Additional file 3: Figure S1. of Dietary intake patterns of children aged 6 years and their association with socioeconomic and demographic characteristics, early feeding practices and body mass index

Direct acyclic graph of the effect of socioeconomic and demographic characteristics, early feeding practices and BMI z-score on dietary intake patterns (Demographic characteristics = maternal age at birth, and child’s sex and skin colour; Early feeding practices = exclusive breastfeeding duration and age of introduction of complementary foods). (DOCX 49 kb

Additional file 1: Table S2. of Dietary intake patterns of children aged 6 years and their association with socioeconomic and demographic characteristics, early feeding practices and body mass index

Multiple linear regression between dietary intake components and BMI z-score at 6 years (BMI as the outcome). Pelotas 2004 birth cohort study (N = 3,427). (DOCX 15 kb

<i>Trypanosoma cruzi</i> acute infection promotes the premature release of CD4⁻CD8⁻ thymocyte cells to the periphery.

<p>BALB/c mice were intrathymically injected with FITC solution or PBS only as a control. After 24 h, lymphoid tissues were harvested and cells stained with anti-CD4 and anti-CD8, for the identification of the specific subsets of recent thymic emigrants (RTEs) by flow cytometry. (<b>A</b>) Graphs represent the absolute numbers of total T cells in the thymus, spleen and subcutaneous lymph nodes (SCLN) during <i>T. cruzi</i> acute infection and uninfected mice. (<b>B</b>) Histograms represent the total numbers of RTEs (FITC⁺ cells) bearing the CD4⁺, CD8⁺, CD4⁺CD8⁺ and CD4⁻CD8⁻ T cell phenotype, obtained from the spleen (<b>right panel</b>) and SCLN (<b>left panel</b>), as indicated. Data are shown as means ± SE (n = 6 mice per group). Differences between uninfected and infected mice are significant * (<i>p</i><0.05), ** (<i>p</i><0.01), *** (<i>p</i><0.001).</p

Splenocytes from <i>T. cruzi</i> infected mice treated with Tc mucin produce low levels of IFN-γ.

<p>BALB/c mice were infected <i>i.p.</i> with 2×10⁵ chemically induced metacyclic forms of <i>Trypanosoma cruzi</i> Dm28c clone. The mice received <i>i.p.</i> injections of Tc Muc (20 µg/mouse) or PBS on alternate days starting at day of infection. Non-infected mice were used as control group. Twenty four days after infection, purified splenocytes were stimulated with PMA and ionomycin, as described in the Methods section, and supernatants were harvested at 24 h for determination of (<b>A</b>) IFN-γ and (<b>B</b>) TNFα by ELISA. The <i>y</i>-axis represents the levels of cytokines, detected by a specific ELISA assay, expressed in ng/ml. Asterisks represent statistical significance (p<0.05) as determined by the Student t test. To access the T cell activation profile CD69 expression on both CD4⁺ (<b>D</b>) and CD8⁺ (<b>E</b>) T cells were analysed by FACS analysis; the frequency of IFN-γ producing T cells from splenocytes polyclonally stimulated with PMA/ionomycin, and the percentages of both IFN-γ producing CD4⁺ T cells (<b>F</b>) and CD8⁺ T cells (<b>G</b>)<b>,</b> were determined by intracellular cytokine FACS-staining. #Infected group are statistically different from non-infected control mice (<i>P</i>≤0.05). Asterisks represent statistical differences between Tc mucin treated <i>versus</i> non-treated mice from infected groups as determined by the Student t test (<i>P</i>≤0.05). All experiments were repeated at least 3 times.</p

Increased numbers of CD4⁻CD8⁻ T lymphocytes in peripheral blood from chronic chagasic patients.

<p>Peripheral whole blood from non-infected individuals, patients at the indeterminate and cardiac forms of Chagas disease were analysed by four-color flow cytometry for expression of CD3, CD4, CD8 and HLA-DR markers. (<b>A</b>) The frequency of circulating human CD4⁻CD8⁻ T cells was measured in peripheral blood from non-infected individuals (n = 10), patients at the indeterminate (n = 12) and cardiac forms of Chagas disease (n = 11). Cells were analyzed by three-color flow cytometry for expression of CD3, CD4 and CD8. The percentages of CD4⁻CD8⁻CD3⁺ T lymphocytes are indicated for each histogram. (<b>B</b>) The activation profile of CD4⁻CD8⁻CD3⁺ T are shown by assessing the expression of HLA-DR. The significant differences between the groups were considered with # (<i>p</i><0.05), * (<i>p</i><0.05) and *** (<i>p</i><0.001).</p

General features of genes whose expression was evaluated by quantitative RT-PCR analysis^*.

*<p>Symbols, GenBank accession numbers and descriptions of the genes mentioned in the text, as well as the corresponding nucleotide sequences of primers used for the analysis of transcripts.</p><p>General features of genes whose expression was evaluated by quantitative RT-PCR analysis^{<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003203#nt101" target="_blank">*</a>}.</p

Down-modulation of intrathymic levels of S1P kinase activity in <i>T. cruzi</i> acutely-infected mice is associated with catabolic gene expression of the S1P pathway.

<p>Thymuses were collected from normal or <i>T. cruzi</i>-infected BALB/c on days 7, 10 and 14 post-infection (dpi) for measurements of the expression of S1P metabolic genes by real-time PCR and the levels of S1P levels by TLC. (<b>A</b>) mRNA levels of murine S1P kinases (SPHK1/2), S1P phosphatases (SGPP1/2) and the S1P lyase (SGPL1) from normal and <i>T. cruzi</i>-infected thymuses. Quantitative PCR results were normalized to values obtained from uninfected thymuses. Gene expression was assessed by comparing the expression of each to the normalizer GAPDH using the Ct method and is represented as mean ± SE of three independent experiments (n = 3 mice per group). (<b>B</b>) S1P kinase activity in thymuses and sera from normal or infected mice were measured by TLC, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003203#s2" target="_blank">Materials and Methods</a>. C₁₇-S1P was used as the internal standard. Graphs show the average values from two independent experiments (n = 6 mice <i>per</i> group), with error bars representing the standard error. Differences between uninfected and infected mice are significant * (<i>p</i><0.05), ** (<i>p</i><0.01), *** (<i>p</i><0.001).</p

Inhibition of CD4⁺ T cell proliferation is partially recovered upon neuraminidase-treatment of <i>T. cruzi</i> mucin.

<p>Purified CD4⁺ T cells from naïve spleens were stimulated with plate bound anti-CD3 for 72 hr, in the presence or absence of increasing concentrations of native or desialylated Tc Muc (10 and 20 µg/mL). Proliferation was measured 72 h after stimulation by [³H]thymidine incorporation. *Differences between native or desialylated Tc Muc treatment <i>versus</i> anti-CD3 stimulated positive controls are significant (<i>P≤</i>0.05). #The inhibition of proliferation by Tc Muc was partially recovered when <i>T. cruzi</i> mucin was desialylated by previous treatment with neuraminidase (<i>P</i> = 0.0023). Results are the means ±SE of triplicate cultures. This experiment was repeated three times, with similar results each time.</p

<i>T. cruzi</i> acute infection increases expression of S1P receptors in CD4⁻CD8⁻ thymocytes and thymic cortical compartment.

<p>(<b>A</b>) Thymuses were stained with anti-S1PR1 monoclonal antibody and analyzed by immunofluorescence. Representative confocal microscopy fields of thymuses stained with anti-S1PR1 monoclonal antibody and goat-anti rabbit alexa-488 secondary antibody are shown in (upper left) normal thymus and (upper right) atrophic thymus at day 14 post-infection. The respective staining controls without the primary antibodies are shown in (lower left) and (lower right). Scale bar = 100 µm. (<b>B</b>) Graphs correspond to relative quantitation analysis of S1PR1 deposition from 5 microscopic fields of thymuses from control (n = 3) or infected animals (n = 3). It is clear the increase in S1PR1 within the cortex of thymic lobules from infected animals. (<b>C</b>) CD4⁻CD8⁻ and CD4⁺CD8⁺ T cells from normal and acutely-infected mice (14 dpi) were purified from thymus, by cell sorting using flow cytometry to assess the transcriptional levels of S1P receptors. Plots show the high purity of isolated CD4⁻CD8⁻ and CD4⁺CD8⁺ T cells (>95%). (<b>D</b>) Total mRNA from purified CD4⁻CD8⁻ and CD4⁺CD8⁺ T cells were processed for RT-PCR. The results represent the amount of transcripts, depicted in relation to the housekeeping gene GAPDH as 2^−ΔCt. There is a significant increase in the contents of both S1PR1and S1PR3 transcripts when comparing control <i>versus</i> infected CD4⁻CD8⁻ T cells, whereas only S1PR3 transcripts increased in the CD4⁺CD8⁺ T subset relative to the controls. Data are means ± SE of two independent experiments using six mice <i>per</i> group. Differences between control and infected mice are significant * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001).</p

Tc Muc induced G1 cell cycle arrest of CD4⁺ T cells correlates with upregulation of Cyclin D3 and downregulation of p27^kip1.

<p>Purified CD4⁺ T cells from naïve spleens were stimulated with plate bound anti-CD3 in the presence or absence of native or desialylated <i>T. cruzi</i> mucins (20 µg/ml). After 3 days, cell cycle analysis (A) was evaluated by flow cytometry based on propidium iodide (PI) intercalation into the cellular chromatin (for details see Materials and methods). The histograms represent the fluorescence intensity of PI for the indicated groups. (B) Flow cytometry cell cycle analysis revealed that the population of cells in the S and G2/M phase was remarkably decreased by Tc muc (<i>P</i>≤0.05). For determination of the cell cycle checkpoint regulators, cells were harvested after 3 days and whole-cell lysates were prepared for immunoblotting with specific atibodies against cyclin D3, p27^kip1, and actin antibodies used to assure uniform loading (bottom row). Optical densitometry of the western blots used NIH Image software, where cyclin D3 and p27^kip1 expression was normalized with the actin expression. (C) Expression of cyclin D3 was increased in anti-CD3 activated CD4⁺ T cells as compared to controls (<i>P≤</i>0.05); this increase was not observed when stimulation was done in the presence of Tc Muc (<i>P</i> = 0.0383); *the sialylation abolished the property of Tc Muc to induce downmodulation of cyclin D3 expression (<i>P = </i>0.0067). Expression of p27^kip1 was decreased in anti-CD3 activated CD4⁺ T cells as compared to controls (<i>P≤</i>0.05); this decrease was not observed when stimulation was done in the presence of Tc Muc (<i>P</i>≤0.05); # the sialylation abolished the property of Tc Muc to induce upregulation of p27^kip1 expression (<i>P≤</i>0.05). These data are representative of two independent experiments.</p