Differential Adhesive Properties of Sequestered Asexual and Sexual Stages of Plasmodium falciparum on Human Endothelial Cells Are Tissue Independent

The protozoan parasite Plasmodium falciparum, responsible for the most severe form of malaria, is able to sequester from peripheral circulation during infection. The asexual stage parasites sequester by binding to endothelial cell receptors in the microvasculature of various organs. P. falciparum gametocytes, the developmental stages responsible for parasite transmission from humans to Anopheles mosquitoes, also spend the almost ten days necessary for their maturation sequestered away from the peripheral circulation before they are released in blood mainstream. In contrast to those of asexual parasites, the mechanisms and cellular interactions responsible for immature gametocyte sequestration are largely unexplored, and controversial evidence has been produced so far on this matter. Here we present a systematic comparison of cell binding properties of asexual stages and immature and mature gametocytes from the reference P. falciparum clone 3D7 and from a patient parasite isolate on a panel of human endothelial cells from different tissues. This analysis includes assays on human bone marrow derived endothelial cell lines (HBMEC), as this tissue has been proposed as a major site of gametocyte maturation. Our results clearly demonstrate that cell adhesion of asexual stage parasites is consistently more efficient than that, virtually undetectable of immature gametocytes, irrespectively of the endothelial cell lines used and of parasite genotypes. Importantly, immature gametocytes of both lines tested here do not show a higher binding efficiency compared to asexual stages on bone marrow derived endothelial cells, unlike previously reported in the only study on this issue. This indicates that gametocyte-host interactions in this tissue are unlikely to be mediated by the same adhesion processes to specific endothelial receptors as seen with asexual forms

Adhesion of asexual and sexual stages of AQ104 and 3D7 on TNF-alpha stimulated HDMEC, HUVEC and HBMEC cell lines.

<p>Static assays were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031567#s4" target="_blank">Material and methods</a> section. Values are expressed as % of bound parasites per mm² of the ItG control. Data shown are the mean number of iRBC per mm² ± S.E. of at least 3 biological replicates was counted and expressed as % of bound parasites per mm² of the ItG control.</p

Adhesion of asexual stages of ItG and 3D7 on stimulated and non stimulated HDMEC, HUVEC and HBMEC endothelial cell lines.

<p>A) Data shown are the mean number of iRBC per mm² ± S.E. of 3 to 5 biological replicates. Static assays were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031567#s4" target="_blank">Material and Methods</a> section. B) Giemsa-stained infected erythrocytes bound to TNF-alpha activated endothelial cells (HDMEC). Scale bar: 25 µm.</p

Adhesion of asexual and sexual stages of 3D7 on TNF-alpha stimulated HDMEC, HUVEC and HBMEC cell lines.

<p>Static assays were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031567#s4" target="_blank">Material and methods</a> section. The mean number of iRBC per mm² ± S.E. of 3 to 5 biological replicates were counted, and are expressed as % of bound parasites per mm² of the ItG control.</p

Erythrocyte remodelling by malaria early gametocytes

In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains instead obscure how sequestration is established, and how the earliest sexual stages, morphologically similar to asexual trophozoites, modify the infected erythrocytes and their cytoadhesive properties at the onset of gametocytogenesis. Here, purified P. falciparum early gametocytes were used to ultrastructurally and biochemically analyse parasite-induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do not modify its surface with adhesive 'knob' structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface by these parasites, and the expression of the var gene family, which encodes 50-60 variants of PfEMP1, is dramatically downregulated in the transition from asexual development to gametocytogenesis. Cytoadhesion assays show that such gene expression changes and host cell surface modifications functionally result in the inability of stage I gametocytes to bind the host ligands used by the asexual parasite to bind endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites

Mutant DNMT3A: a marker of poor prognosis in acute myeloid leukemia

The prevalence, the prognostic effect, and interaction with other molecular markers of DNMT3A mutations was studied in 415 patients with acute myeloid leukemia (AML) younger than 60 years. We show mutations in DNMT3A in 96 of 415 patients with newly diagnosed AML (23.1%). Univariate Cox regression analysis showed that patients with DNMT3A(mutant) AML show significantly worse overall survival (OS; P = .022; hazard ratio [HR], 1.38; 95% confidence interval [CI], 1.04-1.81), and relapse-free survival (RFS; P = .005; HR, 1.52; 95% CI, 1.13-2.05) than DNMT3A(wild-type) AMLs. In a multivariable analysis, DNMT3A mutations express independent unfavorable prognostic value for OS (P = .003; HR, 1.82; 95% CI, 1.2-2.7) and RFS (P < .001; HR, 2.2; 95% CI, 1.4-3.3). In a composite genotypic subset of cytogenetic intermediate-risk AML without FLT3-ITD and NPM1 mutations, this association is particularly evident (OS: P = .013; HR, 2.09; 95% CI, 1.16-3.77; RFS: P = .001; HR, 2.65; 95% CI, 1.48-4.89). The effect of DNMT3A mutations in human AML remains elusive, because DNMT3A(mutant) AMLs did not express a methylation or gene expression signature that discriminates them from patients with DNMT3A(wild-type) AML. We conclude that DNMT3A mutation status is an important factor to consider for risk stratification of patients with AML. (Blood. 2012;119(24):5824-5831

A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages.

Item does not contain fulltextAchievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission