Prothrombotic autoantibodies targeting platelet factor 4/polyanion are associated with pediatric cerebral malaria.

BACKGROUNDFeatures of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM.METHODSPlasma from children with uncomplicated malaria (UM) (n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-platelet factor 4/polyanion (anti-PF4/P), anti-phospholipid, anti-phosphatidylserine, anti-myeloperoxidase, anti-proteinase 3, anti-dsDNA, anti-ß-2-glycoprotein I, and anti-cardiolipin. Plasma samples from individuals with nonmalarial coma (NMC) (n = 49) and healthy controls (HCs) (n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses.RESULTSMedian anti-PF4/P and anti-PS IgG levels were elevated in individuals with malaria infection relative to levels in HCs (P < 0.001) and patients with NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in children with CM (median = 0.27, IQR: 0.19-0.41) compared with those with UM (median = 0.19, IQR: 0.14-0.22, P < 0.0001). Anti-PS IgG levels did not differ between patients with UM and those with CM (P = 0.39). When patients with CM were stratified by malaria retinopathy (Ret) status, the levels of anti-PF4/P IgG correlated negatively with the peripheral platelet count in patients with Ret+ CM (Spearman's rho [Rs] = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02-275, P = 0.048). Plasma from patients with CM induced greater platelet activation in an ex vivo assay relative to plasma from patients with UM (P = 0.02), and the observed platelet activation was associated with anti-PF4/P IgG levels (Rs= 0.293, P = 0.035).CONCLUSIONSThrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.

Whole blood transfusion improves vascular integrity and increases survival in artemether-treated experimental cerebral malaria.

Pathological features observed in both human and experimental cerebral malaria (ECM) are endothelial dysfunction and changes in blood components. Blood transfusion has been routinely used in patients with severe malarial anemia and can also benefit comatose and acidotic malaria patients. In the present study Plasmodium berghei-infected mice were transfused intraperitoneally with 200 µL of whole blood along with 20 mg/kg of artemether. ECM mice showed severe thrombocytopenia and decreases in hematocrit. Artemether treatment markedly aggravated anemia within 24 h. Whole blood administration significantly prevented further drop in hematocrit and partially restored the platelet count. Increased levels of plasma angiopoietin-2 (Ang-2) remained high 24 h after artemether treatment but returned to normal levels 24 h after blood transfusion, indicating reversal to quiescence. Ang-1 was depleted in ECM mice and levels were not restored by any treatment. Blood transfusion prevented the aggravation of the breakdown of blood brain barrier after artemether treatment and decreased spleen congestion without affecting splenic lymphocyte populations. Critically, blood transfusion resulted in markedly improved survival of mice with ECM (75.9% compared to 50.9% receiving artemether only). These findings indicate that whole blood transfusion can be an effective adjuvant therapy for cerebral malaria.

IgG acquisition against PfEMP1 PF11_0521 domain cassette DC13, DBLß3_D4 domain, and peptides located within these constructs in children with cerebral malaria.

The Plasmodium falciparum erythrocyte-membrane-protein-1 (PF3D7_1150400/PF11_0521) contains both domain cassette DC13 and DBLß3 domain binding to EPCR and ICAM-1 receptors, respectively. This type of PfEMP1 proteins with dual binding specificity mediate specific interactions with brain micro-vessels endothelium leading to the development of cerebral malaria (CM). Using plasma collected from children at time of hospital admission and after 30 days, we study an acquisition of IgG response to PF3D7_1150400/PF11_0521 DC13 and DBLß3_D4 recombinant constructs, and five peptides located within these constructs, specifically in DBLα1.7_D2 and DBLß3_D4 domains. We found significant IgG responses against the entire DC13, PF11_0521_DBLß3_D4 domain, and peptides. The responses varied against different peptides and depended on the clinical status of children. The response was stronger at day 30, and mostly did not differ between CM and uncomplicated malaria (UM) groups. Specifically, the DBLß3 B3-34 peptide that contains essential residues involved in the interaction between PF11_0521 DBLß3_D4 domain and ICAM-1 receptor demonstrated significant increase in reactivity to IgG1 and IgG3 antibodies at convalescence. Further, IgG reactivity in CM group at time of admission against functionally active (ICAM-1-binding) PF11_0521 DBLß3_D4 domain was associated with protection against severe anemia. These results support development of vaccine based on the PF3D7_1150400/PF11_0521 structures to prevent CM.

Blood-cerebrospinal fluid barrier: another site disrupted during experimental cerebral malaria caused by Plasmodium berghei ANKA.

Cerebral malaria is one of the most severe pathologies of malaria; it induces neuro-cognitive sequelae and has a high mortality rate. Although many factors involved in the development of cerebral malaria have been discovered, its pathogenic mechanisms are still not completely understood. Most studies on cerebral malaria have focused on the blood-brain barrier, despite the importance of the blood-cerebrospinal fluid barrier, which protects the brain from peripheral inflammation. Consequently, the pathological role of the blood-cerebrospinal fluid barrier in cerebral malaria is currently unknown. To examine the status of the blood-cerebrospinal fluid barrier in cerebral malaria and malaria without this pathology (non-cerebral malaria), we developed a new method for evaluating the permeabilization of the blood-cerebrospinal fluid barrier during cerebral malaria in mice, using Evans blue dye and a software-assisted image analysis. Using C57BL/6J (B6) mice infected with Plasmodium berghei ANKA strain as an experimental cerebral malaria model and B6 mice infected with P. berghei NK65 strain or Plasmodium yoelii as non-cerebral malaria models, we revealed that the permeability of the blood-cerebrospinal fluid barrier increased during experimental cerebral malaria but not during non-cerebral malaria. We observed haemorrhaging in the cerebral ventricles and hemozoin-like structures in the choroid plexus, which is a key component of the blood-cerebrospinal fluid barrier, in cerebral malaria mice. Taken together, this evidence indicates that the blood-cerebrospinal fluid barrier is disrupted in experimental cerebral malaria, whereas it remains intact in non-cerebral malaria. We also found that P. berghei ANKA parasites and CD8+ T cells are involved in the blood-cerebrospinal fluid barrier disruption in experimental cerebral malaria. An understanding of the mechanisms underlying cerebral malaria might help in the development of effective strategies to prevent and manage cerebral malaria in humans.

Serum levels of total human Tau associated with axonal damage among severe malaria patients in Central India.

Tau is a microtubule-associated protein (MAP) that is abundant in the axonal part of neurons of the central nervous system. Previous studies among African children and Vietnamese adults suffering from cerebral malaria (CM) showed the pathological significance of measuring circulatory total Tau levels. A pilot investigation was carried out to better characterise neurological pathogenesis among severe malaria patients in Central India. Serum levels of total human Tau (pg/ml) were measured by ELISA following manufacturer guidelines among hospital admitted P. falciparum malaria patients classified with different degree of severity (mild malaria = MM, non-cerebral severe malaria = NCSM, cerebral malaria survivors = CM-S and cerebral malaria non-survivors = CM-NS) using WHO, 2000 definitions, including healthy controls (HC) enroled from the hospital's blood bank. Categorical and numerical variables were analysed by applying appropriate statistical test using Stata 11.0 software. A total of 139 subjects (14 HC, 25 MM, 29 NCSM, 44 CM-S and 27 CM-NS) were included in this preliminary investigation. Serum levels of total human Tau were detected in 0% HC, 4.0% MM, 20.7% NCSM, 43.2% CM-S and 48.2% CM-NS patients. Compared to MM, percent Tau detection was significantly higher among severe malaria patients (p = 0.001). Further, compared to NCSM,% Tau detection was significantly higher in CM-S patients (Chi2 = 3.9, p = 0.048) & CM-NS patients (Chi2 = 4.7, p = 0.030). Percent Tau detection was also significantly higher among severe malaria cases presenting with multiple complications compared to those without multiple complications (p = 0.006). ROC analysis of serum Tau levels (pg/ml) revealed a fair AUC value (0.75) to distinguish CM-NS group (but not CM-S) from NCSM group. In conclusion, serum percent detection of total human Tau is associated with axonal damage among patients with different degree of P. falciparum malaria severity in Central India.

Association of cerebral malaria and TNF-α levels: a systematic review.

BACKGROUND: Cerebral malaria is the most severe form of infection with Plasmodium falciparum characterized by a highly inflammatory response. This systematic review aimed to investigate the association between TNF-α levels and cerebral malaria. METHODS: This review followed the Preferred Reporting of Systematic Review and Meta-analyses (PRISMA) guidelines. The search was performed at PubMed, LILACS, Scopus, Web of Science, The Cochrane Library, OpenGrey and Google Scholar. We have included studies of P. falciparum-infected humans with or without cerebral malaria and TNF-α dosage level. All studies were evaluated using a risk of bias tool and the GRADE approach. RESULTS: Our results have identified 2338 studies, and 8 articles were eligible according to this systematic review inclusion criteria. Among the eight articles, five have evaluated TNF- α plasma dosage, while two have evaluated at the blood and one at the brain (post-Morten). Among them, only five studies showed higher TNF-α levels in the cerebral malaria group compared to the severe malaria group. Methodological problems were identified regarding sample size, randomization and blindness, but no risk of bias was detected. CONCLUSION: Although the results suggested that that TNF-α level is associated with cerebral malaria, the evidence is inconsistent and imprecise. More observational studies evaluating the average TNF-alpha are needed.

Plasma cell-free DNA predicts pediatric cerebral malaria severity.

BACKGROUNDPrediction of adverse outcomes in cerebral malaria (CM) is difficult. We hypothesized that cell-free DNA (cfDNA) levels would facilitate identification of severe and potentially fatal CM cases.METHODSIn this retrospective study, plasma from Malawian children with CM (n = 134), uncomplicated malaria (UM, n = 77), and healthy controls (HC, n = 60) was assayed for cfDNA using a fluorescence assay. Host and parasite cfDNA was measured by quantitative PCR. Immune markers were determined by ELISA, Luminex, or cytometric bead array.RESULTSTotal cfDNA increased with malaria severity (HC versus UM, P < 0.001; HC versus CM, P < 0.0001; UM versus CM, P < 0.0001), was elevated in retinopathy-positive (Ret+) CM relative to Ret- CM (7.66 versus 5.47 ng/µL, P = 0.027), and differentiated Ret+ fatal cases from survivors (AUC 0.779; P < 0.001). cfDNA levels in patients with non-malarial febrile illness (NMF, P = 0.25) and non-malarial coma (NMC, P = 0.99) were comparable with UM. Host DNA, rather than parasite DNA, was the major cfDNA contributor (UM, 268 versus 67 pg/µL; CM, 2824 versus 463 pg/µL). Host and parasite cfDNA distinguished CM by retinopathy (host, AUC 0.715, P = 0.0001; parasite, AUC 0.745, P = 0.0001), but only host cfDNA distinguished fatal cases (AUC 0.715, P = 0.0001). Total cfDNA correlated with neutrophil markers IL-8 (rs = 0.433, P < 0.0001) and myeloperoxidase (rs = 0.683, P < 0.0001).CONCLUSIONQuantifying plasma cfDNA is a simple assay useful in identifying children at risk for fatal outcome and has promise as a point-of-care assay. Elevated cfDNA suggests a link with host inflammatory pathways in fatal CM.FUNDINGNIH NCATS (AK), Burroughs-Wellcome (AK), and National Health and Medical Research Council of Australia (SJR).

Depleted circulatory complement-lysis inhibitor (CLI) in childhood cerebral malaria returns to normal with convalescence.

BACKGROUND: Cerebral malaria (CM), is a life-threatening childhood malaria syndrome with high mortality. CM is associated with impaired consciousness and neurological damage. It is not fully understood, as yet, why some children develop CM. Presented here is an observation from longitudinal studies on CM in a paediatric cohort of children from a large, densely-populated and malaria holoendemic, sub-Saharan, West African metropolis. METHODS: Plasma samples were collected from a cohort of children with CM, severe malarial anaemia (SMA), uncomplicated malaria (UM), non-malaria positive healthy community controls (CC), and coma and anemic patients without malaria, as disease controls (DC). Proteomic two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry were used in a discovery cohort to identify plasma proteins that might be discriminatory among these clinical groups. The circulatory levels of identified proteins of interest were quantified by ELISA in a prospective validation cohort. RESULTS: The proteome analysis revealed differential abundance of circulatory complement-lysis inhibitor (CLI), also known as Clusterin (CLU). CLI circulatory level was low at hospital admission in all children presenting with CM and recovered to normal level during convalescence (p < 0.0001). At acute onset, circulatory level of CLI in the CM group significantly discriminates CM from the UM, SMA, DC and CC groups. CONCLUSIONS: The CLI circulatory level is low in all patients in the CM group at admission, but recovers through convalescence. The level of CLI at acute onset may be a specific discriminatory marker of CM. This work suggests that CLI may play a role in the pathophysiology of CM and may be useful in the diagnosis and follow-up of children presenting with CM.

A Virus Hosted in Malaria-Infected Blood Protects against T Cell-Mediated Inflammatory Diseases by Impairing DC Function in a Type I IFN-Dependent Manner.

Coinfections shape immunity and influence the development of inflammatory diseases, resulting in detrimental or beneficial outcome. Coinfections with concurrent Plasmodium species can alter malaria clinical evolution, and malaria infection itself can modulate autoimmune reactions. Yet, the underlying mechanisms remain ill defined. Here, we demonstrate that the protective effects of some rodent malaria strains on T cell-mediated inflammatory pathologies are due to an RNA virus cohosted in malaria-parasitized blood. We show that live and extracts of blood parasitized by Plasmodium berghei K173 or Plasmodium yoelii 17X YM, protect against P. berghei ANKA-induced experimental cerebral malaria (ECM) and myelin oligodendrocyte glycoprotein (MOG)/complete Freund's adjuvant (CFA)-induced experimental autoimmune encephalomyelitis (EAE), and that protection is associated with a strong type I interferon (IFN-I) signature. We detected the presence of the RNA virus lactate dehydrogenase-elevating virus (LDV) in the protective Plasmodium stabilates and we established that LDV infection alone was necessary and sufficient to recapitulate the protective effects on ECM and EAE. In ECM, protection resulted from an IFN-I-mediated reduction in the abundance of splenic conventional dendritic cell and impairment of their ability to produce interleukin (IL)-12p70, leading to a decrease in pathogenic CD4+ Th1 responses. In EAE, LDV infection induced IFN-I-mediated abrogation of IL-23, thereby preventing the differentiation of granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing encephalitogenic CD4+ T cells. Our work identifies a virus cohosted in several Plasmodium stabilates across the community and deciphers its major consequences on the host immune system. More generally, our data emphasize the importance of considering contemporaneous infections for the understanding of malaria-associated and autoimmune diseases.IMPORTANCE Any infection modifies the host immune status, potentially ameliorating or aggravating the pathophysiology of a simultaneous inflammatory condition. In the course of investigating how malaria infection modulates the severity of contemporaneous inflammatory diseases, we identified a nonpathogenic mouse virus in stabilates of two widely used rodent parasite lines: Plasmodium berghei K173 and Plasmodium yoelii 17X YM. We established that the protective effects of these Plasmodium lines on cerebral malaria and multiple sclerosis are exclusively due to this virus. The virus induces a massive type I interferon (IFN-I) response and causes quantitative and qualitative defects in the ability of dendritic cells to promote pathogenic T cell responses. Beyond revealing a possible confounding factor in rodent malaria models, our work uncovers some bases by which a seemingly innocuous viral (co)infection profoundly changes the immunopathophysiology of inflammatory diseases.

Gene expression profiling in blood from cerebral malaria patients and mild malaria patients living in Senegal.

BACKGROUND: Plasmodium falciparum malaria remains a major health problem in Africa. The mechanisms of pathogenesis are not fully understood. Transcriptomic studies may provide new insights into molecular pathways involved in the severe form of the disease. METHODS: Blood transcriptional levels were assessed in patients with cerebral malaria, non-cerebral malaria, or mild malaria by using microarray technology to look for gene expression profiles associated with clinical status. Multi-way ANOVA was used to extract differentially expressed genes. Network and pathways analyses were used to detect enrichment for biological pathways. RESULTS: We identified a set of 443 genes that were differentially expressed in the three patient groups after applying a false discovery rate of 10%. Since the cerebral patients displayed a particular transcriptional pattern, we focused our analysis on the differences between cerebral malaria patients and mild malaria patients. We further found 842 differentially expressed genes after applying a false discovery rate of 10%. Unsupervised hierarchical clustering of cerebral malaria-informative genes led to clustering of the cerebral malaria patients. The support vector machine method allowed us to correctly classify five out of six cerebral malaria patients and six of six mild malaria patients. Furthermore, the products of the differentially expressed genes were mapped onto a human protein-protein network. This led to the identification of the proteins with the highest number of interactions, including GSK3B, RELA, and APP. The enrichment analysis of the gene functional annotation indicates that genes involved in immune signalling pathways play a role in the occurrence of cerebral malaria. These include BCR-, TCR-, TLR-, cytokine-, FcεRI-, and FCGR- signalling pathways and natural killer cell cytotoxicity pathways, which are involved in the activation of immune cells. In addition, our results revealed an enrichment of genes involved in Alzheimer's disease. CONCLUSIONS: In the present study, we examine a set of genes whose expression differed in cerebral malaria patients and mild malaria patients. Moreover, our results provide new insights into the potential effect of the dysregulation of gene expression in immune pathways. Host genetic variation may partly explain such alteration of gene expression. Further studies are required to investigate this in African populations.

The Liver-Stage Plasmodium Infection Is a Critical Checkpoint for Development of Experimental Cerebral Malaria.

Cerebral malaria is a life-threatening complication of malaria in humans, and the underlying pathogenic mechanisms are widely analyzed in a murine model of experimental cerebral malaria (ECM). Here, we show abrogation of ECM by hemocoel sporozoite-induced infection of a transgenic Plasmodium berghei line that overexpresses profilin, whereas these parasites remain fully virulent in transfusion-mediated blood infection. We, thus, demonstrate the importance of the clinically silent liver-stage infection for modulating the onset of ECM. Even though both parasites triggered comparable splenic immune cell expansion and accumulation of antigen-experienced CD8+ T cells in the brain, infection with transgenic sporozoites did not lead to cerebral vascular damages and suppressed the recruitment of overall lymphocyte populations. Strikingly, infection with the transgenic strain led to maintenance of CD115+Ly6C+ monocytes, which disappear in infected animals prone to ECM. An early induction of IL-10, IL-12p70, IL-6, and TNF at the time when parasites emerge from the liver might lead to a diminished induction of hepatic immunity. Collectively, our study reveals the essential role of early host interactions in the liver that may dampen the subsequent pro-inflammatory immune responses and influence the occurrence of ECM, highlighting a novel checkpoint in this fatal pathology.

Malawian children with uncomplicated and cerebral malaria have decreased activated Vγ9Vδ2 γδ T cells which increase in convalescence.

Malaria is responsible for almost half a million deaths annually. The role of Vγ9Vδ2 γδ T cells in malaria is still unclear. Studies have reported an association between this cell subset and malaria symptoms and severity. Profiles of Vγ9Vδ2 γδ T cells in bigger cohorts with different levels of clinical severity have not been described. Proportion, numbers, and activation status of Vγ9Vδ2 γδ T cells were measured by flow cytometry in 59 healthy controls (HCs), 58 children with uncomplicated malaria (UM) and 67 with cerebral malaria (CM,) during acute malaria and in convalescence 28 days later. Vγ9Vδ2 γδ T cell were lower in children presenting with UM and CM than in HCs. Cell counts did not vary with malaria severity (CM median counts 40 x 103 cells/µL, IQR [23-103]; UM median counts 30 x 103 cells/µL [10-90], P = 0.224). Vγ9Vδ2 γδ T cell counts increased during convalescence for UM (70 [40-60] x 103 cells/µL and CM (90 [60-140] x 103 cells/µL), to levels similar to those in HCs (70 [50-140] x 103 cells/µL), p = 0.70 and p = 0.40 respectively. Expression of the activation markers CD69 and HLA-DR on Vγ9Vδ2 γδ T cells was higher in malaria cases than in controls (HCs vs UM or CM, p < 0.0001) but was similar between UM and CM. HLA-DR expression remained elevated at 28 days, suggesting sustained activation of Vγ9Vδ2 γδ T cells during recovery. Vγ9Vδ2 γδ T cell proportions and cells counts were suppressed in acute disease and normalized in convalescence, a phenomenon previously hypothesized to be due to transient migration of the cells to secondary lymphoid tissue. The presence of highly activated Vγ9Vδ2 γδ T cells suggests that this T cell subset plays a specific role in response to malaria infection.

Malaria inflammation by xanthine oxidase-produced reactive oxygen species.

Malaria is a highly inflammatory disease caused by Plasmodium infection of host erythrocytes. However, the parasite does not induce inflammatory cytokine responses in macrophages in vitro and the source of inflammation in patients remains unclear. Here, we identify oxidative stress, which is common in malaria, as an effective trigger of the inflammatory activation of macrophages. We observed that extracellular reactive oxygen species (ROS) produced by xanthine oxidase (XO), an enzyme upregulated during malaria, induce a strong inflammatory cytokine response in primary human monocyte-derived macrophages. In malaria patients, elevated plasma XO activity correlates with high levels of inflammatory cytokines and with the development of cerebral malaria. We found that incubation of macrophages with plasma from these patients can induce a XO-dependent inflammatory cytokine response, identifying a host factor as a trigger for inflammation in malaria. XO-produced ROS also increase the synthesis of pro-IL-1ß, while the parasite activates caspase-1, providing the two necessary signals for the activation of the NLRP3 inflammasome. We propose that XO-produced ROS are a key factor for the trigger of inflammation during malaria.

Experimental Cerebral Malaria Alters Blood Lipid Levels During Pathogenesis.

Malaria infection threatens millions of people worldwide. Sequestering of Plasmodium-infected erythrocytes within the blood vessels of the brain may lead to a more severe form of disease called cerebral malaria (CM), which is difficult to diagnose and treat. Here we used C57BL/6 mice to establish a model of experimental CM (ECM). Comparing the dosage dependence of ECM induction, we found that inoculation with 1×103 parasitized erythrocytes had higher efficiency at establishing ECM than 1×106 parasitized erythrocytes. However, the percentage of ECM varied in different experimental batches. Infected mice that developed ECM had elevated serum levels of total cholesterol and decreased serum levels of high-density lipoprotein and low-density lipoprotein cholesterol. In addition, ECM mice exhibited liver and kidney dysfunction. ECM induced by low dose inoculation requires additional verification for efficiency. Biochemical analysis of ECM mice revealed characteristic blood lipid levels. These findings provide new clues for the diagnosis and mechanistic probing of CM pathogenesis.

Collider bias and the apparent protective effect of glucose-6-phosphate dehydrogenase deficiency on cerebral malaria.

Case fatality rates in severe falciparum malaria depend on the pattern and degree of vital organ dysfunction. Recent large-scale case-control analyses of pooled severe malaria data reported that glucose-6-phosphate dehydrogenase deficiency (G6PDd) was protective against cerebral malaria but increased the risk of severe malarial anaemia. A novel formulation of the balancing selection hypothesis was proposed as an explanation for these findings, whereby the selective advantage is driven by the competing risks of death from cerebral malaria and death from severe malarial anaemia. We re-analysed these claims using causal diagrams and showed that they are subject to collider bias. A simulation based sensitivity analysis, varying the strength of the known effect of G6PDd on anaemia, showed that this bias is sufficient to explain all of the observed association. Future genetic epidemiology studies in severe malaria would benefit from the use of causal reasoning.

The blood transcriptome of childhood malaria.

BACKGROUND: Transcriptomic research of blood cell lineages supports the understanding of distinct features of the immunopathology in human malaria. METHODS: We used microarray hybridization, validated by real-time RT-PCR to analyze whole blood gene expression in healthy Gabonese children and children with various conditions of Plasmodium falciparum infection, including i) asymptomatic infection, ii) uncomplicated malaria, iii) malaria associated with severe anemia and iv) cerebral malaria. FINDINGS: Our data indicate that the expression profile of 22 genes significantly differed among the investigated groups. Immunoglobulin production, complement regulation and IFN beta signaling, in particular IRF7 and ISRE binding signatures in the corresponding genes, were most conspicuous. Down-regulation in cerebral malaria seems to rely on AhRF, GABP and HIF1 hypoxia transcription factors. ARG1, BPI, CD163, IFI27, HP and TNFAIP6 transcript levels correlated positively with lactatemia, and negatively with hemoglobin concentrations. INTERPRETATION: Differences in gene expression profile reflect distinct immunopathological mechanisms of P. falciparum infection. They emerge as potential prognostic markers for early therapeutic measures and need to be validated further. FUND: This work was supported by a grant of the NGFN (Nationales Genomforschungsnetz 01GS0114) and by a CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil) PhD scholarship for A. B. W. Boldt. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Young Sprague Dawley rats infected by Plasmodium berghei: A relevant experimental model to study cerebral malaria.

Cerebral malaria (CM) is the most severe manifestation of human malaria yet is still poorly understood. Mouse models have been developed to address the subject. However, their relevance to mimic human pathogenesis is largely debated. Here we study an alternative cerebral malaria model with an experimental Plasmodium berghei Keyberg 173 (K173) infection in Sprague Dawley rats. As in Human, not all infected subjects showed cerebral malaria, with 45% of the rats exhibiting Experimental Cerebral Malaria (ECM) symptoms while the majority (55%) of the remaining rats developed severe anemia and hyperparasitemia (NoECM). These results allow, within the same population, a comparison of the noxious effects of the infection between ECM and severe malaria without ECM. Among the ECM rats, 77.8% died between day 5 and day 12 post-infection, while the remaining rats were spontaneously cured of neurological signs within 24-48 hours. The clinical ECM signs observed were paresis quickly evolving to limb paralysis, global paralysis associated with respiratory distress, and coma. The red blood cell (RBC) count remained normal but a drastic decrease of platelet count and an increase of white blood cell numbers were noted. ECM rats also showed a decrease of glucose and total CO2 levels and an increase of creatinine levels compared to control rats or rats with no ECM. Assessment of the blood-brain barrier revealed loss of integrity, and interestingly histopathological analysis highlighted cyto-adherence and sequestration of infected RBCs in brain vessels from ECM rats only. Overall, this ECM rat model showed numerous clinical and histopathological features similar to Human CM and appears to be a promising model to achieve further understanding the CM pathophysiology in Humans and to evaluate the activity of specific antimalarial drugs in avoiding/limiting cerebral damages from malaria.