Development of ALA in EhLPPG treated mice.

<p>24 h prior to intrahepatic challenge with virulent <i>E. histolytica</i> trophozoites, WT mice were treated with 4 µg EhLPPG by i.p. application. Seven days post infection, mice were sacrificed and sizes of abscesses were determined. Shown are sizes of ALA in EhLPPG treated mice relative to untreated controls. Results were obtained from 2 independent experiments comprising 5 to 6 animals each (statistics: Mann-Whitney U test).</p

Structure and NKT cell stimulatory capacity of EhPIs.

<p>(A) The proposed structures of EhPIa (left) and EhPIb (right). The only difference between the EhPIs is the acylation of inositol at position 2 in EhPIb. The fatty acids and the molar ratios are indicated. (B) IFNγ production of NKT cells stimulated by APCs pulsed with α-GalCer, EhLPPG, EhPI, EhPIa and EhPIb. IFN-γ production compared to DMSO control ^*( <i>p</i><0.05);^**( <i>p</i><0.005); ANOVA, Dunnett. The results were obtained from three independent experiments.</p

Role of NKT cells in the control of ALA in mice.

<p>Mice treated with α-GalCer or mice lacking CD1d (CD1d^−/−) were intrahepatically infected with virulent <i>E. histolytica</i> trophozoites. Seven days p.i. animals were sacrificed and sizes of liver abscesses determined. Bars represent sizes of abscess scores relative to those of wildtype control mice (WT). Results were obtained from 3 independent experiments comprising 5 to 7 animals each (statistics: Mann-Whitney U test).</p

EhLPPG is internalized and requires processing or endosomal trafficking for activation of NKT cells.

<p>(A) WT APC were pulsed with 20 µg/ml EhLPPG for 3 h, fixed with 4% PFA and permeabilized with 0.5% saponin; EhLPPG was labeled with mAb EH5 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000434#ppat.1000434-Marinets1" target="_blank">[28]</a> and anti Lamp-1 mAb was used for detection of late endosomes by confocal microscopy. The experiment was performed four times. (B) Treatment of APC with an inhibitor of endocytosis, bafilomycin A1 (10 nM, 30 min), led to a decrease in the IFN-γ production in NKT cells after stimulation with α-GalCer (4 µg/ml) and EhLPPG (10 µg), but not with PamCys (4 µg/ml) and LPS (1 ng/ml). The addition of mAb EH5 (100 µg/ml) to APC pulsed with α-GalCer and EhLPPG specifically inhibited the IFN-γ secretion of cocultured NKT cells.</p

Activation of NKT cells by EhLPPG involves molecules of the TLR signalling pathway.

<p>APC from IL12-p40^−/−, MyD88^−/−, TRIF^−/− (A), CD1d^−/−, TLR2^−/−, 4^−/−, 9^−/− (B), and TLR1^−/−, 2^−/− and 6^−/− (C) were generated, pulsed with EhLPPG and used to stimulate purified T cells from Vα14tg mice. The production of IFN-γ by purifed T cells was determined by ELISA. EhLPPG-dependent NKT cell activation was abrogated in CD1d^−/−, IL-12p40^−/−, MyD88 and TLR 2^−/− and TLR 6^−/− mice. The results were obtained from two independent experiments.</p

Serum cytokine concentrations in HW, HP, ILI and PH1N1 women.

<p>Pro-inflammatory TNF-α (a), IL-1β (b) and IL-6 (c) and anti-inflammatory IL-10 (d) cytokines were quantified using a CBA system with flow cytometry. The Kruskal-Wallis test with Dunn’s multiple comparison post-test was performed using the GraphPad Software. The significance values were *p<0.05, **p<0.01, ***p<0.001.</p

Clinical manifestations in the ILI and PH1N1 women.

<p>ILI: Pregnant women with influenza-like illness.</p><p>PH1N1: Confirmed H1N1pdm2009 virus-infected pregnant women.</p><p>*Fisher’s exact test.</p><p>Clinical manifestations in the ILI and PH1N1 women.</p

Distribution of leukocytes in the blood.

<p>HW. Healthy women.</p><p>HP. Healthy pregnant women.</p><p>ILI. Pregnant women with influenza-like illness.</p><p>PH1N1. H1N1pdm2009 virus-infected pregnant women.</p><p>a. HW vs ILI.</p><p>b. ILI vs PH1N1.</p><p>c. HW vs PH1N1.</p><p>d. HP vs PH1N1.</p><p>e. HW vs HP.</p><p>f. HP vs ILI.</p>±<p>Kruskal-Wallis test with Dunn’s multiple comparison post-test.</p><p>P values: * = p<0.05; ** = p<0.01; *** = p<0.001.</p><p>Distribution of leukocytes in the blood.</p

Higher percentage of CD69+ lymphocytes in H1N1pdm2009 virus-infected pregnant women.

<p>The peripheral blood leukocytes from the HW, HP, ILI and PH1N1 women were immunostained with CD3-, CD19-, CD14- and CD69-specific antibodies and analyzed by flow cytometry. Representative data of each group are shown in the dot plots for CD69+CD3+ cells (a). The distribution of CD69 expression on CD3− (b), CD19− (c) or CD14− (d) gated cells. The Kruskal-Wallis test with Dunn’s multiple comparison post-test was performed using the GraphPad Software. The significance values were *p<0.05, **p<0.01, ***p<0.001.</p

Demographic and obstetric characteristics of the study population.

<p>HW. Healthy women.</p><p>HP. Healthy pregnant women.</p><p>ILI. Pregnant women with influenza-like illness.</p><p>PH1N1. H1N1pdm2009 virus-infected pregnant women.</p><p>Gw. Gestational weeks.</p><p>n/a not applicable.</p><p>*Fisher’s exact test.</p><p>Demographic and obstetric characteristics of the study population.</p