The resistance to physiological shear stresses of the erythrocytic rosettes formed by cells infected with Plasmodium falciparum

Rosetting forces are believed to be an important contributor to the microcirculatory obstruction that occurs in malaria caused by Plasmodium falciparum. In this study, rosettes of erythrocytes from cultures of this parasite were suspended in different media and exposed to shear stresses corresponding to those encountered on the arterial and venous sides of the human circulation. The rosettes formed by infected erythrocytes in malaria culture medium containing 10% AB serum were disrupted easily (approximately 50% being broken) when exposed to very low shear stresses of < 0.5 Pa. However, use of higher concentrations of serum strengthened the rosetting binding forces considerably. Suspension of rosettes in a viscous colloid (e.g. dextran) increased the adherence forces between infected and uninfected red cells. The results indicate that rosettes do resist the physiological shear forces that are encountered in the venular side of the circulation and could thus contribute to microvascular obstruction in falciparum malaria.     

Erythrocytapheresis and sublingual micro-vascular flow in severe malaria

The authors describe clinical cases of two patients admitted to intensive care unit following severe Plasmodium falciparum malaria. Patients' sublingual microcirculation was monitored by on-line Sidestream dark-field imaging before and after treatment with erythrocytapheresis. Before treatment, microcirculatory flow alterations were obvious. Therapy produced a rapid decrease in infected red blood cells with a significant improvement in microcirculatory flows, capillary perfusion and patients' outcome. The present cases emphasize the relevance of microcirculation monitoring in patients with capillary perfusion alterations resulting from severe malaria. As far as we know, this is the first observations of an improvement of capillary perfusion after erythrocytapheresis.     

Effects of malaria heme products on red blood cell deformability

In falciparum malaria, the deformability of the entire erythrocyte population is reduced in proportion to disease severity, and this compromises microcirculatory blood flow through vessels partially obstructed by cytoadherent parasitized erythrocytes. The cause of rigidity of uninfected erythrocytes in not known but could be mediated by malaria heme products. In this study, we show that red blood cell deformability (RBC-D), measured by laser-assisted optical rotational cell analyzer, decreased in a dose-dependent manner after incubation with hemin and hydrogen peroxide but not with hemoglobin or beta-hematin. Hemin also reduced mean red cell volume. Albumin decreased and N-acetylcysteine (NAC) both prevented and reversed rigidity induced by hemin. Hemin-induced oxidative damage of the membrane seems to be a more important contributor to pathology than cell shrinkage because the antioxidant NAC restored RBC-D but not red blood cell volume. The findings suggest novel approaches to the treatment of potentially lethal malaria.     

Direct in vivo assessment of microcirculatory dysfunction in severe falciparum malaria

BACKGROUND: This study sought to describe and quantify microcirculatory changes in the mucosal surfaces of patients with severe malaria, by direct in vivo observation using orthogonal polarization spectral (OPS) imaging. METHODS: The microcirculation in the rectal mucosa of adult patients with severe malaria was assessed by use of OPS imaging, at admission and then daily. Comparison groups comprised patients with uncomplicated falciparum malaria, patients with bacterial sepsis, and healthy individuals. RESULTS: Erythrocyte velocities were measured directly in 43 adult patients with severe falciparum malaria, of whom 20 died. Microcirculatory blood flow was markedly disturbed, with heterogeneous obstruction that was proportional to severity of disease. Blocked capillaries were found in 29 patients (67%) and were associated with concurrent hyperdynamic blood flow (erythrocyte velocity, >750 mm/s) in adjacent vessels in 27 patients (93%). The proportion of blocked capillaries correlated with the base deficit in plasma and with the concentration of lactate. Abnormalities disappeared when the patients recovered. In healthy individuals and in patients with uncomplicated malaria or sepsis, no stagnant erythrocytes were detected, and, in patients with sepsis, hyperdynamic blood flow was prominent. CONCLUSION: Patients with severe falciparum malaria show extensive microvascular obstruction that is proportional to the severity of the disease. This finding underscores the prominent role that microvascular obstruction plays in the pathophysiology of severe malaria and illustrates the fundamental difference between the microvascular pathophysiology of malaria and that of bacterial sepsis.     

Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium falciparum-infected erythrocytes.

We have studied the pathophysiology of the vascular obstruction induced by Plasmodium falciparum-parasitized erythrocytes with the use of an ex vivo microcirculatory preparation perfused with red cells infected with knobless and knobby clones of the FCR-3 strain. We find that parasitized erythrocyte membrane knobs are indispensable for the generation of the circulatory obstruction. Uninfected erythrocytes incubated in culture and erythrocytes infected with early or late forms of the knobless clones or the early forms of the knobby clone all failed to obstruct the microcirculation, although exhibiting various effects on bulk viscosity and peripheral resistance during flow. In contrast, late forms of the knobby clone produced significantly higher peripheral resistance during flow and significant obstruction as detected by changes in time of pressure flow recovery as well as by direct videorecorded microscopic observation. Optical and electron microscopy showed that the adherence of parasitized cells to the endothelium was limited to the venules and involved the knobs in junctions. In addition, we were able to follow the sequence of events during obstruction: initial red-cell adherence to the venular endothelium (sometimes only transitory) followed by progressive recruitment at the venule surface, finally leading to total obstruction that involved parasitized and nonparasitized erythrocytes. Sometimes, retrograde aggregation would extend the obstruction to the capillaries or even precapillary arterioles. These results show that knobs are necessary and sufficient to produce vascular obstruction and that other factors (spleen, immunological, etc.) can only have a modulating role. These results also exclude the possibility that the exclusive adherence to venules is the consequence of "plasma factors" found in the malaric patients.     

The microcirculation in severe malaria

Severe malaria in humans and animals is initiated by interactions between malaria-infected cells, host blood cells (including monocytes, T cells and platelets) and endothelial cells of the microcirculation. Adhesion to vascular cells, and possible vascular obstruction in severe human disease, involves interaction between host receptors and parasite-derived proteins, such as the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Our understanding of how different PfEMP1 variants may target infected erythrocytes to specific sites, such as the placenta, is rapidly increasing. However, in most instances downstream immune-mediated inflammatory processes appear more central than parasite accumulation to development of severe malaria. Using genetically-manipulated animal models of severe malaria, key roles for CD8 T cells and mediators such as lymphotoxin in the pathogenesis of murine disease have been established. Experimental and human studies suggest vascular deposition of activated platelets may have a central role. Here, we review some recent advances in the understanding of severe malaria pathogenesis from human and animal studies, focusing on events at the level of the microcirculation, and highlight the role for activated host cells in initiating the pathology of the disease.     

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.     

Uninfected erythrocytes inhibit Plasmodium falciparum-induced cellular immune responses in whole-blood assays

Whole-blood assays (WBAs) have been successfully used as a simple tool for immuno-epidemiological field studies evaluating cellular immune responses to mycobacterial and viral antigens. Rather unexpectedly, we found very poor cytokine responses to malaria antigens in WBAs in 2 immuno-epidemiological studies carried out in malaria endemic populations in Africa. We have therefore conducted a detailed comparison of cellular immune responses to live (intact) and lysed malaria-infected erythrocytes in WBAs and in peripheral blood mononuclear cell (PBMC) cultures. We observed profound inhibition of both proliferative and interferon-gamma responses to malarial antigens in WBAs as compared with PBMC cultures. This inhibition was seen only for malaria antigens and could not be overcome by increasing either antigen concentration or responder cell numbers. Inhibition was mediated by intact erythrocytes and occurred early in the culture period, suggesting that failure of antigen uptake might underlie the lack of T-cell responses. In support of this hypothesis, we have shown that intact uninfected erythrocytes specifically inhibit phagocytosis of infected red blood cells by peripheral blood monocytes. We propose that specific biochemical interactions with uninfected erythrocytes inhibit the phagocytosis of malaria-infected erythrocytes and that this may impede T-cell recognition in vivo.     

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.     

Glycolysis in Plasmodium falciparum results in modulation of host enzyme activities

BACKGROUND & OBJECTIVES: Plasmodium falciparum, the causative agent of the most serious form of malaria, infects about 5-10% of the world human population per year. It is well established that the erythrocytic stages of the malaria parasite rely mainly on glycolysis for their energy supply. In the present study, the glucose utilisation of erythrocyte population with parasitaemia levels similar to that of malaria patients was measured. The results allowed us to assess the effect of the parasites on the glucose utilisation of the vast majority of uninfected erythrocytes. METHODS: Using [2-13C]glucose and nuclear magnetic resonance (NMR) technique, the glucose utilisation in normal red blood cell (RBC) and P. falciparum infected red blood cell (IRBC) populations was measured. The IRBC population consisted of > 96% RBC and < 4% of parasite infected red blood cells (PRBC). The glycolytic enzymes were assayed to assess the effect of infected red cells on the enzymatic activities of uninfected ones. RESULTS: The rate of glucose utilisation by IRBC was considerably higher than that of RBC. Upon addition of 25% v/v conditioned culture medium (CM) of IRBC, RBCs exhibited a significant decrease in glucose utilisation. The CM could directly inhibit the activities of RBC glycolytic enzymes-phosphofructokinase (PFK) and pyruvate kinase (PK), without interfering with the activity of the pentose phosphate pathway enzyme-glucose-6-phosphate dehydrogenase (G-6-PD). INTERPRETATION & CONCLUSION: The present study showed that the clinical level of P. falciparum infected RBCs (< 4% parasitaemia) significantly enhance the glycolytic flux as well as down-regulate the glucose utilisation rate in the majority of uninfected RBC population. The mechanism of inhibition seems to be direct inhibition of the regulatory glycolytic enzymes-PFK and PK.     

A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes

Severe malaria by Plasmodium falciparum is a potentially fatal disease, frequently unresponsive to even the most aggressive treatments. Host organ failure is associated with acquired rigidity of infected red blood cells and capillary blockage. In vitro techniques have played an important role in modeling cell deformability. Although, historically they have either been applied to bulk cell populations or to measure single physical parameters of individual cells. In this article, we demonstrate the unique abilities and benefits of elastomeric microchannels to characterize complex behaviors of single cells, under flow, in multicellular capillary blockages. Channels of 8-, 6-, 4-, and 2-microm widths were readily traversed by the 8 microm-wide, highly elastic, uninfected red blood cells, as well as by infected cells in the early ring stages. Trophozoite stages failed to freely traverse 2- to 4-microm channels; some that passed through the 4-microm channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-microm channels, trophozoites mimicked "pitting," a normal process in the body where spleen beds remove parasites without destroying the red cell. Schizont forms failed to traverse even 6-microm channels and rapidly formed a capillary blockage. Interestingly, individual uninfected red blood cells readily squeezed through the blockages formed by immobile schizonts in a 6-microm capillary. The last observation can explain the high parasitemia in a growing capillary blockage and the well known benefits of early blood transfusion in severe malaria.     

Nonimmune IgM,but not IgG binds to the surface of Plasmodium falciparum-infected erythrocytes and correlates with rosetting and severe malaria

Recent work suggests that IgG and IgM from nonimmune human serum (natural antibodies) bind to the surface of Plasmodium falciparum-infected erythrocytes and contribute to rosette formation by stabilizing the interaction between infected and uninfected erythrocytes. Here we show, in both laboratory clones and field isolates, that only IgM but not IgG is detected on the surface of infected cells. In field isolates, there was a strong positive correlation between IgM binding and rosette formation (Spearman's rank correlation coefficient p = 0.804, P < 0.001). Both rosette formation and IgM binding were associated with severe malaria, although statistical analysis indicates that rosette formation is the more strongly associated variable. Rosette formation, but not IgM binding, was also associated with malarial anemia. We conclude that IgM is the predominant class of natural antibodies binding to the surface of infected erythrocytes. However, we could not confirm previous suggestions that infected erythrocytes are coated with nonimmune IgG, which could lead to their interaction with host Fcgamma receptors.     

Red blood cell deformability as a predictor of anemia in severe falciparum malaria.

Decreased erythropoiesis and increased clearance of both parasitized and noninfected erythrocytes both contribute to the pathogenesis of anemia in falciparum malaria. Erythrocytes with reduced deformability are more likely to be cleared from the circulation by the spleen, a process that is augmented in acute malaria. Using a laser diffraction technique, we measured red blood cell (RBC) deformability over a range of shear stresses and related this to the severity of anemia in 36 adults with severe falciparum malaria. The RBC deformability at a high shear stress of 30 Pa, similar to that encountered in the splenic sinusoids, showed a significant positive correlation with the nadir in hemoglobin concentration during hospitalization (r = 0.49, P < 0.002). Exclusion of five patients with microcytic anemia strengthened this relationship (r = 0.64, P < 0.001). Reduction in RBC deformability resulted mainly from changes in unparasitized erythrocytes. Reduced deformability of uninfected erythrocytes at high shear stresses and subsequent splenic removal of these cells may be an important contributor to the anemia of severe malaria.     

The role of rosetting in the multiplication of Plasmodium falciparum: rosette formation neither enhances nor targets parasite invasion into uninfected red cells

The effect of rosette formation on the multiplication in vitro of Plasmodium falciparum was studied in order to establish whether rosetting acts as a major virulence factor in the pathogenesis of severe malaria by facilitating invasion of uninfected red cells. Invasion rates for rosetting (R⁺) and non-rosetting (R⁻) parasites selected from the same clone, PA1, of P. falciparum were similar over a range of starting parasite concentrations when assayed in both static cultures and conditions of shear stress comparable with microvascular flow. However, incubation of both R⁺ and R⁻ parasites under simulated conditions of flow led to decreased invasion and fewer multiply-infected red cells as we have previously observed. Studies using fluorescently labelled red cells or reticulocytes demonstrated that rosetting did not alter the rates of invasion or target merozoites into the uninfected cells comprising a rosette. Preferential invasion of reticulocytes occurred regardless of rosetting or conditions of flow. Although the role of rosetting in the pathogenesis of malaria might relate to microvascular obstruction or perhaps the restriction of phagocytosis, our data suggest that rosetting does not play a role in the invasion or targeting of parasites into uninfected cells, eliminating this mechanism to explain the association of virulence with the rosetting parasite phenotype.     

Electron microscopy of the human brain in cerebral malaria

Ultrastructure of erythrocytes infected with Plasmodium falciparum in human brain, obtained 3 hours post mortem revealed gross distortion of host red cells with abnormality of the red cell surface. The superficial alterations of the parasitized cells as knob-like protrusion appear to be the sites of attachment to vascular endothelium. There was evidence of platelets sticking to the injured endothelium. The endothelial vesicular membrane is in close adhesion to the parasitized red cell, and also to the platelets involved in this mechanism. Thus, explaining the sequestration of parasitized red cell and obstruction in cerebral microcirculation, cerebral oedema and low peripheral platelet count. The was no evidence of inflammation, fibrin or thrombus formation observed in our studies.     

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.     

Malarial anemia: of mice and men

Severe malaria is manifest by a variety of clinical syndromes dependent on properties of both the host and the parasite. In young infants, severe malarial anemia (SMA) is the most common syndrome of severe disease and contributes substantially to the considerable mortality and morbidity from malaria. There is now growing evidence, from both human and mouse studies of malaria, to show that anemia is due not only to increased hemolysis of infected and clearance of uninfected red blood cells (RBCs) but also to an inability of the infected host to produce an adequate erythroid response. In this review, we will summarize the recent clinical and experimental studies of malaria to highlight similarities and differences in human and mouse pathology that result in anemia and so inform the use of mouse models in the study of severe malarial anemia in humans.     

Biochemistry of intraerythrocytic parasites. II. Comparative studies in carbohydrate metabolism.

Comparative studies were carried out on the glucose catabolism of mouse erythrocytes infected with Plasmodium berghei, Plasmodium yoelii, Babesia rodhaini, Babesia microti and Anthemosoma garnhami, as well as on uninfected erythrocytes and reticulocytes. The results showed that there was little qualitative difference between the glucose utilization and lactate production of the parasites although quantitative differences between malaria parasites and piroplasms were observed. The rate of glucose utilization of the infected cells was at least an order of magnitude higher than the rate for uninfected erythrocytes. Reticulocytes were also shown to have higher rates of glucose utilization and lactate production than uninfected erythrocytes.     

Cytoadherence of Plasmodium falciparum-infected erythrocytes to human umbilical vein and human dermal microvascular endothelial cells under shear conditions

To investigate the role of hemodynamics in the adherence of Plasmodium falciparum-infected erythrocytes to cerebral endothelium in vivo, we investigated cytoadherence of parasitized erythrocytes to human umbilical vein endothelial cells (HUVEC) under shear conditions in vitro. At 1.0 dyne/cm2 shear stress, parasitized red blood cell (RBC) adherence to HUVEC ranged from 9.9 +/- 1.0 (+/- SEM) to 75.2 +/- 4.8 RBC/mm2 (mean +/- SEM: 35.1 +/- 2.8 RBC/mm2) and was 13-fold greater than uninfected erythrocyte adherence to HUVEC (range 0.1 +/- 0.1 to 6.7 +/- 1.6 RBC/mm2, mean +/- SEM 2.8 +/- 0.8 RBC/mm2). Only erythrocytes infected with trophozoites and schizonts adhered to HUVEC under shear conditions. Parasitized erythrocyte adherence to HUVEC decreased from 28.4 +/- 2.7 RBC/mm2 to 12.7 +/- 2.4 RBC/mm2 when shear stress was increased from 1.0 to 2.0 dynes/cm2. At 4.0 dynes/cm2, parasitized erythrocyte adherence decreased further to 2.0 +/- 1.3 RBC/mm2. In falciparum malaria patients, endothelial cytoadherence predominates in the microcirculation. Therefore, we also investigated adherence of parasitized erythrocytes to human dermal microvascular endothelial cells (MEC). At 1.0 dyne/cm2, cytoadherence of P. falciparum-infected erythrocytes to MEC ranged from 7.9 +/- 1.1 to 60.0 +/- 2.4 RBC/mm2 (mean +/- SEM: 23.0 +/- 1.7 RBC/mm2) and was 10-fold greater than uninfected erythrocyte cytoadherence to MEC (mean +/- SEM: 2.2 +/- 0.6 RBC/mm2). These data indicate that P. falciparum-infected erythrocytes adhere to human umbilical vein and microvascular endothelial cells under shear stress conditions typical of the postcapillary venules in vivo, and that cytoadherence is specific for parasitized erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed Plasmodium falciparum erythrocyte rosettes.

Cerebral involvement in Plasmodium falciparum malaria is associated with sequestration of infected red blood cells and occlusion of cerebral vessels. Adhesion of infected erythrocytes along the vascular endothelium as well as binding of uninfected erythrocytes to cells infected with late-stage asexual parasites (rosetting) may be important in erythrocyte sequestration. We report that the recently discovered rosetting phenomenon shares characteristics with other human cell-cell interactions (heparin sensitivity, temperature independence, Ca2+/Mg2+ and pH dependence). Mono- and polyclonal antibodies specific for PfHRP1, a histidine-rich protein present in the membrane of P. falciparum-infected erythrocytes, disrupt rosettes but do not affect attachment of infected erythrocytes to endothelial cells. The inhibitory anti-PfHRP1 antibodies reacted with rosetting parasites in indirect immunofluorescence and with P. falciparum polypeptides of Mr 28,000 and Mr 90,000 in immunoprecipitation and immunoblotting, respectively. No inhibitory effects on erythrocyte rosetting were obtained with antibodies to related histidine-rich or other antigens of P. lophurae or P. falciparum. Whether the epitope that mediates rosetting, and is recognized by the anti-PfHRP1 antibodies, is located on PfHRP1 or on a crossreactive antigen remains to be established. The results suggest that endothelial cytoadherence and erythrocyte rosetting involve different molecular mechanisms.