We thank Francisco Candal, CDC Technology Transfer Office, Atlanta Georgia for HBEC-5i cells. This work was funded by the Wellcome Trust (4 year PhD studentship to AC and Senior Fellowship in Basic Biomedical Science to JAR, grant number 084226).

General recommendations
Human brain microvascular endothelial cell (HBEC-5i) culture has been previously described in 6, 8. P. falciparum parasites were cultured as in 9. Both HBEC-5i and P. falciparum parasite cultures should be kept in sterile conditions at all times. All reagents should be pre-warmed at 37&deg;C. We recommend to regularly check for mycoplasma contamination10 by PCR (Minevera Biolabs, following manufacturer's instructions). The protocol is summarized in Figure 1.
1. Endothelial cell routine culture
<ol>
 <li>Prepare the necessary reagents.</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td>
 Medium to prepare</td>
 <td>
 Reagents</td>
 <td>
 Quantity</td>
 </tr>
 <tr>
 <td>"DMEM incomplete"</td>
 <td>DMEM-F12 Ham</td>
 <td>500ml</td>
 </tr>
 <tr>
 <td> &nbsp;</td>
 <td>L-glutamine 200mM</td>
 <td>5ml</td>
 </tr>
 <tr>
 <td> &nbsp;</td>
 <td>Penicillin/streptomycin 100X</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td> &nbsp;</td>
 <td>NaOH 1M</td>
 <td>1.3 ml (adjust pH to 7.4)</td>
 </tr>
 <tr>
 <td>"DMEM complete"</td>
 <td>"DMEM incomplete"</td>
 <td>450ml</td>
 </tr>
 <tr>
 <td> &nbsp;</td>
 <td>Foetal Bovine Serum heat-inactivated</td>
 <td>50ml</td>
 </tr>
 <tr>
 <td> &nbsp;</td>
 <td>Endothelial cell growth supplement</td>
 <td>5 ml</td>
 </tr>
 </tbody>
</table>
 
<ol start="2">
 <li>Culture HBEC-5i in a vented 25cm2 flask with 10ml DMEM complete medium in a 37&deg;C incubator with 5% CO2.</li>
 <li>Passage cells when they become confluent. Remove the old medium by suction and wash twice using DMEM incomplete medium or tissue culture grade PBS (Ca2+ and Mg2+ free) pre-warmed to 37&deg;C.</li>
 <li>Add 1ml of pre-warmed Trypsin-EDTA (0.025% Trypsin, 0.5mM EDTA), swirl to cover the entirety of the flask and incubate for &tilde;2 min at 37&deg;C.</li>
 <li>Check under inverted microscope that at least 90% of cells have been detached. If necessary, gently knock the bottom of the flask to dislodge adherent cells. Add 10ml of DMEM complete medium to block the trypsin and transfer cells into a 15ml conical tube. Centrifuge for 4 min at 300 g at room temperature (RT).</li>
 <li>Discard the supernatant and resuspend the pellet with 10ml of DMEM complete medium. Pipette the solution up and down to thoroughly resuspend the cells.</li>
 <li>Add 1 or 2 ml of the cell suspension into a new culture flask and add 8ml of fresh medium to maintain the culture. Assess culture growth every day under an inverted microscope and change medium every 2 or 3 days before it turns yellow.</li>
</ol>
2. Preparing endothelial cells for a selection
<ol>
 <li>Two days prior to the day of the selection, add fibronectin diluted in sterile PBS (2 &mu;g/cm2) in one (or more) 60 mm Petri dish. Incubate the dish for 5 to 20 min at 37&deg;C, then remove the fibronectin solution, which can be stored at 4&deg;C for a month and re-used once.</li>
 <li>Passage cells as described in sections 1.3. to 1.5. Assuming 100% confluent cells were detached and resuspended in 10ml DMEM complete medium (section 1.6., resuspend with an equivalent volume of medium if the confluency is lower, e.g. resuspend with 8ml if the confluency was 80%), add 1.5ml of the suspended cells to the fibronectin coated Petri dish and another 1.5ml of DMEM complete medium.</li>
 <li>Place the seeded Petri dish in the incubator. Note: Ideally, the confluency will be around 90% at the time of selection two days later.</li>
 <li>Optional. To activate HBEC-5i, add the TNF (Tumour Necrosis Factor) cytokine at a final concentration of 50&mu;g/ml 24 hours prior to the selection.</li>
</ol>
3. Plasmodium falciparum routine culture
<ol>
 <li>Prepare the necessary reagents (see table here under). Prepare human red blood cells (RBC) by separating whole blood (group O+) by passage through a leukocyte depletion filter (see "Methods in Malaria Research" publication11 for general malaria parasites culturing). Wash RBC twice by centrifuging at 400 g for 5 min and resuspending them with 10 ml RPMI incomplete. Keep washed RBC at 4&deg;C in incomplete medium at 50% haematocrit.</li>
</ol>
<table border="1">
<tbody>
 <tr>
 <td>Medium 			to prepare</td>
 <td>Reagents</td>
 <td>Quantity</td>
 </tr>
 <tr>
 <td>"RPMI 			incomplete"</td>
 <td>RPMI 1640 (with 			bicarbonate)</td>
 <td>500ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Hepes 1M</td>
 <td>12.5ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Glucose 20%</td>
 <td>5ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>L-glutamine 			200mM</td>
 <td>5ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Gentamycin 			50mg/ml</td>
 <td>250&mu;l</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>NaOH 1M</td>
 <td>0.7 ml (adjust 			pH to 7.2)</td>
 </tr>
 <tr>
 <td>&quot;RPMI 			complete"</td>
 <td>&quot;RPMI 			incomplete"</td>
 <td>450ml</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Pooled human 			(non-immune) serum</td>
 <td>50ml</td>
 </tr>
 </tbody>
</table>
 
<ol start="2">
 <li>Culture P. falciparum infected RBC with RPMI complete medium at 2% haematocrit and incubate at 37&deg;C with 3% CO2, 1% O2, and 96% N2. Make Giemsa smear11 daily to assess the stage of development of parasites (Figure 2).</li>
 <li>Regularly (approximately once a week) synchronize culture by sorbitol treatment12. The day prior the selection assay, aim for a ring-stage culture at 5% parasitaemia or more (ideally at least 10%).</li>
</ol>
4. Selection of P. falciparum for cytoadhesion to endothelial cells
<ol>
 <li>On the day of the assay, the parasite culture should be at pigmented trophozoite stage (Figure 2)(ideally 10% parasitaemia) while HBEC-5i culture should be at 50 to 100% confluency (ideally 90%). 30&mu;l packed cell volume of parasite culture is needed per HBEC-5i Petri dish.</li>
 <li>Wash parasites twice by centrifuging (500g for 5 min) 1.5ml of parasite culture. Discard supernatant and resuspend with 10ml of freshly made, warmed, DMEM incomplete medium. Repeat the wash a second time. Resuspend the 30&mu;l packed cell volume with 1.5ml of DMEM incomplete with 1% BSA.</li>
 <li>Wash the HBEC-5i coated Petri dish twice aspirating medium and adding 3ml of incomplete DMEM.</li>
 <li>Add the solution of parasites to the HBEC-5i dish and incubate at 37&deg;C for 75min. Resuspend parasites twice (after 30 and 60 min) during the incubation by gently rocking the dish in the four directions, as well as clockwise and anti-clockwise.</li>
 <li>After incubation, wash the dish 5 times by aspirating medium, using a plastic Pasteur pipette to add 3 ml of warm DMEM incomplete medium, and gentle rocking. If many dishes are being used, keep them on a warm surface, such as a large flask filled with water at 37&deg;C.</li>
 <li>Check the dish under an inverted microscope. If many uninfected RBC are still visible, do more washes as described above. Handle the Petri dish very carefully to avoid any risk of contamination.</li>
 <li>Remove medium from the dish and add 3ml of warm RPMI complete medium with 40&mu;l packed cell volume of fresh uRBC.</li>
 <li>Place the dish in an airtight incubating chamber, gas it for 3 min and place the chamber in an incubator at 37&deg;C overnight.</li>
 <li>The day after, harvest the parasites by washing with RPMI incomplete medium, in a similar way as described in section 4.4. but more vigorously. Keep all medium used (containing resuspended RBC) in a 15ml conical tube. Check under inverted microscope that all RBC have been removed from the dish.</li>
 <li>Pellet the parasites, discard the supernatant, resuspend in 5ml RPMI complete medium and place the mixture in a flask for normal culturing.</li>
</ol>
5. Representative Results
Unselected parasites show a low level of binding to HBEC-5i (Figure 3A). Thus, after the first round of selection, very few parasites will be harvested and it may take up to a month of culturing to reach a parasitaemia sufficient for the second round of selection. After each round of selection, more and more parasites bind to the endothelial cells and the selections can be repeated at shorter time intervals. After 4-5 rounds of selection, high-binding parasite populations are obtained (Figure 3).
In our hands, the binding of HB3-HBEC to HBEC-5i or TNF activated HBEC-5i was of similar level (data not shown).
The protocol described here was tested with 4 P. falciparum strains: HB3, IT/FCR3, 3D7 and Dd2 (Table 1). Only the latter proved not able to bind to HBEC-5i, even after 5 rounds of selection.
HB3 parasites were also selected for cytoadherence to Human Dermal and Pulmonary Microvascular Endothelial Cells (HDMEC and HPMEC). After 4 rounds of selection, using the method described here, a high-binding population was obtained on both HDMEC and HPMEC (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/3122/3122fig1.jpg" /> 
Figure 1. Overview of the selection process.
<img alt="Figure 2" src="/files/ftp_upload/3122/3122fig2.jpg" /> 
Figure 2. Development stages of P. falciparum infected red blood cells. Giemsa smear visualized under microscope at 1000X magnification.
<img alt="Figure 3" src="/files/ftp_upload/3122/3122fig3.jpg" /> 
Figure 3. Typical example of parasites binding to HBEC-5i. (A) The blue layer is HBEC-5i fixed with glutaraldehyde and stained with Giemsa, visualized under microscope at 1000X magnification. Pictures were taken after uRBC and unbound iRBC were washed away. The left panel shows a single P. falciparum HB3 (unselected) parasite bound to HBEC-5i. In the right panel the HB3-HBEC parasites had been selected for 5 rounds. (B) Data represents the average of two independent experiments, each performed in duplicate. The number of parasites bound per endothelial cell was counted.
<img alt="Table 1" src="/files/ftp_upload/3122/3122table1.jpg" /> 
Table 1. Summary of Plasmodium falciparum strains that were successfully selected for binding to HBEC-5i. Note that HB3 was also selected on TNF-activated HBEC-5i. After 5 rounds of selection, the Dd2 strain showed no increase in binding to HBEC-5i compared to unselected-Dd2.
<img alt="Figure 4" src="/files/ftp_upload/3122/3122fig4.jpg" /> 
Figure 4. Binding of P. falciparum HB3 parasites to dermal (HDMEC) and pulmonary (HPMEC) endothelial cells, before and after 4 rounds of selection. Although the culture medium for HDMEC and HPMEC slightly differs (see supplier's instructions), the protocol used for the selection was identical as with HBEC-5i. Pictures taken at 400X magnification.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DMEM-F12 Ham </td>
 <td>Sigma </td>
 <td>D6421 </td>
 <td>For DMEM complete medium</td>
 </tr>
 <tr>
 <td>L-glutamine 200mM </td>
 <td>GIBCO </td>
 <td>25030 </td>
 <td>For DMEM and RPMI complete medium</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin 100X (10000 units/ml and 10mg/ml) </td>
 <td>ScienCell </td>
 <td>0503 </td>
 <td>For DMEM complete medium</td>
 </tr>
 <tr>
 <td>Foetal Bovine Serum heat-inactivated </td>
 <td>ScienCell </td>
 <td>0025 </td>
 <td>For DMEM complete medium</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA (0.025% Trypsin,&nbsp; 0.5mM EDTA) </td>
 <td>ScienCell </td>
 <td>0103 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Endothelial cell growth supplement </td>
 <td>ScienCell </td>
 <td>1052 </td>
 <td>For DMEM complete medium</td>
 </tr>
 <tr>
 <td>Tissue culture treated 60 mm X 15 mm Petri dishes </td>
 <td>BD </td>
 <td>353002 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Human Fibronectin </td>
 <td>Millipore </td>
 <td>FC010 </td>
 <td>Use at 2 &mu;g/cm2</td>
 </tr>
 <tr>
 <td>TNF </td>
 <td>R&amp;D Systems </td>
 <td>210-TA </td>
 <td>optional, use at 50 &mu;g/ml</td>
 </tr>
 <tr>
 <td>RPMI 1640 </td>
 <td>Lonza </td>
 <td>BE12-167F </td>
 <td>For RPMI complete medium</td>
 </tr>
 <tr>
 <td>Gentamycin 50mg/ml </td>
 <td>Lonza </td>
 <td>17-518Z </td>
 <td>For RPMI complete medium</td>
 </tr>
 <tr>
 <td>Hepes 1M </td>
 <td>Lonza </td>
 <td>BE17-737E </td>
 <td>For RPMI complete medium</td>
 </tr>
 <tr>
 <td>HDMEC </td>
 <td>ScienCell </td>
 <td>2000 </td>
 <td>Primary cell line</td>
 </tr>
 <tr>
 <td>HPMEC</td>
 <td>ScienCell</td>
 <td>3000</td>
 <td>Primary cell line</td>
 </tr>
 <tr>
 <td>HBEC-5i</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Obtained from Francisco Candal&nbsp;(fcandal@cdc.gov)</td>
 </tr>
 </tbody>
</table>
Table 2. Materials

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	<li>Scherf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+variation+in+malaria:+in+situ+switching,+relaxed+and+mutually+exclusive+transcription+of+var+genes+during+intra-erythrocytic+development+in+Plasmodium+falciparum.">Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum.</a> Embo. J. 17, 5418-5426 (1998).</li><li>MacPherson, G. G., Warrell, M. J., White, N. J., Looareesuwan, S., Warrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cerebral+malaria.+A+quantitative+ultrastructural+analysis+of+parasitized+erythrocyte+sequestration.">Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration.</a> Am. J. 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We have nothing to disclose.

The hallmark of cerebral malaria is sequestration of P. falciparum iRBC within the brain microvasculature2, 3. However, in vitro cultures of P. falciparum only poorly cytoadhere to HBEC-5i, a model for human brain microvascular endothelium. Here we developed a straightforward assay to enrich a population for binding to HBEC-5i, a more "in-vivo like" phenotype. Three out of 4 P. falciparum strains were successfully selected using this method. Furthermore, HB3 was also selected on HDMEC and HPMEC, indicating that this protocol can be used for various parasite and endothelial cell types.
Different hypotheses may explain the lack of binding with the Dd2 strain. The most likely being the fact that this parasite line is knobless, which deeply impedes the cytoadherence13, 14.
We recommend culturing unselected parasites alongside the selection process to provide a control for comparison. This will allow, for example, comparing the transcriptome of binding and non-binding parasites, with the hope of discovering parasite ligand candidates.
TNF is a cytokine found at high level in cerebral malaria patients and has been show to induce the expression of many surface proteins of HBEC-5i (Claessens et al, in preparation and 8, 15, 16). In this case, the amount of bound iRBC was similar in normal HBEC-5i compared to activated HBEC-5i.
This "sequestration model" can also be used to study the molecular interaction between the iRBC and the endothelial cell, as well as the effect of putative anti-cytoadherence drugs. In this case, we recommend plating HBEC-5i in smaller wells, such as "8-well chamber slides" (BD 354628) or "CultureWell" (Sigma C7735-20EA).

selection of plasmodium falciparum parasites for cytoadhesion to human brain endothelial cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Watch this Scientific Journal Video about Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells at JoVE.com

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

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18.9K Views.  University of Edinburgh. An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Video: Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

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 ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

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. ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

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 ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...