This study was financially supported in part by the Novartis foundation for medical- and biological research (to PYM), the Gottfried and Julia Bangerter-Rhyner-Stiftung (to MW and PYM), and the research pool of the University of Fribourg (to PYM). Additional grants include the Swiss Government Excellence Scholarships for Foreign Scholars (to KAB and SM). We thank Isabelle Fellay and Solange Kharoubi Hess for technical support.

Human RBCs were obtained from the blood of healthy donors, in accordance with the guidelines of Swissethics (swissethics.ch).
NOTE: P. falciparum parasite cultures (3D7) and EV production were previously described in Mbagwu, et al.11 Because P. falciparum is a human pathogen, consult the local regulations for handling. The cultures should be kept sterile the entire time.
1. Fluorescence Labeling of EVs
NOTE: The following procedure takes advantage of the labeling technology to stably incorporate a green fluorescent dye (PKH67) with large aliphatic tails in the lipid region of the EV membrane. The reaction is performed in a 200 &#181;L final staining volume containing a 20 &#181;M final concentration of PKH67. Perform all steps at ambient temperature (20-25 &#176;C)
<ol>
	<li>Place 100 &#181;g of EVs in 1 mL of PBS in a microcentrifuge tube.</li>
	<li>Centrifuge the EVs in a microcentrifuge tube at maximum speed (20,000 x g) for 7 min to pellet. 
	NOTE: Since hemozoin is present in EVs, a red-dark brownish pellet should be observed. If the supernatant is reddish, then the EVs have lysed.</li>
	<li>After centrifugation, aspirate the supernatant carefully, in order to avoid removing any EVs. 
	NOTE: Minimize the amount of remaining buffer present before resuspending EVs in diluent C in order to increase reproducibility and efficiency of the staining.</li>
	<li>Resuspend the EV pellet in 100 &#181;L of diluent C (2X EV Suspension) and gently pipet to insure complete dispersion.</li>
	<li>Prepare a new microcentrifuge tube and add 4 &#181;L of the PKH67 ethanolic dye solution to 100 &#181;L of diluent C. Vortex well to mix. Prepare this 2x dye solution (40 &#181;M) immediately prior to staining. 
	NOTE: To avoid heterogeneous staining, do not add the PKH67 ethanolic dye solution directly to the EV pellet.</li>
	<li>Rapidly, combine the 100 &#181;L of 2X EV Suspension (step 1.4) with the 100 &#181;L of 2X dye solution (step 1.5). Then, mix the sample by pipetting up and down 10 times.</li>
	<li>Incubate the EV/Dye suspension from step 1.6 for 5 min protected from light and mix by vortexing 3-4 times during the 5 min incubation. 
	NOTE: Incubating the EV/Dye suspension for longer times provides no advantage, because the staining is so rapid.</li>
	<li>Stop the staining reaction by adding an equal volume (200 &#181;L) of serum and incubate for 1 min to allow binding of excess dye. 
	NOTE: Before stopping the staining, deplete the serum from EVs by centrifugation at 20,000 x g for 20 min.</li>
	<li>Wash 3 times with PBS and spin down at 20,000 x g for 5 min. Resuspend the EVs in 100 &#181;L of PBS to reach a final concentration of 1 &#181;g/&#181;L.</li>
</ol>
2. Visualize Uptake of EVs by Confocal Microscopy
NOTE: The following protocol describes the tracking of EV internalization by endothelial cells grown on glass coverslips. The endothelial cells are semi-immortalized human Bone Marrow Endothelial cells as described in12.
<ol>
	<li>Working in a sterile environment, coat coverslips with an excess of 0.01% poly-L-lysine for 10 min at room temperature (RT).</li>
	<li>Aspirate the poly-L-lysine solution and allow the coverslips to dry completely.</li>
	<li>Perform a viable endothelial cell count using Trypan blue exclusion and a hemocytometer.</li>
	<li>Transfer 1 x 105 endothelial cells in 1 mL of complete Endothelial Cell Growth Medium containing the Supplement Mix to the poly-L-lysine-coated coverslips and let the cells grow to a monolayer. 
	NOTE: The medium contains growth factors that allow cells to form a monolayer and to form tight junctions.</li>
	<li>Once the cells form a monolayer, carefully aspirate the medium and add 1 mL of fresh medium to wash the cells. Repeat the wash once more. Add 50 &#181;g of PKH67-labeled EVs and incubate for 4 h. 
	NOTE: To find the optimal time-point for uptake, a time-course can be performed.</li>
	<li>After 4 h, aspirate the medium, and wash the cells 3 times with PBS to remove excess and unbound EVs. Fix the cells with 3% paraformaldehyde solution in PBS for 15 min. 
	NOTE: Methanol can disrupt actin during the fixation process; therefore, it is best to avoid any methanol containing fixatives or other solvent-based fixatives. It is recommended to use methanol-free formaldehyde as a fixative.</li>
	<li>Wash the cells 3 times with PBS. Add carefully 250 &#181;L of 0.1% Triton X-100 in PBS to permeabilize the cells. Incubate at RT for 5 min.</li>
	<li>Wash 3 times with PBS. Add 400 &#181;L of blocking buffer (5% BSA in PBS) for 30 min at RT to block non-specific binding.</li>
	<li>Wash the cells once with PBS. Prepare the staining solution by diluting 5 &#181;L of the phalloidin stock solution in 200 &#181;L PBS/1% BSA per each cover slip. Pipet 200 &#181;L of the staining solution on the coverslip and incubate for 20 min at RT.</li>
	<li>Wash 3 times with PBS. Counterstain the DNA for 30 s with Hoechst 33342 at a final concentration of 2 &#181;g/mL in 200 &#181;L of PBS.</li>
	<li>Wash 2x the coverslip by adding 400 &#181;L of PBS. Carefully grab the coverslip with forceps and invert it on the top of a drop of antifade mounting media on a glass slide. Remove gently the excess media with a tissue and seal each side with nail polish.</li>
	<li>Store the slides protected from the light at 4 &#176;C.</li>
	<li>Visualize the cells using a confocal microscope at laser excitations 405, 488, and 543 nm and a 63X, 1.3 NA oil objective with fluorescent emissions at 450-470 nm (blue), 505-530 nm (green), and 620 nm. 
	NOTE: Typical F-actin staining results are shown in Figure 1.</li>
</ol>
3. Endothelial Cell Permeability
NOTE: Endothelial cell permeability is assessed by measuring the transfer of rhodamine B isothiocyanate-dextran (average MW 70,000) across the endothelial cell monolayer. Dextran provides an excellent tool to study vascular permeability.
<ol>
	<li>Count viable endothelial cells using 0.4 % Trypan blue exclusion and a hemocytometer.</li>
	<li>Plate the endothelial cells at a density of 0.6 x 105 cells to confluence in 24-well tissue culture inserts (0.4 &#181;m pore size) and leave undisturbed for at least 5 days to form differentiated monolayers. Refresh the medium every second day.</li>
	<li>Once the cells reach a monolayer, load the EVs (50 &#181;g in 1 mL) on the upper chamber. Add the rhodamine-dextran to the top well at a final concentration of 20 mg/mL. Incubate the cells at 37 &#176;C with 5% CO2.</li>
	<li>Monitor the transfer of the fluorescent dextran to the bottom well by measuring fluorescence emission from 40 &#181;L medium aliquots. Set up the multi-label microplate reader at 544 nm excitation and 590 nm emission for measurements. 
	NOTE: The amount of diffused dextran can be quantified using calibration curves established with the stock solution. In addition, kinetic experiments can be performed by removing small samples of the outer chamber medium during a time course (Figure 2). Even without any treatment, the dextran will diffuse through the cell monolayer.</li>
</ol>
4. Determine Puromycin Sensitivity
NOTE: Before transducing the cell lines with lentivirus, it is important to determine the kill curve of a selected drug for that particular cell line. To determine the minimum amount of drug concentration necessary to kill all of the cells, perform a dose response experiment using incremental doses of the selected drug. Because each mammalian cell line has a different sensitivity, before experimentation, the optimal concentration of the antibiotic should be determined by developing the kill curve titration as detailed below.
<ol>
	<li>Count the cells using the hemocytometer. Resuspend the endothelial cells at a concentration of 1 x 105 cells/mL in complete Endothelial Cell Growth Medium containing the Supplement Mix.</li>
	<li>Plate 1 x 104 cells into a 96-well plate in a final volume of 100 &#181;L of the Endothelial Cell Growth Medium.</li>
	<li>Add 100 &#181;L of medium containing the antibiotics to obtain a concentration of 0.1-10 &#181;g/mL (see Figure 3).</li>
	<li>Examine the cell viability every 2 days under a light microscope using a 20X objective. Culture the cells for 10-14 days and replace the media containing puromycin every 2-3 days depending on cell growth.</li>
	<li>Add 10 &#181;L/well MTS Reagent into each well, mix, and incubate for 4 h at 37 &#176;C in standard culture conditions.</li>
	<li>Shake the plate briefly on a shaker and measure absorbance of treated and untreated cells using a plate reader at 490 nm (Figure 3).</li>
</ol>
5. Transduction of Endothelial Cells with Lentiviral Vector
<ol>
	<li>Plate 1 x 105 endothelial cells in 1 mL of complete medium the day before transduction. Perform transduction when the cells have reached 50-80% confluency.</li>
	<li>Thaw the lentivirus on ice, and make aliquots of the viral stocks to avoid freeze-thaw cycles. Spin down the material at 200 x g for 30 s, in tubes before opening to avoid spill over.</li>
	<li>Add hexadimethrine bromide to the cells at a final concentration of 8 &#181;g/mL. 
	NOTE: Hexadimethrine bromide helps the transduction efficiency, however in some cases it might be toxic for the cells. Therefore, the optimal concentration should be determined by performing a killing curve before the transduction.</li>
	<li>Swirl the plate gently to mix. Add the appropriate quantity of viral particles (between 0.5-2 x 106 particles) at a suitable multiplicity of infection (MOI) and swirl the plate gently for 30 s to mix.</li>
	<li>Centrifuge the cell-viral particle mix in the centrifuge at RT for 45 min at 300 x g to increase the transduction efficiency. Incubate the cell-viral particle mixture at 37 &#176;C overnight.</li>
	<li>Remove carefully the viral particle-containing medium and discard appropriately. Replace it with fresh, pre-warmed complete culture medium.</li>
	<li>Start the selection with puromycin on the second day: remove the medium and replace it with fresh medium containing the correct concentration of puromycin as determined previously in section 4 (Figure 3).</li>
	<li>Harvest cells after puromycin selection and isolate RNA using an RNA isolating kit following the manufacturer&#39;s instructions.</li>
	<li>Perform cDNA synthesis with the selected miRNAs using a reverse transcription kit (see the Table of Materials).</li>
	<li>Set up the qPCR reaction according to the cited protocol13.</li>
</ol>

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The authors have nothing to disclose.

Several parasites, including Toxoplasma, Trypanosoma, Leishmania, and Trichomonas trigger the release of EVs by the infected host cell. Depending on the pathogens, the released EVs can modulate the host immune response or mediate cellular communication between the parasites6. Yet, there is little evidence suggesting how these small vesicles contribute to malaria disease. Here, we have described several ways to investigate the function of EVs during Plasmodium infection. For instance, the uptake of EVs by recipient cells in vivo can be efficiently monitored by labeling them with the PKH67 fluorescent dye. Several controls have to be performed in order to guarantee the specificity of the labeling. It is important to note that once mixed with the diluent C, the PKH67 dye tends to form aggregates, and it is therefore necessary to include a control with the dye alone without EVs to determine the non-specific staining. Confocal microscopy is the method of choice to differentiate between vesicles bound to the surface and truly internalized by the recipient cells. In addition, it provides an excellent tool to track and monitor the number of vesicles taken up by the cells. However, it remains unclear how EVs are processed in the recipient cells14; therefore, performing a time-course using time-points of 2, 4, and 8 h, may be very useful in order to determine the optimal conditions for uptake. The addition of subcellular compartment markers can be used to track the EVs inside the cells by confocal microscopy. EV uptake has been shown to occur via membrane fusion or endocytosis15. The fusion can be monitored by labeling EVs with octadecyl rhodamine B (R18). The fusion of EVs with the cellular membranes results in the dilution of R18, which leads to an increase in fluorescence16. Uptake of EVs by endocytosis can be studied by using specific inhibitors of actin, microtubule, and dynamin10.
To study the physiology of EVs, it is possible to label the iRBCs directly with the PKH dyes and co-culture the iRBCs with endothelial cells. The problem here is that the cells require different media and gas compositions and the amount of EV transfer might be low. Furthermore, vesicular proteins fused with a fluorescent tag can be expressed in the donor cells in vitro and in vivo. However, so far it has not been tested with iRBCs
Infected RBCs are cytoadherent to the microvasculature of the brain, in a process thought to be responsible to cause cerebral malaria17. Therefore, the transfer of parasite material via EVs is likely to influence endothelial cells and hence vascular function18. The in vitro assay described here, provides an easy way to investigate some of the basic properties of endothelial cells, as for example its permeability19. Additional test such as TEER or immunostaining with ZO-1 can provide more information about the tight junction integrity10.
Furthermore, the in vitro model turns out to be a powerful tool to study the molecular mechanisms behind the cellular communication between parasites and host cells. Besides uptake, this setup allows the investigation of molecular mechanisms involved in this process. For example, we have monitored the transfer of miR451a to the acceptor cells by Real-Time PCR and fluorescent in situ hybridization. Additionally, we describe how to generate cell lines overexpressing a particular miRNA, to directly address the role of these small RNA in vascular function10. However, several controls should be performed including testing non-transduced cells, since the transduction itself might affect the cellular function. Furthermore, miRNA inhibitors can be used to neutralize the effect of the miRNAs.

According to the World Health Organization, there were 212 million new cases of malaria worldwide in 2015 and approximately 429,000 people died, mainly children under five years of age1. The mechanisms leading to severe disease, which is often associated with vascular dysfunction, remain ill-defined2. Plasmodium-iRBCs secrete small bi-lipid membrane spheres known as extracellular vesicles (EVs). It is known that these EVs are potentially relevant to the infection process and to the host immune response to infection; however, little is known about the exact function of these small vesicles during malaria infection3. It is possible that they play two important roles: on one hand, they might contribute to the pathogenesis by activating macrophages4,5; and on the other hand, they might mediate cellular communication between parasites and between parasites and host6,7. In fact, parasites can transfer proteins or nucleic acids between each other via EVs. For example, Trypanosoma brucei rhodesiense EVs can transfer virulence factor Serum Resistance-Associated (SRA), and can target both other T. brucei and host erythrocytes8. Furthermore, P. falciparum-iRBCs communicate between each other by transferring nucleic acids within EVs. This allows the parasites to optimize and synchronize its growth. In fact, EVs might be the major regulator of gametocyte conversion, and therefore contribute to the regulation of the transmission stage7.
Not only do EVs regulate the parasites, they also mediate parasite-host interactions. We recently discovered that EVs from iRBCs contain host-derived microRNAs (miRNAs; small RNA species in the range of 21-25 nucleotides9) that were taken up by human endothelial cells. The miRNAs in the EVs form a stable complex with Ago2 (a member of the RNA-induced silencing complex), which once delivered to the recipient cells, is capable of specifically silencing gene expression and affecting the barrier properties of the cells10. Standard protocols have been developed to investigate the function of EVs. Here, we describe first a protocol that allows the fluorescent labeling of EVs to investigate their uptake by recipient cells. In addition, by using a confocal microscope, it is possible to track the EV&#39;s fate inside the cell. Several fluorescent dyes can be used to track EVs. The amine-reactive dye, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) and Calcein-AM become fluorescent once inside the vesicles. We prefer to use the amphiphilic label, PKH, because it gives a brighter and more uniform signal. This approach provides important information to understand the interactions between EVs and recipient cells. While in some cases EVs bind to the surface of the cells, some vesicles are rapidly taken up. Upon uptake, EVs deliver their cargoes to the cells, in which they exert their regulatory functions.
Here, we describe a protocol to measure the barrier function of the endothelial cells in vitro by quantifying the transfer of a fluorescent dextran through a cellular monolayer. More sensitive tracers can be used such as radiolabeled markers. However, they require special safety precautions for use. Other assays exist to measure the in vitro barrier function such as transendothelial electrical resistance (TEER), which measures tight junction integrity. Finally visualizing ZO-1, a tight junction protein, by immunofluorescence allows assessment of tight junction integrity as well10. Since EVs are complex and heterogeneous entities containing several cargoes with potential regulatory characteristic, it is useful to overexpress a specific RNA to study its effect on the recipient cell. Therefore, we also define a protocol that aims to generate stable cell lines expressing the miRNA of interest10.

<table><tbody><tr><td>PKH67 Green Fluorescent Cell Linker Mini Kit</td><td>Sigma-Aldrich</td><td>MINI67-1KT</td><td></td></tr><tr><td>Diluent C</td><td>Sigma-Aldrich</td><td>G8278</td><td></td></tr><tr><td>poly-L-lysine</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>PBS</td><td>ThermoFisher - Gibco</td><td>10010023</td><td></td></tr><tr><td>Phalloidin CF594</td><td>Biotium</td><td>#00045</td><td></td></tr><tr><td>Hoechst 33342</td><td>ThermoFisher</td><td>H3570</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>ThermoFisher</td><td>P36934</td><td></td></tr><tr><td>Rhodamine B isothiocyanate&amp;ndash;Dextran</td><td>ThermoFisher</td><td>R9379-250MG</td><td></td></tr><tr><td>Insert with PET membrane transparent Falcon&amp;nbsp;for plate 24 wells</td><td>Falcon</td><td>353095</td><td></td></tr><tr><td>Endothelial Cell Growth&amp;nbsp;Medium MV</td><td>Promocell</td><td>C-22020</td><td></td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma-Aldrich</td><td>P9620-10ML</td><td></td></tr><tr><td>MTS Cell Proliferation Colorimetric Assay Kit</td><td>Biovision</td><td>K300-500</td><td></td></tr><tr><td>hexadimethrine bromide</td><td>Sigma-Aldrich</td><td>107689-10G</td><td></td></tr><tr><td>MISSION&amp;nbsp;Lenti microRNA, Human hsa-miR-451a</td><td>Sigma-Aldrich</td><td>HLMIR0583</td><td></td></tr><tr><td>MISSION Lenti microRNA, ath-miR416, Negative Control 1 Transduction Particles</td><td>Sigma-Aldrich</td><td>NCLMIR001</td><td></td></tr><tr><td>MISSION&amp;nbsp;Lenti microRNA, Human</td><td>Sigma-Aldrich</td><td>NCLMIR0001</td><td></td></tr><tr><td>Leica TCS SP5</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>miRNeasy mini Kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>TaqMan MicroRNA Reverse Transcription Kit 1000 reactions</td><td>ThermoFisher</td><td>4366597</td><td></td></tr><tr><td>hsa-mir-451a&amp;nbsp;RT/750 PCR rxns</td><td>ThermoFisher</td><td>001141</td><td></td></tr><tr><td>U6 snRNA</td><td>ThermoFisher</td><td>001973</td><td></td></tr><tr><td>TaqMan Universal Master Mix II, with UNG</td><td>ThermoFisher</td><td>4440038</td><td></td></tr><tr><td>StepOnePlus Real-Time PCR System</td><td>ThermoFisher</td><td>4376600</td><td></td></tr></tbody></table>

evaluation of extracellular vesicle function during malaria infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

Here, we describe protocols to investigate the interactions of EVs with host cells. The uptake of fluorescently labeled EVs is monitored by confocal microscopy (Figure 1). Endothelial cells efficiently take up EVs, however the incubation time with EVs can be optimized to track the uptake. For a better localization of EVs inside the cells, stain actin with phalloidin. Next, we use a filter membrane on top of which a monolayer of endothelial cells grows. Rhodamine B isothiocyanate-dextran is applied on the top chamber of the filter membrane and then the permeability of the endothelial monolayer is measured by monitoring the transfer of the fluorescent dextran into the bottom chamber (Figure 2A). Typically, the permeability of the endothelial cells is affected upon incubation with an increasing amount of EVs (Figure 2B). It is important to grow the cells for several days to make sure they will form a monolayer, otherwise the dye will quickly diffuse through the wells. It is important to note that even without treatment, the dextran will diffuse slowly through the cell monolayer.
MicroRNAs are key components of EVs and after transfer to the recipient cells, they regulate gene expression. In order to investigate specifically the role of miRNAs, it is very useful to overexpress them in the recipient cell line. Here we describe how to transduce miRNAs into endothelial cells. The first step consists of determining the concentration of puromycin that kills endothelial cells in order to select stably transduced cells (Figure 4A). Finally, the level of expression of the miRNAs can be controlled and validated by qPCR (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57067/57067fig1.jpg" /> 
Figure 1: EVs are taken up by endothelial cells. Purified EVs were labeled in green with PKH67, and incubated for 4 h with a monolayer of endothelial cells. Untreated endothelial cells are used as a negative control. After extensive washing to remove unbound EVs, the cells were stained with phalloidin to stain actin (red) and Hoechst 33342 to stain the nucleus (blue). The cells were observed by confocal microscopy. Images were taken using a laser confocal microscope and a 63x 1.3 NA oil objective. Hoechst 33342 is excited at 405 nm and with emissions collected at 450-470 nm (blue); PKH67 is excited at 488 nm, emissions collected at 505-530 nm (green); and Phalloidin-594 is excited at 543 nm, emissions collected at 620 nm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57067/57067fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57067/57067fig2.jpg" /> 
Figure 2: Effect of EV uptake on endothelial barrier. (A) Schematic of the diffusion of rhodamine-labeled dextran: endothelial cells are grown to confluence on the top of a filter membrane, the fluorescent dextran is applied to the top chamber and diffuses over time through the membrane. The effect of EVs on permeability is assessed after the addition of EVs to the top chamber by measuring the rate of diffusion of the dextran to the bottom chamber. (B) An increasing amount of EVs are incubated with endothelial cells. The transfer of rhodamine-labeled dextran across a trans-well membrane over time allows for permeability measurements. Permeability across the endothelial layer is significantly increased after 2 h of incubation with 50 and 100 &#181;g/mL of EVs. Data represent mean (&#177; s.e.m.) from 3 experiments. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57067/57067fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57067/57067fig3.jpg" /> 
Figure 3: Titration of puromycin. The stock solution of puromycin (10 mg/mL) is diluted in RPMI 1640 supplemented with 10% heat-inactivated (56 &#176;C, 30 min) fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin/streptomycin. A total of 15 &#181;L is added to 10 mL of medium in tube A. Then 7.5 mL of the solution from tube A is mixed to 2.5 mL of RPMI to tube B. Finally, 100 &#181;L of the dilution are added to 100 &#181;L of cells to give a final concentration of 7.5 &#181;L/mL, 5.62 &#181;L/mL and so on. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57067/57067fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57067/57067fig4.jpg" /> 
Figure 4: Generation of endothelial cells overexpressing miR451a. (A) Determination of the killing curve of puromycin on endothelial cells. Endothelial cells were incubated with puromycin at increasing doses. The viability was measured by MTS assay after 72 h. The data represent the mean (&#177; s.e.m.) of one representative experiment. (B) RNAs derived from endothelial cells overexpressing miR451a or a negative control were harvested and the miR451a transcript was quantified by Real-Time PCR. qPCR results are normalized by the 2-Ct method using U6 as a housekeeper gene and expressed as mean (&#177; s.e.m.) (n = 3 experiments). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57067/57067fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Extracellular Vesicle Function During Malaria Infection at JoVE.com

Evaluation of Extracellular Vesicle Function During Malaria Infection

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

evaluation-of-extracellular-vesicle-function-during-malaria-infection

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.6K Views.  University of Fribourg. In dit werk beschrijven we protocollen voor het onderzoek naar de rol van extracellulaire blaasjes (EVs) uitgebracht door Plasmodium falciparum geïnfecteerd erytrocyten. In het bijzonder richten we ons op de interacties van EVW met endotheliale cellen.

Video: Evaluation of Extracellular Vesicle Function During Malaria Infection

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medicine

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

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