The <i>belr1</i> locus controls <i>Plasmodium</i> liver burden.

<p><i>P. berghei</i> 18S rRNA was quantified by real-time PCR both in the livers of parental mouse strains (C57BL/6 and BALB/c) and in B6.C-H2d congenic mice (A) or in C.B10-H2b congenic mice (B) 40 hours after infection with 10⁴<i>P. berghei</i> sporozoites. The observed difference between C57BL/6 and B6.C-H2d is attributable to the congenic region in chromosome 17 of B6.C-H2d mice while the chromosome 17 congenic region in the C.B10-H2b shows no control over malaria resistance phenotype. Statistical significance was evaluated by unpaired t-test (_* p<0.05, _** p<0.01, _*** p<0.001).</p

Genome-wide mapping of liver stage susceptibility trait.

<p>LOD score curves representing the likelihood for linkage of parasite burden trait. Genome-wide linkage is significant for LOD score above 3.3. (A) Shows LOD score curves for 19 mouse autosomes. (B) Shows LOD score curve for chromosome 17 where the X-axis ticks represent the relative position of microsatellite markers. Markers from left to right: D17Mit62; D17Mit103; D17Mit233; D17Igc1; D17Mit24; D17Mit11; D17Igc2; D17Igc3; D17Mit139; D17Mit20; D17mit152; D17Mit193; D17Mit187; D17Mit221.</p

Variance of liver stage susceptibility trait.

<p>Frequency distribution of parasitemia at day 5 post-infection in the 115 (C57BL/6 X BALB/c) F2 genotyped in this study. The observed frequencies are represented in bars, and the predicted t-distribution curve is superimposed.</p

The liver stage susceptibility trait.

<p>Parasitemia was measured at day 5 post-infection with 10⁴<i>P. berghei</i> sporozoites in 10 C57BL/6 (dark squares), 12 BALB/c (white squares), 11 (C57BL/6 X BALB/c) F1 (inverted triangles) and 11 (BALB/c X C57BL/6) F1 (diamonds). Group averages are shown as horizontal bars.</p

Location of <i>belr1</i> locus in chromosome 17.

<p>The diagram represents the physical map of chromosome 17 congenic regions in B6.C-H2d and C.B10-H2b mouse strains compared to the C57BL/6 strain. Delimitation of <i>belr1</i> locus is represented taking into account the malaria resistance phenotypes in the congenic strains.</p

Scale of motor impairments based on spontaneous motor activity.^*

*<p>Modified from Becher <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049022#pone.0049022-Becher1" target="_blank">[19]</a>.</p

Spinal cord parasitism.

<p>After transcardiac perfusion, spinal cord lower lumbar segments were processed for RNA extraction and cDNA production. Real time RT-PCR was performed using primers for <i>T. cruzi</i> 18S rRNA and HPRT as endogenous control. For the absolute quantification, sample values were fitted into a standard curve, constructed from a pattern sample with known number of copies of <i>T. cruzi</i> 18S rRNA. IL-12p40KO mice presented an ascending parasitism while WT ones managed to constrain the infection by the third week, thus decreasing the parasitic load after the fifth week. Representative pictures of HE-stained upper lumbar spinal cord sections, obtained from WT (upper square) and IL-12p40KO mice (lower square) in the sixth week after the infection. Note the high amount of amastigote nests (arrows) in the IL-12p40KO tissue. Scale bars: 20 µm. Mean ± SEM. ***p<0.001 when comparing paired WT and IL-12p40KO groups according to Bonferroni post-tests. n = 4 for each strain in each time point.</p

Astrogliosis and increased macrophage/microglia density in IL-12p40KO spinal cords.

<p>Paralyzed infected IL-12p40KO mice presented higher immunolabeled area to the astrocytic marker GFAP (A) and increased density of macrophages/microglia (B) in the spinal cord when compared to the matched infected WT group. Pictures in Figure A show astrocytes (arrows) in immunocompetent (left panel) and IL-12p40KO (right panel) spinal cords. No <i>T. cruzi</i>-infected astrocyte was found in WT tissue (left panel inset, arrow points an astrocyte), whereas in IL-12p40KO tissue, only a few astrocytes containing <i>T. cruzi</i> amastigotes were seen (right panel inset, arrow points an astrocyte and arrowheads point parasites). Pictures in Figure B show microglial cell (arrow) with long thin processes in WT tissue (left panel) and round-shaped phagocytes (arrows point macrophages or microglia) in a lesion area of an IL-12p40KO spinal cord section (right panel). While no parasite was found in the WT tissue (right panel inset, arrow shows a microglia), more than a third of all IL-12p40KO macrophages/microglias were infected (right panel upper inset, arrow shows a macrophage/microglia and arrowheads point parasites). M2 cells were visualized in the IL-12p40-KO spinal cord only (right panel lower inset, M2 cells in brown). Sections were immunolabeled to GFAP (A) and CD11b (B, also counterstained with Giemsa), GFAP and <i>T. cruzi</i> (A, inset), CD11b and <i>T. cruzi</i> (B, upper insets) or arginase I (B, right panel lower inset, also counterstained with hematoxylin). Scale bars: A and B, 50 µm; A insets and B upper insets, 20 µm; B right panel lower inset, 50 µm. Data on both graphs are presented as the mean ± SEM. **p<0.01, according to the unpaired <i>t</i>-test.</p

Spinal cord inflammation.

<p>A) After WT and IL-12p40KO mouse transcardiac perfusion, spinal cord upper lumbar intumescences were submitted to histopathological analysis. While WT spinal cord displayed preserved morphology (left picture), with few inflammatory cells (inset, arrow), IL-12p40KO spinal cord presented intense morphological disarrangement (right picture), due to the large amount of inflammatory foci (inset, arrows). Representative pictures of HE-stained spinal cord sections of WT and IL-12p40KO mice in the sixth week after infection. Scale bars: 100 µm; insets, 20 µm. B) Once a week during seven weeks, cDNA was obtained from the spinal cord lower lumbar segments of WT and IL-12p40KO mice and submitted to real time RT-PCR procedures, using primers for CD3, TNF-α, IFN-γ, iNOS, IL-10, arginase I and HPRT, β-actin and GAPDH as endogenous control. After the fourth week of infection, the transcription ratio of all genes analyzed tended to remission in the WT tissue, while they continued increasing in the IL-12p40KO spinal cord. Of note, also around the fourth week of infection, a switch in the transcription level of the analyzed genes was observed between the two mouse strains, when IL-12p40KO mice started expressing more RNA than WT ones. Mean ± SEM. **p<0.01; ***p<0.001 when comparing paired WT and IL-12p40KO groups according to Bonferroni post-tests. n = 4 for each strain in each time point.</p

Clinical outcomes of newborns at birth.

<p>Clinical outcomes of newborns at birth.</p