Schematic representation of the cytoadherence assay.

<p>Ring-stage parasites from HbAA and HbAS (or HbAC) children were cultured to trophozoites (a), and then purified by magnetic column and inoculated into wildtype donor RBCs (b). After invasion and maturation to trophozoites expressing PfEMP1 (c), parasites from HbAS or HbAC children were compared for binding to MVECs in parallel with those from HbAA children (d).</p

Schematic representation of the cytoadherence assay.

<p>Ring-stage parasites from HbAA and HbAS (or HbAC) children were cultured to trophozoites (a), and then purified by magnetic column and inoculated into wildtype donor RBCs (b). After invasion and maturation to trophozoites expressing PfEMP1 (c), parasites from HbAS or HbAC children were compared for binding to MVECs in parallel with those from HbAA children (d).</p

Relative cytoadherence of parasitized HbAA, HbAS, and HbAC RBCs to MVECs.

<p>Over three transmission seasons (2008, 2009, and 2010), a total of 31 cytoadherence comparisons were performed between parasites from HbAA and HbAS (or HbAC) children by inoculating them into wild-type donor RBCs and assaying their binding to MVECs in parallel. The ratio of parasitized RBCs (pRBC) bound per MVEC (pRBC/MVEC) was calculated for each sample tested. The pRBC/MVEC ratio from the HbAS- or HbAC-derived parasite in each comparison was then normalized to that of the HbAA-derived parasite from the same comparison to give a measure of the relative cytoadherence. Two comparisons (AS13 <i>vs.</i> AA12 and AC10 <i>vs.</i> AA22) were performed over multiple slides, as shown. Also, several samples (indicated in bold) were used in multiple comparisons.</p

Relationship between cytoadherence, Hb type, and host age.

<p>A Poisson regression model was constructed to examine the effect of Hb type and host age on the cytoadherence of parasitized RBCs to MVECs. Fold-changes and 95% CIs of the relative binding compared to parasites from HbAA (for HbAS and HbAC) children and parasites from >5-year-old (for ≤5-year-old) children are indicated.</p

Characteristics of Malian HbAA, HbAS, and HbAC children from which parasite isolates were obtained.

<p>The number of samples (n), proportion of samples with α-thalassemia (wildtype, WT; heterozygous, HET; not determined, ND) and <i>G6PD*</i>A- (G202A) (absent, ABS; heterozygous, HET; hemizygous, HEM) genotypes, mean age (years), median parasite density (/μl), and proportion of severe malaria cases are shown for <i>all</i> HbAA, HbAS, and HbAC samples used in comparisons, and <i>unique</i> HbAA, HbAS, and HbAC samples used in comparisons. <i>All</i> samples (n = 62) include 10 parasite strains that were used in multiple comparisons, while <i>unique</i> samples include 52 parasite strains that were used in single comparisons. Three <i>unique</i> samples (2 HbAS and 1 HbAC) were classified as severe since they met one or more of these criteria: cessation of eating/drinking, repetitive vomiting, or prostration.</p

Relative cytoadherence and surface PfEMP1 levels of parasitized RBCs. a,

<p>Adherence of parasitized RBCs to MVECs. The numbers of parasitized −α/αα (HE), −α/−α (HO) and −/−α (HH) RBCs adhering to MVECs were normalized to those of parasitized αα/αα RBCs tested in parallel. The mean (± SEM) number of parasitized αα/αα RBCs per 100 MVECs was 260±40, <i>N</i> = 19. Results were obtained from 19 naturally-circulating parasite isolates and 2 laboratory-adapted parasite clones (A4tres and FCR-3), multiple blood donors (5 αα/αα, 2−α/αα, 2 −α/−α and 2−/−α), and 4 MVEC donors (not all combinations tested). This resulted in −α/αα, −α/−α and −/−α samples being compared to αα/αα samples 12, 5 and 4 times. <b>b,</b> Adherence of parasitized RBCs to monocytes. The numbers of parasitized −α/αα, −α/−α and −/−α RBCs adhering to monocytes were normalized to those of αα/αα RBCs tested in parallel. The mean (± SEM) number of parasitized αα/αα RBCs per 100 monocytes was 136±10, <i>N</i> = 12. Results were obtained from 3 naturally-circulating parasite isolates and 3 laboratory-adapted parasite clones (3D7, A4tres and FCR-3), multiple blood donors (5 αα/αα, 3 −α/αα, 2 −α/−α and 2−/−α) and 4 monocyte donors (not all combinations tested). This resulted in −α/αα, −α/−α and −/−α samples being compared to αα/αα samples 20, 3 and 4 times. The αα/αα and −α/αα RBCs were different from those used in endothelial cell adherence assays. <b>c,</b> PfEMP1 expression levels (median fluorescence intensities, MFI) on the surface of parasitized RBCs. The mean (± SEM) MFI of parasitized αα/αα RBCs was 556±153, <i>N</i> = 6. Results were obtained from 2 laboratory-adapted parasite clones (A4tres, FVO and FCR3^CSA), multiple blood donors (4 αα/αα, 6 −α/αα and 2−/−α), and various concentrations of 2 antisera (not all combinations tested). This resulted in −α/αα, and −/−α samples being compared to αα/αα samples 10 and 6 times. The αα/αα and −α/αα RBCs were different from those used in endothelial cell and monocyte adherence assays.</p

Distribution and morphology of knobs on the surface of parasitized RBCs.

<p>Atomic force micrographs (AFMs) of parasitized −α/αα (HE) (<b>a,d</b>) and −α/−α (HO) (<b>b,e</b>) RBCs obtained from naturally-parasitized Malian children with malaria and −/−α (HH) (<b>c,f</b>) RBCs infected with a laboratory-adapted <i>P. falciparum</i> clone showing normal (<b>a,b</b>) or abnormal (<b>c–f</b>) knob distributions and morphologies. AFM images are representative of 32, 10 and 18 images of parasites in −α/αα, −/−αα and −/−α RBCs. Inlays show YOYO-1-stained parasites that correspond to those imaged by AFM. Comparison AFMs of parasitized HbA, HbC and HbS RBCs have been reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037214#pone.0037214-Arie1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037214#pone.0037214-Cholera1" target="_blank">[37]</a>.</p

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

14 septembre 19181918/09/14 (A19,N6445)

Merozoite antigen-specific IgG levels increase with age.

<p>Box and whisker (Tukey) plots of IgG levels in each age group are shown. The levels of IgG to AMA1-3D7 (A), MSP1-3D7 (B), EBA175-3D7 (C) and MSP2-3D7 (D) were quantified. All responses below the limit of detection (44 ELISA units) were assigned a value of 22 ELISA units. IgG levels between the three age groups were compared using a Kruskal-Wallis test followed by Dunn's multiple comparison test (**, p<0.01; ***, p<0.001).</p

Effects of Age, Hemoglobin Type and Parasite Strain on IgG Recognition of <i>Plasmodium falciparum</i>–Infected Erythrocytes in Malian Children

<div><p>Background</p><p>Naturally-acquired antibody responses to antigens on the surface of <i>Plasmodium falciparum</i>-infected red blood cells (iRBCs) have been implicated in antimalarial immunity. To profile the development of this immunity, we have been studying a cohort of Malian children living in an area with intense seasonal malaria transmission.</p> <p>Methodology/Principal Findings</p><p>We collected plasma from a sub-cohort of 176 Malian children aged 3-11 years, before (May) and after (December) the 2009 transmission season. To measure the effect of hemoglobin (Hb) type on antibody responses, we enrolled age-matched HbAA, HbAS and HbAC children. To quantify antibody recognition of iRBCs, we designed a high-throughput flow cytometry assay to rapidly test numerous plasma samples against multiple parasite strains. We evaluated antibody reactivity of each plasma sample to 3 laboratory-adapted parasite lines (FCR3, D10, PC26) and 4 short-term-cultured parasite isolates (2 Malian and 2 Cambodian). 97% of children recognized ≥1 parasite strain and the proportion of IgG responders increased significantly during the transmission season for most parasite strains. Both strain-specific and strain-transcending IgG responses were detected, and varied by age, Hb type and parasite strain. In addition, the breadth of IgG responses to parasite strains increased with age in HbAA, but not in HbAS or HbAC, children.</p> <p>Conclusions/Significance</p><p>Our assay detects both strain-specific and strain-transcending IgG responses to iRBCs. The magnitude and breadth of these responses varied not only by age, but also by Hb type and parasite strain used. These findings indicate that studies of acquired humoral immunity should account for Hb type and test large numbers of diverse parasite strains.</p> </div