Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia

ABSTARCT: For years Plasmodium vivax has been considered the cause of benign malaria. Nevertheless, it has been observed that this parasite can produce a severe disease comparable to Plasmodium falciparum. It has been suggested that some physiopathogenic processes might be shared by these two species, such as cytoadherence. Recently, it has been demonstrated that P. vivax-infected erythrocytes (Pv-iEs) have the capacity to adhere to endothelial cells, in which intercellular adhesion molecule-1 (ICAM-1) seems to be involved in this process

Correlation between <i>var</i> transcript levels and cytoadhesive phenotypes of <i>P</i>. <i>falciparum</i> isolates from Mozambican children.

<p>The relationship between adhesion and transcript levels of <i>var</i>/DCs was assessed by Spearman correlation analysis, with * indicating <i>P</i><0.05 and ** if statistically significant after Benjamini-Hochberg correction for the six adhesive phenotypes tested. PM-agg: platelet-mediated agglutination.</p

Phenotypical and molecular characterization of <i>Pf</i>3D7^gC1qR and <i>Pf</i>3D7^CD36.

<p><b>A)</b> Binding assays of <i>Pf</i>3D7^<i>gC1qR</i> and <i>Pf</i>3D7^<i>CD36</i> over gC1qR, CD36, ICAM1 and CSA receptors. <b>B)</b> Transcriptional analysis of the <i>var</i> gene repertoire of <i>Pf</i>3D7. Transcript levels of <i>var</i> genes were determined by qPCR using primers specific for each of the <i>P</i>. <i>falciparum</i> 3D7 <i>var</i> genes and were expressed as copy number relative to the seryl-tRNA synthetase gene. <b>C)</b> Levels of IgG recognition by plasma from Mozambican children were compared by Wilcoxon matched pair test, with * indicating P≤0.001. Bars represent mean and standard deviation of geometric mean fluorescence intensity (GMFI). D) Domain structure of PFD002c.</p

Breadth of IgG recognition of <i>P</i>. <i>falciparum</i> isolates according to <i>var</i> transcript levels and origin.

<p>Geometric Mean Fluorescence Intensity (GMFI) values from each parasite/plasma combination were scored in relation to the threshold of positivity (GMFI of negative controls plus two standard deviations), with a score of 0 assigned if GMFI values were below the cut-off; 1 if the value was between one- and two-fold the cut-off; 2 if the value was between two- and three-fold the cut-off; and so on until a maximum score of 5. Breadth of IgG recognition (BoR) was calculated as the sum of scores obtained for each parasite and expressed as percentage of the maximum score possible. BoR was compared between A) isolates transcribing <i>var</i>/DCs at low- or high- levels by negative binomial regression models adjusted by age and B) between isolates collected from travelers (n = 3), severe malaria (SM, n = 23), uncomplicated malaria (UM, n = 15) children and adults (Moz adults, n = 4) by test for trend across ordered groups. Bars represent the mean of BoR and standard deviation. * indicates <i>P</i><0.05 and ** <i>P</i>≤0.001.</p

Characteristics of the patients with malaria included in the study

<p>Characteristics of the patients with malaria included in the study</p

Transcript levels of DC8 and DC11 by severe malaria symptoms in Mozambican children.

<p>Transcript levels (y axis) correspond to relative copy number of target genes relative to seryl-tRNA synthetase gene copies (X100). Bars represent the median and interquartile range. Transcript levels were compared between matched case/control pairs by Sign-test, with * indicating <i>P</i><0.05. RCN: Relative copy number; DC: Domain Cassette; SM: severe malaria; UM: uncomplicated malaria; SAn: severe anemia; Pro: prostration; ARD: acidosis or respiratory distress.</p

Transcript levels of <i>var</i>/DCs in <i>P</i>. <i>falciparum</i> isolates from travelers, children with uncomplicated malaria and adults.

<p>Transcript levels (y axis) correspond to relative copy number relative to seryl-tRNA synthetase gene copies (X100). Bars represent the median and interquartile range. Transcript levels were compared between groups by Mann-Whitney test, with * indicating <i>P</i><0.05 and ** if statistically significant after Benjamini-Hockberg correction. RCN: Relative copy number; DC: Domain Cassette; Tv: travelers, Ch: Mozambican children, Ad: Mozambican adults.</p

Cytoadhesion to gC1qR through <i>Plasmodium falciparum</i> Erythrocyte Membrane Protein 1 in Severe Malaria

<div><p>Cytoadhesion of <i>Plasmodium falciparum</i> infected erythrocytes to gC1qR has been associated with severe malaria, but the parasite ligand involved is currently unknown. To assess if binding to gC1qR is mediated through the <i>P</i>. <i>falciparum</i> erythrocyte membrane protein 1 (PfEMP1) family, we analyzed by static binding assays and qPCR the cytoadhesion and <i>var</i> gene transcriptional profile of 86 <i>P</i>. <i>falciparum</i> isolates from Mozambican children with severe and uncomplicated malaria, as well as of a <i>P</i>. <i>falciparum</i> 3D7 line selected for binding to gC1qR (<i>Pf</i>3D7^gC1qR). Transcript levels of DC8 correlated positively with cytoadhesion to gC1qR (rho = 0.287, <i>P</i> = 0.007), were higher in isolates from children with severe anemia than with uncomplicated malaria, as well as in isolates from Europeans presenting a first episode of malaria (n = 21) than Mozambican adults (n = 25), and were associated with an increased IgG recognition of infected erythrocytes by flow cytometry. <i>Pf</i>3D7^gC1qR overexpressed the DC8 type PFD0020c (5.3-fold transcript levels relative to Seryl-tRNA-synthetase gene) compared to the unselected line (0.001-fold). DBLβ12 from PFD0020c bound to gC1qR in ELISA-based binding assays and polyclonal antibodies against this domain were able to inhibit binding to gC1qR of <i>Pf</i>3D7^gC1qR and four Mozambican <i>P</i>. <i>falciparum</i> isolates by 50%. Our results show that DC8-type PfEMP1s mediate binding to gC1qR through conserved surface epitopes in DBLβ12 domain which can be inhibited by strain-transcending functional antibodies. This study supports a key role for gC1qR in malaria-associated endovascular pathogenesis and suggests the feasibility of designing interventions against severe malaria targeting this specific interaction.</p></div

DBLβ12 domain of PFD0020c is involved in the interaction with gC1qR.

<p><b>A)</b> Reactivity of PFD0020c domains against gC1qR and EPCR by ELISA-based binding assays. Antibody-mediated inhibition of <b>B)</b> binding to gC1qR or CD36 of <i>P</i>. <i>falciparum Pf3D7</i>^<i>gC1qR</i>, <i>Pf3D7</i>^<i>CD36</i>; <b>C)</b> binding to human brain endothelial cells (HBMEC) of <i>Pf3D7</i>^<i>gC1qR</i> and <b>D)</b> binding to gC1qR of four <i>P</i>. <i>falciparum</i> Mozambican isolates. The gC1qR binding levels in absence of antibodies were 299 IEs/mm² (SD 53) for <i>Pf</i>3D7^gC1qR; 202 IEs/mm² (SD 11) for <i>Pf</i>moz1; 92 IEs/mm² (SD 17) for <i>Pf</i>moz2; 74 IEs/mm² (SD 8) for <i>Pf</i>moz3; and 81 IEs/mm² (SD 7) for <i>Pf</i>moz4. The CD36 binding level in absence of antibodies was 615 IEs/mm² (SD 27) for <i>Pf</i>3D7^CD36. Binding is expressed as the percentage of mean binding in absence of antibodies. Bars represent the mean and standard deviation. * indicates <i>P</i><0.05 and **<i>P</i><0.001.</p