Testosterone modulates murine NKT cell specific IFNγ production.

<p>A) Liver lymphocytes from placebo or testosterone treated naïve female mice were gradient purified, T cells were isolated by magnetic cell sorting and co-cultured for 48 h with αGalCer (2 µg/ml) or EhLPPG (4 µg/ml) pre-pulsed APCs. Medium control comprises NKT cells co-cultured with naive APC’s. IFN<b>γ</b> in the supernatant was quantified using ELISA. B) Liver lymphocytes were isolated from naïve male or orchiectomized male mice and co-cultured with pre-pulsed APCs as described above. IFN<b>γ</b> production was quantified using ELISA. C/D) Identical experiments as performed in A and B but cells were isolated from the livers of ameba-infected female and male mice- Three independent experiments were performed (data express mean +/− SD, statistics: student`s t test).</p

Influence of gonadectomy on the size of ALA in the mouse model for the disease.

<p>Eight weeks old male and female mice were gonadectomized and intrahepatically challenged with 1×10⁵<i>E. histolytica</i> trophozoites seven weeks after castration. The animals were sacrificed 7 days post infection, the sizes of abscesses were determined and transformed into score values (score 0: no abscess; score 1: abscess <1 mm; score 2∶1 to 5 mm; score 3: >5 mm) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055694#pone-0055694-g001" target="_blank">Fig. 1A</a>) or the re-isolation rate of live <i>E. histolytica</i> trophozoites from abscessed liver tissue was determined (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055694#pone-0055694-g001" target="_blank">Fig. 1B</a>). Results were obtained from 3 independent trials each comprising 7 animals (statistics: Mann Whitney U test).</p

Characterization of NKT cells frequencies and NKT cell specific IFNγ production in female and male mice.

<p>A) NKT cell frequencies in the liver of female and male mice at different ages are shown. NKT cell numbers were determined as percentage of αGalCer -CD1d tetramer positive cells to CD3⁺ T cells. B) IFN<b>γ</b> production of NKT cells sorted (αGalCer -CD1d tetramer) from the liver of female or male mice after 48 h of co-incubation with αGalCer (4 µg/ml) or EhLLPG (8 µg/ml) pre-pulsed APCs. Medium control includes NKT cells co-cultured with naive APC’s. IFN<b>γ</b> production was quantified by ELISA (5 independent experiments were performed, a summary of data is expressed as mean +/− SD, statistics: student t test). C) IFN<b>γ</b> production of male or female NKT cells upon stimulation with αGalCer pre-pulsed male or female APCs.</p

Influence of testosterone on the development of ALA.

<p>A) Time schedule of gonadectomy, testosterone substitution and intrahepatic infection with <i>E. histolytica</i> trophozoites. B) Serum testosterone levels in placebo treated naïve female mice, testosterone substituted female mice and male mice measured by ELISA (ng/ml). C) Size of ALA in testosterone treated, ovariectomized (ovx) or naïve female mice. Score values indicative for the size of ALA determined on day 7 post intrahepatic infection with 1×10⁵ virulent <i>E. histolytica</i> trophozoites of naïve female mice treated either with testosterone or placebo are shown (score 0: no abscess; score 1: abscess <1 mm; score 2∶1 to 5 mm; score 3: >5 mm). D) Re-isolation rate of <i>E. histolytica</i> trophozoites from abscessed liver tissue of ovariectomized (ovx) and naïve female mice treated either with testosterone or placebo. Results were obtained from at least 3 independent trials each comprising of 7 animals (statistics: Mann Whitney U test).</p

Characterization of the IFNγ producing gender specific murine NKT cell subpopulation.

<p>NKT cell subsets (PE- αGalCer -CD1d tetramer/CD4⁺) from female and male mice. IFN<b>γ</b> was determined following 48 h of co-cultivation with pre-pulsed APCs as described above (2 independent experiments were performed, a summary of data is expressed as mean +/− SD, statistics: student t test).</p

Median ICC with confidence intervals of serum metabolites.

<p>(A) Metabolites with median ICC-values below 0.65 and (B) metabolites with median ICC values above 0.65 are displayed.</p

Effect of tube type on serum metabolites.

<p>Stars in boxplots indicate significant differences in concentration between methionine sulfoxide in serum W tubes with clotting activator and serum gel-barrier tubes. (Friedman test, significance level p<0.01).</p

Stability of metabolites in plasma during shipment simulation.

<p>Example of (A), (D) decreasing and (B), (C) increasing metabolite concentration of plasma samples at room temperature (RT) and on cool packs (CP). Stars in boxplots indicate significant difference in concentration compared to baseline (0 h). (Wilcoxon signed rank test, significance level p<0.01).</p

Stability of metabolites in serum during shipment simulation.

<p>Example of (A)-(C) increasing and (D) decreasing metabolite concentration during transportation simulation of serum samples on cool packs (CP). Stars in boxplots indicate significant difference in concentration compared to baseline (0 h). (Wilcoxon signed rank test, significance level p<0.01).</p

Impact of transportation simulation on metabolite concentrations in serum samples.

<p>* Metabolites with coefficient of variance across all plates above 25% in reference samples.</p><p>Metabolites that showed significant changes in serum concentration on cool packs for 3, 6 or 24 h compared to baseline (0 h) (Friedman test, p<0.01) and acceptable delay time for each metabolite during transportation (Wilcoxon signed rank test, p<0.01).</p