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Doctoral thesis from the Department of Immunology, The Wenner-Gren Institute, Stockholm University, Sweden Single nucleotide polymorphisms related to immune responses in Plasmodium falciparum malaria Amre Osman Nasr Stockholm 2008 All previously published papers were reproduced with permission from publishers. © Amre Osman Nasr, Stockholm, Sweden 2008 ISBN 978-91-7155-726-1 Printed in Sweden by Universitetsservice AB, Stockholm 2008 Distributer: Stockholm University Library ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ ﻼ ً وﻣﺎ اوﺗﻴﺘﻢ ﻣﻦ اﻟﻌﻠﻢ اﻹ ﻗﻠﻴ “No matter how much you know, there is much to know” To the memory of my mother To my father To my brother To my Sisters To my family To my friends “Do not think of today’s failures, but of the success that may come tomorrow. You have set yourselves a difficult task, but you will succeed if you persevere; and you will find joy in overcoming obstacles. Remember, no effort that we make to attain something beautiful is ever lost’’ Helen Keller SUMMARY A comprehension of the genetics of host resistance to malaria is essential to understanding the complexity of the host immune response and its interaction with the parasite infection. Current research is directed towards dissection of host genetic factors involved in both the host immune response and the malaria disease outcome. First, a possible association between Fc gamma receptor IIa-R/H131 (FcγRIIa-R/H131) polymorphism and anti-malarial antibody responses was investigated in Sudanese patients in relation to clinical outcome of Plasmodium falciparum malaria. The frequency of the R/R131 genotype was significantly higher in patients with severe malaria as compared to those with mild malaria, while the H/H131 genotype showed a significant association with mild malaria. Antimalarial IgG3 antibodies were shown to be associated with reduced risk of clinical malaria in individuals carrying the H/H131 genotype. Low levels of IgG2 antibodies reactive with the Pf332C231 antigen were also associated with lower risk of severe malaria in individuals carrying the H131 allele. The levels of anti-malarial IgG1 and IgG3 antibodies were significantly higher in patients with mild malaria compared with those with severe malaria. Our data suggest the importance of cytophilic antibodies, since IgG2 can act as such in the combination with FcγRIIaH/H131 genotype. However, the role of IgG2 antibodies in malarial immunity is still a subject of controversy, with different levels being suggested to underlie protective properties. Fulani and Masaleit, two sympatric ethnic groups in eastern Sudan, are characterized by marked differences in susceptibility to P. falciparum malaria. We investigated whether the two populations differ in the frequency of immunoglobulin GM/KM allotypes, which are known to influence both humoral and cellular immune responses and to be associated the with susceptibility to and outcome of several infections. The distribution of GM and KM phenotypes differed significantly among the two ethnic groups, with Gm 6 being significantly lower among the Fulani, and the combined frequency of Km 1,3 and Gm 1,17 5,6,13,14 phenotypes was found to be higher among Masaleit. Previously, it has been suggested that sickle cell trait carriage (HbAS) may protect against the most severe forms of malaria. In the present study we investigated whether the two ethnic groups differ in the frequency of FcγRIIa and HbAS genotypes. The frequency of the FcγRIIa- H/H131 genotype and the H131allele was found to be higher in the Fulani, whereas the R/R131 genotype was significantly higher in the Masaleit group. On the other hand, the HbAS genotype was less frequent in the Fulani as compared to Masaleit. Moreover, the Fulani showed higher levels of antimalarial IgG2 and lower IgG1 and IgG3 antibodies when compared to their sympatric non-Fulani neighbours. The complement-reactive protein (CRP) shows a differential binding to the FcγRIIa-R/H131 receptors. A tri-allelic SNP (C/T/A) in the CRP gene was investigated for possible ethnic associations. The A allele, which is associated with higher basal CRP levels, was found to be less frequent in the Fulani compared with non-Fulani ethnic groups both in Sudan and Mali. Our data thus indicate that CRP could be a contributing factor to the relative resistance to malaria seen in the Fulani as compared to other sympatric ethnic groups. In conclusion, our results suggest possible associations between FcγRIIa-R/H131 and CRP genotypes, GM/KM allotypes, and anti-malarial antibody responses and the clinical outcome of P. falciparum malaria. I LIST OF PAPERS This thesis is based on following original papers, which will be referred to by their roman numerals I. Nasr, A.*, Iriemenam, N.C.*, Troye-Blomberg, M., Giha, H.A., Balogun, H.A., Osman,O.F., Montgomery, S.M., ElGhazali, G., Berzins, K.; 2007. FcγRIIa (CD32) Polymorphism and Antibody responses to Asexual Blood-stage Antigens of Plasmodium falciparum Malaria in Sudanese Patients. Scand. J. Immunol. 66, 8796. * The authors A. Nasr and N. C. Iriemenam contributed equally to this article. II. Pandey, J.P., Nasr, A., Rocca, K.M., Troye-Blomberg, M., ElGhazali, G.; 2007. Significant differences in GM allotype frequencies between two sympatric tribes with markedly differential susceptibility to malaria. Parasite Immunol. 29, 267-269. III. Nasr, A., ElGhazali, G., Giha, H., Troye-Blomberg, M., Berzins K; 2008. Interethnic differences in carriage of HbAS and FcγRIIa (CD32) genotypes in children living in eastern Sudan. Acta Trop. 105, 191-195. IV. Nasr, A., Iriemenam, N. C., Giha, H. A., Balogun, H.A., Anders, R. F., TroyeBlomberg, M., ElGhazali, G., Berzins, K. FcγRIIa (CD32), polymorphism and antimalarial IgG subclass pattern among Fulani and their sympatric tribes in eastern Sudan (Manuscript Submitted). V. Israelsson, E., Ekström, M., Nasr, A., Arambepola, G., Maiga, B., Dolo, A., Doumbo, O.K., ElGhazali, G., Giha, H.A., Troye-Blomberg, M., Berzins, K., Tornvall, P. Polymorphism in the CRP gene may contribute to the lower susceptibility to malaria of the Fulani ethnic group in Africa (Manuscript). II TABLE OF CONTENTS SUMMARY ............................................................................................................................................................ I LIST OF PAPERS................................................................................................................................................ II TABLE OF CONTENTS .................................................................................................................................. III LIST OF ABBREVIATIONS ............................................................................................................................IV INTRODUCTION ................................................................................................................................................. 1 THE IMMUNE SYSTEM ......................................................................................................................................... 1 Innate immunity ............................................................................................................................................. 1 Cells involved in innate immunity ............................................................................................................................. 3 Adaptive Immunity ......................................................................................................................................... 4 MHC ........................................................................................................................................................................... 5 Cytokines .................................................................................................................................................................... 5 T Lymphocytes ........................................................................................................................................................... 6 Th1/ Th2 responses ..................................................................................................................................................... 7 B cells ......................................................................................................................................................................... 7 Immunoglobulins ........................................................................................................................................... 8 Ig classes ..................................................................................................................................................................... 9 Ig allotypes................................................................................................................................................................ 11 Fc receptors ................................................................................................................................................. 11 The FcγR family ....................................................................................................................................................... 11 The Structure of FcγRIIa .......................................................................................................................................... 12 Function of FcγRIIa .................................................................................................................................................. 12 Allelic variants of FcγRIIa ....................................................................................................................................... 13 MALARIA ........................................................................................................................................................... 14 CHARACTERISTICS OF MALARIA ....................................................................................................................... 14 Life cycle and morphology .......................................................................................................................... 14 Pre-patient and incubation periods ........................................................................................................................... 15 Signs and symptoms of malaria................................................................................................................... 16 Severe malaria .......................................................................................................................................................... 16 Diagnosis of malaria ................................................................................................................................... 16 Conventional microscopy ......................................................................................................................................... 16 Alternative diagnostic methods ................................................................................................................................ 17 IMMUNITY TO MALARIA ............................................................................................................................. 19 INNATE RESISTANCE.......................................................................................................................................... 19 Haemoglobin variants ................................................................................................................................. 19 Complement receptors................................................................................................................................. 22 ACQUIRED IMMUNITY ....................................................................................................................................... 22 Immune mechanisms against the pre-erythrocytic stage............................................................................ 23 Immune mechanisms against the erythrocytic stage .................................................................................. 23 RELATED BACKGROUND ............................................................................................................................. 26 THE COMPLEX GENETIC BASIS OF RESISTANCE TO MALARIA ......................................................................... 26 The role of IgG in protection against malarial infection and disease ....................................................... 27 FcγRIIa in Malaria ...................................................................................................................................... 27 Ethnic Differences ....................................................................................................................................... 28 IgG allotypes................................................................................................................................................ 28 C- reactive protein (CRP) ........................................................................................................................... 29 THE PRESENT STUDY .................................................................................................................................... 31 OBJECTIVES OF THE STUDY ............................................................................................................................... 31 MATERIAL AND METHODS USED IN THE STUDY ............................................................................................... 31 RESULTS AND DISCUSSION ............................................................................................................................... 31 FcγRIIa polymorphism in relation to malaria (Papers I, III and IV).......................................................... 32 IgG GM/KM allotypes in relation to malaria (Paper II)............................................................................. 33 IgG subclasses in relation to malaria (papers I and IV) ............................................................................. 34 IgG subclasses in relation to FcγRIIa polymorphism (Papers I and IV).................................................... 36 HbAS in relation to malaria (Paper II) ........................................................................................................ 37 CRP in malaria in relation to ethnicity (Paper V)....................................................................................... 38 CONCLUDING REMARKS AND FUTURE PERSPECTIVES .................................................................. 40 ACKNOWLEDGMENTS .................................................................................................................................. 43 REFERENCES .................................................................................................................................................... 45 III LIST OF ABBREVIATIONS ADCI Antibody-dependent cell mediated inhibition APC Antigen presenting cell BCR B-cell receptor CRP C-reactive protein DC Dendritic cell FcR Fc receptor G6PD Glucose-6-phosphate dehydrogenase H Histidine IFN Interferon Ig Immunoglobulin IL Interleukin iRBC Infected red-blood cell ITAM Immunoreceptor Tyrosin-based Activation Motifs ITIM Immunoreceptor Tyrosin-based Inhibiting Motifs MHC Major histocompatibility complex MPL Mannan-binding lectin MSP Merozoite surface protein NK cell Natural-killer cell PCR Polymerase chain reaction PRRs Pattern recognition receptors R Arginine RBC Red-blood cell SNP Single nucleated polymorphism TCR T-cell receptor Th T helper TLR Toll-like receptor IV INTRODUCTION The Immune System Historically, immunity meant protection from disease, and more specifically, against infectious diseases. The cells and molecules responsible for immunity constitute the immune system, and their collective and coordinated response after the introduction of foreign substance is the immune response. We now know that many of the mechanisms of resistance to infections also involve the individual’s response to non-infectious foreign substances 1. However, mechanisms that normally protect individuals from infections and eliminate foreign substances are themselves capable of causing tissue injury and disease in some situations. Therefore, a more inclusive and modern definition of immunity is a reaction to foreign substances, including microbes, as well as macromolecules, such as proteins and polysaccharides, without implying a physiologic or pathologic consequence of such a reaction 2. Immunology is the study of immunity in this broader sense, and of the cellular and molecular events that occur after an organism encounters microbes or other foreign macromolecules. The immune system is branched into two arms, governing the innate (natural or nonspecific) and the acquired (adaptive or specific) immune responses 1. Innate immunity Most encounters with microorganisms do not result in disease. The few microbes that manage to cross the barriers of skin, mucus, cilia, and pH are usually eliminated by innate immune mechanisms, which commence immediately upon pathogen entry 2. If phagocytosis cannot rapidly eliminate the pathogen, inflammation is induced with the production of cytokines and acute phase proteins. This early induced response is not antigen-specific and does not generate immune memory 2. Only if the inflammatory process is unsuccessful at eliminating the pathogen will the adaptive immune system be activated, a process which requires several days to produce armed effector cells. Pathogens infect at specific locations in the host and damage the host by several mechanisms. Extracellular pathogens and their exotoxins are susceptible to destruction by phagocytes, either directly or in cooperation with antibodies elicited by the acquired immune system, so called opsonization. Intracellular pathogens may be eliminated by lysis of the infected cell by natural-killer (NK) cells 3 or dendritic cells. Pathogens within macrophages may be killed by the activation of these cells by IFN-γ derived from Th1 cells. __________________________________________________________________________ Amre Nasr, 2008 1 The immune response can also damage the body by means of inflammation and cytotoxicity4. Innate immunity covers many areas of the host defence against pathogenic microbes, including the recognition of pathogen-associated molecular patterns (PAMPs) present on the microbes5. In vertebrates, which are the only phylum that possesses an adaptive immune system, there are also mechanisms that inhibit the activation of the innate immunity. One example is the inhibition of killing by NK cells, which are known to receive an inhibitory stimulus from major histocompatibility complex (MHC) class I molecules. The immune system uses a variety of pattern-recognition receptors (PRRs) that can be expressed on the cell surface, in intracellular compartments, or secreted into the bloodstream and tissue fluids2. The principal functions of PRRs include opsonisation, activation of complement, coagulation cascades, phagocytosis, activation of proinflammatory signalling pathways, and induction of apoptosis. In brief, the classical complement pathway of activation is initiated by complement protein 1 (C1) binding to antigen-antibody complexes, while the alternative pathway is initiated by C3b binding to various activating surfaces, such as microbial cell walls. The C3b involved in the alternative pathway initiation may be generated in several ways, including spontaneously, by the classical pathway, or by the alternative pathway itself. Both pathways converge and lead to the formation of the membrane attack complex. C-reactive protein (CRP, for further details see section under Related Background), mannan-binding lectin (MBL), and serum amyloid protein (SAP) are secreted pattern recognition molecules, produced by the liver during the acute phase response at early stages of infection 6-8. Toll-receptors are important components of the innate immunity. They are evolutionarily conserved and homologous receptors found in plants, insects, worms and vertebrates. The first member of this family, named Toll, was initially identified in the fruit fly Drosophila melanogaster 9. This receptor has been shown to play an important role in protection against fungi in the fruit fly 10 . In humans, 11 Toll-like receptors (TLRs) have been identified until now. Based on the cellular localization, TLRs are classified into two groups. TLR 3, 7, 8 and 9 are expressed in endosomes, while the second group, including TLR1, 2, 4, 5 and 6, are present on the surface of many different cells 11, 12 . Upon recognition of their cognate ligands, TLRs dimerize and initiate a signalling cascade that leads to activation of a proinflammatory response 13, 14 . Ligand binding induces two __________________________________________________________________________ 2 signalling pathways, one is MyD88-dependent and the other is MyD88-independent, inducing the production of proinflammatory cytokines and type I interferons 13, 14. Cells involved in innate immunity Monocytes/Macrophages and Neutrophils Macrophages are generally a population of ubiquitously distributed mononuclear phagocytes responsible for numerous homeostatic, immunological, and inflammatory processes. Their wide tissue distribution makes these cells well suited to provide an immediate defence against foreign elements prior to lymphocyte immigration. Because macrophages participate in both the specific immunity via antigen presentation, and in the non-specific immunity against bacterial, viral, fungal, and neoplastic pathogens, it is not surprising that macrophages display a range of functional and morphological phenotypes 15. Macrophages have an innate capacity to recognize pathogen-expressed surface structures and play an important role in immunity to infections. The macrophages are phagocytic cells, and the phagocytosis is facilitated by Fc receptors for IgG and IgE present on their cell surface 16 . They process and present antigens to T cells in association with MHC antigens, initiating the immune response. In addition, macrophages are also involved in the secretion of many products, some of which are toxic. The macrophage activity is enhanced by cytokines, notably IFN-γ, secreted by T- helper (Th) cells. Activated macrophages are more effective than resting ones in eliminating potential pathogens, and they also express higher levels of MHC class II molecules, allowing them to function more effectively as antigen-presenting cells (APCs). Activated macrophages are producing a number of reactive oxygen intermediates and reactive nitrogen intermediates that have potent antimicrobial activities. When macrophages are activated by LPS together with IFN-γ, they begin to express high levels of nitric oxide synthase (NOS), an enzyme that oxidizes L-arginine to yield nitric oxide (NO). Recent evidence suggests that much of the antimicrobial activity of macrophages is due to NO 17. Neutrophils can phagocyte foreign substances, and they also use both oxygen dependent and independent pathways to generate antimicrobial substances. __________________________________________________________________________ Amre Nasr, 2008 3 NK cells and Dendritic cells (DCs) NK cells have cytolytic activity against tumours or pathogen-infected cells. They can also release cytokines, including IFN-γ, TNF, granulocyte-macrophage colony stimulating factor (GM-CSF) and chemokines 18, 19. The functions of NK cells are regulated by a complex balance between inhibitory and activating signals, that are generated by an array of different cell-surface receptors after engagement by their specific cellular ligands18. Two major subsets of NK cells are distinguished by their low or high expression of CD56 (CD56bright and CD56dim respectively). CD56dim NK cells constitute the majority of NK cells among PBMCs and excel in cytotoxicity. CD56bright NK cells are less effective in cytotoxicity but are very potent cytokine producers 20, 21. DCs and their precursors are considered to be sentinels of the immune system; they can circulate through the blood and non-lymphoid peripheral tissues, where they eventually become resident cells 22. DCs are well known as professional APC, able to activate naïve T cells, and are thus critical for the development of adaptive immune responses 23 . DCs also have important effector functions during the innate immune response, such as pathogen recognition and cytokine production. In fact, various subsets of DCs have been described, that are associated with specific pathogen recognition mechanisms, locations, phenotypes, and types of Ag presentation and cytokine production 24. The DCs also up-regulate the costimulatory molecules that are required for an effective interaction with T cells24. Both NK cells and DCs are specialised cells of the innate immune system, which play a crucial role during the early phase of the inflammatory reaction. The reciprocal interaction of these cells results in a potent, activating cross-talk 18, 22 . DCs can prime resting NK cells, which, in turn after activation, might induce DC maturation 25. However, NK cells negatively regulate the function of DCs also by killing immature DCs in peripheral tissues 21. Thus, NK cells have a role in the editing of mature DCs, based on the selective killing of mature DCs that do not express optimal surface densities of MHC class I molecules 21. Adaptive Immunity The adaptive immune responses are mediated by lymphocytes. These cells recognize individual pathogens specifically and they possess memory. Lymphocytes fall into two major categories: T and B lymphocytes. These two types of cells recognize particular structures via their receptors that are responsible for the enormous diversity seen in the __________________________________________________________________________ 4 adaptive immune system. B cells recognize antigens through the surface immunoglobulins (Ig), B-cell receptors (BCR), and the T cells through the T-cell receptors (TCR), these receptors have unique specificities. The recognition of antigen by the TCR is dependent of a their presentation in the context of MHC molecules. T and B-cells are mainly responsible for cell-mediated and humoral immunity, respectively. The differentiation of the cells of the adaptive immune system is dependent on a cross talk with cells of the innate immune system, and cytokines constitute important mediators in this context. MHC The MHC codes for glycoproteins, which are highly specialised molecules with the ability to form unusually stable complexes with peptide antigens, to be presented on the cell surface of APCs, required for the activation of T cells system 26. There are two classes of MHC molecules, class I and class II, which present antigenic peptides that are processed by the proteasomal pathway and the lysosomal pathway, respectively 26. The MHC alleles are highly polymorphic, different hosts usually having different MHC genotypes, and they therefore recognize different spectrums of antigen epitopes 27. MHC plays an important role in immune regulation. Hosts vary genetically in many of the factors controlling the immune responses. Many studies have shown associations between MHC polymorphism and disease susceptibility 28-31. Cytokines Interactions between cells in the immune system can be mediated by soluble proteins, cytokines. All cytokines are produced in small amounts in response to external stimuli, such as microbes, and generally have a short half-life. Cytokines bind to highaffinity receptors on target cells. Most cytokines act on the cells that produce them (called autocrine actions) or on adjacent cells (paracrine actions). A minority of cytokines enters the circulation and acts on distant tissues (endocrine hormonal actions). Cytokines are involved in the regulation of the innate and adaptive immunity. They play an important role in activating macrophages to produce large amounts of cytokines 34 , also activating and regulating lymphocytes 35 32- . Depending on the effect of the immune response, some cytokines may be referred to Th1 or Th2-associated cytokines. __________________________________________________________________________ Amre Nasr, 2008 5 T Lymphocytes Early T cell precursors do not express the CD4 or CD8 co-receptors (doublenegative) and begin the recombination of the TCR gene segments, of which there are 4: α, β, γ, and δ. Recombination begins at the δ, γ, and β loci, and if expression of the γδ TCR is successful, commitment to the γδ T cell lineage results 36 . Most γδ T cells remain double- negative and leave the thymus, populating lymphoid tissues and epithelia. Antigen recognition by γδ TCR expressing T cells does not require processing and is not MHC restricted 37. The γδ T cells represent a relatively rare type of T cells in humans, and their function appears to be distinct from that αβ T cells 38 . Alternatively, a successful β locus recombination results in β TCR expression, which pairs with the surrogate α receptor (preTα) and forms the pre-TCR. With the immunotyrosine-based activation motif (ITAM) rich CD3-signaling machinery, the pre-TCR provides ligand-independent signals, enabling commitment to the αβ T-cell lineage and CD4/CD8 coexpression (double-positive; DP). T cells are further classified by the type of TCR they express on their surface, having either the αβ or γδ combination 39 . The αβ+ TCR expressing T cells have evolved primarily to recognize antigens as peptide fragments in association with MHC antigens present on antigen-presenting cells (APCs) 40. The αβ+ T cells may differentiate into several subsets, all with the accessory marker CD3, in the TCR/CD3 complex, combining with MCH class I (CD8+) or MHC class II (CD4+)41, 42. The CD4+ T cells, designated T helper (Th) cells, recognize peptides, which are bound to MHC class II molecules. Th cells are polarized towards either Th1 or Th2 subsets, depending on the nature of the cytokines they produce at the site of activation 43. CD8+ T cells, referred as CTLs, recognize antigens bound to MHC class I molecules. CD8+ T cells produce cytotoxic proteins, including perforin and granzymes, and secrete them at the point of contact with the target cell, the immunologic synapse, which results in specific killing without bystander-cell damage. Perforin is a membrane-disrupting protein that facilitates the ability of granzymes to induce apoptosis in the target cell. In addition to cytolysis, CD8+ effectors produce IFN-γ and TNF 44. Classically, CD4+ effectors were viewed in the context of the Th1-Th2 paradigm, but other subsets have emerged, including IL-17–producing T cells (Th17) and T cells with regulatory function (Treg) 45. The factors that determine the differentiation of these subsets in vivo are not completely understood, although it seems that the cytokine milieu may be a major factor. __________________________________________________________________________ 6 Th1/ Th2 responses Both Th1 and Th2 subsets are generated from a naïve T cells 46 . The Th1/Th2 concept rests largely on a dichotomy in their cytokine profiles, reviewed in recent research involving human subjects 47. Th1 cells specialize in macrophage activation by IFN-γ production and contactdependent stimulation by using a variety of cell-surface co-stimulatory ligands and, thus playing a major role in intracellular pathogen clearance and delayed-type hypersensitivity. Reactions of Th1 differentiation is directed by IFNs generated by the innate response to infection, which ultimately leads to up-regulation of T-bet, the master regulator of Th1 differentiation. T-bet directs IFN-γ production and IL-12 receptor expression. The presence of IL-12 results in STAT4 activation, further enhancing IFN-γ production and Th1-effector formation. Th2 cells produce IL-4, IL-5, and IL-13, and specialize in regulating B-cell antibody responses. They drive B-cell proliferation by IL-4 and contact-dependent CD40 ligand: CD40 binding, augmenting humoral defences against extracellular pathogens. Furthermore, IL-4 and IL-5 enable IgE production and eosinophilic inflammation, important for the clearance of helminthic infections, but also highly relevant in the allergic response. Th2 differentiation is initiated by weak TCR signals coupled with IL-4 receptor signaling and signal transducer and activator of transcription-6 (STAT-6) activation. This results in upregulation of the transcription factor GATA-3, the master regulator of Th2 differentiation. GATA-3 enhances Th2-cytokine production and inhibits Th1 pathways. B cells B cells provide the humoral immunity against extracellular pathogens through antibody production. Antibodies neutralize pathogens and toxins, facilitate opsonization, and activate complement. In addition to binding soluble peptides, the BCRs are capable of binding conformational epitopes, including protein epitopes discontinuous in the primary structure, as well as non-peptide antigens, such as polysaccharides and nucleic acids. In most cases, primary infection or vaccinations result in a prolonged production of highaffinity specific antibodies, the basis of the adaptive humoral immunity. So-called natural IgM antibodies, produced in the absence of infection, are of lower affinity, often polyreactive and weakly auto-reactive, and bind to many conserved pathogen-associated polysaccharides. These antibodies play a role in the first-line defence against bacterial infections and assist in the clearance of endogenous cellular debris. __________________________________________________________________________ Amre Nasr, 2008 7 Naïve follicular B cells reside in the follicles of secondary lymphoid tissues. Antigen arrives in lymphoid organs as soluble molecules or as immune complexes, or is transported there by DCs 48. BCR cross-linking initiates receptor-mediated endocytosis and antigen processing. Antigen-engaged follicular B cells migrate to the T-B interface, the border between the T-cell zone and B-cell follicle, increasing the probability of encounter with primed helper T cells of cognate specificity. On such an encounter, signals from Tcell–derived cytokines and CD40L:CD40 binding sustain B-cell activation and promote immunoglobulin class switch recombination (CSR). Immunoglobulins Immunoglobulins (Ig) generally assume one of two roles: Ig may act as plasma membrane bound antigen receptors on the surface of B cells or as antibodies free in cellular fluids functioning to intercept and eliminate antigenic determinants or pathogens. In either role, antibody function is intimately related to its structure. When bound on the surface, the Ig functions as a receptor involved in differentiation, activation and apoptosis, while the secreted form can neutralize foreign antigens and recruit other effector components 49 . Ig are composed of four polypeptide chains: two "light" chains (lambda or kappa), and two "heavy" chains (alpha, delta, gamma, epsilon or mu). The type of heavy chain determines the Ig isotype (IgA, IgD, IgG, IgE, IgM, respectively). Light chains are composed of 220 amino acid residues, while heavy chains are composed of 440-550 amino acids. Each chain has "constant" and "variable" regions as shown in the (Figure 1). Figure 1: A simplified structure of the Ig molecule *Modified from Ollila and Vihinen 50 __________________________________________________________________________ 8 Structural differences between Ig are used for their classification. Several different types of heavy chain exist that define the class or "isotype" of an antibody. These heavy chain types vary between different animals. More specifically, the isotype is determined by the primary sequence of amino acids in the constant region of the heavy chain, which in turn determines the three-dimensional structure of the molecule. Since Igs are proteins, they can act as antigens, eliciting an immune response that generates anti-Ig antibodies. However, the structural (three-dimensional) features that define isotypes are not immunogenic in an animal of the same species, since they are not seen as "foreign". For example, the five human isotypes, IgM, IgD, IgG, IgE and IgA are normally found in all humans. Another means of classifying Ig is defined by the term "allotype". Like isotypes, allotypes are determined by the amino acid sequence and corresponding three-dimensional structure of the constant region of the Ig molecule. Unlike isotypes, allotypes reflect genetic differences between members of the same species. This means that not all members of the same species will possess any particular allotype. Therefore, injection of any specific human allotype into another human could possibly generate antibodies directed against the structural features that define that particular allotypic variation. A third means of classifying Ig is defined by the term "idiotype". Unlike isotypes and allotypes, idiotypes are determined by the amino acid sequence and corresponding three-dimensional structure of the variable region of the Ig molecule. In this regard, idiotypes reflect the antigen-binding specificity of any particular antibody molecule. Idiotypes are so unique that an individual person is probably capable of generating antibodies directed against their own idiotypic determinants. Ig classes Upon antigen activation naïve B cells secrete antibodies of the IgM class. Subsequent to primary IgM production, the B cells may terminally differentiate into IgM producing plasma cells, or switch to production of other isotypes than IgM. In the complex process of Ig synthesis, rearrangements in gene segments (VHDJH in heavy chain and VLJL in light chain) and somatic mutations produce variations in amino acid sequences in the amino-terminal portions of the chain. The concentrations of the five isotypes in serum vary widely, reflecting both different numbers of B cells producing each isotype and different intrinsic half-lives of the Ig classes. __________________________________________________________________________ Amre Nasr, 2008 9 IgM antibodies are the largest Ig (900 kilodaltons), which can exist as a monomeric form on the membrane of B cells, but is normally secreted by plasma cells as a pentamer together with a J-chain 51 . IgM is found in blood and lymph fluid and is the first type of antibody made in response to an infection. Furthermore, IgM may cause other cells of immune system to destroy foreign substances 52 . During an immune response, B lymphocytes can switch expression of Ig class (isotype) from IgM to IgG, IgE, or IgA 51 . IgM appears to have a role in the defence against parasitic infections 53. IgD is a monomer and presented on B cells during early stages of differentiation. Very little is found in the serum. IgD antibodies have a role in B cell signalling, but no other apparent effector role in the host defence is known 54. IgG antibodies are monomeric, found in all body fluids, and are the dominating isotype in humoral immune responses, constituting 75% to 80%) of all antibodies in the serum. Human IgG is divided into four subclasses, IgG1- IgG4, where IgG1 is the most common (66%), followed by IgG2 (24%), IgG3 (7%) and IgG4 (3%) 55 . IgG1 and IgG3 antibodies are predominantly produced in response to protein antigens, stimulation may result in an increased proportion of IgG4 57 56 but chronic . In contrast, carbohydrate 56 antigens most often induce IgG2 responses . The different subclasses differ in their ability to activate complement, IgG1 and IgG3 being the most effective ones, and IgG2 is a weak activator, whereas IgG4 does not activate complement. IgG2 antibodies are very important in targeting encapsulated bacteria. IgG antibodies are the only isotype that can cross the placenta in the pregnant woman to help protecting the fetus. IgA provides mucosal immune protection as a result of its ability to interact with the polymeric Ig receptor (pIgR), an antibody transporter expressed on the basolateral surface of epithelial cells 58 . IgA antibodies protect mucosal surfaces that are exposed to outside foreign substances. In humans there are two heavy chain subclasses of IgA: IgA1 and IgA2. IgA1 is monomeric primarily found in the serum. IgA2 can polymerize into multimers linked by a J piece. IgA2 is transported into the secretions by the cooperative effort of epithelial cells and lymphocytes 59. Dimeric IgA, produced by sub-mucosal B cells, binds to an epithelial cell-membrane protein called secretary IgA (SIgA) 59. One important role of IgA is to bind food antigens in the intestinal tract and prevent the triggering of pro-inflammatory responses. The relative inability of IgA to initiate __________________________________________________________________________ 10 inflammatory responses allows food antigens to sequester without deleterious consequences 60 . IgE antibodies are found in the lungs, skin, and mucous membranes. Very low amounts of IgE are present in the serum. Mast cells have high affinity Fc-epsilon receptors (FcεR) that scavenges IgE so quickly that its serum half-life is only 2 days 61. IgE displayed on the mast cell surface mediates immediate hypersensitivity or allergic reactions 62 . IgE appears also to have a role in defence against parasitic infections 63. Ig allotypes The Ig GM and KM allotypes are hereditary antigenic determinants of the Ig heavy chains (γ) and the κ-type light chains, respectively 64 . The striking qualitative and quantitative differences in the distribution of these determinants among different races, raise questions concerning the nature of the evolutionary selective mechanism that maintains this variation. Associations between Ig allotypes and specific antibody responses could be a selective force for the maintenance of various haplotypes and their frequencies. Significant associations between certain GM and KM allotypes and immune responsiveness to particular infectious pathogens and to polysaccharide vaccines were reported 62 . The importance of GM and KM allotypes will be discussed under the “Related Background” section. Fc receptors Cell surface receptors for the Ig Fc domain of immunoglobulin (Ig) are known to be expressed on all cells of the immune system. Fc receptors (FcR) play an important role in immune regulation, as they serve to link antibody-mediated immune responses with cellular effector functions. Specific FcRs exist for all Ig classes, including IgA (FcαR), IgD (FcδR), IgE (FcεR), IgG (FcγR), and IgM (FcμR). As the work in this thesis deals with IgG isotypes, allotypes and Fc receptors particular Fc gamma RIIa, there are more details on each of them in the following sections. The FcγR family The cell-surface receptors for Fc portion of IgG molecules are now known to consist of three sub-families of related molecules, designated FcγRI, II and III 65 . FcγRI (CD64), which includes FcγRIa FcγRIb FcγRIc, is derived from a single gene in humans. The functional receptor consists of a single trans-membrane polypeptide of about 40 KD, __________________________________________________________________________ Amre Nasr, 2008 11 FcγRI is the only high- affinity receptor for the Fc domain of IgG molecules, and binds human IgG1 or IgG3 antibodies with a Kd of 10-8 to 10-9 M 66 . The affinity for IgG2 or IgG4 antibodies is much lower. In the case of the FcγRII (CD32) gene subfamily, which includes FcγRIIa and FcγRIIb, it is encoded by at least three separate genes in humans. The affinity of FcγRII for human IgG1 or IgG3 is relatively weak, for example Kd greater than 10-7 M, and is essentially nonexistent for IgG2 and IgG4. Members of the FcγRIII (CD16) subfamily, including FcγRIIIa and FcγRIIIb, are encoded by two genes in humans. The affinity of FcγRIII for IgG molecules is similar to that FcγRII65. The Structure of FcγRIIa FcγRII identifies a family of type I (cytoplasmic carboxyl terminus) membrane glycoprotein isoforms of 40 kDa. At least nine human FcγRII isoforms may be produced, deriving from three genes, FcγRIIa, FcγRIIb and FcγRIIc, located at chromosome 1q23. The major differences between the isoforms are in the cytoplasmic sequences, although there are also differences in the extracellular regions, so that some antibodies distinguish FcyRIIa from FcyRIIb isoforms (Figure 2). In the intracellular region, the FcγRIIb isoforms have an immunoreceptor tyrosine-based inhibitory motif (ITIM) 67 , while FcγRIIa has an immune-receptor tyrosine-based activation motif (ITAM) 68. Figure 2: The family of Fc receptors for human IgG* *Modified from Falk Nimmerjahn and Jeffrey V. Ravetch, 2008 69. Function of FcγRIIa FcγRII is expressed strongly on B cells, monocytes, macrophages, granulocytes mast cells and platelets. A proportion of T cells express lower levels of CD32 70. There are characteristic, and functionally significant, differences in isoform expression between __________________________________________________________________________ 12 different cell types. Interestingly, B and T lymphocytes express the ITIM isoforms, while in the other haematopoietic cells the ITAM form is expressed almost exclusively. It is a heterogeneous family of molecules (Figure 2) that plays a critical role in immunity, by linking the humoral and the cellular responses 65 . Depending on their cytoplasmic region and/or their associated chains, FcγR display coordinate and opposing roles in the immune system. The activating FcγR contains an ITAM in their cytoplasmic region or in their associated signal transducing units 65, which may initiate inflammatory, cytolytic and phagocytic activities of immune effector cells contain an ITIM in their cytoplasmic tail 73 71, 72 . The inhibitory FcγR , which upon cross-linking with ITAM- containing receptors, down regulate immunological responses. Allelic variants of FcγRIIa Functional consequences of FcγRIIa, and implications for the host defence and host susceptibility to disease, have both acquired and inherited components. FcγRIIa gene has two single-nucleated polymorphisms (SNPs) at position 27 and 131 on the amino acid sequence 74. The SNP in the FcγRIIa gene (G494A) (rs1801274) is of particular interest as it has functional consequences. FcγRIIa, expressed on mononuclear phagocytes, neutrophils, and platelets, has two co-dominantly expressed alleles, H131 and R131, which differ at amino acid position 131, in the extracellular domain (histidine or arginine, respectively), an area which strongly influences ligand binding. The allelic variants differ substantially in their ability to bind human IgG2 49, 75. H 131 is a high-binding allele and R131 is low binding. Because IgG2 is a poor activator of the classical complement pathway, FcγRIIa-H131 is essential for handling IgG2 immune complexes. The genotype distribution of FcγRIIa in Caucasian and African American populations is ~ 25% homozygous for H131 ~ 50% heterozygous, and ~ 25% homozygous for R131. Among Asians, the frequency of the R131 allele is much lower, and <10% of the population are homozygous for R131 76. __________________________________________________________________________ Amre Nasr, 2008 13 MALARIA Malaria remains a devastating global health problem. Worldwide, an estimated 300– 500 million people contract malaria each year, resulting in 1.5–2.7 million deaths annually77, 78. Malaria has a broad distribution in both the subtropics and tropics, with many areas of the tropics endemic for the disease 79. Malaria is estimated to cost Africa more than $12 billion annually and it accounts for about 25% of all deaths in children under the age of five on that continent 80 . Because of the increase in global travel to, and immigration of people from areas endemic for malaria, the incidence of imported cases of malaria in developed countries has risen. In addition, drug-resistant P. falciparum malaria continues to spread and at present involves almost all areas of the world. An increasing number of travellers are exposed to drug-resistant plasmodia. Malaria is caused by obligate intra-erythrocytic protozoa of the genus Plasmodium. Humans can be infected with one (or more) of the following five species: Plasmodium falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. Plasmodia are primarily transmitted by the bite of an infected female Anopheles mosquito, but infections can also occur through exposure to infected blood products (transfusion malaria) and by congenital transmission. Among the five species, P. falciparum is the predominant one, and responsible of most of malaria-related morbidity and mortality. Characteristics of malaria Life cycle and morphology When the infected anopheline mosquito takes a blood meal, sporozoites are inoculated into the bloodstream (Figure 3). Within 2-30 minutes sporozoites enter hepatocytes and begin to divide into exo-erythrocytic merozoites (tissue schizogony). For P. vivax and P. ovale, dormant forms called hypnozoites may typically remain quiescent in the liver until a later time; P. falciparum does not produce hypnozoites. Once merozoites leave the liver, they invade erythrocytes and develop into early trophozoites, which are ring shaped, vacuolated and uninucleated. Once the parasite begins to divide, the trophozoites are called schizonts, consisting of many daughter merozoites (blood schizogony). Eventually, the infected erythrocytes are ruptured, releasing merozoites, which subsequently invade other erythrocytes, starting a new cycle of schizogony. The duration of each cycle in P. falciparum is about 48 hours. In non-immune humans, the infection is amplified about 20-fold each cycle. After several cycles, some of the merozoites develop into gametocytes, the sexual stages of malaria, which cause no symptoms, but are infective for mosquitoes 81. __________________________________________________________________________ 14 Within the mosquito gut, zygotes are formed, which develop further and eventually become sporozoites that reside in the salivary glands. These sporozoites are released into the blood stream of a new human victim when the female mosquito takes a blood meal. Pre-patient and incubation periods In non/semi-immune individuals with P. falciparum infection, the median prepatient period (time from sporozoite inoculation to detectable parasitaemia) is 10 days (range 5–10 days), and the median incubation period (time from sporozoite inoculation to development of symptoms) is 11 days (range 6–14 days). The incubation period may be significantly prolonged by the level of immunity acquired through previous exposures, by anti-malarial prophylaxis, or by prior partial treatment, which may mitigate, but not prevent the disease 82. For non-falciparum malaria, the incubation period is usually longer (median 15–16 days), and both P. vivax and P. ovale malaria may relapse months or years after exposure, due to the presence of hypnozoites in the liver. The longest reported incubation period for P. vivax is 30 years 83. Figure 3: The life cycle of P. falciparum in the human (host) and mosquito (vector)*. *Adopted from Struik and Riley, 2004 89 __________________________________________________________________________ Amre Nasr, 2008 15 Signs and symptoms of malaria The clinical symptoms of malaria are primarily due to schizont rupture and destruction of erythrocytes. Malaria can have a gradual or a fulminate course with nonspecific symptoms. The presentation of malaria often resembles those of common viral infections; this may lead to a delay in diagnosis 84. The majority of patients experience fever (>92% of cases), chills (79%), headaches (70%), and diaphoresis (64%) 85 . Additional common symptoms include dizziness, malaise, myalgia, abdominal pain, nausea, vomiting, mild diarrhea, and dry cough. Physical signs include fever, tachycardia, jaundice, pallor, orthostatic hypotension, hepatomegaly, and splenomegaly. Clinical examination in nonimmune persons may be completely unremarkable, even without fever. Severe malaria Almost all severe forms and deaths from malaria are caused by P. falciparum. Rarely, P. vivax or P. ovale produce serious complications, debilitating relapses, and even death 86 . In 1990, the World Health Organization (WHO) established criteria for severe malaria in order to assist future clinical and epidemiological studies 87. In 2000, the WHO revised these criteria to include other clinical manifestations and laboratory values that portend a poor prognosis based on clinical experience in semi-immune patients (Table 1) 88. The major complications of severe malaria include cerebral malaria, pulmonary edema, acute renal failure, severe anaemia, and/or bleeding. Acidosis and hypoglycemia are the most common metabolic complications. Any of these complications can develop rapidly and progress to death within hours or days 88. In tropical countries with a high transmission of malaria (hyperendemic areas), severe malaria is predominantly a disease of young children (1 month to 5 years of age). Severe malaria accounts for approximately 5% of imported malaria cases (range 1–38%) 85. Diagnosis of malaria Conventional microscopy Light microscopy of thick and thin stained blood smears remains the standard method for diagnosing malaria 90 . Thick smears are 20–40 times more sensitive than thin smears for screening of Plasmodium parasites, with a detection limit of 10–50 trophozoites/ μl. Thin smears allow one to identify malaria species (including the diagnosis of mixed infections), quantify parasitaemia, and assess for the presence of schizonts, gametocytes, __________________________________________________________________________ 16 and malarial pigment in neutrophils and monocytes. The diagnostic accuracy relies on the quality of the blood smear and experience of laboratory personnel. The level of parasitaemia may be expressed either as a percentage of parasitized erythrocytes or as the number of parasites per micro-liter of blood. In non-falciparum malaria, parasitaemia rarely exceeds 2%, whereas it can be considerably higher (>50%) in falciparum malaria. In non-immune individuals, hyper-parasitaemia (>5% parasitaemia or >250 000 parasites/μl) is generally associated with severe disease 91. Alternative diagnostic methods Although examination of the thick and thin blood smear is the 'gold standard' for diagnosing malaria, important advances have been made in diagnostic testing, including fluorescence microscopy of parasite nuclei stained with acridine orange, rapid dipstick immunoassay, and polymerase chain reaction assays (PCR). Sensitivity and specificity of some of these methods approach or even exceed those of the thin and thick smear 92. Rapid dipstick immunoassays detect species-specific circulating parasite antigens targeting either the histidine-rich protein-2 of P. falciparum or a parasite-specific lactate dehydrogenase. Although the dipstick tests may enhance diagnostic speed, microscopic examination remains mandatory in patients with suspected malaria, because occasionally these dipstick tests are negative in patients with high parasitaemia, and their sensitivity below 100 parasites/μl is low 90 . Tests based on PCR for species-specific Plasmodium genome are more sensitive and specific than other tests, detecting as few as 10 parasites/μl blood 93 . Antibody detection has no value in the diagnosis of acute malaria. It is mainly used for epidemiologic studies. __________________________________________________________________________ Amre Nasr, 2008 17 Table 1 Indicators of severe malaria and poor prognosis Manifestation Features Initial World Health Organization criteria from 1990 87 Cerebral malaria Unrousable coma not attributable to any other cause, with a Glasgow Coma Scale score ≤ 9. Coma should persist for at least 30 min after a generalized convulsion Severe anemia Hematocrit <15% or hemoglobin < 50 g/l in the presence of parasite count >10 000/μl Urine output <400 ml/24 hours in adults (<12 ml/kg/24 hours in children) and a serum creatinine>265 μmol/l (> 3.0 mg/dl) despite adequate volume repletion Renal failure Pulmonary edema and acute respiratory distress syndrome Hypoglycemia The acute lung injury score is calculated on the basis of radiographic densities, severity of hypoxemia, and positive end-expiratory pressure 94 Whole blood glucose concentration <2.2 mmol/l (<40 mg/dl) Circulatory collapse (algid malaria) Abnormal bleeding and/or disseminated intravascular coagulation Systolic blood pressure <70 mmHg in patients > 5 years of age (< 50 mmHg in children aged 1–5 years), with cold clammy skin or a coreskin temperature difference >10°C Spontaneous bleeding from gums, nose, gastrointestinal tract, or laboratory evidence of disseminated intravascular coagulation Repeated generalized convulsions ≥ 3 convulsions observed within 24 hours Acidemia/acidosis Macroscopic hemoglobinuria Arterial pH <7.25 or acidosis (plasma bicarbonate <15 mmol/l) Hemolysis not secondary to glucose-6-phosphate dehydrogenase deficiency Added World Health Organization criteria from 2000 88 Impaired consciousness Reusable mental condition Prostration or weakness Hyper- parasitaemia Hyperpyrexia Core body temperature >40°C Hyperbilirubinemia *Modified from W.H.O > 5% parasitized erythrocytes or > 250 000 parasites/μl (in non-immune individuals) Total bilirubin >43 μmol/l (> 2.5 mg/dl) 88, 95, 96 __________________________________________________________________________ 18 IMMUNITY TO MALARIA Immunity to malaria infection is complex, mainly due to the complicated life cycle, that involves different parasitic stages in two different cell types, hepatocytes and erythrocytes, which result in a large number of antigens. Despite extensive research, the exact mechanism of immunity related to malaria is not fully understood. It is clear, however, that it includes both specific and non-specific mechanisms, and that both cellular and humoral immune responses are involved 97. Innate resistance Many intrinsic factors govern the ability of malaria parasites to enter and multiply within the erythrocytes, some of which are determined genetically. Individuals that lack the Duffy blood group antigens on their erythrocytes (i.e. Duffy genotype Fya-Fyb-) are resistant to P .vivax infection, because the receptor for the P. vivax merozoites is associated with antigens of the Duffy blood group described in P. knowlesi malaria 98-104 105, 106 . A similar type of resistance has also previously . Heterozygotes for haemoglobinopathies and other disorders of the RBCs (thalassemia, sickle cell anaemia and glucose-6-phosphate dehydrogenase deficiency) promote innate resistance to P. falciparum 107 . Such protection may be triggered by modification of parasite development within the erythrocytes of these individuals. However, it has been proposed that these effects may foster processes that lead to enhancement in the intensity and/or specificity of the adaptive immune response, enabling individuals carrying these genes to acquire clinical immunity to malaria faster than others 108. Haemoglobin variants Haemoglobin S Haemoglobin S (HbS) was the first of the structural haemoglobin variants to be associated with malaria protection, and in the last 60 years it has received more attention than any since described hamoglobinopathy. HbAS is >90% protective against severe and lethal malaria 109, 110 and 50% protective against even mild clinical attacks 110. Furthermore, when children with HbAS do develop clinical malaria, the parasite densities achieved during such episodes are significantly lower than those in their non-HbAS counterparts 110 . Innate mechanisms are almost certainly major determinants of this protection, of which perhaps the most plausible relates to the premature removal of iRBCs through a process __________________________________________________________________________ Amre Nasr, 2008 19 resembling that seen in glucose-6-phosphate dehydrogenase (G6PD) deficiency 111 . However, for some years, it has been suggested that the protection might also involve an immunological component. Recent studies provide strong support for this conclusion. First, in a cohort study of mild clinical malaria, conducted on the coast of Kenya, the protection attributable to HbAS was shown to increase with age 112, consistent with the conclusion that HbAS might accelerate the acquisition of malaria-specific immunity. In a second study conducted in Gabon, Cabrera et al. used a FACS-based assay to show that, compared to normal children, HbAS subjects had significantly higher titers of IgG antibodies to the P. falciparum erythrocytes membrane protein 1 (PfEMP1) 113, 114 . Similarly, HbAS may contribute to a range of immunological responses to other antigens in the same population 115-118 . The mechanisms, by which HbAS exerts such an important degree of protection, therefore have still to be fully resolved. Haemoglobin C The malaria-protective effect of many of the structural haemoglobin variants is well supported by population-genetic data, although clinical evidence has been slow to accumulate. Early studies of haemoglobin C (HbC) were not particularly convincing 119-123, but more recent observations support a marked effect, particularly against rigorously defined severe disease 124-126. In contrast to HbS, in which selection for the carrier state has occurred at the cost of the loss of homozygotes from sickle cell disease, it now appears that the selective advantage of HbC is greatest in homozygotes (HbCC). In a large study conducted in Burkina Faso, Modiano et al. found that, although significant, the protective effect of HbAC was only 30% as compared to >90% in HbCC 126 . A homozygous advantage for HbC is consistent with observations from two smaller studies that reported no episodes of severe malaria in HbCC subjects, although both lacked the power to draw definitive conclusions 124, 125 . These observations square with in vitro studies in which, on balance, effects have been limited to experiments conducted in HbCC RBCs. For many years, the favoured hypothesis regarding the protective mechanism of HbC was that it reduces the ability of P. falciparum parasites to grow and multiply in variant RBCs 127-130 . However, this mechanism is not strongly supported by in vivo data, where in most studies, HbC has not been found to exert an effect on parasite densities 126, 131 125, . A plausible alternative mechanism has now been provided by in vitro studies, which show that the expression of the major parasite-encoded RBC adhesion protein PfEMP1 is reduced in HbC-containing RBCs, an effect that is most marked in homozygotes 127, 132. As __________________________________________________________________________ 20 iRBC cytoadherence has been associated with the pathogenesis of severe malaria, this observation could explain how HbC might influence the incidence of severe disease, yet no discernible impact on less severe outcomes. Nevertheless, like many other abnormal RBCs, HbC RBCs are particularly prone to oxidant stress and it is still possible that these observations might represent some form of in vitro artefact 133 . Further studies are awaited with considerable interest. Haemoglobin E Like most of the other malaria-protective polymorphisms, Haemoglobin E (HbE) was first identified as a candidate on the basis of its distribution in malaria-endemic populations. A recent genetic analysis now suggests that in one Thai population, HbE β26 Glu → Lys, the most frequent variant in Southeast Asia, has reached its current frequency in less than 5000 years, a rate that is compatible with the conclusion that this gene expanded under positive selection 134 . Further support for the conclusion, that malaria was responsible for this selection, has been provided by a hospital-based study conducted in Thailand, which found that the presence of HbE trait (HbAE) was associated with reduced disease severity in adults admitted with acute P. falciparum malaria 135 . Like HbC, the homozygous state of HbE is relatively benign, and it therefore remains possible that selection might favour both heterozygotes and homozygotes. An intriguing in vitro study suggests that this is unlikely. Chotivanich et al. 136 cultured P. falciparum parasites in media containing various mixtures of normal and variant RBCs to show that in subjects with HbAE, only 25% of RBCs are susceptible to invasion, suggesting that the presence of an unidentified membrane defect renders the majority of RBCs relatively resistant to infection. This effect was specific for HbAE and was not seen in cells from subjects with HbEE or various forms of α-thalassaemia. This raises the possibility that HbAE might protect against severe malaria by limiting the ability of infections to achieve high parasite densities. Thalassaemias Despite compelling population-genetic evidence for malaria-protection by the αthalassaemias (summarised in ref. 137 ), all clinical studies conducted to date have proved confusing. There is now consistent evidence from a number of population studies, showing that α-thalassaemia is associated with protection from severe and fatal malaria however, it does not protect against symptom less parasitaemia 140, 142-144 suggest that it even protects against mild episodes of malaria disease 138-141 ; , and few data 140, 142, 144 . In fact, __________________________________________________________________________ Amre Nasr, 2008 21 paradoxically, in two studies conducted in the Pacifics, the incidence of mild malaria was higher in young children with α-thalassaemia than in normal children however, unlike HbAS 110 143, 144 . Interestingly, , α-thalassaemia has no effect on parasite densities 138-140, 143-145 . Moreover, while HbAS protects against all forms of clinical malaria, the effect of αthalassaemia appears to be relatively specific to severe malaria anaemia 141, 144 . Therefore, on the basis of the clinical data available to date, it seems unlikely that α-thalassaemia protects through a similar mechanism as HbAS, but rather protects against the progression of individual malaria infections to the point at which they succumb to severe anaemia. Complement receptors Parasite sequestration is not due only to endothelial binding. Some P. falciparum isolates show a phenomenon known as rosetting, where a parasitized erythrocyte binds to other erythrocytes. One mechanism for rosetting is through PfEMP-1 binding to erythrocyte CR1, encoded by the CR1 gene 146 . Up to 80% of the population in a malarious region of Papua New Guinea have an erythrocyte CR1 deficiency, which has been associated both with polymorphisms in the CR1 gene and with α+ thalassaemia, also common in this population 147 . In the same study, it was found that both CR1 polymorphisms and α thalassaemia were independently associated with resistance to severe malaria. However, studies of CR1 polymorphisms in the Gambia found no evidence of association with disease severity 148, 149 . In Thailand, an restriction fragment length polymorphism (RFLP) of the CR1 gene, that is associated with reduced expression of CR1 on erythrocytes, has been associated (in homozygotes) with susceptibility to severe malaria 150. Acquired immunity The slow development of acquired immunity to malaria may be explained by poor immunogenicity of the parasite and/or that immune responses are strain-specific. A large number of infections would then be required to encounter the whole repertoire of different antigens. The presence of high proportions of asymptomatic infections in highly endemic areas may also be due to strain specific immunity. Early studies of malaria indicated that malaria immunity is specific to the particular strain inducing the host immune response, enabling an individual to resist infection by that particular strain only, but not against heterologous strains 151, 152. Strain diversity in malaria parasites thus apparently delays development of protective immunity in endemic areas. This __________________________________________________________________________ 22 idea is strongly supported by the finding that the malaria parasite species are polymorphic for a number of specific genetic markers 153, 154. Immune mechanisms against the pre-erythrocytic stage Pre-erythrocytic stage immunity was thought to be directed against the sporozoites in the circulation and mediated by antibodies, which neutralized the sporozoite infectivity for hepatocytes 155, 156 . However, since the extracellular sporozoites invade the hepatocyte within 2-30 minutes of inoculation, anti-sporozoite antibodies must be present in the circulation at high titters and exert their activity within minutes of infection. Hence, antibody-mediated protective immunity is unlikely to be completely effective 157 . It is now generally accepted that the P. falciparum parasite developing within the host hepatocyte is the major target of protective immunity directed against the pre-erythrocytic stage. Both + + CD8 and CD4 T-cells recognize parasite-derived peptides presented by MHC class І or class ІІ molecules, respectively, on the surface of infected hepatocytes. Nevertheless, + protection against the pre-erythrocytic stage is mediated primarily by CD8 T cells 158 and involves cytokines and other factors, such as nitric oxide. In vitro treatment of Plasmodium sp, infected hepatocytes with IFN-γ eliminate P. falciparum or P. berghei parasites from the cultures 159, 160. In mice, it has been shown that IL-12 and natural killer cells have an additional role in the protection induced by immunization with either irradiation attenuated sporozoites or plasmid DNA 161. + An interest in effector CD4 T cells in anti-malarial immunity has increased in + recent years. Initial adoptive transfer experiments demonstrated that CD4 T cells of the Th1 phenotype could protect against Plasmodium sp. challenge in vivo, in the absence of any detectable cytotoxicity 162 . It has been demonstrated that active immunization with linear synthetic peptides, derived from P. yoelii proteins, confers solid protective immunity, + which is mediated by CD4 T-cells and is absolutely dependent on IFN- γ 163, 164. Immune mechanisms against the erythrocytic stage Experiments conducted several years ago in animal models identified the importance of mainly Th2 responses, including both antibody-dependent mechanisms of immunity to the erythrocytic stages 167, 168 165, 166 and cell mediated . B cells and antibodies were found to play an important role in immunity against malaria parasites, and mice lacking B __________________________________________________________________________ Amre Nasr, 2008 23 cells are unable to clear infections with the P. chabaudi parasite, rather such mice developed a chronic parasitaemia 169, 170 . However, others have shown that the effector functions of both Th1 and Th2 cells are necessary for parasite clearance in animals models 171, 172 . In humans, although several immune mechanisms have been identified in vitro as potentially parasite-neutralizing, and several antigens have been identified as targets for the antibodies involved, knowledge about their relative importance in vivo during natural infections is still limited. Different mechanisms maybe involved in the protective effects against malaria parasite of anti-malarial antibodies that have. These antibodies may mediate their effector functions against the parasite on their own or in collaboration with effector cells. IgG obtained from the sera of African adults (malaria-immuned), used for passive immunization of P. falciparum-infected non-immune Thai patients, demonstrated a reproducible reduction in parasitaemia and clinical symptoms 173 . On their own, antibodies against merozoite surface-associated proteins may block RBC invasion 174 or block merozoite release from schizonts, either by binding to surface exposed antigens, or by entering iRBC through leaky membrane at the time of rupture 175. Moreover, antibodies can block sequestration of iRBC in internal organs, allowing the parasite to be cleared by the spleen 176. Anti-malarial antibodies may also inhibit rosetting of RBC to iRBC 177 and may therefore protect against severe malaria complications (cerebral malaria). However, in collaboration with other effector immune cells, parasite antigenspecific antibodies may play an important role via a mechanism called antibody-dependent cellular inhibition (ADCI) 178, 179 . Anti-malarial IgG can inhibit parasite growth efficiently in co-operation with normal human monocytes. The parasite-neutralizing effect obtained in the ADCI assay appeared to mainly be mediated by soluble factors released by the monocytes upon their uptake of opsonized merozoites 179 . These factors, possibly TNF-α and nitric oxide, block the division of surrounding intra-erythrocytic parasites. Other studies have shown that monocytes may also kill parasites by phagocytosis of opsonized whole infected erythrocytes 180 . A study conducted in Senegal, using crude parasite extracts, reported that high-avidity cytophilic IgG1 antibodies and IgG3 were predominant in immune subjects 181. A study from Burkina-Faso showed that specific IgG2 antibody levels increased with the age of the subjects, in parallel with the development of protection against infection and malaria disease 182 . These antibodies might mediate phagocytosis or lysis of iRBCs by monocytes. The mechanism responsible for this type of parasite killing was __________________________________________________________________________ 24 suggested to involve capturing of merozoite- or schizont-bound antibodies by FcRs on the surface of monocytes 173 183 . In general, cytophilic antibodies may allow opsonisation and clearance of merozoites or iRBCs by monocytes/macrophages and neutrophils 184, 185. Naturally acquired protection against malaria in adults has been linked to immune responses to particular malaria parasite antigens 186, 187 . Many targets of humoral immune response are polymorphic or clonally variant antigens exposed on the surface of merozoites or iRBCs, respectively 188, 189 . During the acute natural infection, individuals develop specific antibody responses to antigens of the parasite variant to which they are exposed 190, 191 . These antibody responses are often, but not always, highly specific 192, 193 . Therefore, clinical immunity to blood-stage malaria is correlated with the acquisition of a repertoire of antibodies to different variants of parasite antigens, and is related to exposure and age of the host 194, 195 . Several target antigens for antibody-mediated inhibition of parasite growth or invasion have been identified 196, 197 . Antibodies to merozoite-surface antigens (especially MSP-1 and MSP-2), the apical complex organelles of merozoites (EBA-175, Rhop-1–3, RAP-1–3 and AMA-1) and the dense granule antigen Pf155/RESA, show the capacity to inhibit merozoite invasion 196. Two other antigens, SERA and ABRA, which are associated with the merozoite surface at the time of schizont rupture, are found in the immune clusters formed by antibodies inhibiting merozoite dispersal in parasite cultures 198 . In addition, antibodies to SERA have been shown to inhibit P. falciparum growth in vitro and antibodies to ABRA are efficient inhibitors of merozoite invasion 199 . Of the parasite- derived antigens expressed on the surface of infected erythrocytes, only Pf332 has been demonstrated to induce parasite-neutralizing antibodies, inhibiting the intra-erythrocytic growth of the parasite 200. In addition, lymphocytes and neutrophils have been demonstrated to exert antibodymediated killing of P. falciparum in vitro polymorphonuclear leukocytes 204, 205 201, 202 , NK cells . The monocyte-derived macrophages 206 and γδT cells 56 203 , are able to kill late stages of the intra-erythrocytic parasite in the absence of antibodies. iRBCs directly interact with leucocytes such as NK cells, macrophages and DCs. These interactions may depend on the infecting parasite phenotype and host genetic factor(s) and, together with previous exposure and age, influence the balance of immune responses. Pro-inflammatory cytokines, especially TNF-α, IFN-γ and lymphotoxin (LT), have been associated with severe malaria disease, still are crucial for the initial control of parasitaemia in human. The mechanisms are not yet well understood, but secretion of IFN-γ by NK cells, and γδT cells may have cytotoxic effects on parasite growth as well as activate monocytes and macrophages and __________________________________________________________________________ Amre Nasr, 2008 25 enhance non-opsonic phagocytosis of iRBCs. The concentrations of anti-inflammatory cytokines, such as IL-10 and transforming growth factors (TGF)-β, concurrent with those of pro-inflammatory cytokines appear to have a balancing effect. Such cytokines are also important growth factors for B cells responses. RELATED BACKGROUND The Complex Genetic Basis of Resistance to Malaria The host genetic basis for resistance to malaria is complex at several levels. It is likely that many different genes are involved, and that they interact with environmental variables and with parasite genetic factors. A number of further complexities can be considered when studying genetic resistance to malaria, namely the range of phenotypes involved, the practical difficulties of studying families, and the remarkable geographic and ethnic heterogeneity of malaria-resistance factors. Susceptibility and/or resistance to malaria can be measured in several ways. Usually, they are studied in regions with high levels of malaria transmission. When everyone is repeatedly bitten by infected mosquitoes, many children and adults are likely to have parasites in the blood. Children may have two or three episodes of malarial fever each year, and a small minority (e.g. 1%) of the malaria-fever episodes lead to severe malaria. Different genetic factors may determine the risk of an exposed person to developing parasitaemia, the risk of a parasitaemia person to becoming ill with malaria fever, and the risk of a person with malaria fever for develop severe malaria. Parasitaemia and fever can be regarded as quantitative phenotypes that are ascertained by repeated measurements within the community, whereas severe malaria is a qualitative phenotype that is typically ascertained in hospital-based studies. In principle, studies of severe malaria would be expected to detect genetic factors at each stage in the causal chain of disease progression. Severe malaria is the phenotype that matters most to vaccine developers and to those interested in evolutionary selection. Severe malaria is composed of a number of subphenotypes that may occur alone or in combination. In African children, these are cerebral malaria, severe malarial anaemia, and respiratory distress 207. Several factors may associate with reduced or increased the risk of severe malaria, some such factors will be discussed below. __________________________________________________________________________ 26 The role of IgG in protection against malarial infection and disease There is accumulating evidence for a role of IgG antibodies in protection against malarial infection and susceptibility to disease. Kinyanjui et al. 208 described long term antibody responses to malaria in Kenyan children. These responses are very brief. The brevity of the response has been traditionally attributed to a skew in the response towards IgG3. Human IgG3 antibodies have a half life of 8 days, whereas the half-lives of IgG1, IgG2, and IgG4 are up to 23 days. That study found that children have variable kinetics of antibody production and that IgG1 responses increased with age. Although an antibody response was generated to the infection and peaked after 7 days, the antibody response only lasted for approximately 6 weeks. This loss of specific antibody production occurred regardless of the isotype of the antibody measured, and this raised the question as to whether antibody decay was faster in children, or if there was a failure to produce longlived plasma cells capable of to secreting antibodies for extended periods of time. IgM responses were minimal in the children, suggesting that the measured antibodies mainly reflected a secondary antibody response. However, re-infection did not boost antibody responses. This intriguing observation could have several explanations. It could suggest that memory-B cells were deleted; alternatively it could be that high levels of serum antibodies give negative signals to B cells, or that Treg cells block re-activation of memory responses. Finally, the splenic architecture is disrupted following infection, and this could prevent activation of memory-B cells. FcγRIIa in Malaria As mentioned previously, FcγRIIa exhibits a genetic polymorphism, FcγRIIaR/H131 coded for different capacities of IgG binding and phagocytosis 49, 209 . A defence mechanism requiring the association of antigen bound IgG antibodies with FcγRs on monocytes has been proposed to play an important role in protecting immune African adults from malaria 210 . FcγRIIa is an important receptor mediating the phagocytic function of monocytes, macrophages, and neutrophils 37 . Thus, different FcγRIIa alleles affect the ability of phagocytes to internalize IgG-opsonized micro-organisms, indicating that the clearance of parasites opsonized with different IgG subclasses, may vary according to host’s genotype. Considerable differences in the distribution of FcγRIIa alleles among different ethnic groups have been reported 211 . Human IgG2 is the only IgG subclass that binds efficiently to FcγRIIa-H/H131, but not to FcγRIIa-R/R131, while both alleles bind IgG1 and IgG3 with similar efficiency 49. __________________________________________________________________________ Amre Nasr, 2008 27 A study conducted in Kenya, showed that the polymorphism FcγRIIa-R/R131 may have an influence on protection against the disease 212. Another study showed an association between the polymorphism FcγRIIa-H/H131 and susceptibility to cerebral malaria 213 . Follow-up studies in Thailand and the Gambia found that homozygotes for the 131H allele are susceptible to cerebral malaria 213, 214 . In the Thai study, the FcγRIIa association involved interaction with an FcγRIIIb gene polymorphism with cerebral malaria 213. Ethnic Differences Differences in susceptibility to malaria between ethnic groups suggest host genetic factors have a role in determining host susceptibility to malaria 215-218 . In that addition, the patterns of extent ethnic variation in Africa are important for understanding how genetic variation affects infectious diseases. Studies in West Africa showed that there are ethnic groups living in sympatry under the same malaria transmission and exposure, but, surprisingly, one of these groups, the Fulani, is less susceptible to malaria infection compared to the other ethnic groups 219, 220 . These findings offer an important approach to studying the immune genes behind the susceptibility/resistance to malaria. The Fulani people are traditionally nomadic pastoral people, who are found across West Africa, often settled in close proximity to other ethnic groups. Studies in Burkina Faso 221 , and more recently in Mali 219 , have documented a significantly lower prevalence of malaria parasitaemia and fewer clinical attacks of malaria among the Fulani than among other ethnic groups who live in neighbouring villages. The Fulani have a distinctive culture, but detailed epidemiological investigations indicate that their resistance to malaria arises primarily from genetic factors 221 . Importantly, it has also been observed that the Fulani have high levels of anti-malarial antibodies 222, 223 and a low frequency of protective globin variants and other classic malaria-resistance factors 224 . There is therefore much interest in discovering the genetic factors that determine the reduced risk to malaria infection IgG allotypes Immunoglobulin GM and KM allotypes, are associated with humoral and cellular immunological properties, and are thus ideal candidate genetic systems for investigations to identify risk-conferring factors in malaria pathogenesis. GM and KM allotypes are associated with the susceptibility to and outcome of several infectious agents allotypes are strongly associated with IgG subclass concentrations 226 55, 225 . GM , making them relevant to malarial immunity, as the protective anti-P. falciparum antibodies are predominantly of __________________________________________________________________________ 28 IgG1 and IgG3 subclasses 227. Furthermore, several population genetic properties of the GM system – the marked differences in the frequencies of GM allotypes among races, strong linkage disequilibrium within a race, and racially restricted occurrence of GM haplotypes – suggest that differential selection, caused by major selective forces like malaria, may have played an important role in the maintenance of polymorphism at these loci 228. GM/KM markers could directly affect susceptibility to malaria infection and disease outcomes by influencing the titre and affinity of anti-malarial antibodies. Although GM and KM markers are located on the constant region, there is a growing body of evidence for the involvement of these regions in antibody specificity, which usually are associated with the variable (V) region of the Ig molecule. Possible mechanisms include direct contribution to the formation of idiotypic determinants, effect on V-region protein conformation, modulation of antibody-binding affinity, and linkage disequilibrium with alleles coding for the V-region epitopes 55, 229, 230 . Additionally, GM/KM allotypes may influence antibody- dependent cellular inhibition and phagocytosis of malarial parasites by their differential interaction with FcγR. Evidence for such GM allotypic restriction in the binding affinity of the IgG molecules has been presented for certain virally encoded FcγRs 231 . It has been suggested that GM and KM allotypes themselves may not directly influence disease susceptibility, but that the associations may reflect linkage disequilibrium with other polymorphisms in the constant region genes or with specific variable region genes 12. C- reactive protein (CRP) C-reactive protein (CRP) is an acute phase protein that increases rapidly during infections and/or inflammations. CRP has the ability to activate various immune cells and has the capacity to bind to certain FcγRs; and both CRP and IgG share a sequence homology that is important for FcγR binding 232 . The binding of CRP may compete and interfere with the binding of protective malaria-specific IgG antibodies. The R131 allele of FcγRIIa was shown to bind CRP with high affinity 233 . CRP can indirectly mediate activation of the complement and complement receptors, or direct, through receptors for the FcγRs or even a putative CRP-specific receptor activation of immune cells. Interestingly, high levels of CRP were shown to be associated with severity of P. falciparum infection 234, 235 , while asymptomatic malaria infections were shown to be associated with low levels of CRP 236, 237 , these findings suggest that the levels of CRP are not merely an acute phase response related to the presence of the parasite in the body. Although the role of CRP is not well defined in P. falciparum malaria, studies showed that it can be associated with __________________________________________________________________________ Amre Nasr, 2008 29 complement mediated haemolysis of iRBC and subsequent anaemia defence against pre-erythroctic stage of malaria 239 238 , as well as in . In addition, CRP can induce the anti- inflammatory cytokine IL-10 during early stages of malaria infections 240. Current evidence suggests that serum CRP level is a complex trait, influenced by both clinical and genetic factors estimated to be 40-60% 242-244 241 . The genetic influence of circulating CRP levels is . There are several SNPs identified, in the CRP promoter region, and the -286 C<T<A (rs3091244) has been strongly associated with CRP levels 241. However, the extent to which clinical and genetic variables together account for the total variation in serum CRP level is unclear. __________________________________________________________________________ 30 THE PRESENT STUDY Objectives of the study This study aims at addressing possible associations of FcγRIIa, IgG allotypes, IgG subclasses, HbAS and CRP in relation to malaria by investigating: 1. Whether FcγRIIa (CD32) polymorphism, in association with anti-malarial IgG subclass distribution, is associated with susceptibility/resistance to either mild or severe P. falciparum malaria among Sudanese patients. 2. The distribution of GM and KM allotypes in two sympatric ethnic groups; the Fulani, who are relatively resistant to develop malaria, and the Masaleit, who are relatively susceptible to the malaria infection. 3. Whether FcγRIIa (CD32) and HbAS genotypes differ among the two sympatric ethnic groups, the Fulani and the Masaleit. 4. The distribution of FcγRIIa (CD32) genotypes, anti-malaria IgG subclass levels and their associations among sympatric Fulani and non-Fulani groups. 5. Whether a polymorphism in the CRP gene could be associated to the differences in relative resistance/susceptibility to malaria in sympatric ethnic groups in Mali and Sudan. Material and Methods used in the Study The methodologies, study areas and study populations of the included studies are described in detail in the corresponding papers. Results and Discussion This study was performed in eastern Sudan, an area characterized by seasonal and unstable malaria transmission. The predominant malaria parasite species in the area is P. falciparum. The study has two components: Hospital- and village-based investigations. The hospital study was done in New Halfa teaching hospital and it investigated the possible association between FcγRIIa polymorphisms and the outcome of clinical malaria i.e. severe versus mild malaria, while the second part was performed at the Daraweesh village (Fulani ethnic group) and a neighbouring village (non-Fulani, including the Masaleit), as well as in a village south of Gedarif (Tabarak Allah), where also the Fulani and the Masaleit live __________________________________________________________________________ Amre Nasr, 2008 31 together in the same area. The people living in Daraweesh belong to the Fulani ethnic group 245, 246 , which were reported to be less susceptible to develop malaria 219, 220. FcγRIIa polymorphism in relation to malaria (Papers I, III and IV) In this study we investigated whether FcγRIIa (CD32) polymorphism, is associated with P. falciparum malaria outcome in eastern Sudan. Our results (Paper I) provide evidence for a SNP in the FcγRIIa (CD32) gene being associated with susceptibility to severe P. falciparum malaria in the Sudanese study population. Our data demonstrated that the R/R131 genotype and R131 allele were statistically significantly associated with severe malarial disease as compared to mild malaria. Furthermore, the H/H131 genotype was statistically significantly associated with mild malaria, suggesting that the H/H131 genotype might be associated with reduced risk of succumbing to severe malaria. In support for this, a recent study in Ghana, in an area characterized by seasonal malarial transmission, observed similar findings, with the R/R131 genotype being more frequent in patients with severe malarial anaemia and cerebral malaria 247 . Although there are other reports suggesting that the H/H131 genotype is related to protection against malaria 248 , our data are in contrast with earlier studies performed in areas with higher malaria endemicity. In these studies, a correlation between the R/R131 genotype and relative protection from malaria was shown 212-214 . The reasons for these discrepant results are most likely due to genetic differences in the populations studied, as well as differences in gene-environment interactions and in patterns of malaria transmission in the different study areas. In addition, the haplotype structure of the gene might differ, so that a possibly linked causal disease association would be tagged differently by the SNP typed here. Therefore, an in-depth analysis of polymorphisms, flanking this gene and the haplotype structure is required in future larger disease association studies. When comparing Fulani to non-Fulani individuals in the village-based study (Papers III, IV) for the distribution of FcγRIIa genotypes and allele frequencies, there was a significant difference between the two ethnic groups regarding the frequency of homozygotes (H/H131 and R/R131 genotypes). The H/H131 genotype and H131 allele were significantly more frequent in the Fulani, who are considered to be less parasitized, as compared to the non-Fulani ethnic group (including the Masaleit). In contrast, the R/R131 genotype and R131 allele were significantly more frequent in the Masaleit. Thus, the findings show pronounced interethnic differences in allele and genotype frequencies of this FcγRIIa SNP, suggesting distinct genetic backgrounds of the sympatric Fulani and non- __________________________________________________________________________ 32 Fulani ethnic groups. Thus, our study population setting is similar to that previously described in Burkina Faso, where the Fulani were shown to be genetically distant from the sympatric Mossi and Rimaibé ethnic groups, on the basis of HLA class I markers 249. While the impact of the FcγRIIa polymorphism on the susceptibility to protection against malaria remains unclear, this polymorphism has been shown to play an important role in many other infections. For example, the R/R131 genotype has been found to be a risk factor for recurrent bacterial respiratory infections, while the H131 allele appears to play an important role in protection from other infections, in particular those caused by encapsulated bacteria 250 . However, the pattern of the functional activity of FcγRIIa genotype in a population may be concurrent with the pattern of the IgG subclass responses within the respective population. The importance of FcγRIIa in parasite-neutralizing events mediated by monocytes was indicated by Tebo et al., using THP-1 cells transfected with the 131H or 131R allele of the receptor 251 . While cells expressing the 131R allele of the receptor showed enhanced phagocytosis of P. falciparum-infected erythrocytes, with sera dominated by IgG1 antibodies, the phagocytosis by cells expressing the 131H receptor showed a preference for sera containing high IgG3 antibody levels. Furthermore, in a recent study on antibody-dependent cell-mediated inhibition of P. falciparum growth in vitro, the optimal parasite-neutralizing activity by human monocytes was dependent on the cooperation between FcγRIIa and FcγRIII receptors 252. Taken together, our study revealed that the FcγRIIa-R/R131 genotype is associated with increased susceptibility to develop severe malaria in a Sudanese study population, and that the H/H131 genotype, is more likely to be associated with mild malaria in this population. In addition, the Fulani, who appear to be less susceptible to malaria as compared to their sympatric tribes, the H/H131 genotype and H131 allele, are more common. Thus, it is obvious that genetic polymorphisms in FcγRIIa (CD32) might affect the outcome of malaria disease differently in different ethnic groups. IgG GM/KM allotypes in relation to malaria (Paper II) This part of the study was performed in an area South of Gedarif region (Tabarak Allah), eastern Sudan, where Fulani and Masaleit ethnic groups live in sympatry. Studying different malariometric indices (parasite rate, parasite density, and splenomegaly), showed that, the two ethnic groups differed significantly in various malariometric parameters, the Fulani being less parasitized than the Masaleit subjects. This study aimed at investigating __________________________________________________________________________ Amre Nasr, 2008 33 GM/KM allotypes as possible markers of humoral immunity/immunoglobulins that might reflect differences in the susceptibility to develop malaria between Fulani and Masaleit. GM allotypes are strongly associated with IgG concentrations 226 , making them relevant to malaria immunity. These observations led us to hypothesize that the distribution of GM/KM allotypes in Fulani are significantly different from their sympatric ethnic neighbours. Our results showed that the distribution of GM and KM phenotypes is statistically significantly different between the two ethnic groups. The frequency of GM 6 was significantly lower in the Fulani than that in the Masaleit subjects while the distribution of KM phenotypes by itself was not significantly different between the two ethnic groups. In addition, the prevalence of a particular combination of KM and GM phenotypes, however, was significantly different: the combined frequency of KM 1,3 and GM 1,17 5,6,13,14 phenotypes was significantly more frequent in the Masaleit ethnic group than in the Fulani. It is interesting to note that GM 6 is extremely rare or absent in populations other than Negroes, and this marker is commonly present in the Masaleit ethnic group. Furthermore, GM 6 has the most variable frequency (8–62%), possibly a result of differential selection caused by infectious diseases like malaria 228 . GM/KM markers could directly affect the susceptibility to malaria infection and disease outcomes by influencing the titre and affinity of anti-malarial antibodies 226 . As mentioned previously, the GM and KM markers may influence ADCI and phagocytosis of malarial parasites by their differential interaction with FcγRs especially FcγRIIa, which has been implicated in malaria infection and pathogenesis 212-214 . Taken together, our results showed differences in frequencies of GM/KM allotypes in two sympatric ethnic groups, which might contribute to the relative resistance of Fulani to P. falciparum malaria. IgG subclasses in relation to malaria (papers I and IV) In this part of the study the levels of anti-malarial IgG subclasses were compared among patients with different clinical presentation (mild versus severe malaria) from the hospital-based study (Paper I), as well as in the Fulani from Daraweesh and their non-Fulani neighbours (Paper IV). In the study (Paper IV), antibody responses to four P. falciparum blood-stage antigens, the AMA1, two alleles of MSP2 (3D7 and FC27) and the Pf332-C231 antigen, were analysed in the Fulani and non-Fulani ethnic groups. In general, the serum levels of __________________________________________________________________________ 34 IgG1 and IgG3 subclass antibodies were found to be at higher levels than IgG2 and IgG4 antibodies in both ethnic groups. However, the antibody levels varied between the different antigens. When comparing the antibody levels between the two ethnic groups, the nonFulani showed significantly higher levels of IgG1 antibodies reactive with AMA1. The responses of IgG2 antibodies reactive with AMA1 were, however, significantly higher in the Fulani than in the non-Fulani ethnic group. Although antibodies reactive with MSP23D7 showed a similar pattern of interethnic differences in IgG subclass levels as those reactive with AMA1, IgG1 antibodies reactive with MSP2-FC27 were higher in the Fulani as compared to the non-Fulani. Regarding anti-Pf332-C231 antibodies, IgG3 and IgG4 subclasses were at significantly higher levels in the non-Fulani as compared to the Fulani. In paper I, which was a hospital based study, the levels of anti-malarial IgG1 and IgG3 antibodies were statistically significantly higher in the mild malaria patients when compared with the severe malaria patients. Our results are in line with previous data 173, 210, 251, 253 regarding the association between cytophilic antibodies and protection in individuals naturally exposed to P. falciparum malaria. Several studies in Burkina Faso and Mali have shown that the Fulani have significantly higher levels of anti-malarial antibodies than other sympatric ethnic groups 221-223 , including all the IgG subclasses, except IgG4 227 . In contrast, in the present study, the non-Fulani had significantly higher levels anti-malarial IgG1 and IgG3 antibodies for all the malarial antigens studied, except for MSP2-FC27, than the Fulani. The Fulani, however, tended to have significantly higher levels of anti-malarial IgG2 antibodies than the non Fulani group. These discrepant results can most likely be explained by differences in patterns of malaria transmission in the different study areas 254 245, . In Daraweesh an entomological inoculation rate (EIR) of one infective bite/year has been estimated based on surveys in drought-free years 255 . Almost all malaria cases in Daraweesh are clinically uncomplicated, probably because of the continuous monitoring by health team, who visits the village on daily basis and provided free drug treatment 245, 254. It should be recalled that Sudan is a country characterised by hypo/meso-endemicity, with seasonal and unstable malarial transmission, unlike Burkina Faso and Mali, which are hyper/holo endemic 227. Anti-malarial antibodies against a crude P. falciparum extract (F32) and the recombinant Pf332-C231 antigens were assessed in different patient categories. The levels of anti-malarial antibodies of the different Ig isotypes differed between the study groups; being higher in individuals with mild malaria than in those with severe malaria. Thus, the levels of IgG1 and IgG3 antibodies against crude F32 were significantly higher in the mild __________________________________________________________________________ Amre Nasr, 2008 35 malaria patients compared with those seen in the severe malaria patients. High levels of malaria-specific IgG1 and IgG3 antibodies against crude F32 were associated with reduced risk of clinical episodes of malaria. Malaria-specific IgG subclasses against the recombinant Pf332-C231 showed a biased response towards IgG1, IgG2 and IgG3. The levels of antiC231 IgG1 antibodies were found to be significantly higher in patients with mild malaria than in those with severe malaria. The independent effect before and after adjustment for sex, age and ethnicity also showed that higher levels of anti-Pf332-C231 IgG1 antibodies were statistically associated with reduced risk of severe malaria. Low levels of IgG4 antibodies reactive with to the crude malarial antigen (F32) were found to be associated with lower risk of clinical malaria. Taken together, our results revealed that natural acquisition of immunity against clinical malaria appeared to be more associated with IgG1 and IgG3 antibodies, signifying their roles in parasite-neutralizing immune mechanisms. IgG subclasses in relation to FcγRIIa polymorphism (Papers I and IV) In the hospital-based part of the study (Paper I), the allele distribution FcγRIIa polymorphisms and IgG subclass profiles were analysed and our results showed that antimalarial IgG3 antibodies reactive with the crude malarial antigen, in combination with the H/H131 genotype, were associated with a reduced risk of severe malaria. In addition, a correlation between low levels of IgG2 antibodies specific to Pf332-C231 antigen were found to be associated with lower risk of severe malaria in individuals carrying the H131 allele. The village-based study (Paper II) showed that the non-Fulani had significantly higher levels of anti-malarial IgG1 and IgG3 antibodies for all the studied malaria antigens, except for MSP2-FC27, than the Fulani. The Fulani, however, tended to have significantly higher levels of anti-malarial IgG2 antibodies than the non-Fulani group. The levels of IgG2 and IgG3 antibodies were associated with the H/H genotype, while higher levels of IgG1 antibodies were associated with the R/R131 genotype. Similarly, the Fulani showed a higher frequency of the FcγRIIa-H131 allele and higher levels of anti-malarial IgG2 antibodies than the sympatric non-Fulani ethnic groups. A similar higher frequency of the FcγRIIaH131 allele and a higher proportion of IgG2 among anti-malarial antibodies, were recently observed in the Fulani in Mali compared to a sympatric non-Fulani ethnic group (Israelsson et al. in press). There is accumulating evidence for IgG1 and IgG3 antibodies playing an important role in protection against malarial disease 178 . However, the contribution of __________________________________________________________________________ 36 parasite-reactive IgG2 antibodies in protection against clinical malaria susceptibility to disease 210, 256 182 and/or in has been indicated in some epidemiological settings. In Burkina Faso, the association between anti-malarial IgG2 antibodies and protection against malaria, was suggested to be due to the relatively high prevalence of the IgG2 binding H/H131 genotype in that study population 182 . Whether the higher levels of anti-malarial IgG2 antibodies and the higher frequency of the FcγRIIa-H131 allele in the Fulani may contribute to the lower susceptibility to malaria in this ethnic group as compared to sympatric non-Fulani ethnic groups, remains to be investigated in extended epidemiological studies. While the FcγRIIa-R131 molecule shows a similar binding of the cytophilic subclasses IgG1 and IgG3 210, 251, the H131 receptor binds IgG3 more efficiently, and is also the only FcγR that binds IgG2 efficiently 49, 209, 257-259 . We showed that IgG3 antibodies specific to crude malarial antigen were more associated with reduced risk of clinical malaria in individuals with H/H131 than in individuals with R/R131 genotype. In individuals carrying the H131 receptor, IgG2 antibodies should be considered as cytophilic as it binds FcγR more efficiently. Others have shown that lower levels of IgG2 subclass antibodies to several merozoite surface proteins are independently associated with the FcγRIIa-R131 allele and with the carriage of haemoglobin AS in Gabonese children 116 . In addition, the association between IgG2 antibodies and malaria protection in Burkina Faso may be due to the relatively high titre of the IgG2 binding H/H131 genotype in that study population 182. Taken together, our study revealed that the FcγRIIa-R/R131 genotype is associated with higher levels of anti-malarial IgG1 antibodies and that the FcγRIIa-H/H131 genotype is associated with higher levels of IgG2 and IgG3. Thus, the higher IgG2 antibody levels seen in the Fulani may be due to the relatively high prevalence for the H/H131 genotype in this group. However, further studies are needed to elucidate if the FcγRIIa-R/H131 polymorphism may be a contributing factor to the differential susceptibility to malaria seen in different ethnic groups. HbAS in relation to malaria (Paper II) This study was village-based, involving the Fulani living in the Daraweesh village and neighbouring sympatric non-Fulani ethnic groups, where the prevalence of HbAS was analysed. Of the children, 108 carried haemoglobin AA and 32 carried haemoglobin AS. The frequency of the HbAS genotype was lower in the Fulani compared to the Masaleit. For __________________________________________________________________________ Amre Nasr, 2008 37 the Hb A/S allele frequencies, there was a trend between the Fulani and Masaleit, the S allele being more frequent than A allele in the latter ethnic group. A similar finding was reported from Burkina Faso, where the HbAS genotype was less frequent in the Fulani, compared to other sympatric ethnic groups, indicating that this polymorphism is not the reason for the lower susceptibility to malaria seen in the Fulani 224 . Indeed, the protection provided by HbAS carriage mainly concerns the severe complications of the malaria disease, such as cerebral malaria and/or severe anaemia, and not, or to a much lesser extent, the frequency of infection or the incidence of uncomplicated forms 28, 224 , which are unequivocally reduced among Fulani 220, 221, 224, 249. Thus, our findings are in line with the previous observation, that the lower susceptibility to malaria seen in the Fulani does not involve the classical malaria resistance gene HbAS. This suggests that other still unknown genetic factor(s) or other contributing factor(s) are involved in the differential susceptibility to malaria seen between the Fulani and other sympatric ethnic groups living in Sudan. In our study of the FcγRIIa complex in the individuals of the two ethnic groups carrying the HbAS genotype (sickle cell trait patients), the frequency of the FcγRIIaH/H131 genotype was higher, as compared to that of the R/R131 genotype. In a study from USA, an association between FcγRIIa and HbAS has previously been observed, with the H/H131 genotype being overrepresented in black children with sickle cell disease and a history of H. influenzae type b infection 260 . These findings indicate that the FcγRIIa- H/H131 genotype is more likely to be associated with the HbAS genotype, suggesting a selection for the AS and H/H131 genotypes as a result of protection against severe malaria. CRP in malaria in relation to ethnicity (Paper V) In an attempt to define the role of CRP in susceptibility to malaria, as well as to further define potential immunological factors contributing to the ethnic difference in malaria susceptibility, we analyzed the -286 C>T>A CRP genotypes and the circulating CRP levels in relation to malariometric data in sympatric individuals from two countries, Sudan and Mali, with marked different malaria endemicity. For the Fulani groups in both countries, the AA, AT and TT genotypes were the least common and the AC, CT and CC genotypes were the most common. The Malian nonFulani had the highest frequency of AA, AT and AC, while the Sudanese non-Fulani had the highest frequency for AA, CC and AC genotypes. Post-hoc analyses showed that the AA, AT and CC frequencies in Mali and the AA, AT, CT and TT frequencies in Sudan, __________________________________________________________________________ 38 differed significantly between the two ethnic groups. As mentioned previously, the influence of genetic variations on the levels of circulating CRP is estimated to be 40-60% of the concentrations of CRP 242, and a functional polymorphism in the promoter region of the CRP gene (-286 C>T>A) has been strongly associated with the circulating concentrations of CRP 261. Previous studies on this CRP polymorphism has revealed that the A allele is more common in African American than in Caucasian populations, and this difference in genotypes is associated with higher CRP levels in the former population 241 . This suggests that CRP could have been selected for in the African ancestors as a factor beneficial for the survival from infectious diseases. Our study showed that the allele frequencies were different between the two ethnic groups in both Mali and Sudan. The A allele frequency (high producer allele for CRP levels) was higher in the non-Fulani groups from both countries. In both Mali and Sudan, the, the frequency of non-A allele carriers dominated (low producer alleles for CRP levels) in the Fulani groups, while the A-allele carriers were pre-dominantly found in the nonFulani groups. The non-Fulani group presented a high frequency of A allele carriers, and in line with this, there was a tendency for higher CRP levels in the non-Fulani group than in the Fulani of Mali. All individuals in the Sudanese study populations had undetectable CRP levels, most likely because all blood samples were collected before the rainy season and all individuals were negative for malaria parasites. In conclusion, we suggest that the levels of CRP seen in asymptomatic individuals could influence the malaria infection. Also, our data suggest that the ethnic differences seen in the functional -286 CRP genotypes could be a contributing factor for the lower susceptibility to malaria seen in the Fulani from both Sudan and Mali. __________________________________________________________________________ Amre Nasr, 2008 39 CONCLUDING REMARKS AND FUTURE PERSPECTIVES Considerable efforts have been made to identify effector and host genetic factors that contribute to protection and/or pathogenesis of malaria. The very wide range of such factors involved and their diversity present an arduous task in malaria research. Nevertheless, some factors have been identified as “host genetic factors” and therefore worth pursuing to investigate. The data presented in this thesis lend support to earlier suggestions that human monocytes are important effector cells in the innate and adaptive immune responses. In the case of malaria, we have identified that the FcγRIIa-R/H131 polymorphisms may play a role in regulating the clinical outcome of the disease. In the case of severe malaria, the R/R131 genotype was significantly associated with susceptibility to severe, while the presence of H131 allele and H/H131 genotype appeared to be an important factor protecting from severe malarial disease. Binding of antibodies to the merozoites or infected red blood cells results in their opsonization, usually involving the cytophilic IgG1 and IgG3 subclasses. However, in individuals carrying the FcγRIIa-H131 allele, high levels of IgG2 antibodies have been positively associated with protection from malarial infection and disease. The FcγRIIa-H/H131 and high level for IgG2 might be involved in better parasite clearance and also reduce the risk of severe malaria outcome. These results suggest that a protective role of IgG3 and IgG2, which may activate effector cells through FcγRIIa in malarial infection and disease. The distribution of GM phenotypes as a whole, as well as a particular combination of KM and GM phenotypes, differed significantly between the two ethnic groups. The data suggest that GM allotypes may contribute to the genetic aetiology of malaria. The results presented here provide an impetus for large-scale studies to explore the role of GM and KM allotypes with IgG subclass concentrations in the pathogenesis of malaria with differential prevalence in the Fulani as compared with sympatric ethnic groups. It would be interesting to investigate any possible associations between GM and KM allotypes and FcγRIIa in the Fulani as compared with their sympatric non-Fulani neighbours. Our data showed that the Fulani had lower parasite rates, lower malaria susceptibility, lower frequency of the sickle cell trait (HbAS) and higher frequency of the FcγRIIa-H/H131 genotype and H131allele carriers. The results also indicate that the relative resistance to malaria infection seen in the Fulani does not rely mainly on the HbAS trait. Rather, other factor(s), such as FcγRIIa-H/H131 genotype, may play an important role in __________________________________________________________________________ 40 the immune responses, which can control the infection and consequently avoid development of clinical malaria. Our data showed that the Fulani ethnic group had lower levels of anti-malarial antibodies (both IgG1 and IgG3) and higher IgG2 antibody levels compared to their nonFulani neighbours. The higher IgG2 antibody levels seen in the Fulani were shown to be associated with relatively a high prevalence of the H/H131 genotype in this ethnic group. Unexpectedly, the results of the present study suggest that the magnitude and the quality of the anti-malarial humoral response is not the key element for relative resistance to malaria infection observed in the Fulani. Rather, the results suggest that IgG2 when acting as a cytophilic isotype may contribute to parasite clearance. The influence of genetic variations on the levels of circulating CRP is expected to be associated with mutations in the promoter region of the CRP gene. Our data showed that, in the Fulani ethnic group, from both Sudan and Mali, a significantly higher frequency of the low producer alleles (non-A alleles) was observed, while the high producer allele carriers (A allele) were predominantly found in the non-Fulani. Based on these results, we suggest that the lower susceptibility to malaria seen in the Fulani group could be influenced by this polymorphism. A previous study indicated that CRP can induce an anti-inflammatory IL-10 response 240 . However, further studies are needed to investigate the role of IL-10 and other cytokines in relation to protection against malaria in different endemic settings in Africa. Finally, our data provide evidence that the Fulani and the Masaleit/other sympatric non-Fulani ethnic groups are genetically different. Furthermore, the FcγRIIa polymorphism, the GM/KM allotypes and CRP genotype may be involved or contribute as host genetic factor(s), which could be the real determinant of resistance/protection against malaria infection in the Fulani ethnic groups. To address the actual association of FcγRs polymorphism, especially with malaria out-come, analysis of haplotype studies are required. Sequencing of FcγRs genes in individuals who differ significantly in susceptibility/resistance to malaria would improve our knowledge. It will be of interest to study the associations and interactions of FcγRs polymorphisms, GM/KM allotypes and anti-malarial IgG subclasses in the Fulani as compared to the non-Fulani and also among Fulanis with differential susceptibility to develop malaria infection (intra-Fulani differences). In addition, the impact of IL-4, IL-10 and TGF-β polymorphisms on anti-malarial IgG subclasses and susceptibility to develop malaria infection is of importance to investigate. __________________________________________________________________________ Amre Nasr, 2008 41 __________________________________________________________________________ 42 ACKNOWLEDGMENTS I wish to express my warm gratitude to everyone who has contributed in any way in making this thesis a reality and give me support during these intense and fascinating years, especially: My supervisors at Stockholm University: Klavs Berzins, for your patience, careful comments, advice and good sense of humor that has been really helpful, thanks for always having the time and ready to answer my questions with such a great patience and knowledge which really added a lot to this thesis. Marita Troye-Blomberg, for introducing me into the fascinating world of immunology, for her continuous support, patience and encouragement even when life gets tough and Gehad ElGhazali, currently at King Fahad Medical City in Saudi Arabia, my warmest gratitude for always finding solutions, for believing on me and giving me the strength and opportunity to carry on and finish my studies. Thank you all for your great help and concern, really thanks for the support that I got over the years till I developed as a scientist/researcher, and for making me believe in myself. Thanks for always being willing to answer questions and to give hands in all what I request. Former and present seniors at the Department of Immunology: Carmen Fernández, Eva Severinson, Eva Sverremark-Ekström and Manucherhr Abedi Valugerdi. Thank you for sharing your vast knowledge in the field of immunology and for your support and help with my work. Hedvig Perlmann for your inspiration, kindness, friendly character and your motivation in science. I would like to recognize late Peter Perlmann whose generous and dedicated personality has been, and continues to be, an inspiration for researchers in the field of immunology. Ann Sjölund and Margareta Hagstedt “Maggan”; for your excellent technical support and for your responding to any call for help. I have enjoyed working with you and miss our coffee times. Gelana Yadeta and Anna-Leena Jarva, for making the department run smoothly and for excellent administration, always having a smile, and for your readiness to assist. Past and present colleagues at the department of Immunology - Nnaemeka C. Iriemenam, Manijeh Vafa, Salah Eldin Farouk, Elisabeth Israelsson (Lisa), Nancy Awah, Halima Balogun, John Arko-Mensah, Ahmed Bolad, Pablo Giusti, Anna-Karin Larsson, Petra Amoudruz, Jacob Minang, Alice Nyakeriga, Jubayer Rahman, Camilla Rydström, Valentina Screpanti, Ylva Sjögren, Anna Tjärnlund, Khaleda Qazi Rahman, Nina-Maria Vasconcelos, Stefania Varani, Nora Bachmayer, Shanie Saghafian-Hedengren, Ebba, Olga, Irene, Fernando, Maria, Charles, Olivia Simone, Yvonne Sundström and Magdi M. Ali, for making the department a nice and friendly place to work, co-authors, for your contributions, friends, in and out of the university, for the very beautiful time. Additional co-authors, for the great collaboration that made this thesis a reality: Hayder A. Giha, Scot M. Montgomery, Omran F. Osman, Mattias Ekström, Gishanthi Arambepola, Amagana Dolo, Ogobara K. Doumbo, Per Tornvall, Robin F. Anders. __________________________________________________________________________ Amre Nasr, 2008 43 We thank our collaborators at Uppsala University for providing a friendly atmosphere and stimulating discussions that paved the way to yield our novel and interesting finding. We also thank the collaborators at Khartoum University, Faculty of Medicine especially Prof. Mustafa I. Basher, all staff at the MalRC-Biochemistry department for assistance with storage and hosting in Khartoum. We gratefully acknowledge the continued support and collaboration of the people in the study areas. Dr. Ishag Adam, Dr. Azam Ebid, Dr. Aisha Abdo, Abdallh Hifzalla, Ehasan Osman, Mustafa Hamid, Faiz Omer, Adil Ameen, New Halfa Teaching Hospital staff, Halfa Suger Factory staff, are thanked for excellent assistance in the field and laboratory. The villagers of Daraweesh are thanked for kind are generous way they have housed the study. I also would like to thank the donors, their families, New Halfa, for their participation in this study. I would like to greet my teachers, colleagues and friends in Sudan, for great help and support. To the memory of Shadin Abd-Ghafar, for the immense inspiration and tremendous knowledge. We gratefully thank my home meds Amir and Kamal for the healthy home environment and great help and support during the last four years. We also thanks my friends, in Sweden for cheerful times, special thanks to the Sudanese Association in Stockholm & Uppsala, for creating a friendly and supportive community that makes all of us feel home. Special thanks to my friends Noman, Amani, Raja Mirghani, Ahmed, Seham, Babikir Elebaid, Moawia, Samar, Susan, Hana, Nada, Noon, Mazin, Tota, Hadia, Ali, Hind, Abdallh, Amani, Ghada, Zafir, Azza, Nagat, Misara, Amir Al-bodani, Nagat, Mohamed, Hussam, Yassir, Seif Eyazal, Barsham, Randa, Anika, Elain, Liana, Hani, Anna and my friends at KTH and Cassi restraint at Karlplan. Past and present diplomatic staff at Sudanese Embassy Stockholm/ Oslo, special thanks to the ambassadors and their families: Omar Gorani, Dr. Omar Abdalmaged, Zeinab Mahmoud, Dr. Mohamed A. Altom for your encouragement and support. Finally, and most of all, I would like to express my deepest and warmest gratitude to my beloved family, my father, Osman Nasr, my brother Ayman, my sisters Iman, Wisal and Abeer, to all my nephews and my nieces. Words are not enough to express my love and gratitude to you. Whatever I have, whatever I am, whatever I get, it is all because of you. This thesis is in fact yours. The studies were financially supported by the by grants from the Swedish Agency for Research Development with Developing Countries (SIDA, SAREC), the Swedish Medical Research Council (VR), as well as a grant within the BioMalPar European Network of Excellence (LSHP-CT2004-503578). I received a fellowship from the Swedish Institute (SI) during the period of September 2006 to June 2007. __________________________________________________________________________ 44 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Delves, P. J. & Roitt, I. M. The immune system. First of two parts. N Engl J Med 343, 37-49 (2000). Medzhitov, R. & Janeway, C. A., Jr. Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 9, 4-9 (1997). Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. & Salazar-Mather, T. P. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 17, 189-220 (1999). Moretta, A. et al. Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes. Immunol Rev 155, 105-17 (1997). Janeway, C. A., Jr. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol 54 Pt 1, 1-13 (1989). Fraser, I. P., Koziel, H. & Ezekowitz, R. A. The serum mannose-binding protein and the macrophage mannose receptor are pattern recognition molecules that link innate and adaptive immunity. Semin Immunol 10, 363-72 (1998). Gewurz, H., Mold, C., Siegel, J. & Fiedel, B. C-reactive protein and the acute phase response. Adv Intern Med 27, 345-72 (1982). Schwalbe, R. A., Dahlback, B., Coe, J. E. & Nelsestuen, G. L. Pentraxin family of proteins interact specifically with phosphorylcholine and/or phosphorylethanolamine. Biochemistry 31, 4907-15 (1992). Anderson, K. V., Jurgens, G. & Nusslein-Volhard, C. Establishment of dorsalventral polarity in the Drosophila embryo: genetic studies on the role of the Toll gene product. Cell 42, 779-89 (1985). Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M. & Hoffmann, J. A. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973-83 (1996). Coban, C., Ishii, K. J., Horii, T. & Akira, S. Manipulation of host innate immune responses by the malaria parasite. Trends Microbiol 15, 271-8 (2007). Propert, D. Immunoglobulin allotypes and RFLPs in disease association. Exp Clin Immunogenet 12, 198-205 (1995). Akira, S. TLR signaling. Curr Top Microbiol Immunol 311, 1-16 (2006). Kawai, T. & Akira, S. TLR signaling. Cell Death Differ 13, 816-25 (2006). Taylor, P. R. et al. Macrophage receptors and immune recognition. Annu Rev Immunol 23, 901-44 (2005). Unkeless, J. C., Fleit, H. & Mellman, I. S. Structural Aspects and Heterogeneity of Immunoglobulin Fc Receptors. Adv Immunol 31, 247-70 (1981). Lefkowitz, D. L. & Lefkowitz, S. S. Macrophage-neutrophil interaction: a paradigm for chronic inflammation revisited. Immunol Cell Biol 79, 502-6 (2001). Cooper, M. A., Fehniger, T. A. & Caligiuri, M. A. The biology of human natural killer-cell subsets. Trends Immunol 22, 633-40 (2001). Cooper, L. J., Robertson, D., Granzow, R. & Greenspan, N. S. Variable domainidentical antibodies exhibit IgG subclass-related differences in affinity and kinetic constants as determined by surface plasmon resonance. Mol Immunol 31, 577-84 (1994). Dulphy, N. et al. An unusual CD56(bright) CD16(low) NK cell subset dominates the early posttransplant period following HLA-matched hematopoietic stem cell transplantation. J Immunol 181, 2227-37 (2008). __________________________________________________________________________ Amre Nasr, 2008 45 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. Romagnani, C. et al. CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. J Immunol 178, 4947-55 (2007). Banchereau, J. & Steinman, R. M. Dendritic cells and the control of immunity. Nature 392, 245-52 (1998). Banchereau, J. et al. Immunobiology of dendritic cells. Annu Rev Immunol 18, 767811 (2000). Shortman, K. & Liu, Y. J. Mouse and human dendritic cell subtypes. Nat Rev Immunol 2, 151-61 (2002). Gerosa, F. et al. Reciprocal activating interaction between natural killer cells and dendritic cells. J Exp Med 195, 327-33 (2002). Engelhard, V. H. Structure of peptides associated with class I and class II MHC molecules. Annu Rev Immunol 12, 181-207 (1994). Madden, D. R., Garboczi, D. N. & Wiley, D. C. The antigenic identity of peptideMHC complexes: a comparison of the conformations of five viral peptides presented by HLA-A2. Cell 75, 693-708 (1993). Hill, A. V. et al. Common west African HLA antigens are associated with protection from severe malaria. Nature 352, 595-600 (1991). Hill, A. V. et al. Molecular analysis of the association of HLA-B53 and resistance to severe malaria. Nature 360, 434-9 (1992). Hill, G. R. et al. Interleukin-11 promotes T cell polarization and prevents acute graft-versus-host disease after allogeneic bone marrow transplantation. J Clin Invest 102, 115-23 (1998). Wright, K. L. et al. CIITA stimulation of transcription factor binding to major histocompatibility complex class II and associated promoters in vivo. Proc Natl Acad Sci U S A 95, 6267-72 (1998). Belosevic, M., Davis, C. E., Meltzer, M. S. & Nacy, C. A. Lymphokine-induced macrophage resistance to infection with Leishmania major. Adv Exp Med Biol 239, 239-44 (1988). Belosevic, M., Finbloom, D. S., Van Der Meide, P. H., Slayter, M. V. & Nacy, C. A. Administration of monoclonal anti-IFN-gamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major. J Immunol 143, 266-74 (1989). Nacy, C. A. et al. Lymphokine regulation of macrophage effector activities. Adv Exp Med Biol 239, 1-11 (1988). Hopkins, S. J. The pathophysiological role of cytokines. Leg Med (Tokyo) 5 Suppl 1, S45-57 (2003). Lauritsen, J. P., Haks, M. C., Lefebvre, J. M., Kappes, D. J. & Wiest, D. L. Recent insights into the signals that control alphabeta/gammadelta-lineage fate. Immunol Rev 209, 176-90 (2006). Indik, Z., Kelly, C., Chien, P., Levinson, A. I. & Schreiber, A. D. Human Fc gamma RII, in the absence of other Fc gamma receptors, mediates a phagocytic signal. J Clin Invest 88, 1766-71 (1991). O'Brien, R. L., Lahn, M., Born, W. K. & Huber, S. A. T cell receptor and function cosegregate in gamma-delta T cell subsets. Chem Immunol 79, 1-28 (2001). Miescher, G. C., Howe, R. C., Lees, R. K. & MacDonald, H. R. CD3-associated alpha/beta and gamma/delta heterodimeric receptors are expressed by distinct populations of CD4- CD8- thymocytes. J Immunol 140, 1779-82 (1988). Braciale, T. J. et al. Antigen presentation pathways to class I and class II MHCrestricted T lymphocytes. Immunol Rev 98, 95-114 (1987). __________________________________________________________________________ 46 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. de Gast, G. C. et al. Recovery of T cell subsets after autologous bone marrow transplantation is mainly due to proliferation of mature T cells in the graft. Blood 66, 428-31 (1985). Thibault, G. & Bardos, P. Compared TCR and CD3 epsilon expression on alpha beta and gamma delta T cells. Evidence for the association of two TCR heterodimers with three CD3 epsilon chains in the TCR/CD3 complex. J Immunol 154, 3814-20 (1995). Mosmann, T. R. Properties and functions of interleukin-10. Adv Immunol 56, 1-26 (1994). Tschopp, J. & Nabholz, M. Perforin-mediated target cell lysis by cytolytic T lymphocytes. Annu Rev Immunol 8, 279-302 (1990). Weaver, C. T., Harrington, L. E., Mangan, P. R., Gavrieli, M. & Murphy, K. M. Th17: an effector CD4 T cell lineage with regulatory T cell ties. Immunity 24, 67788 (2006). Lafaille, J. J. The role of helper T cell subsets in autoimmune diseases. Cytokine Growth Factor Rev 9, 139-51 (1998). Dent, L. A. For better or worse: common determinants influencing health and disease in parasitic infections, asthma and reproductive biology. J Reprod Immunol 57, 255-72 (2002). Bergtold, A., Desai, D. D., Gavhane, A. & Clynes, R. Cell surface recycling of internalized antigen permits dendritic cell priming of B cells. Immunity 23, 503-14 (2005). Warmerdam, P. A., van de Winkel, J. G., Vlug, A., Westerdaal, N. A. & Capel, P. J. A single amino acid in the second Ig-like domain of the human Fc gamma receptor II is critical for human IgG2 binding. J Immunol 147, 1338-1343 (1991). Ollila, J. & Vihinen, M. B cells. Int J Biochem Cell Biol 37, 518-23 (2005). Kracker, S. & Radbruch, A. Immunoglobulin class switching: in vitro induction and analysis. Methods Mol Biol 271, 149-59 (2004). Perlmann, H. et al. Dissection of the human antibody response to the malaria antigen Pf155/RESA into epitope specific components. Immunol Rev 112, 115-32 (1989). Couper, K. N., Phillips, R. S., Brombacher, F. & Alexander, J. Parasite-specific IgM plays a significant role in the protective immune response to asexual erythrocytic stage Plasmodium chabaudi AS infection. Parasite Immunol 27, 171-80 (2005). Vladutiu, A. O. Immunoglobulin D: properties, measurement, and clinical relevance. Clin Diagn Lab Immunol 7, 131-40 (2000). Pandey, J. P. Immunoglobulin GM and KM allotypes and vaccine immunity. Vaccine 19, 613-7 (2000). Troye-Blomberg, M. et al. Human gamma delta T cells that inhibit the in vitro growth of the asexual blood stages of the Plasmodium falciparum parasite express cytolytic and proinflammatory molecules. Scand J Immunol 50, 642-50 (1999). Jefferis, R. & Kumararatne, D. S. Selective IgG subclass deficiency: quantification and clinical relevance. Clin Exp Immunol 81, 357-67 (1990). Mostov, K. E. & Deitcher, D. L. Polymeric immunoglobulin receptor expressed in MDCK cells transcytoses IgA. Cell 46, 613-21 (1986). Macpherson, A. J. & Slack, E. The functional interactions of commensal bacteria with intestinal secretory IgA. Curr Opin Gastroenterol 23, 673-8 (2007). Stokes, C. R., Soothill, J. F. & Turner, M. W. Immune exclusion is a function of IgA. Nature 255, 745-6 (1975). __________________________________________________________________________ Amre Nasr, 2008 47 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. Wagner, B. et al. Occurrence of IgE in foals: evidence for transfer of maternal IgE by the colostrum and late onset of endogenous IgE production in the horse. Vet Immunol Immunopathol 110, 269-78 (2006). Ng, O. T. et al. Naturally acquired human Plasmodium knowlesi infection, Singapore. Emerg Infect Dis 14, 814-6 (2008). Farouk, S. E. et al. Different antibody- and cytokine-mediated responses to Plasmodium falciparum parasite in two sympatric ethnic tribes living in Mali. Microbes Infect 7, 110-7 (2005). Grubb, R. Advances in human immunoglobulin allotypes. Exp Clin Immunogenet 12, 191-7 (1995). Ravetch, J. V. & Bolland, S. IgG Fc receptors. Annu Rev Immunol 19, 275-90 (2001). Ravetch, J. V. & Kinet, J. P. Fc receptors. Annu Rev Immunol 9, 457-92 (1991). Daeron, M. et al. Regulation of tyrosine-containing activation motif-dependent cell signalling by Fc gamma RII. Immunol Lett 44, 119-23 (1995). van de Winkel, J. G. & Capel, P. J. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol Today 14, 215-21 (1993). Nimmerjahn, F. & Ravetch, J. V. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol 8, 34-47 (2008). Mantzioris, B. X., Berger, M. F., Sewell, W. & Zola, H. Expression of the Fc receptor for IgG (Fc gamma RII/CDw32) by human circulating T and B lymphocytes. J Immunol 150, 5175-84 (1993). Hulett, M. D. & Hogarth, P. M. Molecular basis of Fc receptor function. Adv Immunol 57, 1-127 (1994). Hulett, M. D., Witort, E., Brinkworth, R. I., McKenzie, I. F. & Hogarth, P. M. Multiple regions of human Fc gamma RII (CD32) contribute to the binding of IgG. J Biol Chem 270, 21188-94 (1995). Amigorena, S. et al. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science 256, 1808-12 (1992). Junghans, R. P. Next-generation Fc chimeric proteins: avoiding immune-system interactions. Trends Biotechnol 15, 155 (1997). Clark, M. R., Stuart, S. G., Kimberly, R. P., Ory, P. A. & Goldstein, I. M. A single amino acid distinguishes the high-responder from the low-responder form of Fc receptor II on human monocytes. Eur J Immunol 21, 1911-6 (1991). Lehrnbecher, T. et al. Variant genotypes of the low-affinity Fc gamma receptors in two control populations and a review of low-affinity Fc gamma receptor polymorphisms in control and disease populations. Blood 94, 4220-4232 (1999). Muentener, P., Schlagenhauf, P. & Steffen, R. Imported malaria (1985-95): trends and perspectives. Bull World Health Organ 77, 560-6 (1999). Sachs, J. & Malaney, P. The economic and social burden of malaria. Nature 415, 680-5 (2002). Breman, J. G., Egan, A. & Keusch, G. T. The intolerable burden of malaria: a new look at the numbers. Am J Trop Med Hyg 64, iv-vii (2001). Snow, R. W., Korenromp, E. L. & Gouws, E. Pediatric mortality in Africa: Plasmodium falciparum malaria as a cause or risk? Am J Trop Med Hyg 71, 16-24 (2004). Garcia, L. S. Malaria and babesiosis. In: Garcia LS. , editor. Diagnostic Medical Parasitology. Washington, DC: American Society for Microbiology, pp. 159–204 (2001). __________________________________________________________________________ 48 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. Taylor, T. E. & Strickland, G. T. Malaria. In: Hunter GW, Strickland GT, Magill AJ, Kersey R. , editor. Hunter's Tropical Medicine and Emerging Infectious Diseases. Philadelphia: WB Saunders, pp. 614– 43 (2000). White, N. J. Malaria. In: Cook GC, Zumla AI, Weir J. , editor. Manson's Tropical Diseases. Philadelphia, PA: WB Saunders, pp. 1205– 95 (2003). Murphy, G. S. & Oldfield, E. C., 3rd. Falciparum malaria. Infect Dis Clin North Am 10, 747-75 (1996). Genton, B. & D'Acremont, V. Clinical features of malaria in returning travelers and migrants. In: Schlagenhauf P. , editor. Travelers' malaria. Hamilton: BC Decker, pp. 371– 92 (2001). Svenson, J. E., MacLean, J. D., Gyorkos, T. W. & Keystone, J. Imported malaria. Clinical presentation and examination of symptomatic travelers. Arch Intern Med 155, 861-8 (1995). Organization, W. H. Severe and complicated malaria. World Health Organization, Communicable Diseases Cluster. Trans R Soc Trop Med Hyg 84 Suppl 2, S1- 65 (1990). WHO. Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. Trans R Soc Trop Med Hyg 94 Suppl 1, S1-90 (2000). Struik, S. S. & Riley, E. M. Does malaria suffer from lack of memory? Immunol Rev 201, 268-90 (2004). Moody, A. H. & Chiodini, P. L. Methods for the detection of blood parasites. Clin Lab Haematol 22, 189-201 (2000). Torres, J. R. Malaria and babesiosis. In:Badour LM, Grobach SL, editor. Therapy of Infectionus Diseases Philadelphia, PA: Saunders; pp. 597-613 (2003). Lee, S. H. et al. New strategies for the diagnosis and screening of malaria. Int J Hematol 76 Suppl 1, 291-3 (2002). Hanscheid, T. & Grobusch, M. P. How useful is PCR in the diagnosis of malaria? Trends Parasitol 18, 395-8 (2002). Gachot, B., Wolff, M., Nissack, G., Veber, B. & Vachon, F. Acute lung injury complicating imported Plasmodium falciparum malaria. Chest 108, 746-9 (1995). WHO. Severe and complicated malaria. World Health Organization, Communicable Diseases Cluster. Trans R Soc Trop Med Hyg 84 Suppl 2, S1- 65 (1990). WHO. Tropical Disease Research Progress (1975- 1994). Twelfth Programme Report of the UNDP/World Bank/WHO Tropical Diseases TDR. (1995). Reilly, A. F. et al. Genetic diversity in human Fc receptor II for immunoglobulin G: Fc gamma receptor IIA ligand-binding polymorphism. Clin Diagn Lab Immunol 1, 640-644 (1994). Barnwell, J. W., Nichols, M. E. & Rubinstein, P. In vitro evaluation of the role of the Duffy blood group in erythrocyte invasion by Plasmodium vivax. J Exp Med 169, 1795-802 (1989). Barnwell, J. W. & Wertheimer, S. P. Plasmodium vivax: merozoite antigens, the Duffy blood group, and erythrocyte invasion. Prog Clin Biol Res 313, 1-11 (1989). Miller, L. H. & Carter, R. A review. Innate resistance in malaria. Exp Parasitol 40, 132-46 (1976). Miller, L. H. & Dvorak, J. A. Visualization of red cell membranes of lysed malariainfected cells by differential interference microscopy. J Parasitol 59, 202-3 (1973). Miller, L. H., Dvorak, J. A., Shiroishi, T. & Durocher, J. R. Influence of erythrocyte membrane components on malaria merozoite invasion. J Exp Med 138, 1597-601 (1973). __________________________________________________________________________ Amre Nasr, 2008 49 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. Miller, L. H., Mason, S. J., Clyde, D. F. & McGinniss, M. H. The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N Engl J Med 295, 302-4 (1976). Miller, L. H., McGinniss, M. H., Holland, P. V. & Sigmon, P. The Duffy blood group phenotype in American blacks infected with Plasmodium vivax in Vietnam. Am J Trop Med Hyg 27, 1069-72 (1978). Miller, L. H., Aikawa, M. & Dvorak, J. A. Malaria (Plasmodium knowlesi) merozoites: immunity and the surface coat. J Immunol 114, 1237-42 (1975). Miller, L. H., Mason, S. J., Dvorak, J. A., McGinniss, M. H. & Rothman, I. K. Erythrocyte receptors for (Plasmodium knowlesi) malaria: Duffy blood group determinants. Science 189, 561-3 (1975). Weatherall, D. J. Common genetic disorders of the red cell and the 'malaria hypothesis'. Ann Trop Med Parasitol 81, 539-48 (1987). Bayoumi, R. A. Does the mechanism of protection from falciparum malaria by red cell genetic disorders involve a switch to a balanced TH1/TH2 cytokine production mode? Med Hypotheses 48, 11-7 (1997). Aidoo, M. et al. Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet 359, 1311-2 (2002). Williams, T. N. et al. Sickle cell trait and the risk of Plasmodium falciparum malaria and other childhood diseases. J Infect Dis 192, 178-86 (2005). Ayi, K., Turrini, F., Piga, A. & Arese, P. Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum malaria in sickle trait and beta-thalassemia trait. Blood 104, 3364-71 (2004). Williams, T. N. et al. An immune basis for malaria protection by the sickle cell trait. PLoS Med 2, e128 (2005). Cabrera, G. et al. The sickle cell trait is associated with enhanced immunoglobulin G antibody responses to Plasmodium falciparum variant surface antigens. J Infect Dis 191, 1631-8 (2005). Marsh, K., Otoo, L., Hayes, R. J., Carson, D. C. & Greenwood, B. M. Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection. Trans R Soc Trop Med Hyg 83, 293-303 (1989). Ntoumi, F. et al. Sickle cell trait carriage: imbalanced distribution of IgG subclass antibodies reactive to Plasmodium falciparum family-specific MSP2 peptides in serum samples from Gabonese children. Immunol Lett 84, 9-16 (2002). Ntoumi, F. et al. Influence of carriage of hemoglobin AS and the Fc gamma receptor IIa-R131 allele on levels of immunoglobulin G2 antibodies to Plasmodium falciparum merozoite antigens in Gabonese children. J Infect Dis 192, 1975-80 (2005). Ntoumi, F. et al. Plasmodium falciparum: sickle-cell trait is associated with higher prevalence of multiple infections in Gabonese children with asymptomatic infections. Exp Parasitol 87, 39-46 (1997). Ntoumi, F. et al. Imbalanced distribution of Plasmodium falciparum MSP-1 genotypes related to sickle-cell trait. Mol Med 3, 581-92 (1997). Gilles, H. M. et al. Glucose-6-phosphate-dehydrogenase deficiency, sickling, and malaria in African children in South Western Nigeria. Lancet 1, 138-40 (1967). Guinet, F. et al. A comparison of the incidence of severe malaria in Malian children with normal and C-trait hemoglobin profiles. Acta Trop 68, 175-82 (1997). __________________________________________________________________________ 50 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. Ringelhann, B., Hathorn, M. K., Jilly, P., Grant, F. & Parniczky, G. A new look at the protection of hemoglobin AS and AC genotypes against Plasmodium falciparum infection: a census tract approach. Am J Hum Genet 28, 270-9 (1976). Thompson, G. R. Significance of haemoglobins S and C in Ghana. Br Med J 1, 6825 (1962). Thompson, G. R. Malaria and Stress in Relation to Haemoglobins S and C. Br Med J 2, 976-8 (1963). Agarwal, A. et al. Hemoglobin C associated with protection from severe malaria in the Dogon of Mali, a West African population with a low prevalence of hemoglobin S. Blood 96, 2358-63 (2000). Mockenhaupt, F. P. et al. Hemoglobin C and resistance to severe malaria in Ghanaian children. J Infect Dis 190, 1006-9 (2004). Modiano, D. et al. Haemoglobin C protects against clinical Plasmodium falciparum malaria. Nature 414, 305-8 (2001). Fairhurst, R. M., Fujioka, H., Hayton, K., Collins, K. F. & Wellems, T. E. Aberrant development of Plasmodium falciparum in hemoglobin CC red cells: implications for the malaria protective effect of the homozygous state. Blood 101, 3309-15 (2003). Friedman, M. J., Roth, E. F., Nagel, R. L. & Trager, W. The role of hemoglobins C, S, and Nbalt in the inhibition of malaria parasite development in vitro. Am J Trop Med Hyg 28, 777-80 (1979). Olson, J. A. & Nagel, R. L. Synchronized cultures of P. falciparum in abnormal red cells: the mechanism of the inhibition of growth in HbCC cells. Blood 67, 997-1001 (1986). Pasvol, G. & Wilson, R. J. The interaction of malaria parasites with red blood cells. Br Med Bull 38, 133-40 (1982). Rihet, P., Flori, L., Tall, F., Traore, A. S. & Fumoux, F. Hemoglobin C is associated with reduced Plasmodium falciparum parasitemia and low risk of mild malaria attack. Hum Mol Genet 13, 1-6 (2004). Fairhurst, R. M. et al. Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria. Nature 435, 1117-21 (2005). Duffy, P. E. & Fried, M. Red blood cells that do and red blood cells that don't: how to resist a persistent parasite. Trends Parasitol 22, 99-101 (2006). Ohashi, J. et al. Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection. Am J Hum Genet 74, 1198-208 (2004). Hutagalung, R. et al. Influence of hemoglobin E trait on the severity of Falciparum malaria. J Infect Dis 179, 283-6 (1999). Chotivanich, K. et al. Hemoglobin E: a balanced polymorphism protective against high parasitemias and thus severe P. falciparum malaria. Blood 100, 1172-6 (2002). Flint, J., Harding, R. M., Boyce, A. J. & Clegg, J. B. The population genetics of the haemoglobinopathies. Baillieres Clin Haematol 11, 1-51 (1998). Allen, S. J. et al. alpha+-Thalassemia protects children against disease caused by other infections as well as malaria. Proc Natl Acad Sci U S A 94, 14736-41 (1997). Mockenhaupt, F. P. et al. Alpha(+)-thalassemia protects African children from severe malaria. Blood 104, 2003-6 (2004). Wambua, S. et al. The effect of alpha+-thalassaemia on the incidence of malaria and other diseases in children living on the coast of Kenya. PLoS Med 3, e158 (2006). Williams, T. N. et al. Both heterozygous and homozygous alpha+ thalassemias protect against severe and fatal Plasmodium falciparum malaria on the coast of Kenya. Blood 106, 368-71 (2005). __________________________________________________________________________ Amre Nasr, 2008 51 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. Mockenhaupt, F. P. et al. The contribution of alpha+-thalassaemia to anaemia in a Nigerian population exposed to intense malaria transmission. Trop Med Int Health 4, 302-7 (1999). Oppenheimer, S. J. et al. The interaction of alpha thalassaemia with malaria. Trans R Soc Trop Med Hyg 81, 322-6 (1987). Williams, T. N. et al. High incidence of malaria in alpha-thalassaemic children. Nature 383, 522-5 (1996). Williams, T. N. et al. Negative epistasis between the malaria-protective effects of alpha+-thalassemia and the sickle cell trait. Nat Genet 37, 1253-7 (2005). Rowe, J. A., Moulds, J. M., Newbold, C. I. & Miller, L. H. P. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complementreceptor 1. Nature 388, 292-5 (1997). Cockburn, I. A. et al. A human complement receptor 1 polymorphism that reduces Plasmodium falciparum rosetting confers protection against severe malaria. Proc Natl Acad Sci U S A 101, 272-7 (2004). Bellamy, R., Kwiatkowski, D. & Hill, A. V. Absence of an association between intercellular adhesion molecule 1, complement receptor 1 and interleukin 1 receptor antagonist gene polymorphisms and severe malaria in a West African population. Trans R Soc Trop Med Hyg 92, 312-6 (1998). Zimmerman, P. A. et al. CR1 Knops blood group alleles are not associated with severe malaria in the Gambia. Genes Immun 4, 368-73 (2003). Nagayasu, E. et al. CR1 density polymorphism on erythrocytes of falciparum malaria patients in Thailand. Am J Trop Med Hyg 64, 1-5 (2001). Collins, W. E. & Jeffery, G. M. A retrospective examination of sporozoite- and trophozoite-induced infections with Plasmodium falciparum in patients previously infected with heterologous species of Plasmodium: effect on development of parasitologic and clinical immunity. Am J Trop Med Hyg 61, 36-43 (1999). Jeffery, G. M. Epidemiological significance of repeated infections with homologous and heterologous strains and species of Plasmodium. Bull World Health Organ 35, 873-82 (1966). Anders, R. F. Antigenic diversity in Plasmodium falciparum. Acta Leiden 60, 57-67 (1991). Smythe, J. A. et al. Structural diversity in the Plasmodium falciparum merozoite surface antigen 2. Proc Natl Acad Sci U S A 88, 1751-5 (1991). Nussenzweig, V. & Nussenzweig, R. S. Rationale for the development of an engineered sporozoite malaria vaccine. Adv Immunol 45, 283-334 (1989). Nussenzweig, V. & Nussenzweig, R. S. Circumsporozoite proteins of malaria parasites. Bull Mem Acad R Med Belg 144, 493-504 (1989). Saul, A. Kinetic constraints on the development of a malaria vaccine. Parasite Immunol 9, 1-9 (1987). Good, M. F. et al. Human T-cell recognition of the circumsporozoite protein of Plasmodium falciparum: immunodominant T-cell domains map to the polymorphic regions of the molecule. Proc Natl Acad Sci U S A 85, 1199-203 (1988). White, J. H. & Kilbey, B. J. DNA replication in the malaria parasite. Parasitol Today 12, 151-5 (1996). Zhan, Z., Wang, X. & Yu, J. [Erythrocyte immunity and regulating factors in mice infected with Plasmodium berghei ANKA strain]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 13, 260-3 (1995). __________________________________________________________________________ 52 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. Sedegah, M., Hedstrom, R., Hobart, P. & Hoffman, S. L. Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein. Proc Natl Acad Sci U S A 91, 9866-70 (1994). Renia, L. et al. Effector functions of circumsporozoite peptide-primed CD4+ T cell clones against Plasmodium yoelii liver stages. J Immunol 150, 1471-8 (1993). Charoenvit, Y. et al. Plasmodium yoelii: 17-kDa hepatic and erythrocytic stage protein is the target of an inhibitory monoclonal antibody. Exp Parasitol 80, 419-29 (1995). Wang, R. et al. Protection against malaria by Plasmodium yoelii sporozoite surface protein 2 linear peptide induction of CD4+ T cell- and IFN-gamma-dependent elimination of infected hepatocytes. J Immunol 157, 4061-7 (1996). Freeman, R. R. & Parish, C. R. Plasmodium yoelii: antibody and the maintenance of immunity in BALB/c mice. Exp Parasitol 52, 18-24 (1981). Grun, J. L. & Weidanz, W. P. Antibody-independent immunity to reinfection malaria in B-cell-deficient mice. Infect Immun 41, 1197-204 (1983). Amante, F. H., Crewther, P. E., Anders, R. F. & Good, M. F. A cryptic T cell epitope on the apical membrane antigen 1 of Plasmodium chabaudi adami can prime for an anamnestic antibody response: implications for malaria vaccine design. J Immunol 159, 5535-44 (1997). Amante, F. H. & Good, M. F. Prolonged Th1-like response generated by a Plasmodium yoelii-specific T cell clone allows complete clearance of infection in reconstituted mice. Parasite Immunol 19, 111-26 (1997). Linke, A. et al. Plasmodium chabaudi chabaudi: differential susceptibility of genetargeted mice deficient in IL-10 to an erythrocytic-stage infection. Exp Parasitol 84, 253-63 (1996). von der Weid, T., Honarvar, N. & Langhorne, J. Gene-targeted mice lacking B cells are unable to eliminate a blood stage malaria infection. J Immunol 156, 2510-6 (1996). Helmby, H., Kullberg, M. & Troye-Blomberg, M. Expansion of IL-3-responsive IL4-producing non-B non-T cells correlates with anemia and IL-3 production in mice infected with blood-stage Plasmodium chabaudi malaria. Eur J Immunol 28, 255970 (1998). Langhorne, J. The immune response to the blood stages of Plasmodium in animal models. Immunol Lett 41, 99-102 (1994). Bouharoun-Tayoun, H., Attanath, P., Sabchareon, A., Chongsuphajaisiddhi, T. & Druilhe, P. Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes. J Exp Med 172, 1633-41 (1990). Wahlin, B. et al. Human antibodies to a Mr 155,000 Plasmodium falciparum antigen efficiently inhibit merozoite invasion. Proc Natl Acad Sci U S A 81, 7912-6 (1984). Green, T. J., Morhardt, M., Brackett, R. G. & Jacobs, R. L. Serum inhibition of merozoite dispersal from Plasmodium falciparum schizonts: indicator of immune status. Infect Immun 31, 1203-8 (1981). David, P. H., Hommel, M., Miller, L. H., Udeinya, I. J. & Oligino, L. D. Parasite sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes. Proc Natl Acad Sci U S A 80, 5075-9 (1983). Carlson, J., Holmquist, G., Taylor, D. W., Perlmann, P. & Wahlgren, M. Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed Plasmodium falciparum erythrocyte rosettes. Proc Natl Acad Sci U S A 87, 2511-5 (1990). __________________________________________________________________________ Amre Nasr, 2008 53 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. Bouharoun-Tayoun, H. & Druilhe, P. Plasmodium falciparum malaria: evidence for an isotype imbalance which may be responsible for delayed acquisition of protective immunity. Infect Immun 60, 1473-1481 (1992). Bouharoun-Tayoun, H., Oeuvray, C., Lunel, F. & Druilhe, P. Mechanisms underlying the monocyte-mediated antibody-dependent killing of Plasmodium falciparum asexual blood stages. J Exp Med 182, 409-418 (1995). Celada, A., Cruchaud, A. & Perrin, L. H. Assessment of immune phagocytosis of Plasmodium falciparum infected red blood cells by human monocytes and polymorphonuclear leukocytes. A method for visualizing infected red blood cells ingested by phagocytes. J Immunol Methods 63, 263-71 (1983). Ferreira, M. U., Kimura, E. A., De Souza, J. M. & Katzin, A. M. The isotype composition and avidity of naturally acquired anti-Plasmodium falciparum antibodies: differential patterns in clinically immune Africans and Amazonian patients. Am J Trop Med Hyg 55, 315-23 (1996). Aucan, C. et al. High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human resistance to Plasmodium falciparum malaria. Infect Immun 68, 12521258 (2000). Gysin, J., Dubois, P. & Pereira da Silva, L. Protective antibodies against erythrocytic stages of Plasmodium falciparum in experimental infection of the squirrel monkey, Saimiri sciureus. Parasite Immunol 4, 421-430 (1982). Ndungu, F. M. et al. Naturally acquired immunoglobulin (Ig)G subclass antibodies to crude asexual Plasmodium falciparum lysates: evidence for association with protection for IgG1 and disease for IgG2. Parasite Immunol 24, 77-82 (2002). Taylor, R. R., Allen, S. J., Greenwood, B. M. & Riley, E. M. IgG3 antibodies to Plasmodium falciparum merozoite surface protein 2 (MSP2): increasing prevalence with age and association with clinical immunity to malaria. Am J Trop Med Hyg 58, 406-13 (1998). Hviid, L. Clinical disease, immunity and protection against Plasmodium falciparum malaria in populations living in endemic areas. Expert Rev Mol Med 1998, 1-10 (1998). Hviid, L. Naturally acquired immunity to Plasmodium falciparum malaria in Africa. Acta Trop 95, 270-5 (2005). Kyes, S., Horrocks, P. & Newbold, C. Antigenic variation at the infected red cell surface in malaria. Annu Rev Microbiol 55, 673-707 (2001). Rich, S. M. & Ayala, F. J. Population structure and recent evolution of Plasmodium falciparum. Proc Natl Acad Sci U S A 97, 6994-7001 (2000). Ahlborg, N., Ling, I. T., Howard, W., Holder, A. A. & Riley, E. M. Protective immune responses to the 42-kilodalton (kDa) region of Plasmodium yoelii merozoite surface protein 1 are induced by the C-terminal 19-kDa region but not by the adjacent 33-kDa region. Infect Immun 70, 820-5 (2002). Bull, P. C., Lowe, B. S., Kortok, M. & Marsh, K. Antibody recognition of Plasmodium falciparum erythrocyte surface antigens in Kenya: evidence for rare and prevalent variants. Infect Immun 67, 733-9 (1999). Chattopadhyay, R. et al. Plasmodium falciparum infection elicits both variantspecific and cross-reactive antibodies against variant surface antigens. Infect Immun 71, 597-604 (2003). Franks, S. et al. Genetic diversity and antigenic polymorphism in Plasmodium falciparum: extensive serological cross-reactivity between allelic variants of merozoite surface protein 2. Infect Immun 71, 3485-95 (2003). __________________________________________________________________________ 54 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. Bull, P. C. et al. Plasmodium falciparum-infected erythrocytes: agglutination by diverse Kenyan plasma is associated with severe disease and young host age. J Infect Dis 182, 252-9 (2000). Dodoo, D. et al. Levels of antibody to conserved parts of Plasmodium falciparum merozoite surface protein 1 in Ghanaian children are not associated with protection from clinical malaria. Infect Immun 67, 2131-7 (1999). Berzins, K. & Anders , R. F. The malaria antigens. In: Wahlgren M, Perlmann P, eds. Malaria Molecular and Clinical Aspects. Chur, Switzerland: Harwood Academic Publishers. 181- 216 (1999). Perkins, M. E. Approaches to study merozoite invasion of erythrocytes. Res Immunol 142, 662-5 (1991). Lyon, J. A., Thomas, A. W., Hall, T. & Chulay, J. D. Specificities of antibodies that inhibit merozoite dispersal from malaria-infected erythrocytes. Mol Biochem Parasitol 36, 77-85 (1989). Sharma, P. et al. Characterization of protective epitopes in a highly conserved Plasmodium falciparum antigenic protein containing repeats of acidic and basic residues. Infect Immun 66, 2895-904 (1998). Ahlborg, N. et al. Plasmodium falciparum: differential parasite growth inhibition mediated by antibodies to the antigens Pf332 and Pf155/RESA. Exp Parasitol 82, 155-63 (1996). Brown, J. & Smalley, M. E. Specific antibody-dependent cellular cytotoxicity in human malaria. Clin Exp Immunol 41, 423-9 (1980). Brown, J. & Smalley, M. E. Inhibition of the in vitro growth of Plasmodium falciparum by human polymorphonuclear neutrophil leucocytes. Clin Exp Immunol 46, 106-9 (1981). Jones, K. R., Cottrell, B. J., Targett, G. A. & Playfair, J. H. Killing of Plasmodium falciparum by human monocyte-derived macrophages. Parasite Immunol 11, 585-92 (1989). Kharazmi, A. & Jepsen, S. Enhanced inhibition of in vitro multiplication of Plasmodium falciparum by stimulated human polymorphonuclear leucocytes. Clin Exp Immunol 57, 287-92 (1984). Nnalue, N. A. & Friedman, M. J. Evidence for a neutrophil-mediated protective response in malaria. Parasite Immunol 10, 47-58 (1988). Orago, A. S. & Facer, C. A. Cytotoxicity of human natural killer (NK) cell subsets for Plasmodium falciparum erythrocytic schizonts: stimulation by cytokines and inhibition by neomycin. Clin Exp Immunol 86, 22-9 (1991). Marsh, K. et al. Indicators of life-threatening malaria in African children. N Engl J Med 332, 1399-404 (1995). Kinyanjui, S. M., Conway, D. J., Lanar, D. E. & Marsh, K. IgG antibody responses to Plasmodium falciparum merozoite antigens in Kenyan children have a short halflife. Malar J 6, 82 (2007). Salmon, J. E., Edberg, J. C., Brogle, N. L. & Kimberly, R. P. Allelic polymorphisms of human Fc gamma receptor IIA and Fc gamma receptor IIIB. Independent mechanisms for differences in human phagocyte function. J Clin Invest 89, 12741281 (1992). Groux, H. & Gysin, J. Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses. Res Immunol 141, 529-542 (1990). __________________________________________________________________________ Amre Nasr, 2008 55 211. 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. Rascu, A., Repp, R., Westerdaal, N. A., Kalden, J. R. & van de Winkel, J. G. Clinical relevance of Fc gamma receptor polymorphisms. Ann N Y Acad Sci 815, 282-95 (1997). Shi, Y. P. et al. Fcgamma receptor IIa (CD32) polymorphism is associated with protection of infants against high-density Plasmodium falciparum infection. VII. Asembo Bay Cohort Project. J Infect Dis 184, 107-111 (2001). Omi, K. et al. Fcgamma receptor IIA and IIIB polymorphisms are associated with susceptibility to cerebral malaria. Parasitol Int 51, 361 - 366 (2002). Cooke, G. S. et al. Association of Fcgamma receptor IIa (CD32) polymorphism with severe malaria in West Africa. Am J Trop Med Hyg 69, 565-568 (2003). Bryceson, A. D., Fleming, A. F. & Edington, G. M. Splenomegaly in Northern Nigeria. Acta Trop 33, 185-214 (1976). Greenwood, B. M. et al. Ethnic differences in the prevalence of splenomegaly and malaria in The Gambia. Ann Trop Med Parasitol 81, 345-54 (1987). Hill, A. V. Genetic susceptibility to malaria and other infectious diseases: from the MHC to the whole genome. Parasitology 112 Suppl, S75-84 (1996). Piazza, A. et al. Genetic and population structure of four Sardinian villages. Ann Hum Genet 49, 47-63 (1985). Dolo, A. et al. Difference in susceptibility to malaria between two sympatric ethnic groups in Mali. Am J Trop Med Hyg 72, 243-8 (2005). Modiano, D. et al. Plasmodium falciparum malaria in sympatric ethnic groups of Burkina Faso, west Africa. Parassitologia 37, 255-9 (1995). Modiano, D. et al. Different response to Plasmodium falciparum malaria in west African sympatric ethnic groups. Proc Natl Acad Sci U S A 93, 13206-11 (1996). Modiano, D. et al. Humoral response to Plasmodium falciparum Pf155/ring-infected erythrocyte surface antigen and Pf332 in three sympatric ethnic groups of Burkina Faso. Am J Trop Med Hyg 58, 220-4 (1998). Modiano, D. et al. Interethnic differences in the humoral response to non-repetitive regions of the Plasmodium falciparum circumsporozoite protein. Am J Trop Med Hyg 61, 663-7 (1999). Modiano, D. et al. The lower susceptibility to Plasmodium falciparum malaria of Fulani of Burkina Faso (west Africa) is associated with low frequencies of classic malaria-resistance genes. Trans R Soc Trop Med Hyg 95, 149-52 (2001). Pandey, J. P., Astemborski, J. & Thomas, D. L. Epistatic effects of immunoglobulin GM and KM allotypes on outcome of infection with hepatitis C virus. J Virol 78, 4561-5 (2004). Pandey, J. P. & French, M. A. GM phenotypes influence the concentrations of the four subclasses of immunoglobulin G in normal human serum. Hum Immunol 51, 99-102 (1996). Bolad, A. et al. Distinct interethnic differences in immunoglobulin G class/subclass and immunoglobulin M antibody responses to malaria antigens but not in immunoglobulin G responses to nonmalarial antigens in sympatric tribes living in West Africa. Scand J Immunol 61, 380-6 (2005). Steinberg, S. G. & Cook, C. E. The distribution of human immunoglobulin allotypes. Oxford University Press (1981). Morahan, G., Berek, C. & Miller, J. F. An idiotypic determinant formed by both immunoglobulin constant and variable regions. Nature 301, 720-2 (1983). Torres, M., May, R., Scharff, M. D. & Casadevall, A. Variable-region-identical antibodies differing in isotype demonstrate differences in fine specificity and idiotype. J Immunol 174, 2132-42 (2005). __________________________________________________________________________ 56 231. 232. 233. 234. 235. 236. 237. 238. 239. 240. 241. 242. 243. 244. 245. 246. 247. 248. Atherton, A., Armour, K. L., Bell, S., Minson, A. C. & Clark, M. R. The herpes simplex virus type 1 Fc receptor discriminates between IgG1 allotypes. Eur J Immunol 30, 2540-7 (2000). Bang, R. et al. Analysis of binding sites in human C-reactive protein for Fc{gamma}RI, Fc{gamma}RIIA, and C1q by site-directed mutagenesis. J Biol Chem 280, 25095-102 (2005). Stein, M. P. et al. C-reactive protein binding to FcgammaRIIa on human monocytes and neutrophils is allele-specific. J Clin Invest 105, 369-76 (2000). Abrams, E. T. et al. Malaria during pregnancy and foetal haematological status in Blantyre, Malawi. Malar J 4, 39 (2005). Hurt, N., Smith, T., Teuscher, T. & Tanner, M. Do high levels of C-reactive protein in Tanzanian children indicate malaria morbidity. Clin Diagn Lab Immunol 1, 43744 (1994). Hurt, N. et al. Evaluation of C-reactive protein and haptoglobin as malaria episode markers in an area of high transmission in Africa. Trans R Soc Trop Med Hyg 88, 182-6 (1994). Imrie, H. et al. Low prevalence of an acute phase response in asymptomatic children from a malaria-endemic area of Papua New Guinea. Am J Trop Med Hyg 76, 280-4 (2007). Ansar, W., Bandyopadhyay, S. M., Chowdhury, S., Habib, S. H. & Mandal, C. Role of C-reactive protein in complement-mediated hemolysis in Malaria. Glycoconj J 23, 233-40 (2006). Pied, S. et al. C-reactive protein protects against preerythrocytic stages of malaria. Infect Immun 57, 278-82 (1989). Marnell, L., Mold, C. & Du Clos, T. W. C-reactive protein: ligands, receptors and role in inflammation. Clin Immunol 117, 104-11 (2005). Carlson, C. S. et al. Polymorphisms within the C-reactive protein (CRP) promoter region are associated with plasma CRP levels. Am J Hum Genet 77, 64-77 (2005). MacGregor, A. J., Gallimore, J. R., Spector, T. D. & Pepys, M. B. Genetic effects on baseline values of C-reactive protein and serum amyloid a protein: a comparison of monozygotic and dizygotic twins. Clin Chem 50, 130-4 (2004). Pankow, J. S. et al. Familial and genetic determinants of systemic markers of inflammation: the NHLBI family heart study. Atherosclerosis 154, 681-9 (2001). Retterstol, L., Eikvar, L. & Berg, K. A twin study of C-Reactive Protein compared to other risk factors for coronary heart disease. Atherosclerosis 169, 279-82 (2003). Creasey, A. et al. Eleven years of malaria surveillance in a Sudanese village highlights unexpected variation in individual disease susceptibility and outbreak severity. Parasitology 129, 263-71 (2004). Theander, T. G. Unstable malaria in Sudan: the influence of the dry season. Malaria in areas of unstable and seasonal transmission. Lessons from Daraweesh. Trans R Soc Trop Med Hyg 92, 589-92 (1998). Ogoe, B. M. et al. Studies on the allotypic variants of IgG receptors Fc gamma RIIA and Fc gamma IIIb and their association with severe clinical malaria amonge Ghanaian children. The Third MIM Pan-African Malaria Conference Abstract No: 166, 116- 116. (2002). Ouma, C. et al. Association of FCgamma receptor IIA (CD32) polymorphism with malarial anemia and high-density parasitemia in infants and young children. Am J Trop Med Hyg 74, 573-7 (2006). __________________________________________________________________________ Amre Nasr, 2008 57 249. 250. 251. 252. 253. 254. 255. 256. 257. 258. 259. 260. 261. Modiano, D. et al. HLA class I in three West African ethnic groups: genetic distances from sub-Saharan and Caucasoid populations. Tissue Antigens 57, 128-37 (2001). Sanders, L. A. et al. Fc gamma receptor IIa (CD32) heterogeneity in patients with recurrent bacterial respiratory tract infections. J Infect Dis 170, 854-861 (1994). Tebo, A. E., Kremsner, P. G. & Luty, A. J. Fc gamma receptor-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes in vitro. Clin Exp Immunol 130, 300-306 (2002). Jafarshad, A. et al. A novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of FcgammaRII and FcgammaRIII. J Immunol 178, 3099 - 3106 (2007). Ferreira, M. U., Kimura, E. A. S. & Katzin, A. M. The IgG-subclass distribution of naturally acquired antibodies to Plasmodium falciparum, in relation to malaria exposure and severity. Ann Trop Med Par 92, 245 - 56 (1998). Hamad, A. A. et al. Chronic Plasmodium falciparum infections in an area of low intensity malaria transmission in the Sudan. Parasitology 120 ( Pt 5), 447-56 (2000). Hamad, A. A. et al. A marked seasonality of malaria transmission in two rural sites in eastern Sudan. Acta Trop 83, 71-82 (2002). Taylor, R. R., Smith, D. B., Robinson, V. J., McBride, J. S. & Riley, E. M. Human antibody response to Plasmodium falciparum merozoite surface protein 2 is serogroup specific and predominantly of the immunoglobulin G3 subclass. Infect Immun 63, 4382-8 (1995). Bredius, R. G. et al. Phagocytosis of Staphylococcus aureus and Haemophilus influenzae type B opsonized with polyclonal human IgG1 and IgG2 antibodies. Functional hFc gamma RIIa polymorphism to IgG2. J Immunol 151, 1463-1472 (1993). Parren, P. W. et al. On the interaction of IgG subclasses with the low affinity Fc gamma RIIa (CD32) on human monocytes, neutrophils, and platelets. Analysis of a functional polymorphism to human IgG2. J Clin Invest 90, 1537-1546 (1992). Sanders, L. A. et al. Human immunoglobulin G (IgG) Fc receptor IIA (CD32) polymorphism and IgG2-mediated bacterial phagocytosis by neutrophils. Infect Immun 63, 73-81 (1995). Norris, C. F. et al. Relationship between Fc receptor IIA polymorphism and infection in children with sickle cell disease. J Pediatr 128, 813-9 (1996). Kovacs, A. et al. A novel common single nucleotide polymorphism in the promoter region of the C-reactive protein gene associated with the plasma concentration of Creactive protein. Atherosclerosis 178, 193-8 (2005). __________________________________________________________________________ 58