1.0 Introduction REVIEW OF LITERATURE. Plasmodium causes malaria. Later, in 1897 Ronald Ross demonstrated

Transcription

1 Review of Literature

2 1.0 Introduction Malaria is a widespread parasitic disease transmitted by mosquitoes. Approximately million people worldwide are affected by malaria and nearly 1.5 to 2.7 million people, mainly children in Africa, die from it every year (WHO, 1997). In 1880, Laveran discovered that parasite called Plasmodium causes malaria. Later, in 1897 Ronald Ross demonstrated that the parasite is transmitted from person to person through the bite of a female Anopheles mosquito (Ross, 1897). There are about 380 species of Anopheline mosquito, but only 50 or so are able to transmit the parasite. Today approximately 40% of the world's population mostly those living in the world's poorest countries in Africa, Asia and Latin America are at risk of malaria as shown in Fig. 1.1 (WHO, 1997). There are also worrying indications of the spread of P. Jalciparum malaria into new regions of the world and its reappearance in areas, for example Azerbaijan, Tajikistan, Iraq and Turkey where it had been eliminated (Sherman, 1998). This rise in the number of malaria cases can be attributed to the development of resistance by P. Jalciparum to commonly used anti-malarial drugs, to the resistance of mosquitoes to insecticides, to the economic crisis following military conflicts and possibly to climatic changes. There are four species of human malaria parasites, namely P. malariae, P. o vale, P. vivax and P. Jalciparum. P. Jalciparum is responsible for majority of the morbidity and mortality resulting from malaria. P. Jalciparum malaria is most common in Sub Saharan Africa, and is responsible for the extremely high mortality related to malaria in this region. 1

3 ~.,, -' ". \, - Areas w here rn a l1:llia lre:t n srnlsslon occ u rs Areas with limited risk Areas wllh no majarta Figure 1.1. Malaria prone areas are shaded dark. (Global malaria situation, WHO. 2002) 1.1 Life Cycle of P. falciparum The life cycle of malaria parasite is complex, with asexual reproduction occurring in mammalian host (panel a in Fig. 1.2) and sexual reproduction in the Anopheline mosquito vectors (panel b in Fig. 1.2). The parasite is transmitted to humans in the form of sporozoites through the bite of an infected female Anopheline mosquito. After the mosquito injects haploid sporozoites in the bloodstream of the mammalian host, sporozoites may circulate for up to 45 min before invading hepatocytes, in which they undergo asexual reproduction to form intracellular schizonts. After 5-15 days of sporozoite inoculation depending on the Plasmodium species, each infected swollen hepatocyte releases approximately merozoites into the bloodstream, where they immediately invade the erythrocytes to initiate the erythrocytic cycle. Inside the erythrocyte, the parasite develops within a membrane-bound parasitophorous vacuole, first as a trophozoite and later as a schizont in which parasite undergoes multiple nuclear divisions to produce 2

4 REVIEW OF UTERATURE approximately 8-20 merozoites. After maturation of the schizont, the infected erythrocyte ruptures, liberating merozoites that rapidly invade fresh erythrocytes, thus continuing the erythrocytic life cycle (Fig. 1.2).,"---L-- Red blood cells Trophozoite Figure 1.2. Schematic diagram of life cycle of P.jaldparum Hepatocytic stage: Invasion of hepatocytes by P. falciparum sporozoites The sporozoite is the only stage in P. Jalciparum life cycle, which invades a host cell twice, once it invades mosquito salivary glands and then hepatocytes of the human host. Sporozoites of P. Jalciparum enter hepatocytes of the host shortly after being inoculated into the blood circulation by an infected mosquito. P. Jalciparum sporozoites are transported by the blood to the liver microcirculation where they come in contact with the sinusoidal cell layer composed of endothelial and Kupffer cells, the stationary phagocytes of the liver. These cells separate the sporozoites from their ultimate target cells, hepatocytes. It is not clear how the sporozoites cross the sinusoidal cell layer to reach the 3

5 hepatocytes. Two sporozoite surface proteins, named Circumsporozoite (CS) protein and a thrombospondin related anonymous protein (also named as thrombospondin related adhesive protein, TRAP) or sporozoite surface protein 2 (SSP2) are thought to be involved in this process. CS protein contains two conserved regions, namely region I and region II (also known as region II-plus). The presence of conserved region II in the CS protein in all Plasmodium species suggests that it has a common functional significance. Disruption of region II of CS protein abolished sporozoite motility and dramatically impaired their ability to invade mosquito salivary glands and the vertebrate host (Tewari et al., 2002). It is not clear whether the impairment of salivary gland invasion was the direct consequence of disrupting a crucial receptor-ligand interaction or was due to the lack of motility. Monoclonal antibodies to the circum sporozoite (CS) protein of P. vivax and P. Jalciparum blocked invasion in a species specific manner (Hollingdale et ai., 1984). However, Beier et al., showed that interaction between anti-cs human antibodies and sporozoites in the hemocoel of mosquito did not block sporozoite invasion into salivary glands (Beier et ai., 1989). The specific interaction between CS protein and heparin sulfate proteoglycans (HSPGs) expressed on the basolateral surface of hepatocytes (Cerami et al., 1992; Frevert et al., 1993, 1996) and the finding that intravenously injected CS protein is selectively targeted to the liver (Cerami et al., 1994; Sinnis et al., 1994) have suggested an important role of CS protein in sporozoites' homing to the hepatic site. Although most other cell types, especially vascular endothelial cells, express HSPGs, the remarkably selective targeting of sporozoites to the liver occurs, maybe due to the unique liver specific HSPG chain or HSPG on a liver specific receptor. However, another study demonstrated that the presence of glycosaminoglycans (GAGs) on the target cell surface is not required for sporozoite penetration, although GAGs enhances sporozoite invasion. They have demonstrated that sporozoite invasion into mutant CHO cells expressing undersulfated 4

6 REVIEW OF UTERATURE glycosaminoglycans or no glycosaminoglycans was only inhibited 41-49% or 24-32%, respectively, in comparison to invasion into CHO-K1 cells, which express normal HSPG on the surface. Prior cleavage of glycosaminoglycans (GAGs) from HepG2 surface with heparinase or heparitinase had no significant inhibitory effect on subsequent P. berghei sporozoite invasion (Frevert et al., 1996). In addition to CS protein, the involvement of TRAP in sporozoite invasion into hepatocytes has been demonstrated by elegantly designed experiments. Antibodies against TRAP dramatically blocked parasite motility (Spaccapelo et al., 1997). Transgenic P. berghei sporozoites carrying a disrupted TRAP were not motile, failed to invade mosquito salivary gland and showed a drastic reduction in their ability to infect host hepatocytes (Sultan et al., 1997). From these observations it is now clear that TRAP must be functionally coupled to a CS protein with an intact region II for successful sporozoite invasion (Tewari et al., 2002) Erythrocytic blood stage Invasion of erythrocytes by merozoites Figure 1.3. Schematic diagram showing different steps of erythrocyte invasion by Plasmodium merozoite. 5

7 Erythrocytic stage of parasite's life cycle initiates with the invasion of human erythrocytes by merozoites. Invasion of P. Jalciparum merozoites into erythrocytes is a complex multi-step process as shown in Fig. 1.3 (Chitnis and Miller, 1994). In the first step any part of the merozoite surface can attach to the erythrocyte surface in a reversible manner. Electron microscopic observation on the invasion of erythrocytes by P. knowlesi merozoites has given some insight to the events associated with merozoite invasion (Aikawa et al., 1978). After initial attachment, reorientation of merozoite takes place to bring its apical end in contact with the erythrocyte surface. The apical end is characterized by the presence of membrane-bound organelles such as the rhoptries and micronemes. The rhoptries and micronemes release their contents to make an indentation on the erythrocyte surface at the point of contact, which leads to the development of tight, irreversible junction between apical end of the invading merozoite and erythrocyte surface. (Aikawa et ai., 1978; Bannister et al., 1986; Bannister and Mitchell, 1989; Stewart et al., 1986). The merozoite, then moves into the erythrocyte and the junction becomes a circumferential ring, which moves around the merozoite from the apical end to the posterior end and eventually pinches closed at the posterior end of the merozoite. The merozoite finds itself inside a vacuolar membrane, known as parasitophorous vacuolar membrane (PVM). Erythrocyte invasion by merozoites is largely dependent on actin polymerization as cytochalasin B treatment of the merozoite can block invasion but not the attachment (Miller et ai., 1979). Efforts have been made to identify the molecules present both on the surface of merozoites and erythrocytes, which ensures the high degree of specificity of merozoite invasion into erythrocytes or its precursor cells. Studies have shown that invasion of different species of Plasmodium relies on distinctively different receptor-ligand interactions. For example, the invasion of P. knowlesi and P. vivax merozoites into human erythrocytes is solely dependent on Duffy blood group antigen present on 6

8 erythrocytes, whereas, P. Jalciparum merozoites use multiple receptors on erythrocyte surface for invasion (Mitchell et ai., 1986, Okoyeh et al., 1999). Therefore, development of a successful invasion blocking strategy to prevent P. Jalciparum infection is challenging. Some preliminary studies have shown that the presence of calcium is also important for invasion. McCallum-Deighton and Holder demonstrated using calciumdepleted parasite culture medium that invasion of P. Jalciparum is dependent upon the presence of calcium and that neither magnesium, manganese nor zinc can substitute for it (McCallum-Deighton and Holder, 1992). This suggests that the inhibition effect is calcium specific and not dependent upon a non-specific, charge-based mechanism. Using erythrocyte ghosts and altering the internal and external concentrations of calcium and the specific Ca 2 +-chelator EGTA, it has also been shown that the calcium in the extracellular environment is required for invasion (McCallum-Deighton and Holder, 1992). Extensive efforts are being made to understand the molecular mechanism of complex invasion process in P. Jalciparum with the hope that it might help to develop novel intervention strategies including invasion blocking vaccines to prevent erythrocyte invasion and hence malaria Ring stage After merozoite invasion the parasite attains a thin discoidal, flat ring form, in which a thick rim of cytoplasm houses the major organelles such as nucleus, mitochondria, plastid, most of the ribosomes and endoplasmic reticulum. The centre of the disc is thin and contains few cytoskeletal structures. Giemsa-stained films viewed under the light microscope show the parasite as a signet ring structure, giving the characteristic name to this stage. Heme derivative resulting from hemoglobin catabolism is converted to inert brown pigment 'hemozoin' crystals that accumulate within the pigment vacuole of parasite. This process starts in the ring stage, and continues throughout the 7

9 REVIEW OF UTERATURE erythrocytic phase of parasite life cycle. As the parasite grows, the area of the parasitophorous vacuolar membrane (PVM) surrounding it increases and extends as narrow, finger-like projections into the surrounding RBC. The ring enters into trophozoite stage after hours of invasion into erythrocyte (Bannister et al., 2000). Parasite Cytoplasm '''1'-- -+ Nucleus Figure 1.4. Ring stage of P. jalciparum and an enlarged image of a ring infected RBC. in which parasite cytoplasm and nucleus are marked by arrows Trophozoite stage Trophozoite stage starts roughly 24 hours after merozoite invasion and continues for 8-12 hours. Trophozoite stage is markedly different from the ring stage in terms of cell size and shape. The number of free ribosomes and the size of the endoplasmic reticulum (ER) increase, suggesting an increase in protein synthesis. The surface area of the trophozoite enlarges greatly, with the appearance of irregular bulges and deep tubular invaginations at its surface (Slomianny, 1990; Elford et al., 1995). DNA replication, RNA and protein synthesis as well as organelle biogenesis takes place in trophozoite proportionately for the following stage of schizogony. P. jalciparum in the ring stage pinocytoses hemoglobin-containing erythrocyte cytosol into small vesicles. As parasites develop, increased metabolic activity needs increased uptake of hemoglobin as a source of amino acids. In the trophozoites stage two 8

10 REVIEW OF UTERATURE specialized structures, namely food vacuole and cytostome are developed. These structures are thought to be involved mainly in hemoglobin transport (Bannister et ai., 2000). During its blood stage, the malaria parasite proteolyses host hemoglobin, releasing large amount of heme as a by-product. Free heme has been shown to be toxic to malaria parasites (Orjih et ai., 1981; Pandey et al., 1999 and 2001). For detoxification malaria parasite converts heme derivative, ~-Hematin to a non-toxic polymerized crystalline form, called hemozoin. In the trophozoite stage P. Jalciparum has an ability to express proteins on the surface of the infected erythrocytes which enables the infected erythrocytes to cytoadhere to vascular endothelium in various organt', a process referred to as cytoadherence that implicated in malaria pathogenesis (Cooke et ai.,2000). A ----jf---- Hemozoin pigment E-----jf---- ParaSite cytoplasm Figure 1.5. Trophozoite stage of P. Jalciparum and B shows an enlarged image of a trophozoite infected RBC. in which parasite cytoplasm and hemozoin pigment are marked by arrows. 9

11 REVIEW OF UTERATURE Schizont stage The erythrocytic cycle is completed with a process called schizogony in which mature trophozoites undergo repetitive nuclear divisions to produce 8-30 daughter merozoites that ultimately disrupt the erythrocyte membrane and are released into the blood circulation to invade fresh erythrocytes. This stage prevails for hours. The food vacuole containing hemozoin pigment becomes more condensed and appears as a dark spot. The rupture of erythrocyte membrane during schizont burst involves the action of proteases. One such protease, named falcipain-2, which cleavages ankyrin and protein 4.1. The removal of these proteins from erythrocytes is postulated to cause membrane instability facilitating merozoite release in vivo (Dua et ai., 2001). Hemozoin pigment ~+-- Merozoite Figure 1.6. Schizont stage of P. Jalciparum and an enlarged image of a schizont infected RBC, in which highly condensed hernozoin pigment and merozoite are marked by arrows. 10

12 1.1.3 Sexual stage Some merozoites, after invading erythrocytes take an alternative pathway of differentiation into sexual forms, male and female gametocytes, which are taken up by the mosquito in the midgut during a blood meal. After release from erythrocytes, male and female gametes fuse to form a diploid zygote, which undergo meiosis to form motile ookinetes. The ookinetes cross the midgut wall and form oocysts on the gut wall on the hoemoceal side. Sporozoites develop in the oocyst, which eventually bursts, releasing large number of haploid sporozoites that migrate to the salivary glands of mosquito to await injection into the human host during the next blood meal (Ho and White, 1999). The stimuli that induce gametogenesis are not well established. Xanthurenic acid (XA), a by-product of tryptophan metabolism present both in vertebrate and invertebrate hosts has been identified as an inducer of gametogenesis. XA production is reduced in some Anopheline species with reduced susceptibility to malaria, supporting its predicted importance. 1.2 Development of resistance to anti-malarial drugs Resistance to chloroquine, sulfadoxin-pyrimethamine and mefloquine has emerged as one of the greatest challenge to control malaria, although resistance to artimisinin derivatives has not been found till now. Antimalarial drug resistance has become a major factor for the spread of malaria into new areas and re-emergence in the areas where malaria has been eradicated. Anti-malarial drug-resistance has been reported in two species among four species causing human malaria, P. vivax and P. Jalciparum. Choloroquine (CQ) resistant strain has spread in all the areas where P. Jalciparum transmission is highest. Resistance to sulfadoxinpyrimethamine is prevalent in Africa and occurs frequently in South East Asia and South America (WHO, 1997). Mefloquine resistance is frequent in some areas of South East Asia, Amazon region of South America and 11

13 REVIEW OF UTERATURE sporadically in Africa (Mockenhaupt, 1995). There are number of factors contributing to the rapid emergence of drug resistance in malaria. The molecular mechanism of resistance to different anti-malarials is being investigated. In case of resistance to drugs, targeted to inhibit folate metabolism, Peterson et al., have identified specific point mutation in the parasite genes encoding target enzymes, dihydrofolate reductase thymidylate synthase (DHFR-TS) and dihydropteroate synthase (DHPS) as mediator of resistance to these drugs (Peterson et ai., 1988). Parasites having a mutation from Thr-108jSer-108 to Asn-108 in DHFR-TS are resistant to the pyrimethamine (Peterson et al., 1988). Mutation at 540 codon in DHPS correlated with increased pyrimethamine-sulfadoxine resistance (Plowe et al., 1997). Studies on the mechanism of action of CQ have suggested that upon entering into the food vacuole of parasite it binds to heme and prevents the polymerization of heme to form nontoxic hemozoin. Free heme is toxic to the parasites. Chloroquine resistance (CQR) in P. JaZciparum has been shown to be associated with a point mutation at position 78 from lysine to threonine in PfCRT (Fidock et ai., 2000). PfCRT is an approximately 300 kda Transmembrane protein that likely functions as a transporter in the parasite's digestive vacuole membrane. This mutation in PfCRT enables P. JaZciparum to counter the action of drug by expelling it rapidly from the food vacuole via an unknown mechanism resulting in decreased accumulation of drug in the food vacuole, thereby nullifying the effect of the drug. A better understanding of the mechanism by which malaria parasites develop drug resistance may be useful in the design of new drugs that circumvent or reverse known parasite resistance mechanisms (Su et al., 1997). 1.3 Malaria Pathogenesis Clinical symptoms of malaria are not manifested during the hepatocytic stage of infection in human host. All the clinical symptoms of malaria 12

14 REVIEW OF UTERA TURE result from the asexual blood stage of the parasite. Pathophysiological processes in malaria result from the asexual erythrocytic cycle with the rupture of schizont infected erythrocytes, which releases toxic materials of parasitic origin along with merozoites into the blood circulation. The response of the host to these toxic materials gives rise to symptomatic disease. Fever and chills also occur at the time of rupture of schizonts. Another unique feature of falciparum malaria is that P. Jalciparum infected trophozoites and schizonts can adhere to endothelial cells lining the blood capillaries. This phenomenon is commonly referred to as cytoadherence. Cytoadherence is believed to be a major pathogenic mechanism responsible for severe malaria Parasite sequestration and cytoadherence The erythrocytes infected with P. Jalciparum trophozoites and schizonts disappear from the peripheral blood circulation and only the erythrocytes infected with ring stage are available in the peripheral circulation. This disappearance of trophozoites and schizonts infected erythrocytes is mainly due to the sequestration of these infected erythrocytes on the microvascular endothelial cells of various host organs like heart, lung, brain, liver, intestine, and skin (Pongponratn et al., 1991; Turner et al., 1994). In addition, post mortem reports revealed that patients who die in the acute phase of falciparum malaria have intense sequestration of infected erythrocytes containing mature forms of the parasite in the microvasculature of vital organs such as liver, kidney, brain and placenta (Aikawa, 1990; MacPherson et ai., 1985; 00 et al., 1987). The observation of large numbers of parasites accumulated in specific host organs such as the brain and the placenta associated with adverse clinical outcomes, suggests that organ-specific sequestration of parasite infected erythrocytes is important for pathogenesis of malaria, although direct evidence showing this correlation is yet to be established. Parasite sequestration in brain microvessels is believed to be responsible for the 13

15 pathology of severe form of malaria in brain, called Cerebral Malaria. The exact mechanism of disease severity due to these events is not well established. But, it is believed that sequestration of infected erythrocytes leads to alterations in microcirculatory blood flow resulting in reduced oxygen and substrate supply to the host organs, leading to anaerobic glycolysis and lactic acidosis (White and Ho, 1992). In addition, local microenvironment of the capillaries and post capillary venules may be favorable to the parasite development and optimize the invasion of merozoites released from these sequestered parasitized red blood cells (PRBCs), adding further complications to severe malaria. The consequences of these events may lead to various organ dysfunctions, which may ultimately result in fatality. Besides adhering to endothelial cells, infected erythrocytes can adhere in vitro to platelets, monocytes, lymphocytes, uninfected erythrocytes (rosetting) and even other infected erythrocytes (autoagglutination). Although the extent of these additional interactions occurring in vivo is not clear, the phenomenon of parasite sequestration is critical both for the survival of the parasite as well as the pathophysiology in the host. The sequestration of infected erythrocytes offers two major advantages to the parasite. One, it enables parasite infected erythrocytes to avoid passage through the spleen where they may be marked as defective and destroyed by the reticulo-endothelial system in the spleen. As the parasite sequestration in various host organs is thought to be responsible for the pathological consequences of malaria, intense investigation of the molecular mechanism of sequestration has drawn considerable attention Rosetting Another characteristic property of the infected erythrocyte is its ability to bind uninfected erythrocytes, a phenomenon commonly referred to as "rosetting". It is believed that parasi te derived molecules, PfEMP 1 expressed on the surface of infected erythrocytes bind to the molecules 14

16 REVIEW OF UTERA TURE present on uninfected erythrocytes forming the rosette. To date, five rosetting receptors have been identified on RBCs: blood group antigens A and B (Barragan et al., 2000), CD36 (van Schravendijk et al., 1992), complement receptor 1 (CR1) (Rowe et al., 1997) and Heparin sulfate (HS)-like glycosaminoglycans (GAGs) (Rowe et al., 1994). The high sensitivity of P. Jalciparum rosettes in type 0 blood to heparin and N sulfated glycans has been shown in both laboratory strains and wild isolates (Barragan et al., 1998; Carlson et al., 1992; Carlson and Wahlgren, 1992.). The rosettes of most isolates can be disrupted by heparin sulfate, whereas other related anionic GAGs such as the chondroitin sulphates A, B, and C and hyaluronic acid have no effect on rosette formation (Rowe et al., 1994). These observations suggest that malaria parasites use heparin and heparin sulfate on the surface of RBCs as rosetting receptors. CR1 is also widely distributed on the RBC surface. CR1 seems to be an important rosetting receptor, as some parasites cultured in CR1-deficient RBCs lose their capacity to form rosettes. The conclusion was further strengthened by the ability of soluble CR1 to disrupt rosettes and block rosette reformation by some parasite strains (Rowe et al., 1997). It is not clear why do malaria parasites form rosettes. Merozoites released from the infected erythrocytes could have higher chances of invasion. Rosetting could also be a strategy to evade the cell mediated immunity as the infected erythrocyte is surrounded by uninfected erythrocytes. It is also not known why some isolates form rosettes, whereas others do not. Rosetting in the microvasculature tends to reduce the blood flow and increase sequestration of parasitized erythrocytes on the capillary walls. 15

17 REVIEW OF UTERATURE Figure 1.7. A rosette, in which a parasitized erythrocyte (shown in orange colour at the centre of the picture) is surrounded by three uninfected erythrocytes (shown in red). Reproduced from the website of Dr. Alex Rowe, Wellcome Senior Research Fellow at the Institute of Cell, Animal and Population Biology, University of Edinburgh. 1.4 Severe malaria The most common clinical symptoms of severe malaria are high fever, progressing anemia, multi-organ dysfunction, and unconsciousness, i.e., coma, which is a sign of cerebral malaria and one of the causes of death associated with malaria. (Miller et al., 1994). In non-immune patients, many of the severe complications of P. jalciparum such as cerebral malaria, anemia, hypoglycemia, renal failure and noncardiac pulmonary edema occur in combination or as isolated complications Cerebral malaria In P. jalciparum infection, parasitized erythrocytes adhere to microvascular endothelium leading to severe clinical disease in the brain, called cerebral malaria (CM). Clinically CM refers to a diffuse symmetric encephalopathy. It accounts for the mortality between 20-50% associated with malaria. In Mrican children growing up in malaria endemic areas, CM usually manifests as seizures, impaired consciousness, metabolic acidosis, respiratory distress and severe anemia (Marsh et al., 1995). In Mrica, children rarely develop renal failure or pulmonary edema in CM in 16

18 comparison with adults. In human CM, the attachment of RBC infected with mature-stage parasites to endothelial cells lining the postcapillary venules is not restricted to the brain. Microvessels of the heart, lungs, kidneys, small intestine, and liver are the other principal sites of sequestration. This sequestration is important for the survival of the parasite but can have severe consequences for the host (Grau et ai., 1993). Sequestered cells that clog the brain capillaries may reduce blood flow resulting in confusion, lethargy, and unarousable coma. Although established understanding claims that only mature forms of the parasite would be sequestered, but it has been reported that all stages of P. Jalciparum are sequestered in the brain (Silamut et ai., 1999). Microscopic observation of brain endothelium in CM patients does not demonstrate any visible damage (MacPherson et al., 1985) to endothelium, but immunohistochemical staining suggests endothelial activation for several receptors such as ICAM1, CD31. It is reported that there is disruption of the blood-brain barrier in CM patients (Brown et al., 1999). TNF-a is thought to playa major role in the outcome of CM as it is reported to upregulate the expression of, ICAM-1, the most important cytoadherence receptor on brain endothelium Placental malaria Pregnant women often suffer from severe malaria, known as pregnancy associated malaria (PAM) or placental malaria. The risk of infection is highest for women in first pregnancy (primigravidae) and tends to reduce, although not completely, in subsequent pregnancies (multigravidae). This pattern might be due to the development of immune response against PAM. Placental malaria occurs even in women who have developed clinical immunity to P. Jalciparum. Histopathological examination of infected placental tissue has shown that some infected erythrocytes appear to adhere to the synocytiotrophoblast cell layer, which lines the placental blood spaces, constitutes an extensive area of fetal tissue that 17

19 is in contact with the maternal circulation (Bray and Sinden, 1979; Yamada et al., 1989). Careful molecular analysis revealed that parasite infected erythrocytes can sequester in the placenta through adhesion to Chondroitin Sulfate A (CSA) and Hyaluronic Acid (HA) that are major constituents of a prominent coat of glycosaminoglycans (GAGs) on the surface of syncytiotrophoblast layer. All placental isolates tested showed binding either to CSA or HA or both (Fried and Duffy, 1996; Beeson et ai., 1999 and 2000). Multigravidae women from Kenya, Malawi and Thailand were found to have serum antibodies that inhibited adhesion to CSA in vitro. However, CSA binding of these parasites is insensitive to antisera collected from adult males living in endemic area (Fried et ai., 1998). The variant surface antigens expressed by the parasites selected for strong adhesion to CSA are not recognized by plasma IgG from clinically immune adult males, although parental un selected lines are well recognized (Ricke et al., 2000; Staalsoe et al., 2001). These findings suggest that the parasites causing placental malaria express antigenically distinct variant surface antigens. It was also reported that early ring forms of P. Jalciparum infected erythrocytes could adhere to placental tissue sections through some unidentified receptor (Pouvelle et ai., 2000). Later it was demonstrated that a 42-kDa parasite derived protein known as ring surface protein 2 (RSP-2) identified as the molecule mediating this adherence. It was also demonstrated that RSP-2 coats the surface uninfected erythrocytes and enabling them to adhere to placental sections (Douki et ai., 2003). This observation challenged the widely believed theory that only erythrocytes infected with mature stages are able to sequester Severe anemia Severe anemia is one of the most common consequences of malaria and has been found to be a lethal complication in children suffering from malaria. Hemoglobin of less than 5 g/ dl is considered to represent 18

20 severe anemia. Anemia depends on the degree of parasitemia, duration of the acute illness and the number of febrile paroxysms. P. vivax predominantly invades young red cells and the number of parasites infected rarely exceeds 2%, whereas P. Jalciparum affects red cells of all ages and the parasitemia can be as high as 20-30% or more. Massive destruction of red cells accounts for rapid development of anemia in P. Jalciparum malaria. There are number of factors thought to be responsible for anemia in malaria patients. A significant decrease in erythropoiesis (Abdalla, 1990) of bone marrow and accelerated destruction of non parasitized erythrocytes are the two principal processes that contribute to the outcome of anemia. The major cause of severe anemia during falciparum malaria is the obligatory destruction of infected red cells at schizont rupture. The life span of uninfected erythrocytes is also reduced by this accelerated destruction (Salmon et al., 1997). The factors influencing the removal of uninfected erythrocytes have not been fully characterized. The reduced deformability is considered to be one of the major factors responsible for destruction of uninfected erythrocytes in malaria patients. Infected erythrocytes may also be phagocytosed by macrophages following opsonization by immunoglobulins and/or complement components. IgG has also been found to be attached to uninfected erythrocyte surface in the malaria patients. This, in turn activates IgG mediated phagocytosis, complement mediated lysis of uninfected erythrocytes. In addition to surface antibodies, it was found that reduced expression of the complement regulatory proteins CR1, CD55, and CD59 on the RBC surface of patients with severe malarial anemia. These proteins normally protect the surface of host erythrocytes from complement-mediated hemolysis by blocking the activation of complement system. CR1 and CD55 are removed from the erythrocyte surface during the transfer of immune complexes from cell surface to macrophages as part of the normal process of complement regulation (Reist et ai., 1993). Thus, an 19

21 acquired deficiency of CR1 and CD55 could occur in association with the formation of circulating immune complexes, as is known to happen in malaria (Adam et al., 1981). Drug induced hemolysis can also contribute to the anemia. Other effector cells and mechanisms are less well defined but may include antibody-dependent cytotoxicity and natural killer (NK) cells Other complications Renal failure In severe P. Jalciparum malaria, acute renal failure may develop in % of the malaria patients. Although there is no direct evidence, it is believed that sequestration in the kidney microvasculature is responsible for renal failure. Severe falciparum malaria results in intravascular hemolysis of parasitized as well as non-parasitized red cells and clogging of the tubules by the products of hemolysis, which may be another important cause for renal dysfunction. Drugs like primaquine can also contribute to hemolysis in severe falciparum malaria, and particularly in patients with deficiency of Glucose 6-phosphate dehydrogenase enzyme Hypoglycemia Hypoglycemia is a common clinical symptom of falciparum malaria. It may arise from increased glucose consumption by a large parasite biomass (Schofield and Hackett, 1993), reduced hepatic gluconeogenesis and glycogenolysis, or triggered by parasite derived toxins such as GPI (White and Ho, 1992). In patients treated with quinine, pancreatic p cells get stimulated to secrete more insulin resulting increased glucose uptake by the cells. Hypoglycemia is believed to contribute to nervous system dysfunction associated with CM. 20

22 1.5 Role of cytokines in severe malaria Development of clinical symptoms of severe malaria involves the release of soluble malaria-derived antigens (or toxins) in the blood circulation. As P. Jalciparum grows in a synchronous manner in the human body, most of the schizonts rupture almost at the same time after every 48 hours, which might explain the characteristic periodic fever in malaria. Parasite-derived malaria toxins molecules, which are released from parasites at trophozoite and schizont stages either directly damage host tissues or, more important, stimulate the overproduction of host cytokines or activate T cells to secrete interferon y (INF-y) and facilitate the production of TNF-a by monocytes and macrophages (Jakobsen et ai., 1995; Schofield and Hackett, 1993). Supernatants of P. Jalciparum cultures contain such toxins stimulating the secretion of TNF-a, IL-1, /i-"': I' ~".' and other cytokines from various host cells (Bate et ai., 1989; Wahlgren "'~'... et al., 1995). Glycosylphosphatidylinositol (GPI) was suggested to be the \:?'~.. ~. main component of toxin molecules, since specific antibodies to it could neutralize the toxic effect, but the exact origin of most of the toxins is still not known. Furthermore, the GPI-anchored components of merozoite surface antigens, like MSP-1 and MSP-2, were found to induce TNF-a and IL-1 (Schofield and Hackett, 1993). Studies of P. Jalciparum suggest that GPI anchoring of antigens expressed at trophozoite and schizont stages is the most common form of glycosylation (Gerold et ai., 1994; Gowda and Davidson, 1999). GPI anchors are also thought to induce nitric oxide synthase. This, in turn might produce more nitric oxide (NO), which may cause local damage at sequestered loci (Anstey et al., 1996; Green et al., 1994). Because high circulating levels of TNF -a and high fever occur at the rupture of schizonts, antigens from schizonts might have potential toxic effects (Karunaweera et ai., 1992). Pichyangkul et al. partially purified a 21 T +-1 r! fig... ',.::

23 soluble antigen predominantly associated with schizonts that was not found in other stages of infection (Pichyangkul et ai., 1997). This schizont-associated antigen (SAA) was able to specifically stimulate T cells in secreting IFN -y, IL-1, IL-1 0, and IL-12. This SAA was further characterized as being resistant to protease digestion, heating, and ph changes and containing a phosphate group(s), which was believed to be crucial for its toxic activities. These antigens are all parasite-derived products of which the GPI anchor and phosphate groups are suggested to be important for their biological activities. The metabolites of P. JaZciparum are themselves toxic. During the asexual stages of proliferation in erythrocytes, P. JaZciparum consumes hemoglobin as an energy source and produces hemozoin as the main breakdown product, which is deposited in the cell as pigment. This pigment, released immediately after schizont rupture, has been suggested to induce IL-1 production. Moderate amounts of cytokines such as tumor necrosis factor alpha (TNF-a), gamma interferon (IFN-y), and interleukin-1 (IL-1) are required for the human host to fight invading microorganisms, but the overinduction of host cytokines can be detrimental to the host. TNF-a can cause fever and chills. TNF-a and IFN-y also upregulate some endothelial receptors such as ICAM-1, CD31 and VCAM1 on endothelium, which may augment sequestration, adding further complications. For example, high circulating levels of TNF -a and IFN-y are more often found in patients with severe malaria than in uncomplicated cases (Kwiatkowski et ai., 1990). Extensive overproduction of TNF-a and IFN-y and IL-1 in organs with massive sequestration (especially in the brain) is more frequently seen in patients who died of cerebral malaria (Udomsangpetch et al., 1997). Increased production cytokines may also be responsible for suppression of erythrocyte production in the bone marrow, causing anemia. Reversing 22

24 the deleterious effect of overproduced cytokines might be important for the clinical treatment of severe malaria (Jakobsen et al., 1995; Miller et al., 1994). However, infusion of an anti-tnf-a monoclonal antibody (MAb) into comatose children with malaria infection did not affect the outcome of the disease, although fever was abated (Kwiatkowski et ai., 1993). There is no direct evidence that cytokines cause coma during malaria. Thus, it might be important to further uncover the chemical nature of parasite antigens that is responsible for cytokine stimulation in order to understand the disease and design novel preventive measures. 1.6 Cytoadherence and antigenic variation in malaria The special virulence of falciparum malaria is attributed to the unique ability of P. falciparum infected erythrocytes to cytoadhere and sequester in the vasculature of various host organs. As a result P. falciparum avoids splenic clearance mechanisms. Although evolution of this strategy in P. falciparum is crucial for their survival, it poses a serious threat to the host. Therefore, it is important to understand the molecular basis cytoadherence. Following erythrocyte invasion and development of ring to trophozoite parasite derived high molecular, multidomain proteins, Plasmodium falciparum erythrocyte membrane protein1 (PfEMP1) expressed on the surface of infected erythrocytes, which enable the parasitized erythrocytes to bind to host endothelial cells lining the blood capillaries. This survival strategy of poses another problem to the parasite because highly immunogenic PfEMP 1 molecules become the target of host immune attack, by which the host can eliminate the infected erythrocytes by a variety of mechanisms such as complement mediated lysis and opsonization of the infected red cells. In order to evade the host immune response, parasite has evolved another mechanism by which parasite switches the expression of one PfEMP1 to another. This 23

25 REVIEW OF UTERATURE phenomenon is referred to as antigenic variation. There are approximately 60 different genes in each P. Jalciparum genome, which can produce different PfEMP1 molecules. Each erythrocyte infected with a single parasite expresses only one PfEMP-1, but many different PfEMP1 can potentially be produced by a parasite population having a number of erythrocytes infected with different individual parasite. The PfEMP-1 proteins are encoded in the parasite's genome by a family of genes called the var (for variation) genes. At an early stage in the erythrocytic cycle, before any PfEMP-1 antigen appears on the red cell surface, each individual parasite is making not one, but several var mrnas. But only one of these mrnas eventually appears to be selected for translation into protein and an unknown feedback mechanism appears to silence the transcription of the remaining var genes or degrade the rest other var gene mrnas. At the later stages of infection, therefore, only the one PfEMP1 appears on the surface of the erythrocyte infected with a single parasite (Chen et ai., 1998; Scherf et al., 1998). The total number of var genes in 3D7 genome is estimated to be around 59 (Gardner et ai., 2000). P. Jalciparum undergoes antigenic variation in which it switches the expression of var genes at a rate of nearly 2% per generation (Roberts et al., 1992) with the concomitant changes in adhesive phenotype. The ability to undergo antigenic variation might partially explain why immunity to the severest effects of malaria only arises after repeated exposure. 1.7 Mechanism of antigenic variation Antigenic variation occurs in many microorganisms such as African Trypanosoma, Neisseria gonorrhoeae, Giardia lamblia and P. Jalciparum (Bienz et al., 2001) to evade the continued host immune surveillance. Different microorganisms have evolved distinct mechanisms for antigenic 24

26 variation. For example, in Neisseria gonorrhoeae antigenic variation of the major subunit of pilin occurs by unidirectional, homologous recombination between a silent locus and the expression locus of variant surface glycoprotein (VSG) coat from a repertoire of at least 100 variants (Seifert, 1996). In Trypanosoma it occurs through the switching of the expression. But the mechanism of antigenic variation occurring in P. /aiciparum is quite different. P. /alciparum undergoes antigenic variation with respect to the expression of PfEMP1 on the infected red cell surface. PfEMP 1 is encoded by a family of multicopy genes termed var. There are 59 different var genes present within the haploid genome of each parasite. Switching the expression from one var gene to another account for the antigenic variation in a P. /aiciparum population over the course of an infection. Nuclear run-on experiments using parasites selected for their cytoadherence phenotype showed a single var gene transcribed in trophozoites. Single cell RT-PCR experiments identified transcripts from multiple var genes in early ring stage parasites, but only a single transcript remained in trophozoites. A mechanism of allelic exclusion appears to control the expression of individual genes (Chen et al., 1998; Scherf et al., 1998). Switching in expression was not found to be accompanied by promoter DNA sequence alterations. Silent promoters were shown to become transcriptionally active when removed from the chromosome and placed on transfected episomes. These results indicated that switches in expression were probably not the result of changes in transcription factors. Deitsch et ai., have demonstrated that intron region of var gene can silence the promoter of a var gene, but not of an unrelated gene hrp3 of Plasmodium (Deitsch et ai., 2001). This finding suggests that silencing due to the presence of the var intron was specific to var promoters and was dependent on transition of the transfected parasite through S-phase of the cell cycle. These observations indicate that the control of var expression might be mediated by epigenetic mechanism and associated with changes in chromatin structure. Very 25

27 recent finding by Calderwood et al., has further elucidated this mechanism. They have shown that each var gene contains two functional promoters, the first upstream of var exon I and the second within the intron located between two exons. The upstream promoter contains an initiator element at a transcription start site. Comparative alignments showed that var introns are composed of three conserved regions with distinct base pair composition. Analysis of a typical var intron demonstrates that these regions have different effects on var silencing. The AT-rich central region is alone sufficient to silence the upstream var promoter. This central region is also necessary for the intron's promoter activity. Deletions that remove the intron's promoter activity also impair its ability to function as a silencer (Calderwood et al., 2003). At this point it is not clear how this silencing mechanism singles out the transcription of only one var gene from a repertoire of approximately 60 genes. 1.8 Molecular basis of cytoadherence Variant Surface Antigens and Cytoadherence Ligands Plasmodium falciparum erythrocyte membrane proteinl (PfEMP1) and var genes Plasmodium falciparum erythrocyte membrane protein1 (PfEMP1) has been identified as key molecules mediating cytoadherence of P. Jalciparum infected erythrocytes. PfEMP 1, a family of high molecular weight proteins ( kda) expressed on the surface of the P. Jalciparum infected erythrocytes could be detected by surface iodination of infected erythrocytes (Leech et ai., 1984; Howard et al., 1988; Magowan et al., 1988). Metabolic labeling of the infected erythrocytes could confirm that PfEMP1 molecules are of parasite origin. These proteins are insoluble in non-ionic detergents like, TritonX-100 but are soluble in anionic detergent like SDS, suggesting that these molecules interact with the erythrocyte cytoskeleton. PfEMP1 from different strain 26

28 REVIEW OF UTERATURE of P. jalciparum have distinct molecular masses, indicating that these molecules are highly polymorphic. Strain specific recognition of P. jalciparum infected erythrocytes by antibodies and inhibition of cytoadherence of homologous but not heterologous strains by these antibodies (Marsh et ai., 1989), suggest that PfEMP1 molecules mediate the binding of infected erythrocytes and are often the targets of host immune attack (Bull et ai., 1998). The FfEMP1 molecules are multi-domain proteins consists of four basic building blocks, N-terminal segment (NTS), DBL (Duffy Binding Like, as it is homologous to region II of the P. vivax Duffy binding protein) domains, cysteine rich inter-domain region (CIDR) and C2 domains as shown below. The first two DBL domains of FfEMP1 are usually separated by another distinct cysteine-rich domain, referred to as the conserved inter-domain region (CIDR). Although DBL domains are also found in proteins in other Plasmodium species, CIDR is unique to P. jalciparum Each domain has a characteristic features (Smith et al., 2000). Transmembrane region Encoded by Exon 1 Encoded by Exon2 Figure 1.8. The schematic representation of?temp 1 The FfEMP1 family of proteins is encoded by var genes (for variation). The var genes were cloned using two independent strategies. In one approach, during mapping the chloroquine resistence locus on chromosome 7 of P. jalciparum Su et al identified several open reading frames (ORFs) containing multiple cysteine-rich domains with homology 27

29 to region II, the binding domain of erythrocyte binding proteins of P. Jalciparum, P. vivax and P. knowlesi (Adams et al., 1990; Chitnis and Miller, 1994 and Sim et al., 1994). As the first binding domain to be identified was region II of the P. vivax Duffy binding protein, these homologous domains are referred to as Duffy-binding-like (DBL) domains. The first two DBL domains are usually separated by another distinct cysteine-rich domain, referred to as the conserved inter-domain region (CIDR). In the other approach, the genes encoding PfEMP1 were cloned by immunoscreening of a P. Jalciparum genomic expression library (Baruch et ai., 1995) using rabbit antisera raised against parasite antigens present on the infected erythrocytes that immunoprecipitated radio-iodinated PfEMP1 from Malayan Camp strain of P. Jalciparum. Smith et al., have shown correlation of var gene switching with antigenic variation (Smith et ai., 1995). The var genes consist of two exons (Su et ai, 1995). Exon1 codes for the variable extracellular domain and transmembrane region and Exon2 codes for a highly conserved cytoplasmic or acidic terminal segment (ATS). The var genes are distributed on all the chromosomes except chromosome 14 in 3D7. Although most of the var genes are located in the subtelomeric regions on both ends of the parasite's chromosomes, some var genes are centrally located on chromosomes 4, 7, 8 and 12. Different var genes differ in the number and sequence of DBL domains present. These DBL domains are believed to be the adhesive domains for different receptors. The variable domain composition and extensive sequence polymorphism provide PfEMP1 great flexibility in binding to a wide variety of host receptors. The binding domains on PfEMP 1 for some of the receptors have been mapped as schematically presented in Fig. 4. ICAM-1 binding region has been shown to reside in the DBL-P-C2 tandem (Smith et ai., 2000a,b). CIDR1u has been shown to be the binding domain for CD36 (Baruch et al., 1997). Adhesion to CSA appears to be mediated through DBL-y, but in some isolates adhesion may also be mediated through 28

30 REVIEW OF UTERATURE CIDRla (Reeder et al., 1999 and Degen et ai., 2000), DBL-a has been shown to be the binding domain for CR1, blood group A, heparin and heparin sulfate (V ogt et ai., 2003). The CD31 binding domain has been shown to be located within CIDRla of PiEMPl molecules. IgM is also reported to bind to PfEMPl through CIDRla domain (Chen et ai., 2000). Semi-conserved Tandem association head structure II Tandem association Complement receptor (CR l) Blood Group A Hepartn Hepartn sulfate ~ 1 CD36 CD31 IgM CSA ICAMl l CSA l CD31 Figure 1.9. Schematic diagram representing the binding domains on PiEMPl of different receptor. Here, NTS stands for N-terminal segment, TM for transmembrane domain and ATS for acidic terminal segment, the regions shown at the arrow tail are the domains that represent the binding domains of the receptors shown at the arrow heads Cytoadherence-linked asexual gene 9 (clag9) During in vitro culture, some parasite lines of P. jalciparum lose the ability to produce knobs and they generally lose the ability to cytoadhere as measured by binding to C32 melanoma cells (Kilej ian, 1979). This is a consequence of subtelomeric deletions of the region of chromosome 2 bearing the KAHRP gene (Udeinya et ai., 1983). However, clone B8, derived from ITG2 can adhere to melanoma cells, although it is KAHRP negative and knob negative (Corcoran et al., 1986). Attempts were made to find some other unidentified gene that is important for cytoadherence 29

31 REVIEW OF UTERA TURE of B8 parasite in the absence of KAHRP. Trenholme et al have reported another gene product located on chromosome 9 that is important for cytoadherence to C32 and CD36 (Trenholme et ai., 2000). They named the gene as cytoadherence-linked asexual gene 9 (clag9). Three models for the action of CLAG9 have been proposed. First, CLAG9 may be a component of the PfEMPl/KAHRP complex involved in cytoadherence. Second, it may be an entirely separate molecule that itself mediates binding or third, it may have role in the regulation, transport or chaperoning of PfEMP 1. But, the precise role of clag9 is yet to be elucidated. It is believed that clag9 is located on the surface of parasitized erythrocytes as anti-clag9 antibodies inhibit the binding of parasitized erythrocytes to melanoma cells Other Multigene families relevant to antigenic variation Besides PfEMP1, additional multigene families, named stevor (subtelomeric open reading frame) and rif (repetitive interspersed family) have been identified and proposed to be associated with antigenic variation (Cheng et ai., 1998; Fernandez et al., 1999). From their distribution on chromosomes 2 and 3, the total number of rif genes in the malaria genome was predicted to be about 200. The rif gene family might be the largest multigene family in the malaria parasite. The proteins encoded by rif genes are having molecular weight within kda that can be labeled on the surface of infected erythrocytes and appears to have reactive antibodies in the infected patients' sera. Chromosome mapping clearly showed that rif genes are located in the subtelomeric regions of chromosomes directly adjacent to a var gene (Bowman et al., 1999 and Gardner et ai., 1998). Due to their presence on the PRBC surface, the role of rif gene products in antigenic variation could be significant. Whether rif gene products adhere to host receptors is not known. 30

32 1. 9 Host Receptors mediating Cytoadherence Thrombospondin (TSP) Thrombospondin (TSP) is the first host molecule to be identified as a receptor for cytoadherence of infected erythrocytes to endothelium. Parasitized erythrocytes adhere avidly to surfaces coated with thrombospondin (TSP) but not to surfaces coated with laminin, fibronectin, factor VIII/von Willebrand factor and vitronectin. Preincubation of parasitized erythrocytes with soluble thrombospondin inhibit adherence both to immobilized TSP and melanoma cells. Anti-TSP antibodies but not non-immune immunoglobulins inhibit binding of parasitized erythrocytes with melanoma cells (Roberts et ai., 1985) CD36 CD36, an integral membrane protein of 471 amino acids has been identified to be the most widely used receptor for cytoadherence by P. Jalciparum. Initially, it has been shown that a monoclonal antibody OKM5 inhibits the in vitro cytoadherence of P. Jalciparum infected erythrocytes to specific target cells, like monocytes, human endothelial cells, and human melanoma cell lines (Barnwell et ai, 1985). By immunoprecipitation OKM5 identified an 88 kda glycoprotein, also known, as CD36 or platelet glycoprotein IV is the candidate mediating cytoadherence to these cells. Purified CD36 can inhibit the cytoadherence of infected erythrocytes with C32 cells, suggesting that the adherence to C32 cells involves CD36 as a cell surface receptor. More direct evidence proof came from, Cos cells transfected with CD36 gene enables the transfected cells to cytoadhere to infected erythrocytes, which is readily inhibited by CD36 antibodies and soluble CD36 (Oquendo et al., 1989). It is widely expressed on microvascular 31

33 endothelial cells of most of the organs but not all, such as the brain (MacPherson et ai., 1985) Intercellular adhesion molecule 1 (ICAM1) The parasite sequestration in cerebral microvasculature is mainly due to intercellular adhesion molecule 1 (ICAM1) (Berendt et al., 1989). ICAM1 is a glycoprotein of molecular weight ranging between kda, expressed on vascular endothelium and a number of cells of the immune system where it serves as an endothelial receptor for leukocytes for its homing to sites of inflammation. It is composed of tandemly arranged multiple immunoglobulin-like domains. P. JaZciparum infected erythrocytes adhere to transfected COS-7 cells expressing ICAM-1 on the surface as well as to purified ICAM-1 coated on plastic plates (Berendt et al., 1989). Widespread expression of ICAM1 on the cerebral microvasculature is observed in the patients who died of cerebral malaria. The expression of ICAM1 on endothelial cells is upregulated by tumor necrosis factor alpha (TNF-a), interleukin-1 (IL-1), and interferony (INF-y) (pober et al., 1987). The exact correlation of ICAM-1 binding with severity of malaria is yet to be demonstrated. Molecular studies have shown that PRBC binds to the first immunoglobulin domain of ICAM-1 at a site that is distinct from the binding site of both LFA-1 and rhinovirus (Berendt et al., 1992; Ockenhouse et al., 1992). In addition, the low affinity of most malaria parasites for this receptor indicates that ICAM-1 only mediates PRBC rolling on the endothelial lining and that stable binding most likely occurs synergistically with other receptors such as CD36 (Craig et al., 1997; McCormic et al., 1997). CD36 and TSP receptors are not well distributed on brain endothelium, and their expression is not sensitive to cytokine (IFN-y or TNF -a) stimulation. So, the stability of PRBC binding to ICAM-1 synergized by CD36 is probably not contributing to the parasite sequestration in brain. There is a 32

34 possibility that some other unidentified receptor present on the brain endothelium might be responsible for it VCAM-l and ELAM-l Parasitized erythrocytes can adhere to plates coated with soluble recombinant VCAM-1 and ELAM-l. The antibodies raised against VCAM- 1 and ELAM-1 could partially inhibit this binding. These receptor are not normally expressed in abundance, but can be induced by TNF-a treatment (Ockenhouse et al., 1992). Available literature does not report any evidence to support roles for VCAM-1 and ELAM-1 in malaria pathogenesis CD31/PECAM In vitro, CD36 is the most frequently used endothelial receptor by P. Jalciparum strains from patients with mild as well as severe P. Jalciparum malaria. However, CD36 is expressed at low levels on the cerebral microvasculature and therefore seems unlikely to be involved in cerebral malaria. Treutiger, et al reported that malaria-infected RBCs adhere to platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31) on the vascular endothelium (Treutiger et al., 1997). PRBCs bind to PECAM- 1/CD31 transfected cells, and directly to recombinant PECAM-1/CD31 absorbed onto plastic plates. Fluorescence-labeled soluble recombinant PECAM-1/CD31 (specam-1/cd31) is shown to bind to the surface of P. Jalciparum-infected erythrocytes. Infected erythrocyte binding to PECAM- 1/CD31 is blocked by the addition of the unlabeled specam-1/cd31 in a dose-dependent manner, but not by unrelated receptor-proteins (Heddini et ai., 2001). Soluble PECAM-1/CD31 and monoclonal antibodies specific for the amino-terminal segment of PECAM-1/CD31 (domains 1-4) blocked the binding. Interferon-y (IFN-y) essential for the development of cerebral malaria in the mouse was found to augment adhesion of human PRBCs to PECAM-1/CD31 on endothelial cell 33

35 monolayers. This observation suggests that PECAM-1/CD31 is a virulence-associated endothelial receptor of P. Jalciparum-infected RBCs (Treutiger et al., 1997). A significant correlation was found between the binding of soluble PECAM-1/CD31 to PRBCs and the binding to transfected L cells expressing CD31, as seen with six different P. Jalciparum lines or clones (Casals-Pascual et al., 2001). CD31 is a highly glycosylated 130-kDa multi-domain polypeptide mainly located on the rim of endothelial cells. Antibodies to domains 1 and 2 of CD31 showed specific inhibition of PRBC binding to both CD31-transfected L cells and endothelial cells. Parasite binding to CD31 was also upregulated by IFNy. Thus, CD31 may be one of the receptors associated with the virulence of P. Jalciparum. Kikuchi et ai., have shown that the occurrence of valine and asparagine at particular locations on CD31 is found in patients having cerebral malaria than patients having severe malaria without cerebral malaria and predicted a correlation of susceptibility to cerebral malaria with PECAM-1/CD31 polymorphism in Thai malaria patients (Kikuchi et ai., 2001) Naturally acquired immunity to malaria The pathogenesis of malaria changes with changes in malaria transmission intensity. In hyperendemic areas, for example parts of Sub Saharan Africa, victims of severe malaria are mostly children under 5 years of age, whereas in hypoendemic areas, such as parts of South East Asia, people of all age groups are affected by severe malaria. These observations suggest that development of protective immunity against malaria requires repeated infections and is slow to develop. (Miller et al., 1994). The immune system targets invading microorganisms for destruction by recognizing antigens that appear either on their surface or on the surface of infected cells. 34

36 During its life cycle in the human body, P. Jalcipantm spends most of the time living and multiplying inside red blood cells. Infected RBCs do not induce cytotoxic T lymphocyte (CTL) response due to their lack of major histocompatibility complex (MHC) expression. Secondly, parasite derived PfEMP 1 molecules expressed on the surface of the erythrocytes are highly variable. These molecules undergo high clonal variation at a rate of approximately 2% per generation in culture. (Roberts et ai., 1992). Blood stage Plasmodium affects dendritic cell (DC) functions, inhibiting maturation and the capacity to initiate immune responses. The interaction of blood stage parasites with DCs inhibits the activation of CD8+ T cells in vitro and the suppression of protective CD8+ T cell responses. It was proposed that blood stage infection induces DCs to suppress CD8+ T cell responses in natural malaria infections. This evasion mechanism leaves the host unprotected against reinfection by inhibiting the immune response. [Ocana-Morgner et ai., 2003) Acquisition of protective immunity to the disease in population in the endemic areas is associated with the development of variant surface antigen (VSA) specific agglutinating antibodies (Beinz et ai., 2001; Giha et ai., 1999; Marsh and Howard, 1986; Marsh et ai., 1989). Chattopadhyay et ai., have demonstrated using agglutination assay that in the low endemic adults develop partially cross-reactive antibodies, whereas, adults from hyperendemic area produce antibodies that can recognize diverse PfEMP1 molecules (Chattopadhyay et ai., 2003). Potential mechanisms by which VSA-specific plasma antibodies can protect include opsonization and interference with VSA-specific adhesion Possibility of having unidentified receptors for cytoadherence Parasite infected erythrocytes cytoadhere to the capillaries of various organs using molecules expressed on endothelial cells lining those 35

37 REVIEW OF UTERATURE capillaries. Parasites use large multidomain proteins, PfEMPl as a major ligand for binding to a variety of endothelial receptors. A wide variety of receptor specificity for cytoadherence largely depends on the structure and binding capacity of the domains of different PfEMPl molecules expressed by different parasites. It is usually observed that most of the field isolates bind to CD36 and thrombospondin (TSP), a significant proportion can bind to ICAM-l and a minor proportion to other receptors such as, VCAM1, CD31, HA and CSA, although most of the parasites isolated from pregnant women are reported to bind with CSA. Efforts have been made to look for the diversity in receptor usage by parasitized erythrocytes in Indian field isolates. Most of those isolates could either bind to CD36 or ICAM-l or both. But, interestingly some isolates could not bind to any of the identified receptors, like CD36, ICAM-l and CSA (unpublished observation, Chattopadhyay and Chitnis); suggesting that there might be some other unidentified molecule(s) responsible for adherence of these isolates to endothelium A novel endothelial receptor for cytoadherence Here we plan to investigate the potential role of an endothelial surface protein, hyaluronan binding protein 1(HABP1)/gClqR/p32 as a receptor for cytoadherence by P. [alciparum. HABPl is expressed in almost all the cell types in a wide variety of organs. It has been shown by different investigators that HABPl is expressed on the surface of different endothelial cells, such as human umbilical vein endothelial cells (HUVEC), bone marrow endothelial cells (BMEC) and human dermal microvascular endothelial cells (HDMEC); although it is also found in mitochondria and nucleus in many other cell types. The most convincing evidence came from the observation made by Soltys et al., in which they have done immunogold electron microscopy with different rat tissue sections using polyclonal antibodies raised against HABP1/p32/gClqR. The data showed prominent cell surface labeling on the microvascular 36

38 endothelial cells of pancreas and kidney (shown in Fig. 3) although highly specific labeling of mitochondria was also observed in many rat tissues, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen, testes and thyroid (Soltys et al., 2000). The expression of HABP1 on the surface of endothelial cells is upregulated by inflammatory cytokines, like TNF-a INF-y and lipopolysaccharide (LPS) (Guo et al., 1999), as is found in case of the expression of ICAM1, CD31 and VCAM1 during malaria. For past several years our laboratory has been working on this novel 34 kda cell surface glycoprotein that can specifically bind to Hyaluronan (hyaluronic Acid). Initially it has been purified from rat brain using hyaluronic acid (HA) affinity chromatography and referred to as hyaluronic acid binding protein or HBP (D'Souza and Datta, 1986). Later the human gene encoding HBP was cloned, sequenced, over expressed and purified to homogeneity in our laboratory and renamed as HABP (Deb and Datta, 1996). The recombinant HABP was shown to be functionally active with respect to HA binding. Its human chromosomal localization was determined by FISH (Fluorescence in-situ Hybridization), which revealed that the gene for HABP is located on human chromosome number 17 (Majumdar and Datta, 1998). It is synthesized as a 282 amino acid long pre-proprotein, which is post-translationally processed and the first 73 amino acid is cleaved off, giving a mature protein of 209 amino acid (Honore et ai., 1993). The mature protein is very acidic having a theoretical pi of It has only one cysteine at residue 186 position. Homology search analysis revealed that HABP1 is identical with other two proteins; gc1qr, the receptor for the globular head of complement subcomponent C1q (Ghebrehiwet et ai., 1994) and p32, a protein co-purified with splicing factor SF2 (Krainer et al., 1991). Its crystal structure has been solved and it revealed that gc1qr appears as a unique doughnut-shaped homo trimer (Jiang et al., 1999). The two faces of the doughnut have asymmetric charge distribution with one face 37

39 REVIEW OF UTERA TURE having a significant higher concentration of negatively charged residues, suggesting that each side plays a different functional role. Besides binding to HA, it is reported to bind to a large number of molecules. Cell surface expressed HABP1/gC1qR/p32 binds to several proteins present in the blood such as complement subcomponent C1q (Ghebrehiwet et al., 1994), thrombin (Ghebrehiwet and Peerschke, 1998), fibrinogen (Lu et ai., 1999), vitronectin (Lim et al., 1996), high molecular weight kininogen (Herwald et al., 1996). Evidences suggest that this protein binds to several proteins present on the surface of pathogenic microorganisms. Protein A of Staphylococcus aureus (Nguyen et al., 2000) binds specifically to gc1qr. In addition, gc1qr has been identified as a receptor for internalin B (InIB) mediated invasion of Listeria monocytogenes into many mammalian cells. Antibodies directed against gc1qr have been shown to inhibit the entry of L. monocytogenes into cells. More direct evidence came from the experiment showing that adhesion and internalization oflnlb coated microbeads by GPC16 guinea pig epithelial cells transfected with plasmid expressing gc1qr. (Braun et al., 2000). gc1qr has been shown to interact with several viral core proteins, such as HIV-1 Rev (Luo et al., 1994; Tange et al., 1996), HIV-1 Tat (Yu et ai., 1995; Fridell et al., 1995), core protein V of adenovirus (Matthews and Russell, 1998), Epstein-Barr virus nuclear antigen-1 (EBNA-1) (Wang et ai., 1997), and open reading frame P of herpes simplex virus (Bruni and Roizman, 1996). It is a ubiquitously expressed in various cell types. The precise sub cellular localization of HABP1 IS still controversial. Although this is predominantly present in the mitochondria and nucleus, strong evidences do exist showing its cell surface localization, especially on endothelial cells. The most convincing evidence came from the observation made by Soltys et ai., in which they have done immunogold electron microscopy with different rat tissue sections using polyclonal antibodies raised against HABP1/p32/gC1qR. The data showed 38

40 REVIEW OF LITERA TURE prominent cell surface labeling on the microvascular endothelial cells of pancreas and kidney (shown in Fig. 3) although highly specific labeling of mitochondria was also observed in many rat tissues, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen, testes and thyroid (Soltys et al., 2000). The expression of HABP1 /gc1qr on the surface of endothelial cells is upregulated by inflammatory cytokines, like TNF -u INF -y and lipopolysaccharide (LPS) (Guo et al., 1999). A B Figure P32 (gclqr) localization on the cell surface of microvascular endothelial cells. A Arteriole in the pancreas. Arrows point to labeling of the surface of the endothelial cell that wraps around the entire vessel. B Kidney endothelial cell showing prominent cell surface reactivity (arrows). pm plasma membrane, n nucleus. (Reproduced from Soltys et al., Histochem Cell Biol. 114(3):245-55). 39