Cytoadherence in paediatric malaria: ABO blood group, CD36, and ICAM1 expression and severe Plasmodium falciparum infection.
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ABSTRACT:
SUBMITTER: Cserti-Gazdewich CM
PROVIDER: S-EPMC3470923 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Malaria is associated with significant microcirculation disorders, especially when the infection reaches its severe stage. This can lead to a range of fatal conditions, from cerebral malaria to multiple organ failure, of not fully understood pathogenesis. It has recently been proposed that a breakdown of the glycocalyx, the carbohydrate-rich layer lining the vascular endothelium, plays a key role in severe malaria, but direct evidence supporting this hypothesis is still lacking. Here, the interactions between Plasmodium falciparum infected red blood cells ( PfRBCs) and endothelial glycocalyx are investigated by developing an in vitro, physiologically relevant model of human microcirculation based on microfluidics. Impairment of the glycocalyx is obtained by enzymatic removal of sialic acid residues, which, due to their terminal location and net negative charge, are implicated in the initial interactions with contacting cells. We show a more than twofold increase of PfRBC adhesion to endothelial cells upon enzymatic treatment, relative to untreated endothelial cells. As a control, no effect of enzymatic treatment on healthy red blood cell adhesion is found. The increased adhesion of PfRBCs is also associated with cell flipping and reduced velocity as compared to the untreated endothelium. Altogether, these results provide a compelling evidence of the increased cytoadherence of PfRBCs to glycocalyx-impaired vascular endothelium, thus supporting the advocated role of glycocalyx disruption in the pathogenesis of this disease.
| S-EPMC6303788 | biostudies-literature
Project description:Blood group O is associated with protection against severe malaria and reduced size and stability of P. falciparum-host red blood cell (RBC) rosettes compared to non-O blood groups. Whether the non-O blood groups encoded by the specific ABO genotypes AO, BO, AA, BB and AB differ in their associations with severe malaria and rosetting is unknown. The A and B antigens are host RBC receptors for rosetting, hence we hypothesized that the higher levels of A and/or B antigen on RBCs from AA, BB and AB genotypes compared to AO/BO genotypes could lead to larger rosettes, increased microvascular obstruction and higher risk of malaria pathology. We used a case-control study of Kenyan children and in vitro adhesion assays to test the hypothesis that "double dose" non-O genotypes (AA, BB, AB) are associated with increased risk of severe malaria and larger rosettes than "single dose" heterozygotes (AO, BO). In the case-control study, compared to OO, the double dose genotypes consistently had higher odds ratios (OR) for severe malaria than single dose genotypes, with AB (OR 1.93) and AO (OR 1.27) showing most marked difference (p = 0.02, Wald test). In vitro experiments with blood group A-preferring P. falciparum parasites showed that significantly larger rosettes were formed with AA and AB host RBCs compared to OO, whereas AO and BO genotypes rosettes were indistinguishable from OO. Overall, the data show that ABO genotype influences P. falciparum rosetting and support the hypothesis that double dose non-O genotypes confer a greater risk of severe malaria than AO/BO heterozygosity.
| S-EPMC10522014 | biostudies-literature
Project description:The adhesion of infected red blood cells (IRBCs) to microvascular endothelium is critical in the pathogenesis of severe malaria. Here we used atomic force and confocal microscopy to examine the adhesive forces between IRBCs and human dermal microvascular endothelial cells. Initial contact of the cells generated a mean ± sd adhesion force of 167 ± 208 pN from the formation of single or multiple bonds with CD36. The strength of adhesion increased by 5- to 6-fold within minutes of contact through a signaling pathway initiated by CD36 ligation by live IRBCs, or polystyrene beads coated with anti-CD36 or PpMC-179, a recombinant peptide representing the minimal binding domain of the parasite ligand PfEMP1 to CD36. Engagement of CD36 led to localized phosphorylation of Src family kinases and the adaptor protein p130CAS, resulting in actin recruitment and CD36 clustering by 50-60% of adherent beads. Uninfected red blood cells or IgG-coated beads had no effect. Inhibition of the increase in adhesive strength by the Src family kinase inhibitor PP1 or gene silencing of p130CAS decreased adhesion by 39 ± 12 and 48 ± 20%, respectively, at 10 dyn/cm(2) in a flow chamber assay. Modulation of adhesive strength at PfEMP1-CD36-actin cytoskeleton synapses could be a novel target for antiadhesive therapy.
| S-EPMC3289509 | biostudies-literature
Project description:There is growing epidemiological and molecular evidence that ABO blood group affects host susceptibility to severe Plasmodium falciparum infection. The high frequency of common ABO alleles means that even modest differences in susceptibility could have a significant impact on the health of people living in malaria endemic regions. We performed an association study, the first to utilize key molecular genetic variation underlying the ABO system, genotyping >9000 individuals across three African populations. Using population- and family-based tests, we demonstrated that alleles producing functional ABO enzymes are associated with greater risk of severe malaria phenotypes (particularly malarial anemia) in comparison with the frameshift deletion underlying blood group O: case-control allelic odds ratio (OR), 1.2; 95% confidence interval (CI), 1.09-1.32; P = 0.0003; family-studies allelic OR, 1.19; 95% CI, 1.08-1.32; P = 0.001; pooled across all studies allelic OR, 1.18; 95% CI, 1.11-1.26; P = 2 x 10(-7). We found suggestive evidence of a parent-of-origin effect at the ABO locus by analyzing the family trios. Non-O haplotypes inherited from mothers, but not fathers, are significantly associated with severe malaria (likelihood ratio test of Weinberg, P = 0.046). Finally, we used HapMap data to demonstrate a region of low F(ST) (-0.001) between the three main HapMap population groups across the ABO locus, an outlier in the empirical distribution of F(ST) across chromosome 9 (approximately 99.5-99.9th centile). This low F(ST) region may be a signal of long-standing balancing selection at the ABO locus, caused by multiple infectious pathogens including P. falciparum.
| S-EPMC2657867 | biostudies-literature
Project description:The propensity of isolates of the malaria parasite Plasmodium falciparum to delete a segment of chromosome 9 has provided positional information that has allowed us to identify a gene necessary for cytoadherence. It has been termed the cytoadherence-linked asexual gene (clag9). clag9 encodes at least nine exons and is expressed in blood stages. The hydrophobicity profile of the predicted CLAG9 protein identifies up to four transmembrane domains. We show here that targeted gene disruption of clag9 ablated cytoadherence to C32 melanoma cells and purified CD36. DNA-induced antibodies to the clag9 gene product reacted with a polypeptide of 220 kDa in the parental malaria clone but not in clones with a disrupted clag9 gene.
| S-EPMC18136 | biostudies-literature
Project description:BackgroundThe ability of mature forms of Plasmodium falciparum infected erythrocytes to bind to a range of host receptors including those displayed on endothelial cells has been associated with the pathology of this infection. Investigations into this adhesive phenomenon have used protein and cell-based adhesion assays to quantify the ability of infected red blood cells to bind. These adhesion assays tend to have relatively high inherent variability and so require multiple experiments in order to provide good quantitation. This means that investigators doing these experiments must count many fields of adherent parasites, a task that is time-consuming and laborious. To address this issue and to facilitate cytoadherence research, developed automated protocols were developed for counting parasite adhesion.MethodsParasite adhesion assays were mainly carried out under static conditions using purified receptors, which is the simplest form of these assays and is translatable to the field. Two different software platforms were used, one commercial (Image Pro-Plus (Media Cybernetics)) and one available in the public domain (ImageSXM) based on the freely available NIH Image software. The adhesion assays were performed and parasite binding quantified using standard manual techniques. Images were also captured using video microscopy and analysed using the two automated systems. The results generated by each system were compared using the Bland and Altman method for assessing the agreement between two methods.ResultsBoth automated counting programs showed concordance compared to the 'gold standard' manual counting within the normal range of adhesion seen with these assays, although the ImageSXM technique had some systematic bias. There was some fall-off in accuracy at very high parasite densities, but this can be resolved through good design of the experiments. Cell based assays were also used as inputs to one of the automated systems (ImageSXM) and produced variable, but encouraging, results.ConclusionsThe automated counting programs are an accurate and practical way of quantifying static parasite binding assays to purified proteins. They are less accurate when applied to cell based systems, but can still provide a reasonable level of accuracy to give a semi-quantitative readout.
| S-EPMC3094228 | biostudies-literature
Project description:The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to receptors on different host cells plays a divergent yet critical role in determining the progression and outcome of the infection. Based on our ex vivo studies with clinical parasite isolates from adult Thai patients, we have previously proposed a paradigm for IRBC cytoadherence under physiological shear stress that consists of a recruitment cascade mediated largely by P-selectin, ICAM-1 and CD36 on primary human dermal microvascular endothelium (HDMEC). In addition, we detected post-adhesion signaling events involving Src family kinases and the adaptor protein p130CAS in endothelial cells that lead to CD36 clustering and cytoskeletal rearrangement which enhance the magnitude of the adhesive strength, allowing adherent IRBC to withstand shear stress of up to 20 dynes/cm². In this study, we addressed whether CD36 supports IRBC adhesion as part of an assembly of membrane receptors. Using a combination of flow chamber assay, atomic force and confocal microscopy, we showed for the first time by loss- and gain-of function assays that in the resting state, the integrin ???? does not support adhesive interactions between IRBC and HDMEC. Upon IRBC adhesion to CD36, the integrin is recruited either passively as part of a molecular complex with CD36, or actively to the site of IRBC attachment through phosphorylation of Src family kinases, a process that is Ca²?-dependent. Clustering of ?1 integrin is associated with an increase in IRBC recruitment as well as in adhesive strength after attachment (?40% in both cases). The adhesion of IRBC to a multimolecular complex on the surface of endothelial cells could be of critical importance in enabling adherent IRBC to withstand the high shear stress in the microcirculations. Targeting integrins may provide a novel approach to decrease IRBC cytoadherence to microvascular endothelium.
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Project description:Cytoadherence of red blood cells (RBCs) invaded by Plasmodium falciparum parasites is an important contributor to the sequestration of RBCs, causing reduced microcirculatory flow associated with fatal malaria syndromes. The phenomenon involves a parasite-derived variant antigen, the P. falciparum erythrocyte membrane protein 1 (PfEMP1), and several human host receptors, such as chondroitin sulfate A (CSA), which has been explicitly implicated in placental malaria. Elucidating the molecular mechanisms of cytoadherence requires quantitative evaluation, under physiologically relevant conditions, of the specific receptor-ligand interactions associated with pathological states of cell-cell adhesion. Such quantitative studies have not been reported thus far for P. falciparum malaria under conditions of febrile temperatures that accompany malarial infections. In this study, single RBCs infected with P. falciparum parasites (CSA binding phenotype) in the trophozoite stage were engaged in mechanical contact with the surface of surrogate cells specifically expressing CSA, so as to quantify cytoadherence to human syncytiotrophoblasts in a controlled manner. From these measurements, a mean rupture force of 43pN was estimated for the CSA-PfEMP1 complex at 37°C. Experiments carried out at febrile temperature showed a noticeable decrease in CSA-PfEMP1 rupture force (by about 23% at 41°C and about 20% after a 40°C heat treatment), in association with an increased binding frequency. The decrease in rupture force points to a weakened receptor-ligand complex after exposure to febrile temperature, while the rise in binding frequency suggests an additional display of nonspecific binding molecules on the RBC surface. The present work establishes a robust experimental method for the quantitative assessment of cytoadherence of diseased cells with specific molecule-mediated binding.
| S-EPMC3870279 | biostudies-literature
Project description:The ABO blood group influences susceptibility to severe Plasmodium falciparum malaria. Recent evidence indicates that the protective effect of group O operates by virtue of reduced rosetting of infected red blood cells (iRBCs) with uninfected RBCs. Rosetting is mediated by a subgroup of PfEMP1 adhesins, with RBC binding being assigned to the N-terminal DBL1?? domain. Here, we identify the ABO blood group as the main receptor for VarO rosetting, with a marked preference for group A over group B, which in turn is preferred to group O RBCs. We show that recombinant NTS-DBL1?? and NTS-DBL1??-CIDR1? reproduce the VarO-iRBC blood group preference and document direct binding to blood group trisaccharides by surface plasmon resonance. More detailed RBC subgroup analysis showed preferred binding to group A?, weaker binding to groups A? and B, and least binding to groups A(x) and O. The 2.8 Å resolution crystal structure of the PfEMP1-VarO Head region, NTS-DBL1??-CIDR1?, reveals extensive contacts between the DBL1?? and CIDR1? and shows that the NTS-DBL1?? hinge region is essential for RBC binding. Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBL??. RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins, opening novel opportunities for intervention against severe malaria. By deciphering the structural basis of blood group preferences in rosetting, we provide a link between ABO blood grouppolymorphisms and rosette-forming adhesins, consistent with the selective role of falciparum malaria on human genetic makeup.
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Project description:Little is known about severe imported Plasmodium falciparum malaria in industrialized countries where the disease is not endemic because most studies have been case reports or have included <200 patients. To identify factors independently associated with the severity of P. falciparum, we conducted a retrospective study using surveillance data obtained from 21,888 P. falciparum patients in France during 1996-2003; 832 were classified as having severe malaria. The global case-fatality rate was 0.4% and the rate of severe malaria was ≈3.8%. Factors independently associated with severe imported P. falciparum malaria were older age, European origin, travel to eastern Africa, absence of chemoprophylaxis, initial visit to a general practitioner, time to diagnosis of 4 to 12 days, and diagnosis during the fall-winter season. Pretravel advice should take into account these factors and promote the use of antimalarial chemoprophylaxis for every traveler, with a particular focus on nonimmune travelers and elderly persons.
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