Low anticoagulant heparin disrupts Plasmodium falciparum rosettes in fresh clinical isolates

The binding of Plasmodium falciparum parasitized erythrocytes to uninfected erythrocytes (rosetting) is associated with severe malaria. The glycosaminoglycan heparan sulfate is an important receptor for rosetting. The related glycosaminoglycan heparin was previously used in treatment of severe malaria, although abandoned because of the occurrence of severe bleedings. Instead, low anticoagulant heparin (LAH) has been suggested for treatment. LAH has successfully been evaluated in safety studies and found to disrupt rosettes and cytoadherence in vitro and in vivo in animal models, but the effect of LAH on fresh parasite isolates has not been studied. Herein, we report that two different LAHs (DFX232 and Sevuparin) disrupt rosettes in the majority of fresh isolates from Cameroonian children with malaria. The rosette disruption effect was more pronounced in isolates from complicated cases than from mild cases. The data support LAH as adjunct therapy in severe malaria.     

The squirrel monkey as an experimental model for Plasmodium falciparum erythrocyte rosette formation.

Experimental cerebral malaria was recently found to occur in the squirrel monkey Saimiri sciureus when infected with the human malaria parasite Plasmodium falciparum. This report is concerned with the existence of spontaneous rosette formation ex vivo (infected blood samples) and in vitro (cultured parasites) between red blood cells (RBC) infected with squirrel monkey-adapted P. falciparum isolates and normal squirrel monkey RBC. Transfer of P. falciparum with high rosette formation tendencies (90-100 R+) from one donor monkey to several recipients gave rise to parasites that varied extensively in their ex vivo rosette formation capacity (4-96% R+). However, all individual parasites readily form rosettes after 24 hr of in vitro culture (60-95% R+). Host factors may be involved in the modulation of rosette formation, although it is found to occur both in splenectomized and spleen-intact animals. Cross-rosette formation is seen between parasitized human RBC and normal squirrel monkey RBC and vice versa, and rosettes formed by RBC of the two hosts are similarly affected by pH, temperature, EDTA, trypsin, as well as squirrel monkey and African human hyperimmune IgG. These characteristics of rosette formation are preserved after long-term in vitro culture in human RBC. Rosettes formed by some isolates are highly sensitive to heparin while others are not, suggesting at least two distinct mechanisms of rosette formation. This idea is also supported by the observation that specific squirrel monkey antisera to heparin-sensitive strains does not dissociate rosettes formed by a heparin-resistant strain. The results suggest that rosettes and anti-rosette formation antibodies formed by squirrel monkeys and humans exhibited similar characteristics, and that the squirrel monkey is therefore a good experimental model to study erythrocyte rosette formation and cerebral malaria.     

Spleen in falciparum malaria: ultrastructural study

An ultrastructural study was undertaken of the spleen of 13 year-old-boy who died of falciparum malaria. The spleen revealed the following: both parasitized and non-parasitized erythrocytes are phagocytosed in large numbers by macrophages, littoral and reticular cells. Blood congestion and trapping of parasitized erythrocytes are commonly seen in splenic sinusoids and cords. Erythrocytes forming rosette structure around immuno-presenting cells is observed. The results of this study provide evidence that the mechanisms underlying splenic host defence in malaria include both immunological and non-immunological interaction with erythrocytes. Splenic trapping of parasitized erythrocytes is an important defence mechanism and the phagocytosis of erythrocytes probably accounts for anaemia.     

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.     

Rheological properties of rosettes formed by red blood cells parasitized by Plasmodium falciparum

A proportion of red blood cells parasitized by Plasmodium falciparum form rosettes with non-parasitized red cells. Although these rosettes are thought to impair microcirculatory flow, their rheological characteristics have not been fully described. Using dual-micropipette manipulation to pull apart individual rosettes, we found that the forces binding rosettes together were strong (average force for removal of a cell was 4.4 x 10(-10) N, approximately 5 times that required to detach a parasitized cell adhered to cultured endothelium). If disrupted rosettes were re-formed, cells rosetted immediately on contact, but the strength of attachment increased over minutes, and did not apparently reach its maximal level for hours. All non-parasitized cells tested could adhere to rosette-forming parasitized cells. Rosettes could withstand arterial flow stresses (1.4-1.6 Pa) for minutes without disintegration. To test the effects of rosetting on flow resistance, the time required for entry into a 4.3 microns pipette was measured. Entry times depended strongly on the number of cells in the rosette, and averaged 35 times longer than for non-parasitized cells. Our studies show that the cell-cell attachments within rosettes are strong, and suggest that rosettes might survive both the arterial circulation and passage through microvessels and could contribute to the ischaemic complications of falciparum malaria.     

Severe falciparum malaria with hyperparasitaemia: management without exchange blood transfusion

The risk of complication in falciparum malaria is associated with parasite load. Drug therapy alone may be insufficient, and blood exchange transfusion is indicated when more than 10% of erythrocytes are parasitized with concurrent pulmonary, renal, cerebral or haemostatic complications; without complications, when the parasitized erythrocytes exceed 30%. The successful use of conventional malaria therapy without exchange transfusion in a young woman with severe falciparum malaria is reported.     

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.     

Inhibitory effect of a fava bean component on the in vitro development of Plasmodium falciparum in normal and glucose-6-phosphate dehydrogenase deficient erythrocytes

We examined the hypothesis that G-6-PD deficiency associated with fava bean ingestion confers resistance to malaria by studying the in vitro interactions between malaria parasites (Plasmodium falciparum), human erythrocytes with varying degrees of G-6-PD deficiency, and isouramil (IU), a fava bean extract that is known to cause oxidant stress and hemolysis of G-6-PD-deficient erythrocytes. Untreated G-6-PD-deficient and normal erythrocytes supported the in vitro growth of P. falciparum equally well. However, after pretreatment with IU, G-6-PD-deficient erythrocytes did not support parasite growth in vitro, whereas growth remained high in normal erythrocytes. Parasite growth was proportional to the G-6-PD activity of the IU-treated erythrocytes. In contrast, when parasitized erythrocytes were exposed to IU, parasites even in normal erythrocytes were destroyed. Ring forms were much less sensitive than late trophozoites and schizonts. The results suggest that there are two modes by which IU affects the development of P. falciparum and demonstrate in vitro that G-6-PD deficiency confers resistance against malaria under conditions of fava-bean-associated oxidant stress.     

Rosette formation in Plasmodium falciparum isolates and anti-rosette activity of sera from Gambians with cerebral or uncomplicated malaria.

The ability of Plasmodium falciparum-infected red blood cells (RBC) to form spontaneous erythrocyte rosettes was studied in 130 fresh isolates from Gambian children with cerebral or uncomplicated malaria from August to November 1990. All isolates (24 of 24) from patients with cerebral malaria formed rosettes, but only 61 of 106 isolates from children with uncomplicated malaria formed rosettes. The mean rate of rosette formation in isolates from children with cerebral malaria (28.3%) was significantly greater than that in isolates from children with uncomplicated malaria (8.5%). Giant rosettes were more frequently formed in isolates from patients with cerebral malaria than in those from patients with uncomplicated malaria. Sera of children with cerebral disease generally lacked anti-rosette activity, while many sera from children with uncomplicated malaria showed strong anti-rosette activity when tested against the patients' ow parasites. Some sera that were devoid of autologous rosette-disrupting activity were able to disrupt rosettes formed in other isolates, indicating the presence of different rosette formation mechanisms. Forty percent (6 of 15) of the sera from patients with cerebral malaria caused microagglutination of the patients' own uninfected and infected RBC, while only 10% (3 of 31) of sera from children with uncomplicated disease caused microagglutination. The ability of infected RBC to bind to melanoma cells grown in vitro did not differ between patients with cerebral or uncomplicated malaria. The results of this study, taken in conjunction with our previous findings, establish a strong association between rosette formation in P. falciparum-infected RBC and cerebral malaria.     

A lectin-like receptor is involved in invasion of erythrocytes by Plasmodium falciparum.

Glycophorin both in solution and inserted into liposomes blocks invasion of erythrocytes by the malaria parasite Plasmodium falciparum. Furthermore, one sugar, N-acetyl-D-glucosamine (GlcNAc), completely blocks invasion of the erythrocyte by this parasite. GlcNAc coupled to bovine serum albumin to prevent the sugar entering infected erythrocytes was at least 100,000 times more effective than GlcNAc alone. Bovine serum albumin coupled to lactose or bovine serum albumin alone had no effect on invasion. These results suggest that the binding of P. falciparum to erythrocytes is lectin-like and is determined by carbohydrates on glycophorin.     

Modulation of malaria virulence by determinants of Plasmodium falciparum erythrocyte membrane protein-1 display

PURPOSE OF REVIEW: Plasmodium falciparum malaria parasites carry approximately 60 var genes that encode variable adhesins termed P. falciparum erythrocyte membrane protein-1. Clonal expression of a single P. falciparum erythrocyte membrane protein-1 variant on the surface of the parasitized host erythrocyte promotes binding of the cell to blood elements (including noninfected erythrocytes, leukocytes) and walls of microvessels. These binding events enable parasitized erythrocytes to sequester and avoid clearance by the spleen, and they also contribute to disease by causing microvascular inflammation and obstruction. RECENT FINDINGS: Steps by which P. falciparum erythrocyte membrane protein-1 is exported to the parasitized erythrocyte surface have recently been elucidated. The ability of parasites to cytoadhere and cause disease depends on the variant of P. falciparum erythrocyte membrane protein-1 as well as its amount and distribution at the erythrocyte surface. An example of a host polymorphism that affects P. falciparum erythrocyte membrane protein-1 display is hemoglobin C, which may protect against malaria by impairing the parasite's ability to adhere to microvessels and induce inflammation. Interference with P. falciparum erythrocyte membrane protein-1-mediated phenomena appears to diminish cytoadherence in vivo and to protect against disease in animal models. SUMMARY: Plasmodium falciparum erythrocyte membrane protein-1-mediated sequestration of parasitized erythrocytes plays a central role in malaria pathogenesis. Clinical interventions aimed at reducing cytoadherence and microvascular inflammation may improve disease outcome.     

Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin

Sickle trait, the heterozygous state of normal hemoglobin A (HbA) and sickle hemoglobin S (HbS), confers protection against malaria in Africa. AS children infected with Plasmodium falciparum are less likely than AA children to suffer the symptoms or severe manifestations of malaria, and they often carry lower parasite densities than AA children. The mechanisms by which sickle trait might confer such malaria protection remain unclear. We have compared the cytoadherence properties of parasitized AS and AA erythrocytes, because it is by these properties that parasitized erythrocytes can sequester in postcapillary microvessels of critical tissues such as the brain and cause the life-threatening complications of malaria. Our results show that the binding of parasitized AS erythrocytes to microvascular endothelial cells and blood monocytes is significantly reduced relative to the binding of parasitized AA erythrocytes. Reduced binding correlates with the altered display of P. falciparum erythrocyte membrane protein-1 (PfEMP-1), the parasite's major cytoadherence ligand and virulence factor on the erythrocyte surface. These findings identify a mechanism of protection for HbS that has features in common with that of hemoglobin C (HbC). Coinherited hemoglobin polymorphisms and naturally acquired antibodies to PfEMP-1 may influence the degree of malaria protection in AS children by further weakening cytoadherence interactions.     

Natural protection against severe Plasmodium falciparum malaria due to impaired rosette formation

Genes for two lethal diseases, thalassemia and sickle cell anemia, are favored by evolution because, in their heterozygous form, they protect against cerebral malaria. Rosette formation, the binding of uninfected red cells (RBCs) to Plasmodium falciparum-infected RBCs (PRBCs), has previously been found to be associated with cerebral malaria, the most important severe manifestation of P falciparum malaria. We show here that thalassemic RBCs and, under certain conditions, even hemoglobin S (HbS)-containing RBCs possess an impaired ability to bind to PRBCs, forming small and weak erythrocyte rosettes compared with rosettes formed by normal RBCs. This decreased rosetting ability is associated with the small size of the thalassemic RBCs and with distortion of the mechanical properties of HbS-containing RBCs. The impairment of rosette formation may hinder the development of cerebral malaria by abatement of sequestration.     

Rheological analysis of the formation of rosettes by red blood cells parasitized by Plasmodium falciparum

Red blood cells infected by mature malarial parasites of the species Plasmodium falciparum can adhere to non-parasitized red cells (rosetting) and also to endothelial cells (cytoadhesion). To investigate how the circulation might influence rosetting, we studied formation of rosettes in cell suspensions sheared in a cone-and-plate viscometer, and the ability of flowing non-parasitized cells to bind to parasitized cells already adherent to a surface. After rosettes of strain R29 had been disrupted with fucoidan, they reformed slowly under stationary conditions but more rapidly in suspensions sheared at low stress (about 0.1–0.2 Pa). Strain Malayan Camp gave a lower rosetting frequency which actually increased at low shear. Increasing shear stress was associated with reduction in rosette formation, although rosetting occurred at >1 Pa, suggesting that rosettes could form in the systemic circulation. Rosetting inhibited adhesion of flowing parasitized cells to immobilized platelets (which express the cytoadhesion receptor CD36), as evidenced by increased adhesion after disruption of rosettes. The de-rosetted adherent cells parasitized by R29 supported only a low level of rosetting when non-parasitized cells were flowed over them at a wall shear of 0.1 Pa, with little increase if the stress was decreased to 0.05 Pa. Rosettes formed in the circulation might obstruct microvessels and inhibit cytoadhesion if they reached venules. However, if cytoadhesion occurred before rosetting, then adherent cells should not efficiently form rosettes.     

Involvement of CD36 on erythrocytes as a rosetting receptor for Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (parasitized red blood cells [PRBCs]) can adhere to uninfected erythrocytes (RBCs) to form rosettes, and adhere to the endothelial cell (EC) surface antigen CD36. These adherence phenomena have previously been considered quite different. We show that anti-CD36 monoclonal antibodies (MoAbs) reverse rosetting of PRBCs from both a culture-adapted line (Malayan Camp [MC] strain) and a natural isolate, GAM425. Three MoAbs that block adherence of PRBCs to ECs or C32 melanoma cells also reversed rosetting by greater than 50% at levels of less than 1 microgram/mL (OKM5, OKM8, and 8A6). Two other MoAbs that react with purified CD36 (1D3 and 1B1), but do not react with the surface of C32 cells, failed to reverse rosetting. When rosettes were disrupted and the RBCs and PRBCs were pretreated separately with antibodies before mixing to allow rosette reformation, only pretreatment of RBCs had an effect. MoAb 8A6 pretreatment of RBCs blocked rosette reformation, while MoAb 1B1 pretreatment did not. Rosetting was also reversed by purified human platelet CD36. In conjunction with evidence that CD36 is expressed on normal human erythrocytes (van Schravendijk et al, Blood 80:2105, 1992), we conclude that this CD36 is able to act as a host receptor for rosetting in the MC strain and some natural isolates of P falciparum.     

Rosette formation by Plasmodium coatneyi-infected red blood cells

Animal models are needed for the study of cytoadherence in falciparum malaria. Red blood cell (RBC) rosette formation is one type of cytoadherence and appears to be associated with knob formation, endothelial cell adhesion and sequestration of Plasmodium-infected RBCs. Since Plasmodium coatneyi-infected RBCs develop knobs and sequester, we hypothesized that they also form rosettes. RBCs from P. coatneyi-infected rhesus monkeys (Macaca-mulatta) were collected, allowed to mature overnight in vitro and found to form rosettes as hypothesized. This observation adds to the known falciparum-like characteristics of P. coatneyi, and suggests that the Macaca mulatta-P. coatneyi model may be appropriate for pathophysiologic studies of cytoadherence.     

Comparison of techniques for detecting T-cell acute lymphocytic leukemia.

Three versions of the E-rosette test, one using untreated sheep erythrocytes at 37 degrees C, another using such cells at 4 degrees C, and a third using sheep erythrocytes treated with S-(2-aminoethyl)isothiouronium bromide hydrobromide (AET), were applied to each of 72 bone marrow specimens from as many unselected patients with untreated acute lymphocytic leukemia (ALL). The same specimens were also examined for T-cell antigens, based on reactivity with an antithymocyte serum. Lymphoblasts in eight ALL specimens formed E rosettes at 37 degrees C; no other E-positive specimens were identified when the assay was done at 4 degrees C. With AET-treated erythrocytes, lymphoblasts from these eight specimens and six additional specimens readily formed rosettes. T-cell antigens were detectable in all specimens positive for rosette formation withe untreated erythrocytes, in four of the six specimens positive for rosette formation with AET-treated erythrocytes, and in four specimens that showed no rosette formation under any of the experimental conditions used. Altogether, 18 specimens contained lymphoblasts with one or more surface markers characteristic of T-cell leukemia. These findings indicate that more specimens are likely to be identified at T-cell luekemias when E-rosette tests of increasing sensitivity and assayss for T-cell antigens are used. Some leukemic blasts do not possess the full array of membrane receptors and antigens usually associated with T cells. A combination of E-rosette tests and serologic tests is necessary to determine reliably the relationship of the test specimen to either T-cell ALL or common ALL and to establish the clinical significance of blasts that express membrane properties intermediate between those of T-cell ALL and common ALL.     

Fc-IgG and Fc-IgM receptor-carrying lymphocytes in human blood. Optimization of determining T(M) lymphocytes

Several methods for enumeration of Fc receptor bearing T lymphocytes and mononuclear cells from human peripheral blood were compared. The detection of Fc receptors is based on the formation of EA rosettes by using bovine erythrocytes and purified rabbit IgG or IgM antibodies. As alternative method the mixed rosette assay (EA rosettes plus sheep erythrocyte rosettes) (3) was applied for determining TG lymphocytes without the need of T cell separation. Independent of the method used for T cell separation (preparative rosetting with sheep erythrocytes stabilized by AET or HSA) the number of TG and TM lymphocytes was found to be identical. TG values obtained by use of the mixed rosette assay were significant lower (10 +/- 2%) than those obtained with the classical test (18 +/- 5%) (EA rosettes after T cell separation). Obviously this difference is due to a contamination of T lymphocyte preparations by non-T cells. On freshly isolated T lymphocytes without overnight culture we obtained 29% and 35%, respectively TM lymphocytes after separation of T cells using sheep erythrocyte rosettes stabilized with AET or HSA. The expression of FcIgM receptors was found to be strongly dependent on the composition and pH value of the culture medium. In the presence of human AB serum the maximum of FcIgM receptor expression on isolated T cells was obtained at pH 8.5. Under optimum conditions we found 63% and 66% respectively TM lymphocytes after T cell separation using AET or HSA stabilized sheep erythrocytes.     

Disruption of Plasmodium falciparum erythrocyte rosettes by standard heparin and heparin devoid of anticoagulant activity.

We have studied the ability of heparin to disrupt spontaneous rosettes formed between Plasmodium falciparum-infected and uninfected red blood cells, which has been proposed to have importance in the pathogenesis of cerebral malaria. Substantial variation in this activity was found among six laboratory stains of P. falciparum. Rosettes formed by three of these strains were highly sensitive to heparin (50% disruption at 0.5-25 micrograms/ml; 1 microgram/ml corresponds to 0.15 IU/ml). The rosettes formed by two other strains showed a much lower sensitivity (50% disruption at 700-2,500 micrograms/ml), while the rosettes formed by another strain were almost completely resistant to heparin (20% disruption at 6,500 micrograms/ml). The ability of heparin (65 or 650 micrograms/ml) to disrupt rosettes formed by 54 fresh Gambian isolates of P. falciparum also varied. Rosettes of 27 (50%) of the 54 isolates were disrupted to a significant degree (greater than or equal to 15%), while rosettes of the other 27 isolates remained unaffected at the concentrations tested. Heparin was fractionated by molecular weight and/or affinity for antithrombin III. We found that its property of rosette disruption was associated, to some extent, with size (high molecular weight) but not with its anticoagulant potential (affinity for antithrombin III). A heparin fraction with low affinity for antithrombin III and one with combined high molecular weight and low affinity for antithrombin III were as effective at disrupting rosettes as standard heparin, while a chemically modified (N-acetylated) high molecular weight-heparin fraction, similarly devoid of anticoagulant activity, lacked strong anti-rosette potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology

The pathophysiology of severe falciparum malaria is complex, but evidence is mounting that its central feature is the old concept of a mechanical microcirculatory obstruction. Autopsy studies, but also in vivo observations of the microcirculation, demonstrate variable obstruction of the microcirculation in severe malaria. The principal cause of this is cytoadherence to the vascular endothelium of erythrocytes containing the mature forms of the parasite, leading to sequestration and obstruction of small vessels. Besides, parasitized red cells become rigid, compromising their flow through capillaries whose lumen has been reduced by sequestered erythrocytes. Adhesive forces between infected red cells (auto-agglutination), between infected and uninfected red cells (rosetting) and between uninfected erythrocytes (aggregation) could further slow down microcirculatory flow. A more recent finding is that uninfected erythrocytes also become rigid in severe malaria. Reduction in the overall red cell deformability has a strong predictive value for a fatal outcome. Rigidity may be caused by oxidative damage to the red blood cell membrane by malaria pigment released at the moment of schizont rupture. Anti-oxidants, such as N-acetylcysteine can reverse this effect and are promising as adjunctive treatment in severe malaria.