Cytoadhesion of Plasmodium falciparum ring-stage-infected erythrocytes.

A common pathological characteristic of Plasmodium falciparum infection is the cytoadhesion of mature-stage-infected erythrocytes (IE) to host endothelium and syncytiotrophoblasts. Massive accumulation of IE in the brain microvasculature or placenta is strongly correlated with severe forms of malaria. Extensive binding of IE to placental chondroitin sulfate A (CSA) is associated with physiopathology during pregnancy. The adhesive phenotype of IE correlates with the appearance of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) at the erythrocyte surface (approximately 16 h after merozoite invasion), so that only early blood-stage (ring-stage) IE appear in the peripheral blood. Here, we describe results that challenge the existing view of blood-stage IE biology by demonstrating the specific adhesion of IE, during the early ring-stage, to endothelial cell lines from the brain and lung and to placental syncytiotrophoblasts. Later, during blood-stage development of these IE, trophozoites switch to an exclusively CSA cytoadhesion phenotype. Therefore, adhesion to an individual endothelial cell or syncytiotrophoblast may occur throughout the blood-stage cycle, indicating the presence in malaria patients of noncirculating (cryptic) parasite subpopulations. We detected two previously unknown parasite proteins on the surface of ring-stage IE. These proteins disappear shortly after the start of PfEMP1-mediated adhesion.

Role of nonimmune IgG bound to PfEMP1 in placental malaria.

Infections with Plasmodium falciparum during pregnancy lead to the accumulation of parasitized red blood cells (infected erythrocytes, IEs) in the placenta. IEs of P. falciparum isolates that infect the human placenta were found to bind immunoglobulin G (IgG). A strain of P. falciparum cloned for IgG binding adhered massively to placental syncytiotrophoblasts in a pattern similar to that of natural infections. Adherence was inhibited by IgG-binding proteins, but not by glycosaminoglycans or enzymatic digestion of chondroitin sulfate A or hyaluronic acid. Normal, nonimmune IgG that is bound to a duffy binding-like domain beta of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) might at the IE surface act as a bridge to neonatal Fc receptors of the placenta.

Taxol arrests the development of blood-stage Plasmodium falciparum in vitro and Plasmodium chabaudi adami in malaria-infected mice.

Taxol, a natural product used to treat a variety of human cancers, is shown here to be extremely effective against chloroquine- and pyrimethamine-resistant malaria parasites. Addition of Taxol (1.0 microM) for one cycle to cultures of human erythrocytes infected with Plasmodium falciparum prevents the establishment of new infections. Blood parasitemia is eliminated in mice infected with Plasmodium chabaudi adami when they are given a single intraperitoneal injection of Taxol at 150 mg/m2. The majority of the animals treated immediately preceding parasite schizogony remain free of infection after eight replication cycles. The impressive antimalarial activity of Taxol, at a dosage that has been tolerated in humans, establishes its potential utility for treatment of severe, drug-resistant human malaria.

Recombinant human thrombomodulin(csa+): a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate.

The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-Plasmodium falciparum-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies.

Isolation and characterization of brain microvascular endothelial cells from Saimiri monkeys. An in vitro model for sequestration of Plasmodium falciparum-infected erythrocytes.

The adhesion of parasitized red blood cells (PRBC) to the endothelium (sequestration) may contribute to the pathogenic events in severe human malaria caused by P. falciparum. However, the factors involved in the pathophysiology, especially cerebral malaria are poorly understood. Previously, we have shown that the squirrel monkey Saimiri sciureus is a potential model for human cerebral malaria. In this paper we describe five stable clones of endothelial cell lines isolated immediately postmortem from different regions of the brain of Saimiri monkeys. The endothelial cell characteristics of these clones were confirmed by analyzing their ultrastructural aspects by transmission electron microscopy and by immunodetection of various endothelial cell markers. The Saimiri brain endothelial cell clones (SBEC) varied in their expression of different surface molecules. For example, various combinations of receptors involved in P. falciparum PRBC adherence such as CD36, ICAM-1 and E-selectin, were expressed at baseline values and could be up-regulated by human srTNF-alpha and human srIFN-gamma. One of the SBEC clones showed a strong cytoadherence for various laboratory strains of P. falciparum despite the absence of surface expression of any of the known endothelial receptors implicated in PRBC adherence. This finding suggests the existence of a new and uncharacterized PRBC binding receptor. The use of target organ specific endothelial cell lines expressing a number of different potential P. falciparum PRBC cytoadherence receptors, will be a useful in vitro system for the evaluation of strategies for the development of vaccine and antimalarial drugs to prevent human cerebral malaria.

Characterization of trafficking pathways and membrane genesis in malaria-infected erythrocytes.

The origin of membraneous structures in the cytoplasm of human erythrocytes infected with the malaria parasite, Plasmodium falciparum, was determined by confocal fluorescence imaging microscopy. When infectious merozoites invaded erythrocytes labeled with the fluorescent, lipophilic, non-exchangeable molecules DiIC16 or DiOC16, a ring of fluorescence was observed surrounding the internal parasite, indicating that the parasitophorous vacuolar membrane (PVM) is formed in part from the erythrocyte membrane. As the parasites matured, fluorescent vesicles were seen to be exported into the erythrocyte cytoplasm, beginning at 6 h post-invasion. During intraerythrocytic development, these dyes were transferred from the PVM to the parasite. When fluorescently labeled merozoites were released from these cells and invaded unlabeled erythrocytes, fluorescence was confined to the parasite throughout the entire erythrocytic cycle. Taken together, these results demonstrate that all vesicles/membranous compartments in the erythrocyte cytoplasm of parasitized erythrocytes (IRBC) contain membrane derived from the PVM. Based on this information, we define pathways that the parasite utilizes to export proteins and lipids to the host cell cytoplasm and surface membrane. When IRBC were labeled post-invasion with DiIC16 or DiOC16 and the parasites allowed to mature for one life cycle, the dyes were confined to the erythrocyte membrane, demonstrating that the host cell membrane of IRBC does not endocytose and there is no membrane exchange from the erythrocyte to the parasite. This investigation helps to resolve two long-standing controversies and provides new insights into the transport pathways that malaria parasites utilize during their development within host erythrocytes.

Characterization of macromolecular transport pathways in malaria-infected erythrocytes.

We have previously provided evidence for a pathway in Plasmodium falciparum-infected erythrocytes, coined the parasitophorous duct pathway, which provides serum (macro)molecules direct access to intraerythrocytic parasites . The present study addresses the purity of the fluorescent macromolecules used to define the duct pathway and provides ultrastructural evidence for its presence. The fluorescent tracers used to characterize transport remain intact during their incubation with infected erythrocytes. Transport of macromolecules in the external medium or host cell cytosol to the intracellular parasites is shown to occur by two distinct pathways. Fluorescent dextrans in the erythrocyte cytosol are ingested by the parasite via a specialized organelle, the cytostome, and are transported to the parasite food vacuole. Transport through this pathway occurs throughout the asexual life cycle. By contrast, fluorescent dextrans in the external medium bypass the erythrocyte cytosol, and are internalized by the parasite by a process resembling fluid-phase endocytosis. Serial sections of mature parasites fixed and stained by various methods for transmission electron microscopy reveal areas of apparent membrane continuity between the erythrocyte membrane and the parasitophorous vacuolar membrane that surrounds the parasite, that could leave the parasites exposed to the external medium. Using carboxylate and amidine-modified fluorescent latex spheres and laser scanning confocal microscopy, macromolecules up to 50-70 nm in diameter are found to have direct access to intraerythrocytic parasites. This size exclusion is consistent with the dimensions of the parasitophorous duct pathway revealed by electron microscopy. This investigation reports for the first time the existence of two, distinct macromolecular transport pathways in malaria-infected erythrocytes.

Characterisation of the chondroitin sulphate of Saimiri brain microvascular endothelial cells involved in Plasmodium falciparum cytoadhesion.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to chondroitin-4-sulphate (CSA) is inhibited by soluble CSA in vitro on Saimiri brain microvascular endothelial cells (SBEC) and in vivo in P. falciparum-infected Saimiri monkeys. We tested whether the SBEC model was appropriate for studying CSA-binding IRBC using four cell lines. All SBEC expressed a chondroitin sulphate (CS), with a composition of CSA. The mean sizes of these CSA were 20.5, 22, 23, 32.5 and 36 kDa for SBEC 3A and C2, CHO, SBEC 1D and 17, respectively. We found that cytoadhesion of the Palo-Alto (FUP)1 CSA-binding phenotype, selected by panning on SBEC 17, was specifically inhibited in a dose-dependent manner by all the purified CSA. The extent of inhibition depended on the cellular origin of the tested CSA. SBEC 17 CSA was 33 times more efficient than CHO-CSA and 21 times more efficient than the 50 kDa commercial bovine trachaea CSA. Dynabeads coated with a total extract of SBEC 1D CS-proteoglycans interacted with CSA- but not with CD36- or ICAM-1-binding IRBC. These Dynabeads also interacted specifically with the PfEMP1 DBL-3 domain, on the surface of CHO transfectants, but not with the CIDR-1 domain. Thrombomodulin was involved in IRBC adhesion to all SBEC whereas CD44 was only expressed by SBEC 1D and 17. These two CSA-proteoglycans have also been detected at the surface of human endothelial cells. Thus, the two homologous models, SBEC/Saimiri sciureus, are useful and reliable tools for the evaluation of new anti-CSA adhesion treatments and anti-disease vaccines for pregnant women.

Plasmodium falciparum-infected erythrocytes: a mutational analysis of cytoadherence via murine thrombomodulin.

The pathophysiologic events leading to organ damage in Plasmodium falciparum malaria infections involve adhesion and sequestration of parasite-infected erythrocytes (PRBC) to the vascular endothelium and syncytiotrophoblast. Several potential receptors to which the PRBCs may bind have recently been identified, one of which is thrombomodulin (TM). TM has been implicated particularly in mediating sequestration of P. falciparum-infected erythrocytes in the placenta and brain, two sites of disease associated with high morbidity. In order to establish that binding of parasite-infected red blood cells to TM is dependent on its containing chondroitin-4-sulfate (CSA), we have mutated the CSA-attachment site of murine TM, and expressed this mutant form (TMsergly) in COS-7 cells. In cytoadhesion assays, we demonstrate that, in contrast to wild-type TM which contains CSA and supports the adhesion of 1466 PRBCs/mm2, TMser-gly does not contain CSA and adhesion of PRBCs to those cells expressing TMser-gly is entirely abrogated (200 PRBCs/mm2). These studies further confirm that the CSA of TM may play a role in the pathophysiology of malaria by providing a binding site for PRBCs.

Ultrastructural aspects of Plasmodium falciparum-infected erythrocyte adherence to endothelial cells of Saimiri brain microvasculature.

We have recently shown that some squirrel monkeys (Saimiri sciureus) develop cerebral malaria when experimentally infected with asexual blood stage forms of different Plasmodium falciparum isolates. Since cerebral malaria is neither an inconsistent nor predictable event, several clones of endothelial cells isolated from the squirrel monkey brain microvasculature have been developed. Infected red blood cell (IRBC) adherence involved the knobs and direct membrane interactions through pseudopodes and microvilli on the Saimiri brain endothelial cell (SBEC) surface, similar to that observed with both brain microvascular endothelial cells from a patient who died of cerebral malaria and the rhesus monkey/P. coatneyi cerebral malaria model. The involvement of pseudopodes and microvilli increase the endothelial cell surface for the attachment of IRBCs; however, they are already present before the SBECs are exposed to IRBCs. With some SBEC phenotypes, embedding of IRBCs into the cytoplasma membrane of the endothelial cell was observed, resulting in an extremely close apposition of both SBEC and IRBC membranes during the adherence process. Once IRBCs are adherent, particularly for the embedding type, heterocellular communication-like structures between the cells become apparent. The upregulation of CD36 and intercellular adhesion molecule-1 by soluble recombinant (sr)-tumor necrosis factor-alpha or sr-interferon-gamma did not modify the IRBC interactions with SBECs at the ultrastructural level. The study shows further that the observed differences of IRBC adherence are due to unidentified phenotypic differences of SBECs rather than to a parasite isolate or particular endothelial cell receptor-associated phenomenon. Exploring P. falciparum IRBC cytoadherence in the squirrel monkey using a homologous physiologic target cell model in vitro should be useful for the evaluation of vaccine strategies and drugs to prevent human cerebral malaria.

[Plasmodium falciparum and chondroitin-4-sulfate: the new key couple in sequestration]. / Plasmodium falciparum et chondroitine-4-sulfate: le nouveau couple clé de la séquestration.

Some complications of Plasmodium falciparum infection such as cerebral malaria and pregnancy-associated malaria may be partially due to cytoadherence of erythrocytes infected by mature parasites on microvascular endothelial cells or placental syncytiotrophoblasts. Recently a new cytoadherence receptor, chondroitin-4-sulphate (CSA), was identified first on endothelial cells in primates and then on CHO cells and purified receptors. Further study has implicated CSA in cytoadherence of infected red blood cells to syncytiotrophoblasts in human placenta and Saimiri sciureus monkeys. In solution the minimal size for full inhibitory effect is approximately 9 kDa. Injection of CSA in Plasmodium falciparum-infected Saimiri monkeys resulted in specific release of sequestered erythrocytes infected by mature parasites. An added interest of these findings is that CSA, a glycosaminoglycan, is already in clinical use for treatment of degenerative joint disease. Current data on the parasite ligand for CSA indicates that it is not co-expressed with other cytoadherence ligands and that its binding activity decreases as the parasite matures from the 20th to 40th hour of the cycle. Since one or more var genes encoding the CSA ligand have been identified, it is likely that peptides will be obtained quickly and used either for direct inhibition of cytoadherence on CSA or for development of an anti-sequestration vaccine.

Presence of the parasitophorous duct in Plasmodium falciparum and P. vivax parasitized Saimiri monkey red blood cells.

Although the exchange of metabolites between the intraerythrocytic malaria parasite and the external medium has been studied extensively, the transport of molecules across the erythrocyte cytoplasmic membrane and cytoplasm and the parasitophorous vacuolar membrane needs to be investigated more fully to be completely understood. Recently, the concept of the parasitophorous duct, establishing a continuity between the environment and the vacuolar space surrounding the intraerythrocytic parasite, has been suggested to provide an explanation of how macromolecules can cross two membranes in a cell devoid of an endocytic system. This concept is highly controversial and has been suspected to be an in vitro artefact. In this article, Bruno Pouvelle and Jürg Gysin present evidence of the existence of the parasitophorous duct in Saimiri monkey Plasmodium falciparum- and P. vivax-infected erythrocytes, with a series of ex vivo experiments showing stage and species dependent variations of the characteristics of this structure.

Biological and biochemical characteristics of cytoadhesion of Plasmodium falciparum-infected erythrocytes to chondroitin-4-sulfate.

The cytoadhesion of Plasmodium falciparum laboratory strains and clones to Saimiri brain microvascular endothelial cells (SBEC 17), with chondroitin-4-sulfate (CSA) as the only adhesion receptor, was tested. Only one strain had significant cytoadhesion. However, CSA-specific infected erythrocytes (IRBCs) were detected in all strains after selection of a CSA-specific subpopulation by culturing the few adherent IRBCs. This demonstrates the lack of sensitivity of cytoadhesion microassays for detecting small quantities of CSA-specific IRBCs in cultures or field isolates. Cytoadhesion to CSA is maximal at 24 h of the cycle and decreases with the onset of schizogony, reaching a minimum just before reinvasion. This fluctuation must be taken into account in comparisons of the cytoadhesion of different strains or isolates. The minimum size of CSA for active inhibition was 4 kDa, and a mass of 9 kDa was required for inhibition similar to that obtained with the 50-kDa CSA. In contrast to cytoadhesion to CSA, which is pH independent or maximal at physiological pH (depending on the target endothelial cells), adhesion to CD36 and intercellular adhesion molecule 1 was pH dependent, requiring acidic conditions to be maximal in all cases. Cytoadhesion to CSA may trigger the occlusion of microvessels and cause the acidosis necessary for the other receptors to be fully efficient. If this key role in the mechanisms of sequestration were to be confirmed in vivo, prevalence studies of the CSA cytoadhesion phenotype would have to be reevaluated, because simple cytoadhesion assays do not detect CSA-specific parasites present in very low numbers, and these parasites might then be undetected in the peripheral blood but present in organs in which sequestration occurs, such as the placenta (M. Fried and P. E. Duffy, Science 272:1502-1504, 1996).

Molecular mechanisms of Plasmodium falciparum placental adhesion.

In natural Plasmodium falciparum infections, parasitized erythrocytes (PEs) circulate in the peripheral blood for a period corresponding roughly to the first part of the erythrocytic life cycle (ring stage). Later, in blood-stage development, parasite-encoded adhesion molecules are inserted into the erythrocyte membrane, preventing the circulation of the PEs. The principal molecule mediating PE adhesion is P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the polymorphic var gene family. The population of parasites is subject to clonal antigenic variation through changes in var expression, and a single PfEMP1 variant is expressed at the PE surface in a mutually exclusive manner. In addition to its role in immune evasion, switches in PfEMP1 expression may be associated with fundamental changes in parasite tissue tropism in malaria patients. A switch from CD36 binding to chondroitin sulphate A (CSA) binding may lead to extensive sequestration of PEs in placenta syncytiotrophoblasts. This is probably a key event in malaria pathogenesis during pregnancy. The CSA-binding phenotype of mature PEs is linked to another distinct adhesive phenotype: the recently described CSA-independent cytoadhesion of ring-stage PEs. Thus, a subpopulation of PEs that sequentially displays these two different phenotypes may bind to an individual endothelial cell or syncytiotrophoblast throughout the asexual blood-stage cycle. This suggests that non-circulating (cryptic) parasite subpopulations are present in malaria patients.

Chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of Plasmodium falciparum infected erythrocytes.

BACKGROUND: Chondroitin-4-sulfate (CSA) was recently described as a Plasmodium falciparum cytoadherence receptor present on Saimiri brain microvascular and human lung endothelial cells. MATERIALS AND METHODS: To specifically study chondroitin-4-sulfate-mediated cytoadherence, a parasite population was selected through panning of the Palo-Alto (FUP) 1 P. falciparum isolate on monolayers of Saimiri brain microvascular endothelial cells (SBEC). Immunofluorescence showed this SBEC cell line to be unique for its expression of CSA-proteoglycans, namely CD44 and thrombomodulin, in the absence of CD36 and ICAM-1. RESULTS: The selected parasite population was used to monitor cytoadherence inhibition/dissociating activities in Saimiri sera collected at different times after intramuscular injection of 50 mg CSA/kg of body weight. Serum inhibitory activity was detectable 30 min after injection and persisted for 8 hr. Furthermore, when chondroitin-4-sulfate was injected into monkeys infected with Palo-Alto (FUP) 1 P. falciparum, erythrocytes containing P. falciparum mature forms were released into the circulation. The cytoadherence phenotype of circulating infected red blood cells (IRBC) was determined before and 8 hr after inoculation of CSA. Before inoculation, in vitro cytoadherence of IRBCs was not inhibited by CSA. In contrast, in vitro cytoadherence of circulating infected erythrocytes obtained 8 hr after CSA inoculation was inhibited by more than 90% by CSA. CONCLUSIONS: In the squirrel monkey model for infection with P. falciparum, chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of parasitized erythrocytes.

Ex vivo desequestration of Plasmodium falciparum-infected erythrocytes from human placenta by chondroitin sulfate A.

We performed ex vivo experiments with Plasmodium falciparum-infected human placentas from primi- and multigravida women from Cameroon. All women, independent of their gravida status, had anti-chondroitin sulfate A (CSA) adhesion antibodies which cross-reacted with heterologous strains, such as FCR3 and Palo-Alto(FUP)1, which were selected for CSA binding. These antibodies, directed against the surface of infected erythrocytes obtained by flushing with CSA (IRBC(CSA)), were restricted to the immunoglobulin G3 isotypes. Massive desequestration of parasites was achieved with soluble CSA but not with anti-ICAM-1 and anti-CD36 monoclonal antibodies. All of the CSA-flushed parasites were analyzed immediately by using in vitro assays of binding to Saimiri brain endothelial cells (SBEC) expressing various adhesion receptors. Parasites derived from all six placentas displayed the CSA adhesion phenotype. However, only partial inhibition of adhesion was observed in the presence of soluble CSA or when Sc1D SBEC were treated with chondroitinase ABC. These results suggest that an additional adhesive molecule of IRBC(CSA) which binds to an unidentified receptor is present in the placenta. This new phenotype was lost once the parasites adapted to in vitro culture. We observed additional differences in the CSA adhesion phenotype between placental parasites and in vitro-cultured parasites panned on endothelial cells carrying CSA. The minimum size of fractionated CSA required for a significant inhibition of placental IRBC(CSA) adhesion to Sc1D cells was 1 to 2 kDa, which contrasts with the 4-kDa size necessary to reach equivalent levels of inhibition with panned IRBC(CSA) of this phenotype. All placental IRBC(CSA) cytoadhered to Sc17 SBEC, which express only the CSA receptor. Panning of IRBC(CSA) on these cells resulted in a significant quantitative increase of IRBC cytoadhering to the CSA of Sc1D cells but did not change their capacity for adhesion to CSA on normal placenta cryosections. Our results indicate that the CSA binding phenotype is heterogeneous and that several distinct genes may encode P. falciparum-CSA ligands with distinct binding properties.

Direct access to serum macromolecules by intraerythrocytic malaria parasites.

Trafficking pathways in malaria-infected erythrocytes are complex because the internal parasite is separated from the serum by the erythrocyte and parasitophorous vacuolar membranes. Intraerythrocytic Plasmodium falciparum parasites can endocytose dextrans, protein A and an IgG2a antibody. Here we show that these macromolecules do not cross the erythrocyte or parasitophorous vacuolar membranes, but rather gain direct access to the aqueous space surrounding the parasite through a parasitophorous duct. Evidence for this structure includes visualization of membranes that are continuous between the parasitophorous vacuolar and erythrocyte membranes, and surface labelling of the parasite with fluorescent macromolecules under conditions that block endocytosis. The parasite can internalize by fluid-phase endocytosis macromolecules from the aqueous compartment surrounding it. Thus, surface antigens on trophozoites and schizonts should be considered as targets for antibody-directed parasiticidal agents.

Chondroitin-4-sulphate (proteoglycan), a receptor for Plasmodium falciparum-infected erythrocyte adherence on brain microvascular endothelial cells.

Adherence of Plasmodium falciparum parasitized erythrocytes to the microvascular endothelium is mediated by different receptors expressed by endothelial cells. The study of the adherence of P. falciparum-infected erythrocytes to Saimiri monkey brain microvascular endothelial cells revealed the presence of an additional receptor, which was identified and further characterized. This receptor was also found on the surface of primary human lung endothelial cells (HLEC). We developed two mAbs to this receptor which very efficiently blocked the adherence of parasite strains to Saimiri brain endothelial cells (SBEC). The ability of these mAb to bind to SBEC was partially blocked by chondroitin-4-sulphate (CSA). Competitive inhibition assays on adherence of parasitized red blood cells (PRBC) showed that CSA, but not hyaluronic acid, chondroitin-6-sulphate, dermatan sulphate, keratane sulphate, heparan sulphate or chondroitin-4S-disaccharide, was able to almost completely inhibit PRBC adherence. The same effect was obtained with chondroitinase ABC and AC, but not B, hyaluronidase or heparinase. These results strongly suggest that a member of the chondroitin-glycosaminoglycan family, CSA, represents an additional receptor used by P. falciparum PRBC to cytoadhere to microvascular endothelial cells.

Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum.

Members of the Plasmodium falciparum var gene family encode clonally variant adhesins, which play an important role in the pathogenicity of tropical malaria. Here we employ a selective panning protocol to generate isogenic P.falciparum populations with defined adhesive phenotypes for CD36, ICAM-1 and CSA, expressing single and distinct var gene variants. This technique has established the framework for examining var gene expression, its regulation and switching. It was found that var gene switching occurs in situ. Ubiquitous transcription of all var gene variants appears to occur in early ring stages. However, var gene expression is tightly regulated in trophozoites and is exerted through a silencing mechanism. Transcriptional control is mutually exclusive in parasites that express defined adhesive phenotypes. In situ var gene switching is apparently mediated at the level of transcriptional initiation, as demonstrated by nuclear run-on analyses. Our results suggest that an epigenetic mechanism(s) is involved in var gene regulation.