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Blood First Edition
Paper,
prepublished
12,
2002;
DOI
10.1182/blood-2002-10-3105
Aberrant development of Plasmodium falciparum in hemoglobin CC red cells:
implications for the malaria protective effect of the homozygous state
Rick M. Fairhurst*, Hisashi Fujioka#, Karen Hayton*, Kathleen F. Collins*, and Thomas
E. Wellems*^
Scientific Heading: Clinical Observations, Interventions, and Therapeutic Trials
Running Title: Plasmodium falciparum development in CC red cells
* Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892 and #Institute of Pathology,
Case Western Reserve University, Cleveland, OH 44106, USA.
Word counts: Abstract (244), Text (3,231)
H. Fujioka acknowledges support by the United States Agency for International
Development (#DPE-936-6001-29) and a grant from Merck Research Laboratories.
^Corresponding author:
Thomas E. Wellems, M.D., Ph.D.
Laboratory of Malaria and Vector Research
NIAID/National Institutes of Health
Building 4, Room 126
4 Center Drive
Bethesda, MD 20892-0425
PHONE: 301-496-4021
FAX: 301-402-0079
EMAIL: [email protected]
Fairhurst et al., 12/2/2002
Copyright (c) 2002 American Society of Hematology
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Abstract
Although selection of hemoglobin C (HbC) by malaria has been speculated for decades, only
recently have epidemiological studies provided support for HbC protection against malaria in
West Africa. A reduced risk of malaria associated with the homozygous CC state has been
attributed to the inability of CC cells to support parasite multiplication in vitro. However,
there have been conflicting data and conclusions regarding the ability of CC red cells to
support parasite replication. Reports that parasites cannot multiply in CC cells in vitro
contrast with detection of substantial parasite densities in CC malaria patients. We have
therefore investigated P. falciparum growth in CC cells in vitro. Our data show that the
multiplication rate of several P. falciparum lines is measurable in CC cells, but lower than
that in AA (HbA-normal) cells. A high proportion of ring forms and trophozoites
disintegrates within a subset of CC cells, an observation that accounts for the overall lower
replication rate. In addition, knobs present on the surface of infected CC cells are fewer in
number and morphologically aberrant when compared with those on AA cells. Events in
malaria pathogenesis that involve remodeling of the erythrocyte surface and the display of
parasite antigens may be affected by these knob abnormalities. Our data suggest that only a
subset of CC cells supports normal parasite replication and that components of malaria
protection associated with the CC state may affect the parasite’s replication capacity and
involve aberrant knob formation on CC cells.
Fairhurst et al., 12/2/2002
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Introduction
The geographic coincidence of hemoglobin (Hb) C ( 6 Glu
Lys) and Plasmodium
falciparum malaria in West Africa has been cited as evidence that this mutant allele was
evolutionarily selected for its protection against life-threatening disease1. Early studies found
that parasite prevalence and density among healthy AA (HbA-normal) and AC (HbC-trait)
subjects were equivalent2-5. Subsequent work failed to associate HbC with malaria
protection6,7 although one study demonstrated increased survival of AC subjects5. Recently,
however, two epidemiological studies performed in Sahelian regions of West Africa
demonstrated the malaria protective effect of HbC8,9. The first study, in the Dogon of Mali,
showed that AC was associated with an ~80% reduced risk of severe malaria8. This finding
and the presentation of seven CC subjects with non-severe malaria suggested that the
homozygous CC state also protected against severe disease. The second study, in the Mossi
of Burkina Faso, found that AC and CC were associated with ~30% and ~90% reduced risks
of malaria (both non-severe and severe)9.
Attempts at defining the mechanism by which HbC protects against malaria have relied on in
vitro culture of P. falciparum in HbC-containing red cells. Although parasites replicate in
AA and AC cells equally well in vitro (Ref. 10; Fairhurst, unpublished observations), their
replication in CC cells is relatively inhibited. Some investigators found P. falciparum (strain
FCR-3/FMG) unable to replicate in CC cells10,11, whereas others achieved limited replication
of the same parasite line12,13. Pasvol and Wilson, for example, noted 50% growth inhibition
but normal parasite development in CC cells12. Dei-Cas et al. likewise reported 31-57%
growth inhibition in CC cells13. Furthermore, these investigators observed that the
Fairhurst et al., 12/2/2002
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ultrastructure of P. falciparum in CC cells was normal. The mechanism of growth inhibition
has only been addressed in one study, which found that infected CC cells were more resistant
to osmotic lysis and proposed a mechanism whereby schizont-infected CC cells were unable
to burst and release merozoites11.
CC individuals have mild chronic hemolytic anemia with frequent splenomegaly and
abnormal blood smears containing microspherocytes and target cells14. A pathogenetic
correlate of these findings is the increased mean corpuscular hemoglobin concentration
(MCHC) of CC cells, which leads to intracellular crystallization of HbC15-17. Although AC
individuals do not show these changes, both AC and CC cells are significantly more rigid
than their AA counterparts15,18. Because AC and CC cells can support high parasitemias in
vivo8, the malaria protective mechanism of HbC may involve elements of both perturbed red
cell physiology and non-sterile immunity.
Here we describe studies of P. falciparum development in CC cells. In contrast to previous
work that reported no in vitro propagation10,11, we have observed multiplication of several P.
falciparum lines in CC cells. We also show that P. falciparum lines develop normally in one
subset of CC cells while undergoing a lethal degradative process in another. Electron
microscopic images suggest that the infected red cell surface may be remodeled differently in
AA and CC cells. We discuss the significance of these findings and their potential
contribution to the malaria protective effects of HbC.
Fairhurst et al., 12/2/2002
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Methods
CC red cells. Heparinized blood from two unrelated CC donors and a series of AA donors
was washed three times with RPMI-1640 and the red cells were stored at 50% hematocrit
in RPMI-1640 for up to 3 weeks at 4oC prior to use. Typically, CC red cells were used in
culture experiments within several hours of the blood draw, whereas AA cells were stored
at 4oC for 12-36 hours prior to use. Hb genotypes were determined by cellulose acetate and
citrate agar electrophoresis and confirmed by HPLC analysis. CC red cells tested negative
for G6PD deficiency as determined by commercially available reagents (Sigma, St. Louis,
MO). Blood collection was performed under protocols approved by the Institutional
Review Board of the National Institute of Allergy and Infectious Diseases.
In vitro parasite culture. Established P. falciparumlines (7G8, FMG/FCR -3, TM284,
GB4, ITG, 3D7, Indochina, and FCB) were maintained in O+ human red cells collected
from healthy blood bank donors. Parasites were cultured in settled red cells at 5%
hematocrit in complete medium (RPMI-1640 supplemented with 25 mg/mL HEPES, 2
mg/mL sodium bicarbonate, 50 µg/mL gentamicin, 10% v/v heat-inactivated human O+
serum). In some experiments, Albumax II (0.5% w/v; Gibco-BRL, Grand Island, NY) was
used instead of human serum. The GB4 parasite line was cloned from the Ghana III line, a
kind gift from W. E. Collins, Centers for Disease Control and Prevention, Atlanta, GA. All
other parasite lines are available from The Malaria Research Reference Reagent Resource
(MR4; Manassas, VA).
P. falciparum growth experiments. Parasite lines cultured in AA cells were enriched to 95-
Fairhurst et al., 12/2/2002
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99% mature forms by the Percoll-sorbitol method or to approximately 50% by gelatin
flotation19. Infected AA cells were then added to uninfected AA or CC cells in culture so
that the initial parasitemia was 0.1%. Parasites were maintained at 37oC in an atmosphere
of 5% CO2, 5% O2, and 90% N2. To test the effect of oxygen, parasites were maintained in
5% CO2 in a standard tissue culture incubator (20% O2 atmosphere). Culture media were
changed daily and parasite counts were determined by examination of Giemsa-stained
blood smears. Parasitemias (mean ± SD) were calculated from triplicate counts of 1000 red
cells. In all experiments, AA and CC cells were inoculated with the same parasite stock
and cultures were performed simultaneously.
Electron microscopy. After 4 days of culture in either AA or CC cells, parasites were
processed for electron microscopy. Infected red cells were fixed with 2.5% glutaraldehyde
in 0.05 M phosphate buffer, pH 7.4, containing 4% sucrose for 2 h and then post-fixed in
1% osmium tetroxide for 1 h. After a 30 min en bloc stain with 1% aqueous uranyl acetate,
the cells were dehydrated in ascending concentrations of ethanol and embedded in Epon
812. Ultrathin sections were stained with 2% uranyl acetate in 50% methanol and lead
citrate20 and then examined in a Zeiss CEM902 electron microscope (Oberkochen,
Germany).
Immunoblotting. Parasite line FMG/FCR-3 was cultured simultaneously in either AA or
CC cells to 1.5% parasitemia. Infected red cells were lysed in 0.01% saponin in RPMI1640 at 4oC for 10 min. Erythrocyte membrane and parasite proteins were resolved by
SDS-PAGE (4-12% gradient), transferred to polyvinylidine fluoride membranes
Fairhurst et al., 12/2/2002
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(Immobilon P; Millipore, Bedford, MA) and probed with either mAb89 (1:1250 ascites;
Ref. 21) specific for knob-associated histidine rich protein (KAHRP) or affinity-purified
rabbit antiserum (1:1250; Ref. 22) specific for P. falciparum chloroquine resistance
transporter (PfCRT). Bound antibodies were detected using HRP-conjugated secondary
antibodies and a chemiluminescence detection system (Pierce, Rockford, IL). The relative
amounts of KAHRP and PfCRT from AA and CC cell cultures were measured
densitometrically and analyzed using Scion Image software (Scion Corp., Frederick, MD).
Fairhurst et al., 12/2/2002
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Results
Different P. falciparum multiplication rates in AA and CC cells. Under standard growth
conditions, in vitro propagation of several parasite lines was readily achieved in CC cells.
Multiplication rates were lower in CC than in AA cells (Fig. 1). In a set of five
representative growth experiments, the ratio of mean parasite densities in AA versus CC cells
after 4 days (2 cycles) of culture was ~3:1. These observations were confirmed repeatedly
using eight different parasite lines (7G8, FMG, TM284, GB4, ITG, 3D7, Indochina, and
FCB).
Parasites cultured in vitro eventually reach a limiting parasitemia, become unhealthy and die.
The cause for this phenomenon is unclear, but may be related to the sensitivity of the
parasites to the effects of media depletion or metabolic product accumulation in culture. The
density at which this process occurs in AA cells can vary with parasite lines and
experimental conditions but in our system is usually 8-12% parasitemia. To determine the
upper parasitemia limit in CC cells, we continued to observe cultures after 4 days. On days 5
and 6 of such experiments, parasites in CC cells reached maximum densities of 4-5% and
then began to die (“crash”) (Fig. 2). We were unable to culture parasites in CC cells at
densities greater than 6%. These findings were confirmed for five parasite lines (3D7,
Indochina, GB4, 7G8, and TM284). Dying parasites in CC cultures were characterized by
condensed cytoplasm and pyknotic nuclei. In AA cells, the morphological appearance of
dying parasites at the 8-12% limit was similar.
Fairhurst et al., 12/2/2002
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We were able to establish long-term cultures in CC cells as long as parasite densities were
below crash levels (2-3%). These cultures were maintained by the daily addition of fresh
media and the twice-weekly addition of CC red cells. Using this approach, we were able to
maintain viable cultures of P. falciparum (3D7 and Indochina) continuously beyond 3 weeks.
Frequent spontaneous degradation of P. falciparum in subpopulations of CC cells underlies
the lower parasite multiplication rate. To explore the basis for lower parasite multiplication
rates in CC cells, we examined the ability of five parasite lines (3D7, Indochina, GB4, 7G8,
and FCB) to invade and develop in CC cells. Trophozoite- and schizont-infected AA cells
were separated in Percoll-sorbitol gradients (95-99% parasitemia) and then added to either
CC or AA cells. After allowing 18 hours for schizont rupture and merozoite invasion, we
compared the number of ring forms in CC and AA cells. Parasite invasion of CC cells was
0.93 ± 0.31 that of AA cells (17 invasion assays, Table 1). Based on these data, reduced
invasion of CC cells could not account for the dramatic reduction in parasite multiplication
rates.
Microscopic examination of maturing parasites, however, provided a possible explanation for
lower multiplication rates in CC cells. The appearance of parasite clone GB4 in AA and CC
cells was representative of eight parasite lines tested. Blood smears routinely showed
approximately 50% healthy forms in CC cells, while the remaining 50% appeared unhealthy
(Fig. 3). Fragmentation of late rings and blurring of trophozoites and schizonts, suggesting
the loss of compartmentalized boundaries, characterized the unhealthy forms.
Approximately half of the parasite population showed evidence of deterioration with each
Fairhurst et al., 12/2/2002
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new replication cycle, even when parasites were maintained continuously in CC cells for 3
weeks. By light microscopy, there were no obvious differences between CC cells that did
and did not support healthy parasite development.
The observation that 50% of parasites showed evidence of deterioration in each replication
cycle was consistent with lower parasite multiplication rates in CC cells. We observed
deteriorating parasites at both the ring and trophozoite stages of maturation. There was no
accumulation of particular forms, indicating that P. falciparum development in CC cells was
not blocked at a specific stage of the asexual life cycle. The healthy parasite forms appeared
completely normal in their maturation through the schizont stage and production of new ringinfected red cells.
Parasite multiplication rates in AA and CC cells do not differ significantly in 5% and 20%
oxygen atmospheres. Hb aggregates and crystals have been reported to form in CC but not
AA cells treated with hypertonic saline, a process facilitated by the relatively high MCHC of
CC cells15-17. Decreased solubility of HbC that occurs upon deoxygenation may contribute to
this aggregation16,23. To explore whether the presence of increased amounts of oxygen in the
culture atmosphere would enhance parasite multiplication rates in CC cells, we repeated
growth experiments with parasites able to grow in gas atmospheres containing 20% oxygen.
No significant difference was found in the ability of these parasite lines to multiply in CC
cells in 5% or 20% oxygen (Fig. 4).
Loss of subcellular compartmentalization accompanies parasite death in CC cells. To
Fairhurst et al., 12/2/2002
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further characterize the process of parasite deterioration in CC cells, we compared the
morphological detail of infected CC and AA red cells by electron microscopy. In agreement
with light microscopy observations, approximately half of the parasites appeared to be
following a healthy path from rings (Fig. 5A) to schizonts (Fig. 5B), whereas the remaining
parasites appeared to be disintegrating within intact red cells. This disintegration was
characterized by vacuolization, parasite membrane dissolution, and organelle destruction
(Figs. 5C-E). Disintegrated parasites were found in ring stages as well as more mature
trophozoite forms. Table 2 summarizes the results of 1,795 observations made from seven
different P. falciparum lines in CC cells and AA cells. The average proportion of
disintegrating parasites in CC cells was 46.8 ± 7.3% (mean ± SD, N = 1,163) whereas only
0.57 ± 0.73% (N = 632) of parasites were found disintegrating in AA cells. The fraction
(range, 36-54%) and appearance of these disintegrating forms were similar in CC cells
obtained from two unrelated donors.
The quantity and morphology of knobs differ between parasite-infected AA and CC cells.
Our observations by electron microscopy also detected differences in the electron dense
protrusions (“knobs”) on the surface of P. falciparum-infected red cells. These knobs
mediate cellular interactions important in the cytoadherence and sequestration of parasites24.
Our counts indicated statistically fewer knobs on CC versus AA cells for each of five knobpositive parasite lines (Fig. 6, Table 2). Averaged over cross sections of similar size, the
number of knobs on AA cells was 22.6 ± 9.2 (mean ± SD, N = 36) and on CC cells was 8.2 ±
5.3 (N = 41) (Mann-Whitney test, P < 0.0001). Knobs on CC cells from both unrelated
donors appeared to be larger and more protuberant than those on AA cells (Fig. 6).
Fairhurst et al., 12/2/2002
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The amounts of a knob-associated protein in AA and CC cells do not differ significantly.
To test whether altered knob morphology in CC cells was associated with reduced
expression of knob-associated parasite proteins, we performed semiquantitative
immunoblot analyses. PfCRT is a protein localized to the parasite digestive vacuolar
membrane and is not present in knobs. To control for loading variations, the amounts of
protein extracts were adjusted so that the PfCRT signal in CC versus AA cells was
approximately 1.0 (Fig. 7). These normalized loadings were then analyzed for amount of
KAHRP, a knob-associated parasite protein. Data from five separate immunoblots showed
that the relative amounts of KAHRP in CC versus AA cells were not significantly different
(Fig. 7; optical density ratio 1.05 ± 0.39).
Fairhurst et al., 12/2/2002
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Discussion
Multiple factors influence the occasional progression of malaria into severe and fatal
complications25. High P. falciparum densities are associated with severe malaria and the
rapidity with which parasites multiply in the bloodstream is also thought to increase the risk
of progression to severe disease26,27. Chotivanich et al. have shown that P. falciparum
isolates causing severe malaria were less selective in their invasion of new red cells and
therefore multiplied at higher rates than those causing uncomplicated disease28. Reduced
parasite invasion of HbAE red cells has also been reported to decrease the parasite burdens
associated with severe malaria,29 which may mediate protection conferred by HbE30. The
reduced multiplication rate of P. falciparum in CC cells may be responsible for a comparable
mechanism of protection against severe malaria, but by an alternative mechanism that affects
the maturation and viability of parasites in red cells.
Parasite multiplication rates are determined by the efficiency of red cell invasion, the ability
of parasites to complete their normal development in the red cell, and the successful release
of mature merozoites from the red cell. Various host cell conditions may affect some of
these processes31-37. Our evidence from this study suggests that abnormal intraerythrocytic
development affects parasite multiplication in CC cells. Parasites invaded AA and CC cells
to a similar extent and we did not observe an accumulation of schizonts that would have
indicated a block in merozoite release. Instead, a substantial number of parasites in CC cells
disintegrated within intact red cells. Parasite death was found at all stages of maturation and
was independent of parasite line or CC cell donor. These findings accounted for the lower
rates of parasite multiplication in CC cells versus AA cells.
Fairhurst et al., 12/2/2002
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To explore the mechanism of death, we examined parasites in CC cells by electron
microscopy. While approximately half of parasites in CC cells showed normal
morphology, the remaining half showed evidence of disintegration within intact CC red
cells. These findings were in agreement with those from light microscopy. Images were
suggestive of widespread damage to parasite membranes. Since healthy and dying
parasites were observed alongside one another in CC cell cultures, it seems unlikely that
the disintegration process was due directly to the HbC molecule itself. Instead, the health
of intraerythrocytic parasites may have been affected by different conditions within subsets
of CC cells.
HbC is less soluble than HbA, especially when deoxygenated16,23, and spontaneous
crystallization is observed in vivo as well as in vitro when CC cells are osmotically
dehydrated15-17. Intracellular aggregation of HbC occurs on deoxygenation even when no
crystal formation is detected by morphological methods16. We therefore conducted growth
experiments in atmospheres containing 20% (atmospheric) and 5% oxygen (with parasites
able to grow under these conditions) to determine whether there was a relative growth
inhibition in 5% oxygen. This increase of oxygen concentration, however, failed to
improve the growth of parasites in CC cells.
In vivo, subsets of CC cells exist that differ in age, MCHC, mean corpuscular volume,
viscosity, and membrane filterability15. These subsets include predominantly target cells
and microspherocytes, in part because cells containing intracellular crystals are removed
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from the spleen15-17. CC cells that have a higher MCHC than AA cells have increased
rigidity and consequent hemolytic damage in the microvasculature15,16. A “precrystalline”
state of intracellular HbC, in which crystallization does not occur even though the MCHC
exceeds the solubility of HbC in hemolysates15, may also contribute to increased rigidity of
CC cells. The subsets of CC cells that we have observed not to support parasite
development in vitro may include fractions that have sustained damage from these effects.
In addition to contributing to lower parasite multiplication rates, the effects of the CC cell
on its remodeling by the intraerythrocytic parasite may contribute to malaria protection.
These effects may include altered structure and function of the knobs created by the
parasite at the red cell surface. Our results with five different parasite lines showed that
knobs were fewer in number and more protuberant on CC than on AA cells. The relative
amounts of KAHRP, a principal knob-associated protein, were found not to differ
significantly between AA and CC cells, consistent with larger deposits over fewer knobs in
CC cells. The antigenically variant cytoadherence molecule PfEMP-1 is a principal target
for agglutinating antibodies that correlate with malaria protection38-40. Display of this
molecule (which is anchored by KAHRP) is likely to be affected by alterations of knobs,
with possible impacts on cytoadherence functions and immune responses that have
important roles in the pathogenesis of severe malaria (reviewed in 41).
These results provide possible explanations for previous in vivo and in vitro observations that
have been difficult to reconcile. Although investigators were initially unable to culture P.
falciparum (line FCR-3/FMG) in CC cells10,11, other investigators were able to achieve
Fairhurst et al., 12/2/2002
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limited growth12,13. Our observations with several lines of P. falciparum suggest these
different findings may be at least partly due to the fact that parasites replicate in some subsets
of CC cells but not in others. The relative proportions of CC cell subsets in an individual
may reduce parasite multiplication in vivo to varying degrees and may help to explain why
some CC individuals have been found with high parasitemias8. Other factors involved in
malaria protection may include altered knob formation at the surface of host CC cells, with
consequent effects on surface antigen display and cytoadherence. Disease outcome
accordingly will depend on the relative contribution of the factors that determine parasite
multiplication rate, intrinsic parasite virulence and host immunity in different CC individuals.
Acknowledgements
The authors wish to thank our CC blood donors and Dr. James F. Casella, Natalie Murray,
and Melissa Law for their efforts in support of this work.
Fairhurst et al., 12/2/2002
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29. Chotivanich K, Udomsangpetch R, Pattanapanyasat K, Chierakul W, Simpson J,
Looareesuwan S, White N. Hemoglobin E: a balanced polymorphism protective against
high parasitemias and thus severe P falciparum malaria. Blood 2002;100:1172-1176.
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VR. Influence of hemoglobin E trait on the severity of falciparum malaria. J Infect Dis.
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of the growth of Plasmodium falciparum in HbEE erythrocytes. J Clin Invest. 1981;68:303305.
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Plasmodium falciparum in red cells containing haemoglobin E, a role for oxidative stress,
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34. Bunyaratvej A, Butthep P, Kaewkettong P, Yuthavong Y. Malaria protection in
hereditary ovalocytosis: relation to red cell deformability, red cell parameters and degree of
ovalocytosis. Southeast Asian J Trop Med Public Health. 1997;28 Suppl 3:38-42.
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malaria. Lancet. 1976;1:1269-1272.
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Nagel RL. Transgenic mice expressing human fetal globin are protected from malaria by a
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adherence receptor on the surface of parasitized human erythrocytes. Cell. 1995;82:77-87.
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against infection. Trans R Soc Trop Med Hyg. 1989;83:293-303.
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2002;415:673-679.
Fairhurst et al., 12/2/2002
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Pf Line
3D7
3D7
3D7
3D7
3D7
3D7
3D7
3D7
Indochina
Indochina
GB4
GB4
GB4
7G8
7G8
7G8
FCB
No. ring-infected
RBCs/1000 RBCs
AA
CC
19
24
14
22
8
5
10
9
19
10
10
11
17
12
30
32
44
24
10
8
10
7
12
14
8
6
8
8
4
6
8
4
24
26
Average ± SD
Ratio
CC/AA
1.26
1.57
0.63
0.90
0.53
1.10
0.70
1.07
0.54
0.80
0.70
1.17
0.75
1.00
1.50
0.50
1.08
0.93 ± 0.31
Table 1. P. falciparum invasion of AA and CC cells. Trophozoite- and schizont-infected
AA cells were added simultaneously to AA or CC cells. After allowing 18 hours for
schizont rupture, merozoite invasion of new red cells and maturation to the ring stage,
blood smears were examined. The number of ring-infected red cells per 1000 red cells in
duplicate samples was counted and averaged. The ratio of ring-infected CC to AA cells is
shown.
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No. degraded forms (%)
No. knobs per infected red cell
(mean ± SD)
AA
CC
Parasite line
AA
CC
3D7
0/98 (0%)
52/99 (52%)
ND
ND
Indochina
1/64 (1.6%)
48/100 (48%)
ND
ND
GB4
1/72 (1.4%)
63/112 (56%)
ND
ND
ITG
0/78 (0%)
46/108 (43%)
14.9 ± 2.7
5.1 ± 4.0
7G8
1/105 (1.0%)
97/206 (47%)
14.4 ± 7.1
3.9 ± 2.1
TM284
ND
74/195 (38%)
ND
7.4 ± 6.2
FMG
0/105 (0%)
53/145 (36%)
28.8 ± 9.2
7.2 ± 2.9
GB4
0/110 (0%)
108/198 (54%)
32.2 ± 8.7
17.4 ± 7.6
Average
0.57 ± 0.73
46.8 ± 7.3
22.6 ± 9.2
8.2 ± 5.3
Table 2. P. falciparum degradation in CC cells. Parasite lines were cultured
simultaneously in either AA or CC red cells (2 donors) for 4 days. The number of healthy
and degraded forms (rings, trophozoites) was then determined by electron microscopy. For
each parasite culture in AA or CC cells, the number of knobs present on cross sections of
nine trophozoite-infected red cells was counted. ND, not determined.
Fairhurst et al., 12/2/2002
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Figure 1
No. infected RBCs/1000
RBCs
7G8
FMG
50
AA
CC
40
50
50
40
40
30
30
30
20
20
20
10
10
10
0
0
0
1
2
3
4
0
0
1
2
3
4
0
1
2
Days
Days
GB4
No. infected RBCs/1000
RBCs
TM284
60
3
4
Days
ITG
30
20
60
50
50
40
40
30
30
20
10
20
10
10
0
0
0
0
1
2
3
4
Days
0
1
2
3
4
AA
CC
Days
Figure 1. Growth of P. falciparum in AA and CC cells. Parasite lines were cultured for 4
days (2 cycles) in either AA or CC cells. Initial parasitemias were 0.1%. Parasite densities
were counted after 3 and 4 days of culture. Mean parasitemias achieved by this set of five
parasite lines after 4 days of culture in either AA or CC cells are compared in the bar graph.
Fairhurst et al., 12/2/2002
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Figure 2
crash
No. of infected
RBCs/1000 RBCs
60
GB4
50
TM284
40
7G8
30
20
10
0
0
1
2
3
4
5
6
7
8
9
10
Days
Figure 2. Growth of P. falciparum in CC cells. Parasite lines were cultured for 4-6 days
(2-3 cycles) in CC cells. Initial parasitemias were 0.1%. Parasite densities were counted
daily. At 5% parasitemia, all cultures “crashed” as evidenced by a large number of parasite
forms with condensed cytoplasm and pyknotic nuclei.
Fairhurst et al., 12/2/2002
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Figure 3. Morphological appearance of P. falciparum in CC and AA cells. Parasite clone
GB4 was cultured for 4 days in either AA or CC cells, stained with Giemsa, and observed
by light microscopy (1000x). Although healthy forms (asterisks) were noted in CC cells,
typically half of all parasites appeared to be unhealthy, as indicated by fragmentation of
ring forms and blurring of trophozoites and schizonts (top panels, arrows). The healthy
condition of parasites in AA cells is shown for comparison (bottom panels).
Fairhurst et al., 12/2/2002
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Figure 4
FMG
No. infected RBCs/1000
RBCs
7G8
50
50
40
40
30
30
20
20
10
10
0
0
0
1
2
3
4
40
30
20
10
0
5
1
2
3
4
5
0
0
1
2
3
4
5
6
ITG
GB4
No. infected RBCs/1000
RBCs
TM284
50
50
50
40
40
30
30
20
20
10
10
40
30
20
10
0
0
0
1
2
3
4
5
6
0
0
1
2
3
4
5
5% O2
20% O2
Days
Figure 4. Growth of P. falciparum in oxygenated CC cells. Parasite lines were cultured
for 4 days (2 cycles) in CC cells under atmospheres of either 5% (filled squares) or 20% O2
(open squares). Initial parasitemias were 0.1%. Parasite densities were counted daily.
Mean parasitemias achieved by this set of five parasite lines after 4 days in culture in
different O2 atmospheres are compared in the bar graph.
Fairhurst et al., 12/2/2002
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Figure 5. Ultrastructure of P. falciparum in CC cells. Parasite clone GB4 was cultured
for 4 days in CC cells and then observed by electron microscopy. Healthy forms (ring,
panel A; schizont, panel B) and degraded forms (Panels C-E) are shown for comparison.
Parasite death is associated with disintegration of parasite membranes, organelles and
nuclei, as well as the absence of knobs. Bars represent 1 µm.
Fairhurst et al., 12/2/2002
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Figure 6. Electron micrographs of P. falciparum knobs in AA and CC cells. Parasites
were cultured for 4 days in AA or CC cells and trophozoite-infected cells observed by
electron microscopy. Knobs (arrows) were fewer in number and more protuberant on CC
than on AA cells. Bar represents 1 µm.
Fairhurst et al., 12/2/2002
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Figure 7
Optical Density
1.5
1.2
0.9
0.6
0.3
0.0
PfCRT
KAHRP
Figure 7. Relative amounts of parasite proteins in CC and AA cells. The amounts of
PfCRT (a parasite protein confined to the digestive vacuolar membrane) and KAHRP (a
parasite protein located within knobs on the erythrocyte membrane) were estimated by
immunoblot analysis and densitometry. After normalization of PfCRT signal in CC and
AA cells to approximately 1.0 (1.11 ± 0.31, mean ± s.d.), the relative amounts of KAHRP
were determined to be 1.05 ± 0.39. The data were calculated from five separate
immunoblots.
Fairhurst et al., 12/2/2002
32
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Prepublished online December 12, 2002;
doi:10.1182/blood-2002-10-3105
Aberrant development of Plasmodium falciparum in hemoglobin CC red
cells: implications for the malaria protective effect of the homozygous state
Rick M Fairhurst, Hisashi Fujioka, Karen Hayton, Kathleen F Collins and Thomas E Wellems
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