INFECTION AND IMMUNITY, Mar. 2011, p. 1244–1253
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 3
Molecular Correlates of Experimental Cerebral Malaria
Detectable in Whole Blood?†
Miranda S. Oakley,1Vivek Anantharaman,2Thiago M. Venancio,2Hong Zheng,3Babita Mahajan,3
Victoria Majam,3Thomas F. McCutchan,4Timothy G. Myers,5L. Aravind,2and Sanjai Kumar3*
Division of Bacterial, Parasitic, and Allergenic Products Center for Biologics Evaluation and Research, Food and Drug Administration,
Rockville, Maryland1; National Center for Biotechnology Information, National Library of Medicine, National Institutes of
Health, Bethesda, Maryland2; Division of Emerging and Transfusion Transmitted Diseases, CBER, FDA, Rockville,
Maryland3; and Laboratory of Malaria and Vector Research4and Microarray Research Facility,
Research and Technologies Branch,5NIH, Bethesda, Maryland
Received 3 September 2010/Returned for modification 29 September 2010/Accepted 2 December 2010
Cerebral malaria (CM) is a primary cause of deaths caused by Plasmodium falciparum in young children in
sub-Saharan Africa. Laboratory tests based on early detection of host biomarkers in patient blood would help
in the prognosis and differential diagnosis of CM. Using the Plasmodium berghei ANKA murine model of
experimental cerebral malaria (ECM), we have identified over 300 putative diagnostic biomarkers of ECM in
the circulation by comparing the whole-blood transcriptional profiles of resistant mice (BALB/c) to those of two
susceptible strains (C57BL/6 and CBA/CaJ). Our results suggest that the transcriptional profile of whole blood
captures the molecular and immunological events associated with the pathogenesis of disease. We find that
during ECM, erythropoiesis is dysfunctional, thrombocytopenia is evident, and glycosylation of cell surface
components may be modified. Furthermore, analysis of immunity-related genes suggests that slightly distinct
mechanisms of immunopathogenesis may operate in susceptible C57BL/6 and CBA/CaJ mice. Furthermore,
our data set has allowed us to create a molecular signature of ECM composed of a subset of circulatory
markers. Complement component C1q, ?-chain, nonspecific cytotoxic cell receptor protein 1, prostate stem cell
antigen, DnaJC, member 15, glutathione S-transferase omega-1, and thymidine kinase 1 were overexpressed in
blood during the symptomatic phase of ECM, as measured by quantitative real-time PCR analysis. These
studies provide the first host transcriptome database that is uniquely altered during the pathogenesis of ECM
in blood. A subset of these mediators of ECM warrant validation in P. falciparum-infected young African
children as diagnostic markers of CM.
Cerebral malaria (CM) is a primary cause of deaths caused
by Plasmodium falciparum, with the majority of cases occurring
in young children living in sub-Saharan Africa. CM is charac-
terized by coma, presence of peripheral asexual P. falciparum
parasites, and exclusion of other causes of encephalopathy.
Early and rapid administration of malaria chemotherapeutic
agents (quinine or artemisinins) is currently the only effective
treatment for CM. However, even with the best treatment,
patients with CM have a case fatality rate of 15 to 20% (29, 37).
Novel methods for early prognosis and differential diagnosis
are critically needed to reduce the high mortality rate of CM.
The need for reliable indicators of clinical disease was first
underscored by an autopsy study that demonstrated that the
standard clinical case definition of CM is incorrect approxi-
mately 25% of the time (38). Recently, clinical studies have
shown that the accuracy of a CM diagnosis can be increased by
investigation of malarial retinopathy in the differential diagno-
sis of CM (2, 37). However, additional measures (i) to predict
which cases of uncomplicated malaria progress into cerebral
malaria and (ii) to discriminate cases of CM from other causes
of encephalopathy will be required to improve the clinical
outcome of CM.
Cancer prognosis and diagnosis typically rely on the ability
to monitor the composition and size of the tumor. However,
efforts to establish noninvasive approaches to monitor cancer
are currently being explored in the field of oncology. In a study
of pancreatic cancer, patients could be discriminated from
healthy subjects by a signature of 19 serum proteins (18). In
another study, efforts to develop a highly sensitive noninvasive
test for colorectal cancer were met with some success by using
a panel of five genes detectable in whole blood (16). Likewise,
in recent years, some progress has been made in the identifi-
cation of correlates of CM in blood (22, 26). However, a robust
test incorporating a large panel of molecules will likely be
required for the accurate prognosis and differential diagnosis
of CM. Interestingly, a recent study conducted on a dengue
cohort in Brazil demonstrated that microarray analysis of pe-
ripheral blood mononuclear cells could be used to design gene
expression signatures that accurately predict which cases of
acute febrile dengue progress into dengue hemorrhagic fever
Previously, to better understand the pathogenic mechanisms
operating during ECM, we used the Plasmodium berghei ANKA
murine model of experimental cerebral malaria (ECM), a use-
ful surrogate of human CM (9), to identify over 200 brain-
specific biomarkers of ECM by comparing the tissue transcrip-
* Corresponding author. Mailing address: Malaria Research Pro-
gram, Laboratory of Emerging Pathogens, DETTD, CBER, FDA,
Rockville, MD 20852. Phone: (301) 827-7533. Fax: (301) 827-4622.
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 13 December 2010.
tional profiles of moribund mice to those of nonmoribund mice
and three types of resistant mice (32). In subsequent studies,
we assessed the biological relevance of a small subset of these
biomarkers and demonstrated that two molecules, CD14 and
galectin-3, play a direct role in the pathogenesis of disease
(31). In the present study, we have identified circulatory bio-
markers of ECM that could be of prognostic/diagnostic value
and may suggest novel therapeutic candidates for human CM.
We compared the whole-blood gene expression profiles of two
susceptible strains of mice (C57BL/6 and CBA/CaJ) with ECM
to that of resistant mice (BALB/c). Using this approach, we
identified 386 potential biomarkers of ECM (45% upregu-
lated, 55% downregulated) detectable in the blood. Further-
more, our analysis of the gene expression patterns provides
novel functional clues regarding the molecular mechanisms
that culminate in the pathogenesis of CM and candidate mol-
ecules for diagnostic purposes.
MATERIALS AND METHODS
Mice and parasite infections. Six- to 8-week-old female C57BL/6, CBA/CaJ,
and BALB/c mice were purchased from the National Cancer Institute (NCI)
breeding facility (Frederick, MD). Parasite infections were performed with an
uncloned line of P. berghei ANKA parasites stored in liquid nitrogen as frozen
stabilities. Thawed parasites were injected and expanded in donor mice. Exper-
imental mice were then infected by intraperitoneal injection of 106P. berghei
ANKA parasites, and mice were monitored for symptoms of ECM beginning on
day 3 postinfection. Thin blood films were prepared for each mouse prior to
sample collection, and percentages of parasitemia (calculated as number of
parasitized erythrocytes/total erythrocytes ? 100) were enumerated by examina-
tion of Geimsa-stained blood films.
Measurement of hematocrit. The hematocrit of whole blood was determined
by using the Autocrit Ultra-3 direct reading centrifuge (BD Biosciences, San
Jose, CA). Briefly, whole blood was collected in a heparinized capillary tube,
sealed with Seal-Ease tube sealer, and then centrifuged at a fixed speed of 11,700
rpm for 3 min. The hematocrit was then determined with a centrifuge-scale plate.
Preparation of whole-blood RNA. Whole blood was collected directly into a
tube preloaded with RNAlater for immediate stabilization of RNA, mixed thor-
oughly, and stored for no longer than 10 days at ?20°C until use. RNA was
prepared using the mouse RiboPure blood isolation kit (Ambion, Inc., Austin,
TX). Briefly, supernatant was removed from thawed samples after centrifugation
at high speed. RNA was then isolated by lysis of sedimented blood with a
guanidinium-based solution, extraction with acid-phenol-chloroform, and puri-
fication with a glass fiber filter cartridge. RNA loaded onto the filter cartridge
was then washed twice, eluted in nuclease-free water, and concentrated by
ultrafiltration with a Vivaspin 500 column (Sartorius Stedim Biotech, Goettin-
gen, Germany) to approximately 3 ?g/?l for subsequent direct labeling.
Microarray and gene expression analysis. Peripheral blood global gene ex-
pression profiles of two susceptible strains of mice (C57BL/6 and CBA/CaJ) were
compared to those of resistant BALB/c mice. Expression profiles were deter-
mined from RNA isolated from five mice per group, and importantly, RNA
samples isolated from susceptible and resistant mice were matched not only by
day of blood collection (when susceptible mice exhibited symptoms of ECM) but
also by percent parasitemia (since parasite RNA is likely a significant component
of infected peripheral blood total RNA). Furthermore, microarray studies were
performed using samples collected on day 6 or 7 postinfection in order to
minimize variations that may arise due to the stage of infection. Synthesis,
incorporation of label, and purification of cDNA were performed as previously
described (30, 32), and then labeled cDNAs from matched samples (approxi-
mately 30 pmol each) were combined and hybridized to a custom-designed
murine oligonucleotide chip containing 37,500 probes (Agilent Technologies,
Santa Clara, CA).
Only spots with valid measurements in 80% of the arrays were considered in
our analysis. Differentially expressed genes were assessed by two different crite-
ria: (i) fold change of ?2 in 80% (4/5) in each of the susceptible strains; (ii)
differential expression detected by the significance analysis of microarray (SAM)
method (one-class mode; false discovery rate (FDR) ? 0.011) (39). In the latter
case, the genetic background of the susceptible mice was disregarded, and SAM
was performed in the one-class mode using 10 data points. SAM was also used
to find genes with differential expression between the susceptible strains (two-
class unpaired mode; FDR ? 0.032). The list of differentially expressed probes
was mapped to Entrez Gene (27), and a single probe was manually selected for
each gene based on fold change and standard deviation. Differentially expressed
probes that did not map to an Entrez Gene ID or UniGene Gene were excluded
from our analysis. Gene Ontology (GO) enrichment analysis was performed
using FuncAssociate (3) (1,000 permutations and corrected P value threshold of
Real-time PCR. RNA was first subjected to two rounds of rigorous DNase
treatment with 6 U of Turbo DNA-free (Ambion, Inc., Austin, TX) at 37°C for
30 min, and absence of DNA contamination was verified by gel electrophoresis
analysis of PCR product amplified from RNA template with primers specific for
the mouse ?-actin gene. To increase the sensitivity of the assay, globin mRNA,
which represents as much as 70% of the total mRNA population, was removed
from whole-blood RNA by using GLOBINclear-mouse/rat (Ambion, Inc., Aus-
tin, TX). cDNA was then synthesized from 1 ?g of enriched RNA at 42°C for 30
min in a 20-?l reaction volume containing iScript reverse transcriptase, random
primers, deoxynucleoside triphosphates (dNTPs), and magnesium chloride (Bio-
Rad Laboratories, Hercules, CA). Quantitative real-time PCR (qRT-PCR) was
performed in a 25-?l reaction volume containing 2 ?l of cDNA, 12.5 ?l iQ SYBR
green supermix (Bio-Rad Laboratories, Hercules, CA), and 10 ?M gene-specific
primers. Amplification and detection of specific product were performed with
the iCycler iQ5 real-time PCR instrument (Bio-Rad Laboratories, Hercules, CA)
with the following cycle profile: 1 cycle at 95°C for 10 min; 5 cycles with 1 cycle
consisting of 20 s of denaturation at 94°C, 15 s of annealing at 48°C, and a 30-s
extension at 68°C; and 40 cycles with 1 cycle consisting of 20 s of denaturation at
94°C, 15 s of annealing at 57 or 60°C, and a 30-s extension at 68°C. A standard
curve derived from the PCR products of 10-fold serial dilutions of plasmid
containing a mouse ?-actin gene fragment was used to determine the absolute
concentration of RNA. Real-time PCR was performed on three mice per group
twice in separate reactions.
Two distinct susceptible strains (C57BL/6 and CBA/CaJ)
and one resistant strain (BALB/c) of mice were used in our
study. We selected a study design that included two susceptible
strains of mice to determine the effects of host genetic back-
ground on the expression of ECM in mice. The mortality rates
from ECM in C57BL/6 and CBA/CaJ mice were 91.7% (11 of
12 mice) and 95.0% (19 of 20 mice), respectively, with the
majority of mice developing symptoms of ECM on day 6 or 7
after infection (Fig. 1). In contrast, although BALB/c mice
developed systemic parasitemia and anemia, they did not ex-
hibit symptoms of ECM. Interestingly, although both suscep-
tible mouse strains had similar mortality rates, we observed
noticeable differences in the clinical symptoms of ECM. We
found that in C57BL/6 mice, progression of disease was grad-
ual and the majority of mice displayed symptoms of irreversible
FIG. 1. Susceptibility of CBA/CaJ and C57BL/6 mice to ECM.
Eleven of 12 (91.7%) C57BL/6 mice and 19 of 20 (95%) CBA/CaJ
mice developed symptoms of ECM by day 9 postinfection.
VOL. 79, 2011 HOST BIOMARKERS OF ECM IN BLOOD1245
coma during the terminal phase of disease, whereas in CBA/
CaJ mice, the onset of disease was comparatively more sudden
and symptoms of rigorous seizures were a more prominent
feature of disease. In order to investigate this observation at
the molecular level, we separately analyzed similarities and
differences in gene expression during ECM between the two
Identification of shared molecular features of susceptible
strains during experimental cerebral malaria. To identify
the common denominator in the molecular signatures of ECM
in two susceptible strains exhibiting slightly different clinical
symptoms, we compared the global expression profiles in pe-
ripheral blood of the C57BL/6 and CBA/CaJ strains of mice
and clustered them as a single class versus those of the resistant
BALB/c mice. A gene was considered differentially expressed
when meeting at least one of the following criteria: (i) 2-fold
change in at least 80% of the samples combining both suscep-
tible strains; or (ii) differential expression detected by the SAM
method (see Materials and Methods for details). Using the
criteria outlined above, we found a total of 1,379 differentially
expressed genes; of these 617 were upregulated and 762 were
downregulated (?? 1.88; FDR ? 0.011) (see Tables S1, S2,
and S3 in the supplemental material). We then explored
differentially expressed genes by checking for enrichment of
Gene Ontology (GO) terms (see Materials and Methods for
details). A major outcome of this analysis was that certain
potential chromatin-related functions were significantly up-
regulated, whereas those related to heme biosynthesis were
significantly downregulated. To further investigate these data,
we performed a more in-depth analysis of the genes showing
the most drastic expression changes (?4-fold): a set of 176
upregulated and 210 downregulated genes. Using sequence
profile searches, analysis of literature, and protein-protein/
genetic interactions, we functionally classified these genes to
get a better picture of the transcriptional landscape of the
peripheral blood in ECM.
Evidence for dysfunctional erythropoiesis during ECM. In-
terestingly, we found evidence for pervasive erythroid dysfunc-
tion in the susceptible compared to resistant strains of mice,
with at least 23 genes related to erythropoiesis showing greater
than 4-fold downregulation in mice exhibiting symptoms of
ECM. As the initial GO analysis revealed a downregulation of
heme biosynthesis genes in the susceptible mice, we analyzed
this issue in greater detail. Genes encoding three key enzymes
at different points in the heme biosynthesis pathway, namely,
aminolevulinic acid synthase, hydroxymethylbilane synthase,
and ferrochelatase were all downregulated greater than 8-fold
on average. Concomitantly, the Hbb-b1 and Hbb-b2 hemoglo-
bin genes and the alpha hemoglobin-stabilizing protein genes
were also comparably downregulated. Beyond heme biosyn-
thesis, several structural and metabolic proteins specific to the
erythroid lineage were also similarly depressed in regulation.
These included genes that encode red blood cell (RBC) cy-
toskeletal proteins such as spectrin alpha 1, ankyrin 1, ery-
throid protein bands 4.1 and 4.2, and adducin 2 (Table 1) and
cell surface components such as the sialoglycoprotein glyco-
phorin A (notably, a receptor for P. falciparum erythrocyte
binding antigen 175) (5), Kell blood group protein, and the Rh
blood group D antigen. The strong downregulation of several
genes related to glutathione metabolism (Table 1) and genes
encoding proteins such as Isca1 (involved in iron-sulfur cluster
assembly), 2,3-bisphosphoglycerate mutase, and ornithine de-
carboxylase might also be related to the transcriptional depres-
sion of genes associated with erythrocyte metabolic pathways.
Also downregulated were potential erythrocyte regulators such
hemogen and a possible trafficking component, ?-synuclein. In-
terestingly, at least five of these genes recovered as downregu-
lated, namely, SNCA, ALAS2, FECH, AHSP, and HEMGN, are
known to be targets of the major erythroid transcription factor
GATA-1. GATA-1 was not found to be significantly downregu-
lated in the susceptible mice, but its functional partners E2F2
and Dp2, required for terminal cell division and maturation
during erythropoiesis (19), were 5- and 7-fold downregulated,
respectively. Hence, we speculate that the depressed erythroid
development might be a consequence of the depressed expres-
sion of this key transcription factor.
We next wanted to determine a possible relationship be-
tween P. berghei ANKA infection-induced anemia and progres-
sion into ECM in susceptible and resistant mice. We first
measured the hematocrit in uninfected mice to ensure that
there were no strain-specific differences in basal hematocrit
levels. The hematocrits were 47.4 ? 1.4 (C57BL/6) and 44.2 ?
1.9 (CBA/CaJ) in susceptible mice and 48.7 ? 1.2 (BALB/c) in
resistant mice. We then measured the hematocrit of infected
mice during the symptomatic phase (day 6) of disease. Suscep-
tible mice with ECM had hematocrits of 47.3 ? 0.6 (C57BL/6)
and 47.8 ? 1.5 (CBA/CaJ). The hematocrit of resistant
BALB/c mice was similar (47.1 ? 1.1), indicating a lack of
association between anemia and expression of ECM in mice.
These results also suggest dyserythropoiesis observed by mi-
croarray in mice with ECM does not result in altered hemat-
ocrit as measured on day 6 postinfection. These discordant
results may be due to the likelihood that young erythrocytes
are only a small fraction of total erythrocytes in blood.
Downregulation of platelet- and clotting-related genes in
ECM. We observed that among the highly downregulated
genes, there were genes related to platelet and blood-clotting
functions such as those encoding von Willebrand factor, coag-
ulation factor XIII A1 subunit, platelet-derived growth factor
alpha, and coagulation factor V. Additionally, angiopoietin 1,
which stabilizes microvessels against leakage (15) and is re-
quired for the recovery of the brain from hypoxia (33), was also
?4-fold downregulated. Taken together, these results suggest
that the susceptible mice are likely to suffer from greater leak-
age via their microvessels along with reduced clotting leading
to a propensity for hemorrhaging. In light of this, potential
therapeutic regimens which specifically target restoration of
the angiopoietin balance and the depletion of coagulation fac-
tors should be further explored.
Possible alterations in the signature of cell surface glycosyl-
ation in ECM. Our data also presented evidence for several
distinct enzymes involved in modification of cell-surface poly-
saccharides to be strongly differentially regulated between sus-
ceptible and resistant strains. Of the downregulated genes are
those coding for the enzymes ST3 ?-galactoside ?-2,3-sialyl-
transferase 2 and 5, which are involved in the synthesis of
glycosphingolipids such as the gangliosides GM3 and GM4
(7). Consistent with this, the cytidine monophospho-N-acetyl-
neuraminic acid synthetase, which synthesizes a potential sub-
strate for the above glycosyltransferases, is also concomitantly
1246 OAKLEY ET AL.INFECT. IMMUN.
TABLE 1. Circulatory biomarkers of ECM categorized by function
Gene transcript Function/description
Aminolevulinic acid synthase 2, erythroid
Alpha hemoglobin stabilizing protein
Hemoglobin, beta adult major chain
Hemoglobin, beta adult major chain
Spectrin alpha 1
Ankyrin 1, erythroid
Erythrocyte protein band 4.1
Erythrocyte protein band 4.2
Kell blood group protein
Rh blood group, D antigen
Hydroxyacyl glutathione hydrolase
Glutamate-cysteine ligase, modifier subunit
Glutaredoxin 5 homolog (Saccharomyces cerevisiae)
Thioredoxin reductase 2
Glutathione peroxidase 4
Platelet and clotting-related
Coagulation factor V
Coagulation factor XIII A1 subunit
von Willebrand factor
Platelet-derived growth factor alpha
Cell surface glycosylation-related
Sialoglycoprotein glycophorin A
Cytidine monophospho-N-acetylneuraminic acid synthetase
ST3 beta-galactoside alpha-2,3-sialyltransferase 5
ST3 beta-galactoside alpha-2,3-sialyltransferase 2
Heparan sulfate (glucosamine) 3-O-sulfotransferase 3B1
Immune response-related genes
Histocompatibility 2, class II antigen E alpha
Histocompatibility 2, class II antigen A, beta 1
Defensin, alpha 1
Interferon-inducible GTPase 1
2?-5? Oligoadenylate synthetase 1E
2?-5? Oligoadenylate synthetase 3
Chemokine (C-X-C motif) ligand 2
Chemokine (C-X3-C) receptor 1
Myelin and lymphocyte protein, T-cell differentiation protein
Nonspecific cytotoxic cell receptor protein 1
Cytotoxic T-lymphocyte-associated protein 4
Killer cell lectin-like receptor, subfamily A, member 8
Killer cell lectin-like receptor, subfamily A, member 15
Transcription and chromatin
Transcription factor Dp 2
Signal transducer and activator of transcription 1
E2F transcription factor 2
ETS domain transcription factor SpiB
Recombination signal binding protein for immunoglobulin kappa J region-like
Continued on following page
VOL. 79, 2011 HOST BIOMARKERS OF ECM IN BLOOD1247
downregulated in ECM. Another enzyme, hexosaminidase A,
involved in the degradation of gangliosides such as GM2, is
also downregulated. Cell-surface gangliosides have previously
been observed to be major targets for interaction with patho-
gen proteins in the process of invasion of eukaryotic host cells
by diverse pathogens (21). These transcriptional changes could
in effect result in the reduced presentation of a particular type
of ganglioside on the host cell surface and thereby modulate a
specific host-parasite interaction. Hence, it is conceivable that
their transcriptional regulation might be a defensive response to
regulate host-parasite interaction. In contrast to these genes, the
Hs3st3b1 gene, involved in sulfation of glucosamine 3-OH in
cell-surface heparan sulfate, is upregulated strongly. We have
previously observed that the polysaccharide-binding protein
galectin-3 has a notable role in cerebral malaria progression,
but it does not appear to directly bind any pathogen moiety
(31). Given this observation, it is also possible that the alter-
ations in host cell-surface polysaccharides by downregulation
of certain pathways and upregulation of others might have a
role in regulating endogenous cell adhesion reactions. Future
studies that could directly assess differences in cell-surface
glycosylation may allow investigation of its utility as a potential
prognostic marker in cerebral malaria.
Alteration in the immune response in relation to ECM. Not
surprisingly, the functional class showing the largest number of
genes with more than 4-fold-altered expression in susceptible
mice relative to the resistant mice is that pertaining to the
immune response. This group includes 65 genes, with 26 genes
downregulated and 39 genes upregulated. We observed that
the ETS domain transcription factor SpiB, which is a key reg-
ulator of the development of a specific class of immune cells,
the plasmacytoid dendritic cells (natural interferon-producing
cells) (35), is downregulated approximately 5-fold in ECM-
susceptible mice. Consistent with this, we observed a major
downregulation of genes associated with such cells or those
genes induced by the cytokines secreted by these cells (e.g., the
STAT1, IIGP1, DEFA1, ISG20, OAS1E, and OAS3 genes)
(Table 1). The primary role of plasmacytoid dendritic cells is
believed to be in innate immunity against viruses. deWalick et
al. have shown that conventional but not plasmacytoid den-
dritic cells play a key role in the pathogenesis of ECM; deple-
tion of conventional dendritic cells early in P. berghei ANKA
infection with B6.CD11c-DTR transgenic mice prevented symp-
toms of ECM, whereas depletion of plasmacytoid dendritic
cells by using the 120G8 monoclonal antibody (MAb) had no
effect on susceptibility to ECM (10). Given that SpiB promotes
the development of these dendritic cells at the expense of T, B,
and NK cells, it is conceivable that downregulation of this
transcription factor might have a role in allowing alternative
developmental programs for blood cells which are required for
the parasite response. Indeed, several genes of cytotoxic T and
NK cell receptors (e.g., the Klra15, Nccrp1, Ctla4, Klra8, and
MAL genes) are upregulated in our data set. Nevertheless, it is
Gene transcript Function/description
Ubiquitin system-related genes
Tripartite motif-containing 10
Ubiquitin-conjugating enzyme E2L 6
Ring finger protein 10
F-box protein 25
Ubiquitin specific peptidase 32
Membrane-associated ring finger (C3HC4) 2; E3 ligase
Membrane-associated ring finger (C3HC4) 8; E3 ligase
F-box protein 9
Ubiquitin-conjugating enzyme E2R 2
RAD23a homolog (S. cerevisiae)
Ubiquitin-associated domain containing 1
Autophagy-related 4A (yeast)
BCL2A1A B-cell leukemia/lymphoma 2-related protein A1a4.0
Cytoskeletal keratin genes
Iron-sulfur cluster assembly 1 homolog
1248OAKLEY ET AL.INFECT. IMMUN.
conceivable that this observation provides a means of distin-
guishing an infection that results in ECM.
Another novel observation emerging from our data set is
extraordinary downregulation (292-fold on average) of the
neuropeptide Y gene (NPY). Npy has been shown to be se-
creted in the peripheral blood by macrophages and B cells and
shown to regulate many aspects of the immune response, such
as macrophage behavior, antibody secretion by B cells, T-cell
proliferation, and uptake of antigens by dendritic cells (43).
Given this pervasive role, the drastic downregulation of Npy
could potentially alter the landscape of the immune response
in a major way. Hence, further investigation of the role of Npy
in ECM pathogenesis is warranted as it could offer both a
potential therapeutic as well as diagnostic tool. Among the
other secreted signaling molecules showing drastic differences
in expression was the gene encoding the chemokine Cxcl2 (32-
fold upregulated in susceptible mice). This chemokine plays a
major role as a neutrophil attractant and has been implicated in
certain inflammatory diseases (14). It would be of interest to
investigate if this molecule might have a role in the progression
of ECM, especially given its elevated expression. Likewise, the
receptor of the chemokine fractalkine, CX3CR1, is also over-
expressed in susceptible mice (7.5-fold). This chemokine re-
ceptor has been previously implicated in endothelial damage in
other infectious diseases such as cytomegalovirus infection (4).
In light of this, it would be of interest to investigate if CX3CR1
plays a role in facilitating the hemorrhaging observed in CM.
The above observations suggest that several distinct immune
pathways that have not previously been considered in the con-
text of malarial pathogenesis could be worth investigating in
Differential expression of transcription and chromatin struc-
ture-related genes. We observed that 27 genes (15 downregu-
lated and 12 upregulated) encoding proteins with a definitive
role in transcription regulation or chromatin organization were
differentially expressed (?4-fold) in the susceptible versus the
resistant mice. In addition to some of the transcription factors
that were discussed above, we identified certain others which
might play important roles in different blood cells. For exam-
ple, Rbpjl, a transcription factor of the CSL family, is upregu-
lated 17.5-fold in the susceptible mice. A role for this tran-
scription factor, which functions downstream of the Notch
signaling cascade, in hemal development has not been previ-
ously uncovered. Given its dramatic expression pattern, we
suggest that it might be involved in an asymmetric cell division
or differentiation of immune cells downstream of an as yet
unclear wing of the Notch pathway. Similarly, the homeodo-
main transcription factor, Hopx, has not been implicated in a
blood cell-related role. Its emergence as a prominently upregu-
lated gene product in our study might implicate it as an as yet
undiscovered transcription factor in a hemal differentiation
process. Several chromatin proteins were found to be sys-
tematically downregulated (Table 1), suggesting that these
chromatin-level alterations might be linked to the differen-
tial gene expression patterns observed in our study. How-
ever, the ubinuclein-2 (Ubn2) gene was notably upregulated
(16-fold). We had recently indentified this protein to be a novel
histone chaperone that functions in conjunction with the Hir
complex (1). Given its detection in our experiments, it would
be of interest to investigate if Ubn2 might have a specific role
in the differential expression of genes during the immune re-
sponse or hemocyte development via the reorganization of
Other miscellaneous genes of interest showing differential
expression. While the above functional categories contain
most of the genes of interest showing marked differential ex-
pression, we found some other genes with unusual expression
differences which might have functional relevance to ECM
pathogenesis. Interestingly, 13 of the 16 genes related to the
ubiquitin system that showed greater than 4-fold expression
change were downregulated. This significantly biased down-
regulation was seen across the Ub pathway, including genes
coding for at least 5 E3 ligases, 2 E2 ligases and two debuiq-
uitinating enzymes (see the supplemental material). Of partic-
ular interest are the March2 and March8 E3 ligases that have
been recently implicated in the Ub-dependent trafficking of
major histocompatibility complex (MHC) class II molecules
into secretary vesicles (11). Their downregulation is consistent
with the concomitant downregulation of various class II MHC
proteins such as H2-Ab1 (11-fold) and H2-Ea (53-fold) (Table
1). In light of this, it would be worthwhile for future investi-
gations on CM pathogenesis to consider the role of the down-
regulation of multiple Ub system genes in the context of de-
pressed class II MHC expression. Also of interest was the
upregulation of the Bcl2 paralog, Bcl2a1a, which has been
implicated in the survival of double-positive thymocytes (41). It
remains to be seen if the overexpression of this antiapoptotic
factor has a significant role in altering the immune cell devel-
opment between susceptible and resistant strains. Notably, five
distinct cytoskeletal keratin genes (Table 1) are between 11-
and 20-fold overexpressed in the susceptible strains. The cell
types in which these might be overexpressed are unknown, but
they could potentially serve as a diagnostic marker.
Identification of the genes differentially expressed between
susceptible strains during the ECM. We also compared the
susceptible strains against each other to find genes with differen-
tial expression between two genetically different backgrounds
(see the supplemental material). In this analysis, the BALB/c
(ECM-resistant) strain was considered a common reference to
find altered expression between the susceptible strains. We
found significantly altered expression in 93 genes. There was a
remarkable overrepresentation of immune-related genes, espe-
cially those related to the MHC (see the supplemental material).
Twenty-oneofthese93geneswerealsofound to be differentially
expressed when comparing susceptible versus resistant strains
(as described above). However, the vast majority of the im-
mune-related genes with significant variation between strains
were not recovered identified in the above analysis treating the
two susceptible strains as one class. This implies that above-
identified genes related to immunity are likely to serve as
useful genetic markers for ECM diagnosis, irrespective of the
strain background. It also suggests that the divergent regula-
tion of several immune response genes might result in distinct
immunity landscapes in the two susceptible strains, which
might have a role in the symptomatic differences observed
Interestingly, while the gene that encodes CD8 antigen, beta
chain 1 (CD8?1) is significantly upregulated (3.9 ? 0.5; P ?
0.0005) in moribund C57BL/6 mice compared to resistant
BALB/c mice, it is markedly downregulated (?7.5 ? 1.2; P ?
VOL. 79, 2011 HOST BIOMARKERS OF ECM IN BLOOD1249
0.0003) in the peripheral blood of moribund CBA/CaJ mice.
Of considerable interest are the diametrically opposite expres-
sion patterns of certain NK cell receptors between the two
susceptible strains: Klra4, Klra21, and Klra18 are all clearly
upregulated in C57BL/6 mice, while being unchanged or mildly
downregulated in CBA/CaJ mice. In contrast, Ncr1 is down-
regulated in only C57BL/6 mice. The exact role of these NK
cell receptors in malaria remains unclear; however, the fact
that some of them are similarly upregulated in both susceptible
strains, whereas others are overexpressed in only one strain,
might suggest some as yet unclear role for them in parasite
Of note is the strain-specific upregulation of genes encoding
two distinct chemokines, Ccl12 and Cxcl11, in the CBA/CaJ
mice. Ccl12 is a chemoattractant that recruits certain types of
regulatory T cells to sites of inflammation. In contrast, Cxcl11
has been previously implicated in a number of human disease
conditions. Of particular interest is its role in neuroborreliosis,
where it has been implicated as a chemoattractant for CD4?T
cells in the cerebrospinal fluid (CSF) (24). It is possible that it
could play a comparable role in the aspects of ECM patho-
genesis specific to the CBA/CaJ mice.
Validation of putative biomarkers by quantitative real-time
PCR. Finally, we wanted to develop a prototype test for the
diagnosis of CM based on the transcriptional analysis of host
biomarkers in blood. To accomplish this, a panel of genes that
were strongly overexpressed (by at least 4-fold) during ECM
and functionally diverse was selected for validation by quanti-
tative real-time PCR comparing C57BL/6 (n ? 3) versus
BALB/c (n ? 3) peripheral blood RNA. Only upregulated (not
downregulated) genes were chosen because it is easier to mea-
sure the upregulation of expression. Furthermore, a function-
ally diverse panel of genes associated with nonoverlapping
pathways was selected to create a molecular signature unique
to the disease state of ECM. Complement component C1q, ?
chain (C1qb) was overexpressed by 17.4 ? 1.3-fold in C57BL/6
(65,800 ? 12,370) versus BALB/c (3,873 ? 430) mice (P ?
0.0075) (Fig. 2A), nonspecific cytotoxic cell receptor protein 1
(nccrp1) by 7.1 ? 0.05-fold in C57BL/6 (174,400 ? 60,980)
versus BALB/c (26,690 ? 7,707) mice (P ? 0.0716) (Fig. 2B),
prostate stem cell antigen (psca) by 5.7 ? 0.3-fold in C57BL/6
(26,920 ? 9,685) versus BALB/c (4,632 ? 1,386) mice (P ?
0.0850) (Fig. 2C), DnaJC, member 15 (DnaJC15) by 3.7 ?
0.04-fold in C57BL/6 (74,300 ? 9,607) versus BALB/c (20,400 ?
2,471) mice (P ? 0.0056) (Fig. 2D), glutathione S-transferase
omega-1 (gsto1) by 3.6 ? 0.5-fold in C57BL/6 (494,200 ?
157,700) versus BALB/c (141,100 ? 32,970) mice (P ? 0.0935)
(Fig. 2E), and thymidine kinase 1 (tk1) by 1.89 ? 0.005-fold in
C57BL/6 (15,020 ? 1,707) versus BALB/c (7,958 ? 983) mice
(P ? 0.0231) (Fig. 2F). In summary, three of the selected genes
(coding for c1qb, DnaJC15, and tk1) had enhanced levels of
expression during ECM that were statistically significant (as
defined by a P value of ?0.05), while the other three genes
(coding for nccrp1, psca, and gsto1) had levels of expression
that approached statistical significance (as defined by a P value
In this study, we identified over 300 potential biomarkers
of ECM detectable in the circulation by comparing the whole-
blood transcriptional profiles of resistant BALB/c mice to
those of two susceptible strains of mice (C57BL/6 and CBA/
CaJ) during the symptomatic phase of disease. While the ma-
jor objective of this study was to identify biomarkers that may
be of prognostic/diagnostic value, we note that the transcrip-
tional profile of the peripheral blood captures the molecular
and immunological events occurring in the brain and spleen
during ECM. To our knowledge, this is the first study that
measured the transcription profile of whole blood during the
FIG. 2. Circulatory biomarkers of ECM as detected by quantitative real-time PCR (qRT-PCR). The expression of a subset of murine
circulatory biomarkers of ECM was measured by comparative qRT-PCR in susceptible C57BL/6 mice (n ? 3) versus resistant BALB/c mice (n ?
3) during the ECM phase of P. berghei ANKA infection. Shown is expression of complement component C1q, ?-chain (A); nonspecific cytotoxic
cell receptor protein 1 (B); prostate stem cell antigen (C); DnaJC, member 15 (D); (E) glutathione S-transferase omega-1 (E); and thymidine
kinase 1 (F). ?, P ? 0.1; ??, P ? 0.05.
1250OAKLEY ET AL.INFECT. IMMUN.
ECM phase in mice; previous studies were performed with
brain and spleen samples.
Several important clinical and biological observations were
made in this study. We noted that the clinical features of ECM
are slightly different between C57BL/6 and CBA/CaJ mice.
While C57BL/6 mice slowly progress into a state of coma,
CBA/CaJ mice experience a more sudden onset of disease with
the obvious signs of seizure prior to coma. Based on the subtle
genetic differences, it is reasonable to argue that the distinct
clinical features induced by P. berghei ANKA infection in these
two inbred mouse strains are regulated by their host genetic
factors. It is important to note that the spectrum of clinical
features seen in young African children experiencing CM is
also known to vary greatly, which is generally attributed to host
genetics. Our study has allowed us to discern some of the host
factors that may be responsible for these various clinical fea-
tures observed during ECM. We also determined if anemia
correlated with the pathogenesis of ECM by comparing the
hematocrits of susceptible mice (C57BL/6 and CBA/CaJ) ex-
hibiting symptoms of ECM to that of resistant BALB/c mice.
We found that on day 6 postinfection, all three strains of mice
had a similar hematocrit, suggesting that ECM occurs inde-
pendently of anemia.
Although there was no significant difference in the hemat-
ocrits in moribund C57BL/6 and CBA/CaJ versus resistant
BALB/c mice, our microarray data suggest that erythropoiesis
is comparatively dysfunctional during ECM, with at least 23
genes related to erythropoiesis downregulated by greater than
4-fold in moribund C57BL/6 and CBA/CaJ mice. Erythropoi-
esis, the differentiation of hematopoietic stem cells into mature
red blood cells, is characterized by the extrusion of nuclei from
orthochromatic erythroblasts to form enucleated reticulocytes
and subsequent mature red blood cells (17). Previously, we
reported simultaneous downregulation of transcription but up-
regulation of translation of the hemoglobin-? gene in brain
tissue of mice with ECM (32). Based on these results, we
hypothesized that low levels of hemoglobin-? RNA were a
result of a paucity of (nucleated) immature erythrocytes within
the brain due to decreased production of erythrocytes, while
elevations in hemoglobin-? protein were caused by an abun-
dance of mature erythrocytes due to excess hemorrhaging
within the brain. The consistent transcriptional repression of
genes associated with erythroid differentiation observed in the
present study may reflect a lack of young nucleated cells of
the erythroid lineage in the peripheral blood and suggests
the importance of suppressed erythropoiesis during ECM.
However, we want to emphasize that since this study in-
cluded only one ECM-resistant strain of mice, it is possible
that robust infection-induced erythropoiesis may be a feature
of the BALB/c strain of mice rather than a phenotype unique
to ECM-resistant mice. Future studies on additional strains of
ECM-resistant mice will be needed to fully address this ques-
In a previous study, transcriptional analysis of uninfected
versus P. berghei ANKA-infected C57BL/6 mice prior to the
onset of symptoms demonstrated suppressed erythropoiesis
in the bone marrow and spleen of mice on days 1, 3, and 5
of infection that translated into reduced levels of peripheral
reticulocytes (36). These results indicate that dysfunctional
erythropoiesis may occur early in the pathogenesis of ECM.
An earlier study that examined bone marrow aspirates of nine
patients with cerebral malaria in Thailand suggested that dys-
erythropoiesis may also be a feature of human CM (44). In this
study, microscopic analysis of the bone marrow aspirates pre-
sented evidence of morphological abnormalities of erythro-
blasts that the authors speculated could be caused by local
release of malaria toxin, hypoxia, or disturbed macrophage
function induced by concomitant cytoadherence of parasitized
erythrocytes to endothelial cells in the bone marrow sinusoids.
Indeed, recent in vitro studies indicate that hemozoin can
directly inhibit erythropoiesis by induction of apoptosis of
erythroid precursors, and macrophages actually reduce this
inhibitory effect of hemozoin on erythroid development by
preventing contact between hemozoin and erythroid cells (23).
Interestingly, murine studies have shown that treatment of
mice with erythropoietin reduces mortality from ECM (20,
45). Although the primary function of erythropoietin is reg-
ulation of erythrocyte production, these studies have fo-
cused on its neuroprotective capacity to prevent hypoxia and
apoptosis within the brain. However, a strong association be-
tween high levels of plasma but not CSF erythropoietin and a
reduced risk of neurological sequelae and death was recently
reported in African children with CM (6). Our microarray data
suggest that suppressed erythropoiesis does not alter the he-
matocrit during ECM. These results may be explained by an
earlier observation showing that reticulocytes are only a small
fraction of total erythrocytes during a P. berghei ANKA infec-
tion (36). However, it is plausible that the absence of young
erythrocytes may contribute to decreased brain oxygenation,
which may augment symptoms and potentially result in neuro-
logical sequelae. Future studies will be needed to determine
whether dysfunctional erythropoiesis during CM has a caus-
ative role in the pathogenesis of disease.
functions were significantly downregulated during ECM. Numer-
ous studies have implicated a role for platelets in the patho-
genesis of ECM (40), and recent studies have examined the
role of platelets in human CM. In a study of Malawian chil-
dren, platelet accumulation in the microvessels of brain tissue
was found to be significantly greater in patients with CM than
those with severe malaria anemia or nonmalarial encephalop-
athy (12). In addition, all Malawian pediatric patients with CM
had low platelet counts with the degree of thrombocytopenia
significantly correlating with parasitemia (42). Indeed, previ-
ous clinical studies have indicated that retinal hemorrhaging (a
possible indicator of thrombocytopenia) is significantly associ-
ated with the severity of CM in humans (25), and malaria
retinopathy is currently being explored as a promising tool for
the differential diagnosis of CM (2, 37).
Interestingly, CD8?1 is significantly upregulated (3.9 ? 0.5;
P ? 0.0005) in moribund C57BL/6 mice but markedly down-
regulated (?7.5 ? 1.2; P ? 0.0003) in moribund CBA/CaJ
mice compared to resistant BALB/c mice. This suggests that
the CD8?T-cell population produced during a lethal P. berghei
ANKA infection that culminates in ECM may differ phenotyp-
ically in susceptible C57BL/6 and CBA/CaJ strains of mice.
Alternatively, it is possible that absence of CD8?1 in the cir-
culation may result from preferential sequestration of CD8?1?
T cells within the brain by CBA/CaJ mice. Previously, Randall
et al. reported that CD8?1 is significantly upregulated in brain
VOL. 79, 2011 HOST BIOMARKERS OF ECM IN BLOOD 1251
tissue of moribund C57BL/6 but not CBA P. berghei ANKA-
infected mice (34). In murine studies conducted by Grau et al.
in the early 1980s, depletion of CD8?T cells with an antibody
that targets CD8?1 did not prevent the onset of ECM in P.
berghei ANKA-infected CBA mice (13). However, it was re-
cently shown that anti-CD8? successfully inhibits the progres-
sion of ECM in CBA mice (34). Together, these studies indi-
cate that pathogenic CD8?T cells may differ phenotypically
among susceptible strains of mice.
The primary objective of this study was to identify biomar-
kers detectable in the circulation that could be used for the
prognosis and differential diagnosis of CM. Efforts are under
way to identify host biomarker-based correlates of CM. For
example, previous studies have assessed the prognostic and
diagnostic potential of angiopoietin 1, a marker of the resting
endothelium, and angiopoietin 2, a marker of endothelial ac-
tivation, for CM (26, 8). In a study of Ugandan children and
Thai adults, Lovegrove et al. demonstrated that angiopoietin-1
levels are decreased and angiopoietin-2 levels are increased in
CM versus uncomplicated malaria and healthy controls and
the angiopoietin 2/angiopoietin 1 ratio was useful in predicting
survival in African children with CM.
Among the greater than 300 circulatory biomarkers of ECM
identified by microarray in our study, we selected a subset of
biomarkers for further evaluation by quantitative real-time
PCR (qRT-PCR). Differential expression of six molecules
(c1qb, nccrp1, psca, DnaJC15, gsto1, and tk1) in susceptible
(C57BL/6) versus resistant (BALB/c) mice was assessed by
qRT-PCR. Of these molecules, expression of c1qb, DnaJC15,
and tk1 was significantly higher (as defined by a P value of
?0.05) in moribund C57BL/6 mice compared to resistant
BALB/c mice, with fold increases of 17.3 ? 1.3, 3.7 ? 0.04, and
1.89 ? 0.005, respectively. Nccrp1, psca, and gsto1 had fold
increases in expression of 7.1 ? 0.05, 5.7 ? 0.3, and 3.6 ? 0.5,
respectively, which approached statistical significance (as de-
fined by a P value of ?0.1). These results demonstrate that
circulatory host biomarkers identified by genome-wide gene
expression profiling studies can be used as a diagnostic tool for
ECM. Overall, our ability to detect the host biomarkers in
blood that are closely associated with the clinical state of ECM
offers the prospect of developing diagnostic tests for the prog-
nosis and differential diagnosis of CM in hospital settings in
countries where malaria is endemic. While some biologically
relevant ECM biomarkers identified in our studies could be
validated in clinical studies, extensive gene expression-profiling
studies of blood samples from children during different clinical
states of CM are needed to identify the biomarkers of hu-
We thank Alvaro Godinez and Qin Su from NIAID, NIH, for expert
help with microarray studies.
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Editor: J. H. Adams
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