Lipid metabolites of the phospholipase A2 pathway and inflammatory cytokines are associated with brain volume in paediatric cerebral malaria
© Pappa et al. 2015
Received: 24 June 2015
Accepted: 4 December 2015
Published: 21 December 2015
Cerebral malaria (CM) remains a significant cause of morbidity and mortality in children in sub-Saharan Africa. CM mortality has been associated with increased brain volume, seen on neuroimaging studies.
To examine the potential role of blood metabolites and inflammatory mediators in increased brain volume in Malawian children with CM, an association study was performed between plasma metabolites, cytokine levels and phospholipase A2 (PLA2) activity with brain volume.
The metabolomics analysis demonstrated arachidonic acid and other lysophospholipids to be positively associated with brain swelling. These lipids are products of the PLA2 enzyme and an association of plasma PLA2 enzymatic activity with brain swelling was confirmed. TNFα, which can upregulate PLA2 activity, was associated with brain volume. In addition, CCL2 and IL-8 were also associated with brain volume. Some of these cytokines can alter endothelial cell tight junction proteins and increase blood brain barrier permeability.
Taken together, paediatric CM brain volume was associated with products of the PLA2 pathway and inflammatory cytokines. Their role in causality is unknown. These molecules will need to undergo testing in vitro and in animal models to understand their role in processes of increased brain volume. These observations provide novel data on host physiology associated with paediatric CM brain swelling, and may both inform pathogenesis models and suggest adjunct therapies that could improve the morbidity and mortality associated with paediatric CM.
KeywordsPlasmodium falciparum Cerebral malaria Brain swelling Inflammation Phospholipase A2 Plasma small molecules Cytokines
Infection with Plasmodium falciparum remains prevalent in many areas of the world and is associated with severe disease and mortality, particularly in children living in sub-Saharan Africa . Cerebral malaria (CM) is a severe disease syndrome with mortality rates ranging from 15 to 25 % in research settings . In addition, almost a third of paediatric CM survivors develop long-term neurological complications [2, 3]. Severe brain swelling seen on neuroimaging has been reported in paediatric CM. CM associated brain swelling is associated with poor outcomes in Kenyan children and is a significant predictor of mortality in Malawian children [4–6]. Recently, CM associated brain swelling determined by magnetic resonance imaging (MRI) found that swelling in survivors was readily reversible and that mortality was not associated with peripheral parasitaemia . These observations have provided new insights into CM related morbidity and mortality and now can potentially inform development of adjuvant therapies to reverse or prevent brain swelling.
The mechanism of CM brain swelling is unknown and likely involves several factors including parasite mediated venous obstruction, increased permeability of the blood brain barrier (BBB), cytotoxic oedema or increased blood flow volume [5, 6]. Prior evidence of alterations in BBB permeability includes the observation of fibrinogen leakage into the brain . Moreover, a reduction in endothelial cell tight junction proteins, which maintain the integrity of the BBB, has been reported, providing further support for increases in BBB permeability during CM [8–10]. A variety of systemic factors can lead to transient increase of BBB permeability in other diseases. These include the metabolic derangements associated with diabetic ketoacidosis and elevated concentrations of oxidized phospholipids [11, 12]. Systemic metabolic abnormalities are common in CM, which is often associated with a hyperlactataemia, hypoglycaemia and evidence of marked inflammation [13–16]. Therefore, metabolites measured in a cohort of Malawian children with CM were correlated to brain volume, to examine their role as potential mediators of brain swelling.
Arachidonic acid, other phospholipase A2 (PLA2) lipid metabolites and plasma PLA2 enzymatic activity were associated with brain swelling. Expression of the PLA2 enzyme is upregulated via the nuclear factor-kappa B (NFκB) pathway, which in turn is regulated by pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNFα) . An association of TNFα and other cytokines with brain swelling was found, suggesting that brain swelling is associated with a high inflammatory state. These data provide new biochemical insights into mechanisms of brain swelling in paediatric CM. Further experiments are needed to determine if these associated molecules induce increased brain swelling in the setting of CM.
To identify small molecules associated with brain swelling in paediatric CM, correlations were sought, between brain volume and both host factors and plasma metabolites in Malawian children enrolled in an ongoing study of malaria pathogenesis in the Blantyre Malaria Project (BMP) during the 2009, 2011 and 2013 transmission seasons. The BMP study enrolls children with clinically defined CM , who are between 6 months and 12 years of age. This analysis was restricted to children who were HIV negative, had negative blood and CSF bacterial cultures and evidence of malaria retinopathy [18, 19]. The study was restricted to patients with retinopathy positive CM, as the presence of retinal abnormalities increases the specificity of the clinical diagnosis of CM . Plasma collected from the study subjects on admission, was stored at −80 °C and shipped to Albert Einstein College of Medicine in a liquid nitrogen dry shipper for subsequent metabolic and cytokine profiling. Plasma histidine rich protein 2 (HRP2), a parasite protein that represents total body parasite burden , was measured using ELISA, as previously described . Clinical and laboratory data were extracted from the study database. Informed consent was obtained from the parent or guardian before enrollment into the BMP. This study was approved by the Albert Einstein College of Medicine Institutional Review Board (IRB), Michigan State University IRB, the University of Rochester IRB, and The University of Malawi College of Medicine Research and Ethics Committee.
Neuroimaging was used to assess brain swelling with a 0.35T Signa Ovation Excite MRI scanner (GE Healthcare, Milwaukee, Wisconsin). The scans were read independently by two radiologists as previously reported [5, 6, 22]. Patient brain volume score was assigned based on a consensus interpretation of both radiologists. Brain volume was measured using a 1–8 scoring system, 1 and 2 indicating atrophy, 3-normal brain volume, 4-slightly increased brain volume, 5-mildly increased brain volume, 6-moderately increased brain volume, defined as loss of some sulcal markings, 7-moderately/severely increased brain volume with diffuse sulcal and cisternal effacement universally evident but without herniation present, and 8-severely increased brain volume with the findings of 7 and with evidence of herniation.
Plasma cytokine quantification
Plasma cytokine data were available from a prior study (Feintuch, C.M. personal communication) for patients from 2009 and 2011. Cytokine levels were assessed by Luminex using the Human Cytokine Panel according to manufacturer’s instructions (Millipore) and read on a Magpix Multiplex Reader (Luminex).
Global non-targeted MS metabolomics analysis was performed at Metabolon, Inc. from 100 μL of plasma . This method uses ultra-high performance liquid chromatography/tandem mass spectrometry in both positive and negative ion modes along with gas chromatography/mass spectrometry to maximize compound detection and accuracy. Metabolites were identified by comparing the spectral signatures of the plasma samples to a reference library using software developed at Metabolon .
Plasma PLA2 activity fluorescence assay
To measure plasma PLA2 activity, a PLA2 fluorescence assay was performed as previously described with minor modifications . Briefly, the liposomes were prepared with 2 mg of PG (l-α-Phosphatidyl-DL-glycerol sodium, Sigma) mixed with 14 μg of Red/Green BODIPY® PC-A2 (1-O-(6-BODIPY® 558/568-aminohexyl)-2-BODIPY® FL C5-sn-glycero-3-phosphocholine, Life Technologies) in 1 ml of chloroform, which was subsequently evaporated under argon. The liposomes were added in 1 ml of (250 mM sucrose, 50 mM Tris–HCl pH 7.5 and 0.02 % sodium azide) and the mixture was vortexed for 20 min and sonicated 6 times for 30 s with 1 min intervals on ice. The liposomes were then aliquoted in smaller quantities, stored in −20 °C and used within 30 days of preparation. For each sample 3 μl of patient plasma were mixed with 97 μl of PLA2 assay buffer (10 mM Tris–HCl, pH 7.5 and 10 mM CaCl2) into a black assay plate (Corning). Subsequently, 1 μl of fluorescent phospholipid substrate was mixed with 99 μl PLA2 assay buffer, sonicated for 10 s and added to the assay plate for a final volume of 200 μl. The plate was shaken for 15 s, incubated at 37 °C for 30 min and fluorescence was recorded at 470 nm excitation and 515 nm emission (BioTek). Samples were run in triplicate and the mean values of the relative fluorescence units (RFUs) at 30 min are reported.
To determine whether age, vital signs, HRP2, peripheral parasitaemia, plasma cytokines, complete blood count on admission, or coma resolution time correlated with brain volume, Spearman’s correlations were used. Two sided p-values <0.05 were considered statistically significant.
For the metabolomics analysis, ion counts were generated for each metabolite. A maximum likelihood method was used to impute left-truncated abundance values for each metabolite [26, 27]. First the mean and variance of the log transformed abundance values was estimated for each metabolite. Then the expectation of the left-truncated normal distribution of each metabolite was used to impute censored values. Spearman’s correlations were then performed between each metabolite or clinical factor and brain swelling scores.
The mean plasma PLA2 RFU values at 30 min of incubation were log10 transformed. Spearman’s correlations on the plasma PLA2 activity were then carried out and brain volume scores with p < 0.05 were considered statistically significant. The correlations were performed using Graph Pad Prism 6.03 (GraphPad Software, San Diego, CA, USA).
Clinical and laboratory data of admission in 53 children with retinopathy positive CM and correlation with brain volume
Female sex, no (%)
Blood pressure (mmHg)
Heart rate (beats/min)
Respiratory rate (breaths/min)
Parasitaemia (parasites × 103/μl)
Total WBC (×103/μl)
Coma resolution time (h)
Death, no. (%)
Metabolite correlation with brain volume
To identify plasma small molecules associated with CM brain swelling, a metabolomics analysis was carried out on 30 randomly selected plasma samples from the total study population enrolled in 2011 and 2013. There were no significant differences in the characteristics reported in Table 1 between the metabolomics sub-cohort and the total study population (Additional file 1: Table S1).
Plasma metabolites associated with brain volume in Malawian children with retinopathy positive CM
Lipids associated with the PLA2 pathway
l-urobilin, a metabolite of the hemoglobin catabolism pathway, was highly correlated with brain volume. Among other molecules, the carbohydrate, mannitol was also associated with brain swelling (Table 2).
Plasma sPLA2 activity and CM brain swelling
Cytokine activity and CM brain swelling
To identify potential mediators of brain swelling associated with paediatric CM, plasma metabolite levels of Malawian children with retinopathy positive CM were correlated to brain volume. Associations were identified between PLA2 metabolites, including arachidonic acid, with brain volume. Plasma PLA2 enzymatic activity also correlated to brain volume, suggesting an upregulation of the PLA2 pathway in children with high brain swelling. TNFα can upregulate PLA2 and this study reports that, in conjunction with other inflammatory cytokines, TNFα is correlated with brain volume. l-urobilin, a haem degradation product and mannitol were also associated with brain volume. Collectively, these results suggest a higher inflammatory state in children with increased brain volume. Further studies on how these molecules may be involved in BBB disruption, and why some children sustain a higher inflammatory state are now warranted.
Brain swelling has recently been shown to be the strongest predictor of mortality in paediatric CM, with an adjusted odds ratio of 7.5 (95 % CI 2.1–26.9) for severe brain swelling among patients who died compared to those who survived . Brain swelling induces increased intracranial pressure that may lead to brain-stem compromise and respiratory arrest. The mechanism of increased brain volume is unknown. Multiple mechanisms of increased brain volume may exist in clinical conditions such as CM , including processes that impact BBB function [5, 7, 10]. This study set out to identify circulating biochemical mediators that may contribute to BBB dysfunction. [7, 10].
Positive correlations between brain volume and several lipids that are PLA2 metabolites were identified. The fatty acids released by PLA2, such as arachidonic acid, can be sources of energy, signaling, and of relevance to this study, potent mediators of inflammation. Specifically, arachidonic acid, the precursor of the eicosanoid pathway, can increase the permeability of human brain microvasculature endothelial cell monolayers via prostaglandin E2 activation of EP3 and EP4 receptors . Lysophospholipids are also generated by PLA2 and are important in cell signaling and membrane biology. PLA2 enzymatic activity has been associated with neurological and inflammatory conditions and inhibitors of the PLA2 enzyme are being studied to reduce pathologic inflammation [32–36]. Fatty acids and lysophospholipids can be generated by enzymes other than PLA2, therefore it was confirmed in this study that plasma PLA2 activity was also correlated with brain swelling [37, 38]. These data extend a prior observation where high PLA2 plasma activity was associated with severe malaria and death in Malawian children . Why some children have higher PLA2 enzyme activity is unknown. PLA2 activity is tightly regulated by host responses including TNFα and reactive oxygen species (ROS), both of which can be elevated during severe malaria [15, 40, 41]. Thus the PLA2 pathway and its metabolites may be acting directly on brain microvasculature endothelial cells or indirectly through their effects on cell signaling or energy metabolism.
l-urobilin, a degradation product of haem also correlated with brain volume. Malaria induces erythrocyte lysis and subsequent release of haemoglobin into the circulation. This results in increase of bloodstream ROS levels, which can alter BBB permeability [41–44]. Lysed erythrocytes can increase BBB permeability and result in brain oedema, typically occurring twenty-four hours after haemolysis [45–47]. Direct measurement of ROS and other haemoglobin metabolites are needed to explore the role of erythrocyte lysis in CM associated brain swelling.
Plasma mannitol was also found to have a positive correlation with brain swelling. Exogenous mannitol has been associated with increased brain oedema or brain weight in animal models of vasogenic oedema and brain infarct [50, 51]. Mannitol is used therapeutically for the treatment of increased intracranial pressure following brain injury [48, 49]. Mannitol adjunctive therapy has been studied in CM and was found to either have no significant effect on clinical outcomes in paediatric CM in Uganda  or to potentially have adverse outcomes by prolonging coma resolution time in adults with CM . Further studies to investigate the role of endogenous and exogenous mannitol in CM paediatric brain swelling are necessary.
Inflammatory cytokines were also associated with brain volume and these may directly alter BBB permeability. CCL2 and IL-8 have been previously associated with alterations in tight junction proteins including occludin, ZO-1 and claudin-5 resulting in increased brain endothelial cell permeability [54, 55]. In addition, TNFα has been shown to increase retinal endothelial cell permeability through protein kinase C zeta (PKCζ) and the NFκB pathway by reducing the expression and distribution of claudin-5 and ZO-1 . Interestingly, inhibition of TNFα by soluble TNF p55 receptor attenuates status epilepticus-induced oedema in a rat model, which could be relevant in CM, as seizures are highly prevalent [3, 57]. Furthermore, TNFα can regulate the transcription of PLA2 proteins via the NFκB pathway, providing a link between inflammatory cytokines and the PLA2 pathway . The mechanism, where inflammatory cytokines, increased PLA2 activity and its lipid metabolites converge to disrupt the BBB, in the setting of parasites sequestration to brain endothelial cells in CM, requires further investigation. If the PLA2 pathway is found to play a direct role in BBB dysfunction, PLA2 inhibitors could be evaluated as potential adjunctive therapy.
The higher inflammation and brain volume do not correlate with peripheral parasitaemia or total parasite body load represented by parasite HRP2 levels . The variability in inflammatory responses during infection could be attributed to differences in disease duration, host or parasite genetic polymorphism, prior malaria exposures, uric acid levels or other host or parasite factors [59–65]. Further studies examining how host and/or parasite parameters are mediating inflammation and upregulation of the PLA2 pathway in the setting of increased brain volume could inform therapeutic intervention.
A limitation to the design of this study is that the plasma metabolome was examined after onset of brain swelling. For this reason, it is difficult to know if the metabolic profile is a cause of BBB dysfunction or the result of changes secondary to increased brain volume. A longitudinal study design with repeated metabolic sampling over time in correlation to brain swelling status would provide a more powerful approach to identify potential mediators of BBB permeability. Additionally, there were no metabolomics data from patients with brain volume scores 1, 2 and 8 due to the low prevalence of those brain volume scores. To develop biomarkers of brain swelling for regions without MRI, further plasma analyses that include all of the brain volume groups are warranted. Given the variances observed, larger sample sizes would permit corrections for multiple comparisons.
This study shows associations between brain volume and plasma PLA2 activity, metabolites of the PLA2 pathway, inflammatory cytokines, and other molecules in paediatric CM patients. The role of these small molecules and cytokines as disease mediators or drug target candidates can now be further investigated.
- PLA2 :
tumour necrosis factor
chemokine (C–C motif) ligand 2
magnetic resonance imaging
blood brain barrier
Blantyre Malaria Project
human immunodeficiency virus
infected red blood cell
Institutional Review Board
- Red/Green BODIPY® PC-A2 :
1-O-(6-BODIPY® 558/568-aminohexyl)-2-BODIPY® FL C5-sn-glycero-3- phosphocholine
relative fluorescence unit
reactive oxygen species
protein kinase C zeta
histidine rich protein 2
VP was involved in study design, cytokine and brain swelling correlation analysis, plasma PLA2 analysis and manuscript preparation. KS and TT were involved in human study design, and manuscript preparation. SG carried out the metabolomics analysis and contributed in manuscript preparation. CMF was responsible for human sample handling, cytokine levels measurements and manuscript preparation. MP and SK evaluated the neuroimaging data and were involved in manuscript preparation. AG and RSK contributed in data analysis. MV and LL contributed expertise in BBB permeability. JWB contributed BBB permeability expertise and manuscript preparation. JPD was involved in study design, data analysis and manuscript preparation. All authors read and approved the final manuscript.
We thank Neida Mita-Mendoza and Gretchen Birbeck for critical review of the manuscript. This work was supported by Grant R01 AI077623 (to JPD) from the National Institutes of Health.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Murray CJ, Ortblad KF, Guinovart C, Lim SS, Wolock TM, Roberts DA, et al. Global, regional, and national incidence and mortality for HIV, tuberculosis, and malaria during 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2014;384:1005–70.PubMedPubMed CentralView ArticleGoogle Scholar
- WHO: Guidelines for the Treatment of Malaria. Geneva: World Health Organization; 2010.
- Birbeck GL, Beare N, Lewallen S, Glover SJ, Molyneux ME, Kaplan PW, et al. Identification of malaria retinopathy improves the specificity of the clinical diagnosis of cerebral malaria: findings from a prospective cohort study. Am J Trop Med Hyg. 2010;82:231–4.PubMedPubMed CentralView ArticleGoogle Scholar
- Newton CR, Peshu N, Kendall B, Kirkham FJ, Sowunmi A, Waruiru C, et al. Brain swelling and ischaemia in Kenyans with cerebral malaria. Arch Dis Child. 1994;70:281–7.PubMedPubMed CentralView ArticleGoogle Scholar
- Seydel KB, Kampondeni SD, Valim C, Potchen MJ, Milner DA, Muwalo FW, et al. Brain swelling and death in children with cerebral malaria. N Engl J Med. 2015;372:1126–37.PubMedPubMed CentralView ArticleGoogle Scholar
- Potchen MJ, Kampondeni SD, Seydel KB, Birbeck GL, Hammond CA, Bradley WG, et al. Acute brain MRI findings in 120 Malawian children with cerebral malaria: new insights into an ancient disease. AJNR Am J Neuroradiol. 2012;33:1740–6.PubMedPubMed CentralView ArticleGoogle Scholar
- Dorovini-Zis K, Schmidt K, Huynh H, Fu W, Whitten RO, Milner D, et al. The neuropathology of fatal cerebral malaria in malawian children. Am J Pathol. 2011;178:2146–58.PubMedPubMed CentralView ArticleGoogle Scholar
- Cardoso FL, Brites D, Brito MA. Looking at the blood-brain barrier: molecular anatomy and possible investigation approaches. Brain Res Rev. 2010;64:328–63.PubMedView ArticleGoogle Scholar
- Brown H, Hien TT, Day N, Mai NT, Chuong LV, Chau TT, et al. Evidence of blood-brain barrier dysfunction in human cerebral malaria. Neuropathol Appl Neurobiol. 1999;25:331–40.PubMedView ArticleGoogle Scholar
- Brown H, Rogerson S, Taylor T, Tembo M, Mwenechanya J, Molyneux M, et al. Blood-brain barrier function in cerebral malaria in Malawian children. Am J Trop Med Hyg. 2001;64:207–13.PubMedGoogle Scholar
- Tasker RC, Acerini CL. Cerebral edema in children with diabetic ketoacidosis: vasogenic rather than cellular? Pediatr Diabetes. 2014;15:261–70.PubMedView ArticleGoogle Scholar
- DeMaio L, Rouhanizadeh M, Reddy S, Sevanian A, Hwang J, Hsiai TK. Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells. Am J Physiol Heart Circ Physiol. 2006;290:H674–83.PubMedView ArticleGoogle Scholar
- Clark IA, Budd AC, Alleva LM, Cowden WB. Human malarial disease: a consequence of inflammatory cytokine release. Malar J. 2006;5:85.PubMedPubMed CentralView ArticleGoogle Scholar
- Sasi P, Burns SP, Waruiru C, English M, Hobson CL, King CG, et al. Metabolic acidosis and other determinants of hemoglobin-oxygen dissociation in severe childhood Plasmodium falciparum malaria. Am J Trop Med Hyg. 2007;77:256–60.PubMedGoogle Scholar
- Lyke KE, Burges R, Cissoko Y, Sangare L, Dao M, Diarra I, et al. Serum levels of the proinflammatory cytokines interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor alpha, and IL-12(p70) in Malian children with severe Plasmodium falciparum malaria and matched uncomplicated malaria or healthy controls. Infect Immun. 2004;72:5630–7.PubMedPubMed CentralView ArticleGoogle Scholar
- Taylor TE, Borgstein A, Molyneux ME. Acid-base status in paediatric Plasmodium falciparum malaria. Q J Med. 1993;86:99–109.PubMedGoogle Scholar
- Lee JI, Burckart GJ. Nuclear factor kappa B: important transcription factor and therapeutic target. J Clin Pharmacol. 1998;38:981–93.PubMedView ArticleGoogle Scholar
- Beare NA, Lewallen S, Taylor TE, Molyneux ME. Redefining cerebral malaria by including malaria retinopathy. Future Microbiol. 2011;6:349–55.PubMedPubMed CentralView ArticleGoogle Scholar
- Taylor TE, Fu WJ, Carr RA, Whitten RO, Mueller JS, Fosiko NG, et al. Differentiating the pathologies of cerebral malaria by postmortem parasite counts. Nat Med. 2004;10:143–5.PubMedView ArticleGoogle Scholar
- Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, et al. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med. 2005;2:e204.PubMedPubMed CentralView ArticleGoogle Scholar
- Fox LL, Taylor TE, Pensulo P, Liomba A, Mpakiza A, Varela A, et al. Histidine-rich protein 2 plasma levels predict progression to cerebral malaria in Malawian children with Plasmodium falciparum infection. J Infect Dis. 2013;208:500–3.PubMedPubMed CentralView ArticleGoogle Scholar
- Potchen MJ, Kampondeni SD, Ibrahim K, Bonner J, Seydel KB, Taylor TE, et al. NeuroInterp: a method for facilitating neuroimaging research on cerebral malaria. Neurology. 2013;81:585–8.PubMedPubMed CentralView ArticleGoogle Scholar
- Evans AM, DeHaven CD, Barrett T, Mitchell M, Milgram E. Integrated, nontargeted ultrahigh performance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Anal Chem. 2009;81:6656–67.PubMedView ArticleGoogle Scholar
- Dehaven CD, Evans AM, Dai H, Lawton KA. Organization of GC/MS and LC/MS metabolomics data into chemical libraries. J Cheminform. 2010;2:9.PubMedPubMed CentralView ArticleGoogle Scholar
- Tsao FH, Shanmuganayagam D, Zachman DK, Khosravi M, Folts JD, Meyer KC. A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum. Clin Chim Acta. 2007;379:119–26.PubMedView ArticleGoogle Scholar
- Schneider: Truncated and censored samples from normal populations. New York: Marcel Dekker, Inc.; 1986.
- Millard SP. An R package for environmental statistics. New York: Springer-Verlag; 2013.Google Scholar
- Six DA, Dennis EA. The expanding superfamily of phospholipase A(2) enzymes: classification and characterization. Biochim Biophys Acta. 2000;1488:1–19.PubMedView ArticleGoogle Scholar
- Murakami M, Sato H, Miki Y, Yamamoto K, Taketomi Y. A new era of secreted phospholipase A(2). J Lipid Res. 2015;56:1248–61.PubMedView ArticleGoogle Scholar
- Nag S, Manias JL, Stewart DJ. Pathology and new players in the pathogenesis of brain edema. Acta Neuropathol. 2009;118:197–217.PubMedView ArticleGoogle Scholar
- Dalvi S, Nguyen HH, On N, Mitchell RW, Aukema HM, Miller DW, et al. Exogenous arachidonic acid mediates permeability of human brain microvessel endothelial cells through prostaglandin E activation of EP and EP receptors. J Neurochem. 2015. doi:10.1111/jnc.13117.PubMedGoogle Scholar
- Fonteh AN, Atsumi G, LaPorte T, Chilton FH. Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells. J Immunol. 2000;165:2773–82.PubMedView ArticleGoogle Scholar
- Green JA, Smith GM, Buchta R, Lee R, Ho KY, Rajkovic IA, et al. Circulating phospholipase A2 activity associated with sepsis and septic shock is indistinguishable from that associated with rheumatoid arthritis. Inflammation. 1991;15:355–67.PubMedView ArticleGoogle Scholar
- Farooqui AA, Litsky ML, Farooqui T, Horrocks LA. Inhibitors of intracellular phospholipase A2 activity: their neurochemical effects and therapeutical importance for neurological disorders. Brain Res Bull. 1999;49:139–53.PubMedView ArticleGoogle Scholar
- Ong WY, Farooqui T, Kokotos G, Farooqui AA. Synthetic and natural inhibitors of phospholipases A2: their importance for understanding and treatment of neurological disorders. ACS Chem Neurosci. 2015;6:814–31.PubMedView ArticleGoogle Scholar
- Wang P, Li Y, Shao Q, Zhou W, Wang K. Targeting human secretory phospholipase A2 with designed peptide inhibitors for inflammatory therapy. J Drug Target. 2015;23:140–6.PubMedView ArticleGoogle Scholar
- Bonventre JV, Nemenoff R. Renal tubular arachidonic acid metabolism. Kidney Int. 1991;39:438–49.PubMedView ArticleGoogle Scholar
- Aveldano MI, Bazan NG. Rapid production of diacylglycerols enriched in arachidonate and stearate during early brain ischemia. J Neurochem. 1975;25:919–20.PubMedView ArticleGoogle Scholar
- Vadas P, Taylor TE, Chimsuku L, Goldring D, Stefanski E, Pruzanski W, et al. Increased serum phospholipase A2 activity in Malawian children with falciparum malaria. Am J Trop Med Hyg. 1993;49:455–9.PubMedGoogle Scholar
- Farooqui AA, Horrocks LA. Phospholipase A2-generated lipid mediators in the brain: the good, the bad, and the ugly. Neuroscientist. 2006;12:245–60.PubMedView ArticleGoogle Scholar
- Griffiths MJ, Ndungu F, Baird KL, Muller DP, Marsh K, Newton CR. Oxidative stress and erythrocyte damage in Kenyan children with severe Plasmodium falciparum malaria. Br J Haematol. 2001;113:486–91.PubMedView ArticleGoogle Scholar
- Ferreira A, Balla J, Jeney V, Balla G, Soares MP. A central role for free heme in the pathogenesis of severe malaria: the missing link? J Mol Med (Berl). 2008;86:1097–111.View ArticleGoogle Scholar
- Nanda NK, Das BS. Presence of pro-oxidants in plasma of patients suffering from falciparum malaria. Trans R Soc Trop Med Hyg. 2000;94:684–8.PubMedView ArticleGoogle Scholar
- Haorah J, Ramirez SH, Schall K, Smith D, Pandya R, Persidsky Y. Oxidative stress activates protein tyrosine kinase and matrix metalloproteinases leading to blood-brain barrier dysfunction. J Neurochem. 2007;101:566–76.PubMedView ArticleGoogle Scholar
- Xi G, Hua Y, Bhasin RR, Ennis SR, Keep RF, Hoff JT. Mechanisms of edema formation after intracerebral hemorrhage: effects of extravasated red blood cells on blood flow and blood-brain barrier integrity. Stroke. 2001;32:2932–8.PubMedView ArticleGoogle Scholar
- Xi G, Keep RF, Hoff JT. Erythrocytes and delayed brain edema formation following intracerebral hemorrhage in rats. J Neurosurg. 1998;89:991–6.PubMedView ArticleGoogle Scholar
- Bhasin RR, Xi G, Hua Y, Keep RF, Hoff JT. Experimental intracerebral hemorrhage: effect of lysed erythrocytes on brain edema and blood-brain barrier permeability. Acta Neurochir Suppl. 2002;81:249–51.PubMedGoogle Scholar
- Battison C, Andrews PJ, Graham C, Petty T. Randomized, controlled trial on the effect of a 20 % mannitol solution and a 7.5 % saline/6 % dextran solution on increased intracranial pressure after brain injury. Crit Care Med. 2005;33:196–202.PubMedView ArticleGoogle Scholar
- Wakai A, McCabe A, Roberts I, Schierhout G. Mannitol for acute traumatic brain injury. Cochrane Database Syst Rev 2013, 8:CD001049.
- Kaufmann AM, Cardoso ER. Aggravation of vasogenic cerebral edema by multiple-dose mannitol. J Neurosurg. 1992;77:584–9.PubMedView ArticleGoogle Scholar
- Cho J, Kim YH, Han HS, Park J. Accumulated mannitol and aggravated cerebral edema in a rat model of middle cerebral artery infarction. J Korean Neurosurg Soc. 2007;42:337–41.PubMedPubMed CentralView ArticleGoogle Scholar
- Namutangula B, Ndeezi G, Byarugaba JS, Tumwine JK. Mannitol as adjunct therapy for childhood cerebral malaria in Uganda: a randomized clinical trial. Malar J. 2007;6:138.PubMedPubMed CentralView ArticleGoogle Scholar
- Mohanty S, Mishra SK, Patnaik R, Dutt AK, Pradhan S, Das B, Patnaik J, Mohanty AK, Lee SJ, Dondorp AM. Brain swelling and mannitol therapy in adult cerebral malaria: a randomized trial. Clin Infect Dis. 2011;53:349–55.PubMedPubMed CentralView ArticleGoogle Scholar
- Stamatovic SM, Dimitrijevic OB, Keep RF, Andjelkovic AV. Protein kinase Calpha-RhoA cross-talk in CCL2-induced alterations in brain endothelial permeability. J Biol Chem. 2006;281:8379–88.PubMedView ArticleGoogle Scholar
- Yu H, Huang X, Ma Y, Gao M, Wang O, Gao T, Shen Y, Liu X. Interleukin-8 regulates endothelial permeability by down-regulation of tight junction but not dependent on integrins induced focal adhesions. Int J Biol Sci. 2013;9:966–79.PubMedPubMed CentralView ArticleGoogle Scholar
- Aveleira CA, Lin CM, Abcouwer SF, Ambrosio AF, Antonetti DA. TNF-alpha signals through PKCzeta/NF-kappaB to alter the tight junction complex and increase retinal endothelial cell permeability. Diabetes. 2010;59:2872–82.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim JE, Ryu HJ, Choi SY, Kang TC. Tumor necrosis factor-alpha-mediated threonine 435 phosphorylation of p65 nuclear factor-kappaB subunit in endothelial cells induces vasogenic edema and neutrophil infiltration in the rat piriform cortex following status epilepticus. J Neuroinflammation. 2012;9:6.PubMedPubMed CentralView ArticleGoogle Scholar
- Seydel KB, Fox LL, Glover SJ, Reeves MJ, Pensulo P, Muiruri A, et al. Plasma concentrations of parasite histidine-rich protein 2 distinguish between retinopathy-positive and retinopathy-negative cerebral malaria in Malawian children. J Infect Dis. 2012;206:309–18.PubMedPubMed CentralView ArticleGoogle Scholar
- Lopera-Mesa TM, Mita-Mendoza NK, van de Hoef DL, Doumbia S, Konate D, Doumbouya M, et al. Plasma uric acid levels correlate with inflammation and disease severity in Malian children with Plasmodium falciparum malaria. PLoS One. 2012;7:e46424.PubMedPubMed CentralView ArticleGoogle Scholar
- Orengo JM, Leliwa-Sytek A, Evans JE, Evans B, van de Hoef D, Nyako M, et al. Uric acid is a mediator of the Plasmodium falciparum-induced inflammatory response. PLoS One. 2009;4:e5194.PubMedPubMed CentralView ArticleGoogle Scholar
- Olivier M, Van Den Ham K, Shio MT, Kassa FA, Fougeray S. Malarial pigment hemozoin and the innate inflammatory response. Front Immunol. 2014;5:25.PubMedPubMed CentralView ArticleGoogle Scholar
- Mbugi EV, Meijerink M, Veenemans J, Jeurink PV, McCall M, Olomi RM, et al. Alterations in early cytokine-mediated immune responses to Plasmodium falciparum infection in Tanzanian children with mineral element deficiencies: a cross-sectional survey. Malar J. 2010;9:130.PubMedPubMed CentralView ArticleGoogle Scholar
- Krishnegowda G, Hajjar AM, Zhu J, Douglass EJ, Uematsu S, Akira S, et al. Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem. 2005;280:8606–16.PubMedView ArticleGoogle Scholar
- Portugal S, Moebius J, Skinner J, Doumbo S, Doumtabe D, Kone Y, Dia S, Kanakabandi K, Sturdevant DE, Virtaneva K, et al. Exposure-dependent control of malaria-induced inflammation in children. PLoS Pathog. 2014;10:e1004079.PubMedPubMed CentralView ArticleGoogle Scholar
- Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, Visintin A, Halmen KA, Lamphier M, Olivier M, Bartholomeu DC, et al. Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc Natl Acad Sci USA. 2007;104:1919–24.PubMedPubMed CentralView ArticleGoogle Scholar