In vitro rosetting, cytoadherence, and microagglutination properties of Plasmodium falciparum-infected erythrocytes from Gambian and Tanzanian patients

To understand the molecular mechanisms that lead to sequestration of red blood cells infected with mature stages of Plasmodium falciparum and to examine the relevance of earlier studies on adherence properties of laboratory-derived P falciparum parasites to the natural parasite population, we analyzed Gambian and Tanzanian isolates for in vitro cytoadherence and antibody-mediated microagglutination. Eighteen cryopreserved isolates of ring-stage parasites were cultured for 20 to 30 hours in vitro, in the patients original erythrocytes, to the trophozoite and schizont stage. All parasites were positive in the microagglutination assay with at least one of four African hyperimmune sera. In a rosetting assay, only 2 of the 18 isolates were strongly positive (35% and 41% of parasitized erythrocytes with more than two uninfected cells bound). Thirteen isolates showed either intermediate (5% to 18%) or low (less than 5%) rosetting while three isolates did not form rosettes. Infected cell-binding of the different isolates to immobilized CD36 or thrombospondin, or C32 melanoma cells correlated with the percentage of mature parasites in the blood samples (r = .932 for CD36, r = .946 for thrombospondin, and r = .881 for C32 melanoma cells). There was a high correlation between binding to CD36 and thrombospondin (r = .982). The extent of infected cell rosetting with uninfected cells in these blood samples was not correlated with these other receptor properties. We also observed coexpression of rosetting and cytoadherence receptors on the same parasitized erythrocytes.     

Thrombospondin binding by parasitized erythrocyte isolates in falciparum malaria

Toward understanding the pathogenesis of vascular sequestration in falciparum malaria, we investigated binding of Plasmodium falciparum parasitized erythrocyte isolates to thrombospondin and other adhesive proteins. Blood samples with rings from 12 patients with falciparum malaria were cultured 30 hr until parasites were mature trophozoites and schizonts. All parasitized erythrocyte isolates bound to thrombospondin, but not to fibronectin, laminin, vitronectin, or factor VIII/von Willebrand factor. Parasitized erythrocyte binding varied among isolates, ranging from 192 to 6,725 per mm2, average 2,953. There was good correlation between trophozoite plus schizont % parasitemia and thrombospondin binding (r = 0.884, P less than 0.001). In two patients with stupor, 3,642 and 2,864 parasitized erythrocytes bound per mm2, in proportion to parasitemia, suggesting cerebral malaria is not due to increased binding affinity. These results indicate there is a conserved function among isolates from this geographic region, known to be antigenically diverse at the parasitized erythrocyte membrane surface. These results support the hypothesis that specific binding to an endothelial receptor, possibly involving thrombospondin, plays a role in vascular sequestration in falciparum malaria.     

Thrombospondin mediates the cytoadherence of Plasmodium falciparum- infected red cells to vascular endothelium in shear flow conditions

Cerebral malaria is thought to involve specific attachment of Plasmodium falciparum-infected knobby red cells to venular endothelium. The nature of surface ligands on host endothelial cells that may mediate cytoadherence is poorly understood. We have investigated the effects of soluble thrombospondin, rabbit antiserum raised against thrombospondin, and human immune serum on cytoadherence of parasitized erythrocytes in ex vivo mesocecum vasculature. Preincubation of infected red cells with soluble thrombospondin or human immune serum inhibits binding of infected red cells to rat venular endothelium. Infusion of the microcirculatory preparation with rabbit antithrombospondin antibodies before perfusion of parasitized erythrocytes also resulted in decreased cytoadherence. In addition, incubation of infected cells with human immune sera obtained from malaria patients significantly inhibited the observed cytoadherence. Our results indicate that thrombospondin mediates binding of infected red cells to venular endothelium and may thus be involved in the pathogenesis of cerebral malaria.     

Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.     

Modifications in the CD36 binding domain of the Plasmodium falciparum variant antigen are responsible for the inability of chondroitin sulfate A adherent parasites to bind CD36

Adhesion of mature Plasmodium falciparum parasitized erythrocytes to microvascular endothelial cells or to placenta contributes directly to the virulence and severe pathology of P falciparum malaria. Whereas CD36 is the major endothelial receptor for microvasculature sequestration, infected erythrocytes adhering in the placenta bind chondroitin sulfate A (CSA) but not CD36. Binding to both receptors is mediated by different members of the large and diverse protein family P falciparum erythrocyte membrane protein-1 (PfEMP-1) and involves different regions of the molecule. The PfEMP-1-binding domain for CD36 resides in the cysteine-rich interdomain region 1 (CIDR-1). To explore why CSA-binding parasites do not bind CD36, CIDR-1 domains from CD36- or CSA-binding parasites were expressed in mammalian cells and tested for adhesion. Although CIDR-1 domains from CD36-adherent strains strongly bound CD36, those from CSA-adherent parasites did not. The CIDR-1 domain has also been reported to bind CSA. However, none of the CIDR-1 domains tested bound CSA. Chimeric proteins between CIDR-1 domains that bind or do not bind CD36 and mutagenesis experiments revealed that modifications in the minimal CD36-binding region (M2 region) are responsible for the inability of CSA-selected parasites to bind CD36. One of these modifications, mapped to a 3-amino acid substitution in the M2 region, ablated binding in one variant and largely reduced binding of another. These findings provide a molecular explanation for the inability of placental sequestered parasites to bind CD36 and provide additional insight into critical residues for the CIDR-1/CD36 interaction.     

Inability to detect transferrin receptors on P. falciparum parasitized red cells

The mechanism by which P. falciparum takes up iron from transferrin has been explored. Binding of 125I labelled transferrin to parasitized red cells at 37 degrees C is two-fold greater than to control cells; at 0 degrees C there is no significant difference. The binding is non-specific as judged from the following: it is not saturable; it is not limited to transferrin as lactoferrin (which has iron binding domains) and bovine serum albumin (which does not) also bind in excess to parasitized red cells. A transferrin receptor complex could not be demonstrated when parasitized red cells, to which 125I transferrin was bound, were solubilized in Triton X100. Previous observation showed that uptake of transferrin iron by parasitized red cells is not accompanied by equimolar uptake of transferrin protein. We therefore suggest that nonspecifically bound transferrin is endocytosed, that the protein is degraded and the iron selectively retained.     

Long-term cultured human vascular endothelial cells (EC-FP5) bind Plasmodium falciparum infected erythrocytes

We have established an endothelial cell line, EC-FP5, that binds Plasmodium falciparum infected erythrocytes after several passages in culture and multiple cryopreservations. Binding by the EC-FP5 cells, measured as the percentage that bound infected erythrocytes and the average number of infected erythrocytes bound per endothelial cell, was similar in cells cryopreserved 1, 2, or 3 times. The parasite strain of the infected erythrocytes and culture conditions, including parasitemia and pH of the erythrocyte suspension, significantly affected binding. The capability of the EC-FP5 cells to be cultured in large amounts and cryopreserved in several aliquots will provide flexibility, reduce experimental variation, and enhance the utility of the endothelial cell-dependent cytoadherence assay.     

Inhibitory activity of human lactoferrin and its peptide on chondroitin sulfate A-, CD36-, and thrombospondin-mediated cytoadherence of plasmodium falciparum-infected erythrocytes.

Lactoferrin (LF), a human serum protein, strongly inhibited the adherence of Plasmodium falciparum-infected erythrocytes (PE) to immobilized chondroitin sulfate A (CSA)-conjugated albumin at a concentration of 100 microg/mL and blocked the PE binding to CD36-expressing Chinese hamster ovary (CHO) cells, as well as immobilized CD36 at concentrations of 5 microg/mL and 100 microg/mL, respectively. Biotinylated LF bound to CD36 in a saturable manner, and such binding was inhibited by unlabeled LF and the anti-CD36 monoclonal antibody, 8A6, suggesting specificity of binding. Additionally, LF inhibited PE binding to immobilized thrombospondin (TSP) at a concentration of 100 microg/mL, and specific binding of LF to TSP was confirmed using biotinylated LF. LF inhibited PE binding to C32 amelanotic melanoma cells in a dose-dependent manner. A peptide of LF, Arg-Asn-Met Arg-Lys-Val Arg-Gly-Pro-Pro-Val-Ser-Cys (amino acid residues 25-37 of LF), which has been suggested to contribute to LF binding to various materials, including CSA, inhibited PE binding to immobilized CSA-conjugated albumin, immobilized CD36, CD36-expressing CHO cells, immobilized TSP, and C32 amelanotic melanoma cells, as well as LF itself. These results suggest that LF peptide may provide the basis for developing agents that are able to inhibit CSA-, CD36-, and TSP-mediated cytoadherence of PE.     

Purification and in vitro selection of rosette-positive (R+) and rosette-negative (R-) phenotypes of knob-positive Plasmodium falciparum parasites.

We have developed methods for in vitro selection of Plasmodium falciparum parasites that bear knob protrusions (K+) and are either of the rosette-positive (K+R+) or rosette-negative (K+R-) phenotypes. Cryopreserved parasites from spleen-intact Aotus monkeys that were K+, C32 cell adherence-positive (C+), CD36 adherence-positive, and R- with Aotus erythrocytes were adapted to continuous growth in human erythrocytes, and selected initially for adherence to C32 melanoma cells. In the absence of independent selection for rosettes, K+R-C+ parasites were produced that adhered to both C32 cells and CD36. Without selection for the C+ phenotype, K+R-C- parasites eventually predominated in such cultures. The R+ parasites were selected using differences in sedimentation behavior of rosette-infected cells versus non-rosette-infected cells. Methods were devised for selection of the R+ or R- phenotypes and for the purification of R+ or R- infected cells of high parasitemia that were suitable for molecular studies. With the repeated selection for K+R+ parasites, we were able to maintain the K+R+ phenotype for several months in vitro. These methods will allow systematic study of the molecular basis of the K+R+ and K+R- phenotypes.     

Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: A receptor for human placental infection

Malaria during the first pregnancy causes a high rate of fetal and neonatal death. The decreasing susceptibility during subsequent pregnancies correlates with acquisition of antibodies that block binding of infected red cells to chondroitin sulfate A (CSA), a receptor for parasites in the placenta. Here we identify a domain within a particular Plasmodium falciparum erythrocyte membrane protein 1 that binds CSA. We cloned a var gene expressed in CSA-binding parasitized red blood cells (PRBCs). The gene had eight receptor-like domains, each of which was expressed on the surface of Chinese hamster ovary cells and was tested for CSA binding. CSA linked to biotin used as a probe demonstrated that two Duffy-binding-like (DBL) domains (DBL3 and DBL7) bound CSA. DBL7, but not DBL3, also bound chondroitin sulfate C (CSC) linked to biotin, a negatively charged sugar that does not support PRBC adhesion. Furthermore, CSA, but not CSC, blocked the interaction with DBL3; both CSA and CSC blocked binding to DBL7. Thus, only the DBL3 domain displays the same binding specificity as PRBCs. Because protective antibodies present after pregnancy block binding to CSA of parasites from different parts of the world, DBL-3, although variant, may induce cross-reactive immunity that will protect pregnant women and their fetuses.     

Ultrastructural analysis of fresh Plasmodium falciparum-infected erythrocytes and their cytoadherence to human leukocytes.

Sixty fresh Plasmodium falciparum isolates obtained from Gambian children with mild or cerebral malaria were investigated by transmission electron microscopy for the expression of knob-like protrusions (K+) on the surface of the infected erythrocytes. More than six-hundred infected erythrocytes were analyzed. Knob-forming parasites were present in all 60 isolates. Although knobless parasites (K-) were found in 25 (42%) of the isolates, only 39 were K-, while 577 were K+. Nine of the 39 K- infected erythrocytes that were studied in greater detail appeared to be asexual parasites because they were either segmented or they lacked mitochondrial DNA-like filaments and cristae, which are abundant in immature gametocytes. No difference was observed in the relative frequency of K+K- infected erythrocytes in isolates from patients with mild or cerebral malaria. Binding of both knobby and knobless infected erythrocytes to autologous leukocytes including monocytes, neutrophils, lymphocytes and plasma cells was found in some of the primary in vitro cultures. By using P. falciparum laboratory strains of known phenotypes and leukocytes from healthy blood bank donors, it was established that this novel adherence phenomenon was related to that of cytoadherence to certain melanoma or endothelial cells. Cytoadherent infected erythrocytes that bind to leukocytes enhance antibody-independent phagocytosis and induce cellular aggregation, while non-cytoadherent or rosetting infected erythrocytes do not.(ABSTRACT TRUNCATED AT 250 WORDS)     

A preliminary report on monoclonal antibodies against human uveal melanoma

Mouse Sp2/10 myeloma cells were fused with spleen cells from mice that had been immunized with freshly obtained primary human uveal melanoma cells. Hybrids that produced antibodies binding to the uveal melanoma cells, but not to fibroblasts, uveal or retinal cells of healthy donors, were cloned. Extensive specificity tests showed that the antibodies produced by the ten clones bound strongly to fresh or short-time cultures of primary human uveal melanoma tumor cells (UMEL-H, UMEL-K). Weaker binding occurred with a human uveal melanoma cell line (VUP-1), and with human skin melanoma cell lines (HMB-2, B-HM8), respectively. Binding assays with carcinoma cells, fibroblasts, uveal and retinal cells were negative. An intensive screening of this type is now under way.     

Heparan sulfate on endothelial cells mediates the binding of Plasmodium falciparum-infected erythrocytes via the DBL1alpha domain of PfEMP1

Plasmodium falciparum may cause severe forms of malaria when excessive sequestration of infected and uninfected erythrocytes occurs in vital organs. The capacity of wild-type isolates of P falciparum-infected erythrocytes (parasitized red blood cells [pRBCs]) to bind glycosaminoglycans (GAGs) such as heparin has been identified as a marker for severe disease. Here we report that pRBCs of the parasite FCR3S1.2 and wild-type clinical isolates from Uganda adhere to heparan sulfate (HS) on endothelial cells. Binding to human umbilical vein endothelial cells (HUVECs) and to human lung endothelial cells (HLECs) was found to be inhibited by HS/heparin or enzymes that remove HS from cell surfaces. (35)S-labeled HS extracted from HUVECs bound directly to the pRBCs' membrane. Using recombinant proteins corresponding to the different domains of P falciparum erythrocyte membrane protein 1 (PfEMP1), we identified Duffy-binding-like domain-1alpha (DBL1alpha) as the ligand for HS. DBL1alpha bound in an HS-dependent way to endothelial cells and blocked the adherence of pRBCs in a dose-dependent manner. (35)S-labeled HS bound to DBL1alpha-columns and eluted as a distinct peak at 0.4 mM NaCl. (35)S-labeled chondroitin sulfate (CS) of HUVECs did not bind to PfEMP1 or to the pRBCs' membrane. Adhesion of pRBCs of FCR3S1.2 to platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, mediated by the cysteine-rich interdomain region 1alpha (CIDR1alpha), was found be operative with, but independent of, the binding to HS. HS and the previously identified HS-like GAG on uninfected erythrocytes may act as coreceptors in endothelial and erythrocyte binding of rosetting parasites, causing excessive sequestration of both pRBCs and RBCs.     

Role of the Plasmodium falciparum mature-parasite-infected erythrocyte surface antigen (MESA/PfEMP-2) in malarial infection of erythrocytes

During intraerythrocytic growth of Plasmodium falciparum, several parasite proteins are transported from the parasite to the erythrocyte membrane, where they bind to membrane skeletal proteins. Mature- parasite-infected erythrocyte surface antigen (MESA) has previously been shown to associate with host erythrocyte membrane skeletal protein 4.1. Using a spontaneous mutant of P falciparum that has lost the ability to synthesize MESA and 4.1-deficient erythrocytes, we examined growth of MESA(+) and MESA(-) parasites in normal and 4.1-deficient erythrocytes. Viability of MESA(+) parasites was reduced in 4.1- deficient erythrocytes as compared with that for normal erythrocytes, but MESA(-) parasites grew equally well in 4.1-deficient and normal erythrocytes. Cytoadherence of MESA(+)- and MESA (-)-parasitized normal and 4.1-deficient erythrocytes to C32 melanoma cells was similar, indicating that neither protein 4.1 nor MESA plays a major role in cytoadherence of infected erythrocytes. Localization of MESA in normal and 4.1-deficient erythrocytes was examined by confocal microscopy. MESA was diffusely distributed in the cytosol of 4.1-deficient erythrocytes but was membrane-associated in normal erythrocytes. These findings suggest that MESA binding to protein 4.1 plays a major role in intraerythrocytic parasite viability.     

Binding of Plasmodium falciparum-infected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates

Platelet-endothelial cell adhesion molecule-1 or CD31 (PECAM-1/CD31) is a receptor recognized by Plasmodium falciparum-parasitized erythrocytes (pRBCs). Fluorescence-labeled soluble recombinant PECAM-1/CD31 (sPECAM-1/CD31) is shown to bind to the surface of P. falciparum-infected erythrocytes on up to 70% of the cells. Binding is blocked by the addition of the unlabeled receptor in a dose-dependent fashion, but not by unrelated receptor-proteins. A significant correlation was found between the binding of sPECAM-1/CD31 to pRBCs and the binding to transfected L cells expressing the receptor as seen with six different P. falciparum lines or clones. Panning of cultures on PECAM-1/CD31 transfected L cells was paralleled by an increase in the binding of sPECAM-1/CD31. The pRBCs of 54% of fresh patient-isolates bound sPECAM-1/CD31 with a mean rate of 12.9% (range = 1.1-44%). The data suggest that PECAM-1/CD31 is a common receptor recognized by wild isolates and that the soluble PECAM-1/CD31 suspension assay is a sensitive and reliable way to study PECAM-1/CD31 binding.     

Cytoadherence of Plasmodium falciparum-infected erythrocytes in the human placenta

In Plasmodium falciparum-parasitized pregnant women, erythrocytes infected by mature stages of the parasite sequester into placental intervillous spaces. The presence of parasites in the placenta causes maternal anaemia and low birth weight of the infant. In-vitro studies suggest placental sequestration may involve the cytoadherence of infected erythrocytes to chondroitin sulphate A (CSA) and/or intercellular adhesion molecule 1 (ICAM-1) expressed by human placental syncytiotrophoblast. We identified P. falciparum receptors expressed on the surface of human syncytiotrophoblast using immunofluorescence of placental biopsies from Cameroon, a malaria-endemic area. In all placentas, a strongly positive staining was observed on the syncytiotrophoblast for CSA, but not for ICAM-1, vascular endothelium cell adhesion molecule-1, E-selectin, nor CD36. The cytoadherence ability of parasites from pregnant women and nonpregnant subjects was assessed on in-vitro cultured syncytiotrophoblast. Parasites from pregnant women bound to the trophoblast via CSA but not ICAM-1. Parasites from nonpregnant hosts either did not bind to the trophoblast culture or bound using ICAM-1. Our data support the idea that placental sequestration may result from cytoadherence to placental trophoblast and that pregnant women are parasitized by parasites that differ from parasites derived from nonpregnant host by their cytoadherence ability.     

Variant antigens and endothelial receptor adhesion in Plasmodium falciparum.

Parasite-derived proteins expressed on the surface of erythrocytes infected with Plasmodium falciparum are important virulence factors, since they mediate binding of infected cells to diverse receptors on vascular endothelium and are targets of a protective immune response. They are difficult to study because they undergo rapid clonal antigenic variation in vitro, which precludes the derivation of phenotypically homogeneous cultures. Here we have utilized sequence-specific proteases to dissect the role of defined antigenic variants in binding to particular receptors. By selection of protease-resistant subpopulations of parasites on defined receptors we (i) confirm the high rate of antigenic variation in vitro; (ii) demonstrate that a single infected erythrocyte can bind to intercellular adhesion molecule 1, CD36, and thrombospondin; (iii) show that binding to intercellular adhesion molecule 1 and CD36 are functions of the variant antigen; and (iv) suggest that binding to thrombospondin may be mediated by other components of the infected erythrocyte surface.     

Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI- 1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell- bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.     

Multiple human serum components act as bridging molecules in rosette formation by Plasmodium falciparum-infected erythrocytes

Rosetting, the binding of parasitized erythrocytes to 2 or more uninfected erythrocytes, is an in vitro correlate of disease severity in Plasmodium falciparum malaria. Although cell ligands and receptors have been identified and a role for immunoglobulin M has been suggested, the molecular mechanisms of rosette formation are unknown. The authors demonstrate unequivocally that rosette formation by P falciparum-infected erythrocytes is specifically dependent on human serum, and they propose that serum components act as bridging molecules between the cell populations. Using heparin treatment and Percoll density gradient centrifugation, they have developed an assay in which parasitized erythrocytes grown in serum-containing medium and optimally forming rosettes are stripped of serum components. These infected cells were no longer able to form rosettes when mixed with erythrocytes and incubated in serum-free medium. Rosette formation was restored by the addition of serum or certain serum fractions obtained by concanavalin A (conA) affinity, anti-IgM affinity, anion exchange, and gel filtration chromatography. The authors clearly demonstrate that multiple serum components-IgM and at least 2 others-are involved in rosette formation. Those others consist of 1 or more acidic components of high-molecular mass that binds to conA (but that is not thrombospondin, fibronectin, or von Willebrand's factor) and of at least 1 more basic, smaller component that does not bind to conA. Data on the size and number of rosettes formed support the authors' hypothesis that multiple bridges are involved in this complex cellular interaction. These findings have important implications for the understanding of pathogenic adhesive interactions of P falciparum and host susceptibility to severe malaria. (Blood. 2000;95:674-682)     

Immunity to malarial antigens on the surface of Plasmodium falciparum-infected erythrocytes

An indirect immunofluorescence test with fresh non-fixed infected blood as antigen was used to show that antibody in human sera from the Gambia recognized antigens on the surface of Plasmodium falciparum-infected human erythrocytes. Surface immunofluorescence was detected on 90% of erythrocytes infected with trophozoites and schizonts produced in continuous culture of isolates from the Gambia (FCR 3/K+), Brazil and Thailand. Fluorescence was equally strong with a Gambian parasite clone (FCR 3/K-) that lacked knobs, an ultrastructural modification of the erythrocyte membrane associated with parasite sequestration. Immunofluorescence could not be detected with an isolate from Uganda. The surface antigenicity of parasitized erythrocytes was eliminated by chymotrypsin and trypsin treatment. Fluorescence was specific for the surface of trophozoite- and schizont-infected cells on the condition that fresh erythrocytes were added to cultures every 4-5 days (subculture); if fresh erythrocytes were not added for over 2 weeks, a large percentage of non-infected erythrocytes also bound antibody. Normal erythrocytes incubated with media from these cultures also gave positive surface immunofluorescence. Thus, there are two types of antigenicity on erythrocytes: one expressed on infected erythrocytes and another passively absorbed from media to normal erythrocytes when parasites are not subcultured for long periods.