days IL-5 was tested. Upper panel: % inhibition was calculated based on IL-5 secretion by CD4⁺ T cells in the presence of the relevant peptide (2 ng/ml over 0 ng/ml of background level). The data are representative of two (upper panel) and four (lower panel) experiments and are means of duplicate determination±SD. Responses significantly higher than the blanks (<i>i.e.</i>, the basal levels of cytokines secretion from CD4⁺ T cells in the presence of LCL only) are indicated as: ***, p<0.001 (determined by unpaired, one-tailed Student's t test). <i>Dose-response curves (C)</i>. CD4⁺ T cells were cultured with titrated doses of the relevant peptide in the presence of irradiated APC in a 2-day stimulation assay and tested for IL-5 and IL-13 release. The data are means of duplicate determination±SD. <i>Recognition of the native protein (D)</i>. CD4⁺ T cells were cultured in the presence of irradiated PBMC, as APC, pulsed with either the relevant peptide (10 µg/ml) as positive control, or the purified CEA protein (30 µg/ml) or human IgG (30 µg/ml) as negative control in a 2-day stimulation assay and tested for IL-13 release. The data are means of duplicate determination±SD. Responses significantly higher than the blanks (<i>i.e.</i>, the basal levels of cytokines secretion from CD4⁺ T cells in the presence of PBMC only) are indicated as: **, 0.001TCRVβ expression (E). The test was performed with the IO Test Beta Mark kit. Quadrants were set based on isotype control staining. <i>Surface expression of Th2 (CRTH2 and CCR4) and Th1 (CCR5) markers (F)</i>. Filled histograms represent isotype controls; open histograms samples stained with the indicated markers.</p

Identification of linc-NeD125, a novel long non coding RNA that hosts miR-125b-1 and negatively controls proliferation of human neuroblastoma cells

<div><p>ABSTRACT</p><p>The human genome contains some thousands of long non coding RNAs (lncRNAs). Many of these transcripts are presently considered crucial regulators of gene expression and functionally implicated in developmental processes in Eukaryotes. Notably, despite a huge number of lncRNAs are expressed in the Central Nervous System (CNS), only a few of them have been characterized in terms of molecular structure, gene expression regulation and function. In the present study, we identify linc-NeD125 as a novel cytoplasmic, neuronal-induced long intergenic non coding RNA (lincRNA). Linc-NeD125 represents the host gene for miR-125b-1, a microRNA with an established role as negative regulator of human neuroblastoma cell proliferation. Here, we demonstrate that these two overlapping non coding RNAs are coordinately induced during <i>in vitro</i> neuronal differentiation, and that their expression is regulated by different mechanisms. While the production of miR-125b-1 relies on transcriptional regulation, linc-NeD125 is controlled at the post-transcriptional level, through modulation of its stability.</p><p>We also demonstrate that linc-NeD125 functions independently of the hosted microRNA, by reducing cell proliferation and activating the antiapoptotic factor BCL-2.</p></div

Mir-23a and mir-125b regulate neural stem/progenitor cell proliferation by targeting Musashi1

<div><p>Musashi1 is an RNA binding protein that controls the neural cell fate, being involved in maintaining neural progenitors in their proliferative state. In particular, its downregulation is needed for triggering early neural differentiation programs. In this study, we profiled microRNA expression during the transition from neural progenitors to differentiated astrocytes and underscored 2 upregulated microRNAs, miR-23a and miR-125b, that sinergically act to restrain Musashi1 expression, thus creating a regulatory module controlling neural progenitor proliferation.</p></div