Mouse groups and AmBisome treatment schedules.

a<p>Mice were either non treated (NT) or treated (T) with AmBisome during the acute (A) and/or chronic (C) phases of infection by intraperitoneal injections (<i>i.p.</i>) on alternate days. Some mice of indicated groups received cyclophosphamide administred on alternate days from dpi 60. Data in brackets indicate the first and the last post-inoculation day (dpi) of treatment.</p

Growth of pups either uninfected or congenitally infected with TcII and TcVI.

<p>See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002271#pntd-0002271-g001" target="_blank">Figure 1</a> for group nomenclature; uninfected offspring were born to infected or uninfected mice; * = P<0.05, ** = P<0.01, *** = P<0.001.</p

Reductions of DNA parasite loads in organs of AmBisome-treated mice.

<p>For each mouse group, reduction ratios are expressed in fold decrease compared to the mean of NT group.</p

Intracellular detection of IFN-g in CD56^bright and CD56^dim NK cells after IL-15 and <i>T. cruzi</i> incubation.

<p>Proportion of blood CD56^bright (AB) and CD56^dim (BD) NK cells from neonates (A and C) and adults (B and D), expressing IFN-g after 24 h incubation with <i>T. cruzi</i> live trypomastigotes (ratio 1 parasite per cell) in the presence or not of rhIL-15 (20 ng/mL). Results are shown as Box-and-Whisker plots (n = 36 cord and 13 adult blood samples). Proportions of responding individuals correspond to percentages of samples presenting a proportion of IFN-g positive cells above the cut-off, calculated as the mean+2SD of the % of IFN-g+ cells observed without stimulation. *: p<0.05, **: p<0.005, ***: p<0.0005 as compared with unstimulated cells, ###: p<0.0005 as compared with the effect of IL-15 alone (Wilcoxon paired test), $: p<0.05 as compared to cord blood cells (Mann-Whitney U test).</p

Survival and course of <i>T. cruzi</i> infection in AmBisome -treated BALB/c mice.

<p>BALB/c mice were <i>i.p.</i> inoculated with 1,000 blood trypomastigotes. NT: untreated mice; AmBisome was administred either in early acute phase (TeA), in acute phase (TA), or in chronic phase (TC). TAC mice treated by AmBisome during the acute and the chronic phases of infection. (A) Parasitaemia determined by fresh blood microscopic examination. AmB: AmBisome administration period, CP: cyclophosphamide administration period. (B) Survival curve of <i>T. cruzi</i>-infected mice.</p

Role of myeloid cells and TLRs in IL-15 and <i>T. cruzi</i> driven IFN-g production by NK cells.

<p>Proportion of cord blood CD56^bright NK cells expressing IFN-g after 24 h incubation of CBMC, monocyte- or mDCs-depleted CBMC or reconstituted CBMC cells (depleted CBMC+purified cells) with or without <i>T. cruzi</i> live trypomastigotes at 1∶1 parasite∶cell ratio in presence of IL-15 (20 ng/mL - A). For further investigation (B, C, D), we used only the IL-15+<i>T. cruzi</i> 1∶1 condition. To investigate the need for contact between monocytes and other CBMC in activation of NK cells, purified monocytes were cultured at the upper side of a transwell insert while monocyte-depleted CBMC were at the bottom. As control, reconstituted CBMC were cultured at both sides. Parasites were added at both sides (B). To investigate the requirement of live parasites, CBMC were cultured in the presence of live or lysed trypomastigotes (C). To investigate the requirement of contact between CBMC and parasites, CBMC were cultured at the bottom of a transwell insert in the presence or not of parasites at the bottom (D). To investigate the requirement of TLRs, we added anti-TLR-2 and/or anti-TLR-4 blocking Ab or unrelated IgG during culture (E). Results are shown as Box-and-Whisker plots (n = 5–6). * or #: p<0.05 as compared with total CBMC or reconstituted or unrelated IgG treated CBMC respectively (Wilcoxon paired test).</p

Production of cytokines by cord and adult blood cells in response to <i>T. cruzi</i> and/or IL-15.

a<p>Results are shown as medians (min-max values) of 8 cord blood samples and 6 adult blood samples.</p>b<p>Cord or adult mononuclear cells were culture for 24 h in the presence or not of <i>T. cruzi</i> live trypomastigotes (parasite to cell ratio 1∶1) and/or IL-15 20 ng/mL.</p>*<p>p<0,05 as compared with unstimulated cells (NS), Wilcoxon paired test.</p>#<p>p<0,05 as compared with the effect of IL-15 alone, Wilcoxon paired test.</p>$<p>p<0,05 as compared with cord blood cell response, Mann-Whitney U test.</p

Congenital transmission of parasites in mice infected with TcI, TcII and TcVI.

<p>See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002271#pntd-0002271-g001" target="_blank">Figure 1</a> for group nomenclature; n = number of positive cases; N = total number of examined pups; the 8 congenitally-infected cases were detected by microscopic blood examination on D15 or D30 after birth; all the other examined pups remained negative at microscopic blood examinations on D15 or D30 and blood/heart qPCR studies performed on animals sacrificed after CP-treatment (see the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002271#s3" target="_blank">results</a> section); parenthesis in the congenital transmission rate column indicate the estimated theoretical maximum rate of congenital infection according to the numbers of studied pups per mouse group.</p

Parasitemias in dams acutely or chronically infected with TcI, TcII or TcVI.

<p>ING = infected and non-gravid mice; see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002271#pntd-0002271-g001" target="_blank">Figure 1</a> for nomenclature of IAM, CI and CI² groups; parasitemias were recorded at delivery for IAM (dpi 7–9), CI (dpi 94–96), CI² dams (dpi 9–11 of reinfection) and at dpi 7 for ING mice; parasitemias in TcI acute-infection and TcII and TcVI chronic infections were recorded by qPCR, whereas those of acute TcII and TcVI infections were determined by blood microscopic investigation. * = P<0.05 by comparison to the ING group.</p

Tissue parasite amounts in AmBisome-treated mice.

<p>NT: untreated mice; AmBisome was administred either in early acute phase (TeA), in acute phase (TA), or in chronic phase (TC). TAC mice treated by AmBisome during the acute and the chronic phases of infection. Organs were collected either in acute phase (dpi 21) or in chronic phase (dpi 74) from mice having received or not cyclophosphamide (CP). Both TcZ and GAPDH sequences were quantified individually for each DNA sample. The amounts of parasite DNA in samples were expressed in parasite equivalents per mL of blood (A) or per 50 ng DNA for heart (B), liver (C), spleen (D), muscle (E) and adipose tissue (F). * denotes a significant difference with NT acute mice group, # denotes a significant difference with NT chronic mice group, § denotes a significant difference between treatments, *, #, § P<0.05; **, ##, §§ P<0.01; ***, ###, §§§ P<0.001.</p