Malaria infection decreases maternal serum ANG-1 in pregnant mice (<i>P </i><0.001, 2-way ANOVA).

<p>Serum ANG-1 levels from uninfected (light bars) and <i>P. berghei</i> infected (dark bars) pregnant mice as measured by ELISA. Dots are individual mice; bars represent the median of each group. *** <i>P</i><0.001 (Bonferonni post-test). D, day post infection/control injection; G, gestational day.</p

Experimental model recapitulates the spontaneous abortion and low birth weight outcomes characteristic of PM.

<p><i>(A)</i> Pregnant BALB/c females infected (dark bars) with <i>P. berghei</i> on gestational day (G)13 have an increased rate of abortion and decreased proportion of viable pups per litter compared to uninfected mice (light bars) by G19, day 6 of infection/control injection (D6). Proportion of viable fetuses depicted by solid bars; aborted fetuses, by striped bars. <i>(B)</i> Body weight of viable fetuses is decreased with maternal malaria infection (dark symbols) as compared to weight of fetuses from uninfected mice (light symbols). Dots are individual viable fetuses; bars represent the median of each group. 20–83 fetuses were collected per group (from 4–13 pregnant females per group). *** <i>P</i><0.001 (Mann-Whitney).</p

The presence of peripheral parasitemia during pregnancy correlates with decreased plasma ANG-1 levels (<i>P</i> = 0.031, mixed linear model).

<p><i>(A)</i> Peripheral plasma ANG-1 levels (± SEM) for uninfected primigravid women who had no detectable peripheral or placental parasitemia during the course of the study (n = 8). <i>(B & C)</i> Peripheral plasma ANG-1 levels from two representative primigravid women with PM. The mean levels for uninfected women are shown as reference (in light shade). Boxed data points represent visits where women were peripheral blood-smear positive for <i>P. falciparum</i>.</p

Characteristics of cross-sectional study participants tested for angiopoietin levels.

<p>Dunn's multiple comparison test: *<i>P</i><0.01 cp to PM- NBW;</p><p>**<i>P</i><0.001 cp to PM- NBW;</p><p>§<i>P</i><0.05 cp to PM+ NBW;</p><p>Fisher's exact test: *** <i>P</i><0.0001 cp to PM- NBW;</p><p>^∧<i>P</i><0.0001 cp to PM+ NBW.</p

Characteristics of subjects from prospective study population tested for angiopoietin levels.

a<p>Mean ± SD.</p>b<p>Mann-Whitney test.</p>c<p>Fisher's exact test.</p>d<p>Unpaired t-test.</p

Placental <i>Angpt2</i> mRNA expression is increased by malaria infection (<i>P</i> = 0.0063, 2-way ANOVA).

<p><i>(A</i>) <i>Angpt1</i> and <i>(B</i>) <i>Angpt2</i> transcripts were measured by real-time quantitative PCR using cDNA templates reverse transcribed from placental RNA from pregnant mice uninfected (light bars) and infected with <i>P. berghei</i> (dark bars). Copy number was normalized to housekeeping gene expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009481#s2" target="_blank">Materials and Methods</a>. <i>(C)</i> The relative expression of <i>Angpt2</i>/<i>Angpt1</i> is also increased in placentas associated with viable fetuses of infected mice (<i>P</i> = 0.0032, 2-way ANOVA on log-transformed data). Dots are individual placentas associated with viable fetuses; bars represent the median of each group. 4–6 mice are represented per group. *<i>P</i><0.01 (Bonferonni post-test). D, day post infection/control injection; G, gestational day.</p

Plasma angiopoietin levels at delivery are dysregulated in PM and with LBW outcomes.

<p><i>(A–C)</i> Peripheral plasma and <i>(D)</i> matched placental plasma obtained at delivery of normal birth weight (NBW) or LBW infants from primigravid women with (PM+) or without (PM-) PM were measured for ANG-1 and ANG-2 by ELISA. <i>(A)</i> Mean maternal peripheral plasma ANG-1 is elevated with LBW deliveries in PM- but not PM+ women. Statistical analyses by Mann-Whitney test. <i>(B)</i> Maternal peripheral plasma ANG-2 is elevated with PM. Statistical analyses by Mann-Whitney test. <i>(C)</i> Elevated maternal peripheral plasma ANG-2/ANG-1 ratio at delivery is associated with PM (<i>P</i> = 0.0016) and LBW (<i>P</i> = 0.0406); 2-way ANOVA on log-transformed data. Statistical analyses between groups by t-test on log-transformed data. <i>(D)</i> Placental plasma ANG-2/ANG-1 ratio levels are elevated compared to peripheral plasma levels. Statistical analysis by t-test on log-transformed data. Dots represent individual women, lines represent the median of each group. Peripheral plasma, PM- NBW (n = 47), PM- LBW (n = 44), PM+ NBW (n = 51), PM+ LBW (n = 35). Placental plasma, PM- NBW (n = 41), PM- LBW (n = 34), PM+ NBW (n = 44), PM+ LBW (n = 28). * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, # <i>P</i> = 0.06, ^∧<i>P</i> = 0.486.</p

Angiopoietin-1 levels are increased upon resolution of peripheral parasitemia.

<p>Paired peripheral plasma <i>(A)</i> ANG-1 and <i>(B)</i> ANG-2/ANG-1 levels of primigravid women at consecutive visits: the first, when parasitemic by peripheral blood smear microscopy, and the next, when successfully treated and blood-smear negative. To account for physiological variation in angiopoietin levels with gestational age, values were normalized to mean value of aparasitemic controls at the corresponding gestational age. n = 13 pairs. ** <i>P</i><0.01, ^∧<i>P</i>>0.05 (paired t-test).</p

Placental ANG-1 and ANG-2 protein levels are dysregulated by malaria infection.

<p><i>(A)</i> ANG-1 and <i>(B)</i> ANG-2 protein levels in placentas from pregnant mice uninfected (light bars) and infected with <i>P. berghei</i> (dark bars). Protein levels were detected by Western blot, quantified by densitometry and normalized to intensity of β-actin bands. Dots are individual placentas associated with viable fetuses; bars represent the median of each group. 3–13 mice are represented per group. **<i>P</i><0.01, ∧ <i>P</i> = 0.053 (unpaired t-test). D, day post infection/control injection; G, gestational day.</p

The Antibody Response of Pregnant Cameroonian Women to VAR2CSA ID1-ID2a, a Small Recombinant Protein Containing the CSA-Binding Site

<div><p>In pregnant women, <i>Plasmodium falciparum</i>-infected erythrocytes expressing the VAR2CSA antigen bind to chondroitin sulfate A in the placenta causing placental malaria. The binding site of VAR2CSA is present in the ID1-ID2a region. This study sought to determine if pregnant Cameroonian women naturally acquire antibodies to ID1-ID2a and if antibodies to ID1-ID2a correlate with absence of placental malaria at delivery. Antibody levels to full-length VAR2CSA and ID1-ID2a were measured in plasma samples from 745 pregnant Cameroonian women, 144 Cameroonian men, and 66 US subjects. IgM levels and IgG avidity to ID1-ID2a were also determined. As expected, antibodies to ID1-ID2a were absent in US controls. Although pregnant Cameroonian women developed increasing levels of antibodies to full-length VAR2CSA during pregnancy, no increase in either IgM or IgG to ID1-ID2a was observed. Surprisingly, no differences in antibody levels to ID1-ID2a were detected between Cameroonian men and pregnant women. For example, in rural settings only 8–9% of males had antibodies to full-length VAR2CSA, but 90–96% had antibodies to ID1-ID2a. In addition, no significant difference in the avidity of IgG to ID1-ID2a was found between pregnant women and Cameroonian men, and no correlation between antibody levels at delivery and absence of placental malaria was found. Thus, the response to ID1-ID2a was not pregnancy specific, but predominantly against cross-reactivity epitopes, which may have been induced by other PfEMP1 antigens, malarial antigens, or microbes. Currently, ID1-ID2a is a leading vaccine candidate, since it binds to the CSA with the same affinity as the full-length molecule and elicits binding-inhibitory antibodies in animals. Further studies are needed to determine if the presence of naturally acquired cross-reactive antibodies in women living in malaria endemic countries will alter the response to ID1-ID2a following vaccination with ID1-ID2a.</p></div