Falciparum Malaria: Sticking up, Standing out and Out-standing

Molecular Approaches to Malaria

Falciparum Malaria: Sticking up, Standing out and Out-standing B.M. Cooke, M. Wahlgren and R.L. Coppel Cytoadherence is believed to be fundamental for the survival of Plasmodium falciparum in vivo and, uniquely, is a major determinant of the virulence of this parasite. Despite the widely professed importance of cytoadhesion in the development of severe disease, there are a number of aspects of this highly complex process that remain poorly understood. Recent progress in the understanding of cytoadhesive phenomena was discussed extensively at the Molecular Approaches to Malaria conference, Lorne, Australia, 2–5 February 2000. Here, Brian Cooke, Mats Wahlgren and Ross Coppel consider just how far we have progressed during the past 30 years and highlight what is still missing in our understanding of the mechanisms and clinical relevance of this apparently vital process. Infections with Plasmodium falciparum can be distinguished from other forms of malaria by the propensity to cause severe disease that is associated with high mortality. Accumulation of parasitized red blood cells (PRBC) may cause perturbation or complete obstruction of blood flow in the microvasculature either directly, by binding to the endothelium or indirectly, by binding to other PRBC (autoagglutination) or uninfected RBC (rosetting). Collectively, these phenomena are generally termed ‘cytoadherence’. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a high molecular mass (200–350 kDa) and antigenically variable parasite-derived protein that is expressed on the surface of RBC infected with maturestage malaria parasites1–3. PfEMP1 is now widely believed to be the parasite-derived ligand most likely to mediate most of these adhesive phenomena, through its interaction with a diverse array of receptors that are expressed on the surface of vascular endothelial cells, RBC or platelets. Whether it is the only adhesive ligand and how it is involved in mediating adhesion to a variety of different receptors, however, remain matters of current investigation. Why focus on cytoadherence? We believe that cytoadherence is a key determinant of parasite virulence and is responsible for the development of severe disease in malaria, or at least, that part of severe disease associated with frequently fatal syndromes such as cerebral malaria (CM). By the late 19th century, clinicians had already recognized that P. falciparum, a species that sequestered in the microvasculature, was more virulent in humans than was P. vivax, a species that did not sequester. In 1985, Langreth and Peterson demonstrated that PRBC that had lost the ability to produce the characteristic Brian Cooke and Ross Coppel are at the Department of Microbiology, PO Box 53, Monash University, Victoria 3800, Australia. Mats Wahlgren is at the Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden. Tel: +61 3 9905 4827, Fax: +61 3 9905 4811, e-mail: [email protected] 416

knob-like structures on their surface were no longer cytoadherent and caused only mild or asymptomatic infections in monkeys4. Further, severe and frequently fatal disease syndromes such as CM are associated with the accumulation in the cerebral circulation of an abundance of PRBC, apparently present by virtue of an intimate cytoadherent interaction with the endothelial cells that line the cerebral vasculature5. Given an intensive world-wide research effort, what further can we add to these observations some 15 years later? Clearly, we have come a long way in our understanding of the molecular players in the receptor–ligand interactions involved in cytoadherence that ultimately result in the overall phenomenon that we call sequestration. But are we now in any better a position to understand how this leads to the development of severe disease? This issue was debated in Parasitology Today some time ago, with the two sides arrayed around the opposing positions of vascular obstruction resulting from sequestration or release of cytokines both locally and systemically as the major cause of severe disease6–8. It makes cautionary reading to see that we have, in fact, advanced little in our answers to the fundamental questions. Ian Clark and others in this issue discuss whether the ‘biochemical’ cytokine camp has refined its position. We, in turn, examine the progress in the ‘mechanical’ paradigm of pathogenesis of malaria. Most researchers have chosen to approach this difficult issue in a logical, stepwise manner. They would argue that, if we can understand the precise molecular mechanisms of cytoadherence, we can then compare these interactions in individuals with and without severe clinical disease. However, a precise understanding probably includes the sites of sequestration, the precise identity of receptors, the ‘strength’ and likelihood of these interactions occurring in the haemodynamic environment of the vasculature in vivo, and the detailed mapping of molecular domains involved in adhesion. Molecular studies will need to be linked to field studies performed at a previously unattained level of sophistication. This is difficult to realize, because the few laboratories that exist in malariaendemic areas are frequently under-resourced and poorly equipped to perform such complex studies. Thus, parasites are often collected during periods of intense malaria transmission, cryopreserved, and then shipped to better-furnished laboratories in, for example, Australia, the USA or Europe, then thawed, cultured, albeit for a minimum time, and subjected to sophisticated analyses. The following question then arises: do these parasites still represent those that were isolated from the infected individuals in the field? Certainly, there is at least one cautionary report of altered agglutination phenotype of clinical isolates after cryopreservation9. It is likely then that this will remain a trade-off between the capacity to perform sophisticated analyses and the risks of unpredictable changes in the properties of PRBC.

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Parasitology Today, vol. 16, no. 10, 2000

Molecular Approaches to Malaria Some of these problems would be alleviated if we possessed an appropriate animal model for human malaria. However, at present, we do not know the molecular players involved in systems such as mice infected with P. chabaudi, where sequestration has been observed. However, even with this information, it is questionable whether the importance of sequestration in pathogenesis could be dissected in a system where so many other potentially important parameters differ, such as the kinetics of the infection, peculiarities of the mouse immune system and the precise nature of the pathology10. Similar constraints also apply to and severely limit the utility of primate models showing sequestration11.

Parasitized red blood cell CLAG

KAHRP

PfEMP3

Pfalhesin

RIFIN

PfEMP1

PfEMP1

Parasitized red blood cell membrane

Sequestrin

PfEMP1 ICAM-1 HS

HA

CD36

avb3

PECAM-1

TM TSP

TSP

VCAM-1 E-selectin CD36

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From cytoadherence to clinical Parasitology Today Host cell disease The overwhelming conclusion from Fig. 1. Schematic representation of the molecules implicated in the cytoadhesive instudies over the past five years is that teraction between RBC infected with Plasmodium falciparum and vascular endothecytoadherence is undoubtedly incredilial cells or placental syncytiotrophoblasts (host cell). A region of a PRBC membrane bly complex. We seem to have deincorporating a characteristic knob-like protuberance formed by the deposition of scribed more receptors to which PRBC parasite-encoded proteins such as KAHRP and PfEMP3 under the membrane skeleton of the PRBC is shown. Solid arrows indicate interactions between specific recan bind than we really need to explain ceptors on the surface of the PRBC and the host cell only where these have been the pathophysiology (Fig. 1). Other unequivocally determined. Specific details of these interactions and precise mapping pathogens, including viruses and bacof the binding domains can be found in Refs 35, 42–47. Broken arrows indicate interia, seem to make do with just one or teractions for which there is either less compelling evidence (in the case of setwo receptors for invasion, survival questrin/CD36; Ref. 48) or for which the binding domain has not yet been precisely and propagation in vivo, so why should mapped (TSP/PfEMP1; Ref. 49). Other interactions such as those involved in rosetP. falciparum require such a complex ting, for example between PfEMP1 and CR1 (Ref. 34), the blood group A antigen or ensemble? How do we make sense of IgM (Ref. 46) have been excluded for simplicity. Abbreviations: avb3, avb3 integrin; which are the more important recepCLAG, cytoadherence-linked asexual gene; CR1, complement receptor 1; CSA, tor–ligand interactions in development chondroitin sulphate A; HA, hyaluronic acid; HS, heparan sulphate; ICAM-1, intercellular adhesion molecule 1; IgM, immunoglobulin M; KAHRP, knob-associated Hisof malaria in humans when faced with rich protein; PECAM-1, platelet-endothelial cell adhesion molecule 1; Pfalhesin, the increasingly diverse multitude of parasite-modified form of the native RBC anion transport protein Band 3; PRBC, endothelial-cell-expressed molecules parasitized RBC; RBC, red blood cell; TM, thrombomodulin; TSP, thrombospondin; to which PRBC can adhere? Are they all VCAM-1, vascular cell adhesion molecule 1. equally relevant? Do specific receptor–ligand interactions correlate with particular clinical syndromes? situation. Presumably, cytoadherent interactions will At least in the case of rosetting, there is a reasonable be quantitatively greater or qualitatively different in body of accumulated evidence that enhanced rosetting parasites isolated from individuals with clinically frequency, albeit measured in vitro, correlates with the more severe disease if the entire theoretical framework development of more severe disease, particularly CM has any validity. As an alternative approach, others and severe anaemia12–14. It seems increasingly likely that this will also be the case for autoagglutination, as, have taken the view that if a particular interaction is in a recent study, autoagglutinates of PRBC were important, then we should see the consequences of this significantly more common in African children with in the form of counter-selection in humans in the strucmore severe disease than in those with less severe ture of important receptors in a way that will decrease disease15. the extent of sequestration. Previous studies have idenHowever, cytoadhesive processes per se are not retified a high-frequency polymorphism in the human quired for virulence, as an examination of murine ICAM-1 gene in a malaria-endemic population in Kilifi, malaria species clearly shows that P. yoelii, a nonKenya that appears to be associated with decreased cytoadherent malaria parasite, can be just as virulent, adhesion of PRBC to the variant protein (termed if not more so, than is P. chabaudi, a species that seICAM-1Kilifi), which had been expressed and purified in 16 questers . This may indicate, however, that there are vitro and tested in both static and flow-based adhesion multiple ways in which a malaria parasite can exert its assays17,18. Unfortunately, however, individuals hovirulence: for some species, at least, this may involve mozygous for this polymorphism, at least in this resequestration (eg. P. falciparum), but for other species, gion of Africa, were twice as likely to develop CM19. alternative mechanisms may be used. This study thus challenges the widely held belief that We feel that many answers must come from the apthe degree of adhesion to ICAM-1 influences the deplication of the advances presented here to the clinical velopment of severe clinical syndromes associated Parasitology Today, vol. 16, no. 10, 2000

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Molecular Approaches to Malaria with malaria. However, these adhesion studies were performed using laboratory-adapted parasite lines, which may not accurately reflect those circulating in the field. Perhaps in this particular area of Africa, where malaria transmission is high, compensatory mutants in PfEMP1 may have arisen that bind to ICAM-1Kilifi with much higher avidity. This hypothesis remains to be tested. David Roberts (University of Oxford, UK) presented a further development in this area. He stressed the importance of adhesion of PRBC to platelets, a previously described adhesion phenotype20,21, and showed that platelets with low expression of CD36 do not form aggregates with PRBC in vitro. Recent sequence analyses of African populations showed a surprisingly high frequency of mutations in the CD36 gene that results in loss of expression of CD36 (Ref. 22). One may have expected this to be protective against malaria, given the perceived importance of CD36 in cytoadherence. Surprisingly, however, individuals deficient in CD36 were in fact more susceptible to severe malaria (particularly to CM) than were individuals expressing normal levels of wild-type CD36. Taken together, these two population studies make it difficult to conclude which of these receptors (ICAM1 or CD36) is important, and seem, in fact, to better argue against the role of cytoadherence in the causation of CM. If cytoadherence really is a virulence factor, then preventing or reversing adhesion with anti-adhesive therapeutics should significantly ameliorate the severity of the disease. Such therapeutics, however, have yet to be realized and tested. Moreover, this effect should occur independently of any decrease in parasitaemia: it does not prove that cytoadherence is causing disease if anti-adhesive therapy leads to milder illness at the same time as it decreases parasitaemia, as the observed benefit could be due entirely to a reduction in parasite density. New receptors and ligands Although new studies addressing some unanswered questions were presented at the conference, generally, more questions were generated than definitive answers were gained. At least two further receptors, one new RBC-expressed ligand and one additional cellular interaction were described to add to the complexity of the situation. Beeson and co-workers (The Walter & Eliza Hall Institute, Melbourne, Australia) described the identification of hyaluronic acid (HA) as a new receptor for PRBC in the placenta23. HA joins the closely related glycosaminoglycan chondroitin sulphate A (CSA) as PRBC receptors that appear to be strongly associated with the development of severe malaria during pregnancy24,25. What remains unclear, however, is the relative importance of these two (and perhaps other) receptors in the pathogenesis of malaria during pregnancy. For example, we do not know if individuals infected with parasites capable of adhering to both CSA and HA have more severe placental infection. We have obtained some preliminary evidence that the efficiency of binding of flowing PRBC to HA is lower than to other receptors, including CSA, indicating that they may not be equally as important under physiological flow conditions in vivo26. It would be interesting, however, to quantify precisely the rela418

tive affinities of these two receptor–ligand interactions using sequestered parasites obtained directly from infected placentas. Clearly, we are only just beginning to understand the mechanisms of placental sequestration, yet already the number of putative receptors for PRBC expressed in this organ appears to extend to greater than just CSA and HA27,28. Whether the situation here will become as complex as that for other organs remains to be seen. To further muddy the waters, an additional parasite-encoded protein that might be located on the surface of the PRBC is the product of the cytoadherence-linked asexual gene (clag9). Located on chromosome 9, clag9 is approximately 7 kb and is predicted to be composed of at least 9 exons. It is transcribed in mature-stage parasites and translated into a 220-kDa protein that is distinct from PfEMP1 and can be detected in PRBC by western blotting. Its precise cellular localization remains to be determined, but prediction of the structure of the protein in silico from its hydrophobicity profile reveals four putative transmembrane domains, suggesting that it is found in a membrane and thus may be exposed on the surface of the PRBC29. Preliminary immunofluorescence data supports this hypothesis; however, at this stage its presence on the surface of the PRBC must remain speculative. Nevertheless, recent evidence that adhesion of RBC parasitized by the 3D7 parasite line, which exhibits a stable cytoadherence phenotype, was ablated when the clag9 gene was knocked out is compelling and confirms the essential role of CLAG in cytoadherence, at least to CD36 (Ref. 29). Further, the same group have recently confirmed and bolstered their findings by using a plasmid transfection-based antisense approach30. For the moment, the mechanism by which the loss of CLAG leads to loss of cytoadherence remains unclear, but it will be intriguing to learn whether CLAG is a cell-surface-expressed adhesive ligand or whether it plays an indirect role in adhesion, perhaps by preventing surface expression of other adhesive ligands such as PfEMP1. Recent data derived from the rapidly progressing Malaria Genome Project has now revealed that clag9 is, infact, part of a multigene family with sequences present on a number of other chromosomes throughout the parasite genome. The function of these paralogs remains to be determined. Contrary to the prevailing view that PRBC do not begin to adhere and sequester in the microvasculature until the parasites reach the early trophozoite stage31,32, Jürg Gysin (Université de la Méditerranée, France) showed that RBC infected by young ring-stage parasites (from laboratory-adapted parasite lines that adhere to CSA and from clinical isolates from the placenta) could in fact cytoadhere to syncytiotrophoblasts and brain microvascular endothelial cells. Furthermore, adhesion appeared to be mediated by a novel molecule present on the surface of the ringinfected RBC that was distinct from PfEMP1. The precise identity of this new ligand and the receptor(s) to which it binds is currently under scrutiny. This remains a surprising observation and clearly further work will be needed to reconcile differences with previous studies, however, these findings do suggest that a cryptic sub-population of non-circulating ring-infected RBC may exist in malaria-infected individuals. Parasitology Today, vol. 16, no. 10, 2000

Molecular Approaches to Malaria PfEMP1, RIFINs and Pf60 proteins Much recent effort has also focused on the precise mapping of multiple functional domains of PfEMP1. The complex and highly variable extracellular head structure of the molecule is composed of between two and seven DBL domains and one or two CIDRs (Cysrich-inter-domain regions). Specific and distinct domains of PfEMP1 have now been identified that mediate binding to a number of endothelial-cell-expressed receptors (see Fig. 1) as well as to normal, non-immune human serum immunoglobulins (IgM and IgG), complement-receptor 1 (CR1/CD35), heparan sulphate and the blood Group A antigen. Heparan sulphate and blood group A antigen mediate rosetting via the DBL1 (Duffy-binding-like) domain of PfEMP133,34. Presumably, however, only a subset of DBL1 domains can bind in this way, as rosetting is not a universal feature, even in individuals with this blood group. This exceptional adhesiveness of the PfEMP1 head structure argues that it holds an important role in sequestration and thus in parasite virulence. In terms of translating our knowledge into practical outcomes of benefit to individuals with malaria, evidence was presented that focusing an immune response on domains of PfEMP1 may have a beneficial effect on the course of disease. Dror Baruch [National Institutes of Health (NIH), Bethesda, MD, USA] presented data from their recent vaccine trials using a subfragment from the CIDR of PfEMP1 that has previously been shown to mediate binding to CD36 under both static and flow conditions35–37. Aotus monkeys immunized with this fragment were protected from challenge with homologous parasites. This result supports the idea that immunizing against receptor domains is beneficial, and provides a rationale for the large numbers of mapping experiments that are being reported. Whether such responses will be generally effective against parasites expressing different forms of PfEMP1 or can extend to interfering with other adhesive phenomena is currently unknown. Despite an apparent overwhelming concentration of effort towards elucidating the functional roles and the mechanisms that control transcription and translation of PfEMP1, two further multigene families have also received recent attention. Members of the first of these encode a low molecular mass (20–40 kDa), radioiodinatable, SDS-soluble, Triton-X100 insoluble, and trypsin-sensitive (albeit less than for PfEMP1) cluster of antigens, termed RIFINs, that were recently discovered to be, in part, identical to some of the previously described ‘rosettins’38. Over 200 rif genes (and their purported sub-family stevor39) encoding these RIFINs form what is probably the largest gene family described so far in P. falciparum. The rif genes are transcribed in asexual stages of the parasite and their gene products are transported to the surface of the PRBC where they can be detected 14–16 h after parasite invasion, at the onset of functional and antigenic changes. The RIFINs are a polymorphic group of antigens that vary in size between different parasite isolates and consist of several isoforms (as seen by two-dimensional electrophoresis) that appear to be expressed at high levels on PRBC of both fresh and long-term in vitro cultivated parasites. In contrast to PfEMP1, it seems that several RIFINs may be co-expressed on the surface of a RBC infected with a single clonal parasite. Parasitology Today, vol. 16, no. 10, 2000

Is there then, any relationship between the RIFINs and PfEMP1? The two families of molecules have similar solubility properties, are both size-variable and are expressed at the surface of the PRBC, but they are of widely different molecular weights. Antibodies to PfEMP1 do not seem to precipitate the RIFINs but further characterization is needed, as it is conceivable that the parasite exports several adhesins to the RBC surface. It may need alternative, variable adhesion molecules as the parasite lives and proliferates in the blood, under constant assault from the host immune system. An essential function of RIFINs in the natural cycle of P. falciparum is also suggested by their prominent expression in all clinical isolates examined to date, and by the formidable investment incurred by the parasite in keeping such a variable battery. Their possible role in adhesion remains to be determined. The second new family comprises the Pf60 proteins encoded by the Pf60 multigene family, as discussed by Odile Mercereau-Puijalon (Institut Pasteur, Paris, France). Their role in cytoadherence is unknown and at present the only link to this phenomenon is provided by the sequence similarity between the cytoplasmic tail of PfEMP1 and domains of proteins encoded by the Pf60 family. They are interesting in their own right, however, because of the unusual mechanism that appears to be involved in their translation40. Explaining how transcription of these various gene families is controlled will be a major challenge. For PfEMP1, at least, the current belief is that only a single member of this family of approximately 45 var genes is transcribed at any one time41. The beginnings of attempts to unravel the mechanisms, both for var and for other gene families is clearly crucial not only for our further understanding of cytoadherence but also for the generation of antigenic variation. Recent progress in this area was discussed by Mary Galinski (Emory University, Atlanta, GA, USA) and Artur Scherf (Institut Pasteur, Paris, France). Although the work is clearly still in its early stages, it is already becoming apparent that the mechanisms are highly complex and most likely differ between the different gene families. Transfection of P. falciparum, which is now becoming more widely practised, will prove to be of enormous utility to help unravel these mechanisms during the next few years. Future focus In our view, this area of research is going to become increasingly more complicated, with further interactions, receptors, ligands and functional domains described. The polymorphism of the PfEMP1 family is such that individual members will have measurable affinity with even more molecules than we are currently aware. We should also have a much clearer understanding of how this and other multigene families are controlled. The challenge will be not to lose ourselves in the molecular detail, but remain focused on the role of this phenomenon in pathogenesis of malaria. References 1 Su, X.Z. et al. (1995) The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell 82, 89–100 2 Baruch, D.I. et al. (1995) Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82, 77–87

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Molecular Approaches to Malaria 3 Smith, J.D. et al. (1995) Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82, 101–110 4 Langreth, S.G. and Peterson, E. (1985) Pathogenicity, stability, and immunogenicity of a knobless clone of Plasmodium falciparum in Colombian owl monkeys. Infect. Immun. 47, 760–766 5 MacPherson, G.G. et al. (1985) Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration. Am. J. Pathol. 119, 385–401 6 Berendt, A.R. et al. (1994) Cerebral malaria: the sequestration hypothesis. Parasitol. Today 10, 412–414 7 Clark, I.A. and Rockett, K.A. (1994) The cytokine theory of human cerebral malaria. Parasitol. Today 10, 410–412 8 Grau, G.E. and Dekossodo, S. (1994) Cerebral malaria: mediators, mechanical obstruction or more? Parasitol. Today 10, 408–409 9 Reeder, J.C. et al. (1994) Diversity of agglutinating phenotype, cytoadherence, and rosette-forming characteristics of Plasmodium falciparum isolates from Papua New Guinean children. Am. J. Trop. Med. Hyg. 51, 45–55 10 Landau, I. and Gautret, P. (1998) Animal models: rodent, in Malaria: Parasite Biology, Pathogenesis, and Protection (Sherman, I.W., ed.), pp 401–417, ASM Press 11 Gysin, J. (1998) Animal models: primates, in Malaria: Parasite Biology, Pathogenesis, and Protection (Sherman, I.W., ed.), pp 419–441, ASM Press 12 Carlson, J. et al. (1990) Human cerebral malaria: association with erythrocyte rosetting and lack of anti-rosetting antibodies. Lancet 336, 1457–1460 13 Newbold, C. et al. (1997) Receptor-specific adhesion and clinical disease in Plasmodium falciparum. Am. J. Trop. Med. Hyg. 57, 389–398 14 Rowe, A. et al. (1995) Plasmodium falciparum rosetting is associated with malaria severity in Kenya. Infect. Immun. 63, 2323–2326 15 Roberts, D.J. et al. (2000) Autoagglutination of malaria-infected red blood cells and malaria severity. Lancet 355, 1427–1428 16 Killick-Kendrick, R. and Peters, W. (1978) Rodent Malaria, Academic Press 17 Adams, S. et al. (2000) Differential binding of clonal variants of Plasmodium falciparum to allelic forms of intracellular adhesion molecule 1 determined by flow adhesion assay. Infect. Immun. 68, 264–269 18 Craig, A. et al. (2000) A functional analysis of a natural variant of intercellular adhesion molecule-1 (ICAM-1Kilifi). Hum. Mol. Genet. 9, 525–530 19 Fernandez-Reyes, D. et al. (1997) A high frequency African coding polymorphism in the N-terminal domain of ICAM-1 predisposing to cerebral malaria in Kenya. Hum. Mol. Genet. 6, 1357–1360 20 Cooke, B.M. and Nash, G.B. (1995) Plasmodium falciparum: characterization of adhesion of flowing parasitized red blood cells to platelets. Exp. Parasitol. 80, 116–123 21 Wahlgren, M. et al. (1995) Adhesion of Plasmodium falciparuminfected erythrocytes to human cells and secretion of cytokines (IL-1-beta, IL-1RA, IL-6, IL-8, IL-10, TGF beta, TNF alpha, G-CSF, GM-CSF). Scand. J. Immunol. 42, 626–636 22 Aitman, T.J. et al. (2000) Population genetics: malaria susceptibility and CD36 mutation. Nature 405, 1015–1016 23 Beeson, J.G. et al. (2000) Adhesion of Plasmodium falciparuminfected erythrocytes to hyaluronic acid in placental malaria. Nat. Med. 6, 86–90 24 Fried, M. and Duffy, P.E. (1996) Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta. Science 272, 1502–1504 25 Fried, M. et al. (1998) Maternal antibodies block malaria. Nature 395, 851–852 26 Cooke, B.M. and Coppel, R.L. (1995) Cytoadhesion and falciparum malaria: going with the flow. Parasitol. Today 11, 282–287 27 Maubert, B. et al. (2000) Cytoadherence of Plasmodium falciparuminfected erythrocytes in the human placenta. Parasite Immunol. 22, 191–199 28 Sartelet, H. et al. (2000) Hyperexpression of ICAM-1 and CD36 in placentas infected with Plasmodium falciparum: a possible role of these molecules in sequestration of infected red blood cells in placentas. Histopathology 36, 62–68 29 Trenholme, K.R. et al. (2000) clag9: A cytoadherence gene in Plasmodium falciparum essential for binding of parasitized erythrocytes to CD36. Proc. Natl. Acad. Sci. U. S. A. 97, 4029–4033 30 Gardiner, D.L. et al. (2000) Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA. Mol. Biochem. Parasitol. 110, 33–41

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Website of Interest Malaria Transmission Blocking Vaccines: An Ideal Public Good A document published by WHO under this title can be downloaded from http://www.who.int/tdr/publications/publications/ pdf/tbv.pdf

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