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Inhibition of nitric oxide (NO) signaling may contribute to pathological activation of the vascular endothelium during severe malaria infection. Dimethylarginine dimethylaminohydrolase (DDAH) regulates endothelial NO synthesis by maintaining homeostasis between asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor, and arginine, the NOS substrate. We carried out a community-based case-control study of Gambian children to determine whether ADMA and arginine homeostasis is disrupted during severe or uncomplicated malaria infections. Circulating plasma levels of ADMA and arginine were determined at initial presentation and 28 days later. Plasma ADMA/arginine ratios were elevated in children with acute severe malaria compared to 28-day follow-up values and compared to children with uncomplicated malaria or healthy children (p, Author Summary During a malaria infection, the vascular endothelium becomes more adhesive, permeable, and prone to trigger blood clotting. These changes help the parasite adhere to blood vessels, but endanger the host by obstructing blood flow through small vessels. Endothelial nitric oxide (NO) would normally counteract these pathological changes, but NO signalling is diminished malaria. NO synthesis is inhibited by asymmetric dimethylarginine (ADMA), a methylated derivative of arginine that is released during normal protein turnover. We found the ratio of ADMA to arginine to be elevated in Gambian children with severe malaria, a metabolic disturbance known to inhibit NO synthesis. ADMA was associated with markers of endothelial activation and impaired tissue perfusion. In parallel experiments using mice, the enzyme responsible for metabolizing ADMA, dimethylarginine dimethylaminohydrolase (DDAH), was inactivated after infection with a rodent malaria. Based on these studies, we propose that decreased metabolism of ADMA by DDAH might contribute to the elevated ADMA/arginine ratio observed during an acute episode of malaria. Strategies to preserve or increase DDAH activity might improve NO synthesis and help to prevent the vascular manifestations of severe malaria.

Human malaria parasite species were originally acquired from other primate hosts and subsequently became endemic, then spread throughout large parts of the world. A major zoonosis is now occurring with Plasmodium knowlesi from macaques in Southeast Asia, with a recent acceleration in numbers of reported cases particularly in Malaysia. To investigate the parasite population genetics, we developed sensitive and species-specific microsatellite genotyping protocols and applied these to analysis of samples from 10 sites covering a range of >1,600 km within which most cases have occurred. Genotypic analyses of 599 P. knowlesi infections (552 in humans and 47 in wild macaques) at 10 highly polymorphic loci provide radical new insights on the emergence. Parasites from sympatric long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) were very highly differentiated (FST = 0.22, and K-means clustering confirmed two host-associated subpopulations). Approximately two thirds of human P. knowlesi infections were of the long-tailed macaque type (Cluster 1), and one third were of the pig-tailed-macaque type (Cluster 2), with relative proportions varying across the different sites. Among the samples from humans, there was significant indication of genetic isolation by geographical distance overall and within Cluster 1 alone. Across the different sites, the level of multi-locus linkage disequilibrium correlated with the degree of local admixture of the two different clusters. The widespread occurrence of both types of P. knowlesi in humans enhances the potential for parasite adaptation in this zoonotic system., Author Summary Extraordinary phases of pathogen evolution may occur during an emerging zoonosis, potentially involving adaptation to human hosts, with changes in patterns of virulence and transmission. In a large population genetic survey, we show that the malaria parasite Plasmodium knowlesi in humans is an admixture of two highly divergent parasite populations, each associated with different forest-dwelling macaque reservoir host species. Most of the transmission and sexual reproduction occurs separately in each of the two parasite populations. In addition to the reservoir host-associated parasite population structure, there was also significant genetic differentiation that correlated with geographical distance. Although both P. knowlesi types co-exist in the same areas, the divergence between them is similar to or greater than that seen between sub-species in other sexually reproducing eukaryotes. This may offer particular opportunities for evolution of virulence and host-specificity, not seen with other malaria parasites, so studies of ongoing adaptation and interventions to reduce transmission are urgent priorities.

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (, Author Summary Malaria remains one of the world's most deadly diseases. Life-threatening malaria is linked to a process called rosetting, in which malaria parasite-infected red blood cells bind to uninfected red cells to form aggregates that block blood flow in vital organs such as the brain. Current efforts to develop drugs or vaccines against rosetting are hindered by variation in the parasite rosette-mediating proteins, found on the surface of infected red cells. We studied these parasite-derived surface proteins and discovered that although they are variable, they share some common features. We raised antibodies against the rosette-mediating proteins, and found that they cross-reacted with multiple rosetting parasite strains from different countries around the world, including samples collected directly from African children with severe malaria. These findings provide new insights into malaria parasite interactions with human cells, and provide proof of principle that variable parasite molecules from virulent malaria parasites can induce strain-transcending antibodies. Hence, this work provides the foundation for the development of new therapies to treat or prevent life-threatening malaria.

Heme oxygenase 1 (HO-1) is an essential enzyme induced by heme and multiple stimuli associated with critical illness. In humans, polymorphisms in the HMOX1 gene promoter may influence the magnitude of HO-1 expression. In many diseases including murine malaria, HO-1 induction produces protective anti-inflammatory effects, but observations from patients suggest these may be limited to a narrow range of HO-1 induction, prompting us to investigate the role of HO-1 in malaria infection. In 307 Gambian children with either severe or uncomplicated P. falciparum malaria, we characterized the associations of HMOX1 promoter polymorphisms, HMOX1 mRNA inducibility, HO-1 protein levels in leucocytes (flow cytometry), and plasma (ELISA) with disease severity. The (GT)n repeat polymorphism in the HMOX1 promoter was associated with HMOX1 mRNA expression in white blood cells in vitro, and with severe disease and death, while high HO-1 levels were associated with severe disease. Neutrophils were the main HO-1-expressing cells in peripheral blood, and HMOX1 mRNA expression was upregulated by heme-moieties of lysed erythrocytes. We provide mechanistic evidence that induction of HMOX1 expression in neutrophils potentiates the respiratory burst, and propose this may be part of the causal pathway explaining the association between short (GT)n repeats and increased disease severity in malaria and other critical illnesses. Our findings suggest a genetic predisposition to higher levels of HO-1 is associated with severe illness, and enhances the neutrophil burst leading to oxidative damage of endothelial cells. These add important information to the discussion about possible therapeutic manipulation of HO-1 in critically ill patients., Author Summary HO-1 is an important anti-inflammatory enzyme induced by several stimuli associated with critical illness. In humans, the amount of HO-1 produced is influenced by a genetic polymorphism in the gene promoter region. Using Plasmodium falciparum malaria that can cause a sepsis-like syndrome as an example, we characterize the associations between the (GT)n polymorphism, HO-1 protein levels and HMOX1-mRNA expression with severity of malaria in 307 Gambian children. Our results support the functionality of this polymorphism, demonstrate that P. falciparum infections increase HO-1 levels, and indicate that a genetic predisposition to strongly upregulate HO-1 is associated with severe forms of malaria and increased risk of dying. We identify neutrophils as the main HO-1-producing blood cells, and provide evidence that hemin-mediated induction of HMOX1 in neutrophils in vitro enhances the oxidative burst. In this way sequestered neutrophils may contribute to oxidative damage of endothelial cells, which may be part of a causal pathway explaining the association between short (GT)n repeats and increased disease severity. Our findings imply that the beneficial effects of HO-1 may be limited to a narrow window of concentrations, which should be born in mind when considering the therapeutic potential of manipulating HO-1 induction in critically ill patients.

Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to pathogenesis of severe malaria. To determine whether this balance is maintained by classical regulatory T cells (CD4+ FOXP3+ CD127−/low; Tregs) we compared cellular responses between Gambian children (n = 124) with severe Plasmodium falciparum malaria or uncomplicated malaria infections. Although no significant differences in Treg numbers or function were observed between the groups, Treg activity during acute disease was inversely correlated with malaria-specific memory responses detectable 28 days later. Thus, while Tregs may not regulate acute malarial inflammation, they may limit memory responses to levels that subsequently facilitate parasite clearance without causing immunopathology. Importantly, we identified a population of FOXP3−, CD45RO+ CD4+ T cells which coproduce IL-10 and IFN-γ. These cells are more prevalent in children with uncomplicated malaria than in those with severe disease, suggesting that they may be the regulators of acute malarial inflammation., Author Summary While Tregs have been implicated in regulation of the immune response to chronic infections, their potential in determining disease outcome in acute infections is unclear. In this study we have found that Tregs are unable to control the florid inflammation during acute, severe P. falciparum malaria infections, suggesting that this component of the immunoregulatory arsenal may be rapidly overwhelmed by virulent infections. Further, we identified, for the first time in an acute human infection, a population of IL-10-producing Th-1 effector cells and found that IL-10-producing Th-1 cells were associated with development of uncomplicated as opposed to severe malaria, leading us to suggest that such “self-regulating” Th-1 cells may contribute to clearing malaria infections without inducing immune-mediated pathology. In addition, we found evidence that malaria-induced Tregs may limit the magnitude of malaria-specific memory responses detectable 28 days later, which may reduce the risk of immune-mediated pathology upon reinfection and may explain how immunity to severe disease can be gained after as little as one or two infections. We conclude that vaccines designed to induce cell-mediated responses should be assessed for their ability to induce IL-10 producing Th-1 cells and Tregs.