Symbols, GenBank accession numbers and descriptions of the genes mentioned in the text.

<p>Symbols, GenBank accession numbers and descriptions of the genes mentioned in the text.</p

Metaclopramide diminishes the abnormal appearance of CD4⁺CD8⁺ cells in subcutaneous lymph nodes from <i>T. cruzi</i> infected mice.

<p>The figure shows that in mice treated with the vehicle alone, acute <i>T. cruzi</i> infection did induce the abnormal appearance of CD4⁺CD8⁺ cells in subcutaneous lymph nodes (SCLN). Although such cells were also detected after <i>in vivo</i> treatment metoclopramide (MET, which promotes an increase in circulating PRL levels) their absolute numbers were significantly lower than those recorded in infected counterparts treated with the vehicle alone. Uninfected and infected mice received daily s.c. MET from 10 to 14 dpi or PBS as vehicle. At 15 dpi they were killed and their SCLN removed, homogenized and counted using Neubauer chamber. One million viable cells were immunostained with anti-CD4, anti-CD8 and anti-TCRβ antibodies, and CD4⁺CD8⁺ T cells were thus characterized. Absolute numbers of these CD4⁺CD8⁺ T cells were expressed as mean ± SE. These data are representative of two independent experiments using three mice per group in each experiment. Statistically significant differences (p<0.05) between uninfected versus infected (<b>*</b>) or between 8 and 15 dpi (<b>#</b>) mice. ***p<0.001.</p

Trypanosoma cruzi induces changes in gene expression of glucocorticoid and prolactin receptors in CD4⁺CD8⁺ cells and alters their sensitivity to GC-induced apoptosis.

<p>Panel <b>A</b> (left) shows gene expression levels for the receptors for glucocorticoids (GR) and prolactin (PRLR) in CD4⁺CD8⁺ thymocytes, as ascertained by real time quantitative PCR. There is a progressive decrease in GR expression levels during T. cruzi infection, which were paralleled by an increase in PRLR gene expression. The values were normalized to the endogenous reference transcript RPL-13. Values are expressed as fold changes related to uninfected animals and represented as (2<b>^−ΔΔct</b>). Panel <b>A</b> (right) represents the ratio of GR/PRLR in CD4⁺CD8⁺ thymocytes during infection. These data are representative of three independent experiments using three mice per group in each experiment. Statistically significant differences (p<0.05) between uninfected versus infected (<b>*</b>) or between 8 and 15 dpi (<b>#</b>) mice. Panel <b>B</b> (left) depicts progressive increase in apoptosis (revealed by the relative numbers of Annexin V⁺CD4⁺CD8⁺ cells). We can see that in cells treated with vehicle alone or after inbubation with dexamethasone (DEX) there is an increase in the numbers of apoptotic thymocytes. However, if we evaluate the ratio of vehicle-treated over DEX-treated cells (panel <b>B</b> right) the sensitivity to DEX-induced apoptosis on CD4⁺CD8⁺ thymocytes is significantly decreased as disease progresses. Thymyses were removed from uninfected, 8 and 15 dpi mice and homogenized. One million cells/animal were incubated with DEX (10⁻⁹ M) or RPMI (vehicle) during 8 hours under 37°C at 5% CO₂ atmosphere. Thymocytes were then washed and incubated with anti-CD4/APC, anti-CD8/PercP or Annexin-V/FITC, for the cytofluorometric characterization of apoptosis within each thymocyte subset. These data are representative of two independent experiments using five mice per group in each experiment. Statistically significant differences (p<0.05) between uninfected versus infected (<b>*</b>) or between 8 and 15 dpi (<b>#</b>) mice. *p<0.05; **p<0.01, ***p<0.001.</p

The metoclopramide-induced reestablishment of PRL synthesis in infected mice prevents thymus atrophy.

<p>As shown in panel <b>A</b>, metoclopramide (MET) does increase PRL serum levels, but without affecting the <i>T. cruzi</i>-induced rise in serum corticosterone. Yet, as observed in <b>B</b> the CD4⁺CD8⁺ thymocyte subset profiles were partially reestablished in MET-treated infected animals, and so was the size of their thymuses (representative images seen in panel <b>C</b>; scale bar = 1 cm). Thymic recovery was further confirmed by the partial yet significant restoration of total thymocyte numbers as well as absolute numbers of CD4⁺CD8⁺ cells (<b>D</b>). MET also reduced the percentages of apoptotic CD4⁺CD8⁺ thymocytes, including those exhibiting activated caspase-3 (<b>E</b> and <b>F</b>, respectively). Intrathymic levels of corticosterone were increased in MET-treated mice whereas PRL decreased, as demonstrated in <b>G</b>. Uninfected and infected mice received daily MET (2.5 mg/kg/100 µl, s.c.) from 10 to 14 dpi, or PBS as negative control. On 15 dpi, animals were sacrificed and their thymuses and sera removed for analysis. Cytofluorometric profiles for detection of CD4 and CD8 are shown as dot blots (panel <b>B</b>) with inserted numbers corresponding to percentages of CD4⁺CD8⁺ subset as mean ± SE. CD4⁺CD8⁺ apoptotic cells were characterized as AnnexinV-FITC positive staining, whereas the activity of caspase-3 was ascertained as FITC-VAD-FMK positive cells. The values are given as percentage of CD4⁺CD8⁺ cells with active caspase-3. In all cases, data are representative of two independent experiments using five mice per group. Corticosterone and PRL levels were determined, respectively by radioimmunoassay and ELISA. Intrathymic contents are represented as pg/mg thymus and systemic levels as ng/mL. Statistically significant differences (p<0.05) between uninfected versus 15 dpi (<b>#</b>) mice. *p<0.05; **p<0.01, ***p<0.001.</p

Nucleotide sequences of primers used for the analysis of transcripts and the size of the corresponding amplicons.

<p>Nucleotide sequences of primers used for the analysis of transcripts and the size of the corresponding amplicons.</p

CD4⁺CD8⁺ depletion in <i>T. cruzi</i> infected animals is related to the increased systemic GC levels, subsequent to decrease in serum PRL.

<p>In panel <b>A</b>, the effects of the PRL synthesis inhibitor, bromocriptine (BRC) is shown, whereas the effects of adrenalectomy (thus abrogating adrenal-derived GCs) are seen in panel <b>B</b>. BRC-induced shutdown of pituitary PRL production increases serum GC levels, but adrenalectomy does not promote a reciprocal rise in PRL. Moreover, BRC decreases thymocyte numbers in infected animals, whereas ADX promote a significant increase in the total numbers of these cells. Infected and uninfected animals were daily given BRC (10 mg/kg/100 µl s.c.) or vehicle alone for 15 days, when they were killed under non-stressor conditions. Mice were adrenalectomized one week prior to infection, and sham surgery was performed in control animals. Sera were used for quantifying PRL (by ELISA) and corticosterone (by radioimmunoassay). Simultaneously, thymuses were removed and total thymocyte numbers determined. Data are representative of two independent experiments using five to eight mice per group in each experiment. Statistically significant differences (p<0.05) between uninfected versus infected (<b>*</b>) or between 8 and 15 dpi (<b>#</b>) mice. *p<0.05; **p<0.01, ***p<0.001.</p

The intrathymic production of glucocorticoids and prolactin is differentially modulated by <i>T. cruzi</i> infection.

<p>Panel <b>A</b> reveals the transient decrease of GC production whereas panel <b>B</b> shows the differential gene expression levels of the enzymes 11β-HSD1 and 11β-HSD2. In parallel with the transient decline in GC production, there is a transient increase in prolactin contents as shown in panel <b>C.</b> Intrathymic <i>in situ</i> prolactin labeling is seen in panel <b>D</b>, through representative confocal microscopy images of thymuses from uninfected and infected mice. In this panel, thymuses were stained with the anti-PRL antibody, whereas the lower right box corresponds to the negative control for the immunostaining in which the anti-PRL was replaced by an unrelated goat IgG. The quantification of fluorescence intensity, separately done in the cortex and medulla of thymic lobules, revealed that the rise in PRL contents occured in both regions, as seen in panel <b>E.</b> As expected, bromocriptine (BRC), as an inhibitor of prolactin secretion by the anterior pituitary gland, promotes a shutdown of systemic prolactin levels (compared to the treatment with the vehicle alone), as measured in the blood (panel <b>F</b> left). However, injection of this compound into normal mice does not induce a decrease in the intrathymic PRL contents, as seen in panel <b>F</b>, when comparing BRC- versus vehicle-treated animals. Indeed, a slight (but significant) increase of PRL in the thymus could be detected in BRC-treated animals. Both intrathymic and systemic corticosterone levels remained unchanged in these animals (panel F, <b>right</b>). The intrathymic levels of corticosterone were determined by radioimmunoassay, whereas prolactin contents were measured by ELISA in thymus lysates from normal and infected (8 and 15 dpi) mice. In both cases, results were expressed as pg/mg thymus. The transcript levels of 11β-HSD1 and 11β-HSD2 genes were ascertained by quantitative real time PCR. The results represent the amount of transcripts, in relation to the housekeeping gene RPL-13 as 2^−Δct. As for the confocal microscopy, the original magnification was ×200, with the scale bars = 20 µm. Graphs in panel <b>E</b> correspond to quantification of PRL contents, defined by immunofluorescence in 5 microscopic fields of each thymus from uninfected (n = 3) or infected animals (n = 3), with data being represented as pixels/µm². Male mice were daily given s.c. BRC (100 µg/animal) during three days, when they were sacrificed and their thymuses and plasma obtained. Intrathymic and plasma PRL contents were measured by ELISA, with data being expressed as ng/mL in sera, and as pg/mg in the thymus. Statistically significant differences (p<0.05) between uninfected versus infected (<b>*</b>) or between 8 and 15 dpi (<b>#</b>) mice. *p<0.05; **p<0.01, ***p<0.001.</p