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1.  Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Oxidized Biopterins in Pediatric Falciparum Malaria: Association with Disease Severity 
PLoS Pathogens  2015;11(3):e1004655.
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<0.001), NMC (1.52 [IQR:1.01–2.71];p = 0.002), and HC (1.60 [IQR:1.15–2.23];p = 0.005). Oxidized biopterins were increased, and the BH4:BH2 ratio markedly decreased in CM. In a multivariate logistic regression model, each Log10-unit decrease in urine BH4 was independently associated with a 3.85-fold (95% CI:1.89–7.61) increase in odds of CM (p<0.001). Low systemic BH4 levels and increased oxidized biopterins contribute to the low NO bioavailability observed in CM. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.
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.
PMCID: PMC4357384  PMID: 25764173
2.  Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Dihydrobiopterin in Adult Falciparum Malaria: Association with Disease Severity, Impaired Microvascular Function and Increased Endothelial Activation 
PLoS Pathogens  2015;11(3):e1004667.
Tetrahydrobiopterin (BH4) is a co-factor required for catalytic activity of nitric oxide synthase (NOS) and amino acid-monooxygenases, including phenylalanine hydroxylase. BH4 is unstable: during oxidative stress it is non-enzymatically oxidized to dihydrobiopterin (BH2), which inhibits NOS. Depending on BH4 availability, NOS oscillates between NO synthase and NADPH oxidase: as the BH4/BH2 ratio decreases, NO production falls and is replaced by superoxide. In African children and Asian adults with severe malaria, NO bioavailability decreases and plasma phenylalanine increases, together suggesting possible BH4 deficiency. The primary three biopterin metabolites (BH4, BH2 and B0 [biopterin]) and their association with disease severity have not been assessed in falciparum malaria. We measured pterin metabolites in urine of adults with severe falciparum malaria (SM; n=12), moderately-severe malaria (MSM, n=17), severe sepsis (SS; n=5) and healthy subjects (HC; n=20) as controls. In SM, urinary BH4 was decreased (median 0.16 ¼mol/mmol creatinine) compared to MSM (median 0.27), SS (median 0.54), and HC (median 0.34)]; p<0.001. Conversely, BH2 was increased in SM (median 0.91 ¼mol/mmol creatinine), compared to MSM (median 0.67), SS (median 0.39), and HC (median 0.52); p<0.001, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4 in severe malaria. Overall, the median BH4/BH2 ratio was lowest in SM [0.18 (IQR: 0.04-0.32)] compared to MSM (0.45, IQR 0.27-61), SS (1.03; IQR 0.54-2.38) and controls (0.66; IQR 0.43-1.07); p<0.001. In malaria, a lower BH4/BH2 ratio correlated with decreased microvascular reactivity (r=0.41; p=0.03) and increased ICAM-1 (r=-0.52; p=0.005). Decreased BH4 and increased BH2 in severe malaria (but not in severe sepsis) uncouples NOS, leading to impaired NO bioavailability and potentially increased oxidative stress. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.
Author Summary
Vascular nitric oxide (NO) bioavailability is decreased in severe falciparum malaria and associated with microvascular dysfunction and increased endothelial activation. Nitric oxide synthase (NOS) requires tetrahydrobiopterin (BH4) as a co-factor to convert L-arginine to NO, but when BH4 is low, NOS is “uncoupled” and produces superoxide instead of NO. In conditions of increased oxidative stress, BH4 is converted to dihydrobiopterin (BH2) and biopterin (B0): the resulting BH2 competes with remaining BH4 as a competitive inhibitor of NOS, further decreasing NO production. We measured BH4 and BH2 in the urine of adults with severe and uncomplicated falciparum malaria and compared results to those of controls or those with sepsis. There was a significant decrease in urinary BH4 and increase in BH2 in severe malaria compared to uncomplicated malaria, sepsis, and controls, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4. The BH4/BH2 ratio was associated with increased risk of severe disease, endothelial activation and microvascular dysfunction, likely through impaired NOS function. This additional mechanism of decreased NO in severe malaria suggests that trials evaluating use of adjunctive L-arginine to increase NO in severe malaria may require concurrent therapy to regenerate BH4.
PMCID: PMC4357386  PMID: 25764397
3.  Decreased Endothelial Nitric Oxide Bioavailability, Impaired Microvascular Function, and Increased Tissue Oxygen Consumption in Children with Falciparum Malaria 
The Journal of infectious diseases  2014;210(10):1627-1632.
Endothelial nitric oxide (NO) bioavailability, microvascular function, and host oxygen consumption have not been assessed in pediatric malaria. We measured NO-dependent endothelial function by using peripheral artery tonometry to determine the reactive hyperemia index (RHI), and microvascular function and oxygen consumption (VO2) using near infrared resonance spectroscopy in 13 Indonesian children with severe falciparum malaria and 15 with moderately severe falciparum malaria. Compared with 19 controls, children with severe malaria and those with moderately severe malaria had lower RHIs (P = .03); 12% and 8% lower microvascular function, respectively (P = .03); and 29% and 25% higher VO2, respectively. RHIs correlated with microvascular function in all children with malaria (P < .001) and all with severe malaria (P < .001). Children with malaria have decreased endothelial and microvascular function and increased oxygen consumption, likely contributing to the pathogenesis of the disease.
PMCID: PMC4334794  PMID: 24879801
severe malaria; cerebral malaria; Plasmodium falciparum; endothelial function; microvascular function; oxygen consumption; tissue hypoxia
4.  Relationship of Cell-Free Hemoglobin to Impaired Endothelial Nitric Oxide Bioavailability and Perfusion in Severe Falciparum Malaria 
The Journal of infectious diseases  2009;200(10):1522-1529.
Hemolysis causes anemia in falciparum malaria, but its contribution to microvascular pathology in severe malaria (SM) is not well characterized. In other hemolytic diseases, release of cell-free hemoglobin causes nitric oxide (NO) quenching, endothelial activation, and vascular complications. We examined the relationship of plasma hemoglobin and myoglobin to endothelial dysfunction and disease severity in malaria.
Cell-free hemoglobin (a potent NO quencher), reactive hyperemia peripheral arterial tonometry (RH-PAT) (a measure of endothelial NO bioavailability), and measures of perfusion and endothelial activation were quantified in adults with moderately severe (n = 78) or severe (n = 49) malaria and control subjects (n = 16) from Papua, Indonesia.
Cell-free hemoglobin concentrations in patients with SM (median, 5.4 μmol/L; interquartile range [IQR], 3.2–7.4 μmol/L) were significantly higher than in those with moderately severe malaria (2.6 μmol/L; IQR, 1.3–4.5 μmol/L) or controls (1.2 μmol/L; IQR, 0.9–2.4 μmol/L; P < .001). Multivariable regression analysis revealed that cell-free hemoglobin remained inversely associated with RH-PAT, and in patients with SM, there was a significant longitudinal association between improvement in RH-PAT index and decreasing levels of cell-free hemoglobin (P = .047). Cell-free hemoglobin levels were also independently associated with lactate, endothelial activation, and proinflammatory cytokinemia.
Hemolysis in falciparum malaria results in NO quenching by cell-free hemoglobin, and may exacerbate endothelial dysfunction, adhesion receptor expression and impaired tissue perfusion. Treatments that increase NO bioavailability may have potential as adjunctive therapies in SM.
PMCID: PMC3740798  PMID: 19803726
5.  Plasma Plasmodium falciparum Histidine-Rich Protein-2 Concentrations Are Associated with Malaria Severity and Mortality in Tanzanian Children 
PLoS ONE  2012;7(5):e35985.
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.
PMCID: PMC3346811  PMID: 22586457
6.  Age-related susceptibility to severe malaria associated with galectin-2 in highland Papuans 
The Journal of infectious diseases  2010;202(1):117-124.
Age and host genetics are important determinants of malaria severity. Lymphotoxin-alpha (LTα) has been linked to the development of cerebral malaria (CM) and other severe malaria (SM) syndromes. Mutations in genes regulating LTα production contribute to other acute vascular diseases and may contribute to malaria pathogenesis.
We tested the association between rs7291467, a single nucleotide polymorphism (SNP) in the LTα-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.
The LGALS2 SNP showed significant association with susceptibility to SM (including CM), in children (OR 2.02 [95% CI:1.14-3.57]) but not 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.
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-endemic areas.
PMCID: PMC2880661  PMID: 20500087
Malaria; Galectin-2; Inflammation; Gene regulation; Age; Severe Malaria; Cerebral Malaria
7.  A study of the TNF/LTA/LTB locus and susceptibility to severe malaria in highland papuan children and adults 
Malaria Journal  2010;9:302.
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.
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.
No associations between SNPs in the TNF/LTA/LTB locus and susceptibility to SM in highland Papuan children and adults were found.
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.
PMCID: PMC2978234  PMID: 21029472
8.  Malaria severity and human nitric oxide synthase type 2 (NOS2) promoter haplotypes 
Human genetics  2009;127(2):163-182.
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.
PMCID: PMC2939908  PMID: 19859740
10.  Increased Asymmetric Dimethylarginine in Severe Falciparum Malaria: Association with Impaired Nitric Oxide Bioavailability and Fatal Outcome 
PLoS Pathogens  2010;6(4):e1000868.
Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is a predictor of mortality in critical illness. Severe malaria (SM) is associated with decreased NO bioavailability, but the contribution of ADMA to the pathogenesis of impaired NO bioavailability and adverse outcomes in malaria is unknown. In adults with and without falciparum malaria, we tested the hypotheses that plasma ADMA would be: 1) increased in proportion to disease severity, 2) associated with impaired vascular and pulmonary NO bioavailability and 3) independently associated with increased mortality. We assessed plasma dimethylarginines, exhaled NO concentrations and endothelial function in 49 patients with SM, 78 with moderately severe malaria (MSM) and 19 healthy controls (HC). Repeat ADMA and endothelial function measurements were performed in patients with SM. Multivariable regression was used to assess the effect of ADMA on mortality and NO bioavailability. Plasma ADMA was increased in SM patients (0.85 µM; 95% CI 0.74–0.96) compared to those with MSM (0.54 µM; 95%CI 0.5–0.56) and HCs (0.64 µM; 95%CI 0.58–0.70; p<0.001). ADMA was an independent predictor of mortality in SM patients with each micromolar elevation increasing the odds of death 18 fold (95% CI 2.0–181; p = 0.01). ADMA was independently associated with decreased exhaled NO (rs = −0.31) and endothelial function (rs = −0.32) in all malaria patients, and with reduced exhaled NO (rs = −0.72) in those with SM. ADMA is increased in SM and associated with decreased vascular and pulmonary NO bioavailability. Inhibition of NOS by ADMA may contribute to increased mortality in severe malaria.
Author Summary
Severe falciparum malaria is associated with impaired microvascular perfusion, lung injury and decreased bioavailability of nitric oxide (NO), but the causes of these processes are not fully understood. Asymmetrical dimethylarginine (ADMA), a competitive endogenous inhibitor of nitric oxide synthase (NOS), is an independent predictor of mortality in other critical illnesses, and can impair vascular function in chronic disease. ADMA can be produced by both the host and malaria parasites. The major novel findings of this study in malaria are that ADMA is an independent predictor of death in falciparum malaria, and is associated with decreased availability of nitric oxide in at least two organ systems affected by malaria parasites, the lining of blood vessels and the lungs. This study contributes to knowledge of regulation and availability of pulmonary and endothelial NO in critical illness and identifies pathogenic processes which may contribute to death in severe malaria. Therapies which increase the availability of NO or which reduce ADMA levels may have potential for adjunctive therapy of severe malaria.
PMCID: PMC2858698  PMID: 20421938
11.  Arginine, nitric oxide, carbon monoxide, and endothelial function in severe malaria 
Purpose of review
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.
Recent findings
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.
PMCID: PMC2732119  PMID: 18725795
arginine; carbon monoxide; endothelium; malaria; nitric oxide
12.  Recovery of Endothelial Function in Severe Falciparum Malaria: Relationship with Improvement in Plasma L-Arginine and Blood Lactate Concentrations 
The Journal of infectious diseases  2008;198(4):602-608.
Severe malaria is characterized by microvascular obstruction, endothelial dysfunction, and reduced levels of L-arginine and nitric oxide (NO). L-Arginine infusion improves endothelial function in moderately severe malaria. Neither the longitudinal course of endothelial dysfunction nor factors associated with recovery have been characterized in severe malaria.
Endothelial function was measured longitudinally in adults with severe malaria (n = 49) or moderately severe malaria (n = 48) in Indonesia, using reactive hyperemia peripheral arterial tonometry (RH-PAT). In a mixed-effects model, changes in RH-PAT index values in patients with severe malaria were related to changes in parasitemia, lactate, acidosis, and plasma L-arginine concentrations.
Among patients with severe malaria, the proportion with endothelial dysfunction fell from 94% (46/49 patients) to 14% (6/42 patients) before discharge or death (P <.001). In severe malaria, the median time to normal endothelial function was 49 h (interquartile range, 20–70 h) after the start of antimalarial therapy. The mean increase in L-arginine concentrations in patients with severe malaria was 11 μmol/L/24 h (95% confidence interval [CI], 9–13 μmol/L/24 h), from a baseline of 49 μmol/L (95% CI, 37–45 μmol/L). Improvement of endothelial function in patients with severe malaria correlated with increasing levels of L-arginine (r = 0.56; P =.008) and decreasing levels of lactate (r = −0.44; P =.001).
Recovery of endothelial function in severe malaria is associated with recovery from hypoargininemia and lactic acidosis. Agents that can improve endothelial NO production and endothelial function, such as L-arginine, may have potential as adjunctive therapy early during the course of severe malaria.
PMCID: PMC2709993  PMID: 18605903
13.  Safety Profile of L-Arginine Infusion in Moderately Severe Falciparum Malaria 
PLoS ONE  2008;3(6):e2347.
L-arginine infusion improves endothelial function in malaria but its safety profile has not been described in detail. We assessed clinical symptoms, hemodynamic status and biochemical parameters before and after a single L-arginine infusion in adults with moderately severe malaria.
Methodology and Findings
In an ascending dose study, adjunctive intravenous L-arginine hydrochloride was infused over 30 minutes in doses of 3 g, 6 g and 12 g to three separate groups of 10 adults hospitalized with moderately severe Plasmodium falciparum malaria in addition to standard quinine therapy. Symptoms, vital signs and selected biochemical measurements were assessed before, during, and for 24 hours after infusion. No new or worsening symptoms developed apart from mild discomfort at the intravenous cannula site in two patients. There was a dose-response relationship between increasing mg/kg dose and the maximum decrease in systolic (ρ = 0.463; Spearman's, p = 0.02) and diastolic blood pressure (r = 0.42; Pearson's, p = 0.02), and with the maximum increment in blood potassium (r = 0.70, p<0.001) and maximum decrement in bicarbonate concentrations (r = 0.53, p = 0.003) and pH (r = 0.48, p = 0.007). At the highest dose (12 g), changes in blood pressure and electrolytes were not clinically significant, with a mean maximum decrease in mean arterial blood pressure of 6 mmHg (range: 0–11; p<0.001), mean maximal increase in potassium of 0.5 mmol/L (range 0.2–0.7 mmol/L; p<0.001), and mean maximal decrease in bicarbonate of 3 mEq/L (range 1–7; p<0.01) without a significant change in pH. There was no significant dose-response relationship with blood phosphate, lactate, anion gap and glucose concentrations. All patients had an uncomplicated clinical recovery.
Infusion of up to 12g of intravenous L-arginine hydrochloride over 30 minutes is well tolerated in adults with moderately severe malaria, with no clinically important changes in hemodynamic or biochemical status. Trials of adjunctive L-arginine can be extended to phase 2 studies in severe malaria.
Trial Registration NCT00147368
PMCID: PMC2405947  PMID: 18545693
14.  Impaired nitric oxide bioavailability and l-arginine–reversible endothelial dysfunction in adults with falciparum malaria 
The Journal of Experimental Medicine  2007;204(11):2693-2704.
Severe falciparum malaria (SM) is associated with tissue ischemia related to cytoadherence of parasitized erythrocytes to microvascular endothelium and reduced levels of NO and its precursor, l-arginine. Endothelial function has not been characterized in SM but can be improved by l-arginine in cardiovascular disease. In an observational study in Indonesia, we measured endothelial function using reactive hyperemia–peripheral arterial tonometry (RH-PAT) in 51 adults with SM, 48 patients with moderately severe falciparum malaria (MSM), and 48 controls. The mean RH-PAT index was lower in SM (1.41; 95% confidence interval [CI] = 1.33–1.47) than in MSM (1.82; 95% CI = 1.7–2.02) and controls (1.93; 95% CI = 1.8–2.06; P < 0.0001). Endothelial dysfunction was associated with elevated blood lactate and measures of hemolysis. Exhaled NO was also lower in SM relative to MSM and controls. In an ascending dose study of intravenous l-arginine in 30 more patients with MSM, l-arginine increased the RH-PAT index by 19% (95% CI = 6–34; P = 0.006) and exhaled NO by 55% (95% CI = 32–73; P < 0.0001) without important side effects. Hypoargininemia and hemolysis likely reduce NO bioavailability. Endothelial dysfunction in malaria is nearly universal in severe disease, is reversible with l-arginine, and likely contributes to its pathogenesis. Clinical trials in SM of adjunctive agents to improve endothelial NO bioavailability, including l-arginine, are warranted.
PMCID: PMC2118490  PMID: 17954570
15.  Elevated Plasma Phenylalanine in Severe Malaria and Implications for Pathophysiology of Neurological Complications  
Infection and Immunity  2006;74(6):3355-3359.
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.
PMCID: PMC1479261  PMID: 16714564
16.  Major histocompatibility complex controls the trajectory but not host-specific adaptation during virulence evolution of the pathogenic fungus Cryptococcus neoformans. 
Genes of the major histocompatibility complex (MHC) play a critical role in immune recognition and are the most genetically diverse loci known. One hypothesis to explain this diversity postulates that pathogens adapt to common MHC haplotypes and thus favour selection of new or rare alleles. To determine whether the pathogenic yeast Cryptococcus neoformans adapts to MHC-dependent immune responses, it was serially passaged in two independent replicate lines of five B10 MHC-congenic strains and Balb/c mice. All passaged lines increased in virulence as measured by reduced host survival. MHC influenced the rate (trajectory) of virulence increase during passages as measured by significant differences in mortality rate (p < 0.001). However, when the post-passage strains were tested, no MHC differences in mortality rate remained and only minor differences in titres were observed. Also contrary to expectations, increased virulence in three lines passaged in B10 mice had a larger effect in Balb/c mice, and the evolution of virulence in lines passaged in alternating hosts was not retarded. To our knowledge, these data represent the first experimental test of MHC-specific adaptation in a non-viral pathogen. The failure to observe MHC effects despite dramatically increased virulence and host-genotype-specific adaptation to non-MHC genes suggests that escape of MHC-dependent immune recognition may be difficult for pathogens with unlimited epitopes or that other virulence factors can swamp MHC effects.
PMCID: PMC1691764  PMID: 15306300
17.  Salmonella enterica Serovar Typhimurium RamA, Intracellular Oxidative Stress Response, and Bacterial Virulence  
Infection and Immunity  2004;72(2):996-1003.
Escherichia coli and Salmonella enterica serovar Typhimurium have evolved genetic systems, such as the soxR/S and marA regulons, to detoxify reactive oxygen species, like superoxide, which are formed as by-products of metabolism. Superoxide also serves as a microbicidal effector mechanism of the host's phagocytes. Here, we investigate whether regulatory genes other than soxR/S and marA are active in response to oxidative stress in Salmonella and may function as virulence determinants. We identified a bacterial gene, which was designated ramA (342 bp) and mapped at 13.1 min on the Salmonella chromosome, that, when overexpressed on a plasmid in E. coli or Salmonella, confers a pleiotropic phenotype characterized by increased resistance to the redox-cycling agent menadione and to multiple unrelated antibiotics. The ramA gene is present in Salmonella serovars but is absent in E. coli. The gene product displays 37 to 52% homology to the transcriptional activators soxR/S and marA and 80 to 100% identity to a multidrug resistance gene in Klebsiella pneumoniae and Salmonella enterica serovar Paratyphi A. Although a ramA soxR/S double null mutant is highly susceptible to intracellular superoxide generated by menadione and displays decreased Mn-superoxide dismutase activity, intracellular survival of this mutant within macrophage-like RAW 264.7 cells and in vivo replication in the spleens in Ityr mice are not affected. We concluded that despite its role in the protective response of the bacteria to oxidative stress in vitro, the newly identified ramA gene, together with soxR/S, does not play a role in initial replication of Salmonella in the organs of mice.
PMCID: PMC321585  PMID: 14742546
18.  Major Histocompatibility Complex-Dependent Susceptibility to Cryptococcus neoformans in Mice  
Infection and Immunity  2003;71(8):4815-4817.
To evaluate the role of major histocompatibility complex (MHC) genes in the resistance to Cryptococcus neoformans, we conducted infection experiments in MHC-congenic strains of mice. Significant MHC-dependent susceptibility differences were found among homozygotes and heterozygotes. This study is the first experimental demonstration of MHC-dependent susceptibility to C. neoformans infections in mice and indicates that MHC genes can be important in host resistance.
PMCID: PMC166009  PMID: 12874366
19.  Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication 
Infection and Immunity  2001;69(12):7413-7418.
Upon contact with host cells, the intracellular pathogen Salmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribed Salmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonella chromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104 to 107 bacteria in C3H/HeN and 101 to 104 bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.
PMCID: PMC98829  PMID: 11705915

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