Polarization of CD3⁺ lymphocytes by whole tumour-lysate pulsed DCs.

<p>To examine the nature of T-cell responses induced by whole tumour-lysate pulsed DCs, syngeneic naïve T-cells from MMTV-<i>Ras</i> mice were cultured with either unloaded DCs or TAA-DCs for 1-3-5-7- days and both T cell- and DC-derived cytokines were measured. ELISA quantified released type 1 [IFN-γ (A), IL-12 (C)] and type 2 [IL-10 (B)] cytokines in the supernatants. Results are expressed as mean values ± standard error of three independent experiments. *P value < 0.05; **P value <0.005.</p

Gene expression profile of tumour-lysate loaded DCs.

<p>mRNA expression of genes involved in DC activation and maturation in day-6 DCs following 6-16-24 hour incubation with whole tumour-lysate are shown. The expression of 84 genes involved in antigen uptake/loading (A) and presentation (B), surface receptor signalling (C), signal transduction (D), cytokine (E) and chemokine secretion (F and G) and cytokine and chemokine receptor signalling (H) was assessed by real-time quantitative RT-PCR and shown as fold-change expression from the unloaded DC samples. Controls were also included on each array for genomic DNA contamination, RNA quality, and general PCR performance. Genes showing a twofold change in the level of mRNA expression of the loaded versus unloaded DCs in two of three independent experiments (fold change ≥2) were considered significantly up- or down-regulated. Only the targets showing different expression levels are presented. Mean values ± standard error are shown.</p

CFSE assay for assessment of T-cell function.

<p>The 7 day response of CFSE-labelled syngeneic T-cells from the spleen of MMTV-<i>Ras</i> mice to stimulation with tumour lysate-pulsed DCs. CD3/CFSE dot plot (A) and CFSE histogram (B and C, anti-CD3 activated T cells and unstimulated T-cells, respectively) are shown. All plots were gated on CD3-positive cells, while the histograms were also gated to include both resting lymphocytes and blasts. (A): alterations in light scatter characteristics; (B and C): progressive two-fold dilutions of CFSE that accompanied mitotic cell division (gate D1). Both anti-CD3 activated and unstimulated T-cells were incubated with tumour lysate pulsed-DCs, unpulsed DC (negative control) and LPS-stimulated DCs (positive control). Application of an analysis algorithm (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146622#sec006" target="_blank">Materials and Methods</a>) resulted in a proliferation index of 2,93. One representative of three independent experiments is shown.</p

Phenotype of CD4⁺ and CD8⁺ T-cell subsets after <i>in vivo</i> immunization.

<p>Syngeneic splenocytes from MMTV-<i>Ras</i> mice from both untreated, control and DC-treated mice were CD3 enriched by magnetic separation and specifically stained for CD4 and CD8. (A) expression of CD25 and CD69 by CD8 positive cells; (B) production of IL-10, IL-12, IFN-γ and TNF-α by CD8⁺ T-cells; (C) production of Granzyme B and Perforin by CD8⁺ T-cells; (D) expression of CD25 and CD69 by CD4 positive cells; (E) production of IL-10, IL-12, IFNγ and TNF-α by CD4 cells; (F) percentage of CD4⁺/CD25⁺/FOXP3⁺ T-cells. Mean values ± SD and statistically significant differences are indicated. *P value < 0.05; **P value <0.005.</p

Immunophenotype of day-7 DCs.

<p>Unpulsed immature DCs (iDCs), tumour lysate-loaded DCs (TAA-DCs) and LPS stimulated-DCs, were set up in parallel and harvested at the same time for phenotype analysis. DCs were identified by MHC-DR, CD11c (A) (upper dot blot and lower dot blot showing gating strategy and MHC II expression on CD11c⁺ cells before and after tumour lysate-loading and LPS stimulation, respectively) and the markers shown in the figure (B). The numbers indicate the MFI (mean fluorescence intensity) for the isotype controls (open histograms) and DC surface markers (shaded histograms). Representative results from one out of three experiments are shown here. Fold-increase in expression levels of maturation markers on tumour-lysate pulsed DCs over iDCs was determined by expressing the MFIs as a ratio of the TAA-DCs to unpulsed iDCs (C).</p

Evaluation of tumor onset.

<p>The graph showed the different timing of tumor onset in DC-treated mice versus controls. Mean values ± SD and statistically significant differences are indicated. *P value < 0.05.</p

Diverse selective regimes shape genetic diversity at <i>ADAR</i> genes and at their coding targets

<div><p>A-to-I RNA editing operated by ADAR enzymes is extremely common in mammals. Several editing events in coding regions have pivotal physiological roles and affect protein sequence (recoding events) or function. We analyzed the evolutionary history of the 3 <i>ADAR</i> family genes and of their coding targets. Evolutionary analysis indicated that <i>ADAR</i> evolved adaptively in primates, with the strongest selection in the unique N-terminal domain of the interferon-inducible isoform. Positively selected residues in the human lineage were also detected in the ADAR deaminase domain and in the RNA binding domains of ADARB1 and ADARB2. During the recent history of human populations distinct variants in the 3 genes increased in frequency as a result of local selective pressures. Most selected variants are located within regulatory regions and some are in linkage disequilibrium with eQTLs in monocytes. Finally, analysis of conservation scores of coding editing sites indicated that editing events are counter-selected within regions that are poorly tolerant to change. Nevertheless, a minority of recoding events occurs at highly conserved positions and possibly represents the functional fraction. These events are enriched in pathways related to HIV-1 infection and to epidermis/hair development. Thus, both <i>ADAR</i> genes and their targets evolved under variable selective regimes, including purifying and positive selection. Pressures related to immune response likely represented major drivers of evolution for <i>ADAR</i> genes. As for their coding targets, we suggest that most editing events are slightly deleterious, although a minority may be beneficial and contribute to antiviral response and skin homeostasis.</p></div

Samples, methods and supporting analyses

Samples, methods and supporting analyse

Vitamin D Receptor Gene Polymorphisms Are Associated with Obesity and Inflammosome Activity

<div><p>To explore the mechanisms underlying the suggested role of the vitamin D/vitamin D receptor (VDR) complex in the pathogenesis of obesity we performed genetic and immunologic analyses in obese and non-obese Saudi individuals without other concomitant chronic diseases. Genomic DNA was genotyped for gene single nucleotide polymorphisms (SNPs) of VDR by allelic discrimination in 402 obese (body mass index –BMI≥30 kg/m2) and 489 non-obese (BMI<30 kg/m2) Saudis. Q-PCR analyses were performed using an ABI Prism 7000 Sequence Detection System. The inflammosome pathway was analysed by PCR, cytokines and plasma lipopolysaccaride (LPS) concentrations with ELISA assays. Results showed that the VDR SNPs rs731236 (G) (TaqI) and rs1544410 (T) (Bsm-I) minor allele polymorphisms are significantly more frequent in obese individuals (p = 0.009, β = 0.086 and p = 0.028, β = 0.072, respectively). VDR haplotypes identified are positively (GTA) (p = 0.008, β = 1.560); or negatively (ACC) (p = 0.044, β = 0.766) associated with obesity and higher BMI scores. The GTA "risk" haplotype was characterized by an up-regulation of inflammosome components, a higher production of proinflammatory cytokines (p<0.05) and a lower VDR expression. Plasma LPS concentration was also increased in GTA obese individuals (p<0.05), suggesting an alteration of gut permeability leading to microbial translocation. Data herein indicate that polymorphisms affecting the vitamin D/VDR axis play a role in obesity that is associated with an ongoing degree of inflammation, possibly resulting from alterations of gut permeability and microbial translocation. These results could help the definition of VDR fingerprints that predict an increased risk of developing obesity and might contribute to the identification of novel therapeutic strategies for this metabolic condition.</p></div

VDR polymorphisms association with BMI scores.

<p>Linear regression analyses were used to compare the VDR genotypes adjusted for the co-variants age and gender. Significance was set at p<0.05. Responsible variable: BMI scores, covariates: age and gender (female vs male). Model 1: VDR rs731236(G):AG/GG <i>vs.</i> AA; Model 2: VDR rs1544410(T): CT/TT <i>vs.</i> CC; Model 3: VDR rs7975232(A):AC/AA <i>vs</i>. CC.</p