Search

A layer of glycocalyx covers the vascular endothelium serving important protective and homeostatic functions. The objective of this study was to determine if breakdown of the endothelial glycocalyx (eGC) occurs during malaria infection in children. Measures of eGC integrity, endothelial activation, and microvascular reactivity were prospectively evaluated in 146 children: 44 with moderately severe malaria (MSM), 42 with severe malaria (SM), and 60 healthy controls (HC). Biochemical measures of eGC integrity included plasma syndecan-1 and total urinary glycosaminoglycans (GAG). Side-stream dark field imaging was used to quantitatively assess integrity of eGC. Plasma angiopoietin-2 (Ang-2) was measured as a marker of endothelial activation and also as a possible mediator of eGC breakdown. Our results show that urinary GAG, syndecan-1, and Ang-2 were elevated in patients with MSM and SM compared with HC. Syndecan-1 and GAG levels correlated significantly with each other and with plasma Ang-2. The eGC breakdown products also inversely correlated significantly with hemoglobin and platelet count. In the MSM group, imaging results provided further evidence for eGC degradation. Although not correlated with markers of eGC degradation, vascular function (assessed by non-invasive near infrared spectroscopy [NIRS]) demonstrated reduced microvascular reactivity, particularly affecting the SM group. Our findings provide further evidence for breakdown of eGC in falciparum malaria that may contribute to endothelial activation and adhesion of parasitized red blood cells, with reduced nitric oxide formation, and vascular dysfunction.

Cerebral malaria (CM) from Plasmodium falciparum infection is associated with endothelial dysfunction and parasite sequestration. The glycocalyx (GCX), a carbohydrate-rich layer lining the endothelium, is crucial in vascular homeostasis. To evaluate the role of its loss in the pathogenesis of pediatric CM, we measured GCX degradation in Tanzanian children with World Health Organization–defined CM (n = 55), uncomplicated malaria (UM; n = 20), and healthy controls (HCs; n = 25). Urine GCX breakdown products [glycosaminoglycans (GAGs)] were quantified using dimethylmethylene blue (DMMB) and liquid chromatography–tandem mass spectrometry assays. DMMB-GAG and mass spectrometry (MS)-GAG (g/mol creatinine) were increased in CM and UM compared with HCs (P < 0.001), with no differences in DMMB-GAG and MS-GAG between CM and UM children or between those with and without a fatal outcome. In CM survivors, urinary GCX DMMB-GAG normalized by d 3. After adjusting for disease severity, DMMB-GAG was significantly associated with parasitemia [partial correlation coefficient (P(corr)) = 0.34; P = 0.01] and plasma TNF (P(corr) = 0.26; P = 0.04) and inversely with plasma and urine NO oxidation products [P(corr) = −0.31 (P = 0.01) and P(corr) = −0.26 (P = 0.03), respectively]. GCX breakdown is increased in children with falciparum malaria, with similar elevations in CM and UM. Endothelial GCX degradation may impair endothelial NO production, exacerbate adhesion-molecule expression, exposure, and parasite sequestration, and contribute to malaria pathogenesis.—Yeo, T. W., Bush, P. A., Chen, Y., Young, S. P., Zhang, H., Millington, D. S., Granger, D. L., Mwaikambo, E. D., Anstey, N. M., Weinberg, J. B. Glycocalyx breakdown is increased in African children with cerebral and uncomplicated falciparum malaria.

Background Cerebral malaria (CM) pathogenesis remains incompletely understood. Having shown low systemic levels of tetrahydrobiopterin (BH4), an enzymatic cofactor for neurotransmitter synthesis, we hypothesized that BH4 and BH4-dependent neurotransmitters would likewise be low in cerebrospinal fluid (CSF) in CM. Methods We prospectively enrolled Tanzanian children with CM and children with nonmalaria central nervous system conditions (NMCs). We measured CSF levels of BH4, neopterin, and BH4-dependent neurotransmitter metabolites, 3-O-methyldopa, homovanillic acid, and 5-hydroxyindoleacetate, and we derived age-adjusted z-scores using published reference ranges. Results Cerebrospinal fluid BH4 was elevated in CM (n = 49) compared with NMC (n = 51) (z-score 0.75 vs −0.08; P Conclusion Despite low systemic BH4, CSF BH4 was elevated and associated with increased odds of survival in CM. Coma in malaria is not explained by deficiency of BH4-dependent neurotransmitters. Elevated CSF neopterin was 100% sensitive for CM diagnosis and warrants further assessment of its clinical utility for ruling out CM in malaria-endemic areas.

The low bioavailability of nitric oxide (NO) and its precursor, arginine, contributes to the microvascular pathophysiology of severe falciparum malaria. To better characterize the mechanisms underlying hypoargininemia in severe malaria, we measured the plasma concentrations of amino acids involved in de novo arginine synthesis in children with uncomplicated falciparum malaria (UM; n = 61), children with cerebral falciparum malaria (CM; n = 45), and healthy children (HC; n = 109). We also administered primed infusions of l-arginine uniformly labeled with 13C6 and 15N4 to 8 children with severe falciparum malaria (SM; age range, 4 to 9 years) and 7 healthy children (HC; age range, 4 to 8 years) to measure the metabolic flux of arginine, hypothesizing that arginine flux is increased in SM. Using two different tandem mass spectrometric methods, we measured the isotopic enrichment of arginine in plasma obtained at 0, 60, 90, 120, 150, and 180 min during the infusion. The plasma concentrations of glutamine, glutamate, proline, ornithine, citrulline, and arginine were significantly lower in UM and CM than in HC (P ≤ 0.04 for all pairwise comparisons). Of these, glutamine concentrations were the most markedly decreased: median, 457 μM (interquartile range [IQR], 400 to 508 μM) in HC, 300 μM (IQR, 256 to 365 μM) in UM, and 257 μM (IQR, 195 to 320 μM) in CM. Arginine flux during steady state was not significantly different in SM than in HC by the respective mass spectrometric methods: 93.2 μmol/h/kg of body weight (IQR, 84.4 to 129.3 μmol/h/kg) versus 88.0 μmol/h/kg (IQR, 73.0 to 102.2 μmol/h/kg) (P = 0.247) by the two mass spectrometric methods in SM and 93.7 μmol/h/kg (IQR, 79.1 to 117.8 μmol/h/kg) versus 81.0 μmol/h/kg (IQR, 75.9 to 88.6 μmol/h/kg) (P = 0.165) by the two mass spectrometric methods in HC. A limited supply of amino acid precursors for arginine synthesis likely contributes to the hypoargininemia and NO insufficiency in falciparum malaria in children.

Microvascular function and oxygen consumption affect oxygen homeostasis but have not been assessed in African children with malaria. Microvascular function in Tanzanian children with severe malaria (SM) or uncomplicated malaria were 39% and 72%, respectively, of controls (P < .001). Uncomplicated malaria (P = .04), not SM (P = .06), children had increased oxygen consumption compared with controls.

A cerebrospinal fluid (CSF) biomarker for cerebral malaria (CM) has not been validated. We examined the detection, semiquantification, and clinical use of the Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) as a parasite antigen biomarker for CM. The PfHRP-2 was detected in archival CSF samples from CM patients from Tanzania both by a newly developed sensitive and specific immuno-polymerase chain reaction (72 of 73) and by rapid diagnostic tests (62 of 73). The geometric mean PfHRP-2 CSF concentration was 8.76 ng/mL with no differences in those who survived (9.2 ng/mL), those who died (11.1 ng/mL), and those with neurologic sequelae (10.8 ng/mL). All aparasitemic endemic and nonendemic control samples had undetectable CSF PfHRP-2. In a separate group of 11 matched plasma and CSF cerebral malaria patient samples, the ratio of plasma to CSF PfHRP-2 was 175. The CSF PfHRP-2 reflects elevated plasma PfHRP-2 rather than elevated CM-specific CSF ratios, falling short of a validated biomarker.

Falciparum malaria remains a significant problem worldwide causing over 0.6 million deaths yearly, mostly in children [1, 2]. New insights into malaria pathophysiology and new treatments are needed. Nitric oxide (NO) is important in resistance to severe falciparum malaria, and there is markedly diminished NO bioavailability in falciparum malaria both adults and children [3, 4]. We have identified several processes contributing to the NO insufficiency ([5] for review). Recently, we found Indonesian adults with severe falciparum malaria had elevated plasma levels of endogenous methylated arginines [asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA)] and that ADMA levels correlated with decreased NO production, endothelial dysfunction, disease severity, and mortality [6]. Dimethylarginines are formed primarily as a result of protein breakdown (eg, in muscle or erythrocytes) and release of methylated arginine residues [7, 8]. High levels of ADMA and SDMA are predictive of poor outcomes in a variety of diseases including coronary artery disease, peripheral vascular disease, renal failure, sepsis, and malaria in Asian adults [7, 9, 10]. ADMA is a competitive inhibitor of NO synthases (NOS), and the ratio of plasma arginine to ADMA is a measure of arginine bioavailability to NOS. SDMA is not active as an inhibitor of NOS, but at supraphysiological concentrations it reduces arginine transport into cells by competitive inhibition of the cationic transporter (CAT2) [11] and enhances oxidative stress and inflammatory mediator release by macrophage-like cells [7, 12]. A recent study evaluating whole genome sequences of African children with falciparum malaria found polymorphisms of the enzyme dimethylarginine-dimethylaminohydrolase-1 (DDAH), which metabolizes ADMA to be associated with an increased risk of severe disease. However, no studies to our knowledge have measured concentrations of dimethylarginines in children with malaria to date, and the relationship with disease severity is not known. The purpose of the current study was to determine whether, as in Asian adults, plasma dimethyl­arginines are also increased in African children with falciparum malaria in proportion to disease severity and whether impaired L-arginine bioavailability (reflected by the arginine:ADMA ratio) would be associated with markers of impaired perfusion and endothelial activation.

Background Severe malaria (SM) syndromes caused by Plasmodium falciparum infection result in major morbidity and mortality each year. However, only a fraction of P. falciparum infections develop into SM, implicating host genetic factors as important determinants of disease outcome. Previous studies indicate that tumour necrosis factor (TNF) and lymphotoxin alpha (LTα) may be important for the development of cerebral malaria (CM) and other SM syndromes. Methods An extensive analysis was conducted of single nucleotide polymorphisms (SNPs) in the TNF, LTA and LTB genes in highland Papuan children and adults, a population historically unexposed to malaria that has migrated to a malaria endemic region. Generated P-values for SNPs spanning the LTA/TNF/LTB locus were corrected for multiple testing of all the SNPs and haplotype blocks within the region tested through 10,000 permutations. A global P-value of < 0.05 was considered statistically significant. Results No associations between SNPs in the TNF/LTA/LTB locus and susceptibility to SM in highland Papuan children and adults were found. Conclusions These results support the notion that unique selective pressure on the TNF/LTA/LTB locus in different populations has influenced the contribution of the gene products from this region to SM susceptibility.

We earlier established that nitric oxide (NO) is protective against severe malaria and that arginine and NO levels are reduced in malaria patients. We now show that an M2-like blood monocyte phenotype is significantly associated with hypoargininemia, NO insufficiency, and disease severity in Tanzanian children with falciparum malaria. Compared to control children (n = 106), children with moderately severe (n = 77) and severe falciparum malaria (n = 129) had significantly higher mononuclear cell arginase 1 mRNA, protein, and enzyme activity; lower NOS2 mRNA; lower plasma arginine; and higher plasma IL-10, IL-13, and IL-4. In addition, monocyte CD206 and CD163 and plasma soluble CD163 were elevated. Multivariate logistic regression analysis revealed a significant correlation of risk of severe malaria with both plasma IL-10 and soluble CD163 levels. Monocyte M2 skewing likely contributes to NO bioinsufficiency in falciparum malaria in children. Treatments that reverse the M2 polarization may have potential as adjunctive treatment for malaria.

Nitric oxide (NO) mediates host resistance to severe malaria and other infectious diseases. NO production and mononuclear cell expression of the NO producing enzyme-inducible nitric oxide synthase (NOS2) have been associated with protection from severe falciparum malaria. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) and haplotypes in the NOS2 promoter, to identify associations of these haplotypes with malaria severity and to test the effects of these polymorphisms on promoter activity. We identified 34 SNPs in the proximal 7.3 kb region of the NOS2 promoter and inferred NOS2 promoter haplotypes based on genotyping 24 of these SNPs in a population of Tanzanian children with and without cerebral malaria. We identified 71 haplotypes; 24 of these haplotypes comprised 82% of the alleles. We determined whether NOS2 promoter haplotypes were associated with malaria severity in two groups of subjects from Dar es Salaam (N = 185 and N = 250) and in an inception cohort of children from Muheza-Tanga, Tanzania (N = 883). We did not find consistent associations of NOS2 promoter haplotypes with malaria severity or malarial anemia, although interpretation of these results was potentially limited by the sample size of each group. Furthermore, cytokine-induced NOS2 promoter activity determined using luciferase reporter constructs containing the proximal 7.3 kb region of the NOS2 promoter and the G-954C or C-1173T SNPs did not differ from NOS2 promoter constructs that lacked these polymorphisms. Taken together, these studies suggest that the relationship between NOS2 promoter polymorphisms and malaria severity is more complex than previously described.

Histatins are small molecular weight salivary proteins that are important in the non-immune host defense system. Two frequent cis-linked coding-change mutations were previously described in exon 5 of the HIS2 gene of Blacks. The polymorphic mutant allele was termed HIS22 and the wild-type allele HIS21. We here describe two new non-coding change polymorphisms of the HIS2 gene: a deletion in intron 5 (7183-7198 del) and a CT mutation in exon 5 [CT (7104)] that characterize two new HIS2 alleles, HIS23 and HIS24 respectively. Both mutations occur on a HIS21 background. The HIS23 allele occurred only in Afro-Americans, but not in 67 Japanese, 51 Chinese and 50 Whites. Among 66 random DNA samples from Afro-Americans, frequencies of HIS21, HIS22, HIS23 and HIS24 were 0.67, 0.22, 0.05 and 0.07 respectively, with a heterozygosity of 0.45. The frequencies of the HIS24 allele in 50 Whites and 50 Chinese were 0.06, and 0.1 respectively. In a comparison of 60 matched saliva and DNA samples from the Afro-American population, the DNA-based mutation analysis reliably identified salivary histatin phenotypes. The salivary histatin polymorphism (inferred from PCR analysis) was used to test a biologically plausible hypothesis, that the mutant histatin phenotype (coded by the HIS22 allele) confers relative resistance to severe and fatal malaria. In a study of 185 Black Tanzanian subjects, there were no significant differences in HIS22 allelic frequencies between the various test groups: for 86 cerebral malaria subjects, 54 uncomplicated malaria subjects, and 45 combined asymptomatic parasitemia and health controls, HIS22 frequencies were 0.16, 0.17 and 0.17 respectively. Thus, there was no support for the hypothesis in this population. Hum. Mutat. 10:58–64, 1997. © 1997 Wiley-Liss, Inc.

Elevated levels of methaemoglobin, the ferric form of haemoglobin incapable of oxygen transport, have been previously found during Plasmodium vivax infections and in acidotic infants. We measured methaemoglobin in the following 5 groups of children with P. falciparum malaria admitted to Muhimbili Medical Centre, Dar es Salaam, Tanzania. (i) Cerebral malaria (CM) with unrousable coma (n = 50), including 32 with complete recovery (CMCR) and 18 with death or neurological sequelae (CMDS); (ii) malaria with severe anaemia but without severe respiratory distress (SA; n = 6); (iii) uncomplicated malaria (UM; n = 37); (iv) asymptomatic parasitaemia (AP; n = 5); and (v) healthy controls (HC; n = 34). Mean methaemoglobin levels were elevated in all groups with malaria, forming up to 16.4% of circulating haemoglobin. The degree of methaemoglobinaemia correlated with disease severity and severity of anaemia. Mean methaemoglobin levels in children with AP, UM, SA, CMCR and CMDS were 3.3%, 4.1%, 5.6%, 4.7% and 5.8% respectively; the mean levels in those with clinical disease were significantly higher than those in healthy controls (2.0%). Methaemoglobinaemia > 10% was found in 5.4%, 16.7%, 12.5%, and 22.2% of those with UM, SA, CMCR and CMDS, respectively. In the presence of parasite sequestration, impaired tissue perfusion, and a reduction in oxygen carrying capacity of blood due to anaemia, a further reduction in oxygen carrying capacity from even a modest concentration of methaemoglobin is likely to exacerbate tissue hypoxia, perhaps critically so in a minority of anaemic and acidotic patients with severe falciparum malaria.

Plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) concentrations, a measure of parasite biomass, have been correlated with malaria severity in adults, but not yet in children. We measured plasma PfHRP-2 in Tanzanian children with uncomplicated (n = 61) and cerebral malaria (n = 45; 7 deaths). Median plasma PfHRP-2 concentrations were higher in cerebral malaria (1008 [IQR 342-2572] ng/mL) than in uncomplicated malaria (465 [IQR 36-1426] ng/mL; p = 0.017). In cerebral malaria, natural log plasma PfHRP-2 was associated with coma depth (r = -0.42; p = 0.006) and mortality (OR: 3.0 [95% CI 1.03-8.76]; p = 0.04). In this relatively small cohort study in a mesoendemic transmission area of Africa, plasma PfHRP-2 was associated with pediatric malaria severity and mortality. Further studies among children in areas of Africa with higher malaria transmission and among children with different clinical manifestations of severe malaria will help determine the wider utility of quantitative PfHRP-2 as a measure of parasite biomass and prognosis in sub-Saharan Africa.

BACKGROUND: Age and host genetics are important determinants of malaria severity. Lymphotoxin-alpha (LTalpha) has been associated with the development of cerebral malaria (CM) and other severe malaria (SM) syndromes. Mutations in genes regulating LTalpha production contribute to other acute vascular diseases and may contribute to malaria pathogenesis. METHODS: We tested the association between rs7291467, a single-nucleotide polymorphism (SNP) in the LTalpha-related gene encoding galectin-2 (LGALS2), disease severity, and function in a case-control study of ethnic Highland Papuan adults and children with SM (n = 380) and asymptomatic malaria-exposed controls (n = 356) originating from a non-malaria-endemic region but residing in a lowland malaria-endemic area of Papua, Indonesia. RESULTS: The LGALS2 SNP showed a significant association with susceptibility to SM (including CM), in children (odds ratio, 2.02 [95% confidence interval, 1.14-3.57]) but not in adults. In SM, the C allele at rs7291467 was associated with enhanced galectin-2 transcript levels. In a separate group of Tanzanian children originating from a malaria-endemic region, we found preservation of the major ancestral LGALS2 allele and no association with susceptibility to CM. CONCLUSIONS: Results suggest differences in the inflammatory contribution to the development of SM between children and adults in the same population and potential differences between individuals originating from malaria-endemic and non-malaria-endemic areas.

Parasiticidal therapy of severe falciparum malaria improves outcome, but up to 30% of these patients die despite best therapy. Nitric oxide is protective against severe disease, and both nitric oxide and arginine (the substrate for nitric oxide synthase) are low in clinical malaria. Parasitized red blood cell interactions with endothelium are important in the pathophysiology of malaria. This review describes new information regarding nitric oxide, arginine, carbon monoxide, and endothelial function in malaria.Low arginine, low nitric oxide production, and endothelial dysfunction are common in severe malaria. The degree of hypoargininemia and endothelial dysfunction (measured by reactive hyperemia-peripheral artery tonometry) is proportional to parasite burden and severity of illness. Plasma arginase (an enzyme that catabolizes arginine) is elevated in severe malaria. Administering arginine intravenously reverses hypoargininemia and endothelial dysfunction. The cause(s) of hypoargininemia in malaria is unknown. Carbon monoxide (which shares certain functional properties with nitric oxide) protects against cerebral malaria in mice.Replenishment of arginine and restoration of nitric oxide production in clinical malaria should diminish parasitized red blood cells adherence to endothelium and reduce the sequelae of these interactions (e.g. cerebral malaria). Arginine therapy given in addition to conventional antimalaria treatment may prove to be beneficial in severe malaria.

Decreased bioavailability of nitric oxide (NO) is a major contributor to the pathophysiology of severe falciparum malaria. Tetrahydrobiopterin (BH4) is an enzyme cofactor required for NO synthesis from L-arginine. We hypothesized that systemic levels of BH4 would be decreased in children with cerebral malaria, contributing to low NO bioavailability. In an observational study in Tanzania, we measured urine levels of biopterin in its various redox states (fully reduced [BH4] and the oxidized metabolites, dihydrobiopterin [BH2] and biopterin [B0]) in children with uncomplicated malaria (UM, n = 55), cerebral malaria (CM, n = 45), non-malaria central nervous system conditions (NMC, n = 48), and in 111 healthy controls (HC). Median urine BH4 concentration in CM (1.10 [IQR:0.55–2.18] μmol/mmol creatinine) was significantly lower compared to each of the other three groups — UM (2.10 [IQR:1.32–3.14];p, Author Summary Vascular nitric oxide (NO) bioavailability is decreased in severe falciparum malaria and associated with microvascular dysfunction, increased activation of the cells lining blood vessels (endothelial cells) and increased parasite biomass. Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase (NOS) enzymatic conversion of L-arginine to NO and L-citrulline. But when BH4 is low, NOS is “uncoupled” and produces superoxide instead of NO. In oxidative conditions, BH4 is oxidized to dihydrobiopterin (BH2) and biopterin (B0). BH2 competes with remaining BH4 at its NOS binding site, further decreasing NOS-catalyzed NO production. We measured BH4, BH2 and B0 in the urine of children with coma due to falciparum malaria (cerebral malaria), uncomplicated falciparum malaria, children with non-malaria central nervous system conditions and healthy controls. Urine BH4 was significantly decreased and BH2 significantly increased in cerebral malaria compared to uncomplicated malaria, non-malaria central nervous conditions and healthy controls, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4. Urine BH4 concentration was independently associated with increased risk of cerebral malaria. Given that safe therapies for regenerating BH4 have been studied in chronic vascular disease, this finding of low BH4 in pediatric cerebral malaria offers a new area of investigation for adjunctive therapies aimed at improving NO bioavailability and, consequently, clinical outcomes in severe falciparum malaria.

Cerebral malaria is associated with decreased production of nitric oxide and decreased levels of its precursor, l -arginine. Abnormal amino acid metabolism may thus be an important factor in malaria pathogenesis. We sought to determine if other amino acid abnormalities are associated with disease severity in falciparum malaria. Subjects were enrolled in Dar es Salaam, Tanzania (children) ( n = 126), and Papua, Indonesia (adults) ( n = 156), in two separate studies. Plasma samples were collected from subjects with WHO-defined cerebral malaria (children), all forms of severe malaria (adults), and uncomplicated malaria (children and adults). Healthy children and adults without fever or illness served as controls. Plasma amino acids were measured using reverse-phase high-performance liquid chromatography with fluorescence detection. Several plasma amino acids were significantly lower in the clinical malaria groups than in healthy controls. Despite the differences, phenylalanine was the only amino acid with mean levels outside the normal range (40 to 84 μM) and was markedly elevated in children with cerebral malaria (median [95% confidence interval], 163 [134 to 193] μM; P < 0.0001) and adults with all forms of severe malaria (median [95% confidence interval], 129 [111 to 155] μM; P < 0.0001). In adults who survived severe malaria, phenylalanine levels returned to normal, with clinical improvement ( P = 0.0002). Maintenance of plasma phenylalanine homeostasis is disrupted in severe malaria, leading to significant hyperphenylalaninemia. This is likely a result of an acquired abnormality in the function of the liver enzyme phenylalanine hydroxylase. Determination of the mechanism of this abnormality may contribute to the understanding of neurological complications in malaria.

Immunofluorescent antibody (IFA) testing was performed on sera drawn from 150 pregnant women in the port city of Dar es Salaam, Tanzania. Prevalence of antibodies to Rickettsia typhi was 28%, higher than in any of the 12 other African countries in which serosurveys using IFA testing have been performed. Seroprevalence of antibodies to spotted fever group rickettsiae antigens was 25.3%, comparable with that found in other sub-Saharan countries endemic for Amblyomma ticks. Only 4.7% of women were seropositive for Coxiella burnetii.

Prostaglandins (PGs) are important mediators of macrophage activity, vascular permeability, fever, erythropoiesis, and proinflammatory responses to infection. Our recent studies have shown that plasma levels of bicyclo-PGE2 (a stable end product of PGE2 metabolism) and leukocyte cyclooxygenase (COX)-2 gene expression are suppressed in children with malarial anemia. Since the role of PGs as immunomodulators of human cerebral malaria (CM) has not been examined, we investigated urinary levels of bicyclo-PGE2/creatinine in children with varying clinical outcomes of CM. Among parasitemic children, those with asymptomatic parasitemia had the highest levels of bicyclo-PGE2/creatinine, whereas those with CM had significantly lower levels of bicyclo-PGE2. Systemic levels of bicyclo-PGE2/creatinine were not significantly associated with parasitemia, hemoglobin levels, or levels of the PG-regulatory cytokine tumor necrosis factor- alpha but were positively correlated with levels of interleukin-10. The results presented here show that impaired systemic production of PGE2 is associated with adverse outcomes of CM, whereas elevated levels of PGE2 in asymptomatic parasitemia suggest a potential role for PGs in protective immunity.

Nitric oxide (NO) production and mononuclear cell NO synthase 2 (NOS2) expression are high in healthy Tanzanian children but low in those with cerebral malaria. Factors that downregulate NOS2 also diminish factors involved in cellular uptake and biosynthesis of L-arginine, the substrate for NO synthesis. We therefore postulated that L-arginine concentrations would be low in individuals with cerebral malaria. We measured concentrations of L-arginine in cryopreserved plasma samples from Tanzanian children with and without malaria. L-arginine concentrations were low in individuals with cerebral malaria (mean 46 micromol/L, SD 14), intermediate in those with uncomplicated malaria (70 micromol/L, 20), and within the normal range in healthy controls (122 micromol/L, 22; p

Summary Background Nitric oxide (NO) is a mediator of immunity to malaria, and genetic polymorphisms in the promoter of the inducible NO synthase gene ( NOS2 ) could modulate production of NO. We postulated that NOS2 promoter polymorphisms would affect resistance to severe malaria. Methods We assessed genomic DNA from healthy children and from those diagnosed with malaria from Tanzania (n=47 and n=138, respectively) and Kenya (n=1106) for polymorphisms by single-stranded conformational polymorphism (SSCP) analysis and sequencing. We also measured in-vivo NO production in Tanzanian children. Findings We identified a novel single nucleotide polymorphism, −1173 C/T, in the NOS2 promoter that was significantly associated with protection from symptomatic malaria (odds ratio 0·12, 95% Cl 0·03–0·48, p=0·0006) in 179 Tanzanian children, and significantly associated with protection from severe malarial anaemia (adjusted relative risk 0·25, 95% Cl 0·09–0·66, p=0·0005) in 1106 Kenyan children studied over 5 years. The risk of parasitaemia was not significantly different in wild-type or −1173 C/T individuals. −1173 C/T protection in Tanzanians was independent of the previously recognised NOS2 −954 G/C polymorphism. The (CCTTT) n NOS2 polymorphism (Tanzania and Kenya) was not associated with severe malaria outcomes. −1173 C/T was associated with increased fasting urine and plasma NO metabolite concentrations in Tanzanian children, suggesting that the polymorphism was functional in vivo. Interpretation The NOS2 promoter −1173 C/T single nucleotide polymorphism is associated with protection against cerebral malaria and severe malarial anaemia. Increased NO production in individuals with the −1173 C/T polymorphism lends support to a protective role for NO against these syndromes. Targeted interventions to increase NO delivery or production could provide novel preventive and therapeutic strategies against these major causes of mortality in African children.

The cause of the anemia associated with chronic, intermittent, asymptomatic, low-level parasitemia in children in malaria-endemic endemic areas is not well understood. Nitric oxide (NO) decreases erythropoiesis, and it is likely an important mediator of anemia of chronic disease. Production of NO is decreased in acute uncomplicated and cerebral malaria, but it is increased in asymptomatic Tanzanian children (with or without parasitemia). We hypothesized that chronic overproduction of NO in these asymptomatic children contributes to the anemia associated with subclinical/subpatent malaria. In 44 fasting, asymptomatic, malaria-exposed, Tanzanian children, NO production (measured using fasting urine NOx excretion) was inversely associated with hemoglobin concentration (P = 0.03, controlling for age and gender). Using multiple linear regression, hemoglobin concentration was negatively associated with parasitemia (P = 0.005). After controlling for age and parasitemia, NO was no longer an independent predictor of anemia. One of the mechanisms of parasite-related anemia in such children may be through the adverse hematologic effects of parasite-induced NO production.

Age appears to influence not only the acquisition of clinical immunity to malaria but also the susceptibility to and clinical manifestations of severe malaria. Asymptomatic malaria-exposed Tanzanian children have high production of nitric oxide (NO) and universal expression of leukocyte NO synthase type 2 (NOS2), which may protect against disease. To determine the effects of age and parasitemia on NO production, we measured urine and plasma NO metabolites and leukocyte NOS2 expression in 45 fasting, asymptomatic, malaria-exposed children of different ages, stratifying parasitemia by thick film and polymerase chain reaction (PCR) analysis. Although NO production was significantly higher in thick film-positive children than in thick film-negative children, after adjusting for age and gender, we were unable to detect a difference in NO production in thick film-negative children between those who were PCR positive and PCR negative. The relationship between age and NO production was determined using a generalized additive model adjusted for the effects of gender and parasitemia. Production of NO using all three measures was highest in infancy, decreasing after the first year of life, and then increasing again after 5 years of age. This pattern of age-related NO production is the reverse of the pattern of age-related morbidity from cerebral malaria in coastal Tanzanian children. Elevated production of NO in both infants and older children may be related to age per se and malaria infection respectively, and may be one of the mediators of the anti-disease immunity found most commonly in these two age groups.