Plasmodium falciparum: Gelatin Enrichment Selects for Parasites with Full-Length Chromosome 2. Implications for Cytoadhesion Assays

Waterkeyn, Jacqueline G.; Cowman, Alan F.; Cooke, Brian M.

doi:10.1006/expr.2000.4593

Cited by 26 publications

(20 citation statements)

References 16 publications

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“…Genetic identity of parasite isolates was confirmed by sequencing the ama1 and var2csa genes (55). Parasites were synchronized using sorbitol (Sigma-Aldrich) treatment (56), and knobexpressing parasites were enriched by gelatin (Sigma-Aldrich) flotation (57). P. falciparum isolate E8Bvpkd was generated by transfecting the E8B-ICAM parental isolate (58), which is a clone of IT4, with the plasmid vector pHBupsCB R (59) and culturing in the presence of WR99210 (2.5 nM) to inhibit endogenous var gene expression.…”

Section: Methodsmentioning

confidence: 99%

Targets of antibodies against Plasmodium falciparum–infected erythrocytes in malaria immunity

et al. 2012

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Plasmodium falciparum is the major cause of malaria globally and is transmitted by mosquitoes. During parasitic development, P. falciparum-infected erythrocytes (P. falciparum-IEs) express multiple polymorphic proteins known as variant surface antigens (VSAs), including the P. falciparum erythrocyte membrane protein 1 (PfEMP1). VSA-specific antibodies are associated with protection from symptomatic and severe malaria. However, the importance of the different VSA targets of immunity to malaria remains unclear, which has impeded an understanding of malaria immunity and vaccine development. In this study, we developed assays using transgenic P. falciparum with modified PfEMP1 expression to quantify serum antibodies to VSAs among individuals exposed to malaria. We found that the majority of the human antibody response to the IE targets PfEMP1. Furthermore, our longitudinal studies showed that individuals with PfEMP1-specific antibodies had a significantly reduced risk of developing symptomatic malaria, whereas antibodies to other surface antigens were not associated with protective immunity. Using assays that measure antibody-mediated phagocytosis of IEs, an important mechanism in parasite clearance, we identified PfEMP1 as the major target of these functional antibodies. Taken together, these data demonstrate that PfEMP1 is a key target of humoral immunity. These findings advance our understanding of the targets and mediators of human immunity to malaria and have major implications for malaria vaccine development.

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Section: Methodsmentioning

confidence: 99%

Targets of antibodies against Plasmodium falciparum–infected erythrocytes in malaria immunity

et al. 2012

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“…Infected RBCs were purified by gelatin 16 or magnetic sorting (Miltenyi Biotec, Auburn, CA). Protein samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with mouse ␣GFP (Roche, Indianapolis, IN; 1:1000), mouse ␣KAHRP-His (1:1000), rabbit ␣KAHRP-3Ј repeats (1:2000), or rabbit ␣heat-shock protein 70 (␣hsp70) antiserum (1:6000) and visualized using enhanced chemiluminescence (ECL; Amersham, Uppsala, Sweden).…”

Section: Plasmid Constructs and Transfection Western Blotting And Amentioning

confidence: 99%

“…Transcription of var commences early with maximal levels at 12 hours after invasion 24 and PfEMP1 is first detected on the surface 16 hours after invasion. 16,25 Thus, the chimeric PfEMP1-GFP proteins, expressed from the Pfcrt promoter in the vector pARL, are expressed at a similar stage to endogenous PfEMP1.…”

Section: Pfemp1-gfp Chimeras In P Falciparum-infected Rbcsmentioning

confidence: 99%

Trafficking of the major virulence factor to the surface of transfected P falciparum–infected erythrocytes

Knuepfer

Rug

Klonis

et al. 2005

Blood

127

150

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“…7 Cultures of knob-expressing parasite clones (3D7 and EMP3KO) were subjected to weekly flotation in gelatin to maintain synchrony and expression of knobs, as recently described. 14 Synchronous cultures of the knobless clone (KKO) were maintained by weekly selection for ring-stage parasites using sorbitol, as previously described. 15 Cultures were used for experiments when most (more than 90%) of the parasites were mature, pigmented trophozoites.…”

Section: Parasitized Red Blood Cellsmentioning

confidence: 99%

Contribution of parasite proteins to altered mechanical properties of malaria-infected red blood cells

Glenister

Coppel

Cowman

et al. 2002

Blood

287

233

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Red blood cells (RBCs) parasitized byPlasmodium falciparum are rigid and poorly deformable and show abnormal circulatory behavior. During parasite development, knob-associated histidinerich protein (KAHRP) and P falciparum erythrocyte membrane protein 3 (PfEMP3) are exported from the parasite and interact with the RBC membrane skeleton. Using micropipette aspiration, the membrane shear elastic modulus of RBCs infected with transgenic parasites (with kahrp or pfemp3 genes deleted) was measured to determine the contribution of these proteins to the increased rigidity of parasitized RBCs (PRBCs). In the absence of either protein, the level of membrane rigidification was significantly less than that caused by the normal parental parasite clone. KAHRP had a significantly greater effect on rigidification than PfEMP3, contributing approximately 51% of the overall increase that occurs in PRBCs compared to 15% for PfEMP3. This study provides the first quantitative information on the contribution of specific parasite proteins to altered mechanical properties of PRBCs. IntroductionMalaria caused by Plasmodium falciparum remains the most serious and widespread parasitic disease of humans. Clinical symptoms of malaria occur during the asexual stage of the parasite's life cycle, when it multiplies within red blood cells (RBCs). The extreme virulence of P falciparum and the occurrence of severe, often fatal clinical complications is related to the ability of RBCs parasitized by mature forms of the parasite to accumulate in the microvasculature of a variety of organs. 1 This abnormal circulatory behavior for RBCs appears to be directly related to parasite-induced alteration of its mechanical and adhesive properties.During the last 2 decades, the altered adhesive properties of parasitized RBCs (PRBCs) have been studied intensively (see 2,3 for recent reviews). In contrast, alterations of their mechanical properties, and the molecular mechanisms underpinning these changes, have been relatively ignored. Previous studies have clearly demonstrated that the deformability of intact PRBCs is profoundly reduced. [4][5][6] The overall increase in red cell rigidity is due, in part, to the presence of the large, nondeformable intracellular parasite and to a number of stage-specific parasite-encoded proteins that associate with the RBC membrane skeleton. 3,5,6 Paulitschke and Nash 6 used micropipette aspiration to measure the rigidity of RBCs parasitized by a number of unrelated parasite lines of knobby and knobless phenotypes. In general, the membranes of knobby PRBCs were more rigid than those lacking knobs; however, there was considerable variation in rigidity, particularly between knobby lines, with some knobby PRBCs only slightly more rigid than others infected with knobless parasite lines. Unfortunately, in their study, there was no characterization of the parasite genotype or immunohistochemical analysis of the PRBCs to determine precisely which parasite proteins were or were not expressed in different parasite lines. As such, thoug...

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Plasmodium falciparum: Gelatin Enrichment Selects for Parasites with Full-Length Chromosome 2. Implications for Cytoadhesion Assays

Cited by 26 publications

References 16 publications

Targets of antibodies against Plasmodium falciparum–infected erythrocytes in malaria immunity

Targets of antibodies against Plasmodium falciparum–infected erythrocytes in malaria immunity

Trafficking of the major virulence factor to the surface of transfected P falciparum–infected erythrocytes

Contribution of parasite proteins to altered mechanical properties of malaria-infected red blood cells

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