Modulation of cerebral malaria by fasudil and other immune-modifying compounds

Malaria continues to cause millions of deaths annually. No specific effective treatment has yet been found for cerebral malaria, one of the most severe complications of the disease. The pathology of cerebral malaria is considered to be primarily immunological. We examined a number of compounds with known effects on the immune system, in a murine model of cerebral malaria. Of the compounds tested, only fasudil and curcumin had significant effects on the progression of the disease. Although neither drug caused a reduction in parasitemia, survival of the treated mice was significantly increased, and the development of cerebral malaria was either delayed or prevented. Our results support the hypothesis that an immunomodulator efficient in preventing CM should be administered together with anti-plasmodial drugs to prevent severe malaria disease; curcumin and fasudil should be further investigated to determine efficiency and feasibility of treatment.     

Affinity Proteomics Reveals Elevated Muscle Proteins in Plasma of Children with Cerebral Malaria

Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.     

Candidate Human Genetic Polymorphisms and Severe Malaria in a Tanzanian Population

Human genetic background strongly influences susceptibility to malaria infection and progression to severe disease and death. Classical genetic studies identified haemoglobinopathies and erythrocyte-associated polymorphisms, as protective against severe disease. High throughput genotyping by mass spectrometry allows multiple single nucleotide polymorphisms (SNPs) to be examined simultaneously. We compared the prevalence of 65 human SNP''s, previously associated with altered risk of malaria, between Tanzanian children with and without severe malaria. Five hundred children, aged 1–10 years, with severe malaria were recruited from those admitted to hospital in Muheza, Tanzania and compared with matched controls. Genotyping was performed by Sequenom MassArray, and conventional PCR was used to detect deletions in the alpha-thalassaemia gene. SNPs in two X-linked genes were associated with altered risk of severe malaria in females but not in males: heterozygosity for one or other of two SNPs in the G6PD gene was associated with protection from all forms of severe disease whilst two SNPs in the gene encoding CD40L were associated with respiratory distress. A SNP in the adenyl cyclase 9 (ADCY9) gene was associated with protection from acidosis whilst a polymorphism in the IL-1α gene (IL1A) was associated with an increased risk of acidosis. SNPs in the genes encoding IL-13 and reticulon-3 (RTN3) were associated with increased risk of cerebral malaria. This study confirms previously known genetic associations with protection from severe malaria (HbS, G6PD). It identifies two X-linked genes associated with altered risk of severe malaria in females, identifies mutations in ADCY9, IL1A and CD40L as being associated with altered risk of severe respiratory distress and acidosis, both of which are characterised by high serum lactate levels, and also identifies novel genetic associations with severe malaria (TRIM5) and cerebral malaria(IL-13 and RTN3). Further studies are required to test the generality of these associations and to understand their functional consequences.     

Acquired immunity and postnatal clinical protection in childhood cerebral malaria

By analysing data on the age distribution of cerebral malaria among sites of different transmission intensities, we conclude that the most plausible explanation for the epidemiological patterns seen is that (i) cerebral malaria is caused by a distinct set of Plasmodium falciparum antigenic types; (ii) these antigenic types or 'CM strains' are very common and induce strong strain-specific immunity; and (iii) the postnatal period of protection against cerebral malaria is much longer than the period of protection against other forms of severe disease. The alternative hypothesis that cerebral malaria may be caused by any 'strain' of P. falciparum is compatible with the data only if a single exposure is sufficient to protect against further episodes. This is not consistent with observations on the history of exposure of patients with cerebral malaria. Finally, it is clear that although the delayed peak in incidence of cerebral malaria (with age) can be generated by assuming that subsequent exposures carry a higher risk of disease, such an explanation is not compatible with the observation that severe disease rates are low among infants and young children in areas of high transmissibility.     

Human cerebral malaria and the blood-brain barrier

Malaria represents a continuing and major global health challenge and our understanding of how the Plasmodium parasite causes severe disease and death remains poor. One serious complication of the infection is cerebral malaria, a clinically complex syndrome of coma and potentially reversible encephalopathy, associated with a high mortality rate and increasingly recognised long-term sequelae in survivors. Research into the pathophysiology of cerebral malaria, using a combination of clinical and pathological studies, animal models and in vitro cell culture work, has focussed attention on the blood-brain barrier (BBB). This represents the key interface between the brain parenchyma and the parasite, which develops within an infected red cell but remains inside the vascular space. Studies of BBB function in cerebral malaria have provided some evidence for parasite-induced changes secondary to sequestration of parasitised red blood cells and host leukocytes within the cerebral microvasculature, such as redistribution of endothelial cell intercellular junction proteins and intracellular signaling. However, the evidence for a generalised increase in BBB permeability, leading to cerebral oedema, is conflicting. As well as direct cell adhesion-dependent effects, local adhesion-independent effects may activate and damage cerebral endothelial cells and perivascular cells, such as decreased blood flow, hypoxia or the effects of parasite toxins such as pigment. Finally, a number of systemic mechanisms could influence the BBB during malaria, such as the metabolic and inflammatory complications of severe disease acting 'at a distance'. This review will summarise evidence for these mechanisms from human studies of cerebral malaria and discuss the possible role for BBB dysfunction in this complex and challenging disease.     

The kidney–brain pathogenic axis in severe falciparum malaria

Severe falciparum malaria is a medical emergency and a leading cause of death and neurodisability in endemic areas. Common complications include acute kidney injury (AKI) and cerebral malaria, and recent studies have suggested links between kidney and brain dysfunction in Plasmodium falciparum infection. Here, we review these new findings and present the hypothesis of a pivotal pathogenic crosstalk between the kidneys and the brain in severe falciparum malaria. We highlight the evidence of a role for distant organ involvement in the development of cerebral malaria and subsequent neurocognitive impairment post-recovery, describe the challenges associated with current diagnostic shortcomings for both AKI and brain involvement in severe falciparum malaria, and explore novel potential therapeutic strategies.     

Malaria transmission and morbidity

Stable malaria endemicity is maintained over a wide range of transmission intensities in sub-Saharan Africa. This paper considers variations in the clinical manifestations and their consequences with differences in transmission intensity. Epidemiological approaches to malarial disease have concentrated on two clinical syndromes, severe malarial anaemia and cerebral malaria. Within an area the mean age of children with severe malarial anaemia is always lower than that of those with cerebral malaria. In areas of higher malaria transmission children, on average, encounter malaria at a younger age and the mean age of clinical cases is lower. Malarial anaemia tends therefore to be relatively more important under high transmission settings and cerebral malaria tends to gain in importance under lower transmission settings. In a number of studies the total load of malaria morbidity, whether measured as none severe malaria in the community or as severe malaria admitted to hospital, is low under stable low transmission conditions but is at its highest under moderate intensities of transmission. Thereafter it reaches a plateau, or even falls, at the highest transmission intensities. It is not known whether the same is true for mortality in communities living under different transmission settings. Possible implications for changes in patterns of morbidity and mortality following interventions which lower malaria transmission are discussed. It is concluded that such interventions should play an important role in integrated malaria control programmes but that these should involve concomitant introduction of other interventions, in order to minimise the possible risks of a reduced effect as the immune response of the population re-equilibrates in the face of reduced challenge.     

Elevated cell-specific microparticles are a biological marker for cerebral dysfunctions in human severe malaria

Cerebral malaria (CM) and severe anemia (SA) are the most severe complications of Plasmodium falciparum infections. Although increased release of endothelial microparticles (MP) correlates with malaria severity, the full extent of vascular cell vesiculation remains unknown. Here, we characterize the pattern of cell-specific MP in patients with severe malaria. We tested the hypothesis that systemic vascular activation contributes to CM by examining origins and levels of plasma MP in relation to clinical syndromes, disease severity and outcome. Patients recruited in Douala, Cameroon, were assigned to clinical groups following WHO criteria. MP quantitation and phenotyping were carried out using cell-specific markers by flow cytometry using antibodies recognizing cell-specific surface markers. Platelet, erythrocytic, endothelial and leukocytic MP levels were elevated in patients with cerebral dysfunctions and returned to normal by discharge. In CM patients, platelet MP were the most abundant and their levels significantly correlated with coma depth and thrombocytopenia. This study shows for the first time a widespread enhancement of vesiculation in the vascular compartment appears to be a feature of CM but not of SA. Our data underpin the role of MP as a biomarker of neurological involvement in severe malaria. Therefore, intervention to block MP production in severe malaria may provide a new therapeutic pathway.     

Distinct patterns of cytokine regulation in discrete clinical forms of Plasmodium falciparum malaria

The pathogenesis of two of the most severe complications of Plasmodium falciparum malaria, cerebral malaria (CM) and severe malarial anaemia (SA) both appear to involve dysregulation of the immune system. We have measured plasma levels of TNF and its two receptors in Ghanaian children with strictly defined cerebral malaria (CM), severe malarial anaemia (SA), or uncomplicated malaria (UM) in two independent studies in an area of seasonal, hyperendemic transmission of P. falciparum. Levels of TNF, soluble TNF receptor 1 (sTNF-R1) and 2 (sTNF-R2) were found to be significantly higher in CM than in the other clinical categories of P. falciparum malaria patients. Levels of both receptors depended on clinical category, whereas only sTNF-R1 levels were significantly dependent on parasitemia. Detailed analysis of the interrelationship between these variables resolved this pattern further, and identified marked differences between the patient categories. While levels of TNF, sTNF-R1 and sTNF-R2 correlated with parasitemia in UM, this was not the case in CM and SA. Rather, there was a tendency towards high levels of TNF and its receptors in CM and low levels in SA without significant correlation to parasitemia in either category. This, and the fact that malaria-induced increases in plasma levels of IL-10 are much lower in SA compared to CM, suggest that distinct forms of dysregulation of the immune response to infection contribute to the pathogenesis of CM and SA.     

Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity

In areas of endemic parasite transmission, protective immunity to Plasmodium falciparum malaria is acquired over several years with numerous disease episodes. Acquisition of Abs to parasite-encoded variant surface Ags (VSA) on the infected erythrocyte membrane is important in the development of immunity, as disease-causing parasites appear to be those not controlled by preexisting VSA-specific Abs. In this work we report that VSA expressed by parasites from young Ghanaian children with P. falciparum malaria were commonly and strongly recognized by plasma Abs from healthy children in the same area, whereas recognition of VSA expressed by parasites from older children was weaker and less frequent. Independent of this, parasites isolated from children with severe malaria (cerebral malaria and severe anemia) were better recognized by VSA-specific plasma Abs than parasites obtained from children with nonsevere disease. This was not due to a higher infection multiplicity in younger patients or in patients with severe disease. Our data suggest that acquisition of VSA-specific Ab responses gradually restricts the VSA repertoire that is compatible with parasite survival in the semi-immune host. This appears to limit the risk of severe disease by discriminating against the expression of VSA likely to cause life-threatening complications, such as cerebral malaria and severe anemia. Such VSA seem to be preferred by parasites infecting a nonimmune host, suggesting that VSA expression and switching are not random, and that the VSA expression pattern is modulated by immunity. This opens the possibility of developing morbidity-reducing vaccines targeting a limited subset of common and particularly virulent VSA.     

Magnetic resonance spectroscopy reveals an impaired brain metabolic profile in mice resistant to cerebral malaria infected with Plasmodium berghei ANKA

Malaria is a major cause of morbidity and mortality with an annual death toll exceeding one million. Severe malaria is a complex multisystem disorder, including one or more of the following complications: cerebral malaria, anemia, acidosis, jaundice, respiratory distress, renal insufficiency, coagulation anomalies, and hyperparasitemia. Using a combined in vivo/in vitro metabolic-based approach, we investigated the putative pathogenic effects of Plasmodium berghei ANKA on brain, in a mouse strain developing malaria but resistant to cerebral malaria. The purpose was to determine whether the infection could cause a brain dysfunction distinct from the classic cerebral syndrome. Mice resistant to cerebral malaria were infected with P. berghei ANKA and explored during both the symptomless and the severe stage of the disease by using in vivo brain magnetic resonance imaging and spectroscopy. The infected mice did not present the lesional and metabolic hallmarks of cerebral malaria. However, brain dysfunction caused by anemia, parasite burden, and hepatic damage was evidenced. We report an increase in cerebral blood flow, a process allowing temporary maintenance of oxygen supply to brain despite anemia. Besides, we document metabolic anomalies affecting choline-derived compounds, myo-inositol, glutamine, glycine, and alanine. The choline decrease appears related to parasite proliferation. Glutamine, myo-inositol, glycine, and alanine variations together indicate a hepatic encephalopathy, a finding in agreement with the liver damage detected in mice, which is also a feature of the human disease. These results reveal the vulnerability of brain to malaria infection at the severe stage of the disease even in the absence of cerebral malaria.     

T cell-derived IL-10 and its impact on the regulation of host responses during malaria

Despite intense research, malaria still is the one of the most devastating diseases killing more people than any other parasitic infection. In an attempt to control the infection, the host immune system produces a potent pro-inflammatory response. However, this response is also associated with complications, such as severe anaemia, hypoglycaemia and cerebral malaria. This pronounced production of pro-inflammatory cytokines response is a common feature of malaria caused by parasites infecting humans as well as rodents and primates. A balance between pro- and anti-inflammatory responses may be fundamental to the elimination of the parasite without inducing excessive host pathology. IL-10 is a key cytokine that has been shown to have an important regulatory function in establishing this balance in malaria. Here we discuss which cells can produce IL-10 during infection, and present an overview of the evidence showing that T-cell derived IL-10 plays an important role in regulating malaria pathology. Many different subsets of T cells can produce IL-10, however, evidence is accumulating that it is effector Th1 CD4(+) T cells which provide the crucial source that down-regulates inflammatory pathology during blood-stage malaria infections.     

CTLA-4 blockade differentially influences the outcome of non-lethal and lethal Plasmodium yoelii infections

An immune response against malaria has to be tightly controlled. The production of pro-inflammatory cytokines is required to control parasites but the same cytokines are also involved in severe malaria. We have shown that CTLA-4 expression during Plasmodium berghei malaria dampens the immune response. This strain provokes a pro-inflammatory immune response that is associated with the pathology of cerebral malaria. Accordingly a blockade of CTLA-4 during the blood-stage of P. berghei malaria leads to an exacerbation of disease. To analyze the effects of a CTLA-4 blockade in a malaria model which is not prone to immune pathology we employed P. yoelii infection. Blood-stage infection led to a rapid induction of CTLA-4 on T cells. Using the non-lethal P. yoelii strain Py17NL we found that a blockade of CTLA-4 resulted in an increased T cell activation and IFN-gamma production, which was accompanied by a lower peak parasitemia and earlier parasite clearance. In contrast, blockade of CTLA-4 during infection with a P. yoelii strain exhibiting a higher parasitemia induced markedly increased serum-levels of TNF-alpha, which was associated with severe inflammation and reduced survival.     

Clinical impact of vivax malaria: A collection review

BackgroundPlasmodium vivax infects an estimated 7 million people every year. Previously, vivax malaria was perceived as a benign condition, particularly when compared to falciparum malaria. Reports of the severe clinical impacts of vivax malaria have been increasing over the last decade.Methods and findingsWe describe the main clinical impacts of vivax malaria, incorporating a rapid systematic review of severe disease with meta-analysis of data from studies with clearly defined denominators, stratified by hospitalization status. Severe anemia is a serious consequence of relapsing infections in children in endemic areas, in whom vivax malaria causes increased morbidity and mortality and impaired school performance. P. vivax infection in pregnancy is associated with maternal anemia, prematurity, fetal loss, and low birth weight. More than 11,658 patients with severe vivax malaria have been reported since 1929, with 15,954 manifestations of severe malaria, of which only 7,157 (45%) conformed to the World Health Organization (WHO) diagnostic criteria. Out of 423 articles, 311 (74%) were published since 2010. In a random-effects meta-analysis of 85 studies, 68 of which were in hospitalized patients with vivax malaria, we estimated the proportion of patients with WHO-defined severe disease as 0.7% [95% confidence interval (CI) 0.19% to 2.57%] in all patients with vivax malaria and 7.11% [95% CI 4.30% to 11.55%] in hospitalized patients. We estimated the mortality from vivax malaria as 0.01% [95% CI 0.00% to 0.07%] in all patients and 0.56% [95% CI 0.35% to 0.92%] in hospital settings. WHO-defined cerebral, respiratory, and renal severe complications were generally estimated to occur in fewer than 0.5% patients in all included studies. Limitations of this review include the observational nature and small size of most of the studies of severe vivax malaria, high heterogeneity of included studies which were predominantly in hospitalized patients (who were therefore more likely to be severely unwell), and high risk of bias including small study effects.ConclusionsYoung children and pregnant women are particularly vulnerable to adverse clinical impacts of vivax malaria, and preventing infections and relapse in this groups is a priority. Substantial evidence of severe presentations of vivax malaria has accrued over the last 10 years, but reporting is inconsistent. There are major knowledge gaps, for example, limited understanding of the underlying pathophysiology and the reason for the heterogenous geographical distribution of reported complications. An adapted case definition of severe vivax malaria would facilitate surveillance and future research to better understand this condition.

Elizabeth A Ashley and colleagues describe the main clinical impacts of vivax malaria globally.     

Complement receptor 1 and malaria

Plasmodium falciparum malaria is an intracellular parasite that is transmitted by Anopheles mosquitoes. It is responsible for approximately 1 million deaths per year. Most deaths occur as a result of complications such as severe anaemia or cerebral malaria (coma). The complement receptor 1 is a key complement regulator found on the surface of red cells and most leucocytes. A growing body of evidence suggests that this molecule plays a critical role in the pathogenesis of P. falciparum malaria. Initial reports showed that CR1 enables the binding of infected red cells to uninfected red cells to form rosettes, which can potentially obstruct small capillaries. However, further evidence suggests that CR1 is also important in the control of complement activation and immune complex formation during malaria infection. Most recently, CR1 has also been shown to be a receptor for the invasion of red cells by the parasite. Its polymorphic nature almost certainly has allowed the selection of variants that have helped humankind survive the scurge of malaria. The finding of conflicting reports about the exact role of these variants in severe disease underlies the complexity of the parasite-host interactions and highlights the need for further studies.     

Reduced immune complex binding capacity and increased complement susceptibility of red cells from children with severe malaria-associated anemia

Plasmodium falciparum malaria causes 1-2 million deaths per year. Most deaths occur as a result of complications such as severe anemia and cerebral malaria (CM) (coma). Red cells of children with severe malaria-associated anemia (SMA) have acquired deficiencies in the complement regulatory proteins complement receptor 1 (CR1, CD35) and decay accelerating factor (DAF, CD55). We investigated whether these deficiencies affect the ability of erythrocytes to bind immune complexes (ICs) and regulate complement activation. We recruited 75 children with SMA (Hb < or = 6 g/dL) from the holoendemic malaria region of the Lake Victoria basin, western Kenya, and 74 age- and gender-matched uncomplicated malaria controls. In addition, we recruited 32 children with CM and 52 age- and gender-matched controls. Deficiencies in red cell CR1 and CD55 in children with SMA were accompanied by a marked decline in IC binding capacity and increased C3b deposition in vivo and ex vivo. Importantly, these changes were specific because they were not seen in red cells of children with CM or their controls. These data suggest that the declines in red cell CR1 and CD55 seen in children with SMA are of physiologic significance and may predispose erythrocytes to complement-mediated damage and phagocytosis in vivo.     

Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome

Our understanding of the basis of severe disease in malaria is incomplete. It is clear that pathology is in part related to the pro-inflammatory nature of the host response but a number of other factors are also thought to be involved, including the interaction between infected erythrocytes and endothelium. This is a complex system involving several host receptors and a major parasite-derived variant antigen (PfEMP1) expressed on the surface of the infected erythrocyte membrane. Previous studies have suggested a role for ICAM-1 in the pathology of cerebral malaria, although these have been inconclusive. In this study we have examined the cytoadherence patterns of 101 patient isolates from varying clinical syndromes to CD36 and ICAM-1, and have used variant ICAM-1 proteins to further characterise this adhesive phenotype. Our results show that increased binding to CD36 is associated with uncomplicated malaria while ICAM-1 adhesion is raised in parasites from cerebral malaria cases.     

Severe malaria: metabolic complications

Metabolic complications of severe malaria are some of the most important and potentially treatable manifestations of this deadly disease. The commonest metabolic complications (lactic acidosis and hypoglycaemia) arise from increased host anaerobic metabolism probably due to a mismatch between tissue oxygen supply and requirement. Optimising treatments for these complications should be guided by detailed understanding of their underlying pathophysiology, and may help to reduce the intolerably high case fatality rate of severe malaria.     

Intestinal helminth infections are associated with increased incidence of Plasmodium falciparum malaria in Thailand

In a prospective study of the total population of 5 hamlets on the western border of Thailand, all subjects were screened for helminth infections; during the following year, the incidence of malaria was recorded. Patients were not treated for helminth infections. Among 731 villagers, helminth-infected subjects were more likely to develop falciparum malaria during the following year (adjusted risk ratio 2.24, range 1.4-3.6; P = 0.001). The risk of developing falciparum malaria increased with the number of helminth species (P =0.036). Whereas in other studies helminths were associated with protection from severe complications of malaria, it seemed here that helminth-infected patients were more likely to develop malaria. It is suggested that a helminth-mediated Th2 shift may have complex consequences on malaria, decreasing antisporozoite immunity, but protecting against severe malaria.     

Inhaled nitric oxide reduces endothelial activation and parasite accumulation in the brain, and enhances survival in experimental cerebral malaria

The host immune response contributes to the onset and progression of severe malaria syndromes, such as cerebral malaria. Adjunctive immunomodulatory strategies for severe malaria may improve clinical outcome beyond that achievable with artemisinin-based therapy alone. Here, we report that prophylaxis with inhaled nitric oxide significantly reduced systemic inflammation (lower TNF, IFNγ and MCP-1 in peripheral blood) and endothelial activation (decreased sICAM-1 and vWF, and increased angiopoeitin-1 levels in peripheral blood) in an experimental cerebral malaria model. Mice that received inhaled nitric oxide starting prior to infection had reduced parasitized erythrocyte accumulation in the brain, decreased brain expression of ICAM-1, and preserved vascular integrity compared to control mice.Inhaled nitric oxide administered in combination with artesunate, starting as late as 5.5 days post-infection, improved survival over treatment with artesunate alone (70% survival in the artesunate only vs. 100% survival in the artesunate plus iNO group, p = 0.03). These data support the clinical investigation of inhaled nitric oxide as a novel adjunctive therapy in patients with severe malaria.