Falciparum-infected Red Blood Cells Research Articles

Zwitterionic self-assembled nanoparticles as carriers for Plasmodium targeting in malaria oral treatment

The current decline in antimalarial drug efficacy due to the evolution of resistant Plasmodium strains calls for new strategies capable of improving the bioavailability of antimalarials, especially of those whose lipophilic character imparts them a low solubility in biological fluids. Here we have designed, synthesized and characterized amphiphilic zwitterionic block copolymers forming nanoparticles capable of penetrating the intestinal epithelium that can be used for oral administration. Poly(butyl methacrylate-co-morpholinoethyl sulfobetaine methacrylate) (PBMA-MESBMA)-based nanoparticles exhibited a specific targeting to Plasmodium falciparum-infected vs. parasite-free red blood cells (74.8%/0.8% respectively), which was maintained upon encapsulation of the lipophilic antimalarial drug curcumin (82.6%/0.3%). The in vitro efficacy of curcumin upon encapsulation was maintained relative to the free compound, with an IC50 around 5 μM. In vivo assays indicated a significantly increased curcumin concentration in the blood of mice one hour after being orally fed PBMA-MESBMA-curcumin in comparison to the administration of free drug (18.7 vs. 2.1 ng/ml, respectively). At longer times, however, plasma curcumin concentration equaled between free and encapsulated drug, which was reflected in similar in vivo antimalarial activities in Plasmodium yoelii yoelii-infected mice. Microscopic analysis in blood samples of fluorescently labeled PBMA-MESBMA revealed the presence of the polymer inside P. yoelii yoelii-parasitized erythrocytes one hour after oral administration to infected animals.

A deep learning approach to the screening of malaria infection: Automated and rapid cell counting, object detection and instance segmentation using Mask R-CNN.

Accurate and early diagnosis is critical to proper malaria treatment and hence death prevention. Several computer vision technologies have emerged in recent years as alternatives to traditional microscopy and rapid diagnostic tests. In this work, we used a deep learning model called Mask R-CNN that is trained on uninfected and Plasmodium falciparum-infected red blood cells. Our predictive model produced reports at a rate 15 times faster than manual counting without compromising on accuracy. Another unique feature of our model is its ability to generate segmentation masks on top of bounding box classifications for immediate visualization, making it superior to existing models. Furthermore, with greater standardization, it holds much potential to reduce errors arising from manual counting and save a significant amount of human resources, time, and cost.

Fluorescence Lifetime Imaging as a Noninvasive Tool to Study Plasmodium Falciparum Metabolism.

Fluorescence lifetime imaging (FLIM) measures the characteristic time that a molecule remains in an excited state prior to emitting a photon and returning to the ground state. It is a state-of-the-art and noninvasive technique that has the potential to obtain signature physiological information during malaria blood-stage infection. The use of autofluorescence signals from intrinsic fluorophores obviates the need to tag the cells with synthetic molecules or to modify their gene expression. Furthermore, it permits time-lapse interrogation of the changes that occur from invasion to the point when the parasite takes over the host for its own survival mechanisms, as well as changes in the health of the parasite due to extrinsically applied metabolic disruptors. In this chapter, we present a protocol to investigate the autofluorescence lifetime signals of both normal red blood cells (RBC) and P. falciparum-infected RBCs. The data shared with this protocol reveals that there is a significant overall increase in autofluorescence lifetime in infected erythrocytes compared to the healthy uninfected ones. We include a metabolic experiment that confirms that the signals obtained from this imaging technique are key metabolites in energetics of the parasites. Furthermore, facilitating these protocols makes it possible to identify infected RBC based on FLIM signals alone, which presents a huge potential for the study of energetic effects of antimalarials and fast, noninvasive diagnosing.

Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for carbohydrate binding

VAR2CSA is the placental-malaria–specific member of the antigenically variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. It is expressed on the surface of Plasmodium falciparum-infected host red blood cells and binds to specific chondroitin-4-sulfate chains of the placental proteoglycan receptor. The functional ∼310 kDa ectodomain of VAR2CSA is a multidomain protein that requires a minimum 12-mer chondroitin-4-sulfate molecule for specific, high affinity receptor binding. However, it is not known how the individual domains are organized and interact to create the receptor-binding surface, limiting efforts to exploit its potential as an effective vaccine or drug target. Using small angle X-ray scattering and single particle reconstruction from negative-stained electron micrographs of the ectodomain and multidomain constructs, we have determined the structural architecture of VAR2CSA. The relative locations of the domains creates two distinct pores that can each accommodate the 12-mer of chondroitin-4-sulfate, suggesting a model for receptor binding. This model has important implications for understanding cytoadherence of infected red blood cells and potentially provides a starting point for developing novel strategies to prevent and/or treat placental malaria.

Gravidity-dependent associations between interferon response and birth weight in placental malaria

BackgroundMaternal malarial infection leads to poor perinatal outcomes, including low birth weight from preterm delivery and/or fetal growth restriction, particularly in primigravidas. In placental malaria, Plasmodium falciparum-infected red blood cells cause an inflammatory response that can interfere with maternal–fetal exchange, leading to poor growth. The type I interferon (IFN-I) pathway plays an immunomodulatory role in viral and bacterial infections, usually by suppressing inflammatory responses. However, its role in placental malaria is unknown. This study examines the cytokine responses in placental tissue from subsets of malaria-infected and uninfected women, and attempts to correlate them with particular birth outcomes.Methods40 whole placental biopsy samples were obtained from pregnant women at least 16 years of age recruited to a larger prospective chemoprevention trial against malaria. These were patients at Tororo District Hospital in Uganda, an area of high malaria endemicity where approximately 40% of women have evidence of malaria infection at delivery. They were regularly followed at a local clinic and monitored for fever, with blood smears performed then and at time of delivery to diagnose malaria infection. Placenta biopsies were taken for histological diagnosis of placental malaria, as well as quantitative PCR analysis of genes in the IFN-I pathway (IFN-β, IL-10 and MX-1). Parameters such as infant birth weight and gestational age were also recorded.ResultsHistological analysis revealed placental malaria in 18 samples, while 22 were found to be uninfected. RT-PCR analysis showed a four-fold increase in IFN-β and IL-10 expression in multigravidas with placental malaria when compared to gravidity-matched, uninfected controls. This effect was not observed in primigravidas. Interestingly, linear regression analysis showed a positive association between IFN-β levels and higher birth weights (β = 101.2 g per log2-fold increase in IFN-β expression, p = 0.042). This association was strongest in primigravidas with placental malaria (β = 339.0, p = 0.006).ConclusionsThese results demonstrate differential regulation of the IFN-I pathway in placental malaria according to gravidity, with the greatest anti-inflammatory response seen in multigravidas. The association between IFN-β levels and higher birth weight also suggests a protective role for IFN-I against fetal growth restriction in placental malaria.

Generation of a Peptide Vaccine Candidate against Falciparum Placental Malaria Based on a Discontinuous Epitope.

In pregnant women, Plasmodium falciparum-infected red blood cells adhere to the placenta via the parasite protein VAR2CSA. Two vaccine candidates based on VAR2CSA are currently in clinical trials; however, these candidates failed to elicit strain-transcending antibody responses. We previously showed that a cross-reactive monoclonal antibody (3D10) raised against the P. vivax antigen PvDBP targets epitopes in VAR2CSA. We now aim to design a peptide vaccine against VAR2CSA based on the epitope that generated 3D10. We mapped the epitope to subdomain 1 (SD1) of PvDBP and identified a peptide that contained the minimal sequence. However, this peptide did not elicit cross-reactive VAR2CSA antibodies in mice. When tested against a broader, overlapping peptide array spanning SD1, 3D10 in fact recognized a discontinuous epitope consisting of three segments of SD1. These findings presented the challenge to generate this larger structural epitope as a synthetic peptide since it is stabilized by two pairs of disulfide bonds. We overcame this using a synthetic scaffold to conformationally constrain the SD1 peptide and coupled it to keyhole limpet hemocyanin (KLH). The SD1-KLH conjugate elicited antibodies in mice that cross-reacted with VAR2CSA. This strategy successfully recapitulated a discontinuous epitope with a synthetic peptide and represents the first heterologous vaccine candidate against VAR2CSA.

Extracellular vesicles derived from Plasmodium-infected and non-infected red blood cells as targeted drug delivery vehicles.

Among several factors behind drug resistance evolution in malaria is the challenge of administering overall doses that are not toxic for the patient but that, locally, are sufficiently high to rapidly kill the parasites. Thus, a crucial antimalarial strategy is the development of drug delivery systems capable of targeting antimalarial compounds to Plasmodium with high specificity. In the present study, extracellular vesicles (EVs) have been evaluated as a drug delivery system for the treatment of malaria. EVs derived from naive red blood cells (RBCs) and from Plasmodium falciparum-infected RBCs (pRBCs) were isolated by ultrafiltration followed by size exclusion chromatography. Lipidomic characterization showed that there were no significant qualitative differences between the lipidomic profiles of pRBC-derived EVs (pRBC-EVs) and RBC-derived EVs (RBC-EVs). Both EVs were taken up by RBCs and pRBCs, although pRBC-EVs were more efficiently internalized than RBC-EVs, which suggested their potential use as drug delivery vehicles for these cells. When loaded into pRBC-EVs, the antimalarial drugs atovaquone and tafenoquine inhibited in vitro P. falciparum growth more efficiently than their free drug counterparts, indicating that pRBC-EVs can potentially increase the efficacy of several small hydrophobic drugs used for the treatment of malaria.

System-wide biochemical analysis reveals ozonide antimalarials initially act by disrupting Plasmodium falciparum haemoglobin digestion.

Ozonide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel), are synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a “multi-omics” workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of ozonide-treated P. falciparum infected red blood cells revealed a rapid depletion of short Hb-derived peptides followed by subsequent alterations in lipid and nucleotide metabolism, while untargeted peptidomics showed accumulation of longer Hb-derived peptides. Quantitative proteomics and ABPP assays demonstrated that Hb-digesting proteases were increased in abundance and activity following treatment, respectively. Ozonide-induced depletion of short Hb-derived peptides was less extensive in a drug-treated K13-mutant artemisinin resistant parasite line (Cam3.IIR539T) than in the drug-treated isogenic sensitive strain (Cam3.IIrev), further confirming the association between ozonide activity and Hb catabolism. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in ozonide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short ozonide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate ozonide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage ozonide-induced damage.

Immune sensing of synthetic, bacterial and protozoan RNA by TLR8 requires coordinated processing of RNA substrates by RNaseT2 and RNase2

Abstract Human TLR8 is an essential sensor of bacterial RNA which induces proinflammatory and Th1 cytokines. Crystallography revealed that TLR8 binds both uridine and short single-stranded RNA, but the RNases that process the RNA are still unknown. Herein, we demonstrate that two endosomal endoribonucleases, RNaseT2 and RNase2, can process RNA for TLR8 recognition. In the endosome, RNase2 and -T2 act synergistically to release uridine from oligoribonucleotides, with RNaseT2 cleaving preferentially before and RNase2 after uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic ligands all required RNase processing for TLR8 activation, and uridine supplementation restored RNA recognition in RNASE2−/− or RNASET2−/− but not double knockout cells. Strikingly, peripheral blood mononuclear cells from RNaseT2 hypomorphic patients did not respond to bacterial RNA but did to small-molecule TLR8 agonists. Our data provide a novel insight into TLR8 activation and its differences between cell types and species.

Heparin-Coated Liposomes Improve Antiplasmodial Activity and Reduce the Toxicity of Poupartone B

AbstractPoupartone B is an alkyl cyclohexenone derivative isolated from Poupartia borbonica. This compound demonstrated promising antimalarial activity (IC50 < 1 µg/mL), however, it was not devoid of toxicity. Thus, to reduce the adverse side effects of this natural bioactive molecule, a delivery strategy involving a nanostructure was formulated. Additionally, poupartone B-loaded liposomes were coated with heparin, a glycosaminoglycan that is known to target proteins on the surface of Plasmodium falciparum-infected red blood cells. The quantification of the compound in the formulation was performed by HPLC-DAD, while heparin was quantitated by 1H NMR spectroscopy. The liposomes’ antiplasmodial activity was tested on artemisinin-resistant P. falciparum isolate, and toxicity was evaluated on human HeLa cells and zebrafish embryos. Throughout this research, the formulation demonstrated higher antiplasmodial activities against both P. falciparum strains and a significant decrease of in vitro toxicity. The formulation improved the selectivity index 2 times in vitro and proved to be 3 times less toxic than the compound alone in the zebrafish embryo acute toxicity test. Hence, the use of this strategy to deliver natural products in Plasmodium-infected cells, particularly those with a narrow therapeutic margin, is proposed.

Clearance of Plasmodium falciparum-infected red blood cells by NK cells and monocytes

Abstract Antibodies naturally acquired during repeated exposure to the parasite Plasmodium falciparum are essential for control of blood-stage malaria. NK cells are innate immune cells with an important role in containing viral infections and tumor growth, mainly by direct cytotoxicity and inflammatory cytokine production. Recently, we reported that primary human NK cells lyse Plasmodium falciparum-infected red blood cells (iRBCs) via antibody-dependent cellular cytotoxicity (ADCC) in presence of IgG isolated from adults living in a malaria-endemic region. This NK-mediated lysis results in inhibition of parasite growth in vitro. Live imaging showed release of parasitophorous vacuoles (PV) from iRBCs after incubation with NK cells. Many of these PVs gave propidium iodide-positive signals, suggesting loss of viability of P. falciparum inside the PVs after NK-mediated iRBC lysis. I am interested to know whether and how PVs are eliminated and postulate that monocytes play a role in clearance of PVs, thereby reducing the inflammatory response. To observe the interaction of NK cells and monocytes with PVs, I isolated PVs from iRBCs by mild saponin treatment and mechanical disruption of RBC membranes. Purified PVs bind IgG isolated from adults living in a malaria-endemic region, showing that adults develop antibodies against Plasmodium antigens at the surface of PVs. Preliminary live imaging experiments show phagocytosis of PVs by monocytes. Currently I am testing the contribution of Fc receptors on monocytes to phagocytosis of PVs and the interaction of NK cells with PVs.

Inactivation of Plasmodium falciparum in whole blood using the amustaline and glutathione pathogen reduction technology.

BACKGROUNDRisk of transfusion‐transmitted (TT) malaria is mainly associated with whole blood (WB) or red blood cell (RBC) transfusion. Risk mitigation relies mostly on donor deferral while a limited number of countries perform blood testing, both negatively impacting blood availability. This study investigated the efficacy of the pathogen reduction system using amustaline and glutathione (GSH) to inactivate Plasmodium falciparum in WB.STUDY DESIGN AND METHODSWB units were spiked with ring stage P. falciparum infected RBCs. Parasite loads were measured in samples at time of infection, after 24 hours at room temperature (RT), and after a 24‐hour incubation at RT post‐treatment with 0.2 mM amustaline and 2 mM GSH. Serial 10‐fold dilutions of the samples were inoculated to RBC cultures and maintained up to 4 weeks. Parasitemia was quantified by cytometry.RESULTSThe P. falciparum viability assay has a limit of detection of a single live parasite per sample. Input parasite titer was >5.7 log10 TCID50 per mL. A 24‐hour incubation at RT paused parasite development in controls, but they retained viability and infectivity when tested in culture. In contrast, no infectious parasites were detected in the amustaline/GSH‐treated sample after 4 weeks of culture.CONCLUSIONA robust level of P. falciparum inactivation was achieved in WB using amustaline/GSH treatment. Parasite log reduction was >5.7 log10 TCID50 per mL. Development of such a pathogen reduction system may provide an opportunity to reduce the risk of TT malaria and improve blood availability.

Blood group and size dependent stability of P. falciparum infected red blood cell aggregates in capillaries.

For Plasmodium falciparum related malaria (B50), one of the outstanding host factors for the development of severe disease is the ABO blood group of malaria patients, where blood group O reduces the probability of severe disease as compared to individuals of groups A, B, or AB. In this report, we investigate the stability of rosette aggregates in malaria caused by Plasmodium falciparum in microflows. These flows are created in microfluidic channels with stenosis-like constrictions of different widths down to ones narrower as the rosette's diameter. High speed videos were recorded and analyzed by a MATLAB© based tracking software (SURF: SUrvival of Rosettes in Flow). We find a correlation of rosette size, channel diameter, and blood group regarding the mobility of the rosettes. Following the concept of a thermodynamic model, we find a critical width of the stenosis for rosette rupture during their passage. Our data reveal that under physiologically relevant conditions, rosettes in blood group A have a higher rosette frequency and stability as compared to blood group O (BG O), which constitutes a crucial factor promoting the observed protection in BG O individuals against severe malaria in non-O individuals.

Dual shape recovery of red blood cells flowing out of a microfluidic constriction.

Micropipette aspiration, optical tweezers, rheometry, or ecktacytometry have been used to study the shape recovery of healthy human Red Blood Cells (RBCs) and measure associated relaxation times of the order of 100-300 ms. These measurements are in good agreement with the Kelvin-Voigt model, which describes the cell as a visco-elastic material, predicting that its relaxation time only depends on cell intrinsic properties. However, such mechanical solicitation techniques are far from being relevant regarding RBC solicitation in vivo. In this paper, we report for the first time the existence of two different behaviors of the RBC shape recovery while flowing out of a microfluidic constricted channel. The calculation of the viscous stress corresponding to the frontier between the two recovery modes confirms that the RBC resistance to shear is the elastic property dominating the transition between the two recovery behaviors. We also quantified associated recovery times and report values as low as 4 ms-which is almost two decades smaller than the typical RBC relaxation time-at high viscosity and flow velocity of the carrier fluid. Although we cannot talk about relaxation time because the cell is never at rest, we believe that the measured shape recovery time arises from the coupling of the cell intrinsic deformability and the hydrodynamic stress. Depending on the flow conditions, the cell mechanics becomes dominant and drives the shape recovery process, allowing the measurement of recovery times of the same order of magnitude than relaxation times previously published. Finally, we demonstrated that the measurement of the shape recovery time can be used to distinguish Plasmodium falciparum (causing malaria) infected RBCs from healthy RBCs.

Development of DNA Aptamers Against Plasmodium falciparum Blood Stages Using Cell-Systematic Evolution of Ligands by EXponential Enrichment.

New biomarkers have to be developed in order to increase the performance of current antigen-based malaria rapid diagnosis. Antibody production often involves the use of laboratory animals and is time-consuming and costly, especially when the target is Plasmodium, whose variable antigen expression complicates the development of long-lived biomarkers. To circumvent these obstacles, we have applied the Systematic Evolution of Ligands by EXponential enrichment method to the rapid identification of DNA aptamers against Plasmodium falciparum-infected red blood cells (pRBCs). Five 70 b-long ssDNA sequences, and their shorter forms without the flanking PCR primer-binding regions, have been identified having a highly specific binding of pRBCs versus non-infected erythrocytes. Structural analysis revealed G-enriched sequences compatible with the formation of G-quadruplexes. The selected aptamers recognized intracellular epitopes with apparent Kds in the μM range in both fixed and non-fixed saponin-permeabilized pRBCs, improving >30-fold the pRBC detection in comparison with aptamers raised against Plasmodium lactate dehydrogenase, the gold standard antigen for current malaria diagnostic tests. In thin blood smears of clinical samples the aptamers reported in this work specifically bound all P. falciparum stages versus non-infected erythrocytes, and also detected early and late stages of the human malaria parasites Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. The results are discussed in the context of their potential application in future malaria diagnostic devices.

Multi-omic Characterization of the Mode of Action of a Potent New Antimalarial Compound, JPC-3210, Against Plasmodium falciparum

The increasing incidence of antimalarial drug resistance to the first-line artemisinin combination therapies underpins an urgent need for new antimalarial drugs, ideally with a novel mode of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, potent in vivo efficacy against murine malaria, and favorable preclinical pharmacokinetics including a lengthy plasma elimination half-life. To investigate the impact of JPC-3210 on biochemical pathways within P. falciparum-infected red blood cells, we have applied a "multi-omics" workflow based on high resolution orbitrap mass spectrometry combined with biochemical approaches. Metabolomics, peptidomics and hemoglobin fractionation analyses revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a specific depletion of short hemoglobin-derived peptides, peptidomics analysis revealed a depletion of longer hemoglobin-derived peptides, and the hemoglobin fractionation assay demonstrated decreases in hemoglobin, heme and hemozoin levels. To further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro β-hematin polymerization assays and showed JPC-3210 to be an intermediate inhibitor of β-hematin polymerization, about 10-fold less potent then the quinoline antimalarials, such as chloroquine and mefloquine. Further, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature compared with other known antimalarials. While JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. These studies revealed that the mode of action for JPC-3210 involves inhibition of the hemoglobin digestion pathway and elevation of regulators of protein translation. Importantly, JPC-3210 demonstrated rapid parasite killing kinetics compared with other quinolones, suggesting that JPC-3210 warrants further investigation as a potentially long acting partner drug for malaria treatment.

Assessing the Role of MIF in Plasmodium spp. Infections Using Ex Vivo Models.

Ex vivo techniques are a valuable tool for the investigation of how immune cells respond to Plasmodium spp. antigen, allowing examination of various aspects of the immune response under controlled conditions. Here we describe how to isolate peripheral blood mononuclear cells (PBMC) from donors and coculture them with purified P. falciparum-infected red blood cells (iRBC) to investigate the role of MIF during Plasmodium spp. infection.

Simultaneous Proton Transfer Reaction-Mass Spectrometry and electronic nose study of the volatile compounds released by Plasmodium falciparum infected red blood cells in vitro

The discovery that Volatile Organic Compounds (VOCs) can be biomarkers for several diseases has led to the conception of their possible application as diagnostic tools. In this study, we aimed at defining of diagnostic signatures for the presence of malaria transmissible stages in infected individuals. To do this, we compared VOCs released by asexual and sexual stage cultures of Plasmodium falciparum, the deadliest species of malaria, with those emitted by uninfected red blood cells (RBCs). VOC analysis was carried out with an innovative set-up, where each sample was simultaneously analysed by proton transfer reaction time of flight mass spectrometry (PTR-ToF-MS) and an electronic nose. PTR-Tof-MS results show that sexual stages are characterized by a larger emission of hexanal, compared with uninfected or asexual stage-infected RBCs, which makes them clearly identifiable. PTR-Tof-MS analysis also detected differences in VOC composition between asexual stages and uninfected RBCs. These results have been substantially replicated by the electronic nose analysis and may open the possibility to develop sensitive and easy-to-use devices able to detect sexual parasite stages in infected individuals. This study also demonstrates that the combination of mass spectrometry with electronic noses is a useful tool to identify markers of diseases and to support the development of optimized sensors.

Gamma Delta T Cells and Its Interactions with Extracellular Vesicles during Malaria

Background and ObjectiveExtracellular vesicles (EVs) have been described as microparticles produced by different cells in the body with the capacity of mediating cell‐cell communication. A clear mechanism of how various immune cells function during malaria infection is still undergoing investigations. With various states and roles of gamma delta T cells reported, its role in human cerebral malaria has not yet been defined as a study has suggested that it is essential in the development of murine cerebral malaria through its production of interferon gamma and tumor necrosis factor. However, there is currently no report on interactions between EVs derived from Plasmodium falciparum infected red blood cells (iRC‐EVs) and human gamma delta T cells. Our aim therefore, was to investigate the interactions between iRBC‐EVs and gamma delta T cells and the resulting immunological responses that could play a role in the pathogenesis of cerebral malaria.MethodBy using Ultracentrifugation, EVs derived from Plasmodium falciparum infected red blood cell (iRBC‐EVs) culture were isolated and purified. The iRBC‐EVs were characterized by western blot, Fast protein liquid chromatography and electron microscopy. Gamma delta T cells were generated from peripheral blood mononuclear cells(PBMCs) isolated from human blood and stimulated with Plasmodium falciparum culture supernatant and IL‐2 for 14 days. The generated gamma delta T cells were treated with the iRBC‐EVs and monitored for uptake, proliferation and activation using confocal microscopy and flow cytometry.Results and ConclusionWe found out that gamma delta T cells are capable of taking up iRBC‐EVs and were proliferating on co‐incubation with iRBC‐EVs and Plasmodium falciparum culture supernatant. In conclusion, our results show that there is an influence of iRBC‐EVs on gamma delta T cells which would form a basis for further investigations on the immunological responses mediated by these cells towards the development of cerebral malaria.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enumeration of the Invasion Efficiency of Plasmodium falciparum In Vitro in Four Different Red Blood Cell Populations Using a Three‐Color Flow Cytometry‐Based Method

A novel in vitro culture system using variable concentrations of biotin/streptavidin to label red blood cells (RBCs) that allows for the simultaneous comparison of growth rates in Plasmodium falciparum malaria parasite in four heterogeneous target RBC populations is described. Donor RBCs containing both P. falciparum-infected RBCs and non-infected RBCs at 0.5% parasitemia were first labeled with 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE) followed by co-culture with a mixture of equal numbers of four differentially biotin/streptavidin labeled RBC populations. After two to three schizogonic growth cycles, co-cultures were harvested and stained with streptavidin-phycoerythrin (SA-PE) followed by staining of parasite-infected RBCs with nucleic acid fluorochrome SYBR Green I. To demonstrate the application of this method, some target RBC populations that had sialic acid residues removed using neuraminidase treatment were mixed with RBC populations without enzymatic treatment and incubated with donor parasitized RBCs strain W2 (sialic acid-dependent) or 3D7 (sialic acid-independent). Significant less susceptibility to malaria parasite invasion was obtained with enzyme-treated RBC populations when compared with non-treated RBCs in blood samples from the same individual when using malaria parasite strain W2, whereas no difference in percent parasitemias was noted following infection with malaria parasite strain 3D7. This novel malaria culture method is cheap and provides increased sensitivity for direct comparison of parasite growth over time of any of the four RBC populations under identical conditions and eliminates the experimental bias due to contaminated donor RBCs. The application of biotin-labeled RBCs will therefore provide a better understanding of invasion phenotype-specific host-parasite interactions and the extent of complex malaria invasion mechanism. © 2019 International Society for Advancement of Cytometry.