In Vitro Inhibition of Plasmodium falciparum Rosette Formation by Curdlan Sulfate

Abstract

Spontaneous binding of infected erythrocytes to uninfected erythrocytes to form rosettes is a property of some strains of Plasmodium falciparum that is linked to severe complications of malaria. Curdlan sulfate (CRDS) is a sulfated glycoconjugate compound that is chemically similar to known rosette-inhibiting drugs such as heparin. CRDS has previously been shown to have antimalarial activity in vitro and is safe for clinical use. Here we show that CRDS at therapeutic levels (10 to 100 g/ml) significantly reduces rosette formation in vitro in seven P. falciparum laboratory strains and in a group of 18 African clinical isolates. The strong ability to inhibit rosetting suggests that CRDS has the potential to reduce the severe complications and mortality rates from P. falciparum malaria among African children. Our data support further clinical trials of CRDS.Despite decades of research, malaria still claims an estimated 1 million lives per year in sub-Saharan Africa, and severe complications, such as cerebral malaria, remain relatively common (19). Treatment for malaria involves the use of antimalarial drugs that kill the parasite, along with supportive measures, such as the provision of intravenous fluids and blood transfusion as required. Progress in understanding the pathogenesis of malaria has led to the possibility of designing therapies that target factors that contribute to severe disease in order to ameliorate the clinical complications and reduce the number of deaths from malaria.Rosetting is the spontaneous binding of noninfected erythrocytes to erythrocytes infected with mature asexual bloodstage Plasmodium parasites (reviewed in reference 14). In Plasmodium falciparum malaria, rosetting is associated with high levels of parasitemia (16) and with disease severity in African children (13,17), especially cerebral malaria (2). Other Plasmodium species also rosette, although it is only in P. falciparum that rosetting is linked to severe disease, probably because of the additional cytoadhesion properties that may act in concert with rosetting to cause microvascular obstruction (8,14). Rosetting in P. falciparum is mediated by heterogeneous mechanisms involving specific members of the parasite variant antigen family PfEMP1 (15), which are expressed on the surface of infected erythrocytes and which binding to a variety of receptors on uninfected red cells, such as complement receptor 1 (CR1), heparan sulfate-like sugars, and ABO blood group oligosaccharides (reviewed in reference 14).Previous work has shown that rosettes can be disrupted by sulfated glycoconjugate compounds such as heparin and fucoidan (1, 12), but the anticoagulant effects of these drugs prevent their use as treatments for severe malaria (19). Curdlan sulfate (CRDS; a semisynthetic sulfated 133-␤-D-glucan) has been shown to inhibit P. falciparum invasion in vitro (6) and is safe for use by adult patients with severe malaria (5). It was hypothesized that CRDS might be effective against P. falciparum rosetting (5) because of the chemical similari...

Fucosylated Chondroitin Sulfate Inhibits Plasmodium falciparum Cytoadhesion and Merozoite Invasion

“…Recently, a pivotal role for cytoadherence through the endothelial protein C receptor in the development of severe malaria was described (30). Cytoadhesion of Pf-iEs is mediated by members of the P. falciparum membrane protein 1 (PfEMP-1) family, which mediates parasite interactions with various host receptors, including CD36 (31), intercellular adhesion molecule 1 (ICAM-1) (32), and chondroitin sulfate A (CSA) (33,34), a receptor frequently associated with MiP (15,33,35) Glycosaminoglycans (GAGs), including heparin, have been employed as a strategy to prevent malaria complications due to their abilities to inhibit parasite cytoadhesion, to block invasion, and to disrupt rosettes (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46). However, the side effects of heparin, mostly serious bleeding (47), and the potential risk of contamination (because some GAGs are obtained from mammals) have hampered GAG-based adjunct therapies.…”

Low Anticoagulant Heparin Disrupts Plasmodium falciparum Rosettes in Fresh Clinical Isolates

“…This finding is not surprising but in concordance with the results of previous studies where it has been shown that some isolates are insensitive to heparin, heparin derivatives, and other rosette-disrupting sulfated glycoconjugates, even at very high concentrations. 23,24,44 In the present study, there was a statistically significant difference ( P < 0.05) between the complicated and mild groups, with a higher parasitemia and more isolates forming rosettes in the complicated malaria cases than in the mild malaria cases. Another observation is that the isolates that showed a rosetting phenotype in the mild group were of higher parasitemia and lower hemoglobin levels compared with the isolates in the same group that did not show any rosetting.…”

“…44,46 This compound has been reported to be effective in vitro at the concentration 50 μg/mL in the majority of 18 P. falciparum isolates from Kenyan children. The mechanism of rosette disruption The mean relative rosetting rate (the proportion of remaining rosettes after treatment compared with the control-treated erythrocytes) ± SEM is shown.…”

“…In some rosette disruption studies, frozen and thawed parasite samples or laboratory strains have been used. 19,22,23,44 In a recent study, it was found that the dominant var gene coding for PfEMP1 changed before and after cryopreservation in a number of isolates, which was analyzed by quantitative polymerase chain reaction (Q-PCR) using strain and stage-specific primers. 36 By using fresh isolates, the risk of changes in antigens presented on the erythrocyte surface, including expression of PfEMP1, is limited.…”

The Effect of Anti-Rosetting Agents against Malaria Parasites under Physiological Flow Conditions

“…In vitro studies have identified sulphated glycoconjugate compounds such as heparin, fucoidan and curdlan sulphate as being effective at disrupting rosettes [13], [14], [15], [16]. Some of these sulphated polysaccharides not only disrupt rosetting, but also inhibit adhesion of IEs to host endothelial and placental receptors such as CSA and CD36 [17], [18], [19], [20].…”