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THE ABO POLYMORPHISM AND PLASMODIUM FALCIPARUM MALARIA
by
Kayla Wolofsky
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Institute of Medical Sciences, Department of Medicine University of Toronto
© Copyright by Kayla Wolofsky 2009
ii
The ABO polymorphism and Plasmodium falciparum malaria
Master’s of Science, 2009 Kayla Wolofsky
Institute of Medical Science University of Toronto
Abstract
Malaria has exerted a major selective pressure for red blood cell (RBC) polymorphisms that
confer protection to severe disease. There is a predominance of blood type O in malaria endemic
regions, and several lines of evidence suggest that the outcome of Plasmodium falciparum
infection may be influenced by ABO blood type antigens. Based on observations that enhanced
phagocytosis of infected polymorphic RBCs is associated with protection to malaria in other
RBC disorders, we hypothesized that infected type O RBCs may be more efficiently cleared by
the innate immune system than infected type A and B RBCs. The present work demonstrates
human macrophages in vitro and murine monocytes in vivo phagocytosed P. falciparum infected
O RBCs more avidly than infected A and B RBCs independent of macrophage donor blood type.
This difference in clearance may confer relative resistance to severe malaria in individuals with
blood type O.
iii
Acknowledgments
I would like to thank and extend my sincere gratitude and appreciation to my supervisor, Dr.
Kevin Kain. He has been exceptionally supportive and his guidance, knowledge, and mentorship
made this project not only possible, but also propelled it into new and exciting directions. As a
result of the experience and opportunity to work with Dr. Kain and other gifted researchers, I
was able to be included and contribute to the scientific community in ways I had not thought
were possible for a Master’s student. Thank you for allowing me these opportunities. I would
like to thank members of my committee, Dr. Conrad Liles, Dr. Christine Cserti-Gazdewich, and
Dr. Don Branch for their advice, expertise, and encouragement. You have all taught me that the
most valuable education is not one learned solely by text books, but through collaboration,
experience, and application. Thank you for donating your time and going above and beyond what
was expected of you. I would also like to thank all of my fellow lab members for being
exceptionally supportive and sharing their knowledge. A special thank you to Dr. Ayi Kodjo for
being my mentor in the lab, continually teaching me techniques and encouraging me to believe in
myself and be independent. You have taught me lifelong skills and your guidance helped me
manage and learn from the frustrations and enjoy the successes. Finally, I would like to extend a
sincerely deep gratitude to my parents, Ewa and Stan, and to my sister, Samara, for all of their
support in pursuing my education and believing I will always succeed. Thank you for always
being there to share the great moments.
Thank you all for your encouragement, without it, I would not be where I am today.
iv
TABLE OF CONTENTS
PART I: LITERATURE REVIEW..............................................................................................1
SECTION 1: MALARIA BACKGROUND ................................................................................2
1.1.1 The evolution and global distribution of malaria.....................................................3
1.1.2 Life cycle of Plasmodium falciparum malaria.........................................................6
1.1.3 Pathophysiology of Plasmodium falciparum malaria ..............................................9
1.1.4 Innate immunity to Plasmodium falciparum malaria ............................................14
SECTION 2: THE RED BLOOD CELL ...................................................................................17
1.2.1 Erythropoiesis and physiology of the RBC ...........................................................18
1.2.2 Essential components of the RBC..........................................................................19
1.2.2.1 Hemoglobin .............................................................................................19
1.2.2.2 RBC enzymes ..........................................................................................20
1.2.2.3 RBC membrane and aging (senescence) .................................................20
SECTION 3: ABO BLOOD TYPE ............................................................................................24
1.3.1 History of ABO ......................................................................................................25
1.3.2 Genetics and biochemistry of ABO antigens .........................................................25
1.3.3 ABO subtypes ........................................................................................................29
1.3.4 ABO antibodies......................................................................................................29
1.3.5 ABO and infectious diseases..................................................................................30
1.3.6 Geographic distribution of ABO blood types ........................................................32
SECTION 4: MALARIA, RED CELL POLYMORPHISMS AND NATURAL SELECTION ................................................................................................................................35
1.4.1 RBC polymorphisms..............................................................................................36
1.4.1.1 Hemoglobin mutations ............................................................................37
1.4.1.2 RBC enzymes ..........................................................................................39
v
1.4.1.3 RBC membrane disorders........................................................................39
1.4.2 Inherited disease specific mechanisms of protection .............................................40
1.4.2.1 Invasion and growth ................................................................................41
1.4.2.2 Cytoadherence and rosetting ...................................................................42
1.4.2.3 Clearance of infected polymorphic RBCs ...............................................42
1.4.3 ABO polymorphism and Plasmodium falciparum malaria ...................................44
1.4.4 Potential mechanisms of protection afforded by blood type O..............................48
1.4.4.1 Invasion and maturation ..........................................................................48
1.4.4.2 Rosetting and sequestration .....................................................................49
1.4.4.3 Additional mechanisms of protection......................................................51
SECTION 5- AIMS AND HYPOTHESIS .................................................................................53
PART II :MATERIALS AND METHODS ...............................................................................54
2.1 Reagents .................................................................................................................55
2.2 Methods ..................................................................................................................56
PART III: RESULTS ..................................................................................................................62
PART IV: DISCUSSION ............................................................................................................74
CONCLUSIONS AND FUTURE DIRECTIONS .....................................................................90
vi
LIST OF TABLES
SECTION 1: MALARIA BACKGROUND
Table 1. Prevalence of ABO frequencies in malaria-endemic regions……………………..……..6
Table 2. Factors contributing to the clinical outcome of Plasmodium
falciparum infection………………………………………………………………………….…..10
SECTION 3: ABO BLOOD TYPE
Table 3. The ABO blood type: Genotype, phenotype and antibodies…………………………...26
Table 4. ABO antigen sites on the red blood cell………………………………………………..28
Table 5. ABO frequencies in human ethnic populations………………………………………...34
vii
LIST OF FIGURES
PART I: LITERATURE REVIEW
SECTION 1: MALARIA BACKGROUND
Figure 1. Geographic distribution of Plasmodium falciparum malaria………………...…………4
Figure 2. Life cycle of Plasmodium falciparum malaria…………………………………...…......9
Figure 3. The PfEMP-1 molecule and associated host receptors…………………………….….14
SECTION 2: THE RED BLOOD CELL
Figure 4. Red cell senescence and band 3 aggregation……………………….…………...…….23
SECTION 3: ABO BLOOD TYPES
Figure 5. Structure of ABO antigens…………………………….…………………………..…..26
Figure 6. Biosynthesis of the ABO antigens……………….…………………………...……….28
PART III: RESULTS
Figure 7. Similar invasion and growth of P. falciparum in A, B or O blood type red blood
cells…………………………………………………………….………………………..……….64
Figure 8. Phagocytosis of A, B and O ring- infected RBCs by human monocyte derived
macrophages.…………………………………………………………………….………………66
Figure 9. Increased phagocytosis of schizont infected O red blood cells compared to A and B
infected red blood cells by human monocyte derived macrophages………………..…..……….67
Figure 10. Schizont infected O RBCS are preferentially phagocytosed independent of
macrophage donor blood type……………………………………………………..……………..69
Figure 11. Peritoneal monocytes of C57/B6 mice clear O-infected red blood cells more
efficiently than A or B infected red blood cell……………………………….…………………..71
viii
Figure 12. Increasing phagocytosis of RBCs bearing decreasing A surface antigen……….…...73
ix
ABBREVIATIONS
AMA Apical membrane antigen
APC Antigen presenting cells
ATP Adenosine triphosphate
CFU Colony forming unit
CR1 Complement receptor 1
DARC Duffy antigen receptor for chemokines
EBA Erythrocyte binding protein
EtBr Ethidium bromide
FcR Fc receptor
FITC Fluorescein isothiocyanate
GPI Glycosylphosphatidylinositol
G6PD Glucose-6-phosphate-dehydrogenase
GM-CSF Granulocyte-macrophage colony stimulating factor
Hb Hemoglobin
HbAS Sickle cell trait
HbS Sickle cell anemia
HE Hereditary elliptocytosis
HO Hereditary ovalocytosis
HS Heparan sulfate
x
ICAM-1 Intercellular adhesion molecule-1
IFN Interferon
MHC Major histocompatibility complex
MSP Merozoite surface protein
NO Nitric oxide
PBMC Peripheral blood mononuclear cell
P.falciparum Plasmodium falciparum
PfEMP Plasmodium falciparum erythrocyte membrane protein
PFK Phosphofructokinase
PfRBP Plasmodium falciparum reticulocyte binding protein
PfRh Plasmodium falciparum reticulocyte homologue
PK Pyruvate kinase
PS Phosphatidylserine
P. vivax Plasmodium vivax
RBCs Red blood cells
TNF Tumor necrosis factor
VCAM-1 Vascular cell adhesion molecule 1
VSA Variant surface antigen
WHO World Health Organization
1
PART I
LITERATURE REVIEW
2
SECTION 1: MALARIA BACKGROUND
Malaria is estimated to account for 1-3 million deaths per year with the major burden of disease
occurring in resource poor areas of the world.1Plasmodium falciparum malaria, accounts for the
highest morbidity and mortality, and has a complicated pathogenesis that is influenced by
geographic factors, parasite virulence factors, and host genetic determinants. Although a highly
effective vaccine is not yet available, an increased understanding of the interaction between
P. falciparum and the above determinants will contribute to interventions to achieve near or
complete elimination of this disease.
3
1.1.1 The evolution and global distribution of malaria
Malaria afflicts 300-500 million people each year and remains a leading cause of death
worldwide, killing between 1-3 million people annually.1,2 Malaria is one of the strongest known
forces for evolutionary selection in the recent history of the human genome.1,3,4 Understanding
the historical and global relationship between malaria and human genetic diversity provides
powerful insights into the evolution of the human genome and the pathogenesis of malaria.
There are five Plasmodium species that infect humans; Plasmodium falciparum, Plasmodium
vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi.5 These species
differ in their morphology, immunology, and geographic distribution.6 Among the five species
that cause malaria in humans, Plasmodium falciparum (P. falciparum) is the most virulent
resulting in the greatest number of complications and the great majority of malaria-related deaths
in children under the age of five.7,8 By killing children before they reach reproductive age,
P. falciparum has, in essence, naturally selected for gene variants capable of conferring a
survival advantage. Research which examines the key protective factors against P. falciparum
malaria, could contribute to control of this major global health threat and further our
understanding of human genetics and natural selection.
The evolutionary history and geographical distribution of P. falciparum reflects a three-way
interaction between the parasite, the host, and the Anopheles sp. mosquito (the vector for
transmission). Circa 1900, prior to the widespread use of anti-malarials, the distribution of
malaria reached the geographic latitudes of 64º north and 32º south.9 Efforts during the 20th
century to control malaria restricted the global expansion of disease, however it remains endemic
to climatic regions which facilitate continuous transmission and in the last two decades has
recurred in several regions that had previously eradicated transmission (Figure 1).2 P. falciparum
4
and its Anopheles vector, are normally confined to tropical, subtropical and warm temperate
regions.10 These regions are predominantly resource poor, highly populated areas with limited
access to adequate malaria prevention and treatment programs. However, genetic history and the
co-evolution of P. falciparum with humans suggest this has not always been the geographic
model. The closest relative to the modern day P. falciparum is the chimpanzee malaria parasite,
Plasmodium reichenowi. 3,10,11 It has been argued that P. falciparum is of African origin because
P. reichenowi is a parasite that infects African chimpanzees.10 Despite some controversy, it is
generally accepted that the divergence of these two species of malaria occurred approximately 9-
10 million years ago, prior to the divergence of humans from non-human primate relatives such
as the chimpanzees.3,10-12 It is believed that the major spread of P. falciparum in Africa occurred
during the “Agrarian Revolution” (4000-5000 years ago) when small nomadic groups began to
establish larger settled communities; this lifestyle change provided ideal conditions for sustained
P. falciparum transmission.10 Around 15 A.D.10, malaria arrived in the Americas carried in the
blood of European colonists and their slaves, and thereafter, became indigenous to the tropical
regions of Central and South America, and some southern parts of North America.13
Figure from Snow, R.et al. Nature.2005;434:214-217
Figure 1. Geographic distribution of Plasmodium falciparum malaria.2
Childhood infection prevalence
Hyper-endemic: >50%
Meso-endemic: 11-50%
Hypo-endemic: <10%
Unclassified areas: <6%
5
P. falciparum has been referred to as the “the strongest known force for evolutionary selection in
the recent history of the human genome.” 3,4 P. falciparum malaria and humans have co-evolved
and have adapted to one another, thereby selecting for survival genes simultaneously in both
species. It has been noted that in African populations where P. falciparum is highly prevalent,
certain polymorphic traits of the red blood cell, such as glucose-6-phosphate dehydrogenase
deficiency (G6PD), sickle cell trait (HbAS), and α-thalassaemia, have conferred a survival
advantage against severe malaria and death.14-17
Blood type O, also a polymorphic trait, is present worldwide, which suggests that this trait must
have been present when humans migrated out of Africa.3,18,19 Interestingly, however, there is also
a higher than expected prevalence of blood type O (along with the previously noted specific
RBC polymorphisms) in Africa, especially in areas where P. falciparum is endemic
(Table 1).3,20-25 This suggests that like other RBC polymorphisms, blood type O may confer
protection against severe malarial disease. To understand how the ABO blood types may be
associated with malaria, the life cycle, and pathophysiology of malaria will be discussed. In
addition, the innate immune system of the host, the RBC, and other known protective
polymorphisms will be reviewed.
6
Table 1. Prevalence of ABO frequencies in malaria-endemic regions.3,20-25
Region A B O Sickle cell
trait/
thalassaemia
Malaria
Norway
Portugal
USA
Canada
Nigeria
50%
53%
42%
44%
21.3%
8%
8%
10%
9%
23.3%
38%
35%
44%
36%
51.5%
No
No
No
No
Yes
No
No
No
No
Yes
Ghana
Kenya
Papua New Guinea
23%
19%
27%
-
20%
26%
47%
60%
41%
Yes
Yes
Yes
Yes
Yes
Yes
1.1.2 Life cycle of Plasmodium falciparum malaria
The P. falciparum infection begins when a human host is bitten by an infected female Anopheles
mosquito, and the mosquito injects sporozoites into the subcutaneous tissue of the human host
(Figure 2). 55,26 The sporozoites find their way into the blood stream where, within one hour of
entering the human host, they travel to the liver and infect hepatocytes. 55,26 The duration of the
asymptomatic liver (exo-erythrocytic cycle) stage of the infection is approximately one-two
weeks. During this stage, each sporozoite may yield thousands of merozoites.27
7
Invasion
The hepatocytes rupture releasing the merozoites into the blood stream (the beginning of clinical
disease) where they are able to enter into RBCs by a complex invasion process comprised of
four phases: (a) initial recognition and reversible attachment of the merozoite to the RBC
membrane, (b) reorientation, (c) invagination of the RBC membrane around the merozoite, and
(d) resealing of the RBC membrane after completion of merozoite invasion.5,6 A number of
interactions and organelles have been identified between the RBCs and the merozoite. There are
three organelles on the invasion (apical) end of the parasite, the rhoptries, micronemes and dense
granules all which define the phylum Apicomplexa.5 These three organelles contain the receptors
that mediate invasion of the merozoite. RBC invasion is a rapid process that is governed by
molecular interactions between the merozoites and the host cell surface.28 Primary contact is
initiated by a surface coat of proteins that is largely comprised of glycosylphosphatidylinositol
(GPI)-anchored membrane proteins. There are at least nine recognized GPI anchored proteins
that are predicted to be potential RBC ligands.29 Merozoite surface protein-1 (MSP-1) is the
dominant antigen and is essential for parasite survival as MSP-1 is involved in the initial
recognition of the RBC via sialic acid residues found on the RBC membrane. Other important
proteins are MSP-2, -3 and -4.30 P. falciparum apical membrane antigen-1 (PfAMA-1) is also
essential for successful invasion as it is translocated to the merozoites surface before invasion of
the RBCs, and is also present on the sporozoite for invasion into hepatocytes. Other interactions
between the merozoites and the RBCs include the erythrocyte binding antigen-175 (EBA-175),
EBA- 140 and EBA-181 found on the merozoites which bind to glycophorin A, glycophorin C ,
and band 4.1 (respectively) on the RBC. 6,30,31 EBA-175 binds via sialic acid residues that are in
an α-2,3 conformation, and binds specifically to glycophorin A. 32 P. falciparum is not able to
invade RBCs that are missing glycophorin A.33,34 Although some P. falciparum strains are reliant
8
on sialic acid to invade RBCs several strains have demonstrated the ability to shift to sialic acid-
independent pathways (3D7). 35 This pathway utilizes a different family of ligands called P.
falciparum reticulocyte binding proteins (PfRBP), or P. falciparum reticulocyte protein
homologues (PfRh). 36 PfRh2b and PfRh4 are important in the sialic acid-independent invasion
pathway, however the receptors responsible for bindings these ligands is unknown.36,37
Redundancy in invasion pathways may provide an advantage to the parasite in case it encounters
polymorphisms in host receptors. Once merozoites have successfully bound and invaded the
RBC, the asexual stage of development of P. falciparum begins.
Maturation
Initially, the merozoites develop into an early trophozoite stage known as the “ring form”. The
ring form persists for 24 hours and matures inside the RBC through a highly active metabolic
state. The P. falciparum ring feeds from the host cytoplasm, importing glucose and breaking
down hemoglobin into constituent amino acids.6 Following the ring stage, P. falciparum matures
and develops to a late stage trophozite. The mature trophozoite stage parasite replicates by
nuclear division resulting in schizont stage parasites. Each schizont is comprised of 20-24
merozoites, which are released upon rupture of the infected RBC.6 When the infected RBCs
rupture, merozoites and parasite metabolic waste products such as hemozoin, degradation of
hemoglobin, and parasite toxins are released. The majority of the merozoites will invade other
RBCs continuing the asexual cycle; however, some parasites will form sexual stage forms called
gametocytes which are then transmitted to new hosts by the Anopheles vector.5,26 The asexual
stages are solely responsible for the pathology associated with malaria which may manifest in a
diverse array of pathological conditions.
9
Figure from Miller, L.H et al. Nature.2002;415:673-679
Figure 2. Life cycle of Plasmodium falciparum malaria.5
1.1.3 Pathophysiology of Plasmodium falciparum malaria
Infection with P. falciparum results in considerable morbidity and without treatment may be
fatal. The clinical outcome of malaria depends on many contributing factors including the
parasite’s virulence, the host’s response, geographical, and socio-economic factors (Table 2).5
The combination of these factors result in a range of possible outcomes for the host, including
asymptomatic infection, uncomplicated malaria infection, severe infection (severe malaria
anemia and cerebral malaria) and death.
10
Table 2. Factors contributing to the clinical outcome of P. falciparum infection.
Parasite Factors Host Factors Geographic and Social factors
-Drug Resistance
-Multiplication rate
-Invasion Pathways
-Cytoadherence
-Rosetting
-Malaria toxins (hemozoin)
-Antigenic Variation (PfEMP1)
-Immunity
-Genetics : Sickle cell, thalassaemia,
ABO b lood type (?) etc.
-Age
-Pregnancy
-Pro-inflammatory cytokines
-Transmission intensity
-Culture and economic factors
-Access to treatment
Adapted from Miller, LH et al. Nature. 2002; 415:673-679
Clinical stages of malaria pathogenesis
There are three defined clinical stages of malaria pathogenesis: uncomplicated malaria, severe
malaria, and cerebral malaria. Uncomplicated malaria initially presents with fever and chills,
nausea and headache, sometimes associated with diarrhea and vomiting.7 Unfortunately, because
of the similarity in symptoms, malarial infection is often mistaken for many other infections
including influenza or gastro- intestinal infection and is therefore not properly treated.7 The liver
stage of the infection does not produce symptoms, but once the merozoites are released from the
hepatocytes, symptoms usually occur approximately two weeks after the primary infection. The
fever that individuals experience may become cyclical during the course of illness because of
synchronized release of the merozoites from the RBCs. If left untreated, patients may progress to
severe malaria.
Asymptomatic Clinical Outcome Death
11
In 1990, the World Health Organization (WHO) established criteria for the diagnosis of severe
malaria. The major criteria include neurological involvement (cerebral malaria), pulmonary
edema, acute renal failure, and severe anemia.8 Severe anemia is the second most common
symptom of P. falciparum infection and is caused by the destruction of RBCs and overall
decreased erythropoiesis. 38,39 Acidosis and hypoglycemia are the most common metabolic
complications.8
Cerebral malaria is the most common cause of death in adults and children with severe malaria.40
According to the WHO, the strict definition of cerebral malaria requires the presence of P.
falciparum parasitemia and unarousable coma with a Glasgow Coma score of 9 or less; all other
causes of coma, such as hypoglycemia, bacterial meningitis and viral encephalitis, need to be
excluded.40,41 Typical neurological symptoms include coma, seizures, edema, and brainstem
damage.40,41 Engorgement of cerebral capillaries and venules filled with infected RBCs and non-
infected RBCs are typical histopathological findings in cerebral malaria.41
As the infection progresses, the increasingly detrimental pathogenesis of P. falciparum malaria is
believed to be caused by two main factors: 1) an imbalance of cytokine production; and 2) the
sequestration of infected RBCs in the microvasculature of vital organs.
Inflammatory response
P. falciparum infection results in an increase of both pro- inflammatory cytokines and anti-
inflammatory cytokines.42-44 However, in cerebral malaria, there is an unbalanced and excessive
production of the pro-inflammatory response.45-47 This phenomenon has been studied extensively
but is beyond the scope of this thesis and will only be briefly reviewed.
12
Blood concentrations of pro- inflammatory cytokines, especially tumor necrosis factor (TNF),
interferon gamma (IFN-γ), interleukin-1ϐ (IL-1ϐ), and IL-6, have been shown to be raised in
cerebral malaria.41,46,47 TNF may contribute to malaria pathogenesis including cerebral malaria.
TNF up regulates endothelial cytoadherence receptors such as intercellular adhesion molecule-1
(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. TNF may cause
hypoglycemia and dyserthryopoesis, and has been shown to induce the release of nitric oxide
(NO) which interferes with synaptic transmission.38,41,48
Parasite sequestration
P. falciparum has a unique ability to adhere to host microvasculature endothelium, a process
known as sequestration. Sequestration causes microvascular obstruction and compromises the
blood flow through tissues such as the liver, spleen, lung, and brain.38 The effects of
sequestration include mechanical obstruction (which can lead to hypoxia), metabolic
disturbances and is a central point where parasite toxins and inflammatory mediators
concentrate.38,49 Increased expression of cytoadherence receptors enhances infected RBC
sequestration to the endothelium via parasite derived proteins (expressed on the surface of the
infected RBC ), such as PfEMP-1 (Figure 3).40 It is broadly accepted that ~12 hours after a
merozoite invades a RBC, the principal parasite surface protein and sequestration ligand known
as P. falciparum erythrocyte membrane protein 1 (PfEMP-1), encoded by var genes, is
expressed.50 It is predominantly mature stage parasites (trophozoites and schizonts) that adhere
to the microvasculature. The PfEMP-1 molecule has a pivotal role in the pathogenesis of P.
falciparum as a number of host receptors are recognized by the various extracellular binding
domains of PfEMP-1 (Figure 3), thus permitting the infected RBCs to adhere to host
endothelium.51 In addition, PfEMP-1 binds to a number of different host receptors on both
13
monocytes and other RBCs.44 In the case of cerebral malaria, PfEMP-1 may mediate adhesion to
several adhesion molecules, in particular ICAM-1 which is unregulated on the cerebral vascular
endothelium. 40 Post-mortem studies have shown that sequestration is greater in the brain than
the other organs and correlates with ICAM-1 expression in cerebral vessels.43,52 However, in
some post mortem studies, not all individuals who die from cerebral malaria present with
sequestered parasites.38
In addition, the schizont infected RBC, to avoid clearance from the spleen, may bind to other
schizont infected RBCs (agglutination), to other non-infected RBCs (rosetting), or to platelets
bound to other infected RBCs or the endothelium.53 Binding is mediated by a number of variant
surface antigens expressed on the RBC, such as PfEMP-1, and any abnormality on either the
receptor or ligands may prevent adhesion. Important receptors found on host tissues involved in
sequestration and rosetting are heparan sulphate (HS), complement receptor 1 (CR1),
thrombospondin, A and B blood type antigens, and CD36 (Figure 3). 38,49,54,55 HS is a
proteoglycan found on the RBC, and along with CR1, acts as a receptor in the formation of
rosettes. A and B blood type antigens have also been shown to act as co-receptors in the
formation of rosettes, and depending on the blood type of the person can produce different
rosetting rates and sizes (see section 1.4.4.2).56 The role of CD36 in the pathogenesis of malaria
is controversial and there is little evidence it contributes to cerebral malaria since there is little
expression of CD36 in cerebral vessels.38 CD36 is also found on platelets, monocytes and
dendritic cells.57 Parasite interaction with CD36 on monocytes has been shown to be an
important interaction in non-opsonic phagocytosis and innate clearance of infected RBCs. 58-60
14
In summary, high parasite burdens leading to sequestration and dysregulated immune responses
are thought to make important contributions to the pathogenesis of cerebral malaria and severe
disease.
Adapted from Cserti, CM et al. Blood. 2007; 110:2250-2258
Figure 3. The PfEMP-1 molecule and associated host receptors.
1.1.4 Innate immunity to Plasmodium falciparum malaria
The innate immune response is crucial to the outcome during a P .falciparum infection. Innate
immune responses take effect immediately and provide an early defense until the adaptive
immune response is engaged. In some cases, an infection by P. falciparum may be controlled by
the innate immune system.61 Parasite burdens observed in non- immune individuals with acute P.
falciparum malaria are lower than expected based on parasite replication rates observed in vitro,
suggesting that the innate immune system can contribute to effective control of acute parasite
replication before the adaptive immune response develops.58,59,62 The innate immune system
functions to limit the maximum parasite density, but gradually acquired adaptive mechanisms
complete parasite elimination. The innate immune system is essential for most inflammatory
responses that are triggered by monocytic cells, other leukocytes and mast cells through their
innate sensing receptors.63 Studies have shown that macrophages are important in innate
immunity as they are able to clear parasitized RBCs in the absence of opsonizing malaria-
specific antibodies.59 It is hypothesized that there are two methods of infected RBC uptake by
macrophages. The predominant method of uptake involves the binding of non-specific IgG and
15
complement to the surface of infected RBCs, and increased exposure of senescent RBC markers
such as exposure of phosphatidylserine (PS). This method induces the release of
pro-inflammatory cytokines. The second method of uptake is CD36 mediated, which involves
the binding of CD36 on the macrophage to PfEMP-1 on the infected RBCs. This method does
not involve the release of pro- inflammatory cytokines.64,58
There are three main biochemical pathways that result in activation of the complement system:
the classical complement binding pathway; the mannose-binding lectin pathway; and the
alternative pathway. All three lead to the formation of C3 and C5 convertase which results in the
cleavage of C3 and C5 into C3a, C3b, C5a and C5b, respectively.65 RBCs opsonized by IgG and
complement (C3b) are recognized by the Fc receptor (FcR) and CR1 (respectively), and
phagocytosed by macrophages. 66,67 This method of clearance is effective in senescent and
damaged RBCs, and also in P.falciparum infected RBCs. 67
Ayi et al compared the uptake of ring infected RBCs and mature infected RBCs and found an
overall higher affinity for the uptake of mature stage parasites over ring stage.68 This uptake may
be due to the structural changes within the RBC, resembling RBC senescence. Specifically, in
mature infected RBCs there is an increase in hemichrome deposition and band 3 aggregation as
well as increased expression of PfEMP-1 on the surface of the RBC. 68,69 The exact mechanism
of phagocytosis of infected RBCs by monocytes and macrophages is unknown; however the
primary mechanism is thought to be through complement and IgG binding, and even possibly via
PS dependent pathways. 66,70-72
Any trait of the RBC that enhances clearance of infected RBCs may confer a survival advantage.
This is especially true if there is enhanced phagocytosis of schizont stage parasites, as it will
16
reduce the number of infected RBCs available to bind within the micro-vascular beds of vital
organs.
17
SECTION 2: THE RED BLOOD CELL
At first, RBCs seem to be one of the simplest cells in the human body, with no organelles, no
nucleus and only two major functions, to deliver oxygen and remove carbon dioxide to and from
the tissues.73 However, blood, more specifically, the RBC is often referred to as “the essence of
life”. The RBC is rich in nutrients, continually renewing, and is a shelter not recognized by the
immune response, thereby rendering it the perfect target for a hemotropic pathogen, such as P.
falciparum.
18
1.2.1 Erythropoiesis and physiology of the RBC
RBCs are anucleate cells devoid of typical organelles.74 They are found in the blood stream and
their primary function is to deliver oxygen and remove carbon dioxide from the tissues.
Erythropoiesis is the process by which RBCs are produced in the bone marrow.73 The process of
RBC maturation involves a series of differentiation steps which are tightly regulated. RBCs
develop from the multi-potential progenitor cell (CFU-GEMM) under the influence of
erythropoietin (EPO), granulocyte-macrophage colony stimulating factors (GM-CSF), IL-3, and
IL-4. 74 This gives rise to the erythropoietin sensitive cell CFU-E which when stimulated by
erythropoietin, develops into a RBC.74 At this stage of RBC development, the cell is released
from the bone marrow and is called a reticulocyte. In healthy, non- infected blood, 1-2% of the
total RBC counts are reticulocytes. Reticulocytes have stage-specific surface antigens specific to
this stage of development, making them targets for invasion by Plasmodium vivax (P.
vivax).75After a day of circulating in the blood stream, they develop into mature RBCs.74 The
mature RBC has a biconcave structure that is deformable. It only consists of cytoplasm and a cell
membrane, which allows the RBC to pass through small blood vessels and narrow capillaries.
The disruption of erythropoiesis is important to the development of severe malaria anemia
(SMA). In cases of SMA, low reticulocytosis is observed, suggesting insufficient erythropoiesis
as a major factor for anemia. In one study, researchers found reduced proliferation and terminal
differentiation of erythroblasts to become mature hemoglobin-producing cells.76 The suppression
of initial proliferation, differentiation and maturation, and inadequate reticulocytosis has been
proposed as the basis of insufficient erythropoiesis during malaria.
19
1.2.2 Essential components of the RBC
As mature RBCs do not differ phenotypically, do not contain internal mechanisms of synthesis
or traffic proteins, or express class I or II MHC molecules on their surface, they are the ideal
vehicle for the parasite to evade the immune system of the host.77 The RBC is central to the life
cycle of P. falciparum, and alterations to the RBC may impair parasite growth and replication
and provide a survival advantage to the host. There are three major components to the RBC:
hemoglobin, metabolic enzymes, and the RBC membrane. All three have a pivotal role in
parasite development.
1.2.2.1 Hemoglobin
Hemoglobin is an assembly of 4 globin protein chains (2 -α globins and 2-ϐ globins chains)
arranged into a set of α-helix structures and 4 heme groups. 73,77,78 The hemoglobin within the
RBC transports oxygen from the lungs to the tissues and trades it for carbon dioxide to take back
to the lungs. The binding of the oxygen to the iron molecule in hemoglobin causes the
hemoglobin to undergo a conformational change. There are two main states of hemoglobin - the
deoxyhemoglobin (taut state, no bound oxygen) and the oxyhemoglobin (the relaxed state, bound
oxygen).79 Over time this process causes oxidative stress on the RBC.80
Hemoglobin is central to malarial infection as the parasite internalizes and degrades massive
amounts of hemoglobin from the host RBC during the blood stage of infection.81 Hemoglobin is
hydrolyzed to free amino acids, which are subsequently incorporated into parasites proteins.82
The released heme is stored as a polymerized byproduct called hemozoin. In vitro studies have
shown that macrophages that have engulfed hemozoin, are unable to digest it and are unable to
repeat phagocytosis, or kill bacteria.83 Mutations in the genes for the hemoglobin proteins results
20
in hemoglobin variants, resulting in a group of hereditary diseases termed hemoglobinopathies
(discussed in section 1.4.1.1), which undoubtedly have an impact on P. falciparum malaria.
1.2.2.2 RBC enzymes
Malaria parasites spend virtually all of their asexual life cycle inside the RBCs of their host.
They must continually adapt themselves to the host’s RBC environment, and need a very specific
environment to grow.84 Malaria-infected RBCs are consistently under oxidative stress, and have
high levels of H2O2 and OH- radials, which are primarily produced by the digestion of the
hemoglobin by the parasite.85 There are two important groups of enzymes involved in the
metabolic pathway in the RBC: enzymes involved in energy metabolism, and enzymes to
prevent/reverse oxidative damage. The mature RBC does not have mitochondria or storage
capacity, and therefore utilizes anaerobic glycolysis as a source of energy.79 The enzyme
pyruvate kinase (PK) is a member of the anaerobic pathway and produces a molecule of ATP. 78
Enzyme deficiency in the anaerobic pathway leads to reduced viability of the RBC. The RBC is
continually susceptible to oxidative damage and at high concentrations of radicals or insufficient
protection, oxidative damage leads to a loss of the ability of the RBCs to transfer O2 and CO2
and eventually the cells hemolyze.86 Glucose-6-phosphate dehydrogenase (G6PD), an enzyme in
the aerobic pathway produces NADPH, which is essential for the production of pentoses needed
for the anaerobic pathway and is a major reducing agent in the RBC. 78,87 Two additional
enzymes that prevent and reverse oxidative denaturation of the hemoglobin are methemoglobin
reductase and superoxide dismutase. 86,88
1.2.2.3 RBC membrane and aging (senescence)
Several properties of the RBC membrane are essential to the invasion and pathogenesis of P.
falciparum malaria. The membrane contains the structures required for invasion and gives the
21
parasite a medium to express surface antigens such as PfEMP-1, which are ultimately important
for its survival. The primary function of the RBC membrane is to form a phospholipid bilayer to
protect and contain hemoglobin allowing the RBC to carry oxygen.
Membrane
The RBC membrane is an exceptionally complex membrane with over 50 transmembrane
proteins identified. Key structures on the membrane include the Rh complex, Duffy
glycoprotein, ABO antigen, and band 3.89 The Rh complex has been implicated in RBC structure
and is hypothesized to have a role in ammonium and CO2 transport. 90-92 The Rh complex is
important in blood transfusion, and has a primary role in hemolytic disease of the newborn.90-92
The Duffy glycoprotein is present on the RBC and corresponds to the chemokine receptor DARC
(Duffy Antigen Receptor for Chemokines) which is a general receptor for several chemokines
and removes excessive chemokines from the blood and tissue.93 The ABO antigen is a
carbohydrate based structure, and will be further discussed in section 3. Band 3 is a transport
protein that is associated with RBC senescence as it aggregates and signals for RBC removal.94,95
Senescence
RBC aging is thought to be accelerated by the intracellular presence of P. falciparum.96 This has
been attributed to decreased levels of antioxidants and ATP, coupled with enhanced flux of ions,
especially calcium. P. falciparum induces biochemical modifications in the membranes of
infected RBCs, mimicking the physiologic aging process of the cell, most likely produced
through P. falciparum- induced oxidative stress.97 There are also a number of key structures on
the membrane that are associated with RBC aging and senescence such as band 3. Band 3
comprises 25% of the total membrane protein and while it has a structural and transporter role it
22
also supports a number of RBC antigens (ABO, Diego and RH). Band 3 is important to maintain
the flexibility of RBCs and also has a role in anion exchange as it exchanges Cl- for HCO3-, thus
removing CO2 from tissues.95 Consequently, it is believed to have an essential role in the removal
of aged or defective cells.98 The average life span of RBCs is 120 days after which most are
phagocytosed by macrophages in the spleen and liver.74 The trigger mechanism for senescence is
not completely understood; however there are RBC modifications that occur both internally and
externally that may contribute to senescence. Senescent RBCs are smaller, more rigid, and their
surface is partially desialylated.96 Many of the primary enzymes have reduced activity, thereby
increasing reactive oxygen species (ROS). This leads to accelerated oxidative injury and the
denaturation of hemoglobin to form hemichromes which bind to the cytoplasmic domain of band
3 (Figure 4).94,98,99 The binding of hemichromes to band 3 causes cross- linking of the
cytoplasmic domains resulting in clustering. Clustering is detected by autologous antibodies to
the altered band 3 molecules. Fc and complement receptors on the macrophage recognize these
signals and the RBC is cleared by the macrophages 94,98,99. In addition, it is thought there may be
neo-antigens exposed during band 3 degradation, including increased exposure of PS on the RBC
surface, which collectively transforms the RBC from “self” to “non-self”.60,66,100
23
Figure from Pantaleo, A et al .Autoimmunity Reviews.2008;7:457-462
Figure 4. RBC senescence and band 3 aggregation.
1. Oxidative denaturation of hemoglobin leading to hemichrome formation 2. Hemichrome binds to cytoplasmic domain of band 3 and causes oxidative cross-linking 3. Band 3 dissociates from cytoskeletal proteins and 4. Formation of band 3/hemichrome clusters and opsonization by anti-band 3 antibodies and C3b
24
SECTION 3: ABO BLOOD TYPE
The ABO polymorphism is the most recognized and the most clinically important antigen
classification system to date. Its recognition is central to the practice of transfusion medicine,
because of the immediate recognition and rejection of major incompatible non-self cells. In the
past century since the ABO discovery, scientists have been able to identify an association
between the ABO blood type and a number of infectious diseases, some of which exert genetic
selection. However, the cause of the molecular and geographic diversity, along with the
evolutionary basis for the origin of the ABO blood type, remains a mystery.
25
1.3.1 History of ABO
The ABO blood type was first recognized in 1900 by Karl Landsteiner. 101,102 The ABO blood
type is the most well known and the most clinically important of the 29 blood type systems due
to its significant role in successful blood transfusion and organ transplantation.
In this blood type system, there are three blood type antigens, A, B and O. These antigenic
determinants are oligosaccarchides located on glycol proteins and glycolipids expressed on
RBCs, and are predominantly found on band 3.103,104 ABO antigens are found on other cell
tissues, epithelia, various body fluids, secretions, lymphocytes, and platelets, however they are
expressed most densely on RBCs.104,105 At first glance, this system seems to be relatively simple;
three antigens (A, B, O), six genotypes (AA, AO, BB, BO, OO, AB), and 4 phenotypes (A, B, O,
and AB).102 However, within 30 years of discovery of the ABO blood type, subtypes of each
blood type were discovered (section 1.3.3). Molecular studies on the ABO blood types show that
the O gene appears to be a mutation of the A gene, suggesting that type O appeared later than the
other ABO blood types, and therefore, blood type O is considered to be the “polymorphic”
type.18,19,106
1.3.2 Genetics and biochemistry of ABO antigens
The ABO blood type antigens are subject to strict genetic control and are a perfect example of
Mendelian genetics. Genes A and B each express themselves dominantly with respect to the O
silent gene, but A and B are co-dominant and in a heterozygote, both A and B antigens are
expressed on the RBC (Table 3). 107
26
Table 3. The ABO blood type: Genotype, phenotype and antibodies.108
Genotype Phenotype Antibodies present in serum
OO
AO
AA
BO
BB
AB
O
A
B
AB
Anti-A,B
Anti-B
Anti-A
None
The ABO gene itself does not encode the carbohydrate ABO antigen but rather a
glycosyltransferase, which transfers either a α-1,3 linked N-acetylgalactosamine by α -1,3 N-
acetyl galactosaminyltransferase or a α-1,3 linked galactose by α-1,3 galactosyltransferase to
form A or B antigens, respectively, on the precursor molecule (H) (Figure 5).107,109,110
Figure from Sheffield, WP et al .Transfus Med Rev.2005;19:295-307
Figure 5. Structure of ABO antigens.
The precursor structure (H) to which A and B trisaccharides are attached is genetically
independent of the ABO system. It represents the precursor substance in the biosynthetic
pathway leading to the A and B determinant structures. The O gene represents an “amorph” or
O (H)
A1
B
A2
27
“silent” gene as there is no antigenic expression or glycosyltransferase enzyme formed.108 Based
on this close chemical relationship, the H system is now included in the discussion of the ABO
system, and is referred to as the ABO (H) system.111 The blood type O character is found on
RBCs and in secretions of blood type O individuals, and is considered the product of the O (H)
gene. O( H) molecules are also present in non-O individuals, but to a lesser degree, as the
remainders of chains are left unmodified by the glycosyltransferase activity.112,113
The ABO locus, located on chromosome 9, position 9q34,1-q34.2 has seven exons ranging from
28-688 base pairs, and six introns with 554 to 12983 base pairs.110,114 The last two exons, 6 and
7, which encode 823 of the 1062 base pairs of the transcribed mRNA encode for the catalytic
domain of the ABO glycosyltransferase.114 Most variants of the A and B antigens are encoded by
missense mutations in exon 7.105The blood type O allele has a single nucleotide deletion, found
in G261 in exon 6. This deleted nucleotide is not found in A and B sequences. This deletion
causes a frame shift that alters the protein sequence after amino acid 88 and introduces a stop
codon after nucleotide 352 in the consensus sequence. Enzyme activity is never achieved after
the pre-catalytic translation stop signal at amino acid 117.110 The O (H) locus is localized on the
long arm of chromosome 19 at position q13.3.111 As earlier stated, the ABO antigens are not
confined to RBCs, but are also found in other organs, various secretions, and tissues. The ability
to secret ABO antigens is closely linked to the H locus and is found on chromosome 19q13.3.
This gene is known as the secretor gene (Se/se). Individuals who have the homozygous or
heterozygous Se gene, are able to secrete ABO active material according to their genotype. In
contrast, individuals who are homozygous for the ``se`` gene are not able to secret antigens.103
The formation of the ABO antigens is a multistep process that involves a number of different
enzymes and pathways depending on an individual’s ABO blood type ( Figure 6).115
28
Adapted from Hosoi, EJ Med Invest.2008;55:174-183
Figure 6. Biosynthesis of the ABO antigens.
Although the values somewhat differ, the average number of ABO antigen sites per RBC are in
the range of 1.5-2 million.111 All RBCs, independent of the ABO blood type have O(H) antigen
present and the subtype determines the number of ABO determinant sites per RBC (Table
4).103,111,116
Table 4. ABO antigen sites on the RBC.104,111,116
Blood Type A sites B sites O(H) sites
A1 910,000-1,300,000 - 70,000-170,000
A2 160,000-290,000 - 1,080,000-1,210,000
B - 610,000-830,000 540,000-760,000
O - - 1,590,000-1,740,000
29
1.3.3 ABO subtypes
In addition to the 4 major types A1, B, A1B and O there are additional subtypes. Subtypes of
ABO are distinguished by decreased amounts of A, B, or O(H) antigens on the RBCs and they
are classified based on the differences in the strength of agglutination of RBCs with anti-A, anti-
B, and anti-A,B reagents.117 Blood type A has the most variation and is classified by the quantity
of A-antigen present. A1 is considered the “normal” A blood type with the highest number of A
antigens present.117 In decreasing order by the quantity of A-antigen, other subtypes include: A2,
A3, Ax, Aend, Am, and Ael.111,115 In general 80% of people who are blood type A are A1 and the
remaining 20% are A2.115 A1 is said to be more branched (Figure 5) and has a greater number of
A antigenic sites (Table 4).117 The difference between A1 and A2 can be observed by the
agglutination of A1 RBCs with anti-A1 lectin extract of Dolichos biflorus seeds. To date, studies
have shown that the A transferase of A1 and A2 are both N-acetyl-galactosaminyltransferases,
however their kinetic properties differ slightly. The α-1,3-N-acetylgalactosaminyltransferase
activity is 5-10 times higher in A1 than that of A2.111 The A2 enzyme adds only a single N-
acetylgalactosamine group on the H substance and modifies fewer O (H) antigens.118 Studies
have shown that people of A2 subtype can have antibodies against A1.103,119
Similarly, subtypes of
blood type B are classified by the quantity of B antigen, and the amount of B antigen decreases
in the order B, B3, Bx, Bm, and Bel. 108,111
1.3.4 ABO antibodies
Anti-A and anti-B antibodies of IgM and IgG origin were long thought to be naturally occurring
and produced at birth. However, the discovery of the presence of A and B antigens on plants,
bacteria, and parasites led to the current widely accepted position that initial formation of anti-A
and anti-B antibodies are likely stimulated in newborns by exposure to environmental antigens in
the aerodigestive tract which are similar to or identical in structure to the A and B antigens found
30
on these living organisms.108,120,121 This was demonstrated in a classical experiment performed
on White Leghorn chickens raised in a germ free environment. Initially, the chickens had no
anti-A or anti-B titers. However, when exposed to Escherichia coli (E.coli), which has been
shown to express B antigen, chickens formed anti-B antibodies.108 This model demonstrates how
antibodies directed against RBC antigens may result from “natural” exposure to carbohydrates
that mimic some blood type antigens.
RBC antibodies cause breakdown of the RBCs by either C3b-mediated cell phagocytosis or
through direct lysis due to activation of the terminal complement components (C5b-9). C3b
mediated RBC phagocytosis is referred to as extravascular immune hemolysis and is the most
common.122 This is the removal and destruction of RBCs by macrophages of the spleen and liver.
During the normal aging of RBCs in circulation, RBCs are destroyed and degraded by
macrophages through the extravascular pathway. This pathway is primarily mediated by IgG1
and IgG3 as splenic macrophages have receptors specific for Fc fragments.123
1.3.5 ABO and infectious diseases
Many studies have attempted to establish a relationship between the ABO blood type system and
various infectious diseases, and recent studies have focused on establishing a scientific rational
for these statistical relationships.10,124-128 The most convincing statistical associations have been
found to be between ABO and diseases such as cancer, peptic ulcers, coagulation and infection
from various viruses and pathogens.124,129,130 In 1954, Aird et al reported that type O individuals
were 50% more likely to have duodenal ulcers than type A, B and AB individuals. 130 The
rationale for this observation was that the blood type Leb, which is closely associated with the
ABO system, is lower in individuals of blood type A. Boren et al, found that Leb is a receptor for
Helicobacter pylori.18,131 Another group found that individuals of blood type O were more likely
31
to develop hemorrhage rather than deep venous thrombosis, the latter more common in blood
type A individuals. This phenomenon may be attributed to the fact that type A individuals have
higher levels of factor VIII (anti-hemophilic globulin/von Willebrand factor levels), and thus a
higher than average level of coagulation capacity.18,132
More relevant to the hypothesis of this thesis is the relationship between the ABO blood types
and infectious diseases. It has been shown that many gram-negative organisms, such as E. coli,
have chemical moieties resembling either the A or B antigen. E. coli itself is said to have A and
B -like antigen present on its surface, thereby stimulating anti-A and anti-B antibodies. A study
done in 1978 by Marsh et al demonstrated how intestinal E .coli stimulated the production of
anti-B antibody in a 20 day old child.133 There are a number of very interesting associations and
while some are relatively minor (e.g. E. coli and blood type A and B), some relationships
between ABO blood types and infectious diseases have been speculated to account for the
various worldwide distribution of the ABO blood type. It has been suggested that the distribution
of ABO blood types in various parts of the world may have been influenced by the occurrence of
endemic and epidemic diseases such as small pox and plague.124,134 Association studies have
shown there is an increased incidence of small pox in people of blood type A. Small pox has
been shown to express A-like antigens, and during viremia the virus is thought to be a cognate
target of anti-A antibody. This suggests that individuals with blood type O or B who contract
small pox may have an immunological advantage over those who are blood type A. 124,126,135
Conversely, the pathogenesis of the plague and cholera has been said to be influenced by blood
type O. It has been reported that an “H-like” antigen is expressed on the plague bacilli and
Vibrio cholera. Therefore, individuals with blood type O may tolerate these bacterial infections
better than individuals with blood type A or B.18
32
P. falciparum malaria is an infectious disease that may express A and B-like antigens. It has been
shown that patients with P. falciparum malaria have higher anti-A and anti-B agglutination titer
when compared with those who were test negative for malaria. 125,128,136 Gonzales and
colleagues were the first to compare the anti-A and anti-B titers of sera from blood type O
patients infected with malaria and from individuals blood type O negative for malaria. They
found the anti-A titers were ~250 x higher for individuals infected with P. falciparum, and anti-B
titers were 32 x higher.125 These datas alone suggest that P. falciparum may express A- and B-
like antigens and may be recognized by the immune system as “non-self” sooner in individuals
with blood type O. This may be due to an additional antibody (anti-A and anti-B antibodies)
immune response alongside the traditional immune response to P. falciparum infected RBCs.125
1.3.6 Geographic distribution of ABO blood types
There are striking differences with respect to the frequency and distribution of the ABO blood
types between populations in varied geographic locations. These differences are related to a
number of factors including natural selection, genetic drift, founder effect, and mutations.
Although the frequency of the ABO blood type distribution worldwide is not exactly known, it is
commonly accepted that blood type O is the most common (45%), followed by type A (40%), B
(11%) and AB (4%) .137 However some blood types, like O in Africa, are more prevalent in
certain geographical locations and ethnic groups .This leads to two questions: 1) if blood type O
is determined by the recessive gene, what occurred to make the O phenotype so common?; and
2) if A and B are dominant alleles, why are these two phenotypes not predominant in any given
population? To answer these questions, one must look at the distribution of ABO across the
world and look at the factors that may influence positive selection for the O phenotype. It is
important to approach this question from three perspectives: 1) how do the frequencies of ABO
differ in identified ethnic populations?; 2) where do these ethnic populations predominate
33
geographically?; and 3) what factors (including historical health epidemics) may have occurred
to influence the outcome?
In temperate regions above the tropic of Cancer and below the tropic of Capricorn, blood type A
is more prevalent. In Asia, blood type B is the most common blood type and around the equator,
especially sub-Saharan Africa, blood type O is the most prevalent. When comparing ABO
frequencies in certain parts of the world to the presence or absence of malaria, it appears as
though blood type O is prevalent in areas of endemic malaria where certain RBC polymorphic
traits are also elevated (Table 1, Section 1.1.1).3,20-25
If one is to consider ethnicity alone, amongst Caucasians, the distribution of those with either
blood type A and O is equal (44% each), while those with blood type B is 9% ( Table 4).108 This
implies that neither the A nor O blood type is favored or advantageous. However, there is a
significant difference when comparing the A and O blood types in those of African descent. The
frequency of blood type O is approximately 2 times greater than that of blood type A and 3 times
greater than B (O=49% ,A=27% , B=19%) (Table 5). 108 This suggests that O is being selected
for within this population. These data are even more interesting given that blood type O is a
recessive gene. Clearly, it is important to further examine the relationship between people of
African descent, ABO, and geographic factors (and the infectious diseases that reside there).
34
Table 5. ABO frequencies in human ethnic populations.
Blood Type Caucasian (%) Black African
(%)
A1
A2
B
AB
O
34
10
9
4
44
19
8
19
4
49
Adapted from Gibbs, FL et al. Blood group systems, ABH and Lewis. American Association of Blood Banks.1986.
35
SECTION 4: MALARIA, RED CELL POLYMORPHISMS AND NATURAL SELECTION
Malaria is one of the leading causes of deaths worldwide from a single infectious agent, killing
~1 million people per year, 85% of which are children under the age of five.1 P. falciparum
malaria has made a larger “footprint” on the human genome than any other pathogen. It has co-
evolved with the human population and selected for survival genes by causing fatalities in
individuals before the age of reproduction. 3 The current form of P. falciparum has existed for
over 8 million years and caused massive mortality among modern humans and our hominid
ancestors. Any gene conferring protection from fatal P. falciparum infection has undeniably
been subjected to the true process of natural selection.
36
1.4.1 RBC polymorphisms
The RBC is a complicated cell, with a complex array of intrinsic and extrinsic proteins,
carbohydrates, receptors, transporters, and enzymes, all of which have distinct functions. P.
falciparum spends most of its growth cycle in the intra-erythrocytic stage which is the stage
when disease pathogenesis occurs. The relationship between the RBC and the parasite is
complex and integrated; the parasite is RBC centric. Any defect directly affecting a membrane
component can modify the shape of the RBC and reduce its survival capacity, and ultimately
create an inhospitable environment for the malaria parasite to invade and grow within. 77 The
same effect may result from molecular lesions of cytoplasmic proteins or enzymes, all of which
could contribute to, or even cause, cell death.
Over time, the morbidity and mortality of malaria has produced great selection pressures in
affected populations for mutations that reduce the impact of the disease. There is strong evidence
supporting an association between severity of malaria, RBC polymorphisms, the geographic
distribution of malaria transmission, and the increased frequency of various polymorphic traits.
This is also known as the “malaria hypothesis” as originally proposed by Haldane, which states “
common abnormalities in RBCs have been selected because of the fitness advantage they confer
against malaria”.51
Inherited RBC disorders with altered membrane and functions can be broadly divided into three
classes. The first class encompasses altered functions due to mutations in the various membrane
or cytoskeletal proteins, including hereditary disorders such as elliptocytosis (HE), ovalocytosis
(HO), and stomatocytosis.138 The second major class of disorders include altered function due to
secondary effects on the membrane resulting from mutations in globin gene such as sickle cell
disease (HbS) and α-and ϐ-thalaseemia.139,140 A consistent feature of these two classes is
37
decreased cell deformability. The structural integrity of the membrane allows the RBC to
perform its biological role of oxygen transport and to deform reversibly during flow.77 The final
class of disorders is whereby a key enzyme involved in the glycolysis or the pentose pathway has
an altered structure, thereby impairing the ability of the RBC to withstand oxidative stress
conditions.77 Examples of this condition include G6PD deficiency and pyruvate kinase (PK)
deficiency. 141
Most RBC diseases are detrimental if a person is homozygous for the defect, however,
heterozygosity for disease-causing alleles of several RBC disorders have been shown to confer
protection against severe malaria.
1.4.1.1 Hemoglobin mutations
Sickle cell anemia
Sickle cell anemia (HbS) is an inherited disorder of hemoglobin and is due to a single nucleotide
substitution of ϐ6 valine for glutamic acid in the ϐ-globin gene (known as ϐs-globin).15 In
homozygous sickle cell anemia (HbS), both a lleles of the ϐs-globin are inherited resulting in a
mutant hemoglobin.139 The life span of the RBC in HbS is significantly shortened as sickle cells
are usually cleared after about 10-20 days.142 During the deoxygenation stage, the HbS
polymerizes causing the RBCs to become abnormally rigid and non deformable leading to an
irregular sickled shape.143 Sickled Hb has the ability to undergo auto-oxidation in the presence of
oxygen to produce superoxide and hydroxyl radicals in RBCs. In addition to the increase of
reactive oxygen species, there is also a decrease in antioxidant enzymes and oxygen radical
scavengers (e.g., glutathione peroxidase and superoxide dismutase) which further leads to
abnormalities in the RBC membrane. 144-148 HbS exhibits accelerated auto oxidation at a rate of
38
1.7 times that of normal HbA.149,150 In people who are heterozygotic and only have one sickle
gene, this trait is referred to as HbAS or sickle trait. Sickle cell trait it usually regarded as a
benign condition and rarely has complications. This is due to the fact that 55-75% of the
hemoglobin in circulation is normal, and hemoglobin concentrations and other indices including
RBC life span are normal.16,151
HbS was one of the first of the structural hemoglobin variants to be associated with malaria
protection. It has been estimated that 300 million people worldwide carry the sickle cell trait,
with the highest concentration in the African region where P. falciparum is present.152 In sub-
Saharan Africa, there is an allelic frequency of the HbS gene of 0.15, suggesting that 30% of
people carry the sickle cell trait.153 This apparent natural selection for the sickle gene is referred
to as “balanced polymorphism” in that the polymorphic trait is relatively protected from P.
falciparum. It has been shown that heterozygotes (HbAS) are at least 90% protected from
developing severe and lethal malaria, but are not protected from asymptomatic parasitemia.154,155
Thalassemia
Thalassaemia genes are widely distributed in the world but are found most often among people
in the Mediterranean, the Middle East, and Southern Asia. Thalassaemia is an inherited
autosomal recessive blood disorder where the genetic defect results in the reduction of one of the
globin chains that make hemoglobin. There are two primary types of thalassaemia: α- and ϐ-
thalassaemia. Both are caused by more than 150 different mutations, most of which are single
nucleotide substitutions.13 Both of these disorders involve the loss of either the α- or ϐ-chain of
the molecule.140 Some mutations completely eliminate the α-or ϐ-chain, whereas others dampen,
but do not abolish globin synthesis. J.B.S Haldane was the first to propose that the high gene
frequency of thalassaemia in certain populations reflected protection against severe disease by
39
heterozygous individuals.13 It is believed that the potentially lethal thalassaemia gene is retained
in the population because it provides some protection from malaria in the heterozygous state.
Case controlled studies in Papua, New Guinea and Africa have shown that the homozygous and
heterozygous states for ϐ-thalassaemia both protect against severe disease, where as α-
thalassaemias offer approximately 50% reduction against severe malaria anemia.17,156
1.4.1.2 RBC enzymes
G6PD deficiency
G6PD deficiency is the most common human RBC enzyme deficiency. G6PD provides RBCs
with crucial protection against oxidant damage. G6PD converts glucose-6-phosphate (the initial
product of glycolysis) into 6-phosphogluconate (6PD), while converting NADP to NADPH
which is essential for the NADPH-dependent enzyme methemoglobin reductase. 13 Although
G6PD deficiency is caused by point mutations that reduce enzymatic activity there are a few
mutants where enzymatic activity is normal or even enhanced. There is a striking overlap
between the areas where G6PD deficiency is common and where P. falciparum malaria is
endemic, providing circumstantial evidence that G6PD deficiency confers protection against
severe malaria.157 There have been a number of case-control studies which examined the
relationship between G6PD-deficient individuals and protection from malaria.8,14,157 Amongst
these studies is the study conducted by Ruwende et al in 1998, who demonstrated that there is a
level of protection of 50% against severe malaria in heterozygous females and hemizygous
males.158
1.4.1.3 RBC membrane disorders
RBC Duffy Negative
40
The Duffy negative phenotype is caused by a single nucleotide substitution polymorphism in the
promoter region of the gene for the Duffy antigen. The Duffy antigen has been identified as a
chemokine receptor as well as an essential receptor for P. vivax merozoites.159,160 Individuals that
do not express the Duffy antigen are protected from P. vivax. Barnwell et al showed that P. vivax
merozoites are incapable of invading Duffy negative RBCs.55
South Asian Ovalocytosis
A deletion in the structural protein band 3 results in a condition known as South Asian
ovalocytosis (SAO). This condition is only present in a heterozygous form, as no infants
homozygous for this band 3 gene deletion survive intrauterine development.161 This
polymorphism is highly prevalent in Melanesian populations where P. falciparum is prevalent.162
It has been shown that carriers of this trait are at a reduced risk of infection with P. falciparum
due to impaired invasion and multiplication. 163, 19
1.4.2 Inherited disease specific mechanisms of protection
Based on studies of blood disorders such as those noted above, it is evident that genetic factors of
the host contribute to the variability and disease complexity of P. falciparum and that there is
also a relationship between polymorphic RBCs and protection from disease severity of P.
falciparum malaria. Carriers of HbAS, α- and ϐ- thalassemia, G6PD deficiency, Duffy negative
and ovalocytosis are all protected from developing severe malaria and are at a survival
advantage.
However, there is no clear consensus regarding how hemoglobinopathies and defective RBC
enzymes and membranes are able to confer protection from malaria. Each RBC polymorphism
comes with its own pathology; HbS causes auto oxidation of the hemoglobin, ϐ-thalassemia
41
causes ineffective erythropoiesis, α-thalassemia is responsible for a mild hemolytic state, G6PD
deficiency causes accelerated oxidative membrane damage, and SAO causes sodium and
potassium to leak out of the cell.87,140,143,164 A number of mechanisms of protection have been
proposed, including decreased invasion and growth of P. falciparum malaria in variant RBCs,
decreased rosetting or cytoadherence, and increased removal of infected RBCs by the innate
immune system.
1.4.2.1 Invasion and growth
The process of merozoite invasion and growth involves a complex sequence of events that
includes a number of specific interactions and requires a specific environment to grow. Any
deviation from the normal human RBC might result in inhibition of P. falciparum to invade or
grow within the RBC. Studies on the invasion and maturation of P. falciparum into RBCs
carrying the sickle cell, thalassaemia, G6PD-deficiency and SAO trait have been controversial.
There have been reports of decreased growth of P. falciparum malaria in HbAS, G6PD-
deficiency, and ϐ-thalassemia RBCs. 165-169 Studies have suggested that oxygen radicals formed
in RBCs with the sickle cell trait or are G6PD deficient, slow growth of P. falciparum as sickle
trait and G6PD deficient RBCs produce higher levels of the superoxide anion and hydrogen
peroxide than normal RBCs.168,170 Studies showing inhibition of maturation of P. falciparum in
RBCs with the sickle cell trait were incubated at low oxygen tension which may not represent an
appropriate experimental condition since RBCs from those with the sickle trait do not sickle at
normal oxygen tensions. The growth conditions in these studies also appear to have been
inadequate, as these cells are sensitive to nutrient deprivation and were likely dehydrated even in
the non-parasitized state.171,172 Better designed studies found no difference in parasite growth in
HbAS, G6PD deficient, or ϐ-thalassemia cells. 68,168,173 SAO RBCs have been shown to resist
invasion by malarial parasites in vitro. This has been attributed to greater rigidity of ovalocytic
42
RBCs.174,175 Studies examining the invasion of other species of Plasmodium into RBC variants
have consistently reported that P. vivax malaria cannot invade into RBCs homozygous for a null
Duffy allele, as Duffy negative cells are missing an essential antigen (Fy6) for invasion.160
1.4.2.2 Cytoadherence and rosetting
Decreased cytoadherence, (the process of infected RBC adhesion to the microvascular
endothelium), and decreased rosetting (binding of infected RBCs to uninfected RBCs) of
polymorphic RBCs have been suggested as potential mechanisms of p rotection from severe
malaria. It has been reported that infected HbAS cells display impaired cytoadherence due to
reduced PfEMP-1 expression on the RBC surface.176 Udomsangpetch et al found that there was a
significant decrease in the levels of P. falciparum antigens associated with the membrane in
infected thalassemic RBCs, and as a result, cytoadherence and rosetting of thalassemic RBCs
was greatly reduced.177
1.4.2.3 Clearance of infected polymorphic RBCs
Polymorphic RBCs already have increased susceptibility to RBC membrane damage, targeting
them for clearance. The interaction between the parasite within the defective RBC and the host
innate immune system represents a final putative mechanism of protection to malaria. Variant
RBCs senesce at a faster rate than normal RBCs, and are marked for phagocytosis at a greater
rate.68 A number of studies have compared the phagocytosis of infected polymorphic RBCs to
infected normal controls.68,178-181 There is an growing body of evidence suggesting that infected
variant RBCs are cleared more efficiently by the innate immune system than infected normal
RBCs. It has been consistently shown that there is increased phagocytosis of malaria infected
HbAS, G6PD deficient and thalassemic RBCs than infected normal controls.66,68,178,180,182 Ayi et
al demonstrated accelerated removal of ring stage infected RBCs carrying genes for HbAS,
43
G6PD deficiency and α-and ϐ-thalassaemia.68,180 The RBC surface of non- infected variant RBCs
possess abnormalities that are already recognized by pattern recognition receptors and other
components of the innate immune system, and these are likely to be further modified by malarial
infection, thereby enhancing the possibility of recognition and removal by macrophages. In
another study, it was demonstrated that, compared to the baseline of non-infected controls, non-
infected RBCs from individuals with HbAS and ϐ-thalassemia had elevated membrane bound
hemichromes, aggregated band 3, bound IgG and C3b.68 It was subsequently demonstrated that
ring stage parasitized HbAS and ϐ-thalassemia RBCs displayed even higher levels of aggregated
band 3, bound IgG and C3b, resulting in a dramatic increase in phagocytosis.68 Ring parasitized
HbAS and thalassemia cells are subject to multiple forms of oxidative stress, the first as a result
of the developing parasite and the second generated by the RBC modification itself. However,
phagocytosis of trophozoite- infected normal RBCs and trophozoite infected mutant RBCs was
not significantly different nor were the levels of hemichrome deposition, band 3 aggregation,
IgG or C3b deposition. It is thought that the damage inflicted by the mature parasite is so
significant that it will overshadow the baseline differences in normal and mutant RBCs as the
damage caused by both the polymorphism and the parasite reaches a maximium.68 The uptake of
ring stage parasites vs schizont stage parasites has been shown to be mediated by different
receptors. Ring stage-infected RBCs are phagocytosed similarly to senescent or oxidative
damaged RBCs, and IgG and complement signal for phagocytosis. However, schizonts are
phagocytosed more intensely by IgG, but also have additional markers such as exposure of PS 66.
A similar study by Cappadoro et al found that there was increased phagocytosis of infected
G6PD deficient cells when compared to normal infected cells. All three of these conditions share
a common trait in that they are characterized by increased generation of oxygen radicals or have
a decreased ability to survive oxidative damage. 140,150,180
44
1.4.3 ABO polymorphism and Plasmodium falciparum malaria
The ABO blood types were the first genetic polymorphisms found to vary significantly among
human populations. In the early 1900’s, a number of studies sought to establish an association of
ABO blood types with certain diseases (Section 1.3.5) One of the largest of these studies was
conducted in Russia by Rubaschkin and Leisermann in 1929.183 A χ2 test indicated a significant
association between malaria and the ABO blood types, with AB having the highest frequency of
malaria patients.183,184 However, a subsequent study by Parr et al in 1930 showed no difference
between ABO blood types and the frequency of malaria.183 The results of both studies were
considered to be ambiguous, and further research to establish a relationship between ABO and P.
falciparum was temporarily abandoned.184 However, over the last four decades, the relationship
between the ABO blood type system and P. falciparum disease severity has been revisited.
While the results of earlier studies (1926-1998) were contradictory and heterogeneous in study
design, recent studies have consistently found an association between ABO blood types and
disease severity of P. falciparum malaria.
The study by Rubaschkin and Leisermann, the associations noted between the ABO blood type
and a variety of infectious diseases, and the discovery of the protective effect of Duffy negative
phenotypes against P. vivax infection, renewed the interest of the research community in the
association between ABO and malaria. 125,160,185 Renewed interest in studying the relationship
between ABO and malaria emerged in the late 1970’s in Nigeria21,186 and led to further studies
between 1979-1998. These studies used parasitemia and incidence of malaria infection as
endpoints. In twelve cross-sectional studies and five case-control studies, parasitemia and
infection by P. falciparum were used as end point criteria. Ten cross sectional studies21,187-195
(completed in Africa and Columbia) and four case control studies 186,196-198 (conducted in Brazil ,
Africa, the UK and India) did not find a significant interaction between ABO and malaria risk
45
and overall parasitemia. Only two cross sectional studies 199,200 and one case control201 study
found an interaction between parasitemia levels and infection risk of malaria and ABO blood
type. All three of these studies were completed in India and these found that fewer blood type O
individuals were infected with P. falciparum and had lower parasitemia. From these studies, it
appeared that parasitemia and incidence of P. falciparum malaria were not associated with ABO
blood types. It was argued that parasitemia and incidence of malaria were not good predictors of
disease outcome in P. falciparum infection.
In 1998, investigators began using clinical severity, rather than incidence or parasitemia as end
points. In five well-designed studies relating ABO to the clinical severity, blood type A was
found to be more prevalent in severe malaria and blood type O was underrepresented.202-206
Severe malaria was defined according the World Health Organization criteria including:
unarounsable coma (Blantyre com score of ≤2 with other causes of coma excluded), severe
anemia (< 5g/dl), neurological impairments, repeated seizures, evidence of hepatic and/or renal
failure.8 Fischer et al compared 209 mild malaria outpatients to 280 severe malaria inpatients in
Zimbabwe. 204 They focused on the relationship between severe anemia and risk of coma.
Infected blood type A had overall lower hemoglobin levels than blood type O (p<0.02), and a
higher risk of coma when compared to non-type A(non-A= 3%, A=9%, p=0.008). A case control
study comparing children with severe malaria and uncomplicated controls was conducted in
Gabon by Lell et al in 1998. Blood type, amongst other RBC polymorphisms, was determined in
100 severe malaria cases and 100 mild malaria cases. 3 They found blood type A was
significantly associated with severe malaria (O.R 3.0; p<0.01).203 Pathirana et al analyzed the
ABO blood types in 243 Sri Lankan patients infected with P. falciparum malaria. 205 In total,
there were 163 patients diagnosed with uncomplicated malaria, 80 with severe malaria and 65
with severe non-malarial illness. The frequencies of the occurrence of each ABO blood type
46
were then compared to a population control. Patients with severe malaria were three times more
likely to be of blood type A versus O (p=0.005) and two times more likely to be of type B than O
(p=0.014).205 Another well-designed study was conducted by Rowe et al in 2007 in Mali. This
study assessed the ABO blood type status of 567 blood samples of Malian children. They
compared the distribution of ABO blood type frequencies in severe malaria cases to healthy
controls (N=124 matched triplicates with severe, uncomplicated malaria and healthy controls).
They also compared ABO frequencies in 65 matched triplicates in non-severe hyper-parasitemia
cases to uncomplicated and healthy controls. The distribution of the ABO frequencies in severe
malaria and the matched uncomplicated and healthy controls showed a decrease in the frequency
of blood type O in severe malaria cases when compared to uncomplicated and healthy controls
(SM:21, UM:44.4, HC:45.2%), and an increase of non-O blood types (SM:79.1,UM:55.6,
HC:54.8% ). It was found that blood type O conferred significant protection against severe
malaria when compared with the non-O blood types (O.R 0.34, p=0.0003). 206 In contrast to these
results, they found the frequency of blood type O in non-severe hyperparasitemia cases did not
differ from their matched controls (O.R 1.0, p=1.0), confirming from original studies that ABO
is not related to overall parasitemia levels. These studies all specifically compared ABO
phenotype to disease severity of P. falciparum malaria but did not discriminate between
genotypes, for example AO heterozygotes vs. AA homozygotes. 206
The most recent study examining the relationship between ABO and malaria, took a novel
approach by genotyping the ABO gene and then comparing ABO genotype frequencies to
disease severity. 202 This was the largest of all the ABO association studies using a sample
population of >9000 individuals across three African populations. When comparing ABO
phenotypes alone, this study strongly supported the hypothesis that individuals with blood type O
are protected from severe malaria. Blood type A was associated with increased risk of severe
47
disease (OR 1.33, p=0.0007), however they found AB individuals had the greatest risk of severe
disease (OR 1.59, p=0.006). The contribution of this study to the field is that they also compared
the genotype of ABO individuals to disease severity.202 There was no significant difference in
the risk estimate for severe malaria between AO and AA genotypes and the same was true for
BB vs. BO.
Placental malaria and ABO
The association between ABO blood types and placental malaria is unclear; two studies have
shown risk of active infection between blood types A and O is parity dependent, and the third
showed no difference in past placental infection in women with blood type O vs. non-O.207-209 In
these studies they compared parity, active placental infection, and bir th outcomes. The first study
in Gambia by Loscertales et al found there was a significant difference in the birth weight of
children born to type O mothers when compared to non-O mothers (O:2893±362 g, A: 2639±323
g, p=0.04) . The authors found parity-related susceptibility to P. falciparum placental infection
associated with O phenotype. They found increased prevalence of P. falciparum placental
malaria infection in primigravid women but a reduced prevalence in multigravid mothers with
blood type O. The following year, the same group replicated these findings in Malawian women.
207,209 Subsequently, another group of researchers in the Sudan, investigated the role of ABO
blood types on pregnancy in mothers and placental infection. They found that blood type O was
associated with past placental malaria infection in all gravities. They found a significant
difference in hemoglobin levels between O vs. non-O blood types (11.8 vs. 10.9, respectively,
p<0.05), but did not find a difference in birth parameters such as birth weight.208 It remains
controversial if blood type is associated with significant differences in birth weight, however
both studies found a higher hemoglobin level in mothers of blood type O. 207-209
48
Collectively these studies analyzing disease severity and ABO blood types, together with those
examining the association between ABO and placental malaria, have demonstrated compelling
evidence of a link between ABO polymorphism and P. falciparum disease severity. It appears
that blood type O protects from progression to severe disease and may offer a survival
advantage. More studies are needed to affirm the link between disease severity and ABO,
including studies which include mortality as the clinical end point. Examination of the direct
mechanism(s) by which blood type O offers protection would validate the relationship between
blood type O and disease severity of P. falciparum malaria.
1.4.4 Potential mechanisms of protection afforded by blood type O
As mentioned, there is mounting evidence documenting an association between the ABO blood
type and P. falciparum malaria. The evidence suggests that individuals with blood type O are as
susceptible to falciparum infection as individuals with blood types A and B. However, it appears
as though blood type O may be protected from progression to severe disease. The exact
mechanism of protection is unclear, however it has been postulated that protection may be
achieved through decreased parasite invasion and/or rosetting/cytoadherence of O RBCs
compared to A and B RBCs.
1.4.4.1 Invasion and maturation
Invasion of RBCs involves a number of specific receptor-ligand interactions, some of which are
associated with ABO blood types,(e.g., attachment of MSP-1 and EBA-175 to band 3 and
glycophorin A, both of which express ABO antigens).210 Only one study has examined invasion
of P. falciparum into A, B and O RBCs.211 Chung et al reported an increase in the invasion of A1
RBCs by P. falciparum when compared to invasion of A2, O or B RBCs (p<0.05). This was
shown in both sialic acid-dependent and acid- independent parasite strains. They further treated
49
A1 RBCs with N-acetyl-galactosaminidase to remove the terminal GalNAc on blood type A1.
They demonstrated a significant reduction in the invasion of A1 RBCs but not in “O treated”
cells. Although these findings support the hypothesis that ABO blood types and P. falciparum
are associated, however this type of comparative invasion assay has only been reported once and
needs to be repeated to establish its validity.
1.4.4.2 Rosetting and sequestration
Rosetting and cytoadherence have been implicated in the pathogenesis of cerebral malaria. It is
thought that the strong binding of infected RBCs to both uninfected RBCs and endothelial cells,
may block or impair the perfusion of blood through the microvasculature. This may cause tissue
ischemia and endothelial cell apoptosis, ultimately leading to severe and cerebral malaria. 212
Sequestration of infected RBCs may occur within the vascular beds of the brain and other vital
organs.42 Studies have shown a decrease of rosetting and cytoadherence in blood isolated from
infected individuals with polymorphic blood disorders such as thalassaemia and sickle cell
(Section 1.4.2.2) It has also been shown that the DBL-1α region on the parasitic virulence
antigen PfEMP-1 demonstrates lectin like properties and can bind to A and B antigens.44,56
Rosetting
Previous studies have shown that rosetting is significantly reduced in blood type O RBCs
compared to A, B and AB .56,206,213-216 In each of these studies, blood type O RBCs were able to
form rosettes, however they were smaller, had a lower rosetting rate and were more easily
disrupted. The A and B trisaccarchides are thought to act as receptors on uninfected RBCs and
therefore are able to bind to schizont-infected RBCs.56 Rowe et al demonstrated that when
soluble A antigen was added to culture, rosette formation was inhibited. 56 Furthermore,
treatment of blood type A with α-N acetyl- galactosaminidase decreased their rosetting
50
capacity.56 There are two important concerns with respect to the rosetting hypothesis; rosetting
has yet to be shown to occur in vivo and it is strain specific as there are A and B blood type
preferring strains (as reviewed in Uneke et al, 2007).217
Overall, the evidence implicating a relationship between disease severity, ABO blood type, and
rosetting is strong. One study which provides compelling support for the rosetting hypothesis
was undertaken by Rowe and colleagues. They examined the relationship between the ABO
blood type and P. falciparum infection in Mali. They found rosette frequencies were
significantly lower in parasite isolates from patients with blood type O compared to non-O blood
types , and that non-O blood types with a >5% rosette frequency were at a greater risk of severe
malaria ( OR 15.23 p<0.0001).206 It may be possible that rosetting is one mechanism that confers
protection associated with blood type O.
Sequestration
Another putative mechanism is increased adhesion of A- infected RBCs to the endothelium
(sequestration). A number of soluble adhesion molecules have been associated with ABO blood
type. Circulating levels of the procoagulant von Willebrand factor (vWf) have been shown to
vary between ABO blood types, with type O having lower levels of vWf than non-O types.218
This is most likely due to the fact that vWf of type A individuals seems to be more resistant to
the proteolysis by ADAMTS13 than O individuals.219 It has also been reported that platelet
CD36 also expresses A-antigen. 220 A model proposed by Cserti et al suggests that enhanced
sequestration of infected and non infected blood type A to the endothelium is caused by a
number of different cell types and associations.3 As previously noted, ABO antigens are present
on a number of different cell types, e.g., endothelial cells, and platelets.221 Cserti et al explain
that cytoadherence begins with the A or B uninfected RBC binding to an infected RBC through
51
A and B antigens and PfEMP-1. The infected cell further binds to the endothelium by either
binding to A or B antigens present on the endothelial cells, or by binding to blood type antigens
that are found on platelets or vWf. Therefore, because of a lack of lectin binding and interactions
between the A and B antigens and PfEMP-1, cytoadhesion of blood type O fails.
1.4.4.3 Additional mechanisms of protection
Preference of anopheline mosquitoes for specific blood types
A number of studies have examined the relationship between the feeding habits of the Anopheles
mosquito and a preference to feed on individuals of certain blood type.222-224 Two different
research groups have investigated the association between the attractiveness of individuals with
certain ABO blood types to the biting habits of the Anopheles gambiae mosquito. These groups
reported contradictory results. Wood et al found that the mosquitoes preferentially selected for
hosts of blood type O (Average number of blood meals: O; 4.4 and A; 3.3, p<0.05).223,224 A later
study done by Bryan et al found that there was a preference for blood type A individuals (70% of
all mosquitoes fed on blood type A, compared to the 24% that fed on blood type O, p value
unknown).222
A and B antigens on the surface of malaria
It has been suggested that individuals of blood type O may have a selective advantage as they
have both anti-A and anti-B antibodies in their serum.125 This would be a potential advantage
since certain micro-organisms appear to have antigenic determinants that resemble the A or B
antigen. A number of studies have shown that P. falciparum malaria parasites share blood type A
and (to a lesser extent) B antigens, and therefore would be better tolerated immunologically by
individuals who are blood type A.125,128,136,185,225 There is evidence that supports the presence of
52
A- and B- like determinants on P. falciparum because there is an increase in anti-A and anti-B
titers in patients with chronic malaria when compared to healthy controls. Oliver-Gonzalex et al
found that there was a 250-fold increase in anti-A antibodies in blood type O patients who had
malaria, and an 32-fold increase in the anti-B antibodies. 185 Antibodies directed against A and B
are IgM and IgG antibodies and therefore bind complement, activating the classical pathway. If
P. falciparum does have some antigenic determinants that resemble A and B antigens, this would
support the hypothesis that blood type O confers protection from developing severe malaria.
Several studies over the past two decades have demonstrated a link between the ABO blood
group system and severity of P.falciparum malaria.186,190,192,197,198,202-206,226,227 A recent meta-
analysis of data associating ABO and malaria, found an association between malaria severity and
blood groups A and B while milder disease was associated with group O.210 The mechanism of
protection from malaria associated with blood group O has been the subject of much speculation.
RBC polymorphisms, which are known to confer protection to clinically severe malaria, have
been shown to confer protection by defective invasion, decreased rosetting, and increased
clearance. Mechanisms investigated to understand why blood group O is protected, have been
based on similar RBC polymorphisms mechanisms. While the mechanism underlying the
protective effects of the O blood type remains unclear, the O blood type has been previously
associated with decreased rosetting when compared to A and B infected RBCs and decreased
invasion by P. falciparum. 56,177,206,211,215,228,229
Studies have reported increased phagocytic uptake of P. falciparum parasitized polymorphic
RBCs.68,69,178-180,182 Based upon these findings, we hypothesized that parasitized blood group O
RBCs are phagocytosed more efficiently than A and B parasitized RBCs.
53
SECTION 5- AIMS AND HYPOTHESIS
The purpose of this study was to examine the mechanistic basis by which blood type O may
contribute to protection against severe malaria including alterations in RBC invasion and
maturation as well as enhanced clearance by effector cells of the innate immune system.
1. Based on the interaction between molecules on the merozoite and the RBC (MSP-1:Band 3,
EBA 175:GPA) , we hypothesized that P. falciparum merozoites would preferentially invade
blood type A RBCs.
2. Where enhanced phagocytosis of polymorphic infected RBCs occurred, we hypothesized that
infected type O RBCs may be more efficiently cleared by the innate immune system than type A
and B RBCS.
The aims of this project were to identify novel mechanisms by which blood type O might confer
protection from severe malaria, to confirm that mechanism in vivo, and determine whether the
amount of O antigen present on the RBC correlates with the observed effect.
The present research study showed that, when compared to type A and B infected RBCs, there
was a significant increase in the phagocytosis of schizont- infected O RBCs. We conclude that
enhanced clearance of infected O RBCs may represent an additional putative mechanism by
which blood type O may contribute to protection against severe malaria.
54
PART II
MATERIALS AND METHODS
55
2.1 Reagents
Endotoxin–free RPMI 1640 and gentamicin were purchased from Invitrogen Life Technologies
(Burlington, ON, Canada). Human AB serum was purchased from Wisent Inc (St-Bruno,
Quebec, Canada). Diff–Quik staining kit and fetal calf serum (FCS) were both purchased from
Fisher Scientific (Ottawa, ON, Canada). Both FCS and human AB serum were heat- inactivated
for 30 minutes at 55˚C before use to remove complement activity. Alanine was purchased from
Sigma Aldrich (Oakville, Ontario, Canada). Mycoplasma removal agent was purchased from MP
Biochemical (Solon, Ohio, USA). Ficoll-Paque and Percoll was purchased from GE Healthcare
(Baie D’Urfé, Québec, Canada). NOVACLONE blood grouping reagent was purchased from
Dominion Biologicals Ltd (Dartmouth, Nova Scotia, Canada).
Mice
Male or female C57BL/6 mice aged 6-10 weeks (Charles River, Hollister, CA) were maintained
under pathogen-free conditions, and used for experiments. All experimental procedures
involving mice were conducted in accordance with the animal protocol approved by the
University of Toronto Animal Use Committee or the University Health Network Animal Care
Committee.
Ethics
Experiments involving mice were approved by the University of Toronto Animal Use
Committee or the University Health Network Animal Care Committee, as appropriate, and
performed in accordance with current institutional regulations.
56
P. falciparum culture
P.falciparum strains ITG and 3D7 (mycoplasma-free) were maintained in continuous culture as
previously described by Trager and Jensen.230 Strains were maintained at 2% hematocrit in R-
10G media (RPMI 1640 supplemented with 10% heat-inactivated human serum, 1.8 g/l Na2CO3,
6g/l HEPES, 25 mg/l gentamicin and 1.35 mg/l hypoxanthine, pH 7.4.) On a daily basis media
was changed, and flasks were infused with a slow current of a gas mixture containing 7% CO 2,
5% O2 and 88% N2.
2.2 Methods
RBC and serum isolation
Whole blood was donated from healthy non- immune individuals aged 20-54 years and collected
in BD Vacutainer Glass tubes containing ACD solution as anti-coagulant. RBCs were separated
from whole blood by as previously described. 231 Briefly, whole blood was layered on an 80%
Percoll gradient [80% (w/v) Percoll, 6% (w/v) mannitol, 10 mM glucose and 20% (v/v) PBS
10X] and spun for thirty minutes at 3000 RPM at 24 º C. The isolated RBCs were washed three
times in R-0G media (RPMI 1640 medium supplemented with 10mM glucose and 10g/L
gentamicin) and re-suspended in parasite growth medium R-10G media (RPMI 1640 containing
20 mM glucose, 2 mM glutamine, 6 g/L Hepes, 2 g NaHCO3, 10 g/L gentamicin, 10% human AB
serum and 1.35 mg/L hypoxanthine, pH 7.3) to approximately 20% hematocrit. Blood for serum
was collected in BD Vacutainer Glass tubes with no additive. Serum was separated from RBCs
by centrifugation at 1500 RPM for five minutes at 24ºC, and 200 μl aliquots were stored at -20°C
for future use. To ensure complement activity was maintained, each aliquot was thawed only
once and discarded after use.
57
Blood typing
Blood samples were typed for ABO blood type by standard hemagglutination techniques
according to manufacturer’s instructions. One drop o f DBL anti-A, anti-B, and anti-A,B were
added to labelled test tubes. Serum free, washed RBCs were re-suspended in PBS to a final
hematocrit of 10%. One drop of the RBC suspension was added to each test tube. Each tube was
mixed and then centrifuged for 30 seconds at 3400 rpm. The test tubes were then gently shaken
and the cells were examined for agglutination. For blood type A samples, cells were further
typed using A1 lectin (Dolichos biflorus) to further confirm presence or absence of the A1
antigen.
Monocyte isolation
Human monocytes were isolated and purified from the peripheral blood of healthy donors (non-
immune A or O type) and plated on glass cover slips in 24-well polystyrene plates as previously
described.83,232,233 Briefly, blood was drawn from donors using BD Vacutainers containing
sodium heparin as anticoagulant. Blood was immediately mixed with warm PBS in a 1:1 ratio,
and then carefully layered on Ficoll (25ml/15ml) and centrifuged at 1800 RPM for 30 minutes at
21ºC. The peripheral blood mononuclear cell (PBMC) layer was carefully removed using a
Pasteur pipette and washed three times with cold PBS. The PBMCs were then re-suspended in R-
10G FCS media (RPMI 1640 medium containing L-glutamine and HEPES, supplemented with
10 % heat- inactivated FCS and 25 mg/l gentamicin). The final volume and total number of cells
were adjusted to give a final concentration of 1.25x106 PBMCs/150μl/well. Each coverslip was
plated with 150 μl of suspension and incubated for 5 days at 5% CO2 in a humidified incubator at
37ºC.
58
Re-infection of ABO RBCs
P. falciparum cultures of clones ITG and 3D7 were purified on a Percoll-mannitol gradient
(3ml/6ml). Isolated schizonts were washed twice and then used to infect fresh non- immune
RBCs of different blood types (A, B and O). Final hematocrit and parasitemia used varied
depending on the experiment and will be discussed in further detail in each section. The newly
infected RBCs were incubated with R-10G human AB serum at 37ºC.58,230
Analysis of invasion and maturation
To assess parasite invasion and maturation, purified schizonts at 0.5% parasitemia were mixed
with non- immune A, B or O RBCs at a hematocrit of 2%, as previously described by Ayi et al.68
Slides were prepared by thin blood smears from cultures at 24 h and 72 h to assess invasion and
at 48 h and 96 h to assess maturation. Slides were stained with Diff-Quik, and 1000 RBCs were
examined microscopically. Percent parasitemia was determined as follows: [number of parasites
÷ number of total RBCs counted] x 100 in each field of view.
Parasitized RBC preparation for phagocytosis assay
Ring-stage and mature-stage parasites were obtained 24 hours and 48 hours, respectively, after
rupture of schizonts. The ring culture was spun and re-suspended in alanine-tris solution (300
µM alanine and 0.15 µM tris, pH 7.4 at a ratio 1:18) and incubated for 5 minutes at 37°C,
causing lysis of the mature-stage parasites while leaving the ring-stage parasites intact.234 The
culture was then washed and re-suspended in R-0G media, separated using Percoll-mannitol
gradient, and centrifuged at 1800 RPM for 20 minutes at room temperature. The pellet
containing uninfected RBCs and ring-stage parasites was then washed and incubated with
autologous non- immune serum. The final parasitemia was 15-20%. Mature parasite cultures
were synchronized with alanine for 15 minutes 24 hours after re- infection of ABO RBCs,
59
washed with the R-0G media, and re- incubated with the R-10G media containing the AB serum.
Mature-stage parasites were obtained 48 hours after rupture, and were spun and incubated with
autologous non- immune serum.
Phagocytosis in vitro
Parasitized and non-parasitized cultures were incubated with autologous non- immune serum for
30 minutes at 37˚C in a ratio of 1:1 (culture/serum). Cell suspensions were then washed twice
with the R-0G media, and re-suspended in 1 ml of R-0G media. An aliquot of 500 ul of each
sample (parasitemia 12-20% ring-stage or 5% mature-stage) were incubated in each well
containing approximately 1.25 x 105 macrophages adhered to the glass coverslip in culture plates
at ratio of 20-40:1 (parasitized RBCs: monocyte-derived macrophages). The plates were rotated
gently for 90 min (ring-stages) or 120 minutes (mature-stages) at 37°C in a 5% CO2 humidified
incubator. The ring stage phagocytosis assay was performed over a shorter period (45 minutes)
to prevent complete degradation of the ring stage cytoplasm (to allow accurate counting of
phagocytic uptake). After incubation, all RBCs that had not been internalized were removed by
hypotonic lysis with cold water to prevent counting infected RBCs not phagocytosed.
Macrophages were then fixed and stained using Diff-Quik. Phagocytosis was assessed by
counting the number of internalized parasites in 250 macrophages. Values were expressed in
percentage as: [number internalized parasites ÷ number of total macrophages counted] x 100.
RBC preparation and phagocytosis assays were preformed as previously described, 58,68,235 and
all the experiments were performed in duplicate, repeated at least three times with Plasmodium
falciparum strain ITG and then confirmed using the 3D7 strain.
60
Phagocytosis in vivo
To assess phagocytosis of infected A,B and O RBCs in vivo, 5x107 mature infected RBCs and
non- infected RBCs were injected into the peritoneal cavity of C57BL/6 mice as previously
described by Serghides et al.235 Briefly, 48 hours after re- infection, mature stage parasites were
isolated using a Percoll-mannitol gradient. The purified mature culture and the uninfected RBCs
were then incubated with autologous human serum in a 1:1 ratio (culture/serum), washed using
R-0G media , and then 5x107 opsonized mature infected RBCs and uninfected controls were
injected into the peritoneal cavity of C57BL/6 mice . Three hours after injection, peritoneal cells
were collected, and washed with RPMI-1640. The cells were then separated by centrifugation
and re-suspended in 500 µl of R-0G media. 150 μl aliquots of peritoneal cells from each mouse
were placed on a cover slip in a 24 well plate and allowed to adhere for 30 minutes. Cells on
coverslips were treated by hypotonic lysis to remove RBCs and stained with Diff-quick. In
addition, 200 µl of the suspension was lysed for 45 seconds with cold water, washed, cytospun at
800 RPM for 10 minutes and stained with Diff-quick and used for images. Images were acquired
with an Olympus BX41 microscope and an Infinity2 camera at 100 x magnification.
Statistical analysis
Statistical analysis was performed with Graphpad Prism 4 software (San Diego, CA, USA). To
confirm the normal distribution of data, all data sets were assessed using the Kolmogorov-
Smirnov test. Data sets that displayed normal distribution were analyzed by t-test or one way
ANOVA as appropriate, and data sets that did not display normal distribution were analyzed by
Mann-Whitney rank sum test or Kruskall-Wallis test, as appropriate. A general linear model was
used to analyze experiments with multiple independent variables (e.g., macrophage and RBC
61
type). To test the dose-dependent effect of the A antigen, we used linear regression analysis.
Conditions for all experiments were performed in duplicate, and each experiment was repeated at
least three times with strain ITG. Data are either shown as box plots representing the median,
inter-quartile range and range or as bar graphs representing the mean and ±SD. Differences with
a p<0.05 were considered statistically significant.
62
PART III
RESULTS
63
P. falciparum does not preferentially invade or mature within blood type A, B or O RBCS.
Several studies examining the protective nature of heterozygous and homozygous RBC
polymorphisms from P. falciparum malaria have found impaired invasion and maturation in
RBCs in vitro. This finding was observed in carriers of ovalocytosis, pyruvate kinase deficiency
and in thalassaemia. 68,178-180 Similarly Chung et al. reported a decrease of P. falciparum invasion
into O RBCs when compared to A1.211 Based on these results and the interactions between
merozoites and RBCs (Section 1.1.2), we hypothesized that invasion of the A RBC would be
increased compared to O. To test this hypothesis, we infected A, B and O RBCs and measured
the parasitemia daily for two cycles of growth. As shown in Figure 7, there was no significant
difference in the invasion of P. falciparum into A, B or O RBCs (p>0.05).211 In addition, there
was no significant difference in the maturation of P.falciparum within A, B and O RBCs
(p>0.05). Datas shown represent three independent experiments using P. falciparum strain ITG
and five different blood type A, two blood type B and five blood type O donors. To ensure these
observations were generalizable to other parasite lines, we confirmed these find ings with the P.
falciparum strain 3D7. Our data indicate that the parasitic invasion and growth in O RBCs is
normal, suggesting that protection against malaria in blood type O individuals is not due to
decreased invasion or maturation.
64
Figure 7. Similar invasion and growth of P. falciparum in A, B or O blood type RBCs.
Healthy RBCs from blood types A1, B and O donors were infected with P.falciparum strain ITG
schizonts. The initial inoculum was adjusted to 0.5% parasitemia. Parasitemia for invasion cycles
were assessed by thin blood smears at 24 and 72 hours after re- infection and 48 and 96 hours for
maturation cycles. Parasitemia was determined by counting the number of infected RBCs in
1000 RBCs. Data shown represent three independent experiments (A:n=7, B:n=5, O:n=7). Box
plots depict the median, interquartile range and range. There was no significant difference within
each cycle of invasion and maturation between blood types A, B and O RBCs, p>0.05; Kruskal-
Wallis test).
65
Human monocyte-derived macrophages phagocytose infected O RBCs more efficiently
than infected A and B RBCs.
Mounting evidence suggests that variant RBCs infected with P. falciparum malaria are cleared
more rapidly than normal RBCs.68,69,178-180,182 This has been proposed and examined in RBCs
heterozygous for G6PD, sickle cell anemia, -thalassaemia and pyruvate kinase deficiency.236
These studies compared the phagocytic uptake of polymorphic RBCs and normal RBCs infected
with ring-stage or mature schizont-stage parasites. It was consistently observed that polymorphic
RBCs are phagocytosed significantly more efficiently than normal RBCs, when infected with
ring-stage parasites. A similar trend has been observed in the clearance of mature stage infected
polymorphic RBCs. Based on these observations, we examined the phagocytic uptake of infected
A, B and O RBCs. Infected A, B and O RBCs were incubated with autologous non- immune
serum and then incubated with human monocyte-derived macrophages. We hypothesized that
infected O type RBCs would be preferentially phagocytosed compared to type A and B RBCs.
While there was a slight increase of phagocytosis of the ring stage infected O RBCs when
compared to infected A (p=0.34) and B RBCs (p=1.16), the difference did not reach statistical
significance (Figure 8).
Schizont-parasitized RBCs are more susceptible to phagocytic uptake as they express higher
levels of parasite antigens and are more susceptible to oxidative stress.69,94 This prompted us to
examine the uptake of schizont-stage parasitized A, B and O RBCs. The uptake of schizont-
parasitized O RBCs was approximately 2.5-fold higher than the uptake observed with the
infected A (p=0.0008) and B (p=0.004) RBCs (Figure 9). To ensure that the differences in
uptake were attributable to infection by P.falciparum, we used non- infected A, B and O RBCs as
controls. There was no significant difference in the uptake of non- infected A, B and O RBCs
(Figure 9).
66
Figure 8. Phagocytosis of ring stage infected A, B and O RBCs by human monocyte-derived
macrophages.
P. falciparum ring- infected ABO and non-infected ABO RBCs were incubated with autologous
non- immune serum and incubated in a 40:1 ratio with human monocyte-derived macrophages for
two hours. The phagocytic index for each RBC type was calculated by counting the number of
internalized parasites in 250 macrophages by microscopy and normalized to the average
phagocytic index of parasitized blood type A. Data represent pooled results from three
independent experiments using P. falciparum strain ITG, using at least three different donors in
each blood type (A:n=7, B:n=3, O:n=7). The bar graph represents the mean ± SD. There was no
significant difference in the uptake of infected O RBCs when compared to A or B infected RBCs
(p=0.34, p=1.16, respectively; Mann Whitney rank sum test with Bonferroni correction for
multiple comparisons).
***
**
67
Figure 9. Increased phagocytosis of schizont infected O RBCs compared to infected A and
B RBCs by human monocyte-derived macrophages.
P. falciparum schizont infected ABO and non- infected ABO RBCs were incubated with
autologous non- immune serum and then incubated with human monocyte-derived macrophages
at a 20:1 ratio for two hours. The phagocytic index was calculated by counting the number of
internalized parasites in 250 macrophages by microscopy. Data was then normalized to the
average phagocytic index of parasitized blood type A. Data represent three independent
experiments using P. falciparum strain ITG and each blood type is represented by at least three
different donors, (A:n=9, B:n=4, O:n=9). The box plots represent the median, interquartile and
complete range. There was an increase in the phagocytic uptake of infected O RBCs when
compared to the phagocytic uptake of both A and B infected RBCs (***, p<0.001 and **,
p<0.01, respectively; Mann Whitney rank sum test with Bonferroni correction for multiple
comparisons).
***
**
68
Enhanced phagocytosis of schizont-infected O RBCs is independent of macrophage donor
blood type.
ABO antigens are expressed in multiple cell types and not exclusively on RBCs , hence they are
frequently referred to as histo–blood type antigens.68,178,180,182,237 However, their expression
status on platelets, lymphocytes and macrophages is uncertain.221,238 In order to ensure that
preferential uptake of infected O RBCs was not dependent on the blood type of the macrophage
donor, we performed two phagocytosis assays in parallel, one with macrophages from an A
blood type donor and the other from an O donor. When comparing the uptake of infected A and
O RBCs, we observed that infected O RBCs had a higher phagocytic index (p=0.0001; two-way
ANOVA), independently of the blood type of the macrophage donor (Figure 10). No difference
was observed in the uptake of RBCs between macrophage donors of different ABO blood types
(p=0.552; two-way ANOVA).
69
Figure 10. Schizont infected O RBCS are preferentially phagocytosed independent of
macrophage donor blood type.
Blood was simultaneously drawn from A and O blood donors and monocytes were isolated on a
Percoll-mannitol gradient. Monocytes were then plated on glass coverslips and incubated at 37˚C
for 5 days. Blood donors were ABO blood typed by standard hemagglutinin techniques.
Schizont- infected blood type A and O cultures were incubated with autologous non- immune
serum and incubated with the isolated macrophages at a 20:1 ratio. The phagocytic index was
calculated by counting the number of internalized parasites within 250 macrophages. Data
represent three independent experiments using P. falciparum strain ITG and each blood type is
represented by at least three different donors, (A:n=8, B:n=8). The box plots represent the
median, interquartile and complete range. Data was normalized to the average phagocytic uptake
of infected blood type A by macrophages isolated from A donors. Using two-way analysis of
variance (ANOVA), the blood type of the infected cells was found to influence the uptake of
infected A and O RBCs (***, p<0.001), whereas the blood type of the macrophage donor was
found not to influence the uptake of infected A and O RBCs (p>0.05).
*
*
*** ***
70
Phagocytosis of infected O RBCs is increased in C57BL/6 mice.
To simulate phagocytosis by peripheral blood monocytes in vivo, Serghides et al developed a
method of measuring phagocytosis of P. falciparum using resident monocytes found in the
peritoneal cavity of C57BL/6 mice. Fifty million purified schizont-infected A, B and O RBCs
that had been previously incubated with autologous human serum were injected into the
peritoneal cavity of C57BL/6 mice. Based upon our previous findings, we hypothesized that the
resident monocytes would clear the infected O RBCs at a greater rate than the infected A and B
RBCs. As expected infected blood type O RBCs were phagocytosed more avidly than types A
and B (Figure 11a). There was a threefold increase in the uptake of infected blood type O RBCs
when compared to both infected blood types A and B RBCs (O vs. A p=0.03 and B p=0.04).
This is illustrated in Figure 11b showing internalization of infected non-O and O RBCs by
resident monocytes from C57BL/6 mice. By comparing panels A and B to O, one can observe a
substantial difference in the uptake of infected O RBCs when compared to A and B as there is a
significantly greater density of internalized infected O RBCs contained within the monocyte.
71
A)
B)
Non-O O
Blood Type
Figure 11. Peritoneal monocytes of C57BL/6 mice clear infected O RBCs more efficiently
than infected A or B RBC.
The peritoneal cavity of C57BL/6 mice were injected intraperitoneally with 5.0 x 107 purified
mature stage parasites cultivated in blood donated from A, B and O blood type donors. Three
hours after injection, resident monocytes were collected, washed, and plated on glass coverslips.
A) The phagocytic index was calculated by counting the number of internalized parasites within
250 monocytes and then data were normalized to the average phagocytic index of infected A
**
*
*
* *
72
RBCs. Data represent three independent experiments using P. falciparum strain ITG and each
blood type is represented by at least three different donors (A:n=5,B:n=3, O:n=5). Bar graphs
represent the mean ± SD. There was a significant increase in the phagocytic uptake of infected O
RBCs when compared to the phagocytic uptake of both A and B infected RBCs (*, p<0.05).
Statistical significance was assessed by Mann Whitney rank sum test and corrected using the
Bonferroni multiple comparison test. B) Panels show light-microscopy images of H & E stained
resident monocytes isolated from the peritoneal cavity of C57BL/6 mice that had been injected
with ITG infected A, B and O RBCs.
The amount of A and H antigen present on the RBC influences the phagocytic uptake of
infected A and O RBCs.
There are various subtypes in each ABO blood type, resulting in deviations in the number of
ABO antigens present and the density of the side chains.221 Blood type A has the most variation,
the most common variants being A1 and A2. A1 RBCs have approximately one million A
antigens per cell and they are more branched while A2 has only one forth the amount of A
antigen and is less branched, resulting in a higher number of O antigens119. Alternatively, one
could interpret the differences in subtypes by the number of O antigens present on the RBC as
they increase from O>A2>B>A1.104,117 We postulated that an increase/decrease of the O/A
antigen expression level could be associated with increased phagocytosis, which predicts that the
infected A2 RBCs would display enhanced phagocytosis versus infected A1 RBCs but less
efficiently than the infected O RBCs. A RBC donors were typed using Dolichos biflorus lectin,
which is A1 specific.104,238 In support of our hypothesis, infected A2 RBCs were cleared at an
intermediate rate between the infected A1 and O RBCs (Figure 12). Our finding reveals a dose-
dependent effect of A/H antigen on phagocytosis of P. falciparum infected cells, with decreasing
A antigen and/or increasing H antigen associated with increased phagocytosis (p-value for
trend=0.0049; linear regression).
73
Figure 12. Increasing phagocytosis of RBCs bearing decreasing A and increasing O surface
antigen.
A and O RBCs were typed using standard hemagglutinin techniques. Blood type A was further
classified into A1 or A2 subtype using Dolichos biflorus lectin. Infected A1, A2 and O RBCs were
incubated with autologous non- immune serum, and then incubated with human monocyte-
derived macrophages in a ratio of 20:1. The phagocytic index was calculated by counting the
number of internalized parasites within 250 macrophages and then data was normalized to the
average phagocytic index of infected A1 cells. Data represent three independent experiments
using P. falciparum strain ITG and each blood type is represented by at least three different
donors, (A:n=8, B:n=8,O:n=8). The box plots represent the median, interquartile and complete
range. Data was analyzed using linear regression and it was found that phagocytosis of infected
RBCs was dose-dependent on increased H and decreased A antigen (**, p<0.01).
*
**
**
74
PART IV
DISCUSSION
75
There is strong epidemiological evidence that the ABO phenotype modulates disease severity of
P. falciparum malaria, with blood type A being associated with severe disease and blood type O
with a milder form of disease.202-206 This association is further supported by the increased
prevalence of blood type O in malaria-endemic areas. However, the exact mechanism by which
blood type O protects against severe malaria remains unclear. A number of studies have
indicated that decreased rosetting is likely a major mechanism by which P. falciparum-infected
O RBCs might protect against severe malaria.56,206,213,215 In addition , one study has shown a
preference for P. falciparum invasion into blood type A1 RBCs when compared to A2, B or O
RBCs.211 Although controversy persists 68,167,239,240, there is some evidence that these methods of
protection parallel those of RBC polymorphic traits such as G6PD deficiency, thalassaemia and
sickle cell. In addition to these mechanisms, increased clearance of infected-G6PD deficient,
thalassaemia and HbAS RBCs by human monocyte-derived macrophages has also been
demonstrated in RBCs hemizygote for these traits.66,68,180,182
i) Blood type O protects against severe disease caused by P.falciparum malaria and was
selected for in malaria endemic regions.
The association between ABO and P. falciparum malaria has been studied extensively and
earlier studies generally found that there was no association between ABO blood type and P.
falciparum (Section 1.4.3). 21,186-198 The majority of these earlier studies used parasitemia or
incidence of infection as a clinical endpoint. Parasitemia is a poor predictor of severity, partly
because it does not account for sequestered parasites. Inadequate sample size was a major
criticism of these studies, and many were flawed by inappropriate design or lack of controls.3
However, the most recent studies have found a significant association between ABO and P.
falciparum malaria.202-206 These studies had adequate sample sizes, used clinical severity as the
76
clinical outcome, and included children in their study. These studies concluded that blood type O
protects against severe malaria.
One of the unresolved criticisms regarding the ABO and P.falciparum malaria hypothesis is if
blood type O is a protective mechanism against malaria, then why do blood types A and B
continue to exist in Africa. While natural selection remains the primary explanation for adaptive
evolution, there are a number of factors that could have influenced the distribution of the ABO
blood type. The outcome of natural selection of ABO blood types is also due to other endemic
and epidemic diseases such as the plague and smallpox, which have been demonstrated to
express O and A antigens, respectively. Small pox was endemic in South East Asia and India,
therefore persons of blood type B or O became resistant to small pox infection and developed a
selective survival advantage .241 This would account for the higher proportion of type B
individuals in India but not for the lower incidence of blood type O. The lower incident of type O
in India , has been attributed to the past prevalence of the plague, as the plague posses an antige n
similar to blood antigen O.134 The distribution of blood type antigens may have also been due to
genetic drift and founder effect, but did not nearly have the same impact as natural selection.107
ii) Decreased rosetting of infected O RBCs.
A number of studies have shown that infected A RBCS in vitro form rosettes more readily than
infected- blood type O.56,206,213-215 However, rosette aggregates of infected RBCs to uninfected
RBCs have only rarely been described in post mortem studies in malaria and it is unknown if
rosetting occurs avidly in vivo.242,243 Decreased rosetting of blood type O may have a role in
protection from severe disease , however only strains such as FCR3 and TM form rosettes, and
certain strains have a higher affinity of binding to certain A and B blood types.213 These issues
undermine the rosetting hypothesis and need further exploration. It also remains unclear whether
77
rosetting is important in the pathogenesis of malaria or if it is a marker for some other factor
which mediates the disease process.217 If rosetting occurs in vivo, it may occur in combination
with other mechanisms of protection.
The purpose of the present study was to evaluate other potential mechanisms by which blood
type O offers protection against severe forms of the disease. To do so, we investigated the
growth cycle of P. falciparum in ABO RBCs as well as examined a role for differential clearance
of infected RBCs by macrophages.
iii) Decreased invasion and maturation of P.falciparum were not validated as a mechanism of
blood type O protection.
The purpose of the invasion study was to replicate and evaluate the results of the study by Chung
et al in 2005. This study indicated a significant preference for invasion of P. falciparum
merozoites into A1 RBCs, when compared to A2, B and O RBCs. Based on Chung’s study it was
hypothesized that blood type O may confer protection against severe malaria by reducing
invasion, thereby leading to control of parasitemia. However, the outcome of our invasion
studies demonstrated that P. falciparum invaded and matured similarly in A1, B, and O RBCs for
the first two cycles of parasite growth (Figure 7). There were no trends detected regarding
increased invasion into blood type A RBC. Our data did not support the hypothesis that there is
reduced invasion of P.falciparum into blood type O RBCs. Results of our study parallel findings
of other studies that have examined parasite invasion and growth in normal and polymorphic
(HbS, thalassaemia) RBCs and found no difference.68 In general, the results of studies assessing
invasion and maturation of P. falciparum into RBCs with various polymorphic traits have been
contradictory.
78
These difficulties in reproducing invasion/maturation differences have also been seen in studies
of sickle cell, thalassaemia, and G6PD deficiency. While Ayi et al, observed there was no
difference in the growth of P.falciparum in sickle cell, thalassaemia and G6PD deficient RBCs,
others have found there is a significant decrease in the invasion of HbAS and G6PD deficient
RBCs.68,168,169 The differences have been attributed to different parasite culture conditions, and
inappropriate experimental culture conditions for those studies that did report a difference.244
While it is not clear why the results of Chung et al concerning preferential invasion of A RBC
could not be replicated, there are a number of potential explanations for the discrepancy. It is
unknown whether specific parasite strains may preferentially invade different ABO blood types.
Chung et al used a panel of eight P.falciparum strains (Dd2, D10, K1, FCR3, ITOA4, 3D7, 1916
and 7G8) and combined the data from each separate strain into one outcome. In this study, we
used two different strains P. falciparum ITG (chloroquine resistant, and potentially sialic acid
dependent) and 3D7 (chloroquine sensitive, sialic acid independent) and evaluated each strain as
unique sets of data. Chung et al did not indicate if specific strains preferentially invade ABO
RBCs. Chung et al claimed there was a preference for A1 RBCs in parasites utilizing both sialic
acid dependent and independent pathways, however this data was not shown. A number of
parasite strains use sialic acid dependent invasion pathways (Dd2,D10,K1, FCR3)211 and studies
have shown that membrane sialic acid content is higher in type O blood.245 Chung et al do not
state if the parasites utilizing the sialic acid pathway significantly invaded into A1 RBC. It is
possible that if there was a preference for invasion into A1 RBCs, it was only a minor increase. It
has been shown that parasite strains have specific receptors for certain blood types when
rosetting, for example R+PA1 prefers A RBCs while TM284 prefers B RBCs.213,228 Invasion of
P. falciparum into A, B and O RBCs has not been studied sufficiently enough to know if there
are parasite strains which may invade A,B or O RBCs preferentially.
79
Another potential explanation for the difference between studies could be that they used blood of
unknown age that had been previously frozen and thawed at least ten days prior. Previous studies
from our laboratory have demonstrated that the use of this kind of blood reduces invasion by
50% and parasites do not reliably invade previously frozen blood (unpublished data). As such, in
order to maintain proper control and ensure maximum possible invasion, the blood that was used
for this study was drawn from donors daily and was never frozen.
An additional reason for the discrepancies in the results may have been due to the different
experimental methods that were used to determine overall parasitemia. Chung et al used flow
cytometry and labeled infected RBCs with fluorescein isothiocyanate (FITC), and parasite DNA
with Ethidium Bromide (EtBr). To ensure uninfected RBCs were not being recognized, a control
should have been used whereby uninfected RBCs were stained with both FITC and EtBR, Chung
et al, in their study, used a flow cytometer to measure parasitemia. Therefore, those parasites that
were not synchronized within the culture may have also been counted. The present study used
blind methodology and microscopic techniques (which have been demonstrated to yield reliable
and consistent outcomes -Serghides et al, unpublished data) and parasitemia was determined by
counting the number of internalized parasites in microscopic fields of 1000 total RBCs.
Schellenberg et al along with other researchers have repeatedly demonstrated a lack of
association between case fatality rate in African children and parasitemia.246 Parasitemia is a
poor predictor of severity, partly because it does not account for sequestered parasites.
Furthermore, a number of studies,21,188,189 have confirmed that there is not a relationship between
ABO blood types and parasitemia. This could imply that a decreased invasion of blood type O
RBCs, which Chung et al have demonstrated, would not necessarily influence the fatality rate in
children infected with P. falciparum malaria.
80
Our results are supported by epidemiological data that indicate that: 1) Parasitemia is not a
reliable indicator for disease severity, and 2) there is no correlation between ABO, disease
severity and parasitemia in uncomplicated malaria.206 Blood type O does not protect against
severe malaria by decreased invasion of P. falciparum.
This study could have been improved by increasing the number of P. falciparum strains used
including those that use sialic dependent and independent invasion pathways. Our strains have
been continuously cultured in O RBCs to prevent agglutination with serum in the growth media.
If P. falciparum is utilizing the A antigen then this may have caused a change in gene
expression, to adapt to better invade O RBCs. In vitro studies with P. falciparum clones have
shown that the verified rate of antigen switching is around 2% per generation. 247 For our
experiments it would be best to culture the parasites in A, B and O RBCs prior to the invasion
assay. We also need to increase the numbers of donors, adding A2, and use field strains instead of
laboratory strains.
iv) Increased clearance of infected O RBCs is a novel mechanism by which blood type O
may confer protection against severe malaria.
A primary goal of this study was to identify potential novel mechanisms for the protective effect
of blood type O from developing severe malaria. Because polymorphic RBCs such as G6PD
deficiency, sickle cell and thalassaemia have shown increased clearance of infected polymorphic
RBCs, we hypothesized that there may be increased clearance of malaria infected O RBCs.
There was only a slight increase in the phagocytic uptake of ring- infected O RBCs when
compared to A and B RBCs (Figure 8). However, we found that there was a significant increase
in the phagocytosis of schizont- infected O RBCs by human monocyte-derived macrophages
when compared to schizont-infected A1 and B RBCs (Figure 9).
81
A and B antigens are found throughout the body on epithelial cells, neurons, platelets and
potentially macrophage subpopulations. 248 In this study, to ensure that the preferential uptake of
infected O-RBCs by human monocyte derived macrophages was not due to the ABO blood type
of the macrophage donor, two phagocytosis experiments were conducted simultaneously, one
with macrophages derived from a blood type A donor and the other from a blood type O donor.
Macrophage donor blood type did not influence the preferential uptake of infected O RBC
(Figure 10). This may be attributed to the fact that the macrophages used were cultured in R-10G
FCS (see methods). There are no ABO antigens present in FCS, and leukocyte type cells must
absorb ABO antigens from the serum. This suggests that the effect that is being observed is
independent of macrophage donor blood type. Therefore, increased phagocytosis of infected O
RBCs, is due to the RBC itself and is not a factor of the macrophage.
The in vitro phagocytosis model used in this study is a reductionist system involving only
purified monocyte-derived macrophages. In the human body, the innate immune system is more
complicated and there is a complex mixture of immune cells (dendritic cells, lymphocytes, NK
cells) that communicate with each other. 249 The differences of uptake between blood types could
be minimized due to the interaction between these different cell types. To address this, schizont-
infected A, B and O RBCs were injected into the intra-peritoneal cavity of C57BL/6 mice. There
was a threefold increase in the uptake of infected O RBCs when compared to infected A and B
RBCs by resident monocytes, but no difference in the uptake of non-infected A, B or O RBCs.
This type of experimental technique has been used to evaluate the uptake of CS2 (placental
malaria specific strain) and E8B (non-pregnancy strain)- infected-RBCs and to examine
monocyte responses to infected RBCs in vivo. 235 A weakness of this technique is that it is an
artificial heterologous system. Mouse peritoneal monocytes in vivo are exposed to human P.
falciparum infected RBCs. In addition, the phagocytosis assay is also occurring in the peritoneal
82
cavity of the mouse which would not occur during a natural infection. The advantage of this
technique is that activated monocytes in vivo can be examined, it allows for signals to occur
between different cells of the immune system, and the response to schizont- infected A, B and O
RBCs can be evaluated. Using this technique, a threefold increase in the uptake of O when
compared to schizont infected A and B RBCs (Figure 11) was found which confirmed this
study’s previous results. Although the in vivo model has limitations, it did provide evidence that
when other innate immune cells are present, such as neutrophils, mast cells, NK and
lymphocytes, the effect of increased uptake of O is still present.
Our results have demonstrated that, when compared to A and B RBCs, schizont infected-O
RBCs are preferentially phagocytosed independent of macrophage donor blood type. These
findings were further extended and confirmed in an in vivo like model using C57BL/6 mice.
Although this study confirmed the findings that RBCs with a polymorphic trait (HbAS,
thalassaemia and G6PD deficiency) are preferentially phagocytosed, it should be noted that upon
further comparison between the findings of this study and other relevant studies, an anomaly is
present. The findings of Arese et al, Turrini et al 66,67,95,180 along with the work conducted by Ayi
et al68, have shown that there is increased phagocytosis of ring-stage infected RBCs which are
carriers for the ϐ-thalassaemia trait, sickle cell trait, G6PD deficient and PKD, but not with
infected-mature stage RBCs. However, the present study found that only mature stage
phagocytosis was significantly different. This difference may be explained in part by the fact that
variant RBCs (sickle cell trait, thalassaemia, or G6PD deficiency) senesce at a faster rate than
normal RBCs, and are therefore marked for phagocytosis sooner than normal RBCs.68,95 In
variant RBCs there is an overall increase of membrane-bound hemichromes, IgG and
complement C3b, and aggregated band 3; all physiological signs of an RBC undergoing the
83
process of senescence.68 Ayi et al suggest that ring-stage infected variant RBCs are subjected to
double the oxidative stress when compared to the ring-stage infected normal RBCs. The first
stress is caused by the parasite and the other is specifically generated by the polymorphism. The
lack of difference between the uptake of the mature-stage normal and variant RBCs can be
attributed to two major factors: 1) There is a much higher affinity for the uptake o f schizont-
infected RBCs;250 however, macrophages have an estimated limit of 10 ingested parasites /
macrophage.251 It may be possible that the difference between infected-normal and infected-
polymorphic RBCs was not significant because the limiting factor was the number of
macrophages and the possible total uptake. It would be interesting to have examined the outcome
if the ratio of schizonts to macrophages had been decreased. If the limiting fac tor, the
macrophages, was increased, then perhaps a difference would have been observed. 2) Band 3 has
been shown to have limited mobility in RBCs due to the cytoplasmic moiety participating in
protein-protein interactions.252 Ayi et al have shown that hemichrome levels were significantly
different between schizont- infected polymorphic RBCs and schizont- infected normal RBCs, as
observed with the ring-stage. When band 3 was measured, there was no longer a difference
between the schizont infected–polymorphic RBC and the schizont infected-normal RBC and
therefore, there was no difference in the uptake of infected-polymorphic RBCs.68 The uptake was
also therefore limited by band 3 aggregation.
In contrast to these studies comparing normal RBCs to those with hematological defects (HbAS,
thalassaemia, G6PD),68,180 the present study compared physiologically healthy A, B and O
RBCs. To date, there are no known physiological differences between A, B and O RBCs. It is
currently unknown if A, B, or O RBCs senesce sooner, and if there is greater hemichrome
deposition or band 3 aggregation. It has been shown that the life span of RBCs lacking
carbohydrate ABO antigens (Bombay phenotype) or cells lacking in glycophorin A or B is
84
normal.210 This suggests that there would be no obvious reason for non- infected A, B, and O
RBCs to be physiologically different and to senesce at different rates. Therefore, we may not see
a significant difference in the uptake of the ring- infected A, B and O RBCs as they are only
subjected to a single stress, i.e., the parasite. Ring stage parasites do not express the same variant
surface antigens that are expressed at a later stage of growth, (e.g. full expression of PfEMP-1).
The damage to the membrane may not be significant enough to lead to differential uptake (e.g.,
low levels of aggregated band 3 and PS exposure).
Potential mechanisms underlying the preferential uptake of infected O RBCs
Based on our results, findings of other studies, and other relevant literature, we can speculate as
to why infected blood type O is preferentially phagocytosed.
O antigen is not a ligand for a receptor on human monocyte-derived macrophages.
The increase in uptake of parasitized O-type RBCs is not due to the O antigen acting as a ligand
for a receptor on the macrophages. In all of the above experiments, non- infected A, B, and O
RBCs were used as controls to determine if there was a difference in uptake of non- infected
RBCs. A difference in uptake of the non- infected RBCs would suggest that increased uptake of
infected O RBCs may be due to ABO antigens on the RBC surface, and is not a factor related to
P. falciparum infection. There was no difference in the uptake between non- infected A, B, or O
RBCs, suggesting that the increase in uptake of parasitized O-type RBCs is not due to the O (H)
antigen acting as a ligand for a receptor on the macrophages.
The combined size and number of antigens present on the RBC may influence the uptake of
A, B and O RBCs due to steric hindrance. Preventing binding of an essential ligand on the
RBC to a receptor on the macrophage.
85
In order to determine if increased uptake of O-infected RBCs was due to decreased A antigen,
A1, A2 and O RBCs were infected with P. falciparum and incubated with human monocyte-
derived macrophages. We found schizont- infected A2 cells were phagocytosed at an intermediate
rate when compared to infected A1 and O RBCs. Thus far, there are no epidemiological studies
which have separated blood type A into its subtypes and examined the prevalence of A1 vs. A2 in
severe malaria (or any infectious disease). The frequency of blood type A2 in Africa is very low
(2%).104 An extremely large sample size would be required to determine whether individuals
with A2 blood type were significantly associated with severe malaria. One study did compare
rosetting in A1 and subtype A2, and found rosettes were stronger and increased in A1.213
However, they only compared four donors of A1 to four donors of A2. Although the researchers
in this study did not directly compare A2 in relation to O, comparison of the data provided in the
study leads one to conclude that A2 formed more rosettes than O. Chung et al also found that A2
acts as an intermediary between A1 and O, as there was less invasion of A2 than of A1, but greater
than O. Both of these studies support our data that shows that A2 is a mechanistic intermediate
between A1 and O.
The A2 antigen is less branched and has fewer A antigens present on the RBC.111 The exact size
of the A1, A2, B, and O structure is unknown. It is known however, that when comparing size due
to the additional carbohydrate structures, A1 and B are the largest antigens followed by A2 and
the smallest (base) structure is the O (H) antigen. This corresponds to the phagocytic uptake of
infected ABO RBCs. One may hypothesize that the A and B antigens may be sterically
hindering/blocking a variant surface antigen (e.g. PfEMP-1) or a ligand formed from the
damaged RBC membrane (e.g., PS exposure) from exposure to macrophage receptors.
86
Shared antigens: P. falciparum infected RBCs may express A and B like antigens on the
surface of the RBC.
It could be speculated that the increase of uptake of schizont-infected O RBCs is due to the
presence of a variant surface antigens (VSA) resembling A and B antigens on the surface of the
O RBC. This suggests that people of blood type O may have an immunological advantage over
those who are blood type A and B. 124,126,135 This might explain why there is an increase of
uptake in schizont- infected O RBCs and why this difference is only visible when the parasite is
in the mature form.
It has been shown and reviewed in a number of studies that bacteria and viruses promote the
generation of natural anti-A and anti-B antibodies.18,124,125,253,254 It has been argued that while the
ability to make antibodies is inherited, anti-A and anti-B antibodies develop if the host is
exposed to exogenous cross-reactive antigens. This was demonstrated in a study conducted by
Springer et al, who investigated whether it was possible for human blood type A and B
antibodies to be induced by immunogenic stimuli such as E.coli, which is thought to possess B
antigens. Using methods of inhalation and feeding, both healthy and sick individuals were
exposed to killed E .coli expressing type B antigen. Eighty percent of individuals with blood type
O and A responded with a significant increase of anti-B antibodies.255 The outcome of Springer’s
study supports two possibilities for consideration and further examination: 1) it is feasible for A
and B-like antigens to exist on pathogens and 2) pathogens expressing these antigens would
induce anti-A or B-antibodies resulting in greater opsonic signals for phagocytosis.
It has been demonstrated in a number of studies that P. falciparum malaria induces a rise in the
production of anti A (and to some, extent anti-B) antibodies. 124,125,185 The existence of pre-
formed antibodies (i.e., IgM and IgG) may recognize A-and B-like antigens expressed on the
87
infected O RBCs and these may work synergistically with complement to modify pathogens and
make them more susceptible to phagocytosis. If P. falciparum expresses A-and B- like antigens,
this may also explain why increased phagocytosis of infected O RBCs is not seen until the later
stage of growth. A and B like antigens, similar to other parasite virulence factors such as PfEMP-
1, are not fully expressed until the later erythrocytic stage of growth.50 It has been demonstrated,
however, that there may be some expression of PfEMP-1 antigens in the ring stage, which could
account for the slight increase in phagocytosis of ring-stage infected O RBCs.54 Therefore, if
PfEMP-1 is expressed to some degree in rings, then there may be a possibility that A and B like
antigens are also expressed to some degree.
In this study, prior to phagocytosis, schizont-infected RBCs were incubated with autologous
human serum to allow for non-specific complement and antibody binding. O-type serum
contains anti-A and anti-B antibodies, which would bind to A-and B- like antigen exposed on the
surface of the infected O RBC. This would be beneficial as it would promote increased clearance
of infected O RBCs. While the A serum lacks anti-A antibodies, it does have anti-B antibodies.
As a result, there may be some anti B antibody deposited on the P. falciparum infected A RBC.
This, however, would not be to the same extent as the total amount of anti-A and anti-B antibody
deposited on the O RBC. As there was intermediate phagocytosis of A2, this supports the theory
that there are A-and B-like antigens on P. falciparum because anti-B antibodies and some anti-
A1 antibodies may be deposited on the RBCs. It has been shown that A2 individuals have
antibodies against B antigens and have anti-A1 antibodies.111,119 .
A potential concern with this proposed hypothesis is possible hemolysis of the O-infected RBCs
which would be as a result of activated complement induced intravascular immune hemolysis.
This would be a concern for two reasons: a) parasite products such as GPI could be released
88
generating a high inflammatory response,256 and b) there would not be an increase in the uptake
of O- infected RBCs as the RBCs would have been hemolysed prior to phagocytosis.
However, recognition of A and B antigens also occurs through another antibody-mediated
phagocytosis pathway referred to as extra-vascular immune hemolysis. This pathway is less
severe, is IgG and IgM mediated, and does not activate complement.123
Some antibodies signal for phagocytosis such as IgM and some subtypes of IgG, while other
antibodies, when they bind, lead to complement activation causing hemolysis.123,257
Macrophages have high affinity receptors for IgG3 and IgG1, as well as non-complement
activating IgM antibodies. IgG3, IgG1, and IgM are classes of both anti-A and B antibodies. In
addition, splenic macrophages, which are partly responsible for clearing schizonts, have high
affinity for IgG 1 and IgG3 coated RBCs.258
In the human body, clearance of schizonts would most likely occur by splenic macrophages as
schizonts are typically retained in the spleen. Splenic macrophages have similar characteristics to
circulating monocytes as they are derived from circulating monocytes which become trapped
within the spleen.259 Clearing and removing the schizont from the blood and the tissue could
prevent a number of pathogenic factors caused by mature forms of the parasites. There would be
fewer schizonts available to sequester and cytoadhere in the microvasculature of vital organs,
thereby preventing vascular obstruction and hypoxia. Clearance of schizonts before they rupture
prevents the release of merozoites as well as parasite toxins such as GPI and several other P.
falciparum components that induce the host inflammatory response. While an early
inflammatory response is critical for controlling acute blood stage infection, severe malaria is
associated with dysregulated and excessive inflammation. Minimizing the pro- inflammatory
response could prevent up regulation of cell adhesions molecules such as I-CAM, V-CAM, and
89
E-selectin, and prevent sequestration. Additionally, clearing schizonts could also assist in
maintaining an overall lower parasite burden, and more importantly fewer parasites would be
sequestered to the endothelium.
90
PART V
CONCLUSIONS AND FUTURE DIRECTIONS
91
The present study examined two different avenues by which blood type O may confer protection
from developing sever disease cause by P. falciparum malaria. Comparison of the invasion and
maturation of P.falciparum into A, B, and O RBCs yielded no significant differences. Therefore,
it can be concluded that there is no preferential invasion of P.falciparum into blood type A, B or
O. Individuals who are blood type A are not likely to have a higher parasitemia that B and O
individuals. It is also concluded that blood type O individuals are not protected from developing
severe malaria due to decreased invasion. The examination of the phagocytic uptake of ring-
stage and schizont-stage infected A, B and O RBCs by human monocyte-derived macrophages
yielded a significant difference in the uptake of schizont- infected O RBCs, but only a slight
increase in the ring- infected O RBCs.
Future studies of P. falciparum regarding invasion and maturation of ABO RBCs, may wish to
consider repeating the experiment ensuring that A2, and AB RBCs are included and that a variety
of parasite strains, both sialic acid-dependent and- independent are used. It would also be
important to use P. falciparum strains that have been continuously cultured in a series of
different A, B, and O RBCs.
To investigate the findings of Chung et al in relationship to the results of our study, it would be
important to:
1) Replicate their invasion and maturation experiment,
2) Compare the total amount of parasitemia using flow cytometry and microscopic determination
methods,
3) Include a control group in the experimental design by staining non- infected RBCs with FITC
and EtBr.
92
This study has identified a novel potential mechanism of blood type O protection from
individuals infected with P. falciparum. Schizont- infected O RBCs are preferentially
phagocytosed and cleared when compared to non-O individuals. This is consistent with studies
that individuals with blood type O are protected from developing severe malaria. We propose
that increased clearance of schizont-infected O RBCs reduces overall parasite burden and the
number of schizonts available to sequester to vital organs. Thereby this additional mechanism
could contribute to overall protection to blood type O individuals from developing severe
malaria.
The mechanism underlying the increased clearance of P. falciparum infected O RBCs should
also be further investigated. The presence of A and B like antigens on P. falciparum-infected
RBCs may be inducing an antibody response and opsonization of the infected RBCs by anti-A
and anti-B antibodies and would therefore increase the signal for phagocytosis by human
monocyte derived macrophages. A number of approaches could be used to confirm the
expression of A-and B-like antigens on the infected O RBC. Instead of incubating the infected
RBCs in autologous serum, the infected RBCs could be incubated in AB serum. The AB serum
would not contain anti-A or anti-B antibodies, therefore, no additional antibodies would bind to
the A/B like antigen expressed on the schizont- infected O RBC. If the uptake of infected O
RBCs was still significantly increased, then it could likely be concluded that the increased uptake
was not due to presence of an A or B like antigen on the infected RBC. If the uptake of infected
O was decreased and this resulted in no difference in the uptake of schizont- infected A, B or O
RBCs, this would then suggest that there may be A and B like antigens expressed by P.
falciparum. Another possible approach would be to culture P. falciparum in AB RBCs and
opsonize the schizont- infected AB RBCS in autologous serum. The phagocytosis would be
expected to be even lower than for A or B RBCs as no A or B antibodies are present. If these
93
experiments indicate that A-and B-like antigens are being expressed by P. falciparum, then the
next possible steps would be to confirm the presence of A-and B- like antigens on the surface of
the O RBC or to confirm binding of anti-A and anti-B antibodies to the O RBCs. This could be
achieved by labeling an IgG antibody specific to either A or B antigens with fluorescein and then
measuring the binding of the antibody to the RBCs using flow cytometry.221 This method would
demonstrate if A or B antigen were present on the infected RBCs.122
The presence of blood type A-or B- like antigens could be confirmed by opsonizing the infected
O RBCs with O serum, allowing the anti-A and anti-B antibodies to bind and then removing the
antibodies by elution. There are a number of different elution methods reviewed in Judd et al
whereby the bound antibodies can be dissociated by either heat, ether, changes in pH, exposure
to cycles of freeze-thaw, or treatment with organic solvents. 260 Heating the RBCs is the most
common method to use in the laboratory. Heat increases the thermal motion of the atoms and
molecules, ultimately leading to dissociation of the antibody.261 The exact antibody present could
be determined by a number of methods, such as standard serological techniques (e.g. run the
elution against standard cells to determine the specificity of the antibody) or by using an ELISA.
If anti-A or anti-B antibody were found to be present on infected O RBCs but not on the control
O RBCs, one could speculate that this was due to P. falciparum expressing A-or B- like antigens.
Blood type O RBCs may also be preferentially phagocytosed due to the fact that O RBCs are the
smallest of the antigens with the least branching. The additional branching from the addition of
N-acetyl galactosamine (A) and galactose (B) onto the O antigen may be blocking or hindering
variant surface antigens or other ligands, making them inaccessible to receptors on the
macrophages. To test this hypothesis, A and B RBCs could be treated with α-N-acetyl
galactosaminidase or α-galactosidase, respectively, to remove the A and B antigens. This
94
approach has been used in rosetting experiments by Barragan et al, and the invasion assays by
Chung et al.56,211 It has been observed in both of these studies, that when the A RBCs were
treated with α-N-acetyl galactosaminidase, the A RBCs began to behave like an O RBCs
(decreased rosetting and invasion). We attempted to replicate the removal of the A antigen by the
methods found in both Barragan et al and Chung et al, but this was not successful and the A1
cells continued to agglutinate. Review of the literature indicates there are currently no enzymes
that remove 100% of the A1 antigen.106 Zymequest has recently developed an enzyme which has
been shown to be an improvement over the α-N-acetyl galactosaminidase isolated from chicken
liver.106 It has been shown to efficiently convert the A1 RBC into an O-like RBC. 106 By
removing the A and B terminal sugars, variant surface antigens and ligands which had not been
recognized due to steric hindrance, may be exposed and recognized for clearance. Another
method to determine if the increased phagocytosis of infected O RBCs is due to steric hindrance
would be to insert a synthetic A antigen into the O RBC membrane. In theory, this method would
then cover any exposed variant surface antigens or ligands thereby decreasing phagocytosis of
infected O RBCs. Using the FSL-A antigen manufactured by KOBE, blood type O RBCs could
be “painted” to mimic the A RBC; it would be expected that there would be a decrease in
phagocytosis of the infected O RBCs.
In G6PD deficiency, thalassaemia, and sickle cell, phagocytosis of infected RBCs was increased
due to increased oxidative stress within the RBC, thereby leading to hemichrome deposition, and
band 3 aggregation68,95. It has not previously been shown if there is a difference in the
senescence of A, B or O RBCs. To assess this, one could measure the hemichrome deposition
and band 3 aggregation in non- infected and P. falciparum- infected A, B and O RBCS. Based on
our results of increased phagocytosis, it is predicted that blood type O would likely be under
greater oxidative stress.
95
Severe malaria has been linked to an imbalance between pro- inflammatory (TNF and IFN-γ) and
anti- inflammatory cytokines (TGF-ϐ, IL-10). High levels of TNF-α, IFN-γ and low levels of IL-
10, IL-12 and TGF-ϐ are related with poor outcome in malaria.262,263 In epidemiological studies
correlating ABO and progression of malaria disease, pro-or anti- inflammatory cytokines were
not measured. One may expect that individuals belonging to blood type O would have a better
balance between pro and anti- inflammatory cytokines.
This project involved knowledge and data from a mosaic of disciplines including parasitology,
hematology, immunology, and epidemiology. The outcomes of this study will likely invite
speculation and research from individuals in a number of different fields.
Future research will need to be broadened and expanded into examining ligands expressed on the
infected RBC that may mimic host RBCs ligands in addition to the distinct parasite VSA. The
results of this study have added to increasing evidence that innate immune responses are
essential to controlling the degree of P. falciparum severity. It also suggests that more research
needs to be dedicated to the study of the clearance of mature stage P. falciparum malaria. It is
evident that increased clearance of schizonts by macrophages leads to slowing the progression to
severe disease. This work is especially important as it further suggests the notion that any
enhanced signal for phagocytosis, even if minor, may greatly increase clearance, and therefore,
decreases pathogenesis.
Despite intensive research efforts, the exact mechanism of infected blood type O protection from
malaria remains unclear. This research demonstrates that there is a relationship between the ABO
blood type system, P. falciparum malaria, and the innate immune system.
96
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