Transcriptomic evidence for modulation of host inflammatory responses during febrile Plasmodium falciparum malaria

Identifying molecular predictors and mechanisms of malaria disease is important for understanding how Plasmodium falciparum malaria is controlled. Transcriptomic studies in humans have so far been limited to retrospective analysis of blood samples from clinical cases. In this prospective, proof-of-principle study, we compared whole-blood RNA-seq profiles at pre-and post-infection time points from Malian adults who were either asymptomatic (n = 5) or febrile (n = 3) during their first seasonal PCR-positive P. falciparum infection with those from malaria-naïve Dutch adults after a single controlled human malaria infection (n = 5). Our data show a graded activation of pathways downstream of pro-inflammatory cytokines, with the highest activation in malaria-naïve Dutch individuals and significantly reduced activation in malaria-experienced Malians. Newly febrile and asymptomatic infections in Malians were statistically indistinguishable except for genes activated by pro-inflammatory cytokines. The combined data provide a molecular basis for the development of a pyrogenic threshold as individuals acquire immunity to clinical malaria

Plasmodium falciparum transcription in different clinical presentations of malaria associates with circulation time of infected erythrocytes

Following Plasmodium falciparum infection, individuals can remain asymptomatic, present with mild fever in uncomplicated malaria cases, or show one or more severe malaria symptoms. Several studies have investigated associations between parasite transcription and clinical severity, but no broad conclusions have yet been drawn. Here, we apply a series of bioinformatic approaches based on P. falciparum’s tightly regulated transcriptional pattern during its ~48-hour intraerythrocytic developmental cycle (IDC) to publicly available transcriptomes of parasites obtained from malaria cases of differing clinical severity across multiple studies. Our analysis shows that within each IDC, the circulation time of infected erythrocytes without sequestering to endothelial cells decreases with increasing parasitaemia or disease severity. Accordingly, we find that the size of circulating infected erythrocytes is inversely related to parasite density and disease severity. We propose that enhanced dhesiveness of infected erythrocytes leads to a rapid increase in parasite burden, promoting higher parasitaemia and increased disease severity

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence

Hernies incisionnelles chez l’adulte dans un contexte sous médicalisé.

L’objectif de ce travail était d’étudier les facteurs favorisants et le traitement chirurgical des hernies incisionnelles. Il s’agissait d´une étude rétrospective descriptive portant sur des patients opérés pour hernie incisionnelle dans le service de chirurgie B du CHU du Point G sur une période de 11 ans allant du 1er janvier 2001 au 31 décembre 2011. Il a été enregistré 60 cas de hernies incisionnelles dont 40 femmes avec un sex-ratio de 0,5. L’âge moyen était de 39,8 ans ± 18,3. L’indice de masse corporelle moyen était de 26,3 kg/ m2. Les principaux facteurs favorisants qui étaient statistiquement liés à la survenue de la hernie incisionnelle ont été : âge jeune (≤ 45 ans), le sexe féminin, l’état nutritionnel, l’opération de la pathologie initiale en urgence, l’incision médiane et la longueur de l’incision initiale ≥ 5 cm. La cure a été une herniorraphie par suture aponévrotique dans 96,7% des cas (n=58), la mise en place d’une prothèse dans 3,3% des cas (n=2). Les suites opératoires étaient compliquées de suppuration pariétale dans 15% (n=9). Les récidives étaient de 13,3% des cas (n=8). Les facteurs favorisants de la hernie incisionnelle restent dominés dans notre contexte par les femmes jeunes, opérées en urgence par la laparotomie médiane, surtout sous ombilicale. La pariétorraphie est la technique chirurgicale la plus pratiquée.Mots clés : Hernie incisionnelle, facteurs favorisants, éventrations postopératoires, chirurgie

Superiority of 3 Over 2 Doses of Intermittent Preventive Treatment With Sulfadoxine-Pyrimethamine for the Prevention of Malaria During Pregnancy in Mali: A Randomized Controlled Trial

Background. In 2003, Mali introduced intermittent preventive therapy in pregnancy (ITPp) with sulfadoxine-pyrimethamine (SP) for the control of malaria in pregnancy, consisting of 2 doses of SP given in the 2nd and 3rd trimester. This widely used regimen, although very effective, leaves many women unprotected from malaria during the last 4-to-8 weeks of gestation, which is a pivotal period for fetal weight gain. The aim of the study was to compare the efficacy and safety of 3-dose versus 2-dose IPTp-SP for the prevention of placental malaria and associated low birth weight (LBW). Methods. We conducted a parallel-group, open-label, individually randomized controlled superiority trial involving 814 women of all gravidity, enrolled from April 2006 through March 2008. All women were seen at least 3 times and received either 2 (n = 401) or 3 (n = 413) doses of IPTp-SP. The primary endpoint measured was placental malaria, LBW, preterm births, and maternal anemia were secondary endpoints, and severe maternal skin reactions and neonatal jaundice were safety endpoints. Results. Among the 96% of study subjects who were followed up until delivery, the prevalence of placental malaria was 2-fold lower in the 3-dose group (8.0%) than in the 2-dose group (16.7%); the adjusted prevalence ratio (APR) was 0.48 (95% confidence interval [CI], 0.32–0.71). LBW and preterm births were also reduced; the prevalence of LBW was 6.6% in the 3-dose group versus 13.3% in the 2-dose group (APR, 0.50; 95% CI, 0.32–0.79), and the prevalence of preterm births was 3.2% versus 8.9% (APR, 0.37; 95% CI, 0.19–0.71). No significant reductions in maternal anemia or differences in safety endpoints were observed. Conclusions. Adding a third dose of ITPp-SP halved the risk of placental malaria, LBW, and preterm births in all gravidae, compared with the standard 2-dose regimen, in this area of highly seasonal transmission with low levels of SP resistance

A Molecular Signature in Blood Reveals a Role for p53 in Regulating Malaria-Induced Inflammation

Immunity that controls parasitemia and inflammation during Plasmodium falciparum (Pf) malaria can be acquired with repeated infections. A limited understanding of this complex immune response impedes the development of vaccines and adjunctive therapies. We conducted a prospective systems biology study of children who differed in their ability to control parasitemia and fever following Pf infection. By integrating whole-blood transcriptomics, flow-cytometric analysis, and plasma cytokine and antibody profiles, we demonstrate that a pre-infection signature of B cell enrichment; upregulation of T-helper type 1 (Th1) and Th2 cell-associated pathways, including interferon responses; and p53 activation associated with control of malarial fever and coordinated with Pf-specific IgG and Fc receptor activation to control parasitemia. Our hypothesis-generating approach identified host molecules that may contribute to differential clinical outcomes during Pf infection. As a proof of concept, we have shown that enhanced p53 expression in monocytes attenuated Plasmodium-induced inflammation and predicted protection from fever

A molecular pattern of restrained inflammation and enhanced anti-parasite effector function upon <i>P. falciparum</i> re-exposure.

<p>(<b>A</b>) PBMCs were collected from 34 healthy children with blood smears negative for <i>P. falciparum</i> infection before the malaria season (HB) and 7 days after treatment of their first febrile malaria episode of the ensuing malaria season when malaria symptoms had resolved (d7). RNA was extracted from PBMCs immediately after thawing and hybridized onto Affymetrix GeneChip Human 1.0 ST arrays. RNA from all 68 PBMC samples was of sufficient quantity and quality for microarray analysis. Nine of 68 samples did not pass the microarray quality assessment and were removed from further analysis (see Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004079#ppat-1004079-g001" target="_blank">Figure 1A</a>) such that 25 children with paired RNA samples at the healthy baseline and 7 days after malaria were analyzed. The heat map shows <i>ex vivo</i> RMA-normalized log₂ ratios (d7/HB) of differentially expressed genes (rows) for each child (columns). Genes are grouped and color-coded by function as indicated. (<b>B</b>) PBMCs analyzed by FACS for B cells (CD19⁺), T cells (CD3⁺), CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, and monocytes (CD14⁺) at the healthy baseline and after malaria. (n = 34 children; except CD14⁺ monocytes, n = 30). (<b>C</b>) Ratio of monocyte percentage (d7/HB) versus the ratio of the expression level of monocyte-derived pro-inflammatory cytokines and chemokines (d7/HB). Each point represents an individual subject (n = 21 children with paired samples). (<b>D</b>) RNA was extracted from PBMCs of the same 34 children after 18 h of <i>in vitro</i> stimulation with <i>P. falciparum</i>-infected red blood cell (iRBC) lysate. After stimulation with iRBC lysate, 22 of the 34 children had RNA samples from both time points of sufficient quantity and quality for microarray analysis and also passed the microarray quality assessment. The heat map shows RMA-normalized log₂ ratios (d7/HB) of differentially expressed genes (rows) for each child (columns) in response to <i>in vitro</i> iRBC lysate stimulation. Genes are grouped and color-coded by function as indicated. (<b>E</b>) q-RT-PCR confirmation of the microarray data. The data represent the results of one experiment with 6 genes (<i>IL1B</i>, <i>IL6</i>, <i>IL10</i>, <i>TGFB1</i>, <i>TLR2</i>, <i>CXCL5</i>) from 17 subjects at two time points (d7 and HB) from both the <i>ex vivo</i> unstimulated and <i>in vitro</i> iRBC-stimulated datasets. Each symbol represents a single gene at a given time point. PCR expression computed as antilog₂ –dCT. <i>n</i> = 497 XY pairs. (<b>F</b>) q-RT-PCR expression of genes encoding the pro-inflammatory cytokines IL1-β and IL-6 and the anti-inflammatory cytokine TGF-β in PBMCs of children (n = 17) collected at the healthy baseline (HB) and after resolution of febrile malaria (d7), either directly <i>ex vivo</i> (unstimulated) or after <i>in vitro</i> stimulation with iRBCs for 18 h. ns, not significant (<i>P</i>≥0.05), <i>P</i> values determined by the paired <i>t</i>test (B), Pearson's (C), Spearman's (E) or paired Wilcoxon rank sum test (F). Data are shown as the means ± s.d. (B) or means ± s.e.m. (F).</p

<i>P. falciparum</i>-inducible IL-10 is mainly produced by CD4⁺CD25⁺Foxp3⁻ T cells that co-produce IFNγ and TNF.

<p>(<b>A</b>) PBMCs from the healthy baseline (HB), 7 days after malaria (d7), and at the healthy baseline at the end of the subsequent dry-season (HB′) were stimulated for 18 h with iRBC lysate and assayed for the production of IL-10, IFNγ and TNF by intra-cellular FACS. Results are shown as the ratio of live CD3⁺ CD4⁺ antigen-experienced cells (CD45RO⁺ CD27⁺, CD45RO⁺ CD27⁻, and CD45RO⁻ CD27⁻) producing IL-10, IFN-γ or TNF in response to stimulation with iRBC lysate vs. uninfected RBC (uRBC) lysate (n = 16, 13 paired samples). (<b>B</b>) Overlay of IL-10-producing cells (red) among all live cells (gray) in a CD3 vs. CD4 dot plot (top) (n = 14), and IL-10-producing CD4⁺ T cells (red) with all CD4⁺ T cells (gray) in CD25 vs. FoxP3 dot plot (bottom) (n = 9; representative subject shown). (<b>C</b>) Using SPICE analysis, cytokine-producing CD4⁺ T cells were divided into 7 distinct subpopulations producing any combination of IL-10, IFNγ and TNF (n = 16). (<b>D</b>) Pie chart representation of the combination of cytokines produced by CD4⁺ T cells after iRBC stimulation for 3 representative donors 7 days after malaria (d7). The black arcs indicate the IL-10-producing CD4⁺ T cells. (<b>E</b>) Representative FACS plots of live CD3⁺ CD4⁺ antigen-experienced cells producing IL-10, IFNγ and TNF after iRBC stimulation of PBMCs collected at the healthy baseline (HB), 7 days after malaria (d7) and at the healthy baseline at the end of the subsequent dry-season (HB′). (<b>F</b>) CD4⁺ T cells were isolated from PBMCs which had been collected from children 7 days after malaria and were then stimulated for 18 h with iRBC or uRBC lysate in the absence (CD4⁺T d7) or presence of non-CD4⁺T cells isolated from PBMCs of the same individuals collected at either the healthy baseline (CD4⁺T d7 + nonCD4⁺T HB) or 7 days after malaria (CD4⁺T d7 + nonCD4⁺T d7) (n = 8 paired samples). (<b>G</b>) PBMCs collected from children 7 days after malaria were stimulated for 18 h with iRBC lysate and assayed for the production of IL-10 in the presence (αMHC-II) or absence (isotype) of antibodies specific for HLA-DR, -DQ and -DP (n = 8). ns, not significant (<i>P</i>≥0.05), <i>P</i> values determined by a linear mixed model for repeated measures ANOVA with Tukey HSD post hoc tests (A) and permutation re-sampling tests (F, G). Data are shown as the means ± s.d.</p

Proposed model by which children remain asymptomatic and control parasitemia upon <i>P. falciparum</i> re-exposure.

<p>In children without prior or recent malaria exposure, <i>P. falciparum</i> infection induces a robust pro-inflammatory cytokine and chemokine response (e.g. IL-1β, IL-6, IL-8) whereas effector mechanisms that mediate parasite clearance (phagocytosis, phagolysosome activation, antigen presentation, T cell co-stimulation and IFN-<b>γ</b> production by CD4⁺ T cells) are not readily inducible, leaving children susceptible to fever and other systemic symptoms of malaria as well as poorly controlled parasite replication. In contrast, febrile malaria induces an exposure-dependent regulatory state (shown here) whereby re-exposure to <i>P. falciparum</i> results in reduced production of pro-inflammatory cytokines and chemokines and enhanced expression of regulatory cytokines (e.g. IL-10 production by CD4⁺ T cells) and pathways involved in phagocytosis-mediated clearance of infected red blood cells and activation of adaptive immunity, thus enabling children to remain asymptomatic and control parasite replication in the face of ongoing <i>P. falciparum</i> exposure. In addition, <i>P. falciparum</i>-specific IgG levels are low in children who have not been recently exposed to malaria, but transiently increase in response to <i>P. falciparum</i> infection <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004079#ppat.1004079-Weiss1" target="_blank">[44]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004079#ppat.1004079-Crompton2" target="_blank">[45]</a>, further enhancing exposure-dependent parasite clearance through opsonization and phagocytosis of infected erythrocytes. Arrows indicate the direction of expression observed in this study of molecules at the mRNA and/or protein levels induced by <i>P. falciparum</i> re-exposure after febrile malaria relative to responses induced by <i>P. falciparum</i> exposure at the healthy baseline. Molecules are color-coded by biological function.</p