The mature erythrocyte surface antigen of Plasmodium falciparum is not required for knobs or cytoadherence

Intraerythrocytic Plasmodium falciparum parasites at the trophozoite and schizont stages synthesize a greater than 200-kDa protein, the mature erythrocyte surface antigen (MESA), that is localized at the membrane of infected red blood cells and manifests size polymorphism and antigenic diversity among parasite isolates. Because MESA is localized in the host cell membrane, we examined parasites with differing knob and cytoadherence phenotypes to determine whether MESA expression correlated with knob formation and cytoadherence. A cloned line of P. falciparum that was cultured with repeated selection for the knobbed and cytoadherent phenotypes did not express MESA, due to at least partial deletion of the single-copy MESA gene. In contrast, parasites from the same clone that were cultured without this selection lost the knobbed and cytoadherent phenotypes, but continued to express MESA. These results indicate that MESA is apparently not required for differentiation and multiplication of erythrocyte stage P. falciparum parasites in vitro, or for knob formation and cytoadherence. We speculate that MESA may have a role in evasion of the host immune response by P. falciparum.     

Parasite virulence factors during falciparum malaria: rosetting, cytoadherence, and modulation of cytoadherence by cytokines.

To determine virulence factors of isolates of Plasmodium falciparum and the potential role of cytokines in cerebral malaria, 46 Malagasy patients presenting with cerebral (n = 10), severe (n = 10), and uncomplicated (n = 26) malaria were enrolled in a study. The capacity of 21 of 46 P. falciparum isolates to form rosettes in vitro and to adhere to human umbilical vein endothelial cells (HUVECs) that express intercellular adhesion molecule-1 receptors and to C32 amelanotic melanoma cells that express mainly CD36 receptors was investigated together with the effects of tumor necrosis factor alpha (TNF-alpha), granulocyte macrophage-colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-6 alone and in two-by-two combinations on the cytoadherence of infected erythrocytes to HUVECs. Plasma levels of these cytokines were also measured in the patients at admission. The percentage of rosette formation was higher for the isolates from patients with cerebral (n = 6; 19.5%) and severe (n = 6; 30.5%) malaria than for those from patients with uncomplicated malaria (n = 9; 5%) (P < 0.002). The cytoadherence properties of the isolates did not differ among the three groups whatever the target cell used, but adherence to melanoma cells was systematically higher than that to HUVECs. Adhesion to HUVECs was increased more after TNF-alpha stimulation than after GM-CSF, IL-3, or IL-6 stimulation (P < 0.01). Only the combination of TNF-alpha and IL-3 enhanced cytoadherence more than TNF-alpha used alone (P < 0.02). No difference in the modulation of cytoadherence by cytokines was found in relation to the severity of the disease. TNF-alpha and IL-6 levels in peripheral blood were higher in the patients with cerebral and severe malaria than in the patients with uncomplicated malaria (P < 0.005). Most of the patients' sera contained little or no IL-3 or GM-CSF. Our results challenge the role of intercellular adhesion molecule-1 as the principal receptor mediating the cytoadherence of P. falciparum-infected erythrocytes and contrast with data obtained in the murine model.     

Cytoadherence characteristics of rosette-forming Plasmodium falciparum.

Sequestration of Plasmodium falciparum-infected erythrocytes to the capillary endothelium can cause obstruction and localized tissue damage. Occlusion of vessels in falciparum malaria infection has been related to two properties of the parasite: adhesion to endothelial cells and rosette formation. Our study on P. falciparum isolates from Thailand producing variable numbers of rosettes suggests the involvement of rosettes in capillary blockage caused by direct adhesion of the rosette-forming infected erythrocytes to various target cells, e.g., live human umbilical vein endothelial cells, monocytes, and platelets. These rosettes did not bind Formalin-fixed target cells, nor did they bind to live or fixed C32 or G361 melanoma cells. Classification of the receptors involved in cytoadherence of endothelial cells and monocytes by specific antibody blocking and flow cytometry indicated that CD36 was involved in the adherence of monocytes but that other receptors besides CD36 may be involved in parasite adherence to endothelial cells. The cytoadherence of infected erythrocytes to monocytes was also associated with CD54 (ICAM-1). Further, differentiation of adherent monocytes resulted in an inversion of CD36 and CD54 levels on the cell surface which correlated with a decrease in surface binding of infected erythrocytes. This observation suggests that the state of cell activation and differentiation may also contribute to sequestration of parasites and to the pathogenesis of malaria.     

Rosette formation of Plasmodium falciparum-infected erythrocytes from patients with acute malaria.

Noninfected erythrocytes form rosettes around those infected with trophozoites and schizonts of Plasmodium falciparum in vitro. These rosettes are thought to contribute to the microvascular obstruction which underlies the pathophysiology of severe falciparum malaria. To determine whether the percentage of infected erythrocytes forming rosettes for a parasite isolates in vitro correlates with the in vivo severity of disease, we studied the rosette formation behavior of 35 isolates of P. falciparum from patients with uncomplicated, severe, and cerebral malaria. There was a wide variation in the degree of rosette formation (0 to 53%). Four parasite isolates formed rosettes well (30 to 53%), and seven isolates formed rosettes poorly or not at all (0 to 5%), while the majority of the isolates formed rosettes to various degrees between these two extremes. In this relatively small sample of patients, we were unable to demonstrate a significant association between in vitro rosette formation and patients with cerebral malaria or conscious patients with significant renal (serum creatinine greater than 200 mumol/liter) or hepatic dysfunction (serum bilirubin greater than 50 mumol/liter and aspartate aminotransferase greater than 50 Reitman-Frankel units). However, there was an inverse relationship between rosette formation and cytoadherence (r = -0.575, P less than 0.01) which could not be explained on the basis of steric hindrance. This finding suggests that cytoadherence and rosette formation properties are intrinsic to the parasites, with isolates having a greater propensity for one or the other but not both. Further studies are required to establish the occurrence and pathophysiological role of rosette formation in vivo.     

Plasmodium falciparum: inhibition/reversal of cytoadherence of Thai isolates to melanoma cells by local immune sera.

The effect of sera on the cytoadherence in vitro of Plasmodium falciparum-infected erythrocytes to melanoma cells was examined. Sera from 19 healthy individuals living in endemic malarious areas in Thailand and 24 patients with P. falciparum malaria were tested against four local P. falciparum isolates. Out of 57 sera examined, 12 (21%) showed significant inhibition (greater than 50%) of cytoadherence for at least one isolate. Anti-malarial IgG antibody titres were determined for all 57 sera and although 11 of the 12 inhibitory sera had relatively high titres, 36 out of 47 sera with similarly high titres showed no significant inhibitory activity. Convalescent sera were no more effective than corresponding acute sera in inhibiting the cytoadherence of erythrocytes infected with any of the four heterologous isolates examined. Sera which significantly inhibited cytoadherence were also capable of reversing cytoadherence, and pooled plasma, from healthy individuals living in malarious areas, was effective in significantly reversing the in vitro cytoadherence of all the five parasite isolates examined. The results confirm the antibody mediated strain-specific nature of the inhibition of cytoadherence and stress the difficulty in selecting immune sera potentially useful for the immunotherapy of cerebral malaria patients in Thailand.     

Cytoadherence and ultrastructure of Plasmodium falciparum-infected erythrocytes from a splenectomized patient.

In malarial infections of primates, the spleen has been shown to modulate parasite antigen expression on the surfaces of infected erythrocytes. The processes affected include cytoadherence, which is central to the pathophysiology of severe falciparum malaria, and the related phenomenon of rosette formation. In this study, the cytoadherence and rosette formation behaviors of Plasmodium falciparum-infected erythrocytes from a splenectomized patient were examined during the first erythrocytic cycle in vitro. Ultrastructural studies were also performed. Infected erythrocytes were found to cytoadhere to C32 melanoma cells via leukocyte differentiation antigen CD36 but not intercellular adhesion molecule 1. They also displayed on their surfaces electron-dense knobs similar in structure and density to those on infected erythrocytes from intact hosts. These findings may reflect a stable cytoadherent phenotype of the parasite isolate that is unaffected by the absence of the spleen. Alternatively, the modulating role of the spleen may have been assumed by other organs of the mononuclear phagocytic system in a previously infected individual. No rosette formation was observed, but as not all natural isolates form rosettes, this observation may or may not be related to the asplenic status of the patient. Parasite and host factors appear to be important in determining the effect of splenectomy on cytoadherence and rosette formation in human falciparum malaria.     

A quantitative analysis of the microvascular sequestration of malaria parasites in the human brain.

Microvascular sequestration was assessed in the brains of 50 Thai and Vietnamese patients who died from severe malaria (Plasmodium falciparum, 49; P. vivax, 1). Malaria parasites were sequestered in 46 cases; in 3 intravascular malaria pigment but no parasites were evident; and in the P. vivax case there was no sequestration. Cerebrovascular endothelial expression of the putative cytoadherence receptors ICAM-1, VCAM-1, E-selectin, and chondroitin sulfate and also HLA class II was increased. The median (range) ratio of cerebral to peripheral blood parasitemia was 40 (1.8 to 1500). Within the same brain different vessels had discrete but different populations of parasites, indicating that the adhesion characteristics of cerebrovascular endothelium change asynchronously during malaria and also that significant recirculation of parasitized erythrocytes following sequestration is unlikely. The median (range) ratio of schizonts to trophozoites (0.15:1; 0.0 to 11.7) was significantly lower than predicted from the parasite life cycle (P < 0.001). Antimalarial treatment arrests development at the trophozoite stages which remain sequestered in the brain. There were significantly more ring form parasites (age < 26 hours) in the cerebral microvasculature (median range: 19%; 0-90%) than expected from free mixing of these cells in the systemic circulation (median range ring parasitemia: 1.8%; 0-36.2%). All developmental stages of P. falciparum are sequestered in the brain in severe malaria.     

Intravenous immunoglobulin in the treatment of paediatric cerebral malaria.

Hyperimmune globulin can inhibit and reverse the cytoadherence between Plasmodium falciparum-infected erythrocytes and melanoma cells in vitro. Cytoadherence is believed to mediate disease in cerebral malaria. Therefore we studied the efficacy of i.v. immunoglobulin, purified from the plasma of local semi-immune blood donors, as an adjunct to standard treatment for cerebral malaria in Malawian children. The immunoglobulin preparation (IFAT antimalarial antibody titre 1:5120) recognized erythrocyte-associated antigens of each of 22 Malawian P. falciparum isolates studied, and reversed binding of Malawian isolates to melanoma cells. Immunoglobulin did not reverse binding to human monocytes or to cells of the human histiocytic lymphoma cell line U937. Thirty-one children with P. falciparum parasitaemia and unrousable coma were enrolled. All were treated with i.v. quinine dihydrochloride; in addition patients were randomized to receive either immunoglobulin (400 mg/kg by i.v. infusion over 3 h) or placebo (albumen and sucrose by similar infusion) in a double blind trial with sequential analysis. Of 16 patients receiving immunoglobulin, five (31%) died and five survivors had neurological sequelae. Of 15 patients receiving placebo, one (7%) died and two had sequelae. Parasite clearance, fever clearance and coma resolution times in survivors were similar in the two groups. Although the difference in outcome between the two groups was not significant, the trial was stopped because immunoglobulin was demonstrated not to be superior to placebo.     

Protection by alpha-thalassaemia against Plasmodium falciparum malaria: modified surface antigen expression rather than impaired growth or cytoadherence.

We have attempted to determine the cellular mechanism by which alpha-thalassaemia may protect against Plasmodium falciparum malaria. Invasion and development of P. falciparum in the microcytic red cells of two-gene deletion forms of alpha-thalassaemia when measured morphologically or by [3H]hypoxanthine incorporation were normal compared to controls. Normal invasion rates were also observed following schizogony in thalassaemic red cells. Neither the addition of the oxidant menadione, 30% oxygen, nor modified medium, produced differential damage to parasites within thalassaemic cells. Furthermore, there were no significant differences in the binding of P. falciparum-parasitized alpha-thalassaemic and normal cells to C32 melanoma cells in vitro. However, when neoantigen expression on the surface of infected thalassaemic cells was estimated using a quantitative radiometric antiglobulin assay, clear differences were observed. It was found that alpha-thalassaemic cells bound higher levels of antibody from serum obtained from individuals living in a malaria endemic area than control normal red cells. The binding ratio for thalassaemic compared with controls was 1.69 on a cell-for-cell basis, and 1.97 when related to surface area. The binding of antibody from immune serum increased exponentially during parasite maturation. We also found increased binding of naturally occurring antibody present in non-immune serum to parasitized thalassaemic red cells which also increased during parasite maturation. We conclude that the protection afforded by thalassaemia against malaria may not reside in the ability of parasites to enter, grow or cytoadhere to endothelium in such cells, but may be related to immune recognition and subsequent clearance of parasitized red cells.     

Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA

We have previously shown by targeted gene disruption that the clag9 gene of Plasmodium falciparum is essential for cytoadherence to CD36. Here we report inhibition of the function of clag9 by the use of an antisense RNA vector as an alternative to targeted gene disruption. We transfected an antisense construct of clag9 into the P. falciparum clone 3D7 and when the resulting line was cultured in the presence of pyrimethamine it showed 15-fold lower cytoadherence to C32 melanoma cells than the control. Reversion to wildtype upon removal of the introduced plasmid provides direct evidence that the event responsible for the phenotypic change is not at an unrelated site and this approach provides a valuable new tool in malaria transfection technology.     

Protection against severe clinical manifestations of Plasmodium falciparum malaria among sickle cell trait subjects is due to modification of the release of cytokines and/or cytoadherence of infected erythrocytes to the host vascular beds

It is proposed that the surface ligands of Plasmodium falciparum infected HbAS erythrocytes, not like infected HbAA erythrocytes, are altered due to the sickling that soon takes place once a HbAS erythrocyte gets infected with P. falciparum parasite. This alteration modulates cytoadherence and/or binding of the sickled erythrocytes to the peripheral blood mononuclear cells (PBMCs). Both cytoadherence and binding to PBMCs are responsible for the pathogenesis of malaria. Therefore, subjects of the HbAS genotype experience mild symptoms of malaria. The hypothesis could be tested in vitro by comparing the binding of P. falciparum infected HbAS and HbAA erythrocytes to platelet-endothelial cell adhesion molecule-1 (CD31) and by comparing the levels of tumor necrosis factor (TNF) and interferon gamma (IFN-gamma) following in vitro stimulation of PBMCs by HbAS and HbAA infected erythrocytes.     

Strain variation in tumor necrosis factor induction by parasites from children with acute falciparum malaria.

A small proportion of individuals infected with Plasmodium falciparum develop cerebral malaria. Why it affects some infected individuals but not others is poorly understood. Since tumor necrosis factor (TNF) has been implicated strongly in the pathogenesis of cerebral malaria, here we have compared different parasite isolates for their ability to induce TNF production by human mononuclear cells in vitro. Wild isolates were collected from 34 Gambian children with cerebral malaria and 66 children with uncomplicated malaria fever. Cerebral malaria isolates tended to stimulate more TNF production than mild malaria isolates, but there was considerable overlap between the two groups, and the present data provide only limited support for the hypothesis that cerebral malaria is caused by strains of P. falciparum inducing high levels of TNF. However, it is notable that the amounts of TNF induced by different wild isolates from a single locality differed by over 100-fold. The biological significance of this polymorphism deserves further scrutiny in view of the central role that TNF is believed to play in host defense and in the clinical symptomatology of human malaria.     

Cytoadherence of Plasmodium berghei-Infected Red Blood Cells to Murine Brain and Lung Microvascular Endothelial Cells In Vitro

Sequestration of infected red blood cells (iRBC) within the cerebral and pulmonary microvasculature is a hallmark of human cerebral malaria (hCM). The interaction between iRBC and the endothelium in hCM has been studied extensively and is linked to the severity of malaria. Experimental CM (eCM) caused by Plasmodium berghei ANKA reproduces most features of hCM, although the sequestration of RBC infected by P. berghei ANKA (PbA-iRBC) has not been completely delineated. The role of PbA-iRBC sequestration in the severity of eCM is not well characterized. Using static and flow cytoadherence assays, we provide the first direct in vitro evidence for the binding of PbA-iRBC to murine brain and lung microvascular endothelial cells (MVEC). We found that basal PbA-iRBC cytoadherence to MVECs was significantly higher than that of normal red blood cells (NRBC) and of RBC infected with P. berghei K173 (PbK173-iRBC), a strain that causes noncerebral malaria (NCM). MVEC prestimulation with tumor necrosis factor (TNF) failed to promote any further significant increase in mixed-stage iRBC adherence. Interestingly, enrichment of the blood for mature parasites significantly increased PbA-iRBC binding to the MVECs prestimulated with TNF, while blockade of VCAM-1 reduced this adhesion. Our study provides evidence for the firm, flow-resistant binding to endothelial cells of iRBC from strain ANKA-infected mice, which develop CM, and for less binding of iRBC from strain K173-infected mice, which develop NCM. An understanding of P. berghei cytoadherence may help elucidate the importance of sequestration in the development of CM and aid the development of antibinding therapies to help reduce the burden of this syndrome.     

Expression of the histidine-rich protein PfHRP1 by knob-positive Plasmodium falciparum is not sufficient for cytoadherence of infected erythrocytes.

Six culture-adapted knob-positive Plasmodium falciparum parasites, four of which were nonbinding in an in vitro cytoadherence assay, were tested for the presence of the knob-associated histidine-rich protein PfHRP1. Two knob-positive, strongly cytoadherence-positive P. falciparum strains from Aotus monkeys were also examined. All parasites expressed PfHRP1, suggesting that it plays a structural or functional role related to knobs but is not by itself sufficient for cytoadherence of P. falciparum-infected erythrocytes.     

Host vascular endothelial growth factor is trophic for Plasmodium falciparum-infected red blood cells

Plasmodium falciparum, the protozoan parasite responsible for severe malaria infection, undergoes a complex life cycle. Infected red blood cells (iRBC) sequester in host cerebral microvessels, which underlies the pathology of cerebral malaria. Using immunohistochemistry on post mortem brain samples, we demonstrated positive staining for vascular endothelial growth factor (VEGF) on iRBC. Confocal microscopy of cultured iRBC revealed accumulation of VEGF within the parasitophorous vacuole, expression of host VEGF-receptor 1 and activated VEGF-receptor 2 on the surface of iRBC, but no accumulation of VEGF receptors within the iRBC. Addition of VEGF to parasite cultures had a trophic effect on parasite growth and also partially rescued growth of drug treated parasites. Both these effects were abrogated when parasites were grown in serum-free medium, suggesting a requirement for soluble VEGF receptor. We conclude that P. falciparum iRBC can bind host VEGF-R on the erythrocyte membrane and accumulate host VEGF within the parasitophorous vacuole, which may have a trophic effect on parasite growth.     

Cytoadherence of Babesia bovis-Infected Erythrocytes to Bovine Brain Capillary Endothelial Cells Provides an In Vitro Model for Sequestration

Babesia bovis, an intraerythrocytic parasite of cattle, is sequestered in the host microvasculature, a behavior associated with cerebral and vascular complications of this disease. Despite the importance of this behavior to disease etiology, the underlying mechanisms have not yet been investigated. To study the components involved in sequestration, B. bovis parasites that induce adhesion of the infected erythrocytes (IRBCs) to bovine brain capillary endothelial cells (BBEC) in vitro were isolated. Two clonal lines, CD7(A+I+) and CE11(A+I-), were derived from a cytoadherent, monoclonal antibody 4D9.1G1-reactive parasite population. This antibody recognizes a variant, surface-exposed epitope of the variant erythrocyte surface antigen 1 (VESA1) of B. bovis IRBCs. Both clonal lines were cytoadhesive to BBEC and two other bovine endothelial cell lines but not to COS7 cells, FBK-4 cells, C32 melanoma cells, or bovine brain pericytes. By transmission electron microscopy, IRBCs were observed to bind to BBEC via the knobby protrusions on the IRBC surface, indicating involvement of components associated with these structures. Inhibition of protein export in intact, trypsinized IRBCs ablated both erythrocyte surface reexpression of parasite protein and cytoadhesion. IRBCs allowed to recover surface antigen expression regained the ability to bind endothelial cells, demonstrating that parasite protein export is required for cytoadhesion. We propose the use of this assay as an in vitro model to study the components involved in B. bovis cytoadherence and sequestration.     

Transient supplementation of superoxide dismutase protects endothelial cells against Plasmodium falciparum-induced oxidative stress

The pathogenesis of cerebral malaria, a major complication of Plasmodium falciparum infection, relies on mechanisms such as cytokine production and cytoadherence of parasitized red blood cells (PRBCs) on microvascular endothelial cells. In this way parasites avoid spleen clearance by sequestration in post-capillary venules of various organs including the brain. Infected erythrocytes adhesion has also been shown to have molecular signaling consequences providing insight on how tissue homeostasis could be comprised by endothelium perturbation. Our previous work demonstrated that PRBCs adhesion to human lung endothelial cells (HLEC) induces caspases activation, oxidative stress and apoptosis. Cytoplasmic Cu/Zn superoxide dismutase (SOD1), which provides the first line of defense against oxidative stress within a cell, is now used as a treatment of numerous diseases including traumatic brain injury and ischemic stroke. In this report, we demonstrated that transient supplementation of SOD1 protects endothelial cells against P. falciparum induced oxidative stress and apoptosis. We also showed a significant decrease in PRBCs cytoadherence through a downregulation of ICAM-1 and an induction of iNOS. Protection of endothelium via antioxidant delivery may constitute a relevant strategy in cerebral malaria treatment.     

Specific antibody-dependent cellular cytotoxicity in human malaria.

A micromethod for the study of specific antibody-dependent cellular cytotoxicity (ADCC) in human malaria is described, using cultured, asexual Plasmodium falciparum parasites as viable target cells. Lymphocytes from children with acute malaria, uninfected immune adult Gambians and adult Gambians infected with P. falciparum were capable of killing P. falciparum in vitro in the presence of malaria antibody. A parasite growth-promoting factor, produced by lymphocytes in non-immune serum and at a lymphocyte--parasite ratio of 10:1, in immune serum, was found to produce three-fold increases in growth of P. falciparum. The mechanisms by which ADCC may occur are also discussed.     

Complement factor D, albumin, and immunoglobulin G anti-band 3 protein antibodies mimic serum in promoting rosetting of malaria-infected red blood cells

Rosetting of Plasmodium falciparum-infected red blood cells (parasitized RBC [pRBC]) with uninfected RBC has been associated in many studies with malaria morbidity and is one form of cytoadherence observed with malarial parasites. Rosetting is serum dependent for many isolates of P. falciparum, including the strains FCR3S1.2 and Malayan Camp studied here. We identified the three naturally occurring components of sera which confer rosetting. Complement factor D alone induced 30 to 40% of de novo rosetting. Its effect was additive to that of 0.5 mg/ml albumin and to that of 15 ng/ml of naturally occurring antibodies to the anion transport protein, band 3. The three components together mediated rosetting as effectively as 10% serum. De novo rosetting experiments showed that naturally occurring anti-band 3 antibodies as well as factor D were effective only when added to pRBC. Factor D appeared to cleave a small fraction of a protein expressed on the surface of pRBC.     

A model for sequestration of the transmission stages of Plasmodium falciparum: adhesion of gametocyte-infected erythrocytes to human bone marrow cells

With the aim of developing an appropriate in vitro model of the sequestration of developing Plasmodium falciparum sexual-stage parasites, we have investigated the cytoadherence of gametocytes to human bone marrow cells of stromal and endothelial origin. Developing stage III and IV gametocytes, but not mature stage V gametocytes, adhere to bone marrow cells in significantly higher densities than do asexual-stage parasites, although these adhesion densities are severalfold lower than those encountered in classical CD36-dependent assays of P. falciparum cytoadherence. This implies that developing gametocytes undergo a transition from high-avidity, CD36-mediated adhesion during stages I and II to a lower-avidity adhesion during stages III and IV. We show that this adhesion is CD36 independent, fixation sensitive, stimulated by tumor necrosis factor alpha, and dependent on divalent cations and serum components. These data suggest that gametocytes and asexual parasites utilize distinct sets of receptors for adhesion during development in their respective sequestered niches. To identify receptors for gametocyte-specific adhesion of infected erythrocytes to bone marrow cells, we tested a large panel of antibodies for the ability to inhibit cytoadherence. Our results implicate ICAM-1, CD49c, CD166, and CD164 as candidate bone marrow cell receptors for gametocyte adhesion.