Yonago Acta medica 2004;47:7–15

Regulation of TNF-α, IL-1β and ICAM-1 Gene Expression in
THP-1 Monocytes Stimulated with Plasmodium falciparumCultured Medium by Excretory/Secretory Products from
Spirometra erinaceieuropaei Plerocercoids
Paramasari Dirgahayu, Soji Fukumoto, Sayuri Tademoto and Kazumitsu Hirai
Division of Molecular Medical Zoology, Department of Microbiology and Pathology, School of
Medicine, Tottori University Faculty of Medicine, Yonago 683-8503 Japan
We have reported that excretory/secretory (ES) products from Spirometra erinaceieuropaei
plerocercoids suppress production and gene expression of tumor necrosis factor (TNF)-αin murine macrophages stimulated with lipopolysaccharide. The present study demonstrates that ES products suppress TNF-α, interleukin (IL)-1β and intercellular adhesion
molecule (ICAM)-1 gene expression in human monocytic leukemia cell line THP-1 stimulated with Plasmodium falciparum-cultured medium (Pf-CM). Inhibition of extracellularsignal regulated protein kinase 1/2 (ERK1/2) with PD98059 reduced TNF-α, IL-1β and
ICAM-1 gene expression; on the contrary inhibition of p38 mitogen-activated protein kinase(MAPK) with SB203580 increased the expression of these genes. These findings indicatedthat ERK1/2, but not p38 MAPK pathway is important for induction of TNF-α, IL-1β and
ICAM-1 gene expression in Pf-CM stimulated THP-1 monocytes. ES products additionallysuppress IL-1β, but not TNF-α and ICAM-1 gene expression in Pf-CM stimulated THP-1
cells treated with PD98059. We hypothesize that ES products may be useful in reducing
falciparum malaria-induced inflammatory response and sequestration in the late stage ofmalarial diseases such as cerebral malaria.
Key words: human monocytes; mitogen-activated protein kinase; Plasmodium falciparum; Spirometra
erinaceieuropaei; tumor necrosis factor-α

Plasmodium falciparum malaria is the most prominent parasitic infection in humans. It has been
known that the pathogenesis of Plasmodium falciparum malaria depends on many factors, including
the destruction of erythrocytes, drug resistance,
cytoadherence and immunological responses (Clark
et al., 1989; Aikawa et al., 1990). In malaria,
although complement and antibody-mediated im-

mune responses are important (Rigione et al., 1996;
Guevara et al., 1997; Kumaratilake et al., 1997),
cell-mediated immunity is also involved in the protection and pathogenesis of this disease and some
reports suggest that the host immunological responses, especially those mediated through thecytokine cascade, may determine the severity ofmalaria (Artavanis and Riley, 2002). Severe falci-

Abbreviations: dNTPs, deoxyribonucleotide triphosphate; ERK1/2, extracellular-signal regulated protein kinase 1/2;
ES, excretory/secretory; ICAM, intercellular adhesion molecule; IL, interleukin; LPS, lipopolisaccharide; MAPK,
mitogen-activated protein kinase; M-MLV RT, Moloney mouse leukemia virus reverse transcriptase; Pf-CM,
Plasmodium falciparum-cultured medium; PMA, phorbol 12-myristate-13 acetate; RT, reverse transcription; TNF,
tumor necrosis factor

7

P. Dirgahayu et al.

parum malaria is usually accompanied by serious
secondary complication, such as cerebral malaria,
severe anemia, hypoglycemia and renal impairment(Aikawa et al., 1990; Kwiatkowski, 1990). Several
lines of evidences implicate that tumor necrosis factor (TNF)-α is a major mediator for these complications. Indeed, it has been reported there is an elevation in serum levels of not only TNF-α, but also interleukin (IL)-1β and IL-6 in patients with falciparum
malaria (Kwiatkowski et al., 1989, 1990; Kwiatkowski,1990), which are correlated with severity of thedisease and death (Grau et al., 1989; Clark et al.,
1990; Kwiatkowski et al., 1990; Kurtzhals et al.,1998). A study by Udomsangpetch et al. (1997) showed that there were localized high levels of inflammatory cytokines TNF-α, IL-1β and interferon-γ inthe brain tissue of a patient who died from cerebral
malaria, in which an up-regulation of adhesion
molecules such as CD36 or intercellular adhesion
molecule (ICAM)-1 and sequestration of parasitized erythrocytes, lymphocytes and monocytes tothe endothelial cells were associated. Identificationof ICAM-1 as a major sequestration receptor, especially in the brain, has led to the assumption that it
contributes to the pathophysiology of severe malaria (Aikawa et al., 1990). In fact, in the early stagesof malaria, the mild and moderate amount of inflammatory cytokines, such as TNF-α or IL-1β, arebeneficial for the host by suppressing and killing
the parasite (Grau et al., 1989; Clark et al., 1990;
Kwiatkowski, 1990; Kwiatkowski, et al., 1990).
However, in later stages, an excessive production ofthese cytokines are detrimental, since activation ofphagocytes to kill intracellular or extracellularparasites requires a great amount of inflammatory
cytokine production which can cause adverse systemic effects such as severe anemia and cerebral
malaria (Kwiatkowski, 1990; McGuire et al., 1994;
Luty et al., 2000).
The plerocercoids of Spirometra erinaceieuropaei, one species of parasitic tapeworm, has been
known to produce anti-inflammatory like substance(s) which suppressed the nitrite production
and gene expression of inducible nitric oxide synthase (Fukumoto et al., 1997) and the TNF-α inmurine macrophages stimulated with lipopolysaccharide (LPS) (Miura et al., 2001; Dirgahayu et al.,2002). Based on these findings and on what we
now understand about malaria pathogenesis, thepresent study was conducted to investigate whether
excretory/secretory (ES) products from these plerocercoids might suppress TNF-α, IL-1β and ICAM1 mRNA expression of the human monocytic leukemia cell line THP-1 induced by Plasmodium falciparum-cultured medium (Pf-CM). A possible potential role of ES products in malaria pathogenesis
will be discussed.

Materials and Methods

Plerocercoids of Spirometra erinaceieuropaei and their ES products
Plerocercoids of Spirometra erinaceieuropaei werecollected from 2 species of snakes (Elaphe quadrivirgata and Rhabdophis tigrinus) in the southernpart of Ehime Prefecture, Japan and stored for over
6 months in the subcutaneous tissues of golden hamsters, which were housed and maintained according
to the guidelines for proper treatment of animals at
the Division of Laboratory Animal Science, ResearchCenter for Bioscience and Technology, Tottori University, Japan. ES products were obtained as described previously (Miura et al., 2001). The protein
concentration was assayed using a Bio-Rad protein
assay kit (Bio-Rad, Hercules, CA) and adjusted to 50µg/mL. We made separate lots for ES products
from each golden hamster in order to perform several experiments independently. One representative experiment is shown here.

Preparation of Pf-CM
The Pf-CM was obtained from the continuous culture of Plasmodium falciparum FCR-3 strain which
was maintained according to the method of Trager
and Jensen (1976), kindly provided by the Department of Molecular Parasitology, School of Medicine,
Ehime University, Japan. The Pf-CM was obtained

8

TNF-α and ICAM-1 regulation by parasites

from the culture when the parasite reached an initialhematocrit of 5% in RPMI 1640 medium (Sigma
Chemical, St. Louis, MO) supplemented with HEPES
buffer (25 mM) and 0.2% NaHCO3 with 10% heatinactivated human serum (albumax) (Gibco BRL,
Paisley, United Kingdom). The cultured-medium
were then treated as previously described (Ballet etal., 1981; Abdalla and Wickramasinghe, 1985). Briefly, 40 mL of Pf-CM (20% parasitemia) was centrifuged for 3000 rpm for 20 min at 4˚C prior to dialyzation against cold distilled water for 48 h. Afterbeing lyophilized, the sample was then re-suspended in 4 mL RPMI 1640. The Pf-CM was then passed through a 0.45-µm-pore-size membrane filterand stored at –30˚C until use. Thawed media were
filtered again through a 0.45-µm-pore-size membrane filter before being added to culture.

Reverse transcription (RT)-PCR analysis
Total RNA was prepared from Pf-CM-stimulated orcontrol macrophages using an ISOGEN Kit (Nippon
Gene, Tokyo) according to the manufacturer’s protocol. Approximately 1 µg of total RNA was reversetranscribed in a 20 µL reaction volume according to
the manufacturer’s protocol. Approximately 1 µgof total RNA was reverse-transcribed in a 20 µL reaction volume consisting of 2.5 µL random hexamers (50 pM), 4 µL 5× Moloney mouse leukemia
virus reverse transcriptase (M-MLV RT)-reaction
buffer, 2.5 µL deoxyribonucleotide triphosphate(dNTPs) (2.5 mM each) (Nippon Gene, Tokyo), 1 µL
of RNase-inhibitor and 1 µL of M-MLV RT enzyme (all reagents were purchased from Promega,
Tokyo). PCR reactions were run on a Perkin Elmer
DNA Thermal Cycler (Takara, Kyoto, Japan), in 20µL total volume per reaction mixture containing 2µL of cDNA RT products, 1.6 µL dNTPs mix (2.5mM each), 2 µL 10× Gene Taq Universal buffer(Nippon Gene, Tokyo), 1 µL of each primer (diluted
to 20 µM) and 0.1 µL Taq Gold. Primer sequencesused were as follows: TNF-α: (sense: 5'-AAG CCTGTA GCC CAT GTT GT-3' and antisense: 5'-CAG
ATA GAT GGG CTC ATA CC-3'), ICAM-1:(sense: 5'-AAT GCC CAG ACA TCT GTG TC-3'
and antisense: 5'-CAG TTC AGT GCG GCA CGAGAA AT-3'), IL-1β: (sense: 5'-GCT CCT TCC
AGG ACC TGG AC-3' and antisense: 5'-CGTGCA CAT AAG CCT CGT TA-3') and β-actin:(sense: 5'-CCA GAG CAA GAG AGG CAT CC-3'
and antisense: 5'-GTG GTG GTG AAG CTG TAG
CC-3'). The PCR cycles consisted of denaturation
at 94˚C for 30 s, annealing at 60˚C for 1 min andextension at 72˚C for 1 min for 27 cycles (TNF-α),
25 cycles (ICAM-1), 23 cycles (IL-1β) and 21 cycles (β-actin) for semi-quantitative analysis. A 15
µL aliquot of each of the PCR products was separated by electrophoresis on a 1.5% agarose gel,stained with ethidium bromide and photographed.
For quantification, photographs showing PCR products were scanned and analyzed using Densitographversion 4.0 software (ATTO, Tokyo). For each sample, the PCR product values were normalized to theβ-actin PCR product values.

Cell culture
The human monocytic leukemia cell line THP-1(TIB-202) was purchased from RIKEN cell bank
(Tokyo, Japan). THP-1 cells were grown in RPMI
1640 medium supplemented with 10% fetal bovine
serum (Gibco BRL, Gaithersburg, MD) containing
100 U/mL penicillin G (Banyu Pharmaceutical,
Tokyo) and 100 µg/mL streptomycin (Meiji Seika.,
Tokyo), plated in tissue culture dishes (Greiner,
Frickenhausen, Germany), and incubated at 37˚C inan atmosphere of 5% CO2 in a humidified incubator. We differentiated the THP-1 cells by re-suspending them for 24 h in medium containing 200 nM ofphorbol 12-myristate-13-acetate (PMA) (Wako,
Tokyo), as previously described by Reyes et al.
(1999). Such treatment with PMA induces the conversion of THP-1 into mature cells as macrophages(Tsuchiya et al., 1980), designated “THP-1-derived
macrophages”. The macrophages were washedwith phosphate buffered saline and were left overnight before treatment. We used polymyxin B (10µg/mL) (Sigma Chemical) for treatment of ES products for all subsequent experiments prior to stimulation with Pf-CM.

9

P. Dirgahayu et al.

Results

sions. As seen in Fig. 1, Pf-CM induced TNF-α,
IL-1β and ICAM-1 mRNA accumulation in THP-1
cells (lane 4). Although LPS-induced gene expression of TNF-α, IL-1β and ICAM-1 mRNA was reduced in the presence of polymyxin B (lane 3), PfCM-induced expression of these genes was not prevented in the presence of polymyxin B (lane 5). No
appreciable band was observed in controls and hu-

Induction of TNF-α, IL-1β and ICAM-1 mRNA
expressions of THP-1 cells in response to
Pf-CM
We first examined the effect of Pf-CM on the induction of TNF-α, IL-1β and ICAM-1 mRNA expres-

A

LPS
PoB
Lane

–
1

–
2

+
3

Pf-CM
–
4

+
5

Pf-CM

A

HS

ES
Lane

–
6

–
1

–
2

+
3

–
4

HS
5

TNF-α

TNF-α
IL-1β

IL-1β

GAPDH

GAPDH

0

100

B-3

ICAM-1 mRNA

50

0Fig. 1. TNF-α, IL-1β and ICAM-1 gene expressions inhuman monocytes stimulated with LPS or Pf-CM. THP1 monocytes were left unstimulated or stimulated withLPS (100 ng/mL), Pf-CM or heat-inactivated human serum (HS) for 4 h in the absence or presence of polymyxin B (10 µg/mL). Total RNA (1 µg/lane) was obtained and the levels of TNF-α, ICAM-1, IL-1β and βactin mRNA expression were assessed by semiquantitative RT-PCR. One representative of 3 experiments isshown. PoB, polymyxin B.

Relative expression

50

Relative expression

B-2

IL-1β mRNA

100

(%)

Relative expression

0(%)

Relative expression

50

TNF-α mRNA

100

50

0

IL-1β mRNA

100

50

0

(%)

B-1

TNF-α mRNA

(%)

B-3

(%)

B-2

100

(%)

B-1

Relative expression

ICAM-1

Relative expression

ICAM-1

100

ICAM-1 mRNA

50

0Fig. 2. Effect of ES products on the Pf-CM-induced
TNF-α, IL-1β and ICAM-1 gene expression in humanmonocytes. THP-1 monocytes were left untreated or
pretreated with ES products (5 µg/mL) and polymyxin B(10 µg/mL) for 6 h, washed, and then the medium was
replaced before the stimulation with 250 µL of Pf-CM or
human serum (HS) for 4 h. Total RNA (1 µg/lane) was
obtained and the levels of TNF-α, ICAM-1, IL-1β and βactin mRNA was analyzed by semiquantitative RT-PCR.
One representative of 3 experiments is shown.

10

TNF-α and ICAM-1 regulation by parasites

before the addition of 250 µL Pf-CM in the final volume of 3 mL for further 3 h incubation. As shown in
Fig. 3, PD98059 treatment suppressed TNF-α, IL1β and ICAM-1 mRNA expression by 83%, 75%

man serum-treated monocytes in the absence of
LPS or Pf-CM (lanes 1 and 6). These results indicate that Pf-CM has the ability of some stimulatory
activity to induce the gene expression of pro-inflammatory cytokines and ICAM-1, and this activity exhibited in Pf-CM-cultured THP-1-cells is a Plasmodium falciparum origin, without a trace of endotoxin contamination.

A

Pf-CM (3 h)
SB

–

ES
Lane

Treatment of THP-1 cells with ES products
prior to stimulation with Pf-CM prevent induction of TNF-α, IL-1β and ICAM-1 gene expression

–
1

–
2

+
3

–
4

+
5

PD
–
6

+
7

SB
PD
–
8

+
9

TNF-α
IL-1β
ICAM-1

In order to evaluate the impact of ES products on
TNF-α, IL-1β and ICAM-1 gene expressions of
THP-1 cells, these monocytes were left untreated ortreated with 5 µg/mL ES products and polymyxin B
prior to stimulation with 250 µL Pf-CM in the final
medium volume of 3 mL. As shown in Fig. 2, ES
products potently reduced TNF-α mRNA (by 76%),
IL-1β (by 85%) and ICAM-1 mRNA expression (by60%) in response to Pf-CM (lane 3). THP-1 cells
treated with human serum showed a band patternsimilar to the controls (lanes 1 and 5). The levels of
constitutively expressed β-actin mRNA, however,
did not change with the treatment of ES products.
This result shows that ES products are a potent inhibitor for TNF-α, IL-1β and ICAM-1 gene expression in human monocytes in response to Pf-CM.

GAPDH

Using well-known pharmacological inhibitors of
the ERK1/2 and p38 MAPK pathways, PD98059
and SB203580, respectively (Dudley et al., 1995),
we investigated the suppressive mechanisms of ES
products on TNF-α, IL-1β and ICAM-1 gene expressions in human monocytes stimulated with PfCM. THP-1 cells were pretreated with 5 µg/mL ES
products and polymyxin B for 6 h, washed and thenthe medium was replaced. The monocytes werethen treated with SB203580 and/or PD98059 for 1 h

Relative expression
Relative expression
Relative expression

Role of ERK1/2 and p38 MAPK pathways in
the induction of TNF-α, IL-1β and ICAM-1
gene expression in Pf-CM-stimulated THP-1
cells

160

160

TNF-α mRNA

120

80

40
0

IL-1β mRNA
120

80

40
0

(%)

B-3

160

(%)

B-2

(%)

B-1

ICAM-1 mRNA
120

80

40
0

Fig. 3. MAPK inhibitors differentially modulate the PfCM-induced TNF-α, IL-1β and ICAM-1 mRNA expressions in THP-1 monocytes. THP-1 monocytes were pretreated with 5 µg/mL ES products and 10 µg/mL of polymyxin B for 6 h. The cells were then washed and the
medium was replaced. The cells were then either treatedwith PD98059 (50 µM), SB20358, (10 µM) or a combination of these inhibitors for 1 h prior to the stimulation
with 250 µL Pf-CM for an additional 3 h incubation.
Total RNA (1 µg/lane) was obtained and TNF-α, ICAM1 and β-actin mRNA was assessed by semiquantitative
RT-PCR. One representative of 3 experiments is shown.

11

P. Dirgahayu et al.

and 73% (lane 6), respectively; however, SB203580
treatment induced approximately 1.5-fold levels of
the TNF-α, IL-1β mRNA and ICAM-1 mRNA expression (lane 4), compared to the positive controls(lane 2). Combination of PD98059 and SB203580
profoundly reduced the 3 genes to near control levels (lane 8). These data indicate that the ERK1/2
MAPK pathway, but not p38 MAPK is importantfor induction of TNF-α, IL-1β and ICAM-1 mRNA
expression in THP-1 cells stimulated with Pf-CM.
We observed an additional suppressive effect by ES
products on IL-1β, but not TNF-α, and ICAM-1
mRNA expression in the cells treated with PD98059(lane 7).

and induction of TNF-α and IL-1β gene expressionof murine macrophages. In agreement with several
previous studies (Abdalla and Wickramasinghe, 1985;
Taverne et al., 1990a, 1990b), we demonstrated that
Pf-CM induced TNF-α, IL-1β and ICAM-1 mRNA
expression in THP-1 cells, which was not abolishedby the treatment of 10 µg/mL polymyxin B (Fig. 1),
suggesting that the stimulatory activity of Pf-CM
was of parasite origin. We found that pretreatment
of THP-1 monocytes with ES products prior to
stimulation by Pf-CM reduced TNF-α, IL-1β and
ICAM-1 gene expression (Fig. 2). We are confident
that the results are not a consequence of toxicity
since treatment of macrophages by ES products forover 24 h neither inhibited the growth of cell numbers (unpublished data) nor affected the expression
level of the house-keeping gene β-actin.
Further investigation through the MAPK pathway showed that pretreatment of THP-1 cells with
PD98059 partially inhibited TNF-α, IL-1β and
ICAM-1 mRNA expression, whereas SB203580,
instead of inhibition, induced all gene expressions
approximately 1.5 times compared to the positive
controls (Fig. 3) and a combination of PD98059 andSB203580 profoundly reduced TNF-α, IL-1β and
ICAM-1 mRNA expression to near control levels,
indicating that the ERK1/2 pathway is important for
optimal induction of TNF-α, IL-1β and ICAM-1
mRNA expression. We observed that ES products
additionally suppress IL-1β mRNA expression in
LPS-stimulated THP-1 cells treated with PD98059,
suggesting that ES products may suppress IL-1βmRNA by inhibiting phosphorylation of the ERK1/2
pathway. The reason for this additional suppressive
effect only observed for IL-1β mRNA expression isstill unclear.
Paroxysmal fever is the hallmark of malaria.
Several observations indicate that TNF-α is a critical mediator of malarial fever: i) it is a potent endogenous pyrogen (Dinarello et al., 1986); ii) when P.
falciparum is cultured with human monocytes, therate of TNF production rises sharply at the time ofschizont rupture (Kwiatkowski, 1990; Kwiatkowski etal., 1990); iii) in P. vivax infection, paroxysmal fever
is associated with a sharp rise in circulating TNF
levels (Karunaweera et al., 1992) and iv) anti-TNF

Discussion
A complex parasite such as human Plasmodium islikely to generate a variety of substances that injure
the hosts directly or cause immunopathology. It has
been widely known that severe malaria is associated with a failure of the host defense system to control parasite replication, excessive secretion of proinflammatory cytokines and sequestration of parasitized-erythrocytes in vital organs, such as the kidney, the lungs, the brain, etc. The excessive secretion of pro-inflammatory cytokines, such as TNF-α,
IL-1β and IL-6 (Aikawa et al., 1990) has been assumed because of the stimulation of effector cells bycertain parasite-derived toxins [substance(s)], released by ruptured schizonts that cause paroxysmsof fever. This substance(s) has been shown to contain glycosylphosphatidylinositol (Schofield andHacket, 1993; Schofield et al., 1996; Tachado et al.,1996) and malarial pigments (Pichyangkul et al.,1994). Several studies have shown that glycosylphosphatidylinositol anchor, a membrane attachment mechanism for cell surface proteins widelyused in eukaryotes, plays a crucial role in the pathology of malaria by inducing pro-inflammatory
cytokines, including iNOS (Schofield et al., 1996;
Tachado et al., 1996), TNF-α (Schofield et al., 1993)
and ICAM-1 (Schofield et al., 1996). Pichyangkul etal. (1994) demonstrated that sequestered malaria pigment in the microvasculature also induced production
12

TNF-α and ICAM-1 regulation by parasites

therapy inhibits fever in children with cerebral malaria (Kwiatkowski et al., 1993). However, TNF-α
is not the only mediator of malaria fever. Other
cytokines also have pyrogenic properties, including
IL-1β (Rocket et al., 1994), IL-1α (Kwiatkowski etal., 1990) and IL-6 (Kern et al., 1989), which areproduced mainly by the monocytes/macrophages
series. In addition to the pyrogenic effect, TNF-α,
has been shown to induce ICAM-1 gene expressionof the endothelial cell, the major adherence receptor
in the brain (Aikawa et al., 1990; Udomsangpetch etal., 1997). A study by Udomsangpetch et al. (2002)
demonstrated that febrile temperatures in the range
usually encountered during malaria induced cytoadherence of the ring-stage of Plasmodium falciparum-infected erythrocytes in vitro. This feverinduced cytoadherence was shown to be associatedwith increased expression of the Plasmodium falciparum-erythrocyte membrane protein-1, a protein
which is exported from the parasite and linked tothe erythrocyte cytoskeleton during the 2nd half ofthe life cycle, on the surface of the infected red
blood cell (Sharma, 1991). Up-regulating ICAM-1
expression of the endothelial cells allows parasites
to bind and sequester in cerebral vessels, disrupting
the function of the central nervous system (Adams
et al., 2000), and in turn, leading to cerebral malaria. Based on the understanding of the pathogenesisof malaria, we suppose that suppression of TNF-α
and IL-1β mRNA expression in THP-1 cells by ES
products would, in turn, reduce the expression of
ICAM-1 of endothelial cells by decreasing fever indirectly. In addition, ES products might also directly
suppress ICAM-1 expression in monocytes.
In epidemic areas, helminthic infections commonly coexist with malaria in an individual. The
significance of such concurrent infection, however,is not yet clearly understood. However, results
have revealed that worm infection is associatedwith protection from cerebral malaria (Nacher etal., 2000), acute renal failure and jaundice (Nacher
et al., 2001). Thus, worms might be central to natural anti-malarial immunity. Worms, which can induce a bias towards Th-2 cytokines and depress
other cytokines could render the host more susceptible to liver-stage parasites and favor P. falciparum

reproduction but protect the host from severe complication of blood stage malaria. The beneficial or
detrimental role of TNF-α, IL-1β and ICAM-1 during malaria attack could have important consequences, and modulation of these mediators during
acute disease could lead to a novel approach in thetreatment of malaria. Although co-infection of malaria and Spirometra erinaceieuropaei is unusual,the evidence presented here points to the possibility
that ES products might be useful in reducing inflammatory responses and the phenomenon of sequestration in severe malaria, especially in cerebral
malaria.
Acknowledgments: We thank Professor Motomi Torii
and Dr. Osamu Kaneko, the Department of Molecular
Parasitology, School of Medicine Ehime University, Japan, for kindly providing Plasmodium falciparum-cultured medium.
This work was supported by a Grant-in-Aid for Scientific Research (numbers 12670231, 09670258 and13670246) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
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Received January 5, 2004; accepted January 28, 2004
Corresponding author: Kazumitsu Hirai, MD, PhD

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