Rosetting: a new cytoadherence property of malaria-infected erythrocytes

Plasmodium fragile infection of the toque monkey is a natural host-parasite association in which parasite sequestration occurs as during P. falciparum infection of humans. We have studied parasite sequestration of P. fragile and demonstrated the existence of a new property of cytoadherence of infected erythrocytes, "rosetting," which is defined as the agglutination of uninfected erythrocytes around parasitized erythrocytes. Rosetting in vitro and sequestration in vivo appear simultaneously as the parasite matures. The spleen plays a role in modulating cytoadherence; both sequestration and rosetting, which occur with cloned parasites from spleen-intact animals, are markedly reduced in splenectomized animals infected with parasites derived from the same clone. Sequestration and rosetting can be reversed by immune serum. Protease treatment of infected blood abolishes rosetting; however, if treatment is performed at an early stage of schizogony, rosetting reappears if parasites are allowed to further develop in the absence of protease. These results indicate that with P. fragile in its natural primate host, rosetting and sequestration are related to the presence on the infected erythrocyte surface of a parasite-derived antigenic component, the expression of which is modulated by the spleen.     

Levamisole inhibits sequestration of infected red blood cells in patients with falciparum malaria

BACKGROUND: Sequestration of infected red blood cells (iRBCs) in the microcirculation is central to the pathophysiology of falciparum malaria. It is caused by cytoadhesion of iRBCs to vascular endothelium, mediated through the binding of Plasmodium falciparum erythrocyte membrane protein-1 to several endothelial receptors. Binding to CD36, the major vascular receptor, is stabilized through dephosphorylation of CD36 by an alkaline phosphatase. This is inhibited by the alkaline phosphatase-inhibitor levamisole, resulting in decreased cytoadhesion. METHODS: Patients with uncomplicated falciparum malaria were randomized to receive either quinine treatment alone or treatment with a single 150-mg dose of levamisole as an adjunct to quinine. Peripheral blood parasitemia and parasite stage distribution were monitored closely over time. RESULTS: Compared with those in control subjects, peripheral blood parasitemias of mature P. falciparum parasites increased during the 24 h after levamisole administration (n=21; P=.006). The sequestration ratio (between observed and expected peripheral blood parasitemia) of early trophozoite and midtrophozoite parasites increased after levamisole treatment, with near complete prevention of early trophozoite sequestration and >65% prevention of midtrophozoite sequestration. CONCLUSION: These findings strongly suggest that levamisole decreases iRBC sequestration in falciparum malaria in vivo and should be considered as a potential adjunctive treatment for severe falciparum malaria. TRIAL REGISTRATION: Current Controlled Trials identifier: 15314870.     

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.     

Expression of strain-specific surface antigens on Plasmodium falciparum-infected erythrocytes

Antigenic determinants present on the surface of squirrel monkey erythrocytes infected with late developmental stages of P. falciparum were recognized through their ability to bind antibodies present in the serum of immune monkeys. Two different assays were used to demonstrate the presence of these antigenic determinants on the cell surface: the fluorescent labelling of intact cells in suspensions and the binding to protein A-Sepharose. Such determinants appear to be strain-specific and have, for a given strain, a variable expression according to whether the parasites are taken from intact or splenectomized animals or whether they have been grown in vitro.     

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.     

Factors influencing invasion of erythrocytes by Plasmodium falciparum parasites: the effects of an N-acetyl glucosamine neoglycoprotein and an anti-glycophorin A antibody

When schizont-infected erythrocytes were incubated with N-acetyl glucosamine coupled to bovine serum albumin (GluNAc-BSA), the number of new ring forms which appeared several hours later was reduced and the number of abnormal and unruptured schizont-infected erythrocytes was increased compared with controls, indicating that GluNAc-BSA prevents invasion by a toxic effect on schizonts rather than by receptor blockade. Invasion of erythrocytes by Plasmodium falciparum was inhibited by a monoclonal antibody against glycophorin A, but inhibition also occurred with P. knowlesi, a parasite that is known to invade independently of glycophorin A. Inhibition of invasion with anti-glycophorin A is unlikely to be related to receptor blockade and is probably related to decreased deformability of the erythrocyte membrane caused by the binding of this antibody. Previous studies suggesting that GluNAc-BSA and anti-glycophorin A antibodies inhibit invasion by receptor blockade should be reevaluated. Erythrocytes deficient in glycophorin C and band 4.1 were also resistant to invasion by both P. falciparum and P. knowlesi.     

The distribution and intensity of parasite sequestration in comatose Malawian children

BACKGROUND: The sequestration of Plasmodium falciparum-infected erythrocytes in capillary beds is a characteristic feature of severe malaria and is believed to be central to disease pathogenesis. Sequestration occurs in all P. falciparum infections, including those in asymptomatic individuals. Therefore, sequestration cannot be the sole determinant of severe disease; the intensity or distribution of infected erythrocytes may also contribute. Discerning the relationship between sequestration and well-defined clinical syndromes may enhance understanding of disease mechanisms. METHODS: We measured the concentration of parasite-derived lactate dehydrogenase (pLDH) in tissue samples obtained at autopsy from patients with clinically defined cerebral malaria. On the basis of the autopsy findings, patients were divided into 2 groups: those with an identifiable, nonmalarial cause of death and those without, who were presumed to have died of cerebral malaria. The concentration of pLDH, as determined by enzyme-linked immunosorbent assay, was used to estimate parasite load in different organs. RESULTS: When pLDH could be detected, the parasite load was higher in patients with presumed cerebral malaria than in parasitemic patients with assumed cerebral malaria with a nonmalaria cause of death identified at autopsy (P<.05 for brain, intestine, and skin). CONCLUSIONS: These findings suggest that sequestration in patients with fatal cerebral malaria occurs in multiple organs and does not reflect a predilection in the parasite for the cerebral vasculature.     

Sequestration and microvascular congestion are associated with coma in human cerebral malaria

The pathogenesis of coma in severe Plasmodium falciparum malaria remains poorly understood. Obstruction of the brain microvasculature because of sequestration of parasitized red blood cells (pRBCs) represents one mechanism that could contribute to coma in cerebral malaria. Quantitative postmortem microscopy of brain sections from Vietnamese adults dying of malaria confirmed that sequestration in the cerebral microvasculature was significantly higher in patients with cerebral malaria (CM; n = 21) than in patients with non-CM (n = 23). Sequestration of pRBCs and CM was also significantly associated with increased microvascular congestion by infected and uninfected erythrocytes. Clinicopathological correlation showed that sequestration and congestion were significantly associated with deeper levels of premortem coma and shorter time to death. Microvascular congestion and sequestration were highly correlated as microscopic findings but were independent predictors of a clinical diagnosis of CM. Increased microvascular congestion accompanies coma in CM, associated with parasite sequestration in the cerebral microvasculature.     

Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration

Sequestration of malaria-parasite-infected erythrocytes in the microvasculature of organs is thought to be a significant cause of pathology. Cerebral malaria (CM) is a major complication of Plasmodium falciparum infections, and PfEMP1-mediated sequestration of infected red blood cells has been considered to be the major feature leading to CM-related pathology. We report a system for the real-time in vivo imaging of sequestration using transgenic luciferase-expressing parasites of the rodent malaria parasite Plasmodium berghei. These studies revealed that: (i) as expected, lung tissue is a major site, but, unexpectedly, adipose tissue contributes significantly to sequestration, and (ii) the class II scavenger-receptor CD36 to which PfEMP1 can bind is also the major receptor for P. berghei sequestration, indicating a role for alternative parasite ligands, because orthologues of PfEMP1 are absent from rodent malaria parasites, and, importantly, (iii) cerebral complications still develop in the absence of CD36-mediated sequestration, dissociating parasite sequestration from CM-associated pathology. Real-time in vivo imaging of parasitic processes may be used to evaluate the molecular basis of pathology and develop strategies to prevent pathology.     

Cytoadherence in cerebral malaria as particular example of pathology in host--parasite system

Cerebral malaria in man and in animals is the consequence of a cascade of events, involving the patological changes, leading to the amplification of the expression of the receptors for cytoadherence on brain capillary endothelial cells. Sequestration is the process by which erytrocytes infected with the mature forms of the malaria parasite Plasmodium falciparum disappear from circulation and accumulate within venules and capillaries of various organs and tissues. The molecular mechanism in sequestration is one of the most rapidly advancing fields in malaria research. The several specific aspects considered in this paper. Our electron micrographs show cytoadherence in own model of cerebral malaria in rats infected with Plasmodium berghei.     

Diversity of antigens expressed on the surface of erythrocytes infected with mature Plasmodium falciparum parasites in Papua New Guinea

Antigens were detected on the surface of erythrocytes from children with acute falciparum malaria in Madang, Papua New Guinea. These parasite-induced erythrocyte surface antigens (PIESA) were serotyped with convalescent sera from children and hyperimmune sera from adults in parasite infected cell agglutination assays (PICAs) and by inhibition of binding of infected cells to melanoma cells. Extensive serological diversity of PIESA was demonstrated. A significant correlation between serotypes defined by reactivity of immune sera in PICA and inhibition of melanoma cell binding (MCB) was observed. This suggests that both assays measure antibody responses to the same antigen(s). Increased recognition of different PIESA specificities with age is consistent with the hypothesis that repeated exposure to malaria confers immunity against a range of PIESA serotypes and parallels the development of clinical immunity to malaria in this area of Papua New Guinea.     

Plasmodium chabaudi: a rodent malaria model for in-vivo and in-vitro cytoadherence of malaria parasites in the absence of knobs

The ability of Plasmodium chabaudi infected erythrocytes to bind to endothelial cells in vivo and to various murine cell lines in vitro is described. A procedure for the selective recovery of a sequestering subpopulation of schizont-infected erythrocytes from hepatic sinusoids of BALB/c mice, using a combination of body perfusion, trypsin treatment and Percoll density centrifugation was developed. The serial subinoculation of such a subpopulation was used to select for a clone of parasites (strain AJJ) with considerably better cytoadherent characteristics than the parent line (strain AJ). In contrast, it was demonstrated that another clone (PC7), showed no cytoadherence in vivo or in vitro. This study shows that parasite induced alterations occurred on the surface of erythrocytes infected with late developmental stages of P. chabaudi. The antigenicity of these molecules in the infected mouse was demonstrated using immune serum and affinity chromatography. Cytoadherence and surface antigenic changes in P. chabaudi schizont-infected erythrocytes were demonstrated in the absence of the submembranous 'knobs' associated with cytoadherence in P. falciparum.     

Haemolysis of Plasmodium falciparum trophozoite-infected erythrocytes after artemisinin exposure

This study has examined in vitro , how exposure to the antimalarial drug artemisinin affects Plasmodium falciparum and its host erythrocytes. Factors examined include: cell morphology, intracellular haemoglobin levels, and haemoglobin catabolism (haemozoin production). To avoid uninfected erythrocytes complicating the study, P. falciparum ring-infected erythrocytes were concentrated to 99% parasitaemia, by saponin haemolysis, before the parasites were grown with or without artemisinin. Without artemisinin, the parasites completed their life cycle in the normal time (40 h), during which a mean of 980 pmol of ferriprotoporphyrin IX from haemoglobin was incorporated into haemozoin per 10⁶ parasitized erythrocytes, and intracellular haemoglobin level decreased by 90%. Exposure of ring-infected erythrocytes to artemisinin (250 ng per ml of culture medium) inhibited parasite growth completely, haemozoin production by 95%, and decreased the intraerythrocytic haemoglobin level by 90%; the infected erythrocytes remained intact during the 64 h of study. Haemozoin production was also inhibited when the drug was administered at the trophozoite stage of parasite growth, but the infected erythrocytes haemolysed. These findings may contribute to understanding of antimalarial actions of artemisinin that promote parasite clearance.     

Studies of the receptors on melanoma cells for Plasmodium falciparum infected erythrocytes

We investigated whether thrombospondin plays a role in the binding of Plasmodium falciparum parasitized erythrocytes to C32 melanoma cells. Twelve patient isolates bound variably to melanoma cells, with good correlation between the degree of binding to cells and binding to thrombospondin. With a synchronous preparation of asexual parasites, acquisition of the capacity to bind to thrombospondin occurred at the same parasite stage as binding to melanoma cells. Development of parasites to trophozoites and schizonts correlated with binding of parasitized erythrocytes to thrombospondin and melanoma cells. The infected erythrocyte receptor for thrombospondin was destroyed by mild trypsinization, as was the receptor for melanoma cells. Although these results suggest similarity in the melanoma cell receptor and thrombospondin receptor for infected cells, other results showed that thrombospondin cannot alone be the melanoma cell receptor. Binding to other melanoma cell lines did not correlate with thrombospondin secretion: the RPMI 8252 and G361 cell lines bound few or no infected cells, yet secreted 50-100% as much thrombospondin as C32 cells. Iodinated thrombospondin bound in similar amounts to C32 cells and to noncytoadherent C361 melanoma cells. Binding and nonbinding melanoma cells did not differ in quantity of surface thrombospondin by radioimmunoassay. Thus, although purified, immobilized, thrombospondin binds parasitized erythrocytes, expression of thrombospondin alone on melanoma cells is not sufficient to mediate adherence.     

In vitro and in vivo adaptation of the Geneve/SGE-1 strain of Plasmodium falciparum to growth in a squirrel monkey (Saimiri sciureus) model

Human erythrocytic culture-adapted parasites of the Geneve/SGE-1 strain of Plasmodium falciparum were successfully adapted to grow in an in vitro culture system containing squirrel monkey erythrocytes and serum. These monkey culture-adapted organisms were then used to produce a patent infection in a splenectomized squirrel monkey. Fresh infected blood from this animal was introduced into another splenectomized monkey and was subsequently serially passed through seven intact squirrel monkeys. High level parasitemias (greater than 10%) were obtained in the animals from the last two passes following inoculation of moderate numbers of parasites. It is anticipated that this squirrel monkey-adapted Geneve/SGE-1 strain of P. falciparum will continue to produce high level parasitemias in intact Bolivian Saimiri, and consequently will be suitable for challenge of these monkeys.     

Sequestrin, a CD36 recognition protein on Plasmodium falciparum malaria-infected erythrocytes identified by anti-idiotype antibodies.

The CD36 molecule expressed by human endothelial cells is a receptor for the adhesion of erythrocytes infected with the human malaria parasite Plasmodium falciparum. A CD36-specific monoclonal antibody, OKM8, inhibits the adhesion of malaria-infected erythrocytes (IRBC) to purified CD36 and cells expressing CD36. Monospecific polyclonal anti-idiotype (anti-Id) antibodies, raised against monoclonal antibody OKM8, expressed determinants molecularly mimicking the CD36 binding domain for the adhesion of IRBC. Purified rabbit anti-Id antibodies reacted with the surface of IRBC by immunofluorescence, directly supported the adhesion of wild-type P. falciparum malaria isolates, and inhibited IRBC cytoadherence to melanoma cells. An approximately 270-kDa protein was immunoprecipitated by the anti-Id antibodies from surface-labeled and metabolically labeled IRBC and was competitively inhibited by soluble CD36. These results support the hypothesis that CD36 is a receptor and the approximately 270-kDa protein, sequestrin, is a complementary ligand involved in the adhesion of IRBC to host-cell endothelium. Sequestrin is a candidate malaria vaccine antigen, and anti-Id antibodies that recognize this molecule may be useful for passive immunotherapy of cerebral and severe P. falciparum malaria.     

Calcium and calmodulin antagonists inhibit human malaria parasites (Plasmodium falciparum): implications for drug design

The malaria parasite has an obligate calcium requirement for normal intracellular growth and invasion of host erythrocytes. Calmodulin (CaM) is a vital calcium-dependent protein present in eukaryotes. We found by radioimmunoassay that free parasites contain CaM. Schizont-infected erythrocytes had CaM levels of 23.3 +/- 2.7 ng per 10(6) cells compared to normals (11.2 +/- 1.5 ng per 10(6) cells). CaM levels were proportional to parasite maturity. Immunoelectron microscopy identified CaM diffusely within the cytoplasm of mature parasites and at the apical end of merozoites within the ductule of rhoptries, which may explain the calcium requirement for invasion. Cyclosporin A (CsA) was also found by electron microscopic autoradiography to concentrate in the food vacuole, as do chloroquine and mefloquine, and to distribute within the cytoplasm of mature parasites. The binding of dansylated CsA to schizont-infected erythrocytes was higher than to normal erythrocytes as analyzed by flow cytometry. Kinetic analysis revealed that binding was saturable for normal and infected erythrocytes and possibly free parasites. Competition for binding existed between dansylated CsA and native CsA as well as the CaM inhibitor W-7 and the classic antimalarial chloroquine. The in vitro growth of Plasmodium falciparum was sensitive to CaM antagonists, and in large part inhibition of the parasite was proportional to known anti-CaM potency. Antagonism existed between combinations of these drugs in multi-drug-resistant strains of P. falciparum, suggesting possible competition for the same binding site. In addition, the malaria parasite was also susceptible to calcium antagonists.     

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria

Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria.     

Growth and immunity conferred by a Plasmodium falciparum temperature sensitive mutant in Panamanian owl monkeys

We have compared the growth of the wild type Plasmodium falciparum strain Honduras 1 and a previously isolated temperature sensitive mutant of it, AP1-16, in Panamanian owl monkeys. We examined serially infected splenectomized and normal animals that were initially infected with cultured parasites that had been grown in a mixture of owl monkey and human erythrocytes. Initial infections in splenectomized monkeys were marked by multiple recrudescences. The mutant grew less well than the wild type in the splenectomized monkeys, as determined by lower peak and total parasitemias. In the splenectomized monkeys tested by rechallenge with the wild type parasite, the mutant stimulated a comparable degree of protection. That protection was manifested in 2 ways. There was a marked reduction in the level of the primary parasitemia in the rechallenged monkeys and an absence of recrudescent parasitemias after the primary parasitemia. The potential value of generating and studying temperature sensitive P. falciparum strains that show attenuated growth is considered.     

Uninfected erythrocytes form "rosettes" around Plasmodium falciparum infected erythrocytes

The human malaria parasite, P. falciparum, exhibits cytoadherence properties whereby infected erythrocytes containing mature parasite stages bind to endothelial cells both in vivo and in vitro. Another property of cytoadherence, "rosetting," or the binding of uninfected erythrocytes around an infected erythrocyte, has been demonstrated with a simian malaria parasite P. fragile which is sequestered in vivo in its natural host, Macaca sinica. In the present study we demonstrate that rosetting occurs in P. falciparum. Rosetting in P. falciparum is abolished by protease treatment and reappears on further parasite growth indicating that, as in P. fragile, it is mediated by parasite induced molecules which are protein in nature. P. vivax and P. cynomolgi, which are not sequestered in the host, did not exhibit rosetting. Rosetting thus appears to be a specific property of cytoadherence in malaria parasites.