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>

<ol>
	<li>Graham, A. L., Cattadori, I. M., Lloyd-Smith, J. O., Ferrari, M. J., Bj&#248;rnstad, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+consequences+of+coinfection:+cytokines+writ+large.">Transmission consequences of coinfection: cytokines writ large.</a> Trends in parasitology. 23 (6), 284-291 (2007).</li><li>French, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+rates+of+malarial+fever+with+deteriorating+immune+status+in+HIV-1-infected+Ugandan+adults.">Increasing rates of malarial fever with deteriorating immune status in HIV-1-infected Ugandan adults.</a> AIDS (London, England). 15 (7), 899-906 (2001).</li><li>Hoffman, I. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+Plasmodium+falciparum+malaria+on+HIV-1+RNA+blood+plasma+concentration.">The effect of Plasmodium falciparum malaria on HIV-1 RNA blood plasma concentration.</a> AIDS (London, England). 13 (4), 487-494 (1999).</li><li>Kamya, M. R., et al. <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+HIV-1+infection+on+antimalarial+treatment+outcomes+in+Uganda:+a+population-based+study.">Effect of HIV-1 infection on antimalarial treatment outcomes in Uganda: a population-based study.</a> The Journal of infectious diseases. 193 (1), 9-15 (2006).</li><li>Kublin, J. G., et al. <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+Plasmodium+falciparum+malaria+on+concentration+of+HIV-1-RNA+in+the+blood+of+adults+in+rural+Malawi:+a+prospective+cohort+study.">Effect of Plasmodium falciparum malaria on concentration of HIV-1-RNA in the blood of adults in rural Malawi: a prospective cohort study.</a> Lancet. 365 (9455), 233-240 (2005).</li><li>Mermin, J., et al. <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+co-trimoxazole+prophylaxis,+antiretroviral+therapy,+and+insecticide-treated+bednets+on+the+frequency+of+malaria+in+HIV-1-infected+adults+in+Uganda:+a+prospective+cohort+study.">Effect of co-trimoxazole prophylaxis, antiretroviral therapy, and insecticide-treated bednets on the frequency of malaria in HIV-1-infected adults in Uganda: a prospective cohort study.</a> Lancet. 367 (9518), 1256-1261 (2006).</li><li>Patnaik, P., et al. <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+HIV-1+serostatus,+HIV-1+RNA+concentration,+and+CD4+cell+count+on+the+incidence+of+malaria+infection+in+a+cohort+of+adults+in+rural+Malawi.">Effects of HIV-1 serostatus, HIV-1 RNA concentration, and CD4 cell count on the incidence of malaria infection in a cohort of adults in rural Malawi.</a> The Journal of infectious diseases. 192 (6), 984-991 (2005).</li><li>Whitworth, J., et al. <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+HIV-1+and+increasing+immunosuppression+on+malaria+parasitaemia+and+clinical+episodes+in+adults+in+rural+Uganda:+a+cohort+study.">Effect of HIV-1 and increasing immunosuppression on malaria parasitaemia and clinical episodes in adults in rural Uganda: a cohort study.</a> Lancet. 356 (9235), 1051-1056 (2000).</li><li>Xiao, L., Owen, S. M., Rudolph, D. L., Lal, R. B., Lal, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+antigen-induced+human+immunodeficiency+virus+type+1+replication+is+mediated+through+induction+of+tumor+necrosis+factor-alpha.">Plasmodium falciparum antigen-induced human immunodeficiency virus type 1 replication is mediated through induction of tumor necrosis factor-alpha.</a> The Journal of infectious diseases. 177 (2), 437-445 (1998).</li><li>Francesconi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV,+malaria+parasites,+and+acute+febrile+episodes+in+Ugandan+adults:+a+case-control+study.">HIV, malaria parasites, and acute febrile episodes in Ugandan adults: a case-control study.</a> AIDS (London, England). 15 (18), 2445-2450 (2001).</li><li>Finney, C. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+infection+deregulates+Tim-3+expression+on+innate+cells:+combination+antiretroviral+therapy+results+in+partial+restoration.">HIV infection deregulates Tim-3 expression on innate cells: combination antiretroviral therapy results in partial restoration.</a> Journal of acquired immune deficiency syndromes. 63 (2), 161-167 (2013).</li><li>Finney, C. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+infection+deregulates+innate+immunity+to+malaria+despite+combination+antiretroviral+therapy.">HIV infection deregulates innate immunity to malaria despite combination antiretroviral therapy.</a> AIDS (London, England). 27 (3), 325-335 (2013).</li><li>Ljungstrom, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Malaria Research. , (2013).</li><li>Heid, C. A., Stevens, J., Livak, K. J., Williams, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+quantitative+PCR.">Real time quantitative PCR.</a> Genome Res. 6 (10), 986-994 (1996).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2-DDCT+method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Hensmann, M., Kwiatkowski, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+basis+of+early+cytokine+response+to+Plasmodium+falciparum.">Cellular basis of early cytokine response to Plasmodium falciparum.</a> Infection and immunity. 69 (4), 2364-2371 (2001).</li><li>Artavanis-Tsakonas, K., Riley, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+response+to+malaria:+rapid+induction+of+IFN-gamma+from+human+NK+cells+by+live+Plasmodium+falciparum-infected+erythrocytes.">Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum-infected erythrocytes.</a> J Immunol. 169 (6), 2956-2963 (2002).</li><li>Lambros, C., Vanderberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Plasmodium+falciparum+erythrocytic+stages+in+culture.">Synchronization of Plasmodium falciparum erythrocytic stages in culture.</a> The Journal of parasitology. 65 (3), 418-420 (1979).</li><li>Ludlow, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+inhibits+phagocytosis+and+inflammatory+cytokine+responses+of+human+monocyte-derived+macrophages+to+P.+falciparum+infected+erythrocytes.">HIV-1 inhibits phagocytosis and inflammatory cytokine responses of human monocyte-derived macrophages to P. falciparum infected erythrocytes.</a> PloS one. 7 (2), e32102 (2012).</li><li>Weinberg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+and+functional+activity+of+cryopreserved+mononuclear+cells.">Viability and functional activity of cryopreserved mononuclear cells.</a> Clinical and diagnostic laboratory immunology. 7 (4), 714-716 (2000).</li><li>Fowke, K. R., Behnke, J., Hanson, C., Shea, K., Cosentino, L. 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...

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 border="0" cellpadding="0" cellspacing="0">
	<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 (10mg/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 (10mM)</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 (100mM)</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% CO2, 1% O2, balance N2</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 gra...

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. ...

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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 Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.