Name: an in vitro model for measuring immune responses to malaria in the context of hiv co-infection
Uploaded: 2015-10-06T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection at JoVE.com

C.A.M.F. and L.S. participated in protocol design, acquisition and analysis of data, and drafting of the article. 
The authors wish to thank Dr. Kain, Dr. Loutfy, Dr. Wasmuth, and Dr. Ayi for their contributions.
C.F. was supported by a CTN/Ontario HIV Treatment Network (OHTN) postdoctoral fellowship. L.S. is supported by an OHTN Junior Investigator Development Award. The present work was supported by a Canadian Institutes of Health Research (CIHR) operating grant (MOP-13721 and 115160), and a CIHR New Investigator Catalyst grant.

This protocol requires the recruitment of donors for serum and RBC to be used for parasite culture, and HIV(+) and uninfected donors for PBMC isolation. Institutional Review Boards must approve all studies and all donors must provide informed consent prior to blood draw.
CAUTION: Working with human blood samples and human malaria parasites requires precautionary measures. Always wear a lab coat, gloves, and work in a level 2 Biosafety cabinet. In the event of accidental percutaneous exposure to human malaria, report to Health and Safety for prophylactic treatment. Additional safety consideration should also be put in place for working with HIV(+) blood. Wear a back closing lab coat. Double glove (top glove should be latex). Perform all handling in a level 2 Biosafety cabinet. Do not use any glass or sharp objects. Do not use aspirator. Place all contaminated items in virox solution or bleach for a minimum of 1 hr prior to discarding. Wash all surfaces with virox and UV for 1 hr following use. Note that each institution will have their own specific biosafety regulations that need to be followed. Report all accidental exposures to HIV-infected blood to Health and Safety for evaluation and consideration of possible post-exposure prophylaxis.
Note: Different strains of P. falciparum malaria parasites exist. ITG was used for these experiments, but different strains can be used. Excellent instructions on freezing and thawing Plasmodium falciparum parasites are available at the MR4 website 13.
1. Making RPMI-A for Malaria Parasite Culture
<ol>
	<li>Make RPMI-0 by mixing 950 ml of ddH2O, 1 packet of RPMI-1640 powder, 6 g of HEPES, 2 g of sodium bicarbonate, and 1.35 mg of hypoxanthine.</li>
	<li>Thaw heat inactivated human serum from two different donors. AB donors are best but any can be used if parasites are grown in O type red blood cells (RBC). Invert tubes to mix. If serum contains particulate matter or is thick spin at 2,000 rpm and then filter liquid portion using a 0.45 &#181;m filter unit.</li>
	<li>Using a 0.2 &#181;m filter unit filter 180 ml of RPMI-0, 20 ml of human serum (10 ml from each donor), and 0.5 ml of 10 mg/ml gentamycin. 
	Note: Human serum can clog the filter so more than one filter unit may be required.</li>
	<li>Label the medium bottle with RPMI-A, the date, and the source of the serum. Refrigerate until required. RPMI-A may turn cloudy when refrigerated. This is normal, but increasing cloudiness is a sign of contamination. 
	Note: Parasite growth may vary in different donor serum. It is a good idea to test all human serum batches for good parasite growth before using.</li>
</ol>
2. Preparing Human Red Blood Cells for Parasite Culture
Note: Blood donors should be type O.
<ol>
	<li>Collect 7-10 ml of blood into acid-citrate-dextrose (ACD) tubes. Write the donor&#8217;s ID and date of collection on the label.</li>
	<li>Store blood at 4 oC until needed. Use blood for parasite cultures within 1 month.</li>
	<li>Wipe the top of the tube with 70% ethanol. Carefully remove stopper and discard. Transfer blood into a 15 ml tube. Spin for 3 min at 1,000 x g. Remove plasma by aspiration.</li>
	<li>Suspend RBC with equal volume of warm RPMI-0. Spin for 5 min at 1,000 x g. Remove the buffy coat by aspiration, resuspend in 5 ml of RPMI-0 and repeat the wash 2 more times.</li>
	<li>Remove the supernatant and add enough RPMI-A to produce a mixture that is 50% RBC by volume.</li>
	<li>Store at 4 oC until needed.</li>
</ol>
3. Maintaining Parasite Cultures
<ol>
	<li>Pre-warm RPMI-A to 37 oC.</li>
	<li>Place thawed parasites (see MR4 protocol for thawing procedure) into a T25 flask with 5 ml of RPMI-A and 75 &#956;l of washed human RBC for a hematocrit of ~3%. Note: Hematocrit measures the volume of RBC compared to total volume. To calculate hematocrit measure the volume of packed RBC to the total volume of medium plus RBC. Assume that the thawed parasites will contribute 75 &#956;l of RBC. By adding 150 &#956;l of RBC stock (which is equivalent to adding 75 &#956;l of packed RBC) to the flask there is a total of 150 &#956;l of RBC into 5 ml of medium, which equals to a hematocrit of 3% (150 &#956;l/5,000 &#956;l x 100% = 3%).</li>
	<li>Gas the flask for 30 sec with parasite gas mixture (1% O2, 3% CO2, balance N2). Seal and place the flask wide side down in a 37 oC incubator as to allow for the greatest surface area for gas exchange.</li>
	<li>To change the medium and check for parasitemia (needs to be done daily), carefully move the flask as to not disturb the RBC layer. Using a sterile unplugged Pasteur pipette draw off the culture medium without disturbing the blood layer.</li>
	<li>To check parasitemia, remove a 10 &#956;l sample from the blood layer, place it on a glass slide and using a second glass slide create a thin blood film. Allow slide to dry.</li>
	<li>Place 4 ml of fresh RPMI-A into the flask, gently mix, gas for 30 sec, and place in incubator at 37 &#176;C.</li>
	<li>Stain the slide using the Hema3 staining kit according to manufacturer&#8217;s protocol.
	<ol>
		<li>For optimal staining, dip slides in the fixing solution for 10 sec, solution I for 10 sec, and solution II for 30 sec. Rinse in water and leave slides to dry.</li>
	</ol>
	</li>
	<li>Calculate parasitemia by counting the number of PfRBC (stained dark purple) versus the total number of RBC (stained pink). Count a total of 300 cells to ensure accuracy.</li>
	<li>When parasites have reached a parasitemia of 5%, expand the culture into a T75 flask, using 40 ml of RPMI-A, and 500 &#956;l of RBC.</li>
</ol>
4. Parasite Synchronization
Note: The day before the experiment, synchronize the parasite culture by treating with alanine. Only ring stage parasites and uninfected RBC will survive this treatment. Alanine synchronization will give you a pure trophozoite culture the next day that can be used in the co-culture experiments. Make sure to start with a parasite culture that contains a majority of ring stage parasites.
<ol>
	<li>Prepare alanine solution by mixing 8.01 g of alanine (300 mM) and 0.365 g of Tris (10 mM) into 300 ml&#160;of ddH2O. Bring pH to 7.4. Filter sterilize using a 0.2 &#956;m filter unit.</li>
	<li>Pre-warm alanine solution to 37 oC.</li>
	<li>Spin down the parasite culture (5 min x 1,000 x g), and remove medium.</li>
	<li>Resuspend pellet in 19 volumes of alanine solution (1 ml packed RBC to 19 ml alanine solution). Incubate for 15 min at RT.</li>
	<li>Spin 5 min x 1,000 x g. Aspirate supernatant. Wash in RPMI-0 once. Aspirate supernatant and resuspend in RPMI-A and adjust hematocrit to ~3%. Gas flask and return to 37 oC. 
	Note: For co-culture experiments a minimum parasitemia of 5% trophozoites is needed, with 10% parasitemia considered optimal.</li>
</ol>
5. Parasite and RBC Preparation for Co-culture Experiments
<ol>
	<li>Use a ratio of 3 PfRBC per PBMC in co-culture experiments to induce an inflammatory response. To calculate total PfRBC, quantify parasitemia by making a thin blood smear as described in 3.5, and hematocrit by counting the number of RBC per mL of parasite culture using a hemocytometer. 
	Number of PfRBC = % parasitemia x total RBC/ml x ml of culture. For example: 10 ml of culture at 10 x 106 RBC/ml and 10% parasitemia is equal to 0.1 x 106/ml x 10 ml = 10 x 106 PfRBC.</li>
	<li>Spin the parasite culture for 5 min at 1,000 x g (RT). Aspirate medium, and resuspend at 6 x 106 PfRBC per ml in RPMI-S+ (500 ml RPMI-1640 supplemented with L-glutamine and HEPES, 10% heat inactivated FBS, 1.5 ml gentamicin, 5 ml of 100 mM sodium pyruvate, 5 ml of 10 mM MEM non-essential amino acids, 5 ml of 5mM &#946;-mercaptoethanol).</li>
	<li>Take control blood (uninfected RBC from the same donor used for maintaining the parasite culture), calculate hematocrit and resuspend in RPMI-S+ at the same number of RBC per ml as the parasite culture.</li>
</ol>
6. Isolation of Human Peripheral Blood Mononuclear Cells (PBMC)
<ol>
	<li>Collect venous blood in sodium heparin tubes (green top) from both a chronic HIV(+) donor and an HIV(-) control. Do not use EDTA as an anti-coagulant as EDTA&#8217;s calcium chelating action affects cell function. On average expect between 5-10 x 106 PBMC per 10 ml of blood. For a typical experiment collect 30 ml of blood from each donor. The blood volume will need to be adjusted according to the experimental requirements.</li>
	<li>As quickly as possible and definitely within an hour of blood being collected, remove the blood from the tubes and place into a plastic 50 ml tube. Monocytes in particular will be activated and stick to glass, so it is important that if glass blood collection tubes are used cells are removed as quickly as possible.</li>
	<li>Spin the blood at 1,000 x g for 15 min and collect plasma (this can be used for other studies if required &#8211; if not this step can be skipped). Dilute the blood in equal volume of cold DPBS.</li>
	<li>Place 15 ml of RT Ficoll into a 50 ml tube. Slowly, using a sterile plastic transfer pipette, layer the blood/DPBS solution over the Ficoll without any mixing. A maximum of 25 ml of blood/DPBS solution can be layered over each 15 ml of Ficoll.</li>
	<li>Spin the gradients for 30 min at RT at 600 x g. Make sure the &#8216;no brake&#8217; setting is on.</li>
	<li>Once the cells have spun, look for the interface between the two phases. This is where the PBMC are. Using a sterile plastic transfer pipette collect the PBMC from the interface (see Figure 1). Minimize contamination by RBC.</li>
	<li>Place collected cells in 50 ml tubes, with no more than 20 ml per tube. Top tube up to 50 ml with sterile cold DPBS.</li>
	<li>Spin the cells for 10 min at 4 oC at 300 x g.</li>
	<li>Discard supernatant, resuspend pellet in 5 ml sterile cold DPBS and pool all cells from the same donor into one tube. Top up to 50 ml with sterile DPBS, and repeat the wash steps 2 more times.</li>
	<li>Resuspend cells in 10 ml RPMI-S+ medium.</li>
	<li>Remove a 20 &#956;l aliquot and mix with 20 &#956;l of trypan blue. Pipette 10 &#956;l in a haemocytometer and count the living cells (cells that have not taken up the blue dye &#8211; all blue cells are dead). Use a haemocytometer to calculate cell number in a solution as follows. 
	Note: A haemocytometer has a grid of specified dimensions so that the area covered by the lines is known.
	<ol>
		<li>Make sure the haemocytometer is clean. Place the coverslip over the counting area. Load 10 &#956;l of cell solution by placing the pipette tip into one of the V-shaped wells. The area under the coverslip will fill by capillary action.</li>
		<li>Place the loaded haemocytometer under a microscope. The full grid of the haemocytometer contains 9 squares of 1 mm2 each. Count all cells within each large square. If too many cells are present, dilute the solution further and recount.</li>
		<li>Calculate cell concentration as follows: Total cells/ml = (total cells counted/# of squares) x dilution factor x 10,000 cells/ml, e.g., (500 cells/5 squares) x dilution factor of 20 x 10,000 cells/ml = 20 x 106 cells total.</li>
	</ol>
	</li>
	<li>Adjust medium to a final concentration of 10 x 106 per ml (RPMI-S+ medium).</li>
</ol>
7. Malaria/HIV Co-infection Culture
<ol>
	<li>Plate 100 &#956;l of isolated PBMCs and 400 &#956;l of RPMI-S+ medium to a 24-well plate (1 x 106 PBMC per well). Ensure that wells are set up in triplicate: 3 wells for uninfected RBC, 3 wells with PfRBC, 3 wells with medium, and 3 wells with PMA/Ionomycin. This is both for the HIV(+) and HIV(-) sample.</li>
	<li>For the PfRBC wells, place 3 x 106 PfRBC in each of the wells (500 &#956;l of parasite culture prepared in 5.2).</li>
	<li>For uninfected RBC wells, place 500 &#956;l of the uninfected RBC culture prepared in 5.3 in each of the wells.</li>
	<li>For medium wells, place 500 &#956;l of RPMI-S+ medium in each of the wells.</li>
	<li>For PMA/Ionomycin wells, prepare PMA/Ionomycin solution (2.5 pg/ml, 250 pg/ml respectively). Place 500 &#956;l of this solution in each well. 
	Note: As potent stimulators of T cell cytokine secretion, PMA and Ionomycin are used as a positive control to ensure cells are functional.</li>
	<li>Place plate at 37 oC in a 5% CO2 incubator.</li>
	<li>Optional: use excess cells for phenotypic analysis using flow cytometry or store in an RNA stabilization solution for future mRNA expression analysis.</li>
</ol>
8. Detection of Malaria Immune Responses
Note: Perform co-culture experiments for as long as 4 days. No medium change is required during this time. The optimal time point will depend on the cell type of interest and the question asked. An incubation of 12-48 hr is optimal for monocytic responses to PfRBCs, while lymphocyte responses were best observed at 72-96 hr. The time points will need to be optimized based on the experimental question. Shorter periods (2-4 hr) may be used if interaction between intact PfRBC and PBMCs is of interest.
<ol>
	<li>Spin plate at 300 x g for 3 min to pellet cells. Collect 700 &#956;l of culture supernatant from each well.</li>
	<li>Spin supernatant at 1,000 x g for 5 min to clear of any debris.</li>
	<li>Aliquot cleared supernatant as needed, label, and freeze at below -20 &#176;C until analysis of secreted factors is to be performed.</li>
	<li>Analyze cytokine/chemokine responses by ELISA or by bead array (follow the manufacturer&#8217;s suggested protocols).</li>
	<li>Collect cells remaining in the plate into an RNA stabilizing solution and utilize for mRNA expression analysis by quantitative real-time PCR 14-16.</li>
</ol>
9. Intracellular Flow Cytometry for Cell-specific Ccytokine Responses Using PBMC Co-cultured with P. falciparum Infected RBC
Note: As mentioned above the length of co-culture will depend on the cell type of interest. If interested in monocytic responses to PfRBC a shorter incubation period is needed. Times will be longer for innate lymphocyte responses &#947;&#948; T cells, NK cells, NKT cells), and longer still for CD4 and CD8 T cells. Optimization will be required.
<ol>
	<li>6-8 hr prior to analysis add 1 &#956;l of 1,000x brefeldin A to all wells. Return the plate to 37 oC incubator. After 6-8 hr incubation, place plate on ice for 15 min.</li>
	<li>Remove cells from the wells using vigorous pipetting and place them into labeled microcentifuge tubes. Scraping may be required to remove monocytes.</li>
	<li>Spin cells for 5 min at 1,000 x g. Remove supernatants. Resuspend cells in 500 &#956;l flow cytometry buffer (1x DPBS, 2% heat inactivated FBS, 0.02% sodium azide).</li>
	<li>Divide cells up so that each sample has: one tube for full antibody staining (240 &#956;l), and one tube for fluorescent minus one (FMO) control antibody staining (240 &#956;l). Pool a small amount of each sample (20 &#956;l) for the unstained flow cytometry control (this will be used in setting up the flow cytometer).</li>
	<li>Spin cells for 5 min at 500 x g. Remove supernatants.</li>
	<li>Incubate cells with unconjugated anti-human CD16/CD32 (5 &#956;g/ml) in 50 &#956;l flow cytometry buffer for 15 min at 4 oC to block Fc receptors.</li>
	<li>Add 1 ml of flow cytometry buffer to each tube. Spin cells for 5 min at 500 x g. Remove supernatants.</li>
	<li>Resuspend cells in 50 &#956;l of flow cytometry buffer with a pre-titrated concentration of fluorophore-conjugated antibodies to desired cell surface markers. Pre-mix enough antibody solution for all samples to be stained. Incubate cells at 4 oC for 20 min. Protect from light.Note: Amount of each antibody will have to be optimized. We routinely stain for CD56, CD3, &#947;&#948;,&#160;CD4, CD8, CD14. Titrated volumes for these antibodies are shown in Table 1.</li>
	<li>Add 1 ml of flow cytometry buffer to each tube. Spin cells for 5 min at 500 x g. Remove supernatants.</li>
	<li>Add 100 &#956;l of cytofix/cytoperm solution to each tube. Incubate cells for 20 min at 4 oC. Protect from light.</li>
	<li>Add 1 ml of perm/wash buffer (provided as 10x concentrate &#8211; dilute to 1x using ddH2O) to each tube. Spin cells for 5 min at 500 x g. Remove supernatants.</li>
	<li>Add 100 &#956;l of perm/wash buffer to FMO control antibody staining tubes and mix to resuspend cells.</li>
	<li>Add 100 &#956;l of perm/wash buffer with a pre-titrated concentration of fluorophore-conjugated antibodies to desired intracellular markers (i.e., cytokines/chemokines) to full antibody staining tubes. 
	Note: Amount of each antibody will have to be optimized. We routinely stain with anti-TNF and anti-IFN&#947; antibodies. Titrated volumes for these antibodies are shown in Table 1.</li>
	<li>Incubate all tubes for 30 min at 4 oC. Protect from light.</li>
	<li>Add 1 ml of Perm/Wash buffer to each tube. Spin cells for 5 min at 500 x g. Remove supernatants.</li>
	<li>Resuspend cells in 300 &#956;l flow cytometry buffer with 1% paraformaldehyde. Let sit for a minimum of 15 min to neutralize HIV.</li>
	<li>Prepare single antibody stained samples using compensation beads, to be used for compensation set up on the flow cytometer. The type of compensation beads will depend on the antibodies used (i.e., anti-mouse Ig, anti-rat/hamster Ig). Vortex beads. Add one drop of beads per antibody. Add equal amounts of antibody as used for staining. Add 200 &#956;l of flow cytometry buffer. These are now ready for use. There is no need to wash the beads.</li>
	<li>Acquire samples on a flow cytometer as soon as possible and within 24 hr for best results. Tandem dyes are susceptible to dissociation with storage so we recommend immediate acquisition if possible.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+a+method+for+evaluating+the+cryopreservation+of+whole+blood+and+peripheral+blood+mononuclear+cells.">Apoptosis: a method for evaluating the cryopreservation of whole blood and peripheral blood mononuclear cells.</a> Journal of immunological. 244 (1-2), 139-144 (2000).</li><li>Jeurink, P. V., Vissers, Y. M., Rappard, B., Savelkoul, H. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+responses+in+fresh+and+cryopreserved+peripheral+blood+mononuclear+cells:+kinetics+of+cell+viability,+cellular+subsets,+proliferation,+and+cytokine+production.">T cell responses in fresh and cryopreserved peripheral blood mononuclear cells: kinetics of cell viability, cellular subsets, proliferation, and cytokine production.</a> Cryobiology. 57 (2), 91-103 (2008).</li><li>Kvarnstr&#246;m, M., Jenmalm, M. C., Ekerfelt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cryopreservation+on+expression+of+Th1+and+Th2+cytokines+in+blood+mononuclear+cells+from+patients+with+different+cytokine+profiles,+analysed+with+three+common+assays:+an+overall+decrease+of+interleukin-4.">Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4.</a> Cryobiology. 49 (2), 157-168 (2004).</li><li>Venkataraman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+of+immune+responses.+XI.+Heightened+secretion+of+tumor+necrosis+factor-alpha+by+frozen+human+peripheral+blood+mononuclear+cells.">Effects of cryopreservation of immune responses. XI. Heightened secretion of tumor necrosis factor-alpha by frozen human peripheral blood mononuclear cells.</a> Cryobiology. 34 (3), 276-283 (1997).</li><li>Venkataraman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+immune+responses.+X.+Decrease+in+interleukin-12+production+by+frozen+human+peripheral+blood+mononuclear+cells+is+mediated+by+the+endogenously+hypersecreted+interleukin-10.">Effects of cryopreservation on immune responses. X. Decrease in interleukin-12 production by frozen human peripheral blood mononuclear cells is mediated by the endogenously hypersecreted interleukin-10.</a> Cryobiology. 33 (5), 581-588 (1996).</li><li>Reimann, K. A., Chernoff, M., Wilkening, C. L., Nickerson, C. E., Landay, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+lymphocyte+immunophenotype+and+proliferative+responses+in+cryopreserved+peripheral+blood+mononuclear+cells+from+human+immunodeficiency+virus+type+1-infected+donors:+implications+for+multicenter+clinical+trials.">Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected donors: implications for multicenter clinical trials.</a> The ACTG Immunology Advanced. Clinical and diagnostic laboratory immunology. 7 (3), 352-359 (2000).</li><li>Bennett, S., Breit, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variables+in+the+isolation+and+culture+of+human+monocytes+that+are+of+particular+relevance+to+studies+of+HIV.">Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV.</a> Journal of leukocyte biology. 56 (3), 236-240 (1994).</li></ol>

The authors have nothing to disclose.

Our protocol has been optimized in order to most realistically study HIV-malaria co-infection in vitro. First, fresh human RBCs and serum are required for malaria parasite culture. This is vital to obtain a healthy population of malaria parasites. Parasite lysates cannot be substituted for live parasites as cytokine production is much more rapid and intense when using live P. falciparum infected RBC (PfRBC) 17,18. In addition, activation of cell types like NK cells, requires whole PfRBCs, and does not work efficiently with parasite lysates. This may be due to the need for direct contact between the PfRBC and the leukocyte or may be due to the unstable nature of the parasite-derived ligands that interact with cell surface receptors 18. Malaria PfRBC cultures must also be well synchronized (Protocol 4) prior to the experiment. Synchronized PfRBCs improve the reproducibility of the experimental data. Although this protocol describes the use of trophozoite stage PfRBC for co-culture, it can easily be modified for the study of ring stage PfRBC.
Human leukocytes can be artificially infected with HIV, and this method has been used in studies of malaria-HIV co-infection research 20. However, this does not model the immune dysregulation that results from chronic HIV infection. In this co-culture system we use PBMCs isolated from human participants that are chronically infected with HIV-1. When participants are required for a study, it is important to carefully select this population. Inclusion and exclusion criteria are vital to ensure minimum variability and maximum reproducibility. Our criteria included a clear definition of chronic HIV infection (HIV-infected for &#62;1 year, with CD4+ T-cell count decline of &#62;50 cells/mm3/year) and the exclusion of anyone with a concurrent infection. This ensured that the results obtained could be attributed solely to the effect of chronic HIV infection, and not another infection. Additionally, since we were interested in innate immune responses to malaria we excluded donors that had previous malaria infection.
HIV-uninfected controls are also used for each HIV(+) donor, at every sampling time point, to allow for data normalization. An attempt should be made to match controls to their respective HIV(+) donor for at least age but preferably also sex (although in our previous study 12 we did not observe a significant difference in cell subsets or cytokine responses between female and male HIV-uninfected participants). If prospective sampling is planned maintaining the same HIV-uninfected control for each sampling time point is beneficial. Responses can vary from experiment to experiment due to multiple factors including the health of the PfRBCs, their degree of synchronization, parasitemia level and stage of maturity of PfRBCs, and hematocrit level. Care must be taken to keep as many of these consistent between experiments. Normalizing to an HIV-uninfected control allows for some accounting for these variables.
Using fresh cells is paramount to avoid artificial results. Freezing and thawing samples can have a significant impact on cell viability 21,22, cytokine production 23-26 and cell surface phenotypic markers 27.
If monocytes are of particular interest, it is important that glass is not used during the protocol. Monocytes adhere to glass and many other plastics 28. We use polypropylene plastic pipettes, transfer pipettes, and tubes throughout to minimize monocyte adherence and ultimate removal from our study cell populations.
When setting up the flow cytometry assay, any combination of antibodies can be used depending on the cells of interest. We were particularly interested in IFN&#947; and TNF production from NK cells, NKT cells and &#947;&#948; T cells. This defined our antibody panel. However, if using a different combination, it is important to optimize the amounts of antibody for each panel. This is vital to avoid erroneous data. Also, to avoid variability and ensure validity, FMOs are required to assess background cytokine levels for each condition (RBC, PfRBC, medium, and PMA/Ionomycin) and participant population (HIV(-) and HIV(+), Figure 2). Measuring in vitro intracellular cytokine production usually results in high background levels, especially when cells have been cultured prior to staining. Since the culture conditions affect background levels, appropriate FMOs ensure knowledge of the background level for each sample. Our supply of cells was limited, therefore we designed our FMOs to contain all surface stains but no intracellular stains. This allows for the specific comparison of cytokine background levels on all the different cell types studied. We also ran one single stain per experiment for each of the surface markers to confirm their background levels.
The protocol described is a versatile one, which can be used to examine responses within hours or days depending on the cells of interest. For optimal PfRBC-induced cytokine production from innate lymphocytes, we measured flow cytometry output after 2 or 3 days. When looking at monocytes, earlier time points (1 or 2 days) are recommended. This system allows for multiple cell types and cell responses to be distinguished by flow cytometry, secretory responses to be measured in cell supernatants, and expression profiles to be assessed in extracted RNA. Further, use of antibodies to block specific receptors or neutralize cytokines can be utilized in this system to further dissect mechanisms. We have successfully used IL-18 receptor blockade to implicate IL-18 receptor in PfRBC-induced IFN&#947; responses 12. This system represents a realistic method by which to assess a number of innate immune responses to malaria in the context of HIV infection.

Co-infection, infection with multiple concurrent infections, is the norm in natural environments. Co-infection can have a major impact on disease pathology and on the clinical management of each infection. In the context of co-infection, vaccine and drug efficacy, as well as diagnostic testing, can be negatively impacted (reviewed in 1). However, despite its importance, the majority of pathogen research considers only single infections.
Malaria and HIV-1 (HIV) are leading causes of morbidity and mortality globally. Areas of malaria and HIV endemicity share a wide geographic overlap, putting millions of people at risk of co-infection and consequently at risk for more severe clinical disease 2&#8211;10. The two diseases negatively interact. In HIV-infected individuals, higher HIV viral loads and temporary decreases in CD4+ T-cell counts can be seen during a malaria infection, while malaria parasite burdens and risk of clinical and severe malaria are higher in co-infected individuals 2,3,5,7,8,10. The mechanisms by which HIV increases malaria severity are not fully understood and warrant further investigation.
Here we describe a method by which malaria and HIV co-infection can be studied in vitro. Specifically, this method allows for the examination of malaria-specific immune responses in the context of HIV infection. Our protocol describes a versatile co-culture system using freshly isolated peripheral blood mononuclear cells (PBMCs) isolated from chronically HIV infected donors and in vitro cultured P. falciparum parasitized erythrocytes (PfRBC). The impact of HIV antiretroviral therapy on these responses can also be examined using prospectively collected PBMCs from HIV(+) donors pre- and post-therapy.
We have used this system to investigate the impact of HIV infection on malaria-specific innate immune responses 11,12, and were able to determine that malaria-specific IFN&#947; and TNF responses are impaired in NK cells, NKT cells, &#947;&#948; T cells from HIV(+) donors pre- and post-HIV antiretroviral therapy. Additionally, we were able to use this system to determine that monocytic functions are also impaired in HIV(+) donors, but recover post-HIV antiretroviral therapy.

<table><tbody><tr><td>alanine</td><td>Sigma</td><td>A7377</td><td></td></tr><tr><td>antibodies (see other table)</td><td></td><td></td><td></td></tr><tr><td>BD Cytofix/Cytoperm with BD GolgiPlug</td><td>BD</td><td>555028</td><td>includes brefeldin A, cytofix/cytoperm buffer and perm/wash buffer</td></tr><tr><td>BD Vacutainer ACD Solution A</td><td>BD</td><td>364506</td><td></td></tr><tr><td>BD Vacutainer Sodium Heparin</td><td>BD</td><td>17-1440-02</td><td></td></tr><tr><td>DPBS (no calcium, no magnesium)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F1051</td><td>heat inactivate before use</td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>17-1440-02</td><td></td></tr><tr><td>gentamycin (10 mg/ml)</td><td>Gibco</td><td>15710-064</td><td></td></tr><tr><td>Hema3 Staining Set</td><td>Fisher</td><td>122-911</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma</td><td>H9636</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma</td><td>I3909</td><td></td></tr><tr><td>MEM non-essential amino acids (10 mM)</td><td>Gibco</td><td>11140</td><td></td></tr><tr><td>PMA</td><td>Sigma</td><td>P8139</td><td></td></tr><tr><td>RPMI-1640 powder</td><td>Life-Technologies</td><td>31800-022</td><td></td></tr><tr><td>RPMI-1640 with L-glutamine and HEPES</td><td>Thermo Scientific</td><td>SH30255.01</td><td></td></tr><tr><td>sodium bicarbonate (powder, cell culture)</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Gibco</td><td>11360</td><td></td></tr><tr><td>Tris (Trizma base)</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>Trizol</td><td>Ambion</td><td>15596018</td><td></td></tr><tr><td>Trypan Blue (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>BD CompBead</td><td>BD</td><td>552843, 552845</td><td>depends on antibodies used</td></tr><tr><td>Parasite Gas Mixture</td><td>By special order</td><td></td><td>3% CO&lt;sub&gt;2&lt;/sub&gt;, 1% O&lt;sub&gt;2&lt;/sub&gt;, balance N&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2 &amp;#956;m filter unit</td><td></td><td></td><td></td></tr><tr><td>Glass slides</td><td></td><td></td><td></td></tr><tr><td>T25 flasks</td><td></td><td></td><td></td></tr><tr><td>T75 flasks</td><td></td><td></td><td></td></tr><tr><td>50 ml tubes</td><td></td><td></td><td></td></tr><tr><td>24 well culture plates</td><td></td><td></td><td></td></tr><tr><td>unplugged Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>plugged Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>sterile transfer pipettes</td><td></td><td></td><td></td></tr><tr><td>hemocytometer</td><td></td><td></td><td></td></tr><tr><td>flow cytometer (3 laser)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibodies used for flow cytometry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-TNF</td><td>eBiosciences</td><td>551487</td><td>FITC (fluorophore)
2 &amp;#956;l</td></tr><tr><td>Anti-IFN&amp;#947;</td><td>Biolegend</td><td>506507</td><td>PE (fluorophore) 
20 &amp;#956;l</td></tr><tr><td>Anti-CD8</td><td>Biolegend</td><td>300914</td><td>PE-Cy7 (fluorophore) 
1 &amp;#956;l</td></tr><tr><td>Anti-CD14</td><td>Biolegend; BD Biosciences</td><td>325624; 551487</td><td>Biotin (fluorophore) 
0.5 &amp;#956;l
Streptavin PE-TR (fluorophore) 
0.1 &amp;#956;l</td></tr><tr><td>Anti-CD56</td><td>Biolegend</td><td>318322</td><td>PerCP/Cy5.5 (fluorophore) 
5 &amp;#956;l</td></tr><tr><td>Anti-&amp;#947;&amp;#948;</td><td>Biolegend</td><td>331212</td><td>APC (fluorophore)
5 &amp;#956;l</td></tr><tr><td>Anti-CD3</td><td>Biolegend</td><td>300426</td><td>APC-Cy7 (fluorophore) 
5 &amp;#956;l</td></tr><tr><td>Anti-CD4</td><td>Biolegend</td><td>317424</td><td>Pacific Blue (fluorophore)
1 &amp;#956;l</td></tr></tbody></table>

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.

The graphs depict the levels of IFN&#947; production from NKT cells (Figure 2), using CD56+CD3+&#947;&#948;- gates to obtain the NKT cells population (data not shown). The cells were cultured for 72 hr prior to staining. Once stained, 100,000 CD3+ cells were acquired on the flow cytometer to obtain large enough populations of NK, NKT and &#947;&#948; cells (cells of interest). A minimum of 5,600 NKT cells are displayed on each graph. TNF production is obtained in the same manner (data not shown). The graphs clearly demonstrate that IFN&#947; production is lower in cells from HIV(+) individuals compared to HIV(-) individuals exposed to PfRBC.
Flow cytometry analysis is very subjective. It is therefore essential to have all the appropriate controls for each experiment (see Figure 2). Background levels are calculated using the FMO samples, which allows for a true representation of the cytokine staining. Cells stimulated with PMA/Ionomycin were used as a positive control (data not shown). PMA and Ionomycin are strong stimulators of T cell cytokine production. A lack of IFN&#947; production in these samples would most likely indicate a problem with the staining protocol. However, other variables such as cell viability or inactive reagents may also be at fault.
<img alt="Figure 1" src="/files/ftp_upload/52969/52969fig1.jpg" /> 
Figure 1.&#160;Depiction of Ficoll gradient post spin demonstrating the position of the PBMCs.
<img alt="Figure 2" src="/files/ftp_upload/52969/52969fig2.jpg" /> 
Figure 2.&#160;IFN&#947; production by Natural Killer T cells. The flow diagrams were obtained by gating on CD56+CD3+&#947;&#948;- cells (minimum of 5,600 events). IFN&#947; production is detectable in the HIV(-) sample stimulated with P. falciparum infected red blood cells. This cytokine response is no longer apparent in the context of a chronic HIV infection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52969/52969fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection at JoVE.com

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.

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. ...

an-vitro-model-for-measuring-immune-responses-to-malaria-context-hiv

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.2K Views.  University of Calgary. 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. 

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

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 ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Medicine

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 ...

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 ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

Generation of an In Vitro Cell Culture Model of Malaria-HIV Co-Infection

Source: Finney, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52969">An In Vitro&#160;Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates the generation of an in vitro malaria-HIV co-infection cell culture model. HIV-infected peripheral blood mononuclear cells co-cultured with Plasmodium falciparum parasitized erythrocytes potentially support each other&#39;s growth and provide insights into the immune responses and disease progression in HIV and malaria co-infections.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
CAUTION: Working with human blood ...

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

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