RSV A2 was kindly provided by Dr. R. de Swart (Erasmus MC, Rotterdam, The Netherlands). MV and GF are supported by the Virgo consortium, funded by the Dutch government project number FES0908, and by the Netherlands Genomics Initiative (NGI) project number 050-060-452. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Note:
Experiments with HRSV must be performed in a biological safety cabinet in a biosafety level 2 laboratory. Collect all viral waste and material that has been in contact with the virus for proper disposal due to biological hazards.
Special precaution must be taken when using potentially infected human blood.
It is crucial to use endotoxin-free reagents throughout all protocols.
1. Culturing of HRSV A2
<ol>
	<li>Culture HeLa or HEp-2 cells in DMEM medium + 10% fetal calf serum (FCS) + 1% penicillin/streptomycin (P/S) in a 175 cm2 culture flask until they are approximately 50% confluent.</li>
	<li>Wash the cells three times with room temperature phosphate buffered saline (PBS).</li>
	<li>Thaw a vial with HRSV seeding stock virus at 37 &#176;C in a water bath. Dilute the virus stock so to keep the MOI 0.1 or below. This will prevent the generation of defective interfering particles (DIPs). Dilute the virus stock in 4 ml/flask infection medium (DMEM + 1% P/S) and add to the HeLa or HEp-2 cells. Keep this volume as low as possible without causing the cells to dry out. The lower the volume the higher the infection rate will be.</li>
	<li>Incubate 2-4 hr at 37 &#176;C, 5% CO2. Swirl the flask every 15 min to redistribute the medium.</li>
	<li>Take off the inoculum and wash one time with PBS.</li>
	<li>Add 15 ml culture medium (DMEM + 10%FCS + 1% P/S) and incubate for 3-5 days at 37 &#176;C, 5% CO2. Observe the cells with normal bright field microscopy for virus-induced cytopathological changes, e.g. syncytia formation and death and floating cells.</li>
	<li>When cytopathology appears, harvest HRSV by scraping cells and collect the virus suspension in a sterile 50 ml tube. Vortex the cell suspension. Scraping and vortexing of the cells releases more viral particles.</li>
	<li>Centrifuge the virus suspension for 10 min at room temperature at 1,800 x g&#160;to pellet the cell debris. HRSV in the supernatant does not pellet at this speed and the supernatant can be directly used to infect multiple 175 cm2 flasks or can be stored at -80 &#176;C as seeding stock in 1 ml aliquots for future use. Always snap-freeze HRSV in liquid nitrogen to prevent loss of infectious virus particles.</li>
	<li>As stated above, the seeding stock made with the above protocol is used to infect multiple 175 cm2 culture flasks. Repeat the exact protocol to produce more virus for purification. Purify up to 180 ml of virus suspension in one ultracentrifugation cycle.</li>
	<li>When cytopathology appears, harvest HRSV by scraping cells and collect supernatant. Vortex the virus suspension. Centrifuge for 10 min at room temperature at 1,800 x g&#160;to pellet the cell debris. HRSV in the supernatant does not pellet at this speed and the supernatant is aliquotted in 15 ml per 50 ml tube. Snap-freeze and store at -80 &#176;C until purification.</li>
</ol>
2. Purification of HRSV
Note: Before using an ultracentrifuge, be aware that it is a potentially dangerous piece of equipment. Read the manuals and perform the first ultracentrifugation spin under supervision of someone with experience.
<ol>
	<li>Clean ultracentrifugation buckets with 70% ethanol and let them dry in a biological safety cabinet.</li>
	<li>Place a sterile centrifuge tube in each bucket.</li>
	<li>Prepare 30% (w/v) sucrose in PBS solution, filter sterilize (0.2 &#956;m filter) and add 5 ml to the bottom of each centrifuge tube. For a faster protocol, the tubes with sucrose solution can be frozen. Be sure to keep them sterile.</li>
	<li>If the sucrose solution was not frozen, very carefully add 30 ml of HRSV solution on top of this sucrose solution. Pipette slowly to avoid disturbing the sucrose solution. Keep a clear demarcation. If the tubes were frozen, pour the viral suspension on top of the frozen sucrose solution and then let it thaw.</li>
	<li>Very carefully balance the six ultracentrifugation buckets with sterile PBS. Put the scale into a flow cabinet to keep everything sterile. Fill the tubes until 3-5 mm below the edge otherwise the centrifuge tubes can collapse. Balance the buckets to within 0.1 g and include the screw-on cap, just in case the caps are not perfectly matched.</li>
	<li>Centrifuge in the ultracentrifuge for 1.5 hr, 4 &#176;C at 20,000 rcf.</li>
	<li>Carefully remove the centrifuge tube from the bucket using sterile forceps. Virus particles will have passed through the sucrose layer and form a pellet. Conversely, cell-secreted soluble factors (e.g. cytokines) will stay in solution. Tip the centrifuge tube to drain all the liquid. Leave it upside-down on a sterile surface, e.g. a culture plate, to dry but don&#39;t let the pellet completely dry out.</li>
	<li>Carefully add 1 ml Hank&#39;s Balanced Salt Solution (HBSS) to the pellet and tip the centrifuge tube to drain the HBSS. Again let the tube dry upside-down on a sterile surface. Don&#39;t let the pellet completely dry out.</li>
	<li>Resuspend the pellet in 1 ml HBSS by pipetting up and down 40x. Try to prevent air bubble formation as this can destroy the virus.</li>
	<li>Aliquot the virus, snap-freeze in liquid nitrogen and store at -80&#160;&#176;C until further use. Use a new aliquot of virus for each experiment, as repeated freeze-thawing will reduce the infectious titer. When thawing, always thaw the virus at 37 &#176;C as HRSV is less stable between -20&#160;and&#160;15 &#176;C.</li>
</ol>
3. Quantification of HRSV
Note: To ensure reproducible data, repeat the quantification at least 3x in duplicate.
3.1. Infection of A549 cells
<ol>
	<li>Culture A549 cells in a 24-well plate until confluent.</li>
	<li>Wash the A549 cells two times with 1 ml of PBS at room temperature.</li>
	<li>Prepare 5-fold serial dilutions of HRSV in Reduced Serum Medium. Add 100 &#956;l of diluted virus to both wells. Make a minimum of 4 dilutions (e.g. 4-, 20-, 100-, and 500-fold). Include two extra wells infected with the highest dose of virus (these will be used for the isotype control) and include two mock-infected wells.</li>
	<li>Incubate the cells for 2 hr at 5% CO2, 37 &#176;C. Rock the 24-well&#160;plates every 15 min to prevent drying out.</li>
	<li>Remove the virus suspension and wash the cells 1x with 1 ml of PBS at room temperature.</li>
	<li>Prepare 4% (w/v) bovine serum albumin (BSA) in Reduced Serum Medium. Filter-sterilize (0.2 &#956;m filter) and add 500 &#956;l to each well.</li>
	<li>Incubate at 5% CO2, 37 &#176;C for 22 hr.</li>
</ol>
3.2. Preparation for FACS
<ol>
	<li>Remove medium and wash cells one time with 1 ml of room temperature PBS.</li>
	<li>Add 200 &#956;l of prewarmed trypsin (37 &#176;C) to each well and incubate for 10 min at 37 &#176;C, 5% CO2.</li>
	<li>Check cells for detachment under the microscope. If cells are detached, add 800 &#956;l DMEM + 10% FCS + 1% P/S to neutralize the trypsin. Resuspend the cells.</li>
	<li>Transfer the cells to a conical, nontissue culture treated 96-well plate. Centrifuge 5 min at 400 x g&#160;at room temperature. Resuspend the pellets in 200 &#956;l cold PBS.</li>
	<li>Centrifuge 5 min at 400 x g&#160;at 4 &#176;C. Decant the supernatant and resuspend in 200 &#956;l cold PBS.</li>
	<li>Centrifuge 5 min at 400 x g&#160;at 4 &#176;C. Decant the supernatant and resuspend in 150 &#956;l 80% ice-cold acetone.</li>
	<li>Fixate the cells at room temperature for 15 min.</li>
	<li>Centrifuge 5 min at 400 x g&#160;at 4 &#176;C. Decant the supernatant and resuspend in 200 &#956;l cold PBS + 0.1 % BSA.</li>
	<li>Centrifuge 5 min at 400 x g&#160;at 4 &#176;C. Decant the supernatant and resuspend in 50 &#956;l anti-NP-RSV-FITC (1:50 diluted in PBS + 1% BSA) or 50 &#956;l IgG2 anti mouse-FITC (this is an isotype control; therefore use same antibody concentration).</li>
	<li>For the remainder of the protocol, keep tubes in dark. Incubate at least 1 hr at 4 &#176;C but this can be extended to overnight.</li>
	<li>Add 150 &#956;l cold PBS + 0.1% BSA.</li>
	<li>Centrifuge 5 min at 400 x g&#160;at 4 &#176;C. Decant the supernatant and resuspend in 200 &#956;l cold PBS + 0.1% BSA.</li>
	<li>Centrifuge 5 min at 400 x g&#160;at 4 &#176;C. Decant the supernatant and resuspend in 200 &#956;l cold PBS + 0.1% BSA. Keep plate on ice and in the dark until FACS measurement.</li>
	<li>Measure the infected cells on a flow cytometer.</li>
</ol>
3.3. FITC-50 calculation
<ol>
	<li>Use a flow cytometry analysis program to set the cutoff using the negative control (isotype control). Use the Pseudocolor graph type. Draw a quadrant gate, where the positive cells are in field Q3. The percentage of false positives should be below 0.2% in both isotype controls.</li>
	<li>Export the data to a spreadsheet program and prepare a table with the percentage of FITC+ cells for each dilution. Subtract the average value of false positives (as determined by the isotype control) for each experiment for each value.</li>
	<li>Open a new file (XY scatter, Single X and multiple Y columns) in a statistics program. Copy data to this program: X = dilution, Y = % infected cells, n = 3, use the average of two measurements for each dilution. Perform a nonlinear regression, one-phase exponential decay statistical analysis. Check if the curve fits the data points, if not, choose a different statistical analysis.</li>
	<li>Copy the equation for the curve and the values for the equation to a spreadsheet program. Calculate the X value for Y = 50%. Accordingly determine the FITC-50 value by calculating the FITC-detected Infectious Particles/ml (FIP/ml) at the dilution at which there is 50% infection, using the following equation: 
	<img alt="Equation 1" fo:src="/files/ftp_upload/50766/50766eq1highres.jpg" src="/files/ftp_upload/50766/50766eq1.jpg" /> 
	A = average number of A549 cells/well 
	B = &#956;l virus per well used for infection 
	C = dilution at which there is 50% infection</li>
</ol>
4. Peripheral Blood Mononuclear Cell (PBMC) Isolation from Healthy Volunteer Blood
<ol>
	<li>Draw venous blood in 10 ml EDTA tubes from healthy volunteers upon informed consent. A volume of 10 ml of blood will yield approximately 107 PBMCs.</li>
	<li>From now on, work sterile in a flow-cabinet.</li>
	<li>Dilute the blood 2x with room temperature PBS in a 50 ml tube. Per 50 ml tube a maximal total volume of 35 ml of diluted blood can be used.</li>
	<li>Take a 50 ml sterile syringe and add a sterilized Luer lock needle. Take up density gradient media in the syringe. Place the needle on the bottom of a tube with diluted blood and carefully pipette 15 ml density gradient media under the diluted blood. Keep a clear demarcation between density gradient media and blood.</li>
	<li>Centrifuge at 800 x g&#160;at room temperature for 20 min with acceleration at 9 (max) and the brake off. Set the temperature of the centrifuge to 4 &#176;C after use.</li>
	<li>The density gradient centrifugation will result in multiple layers. On the bottom a pellet with erythrocytes is present with a silvery layer on top, which contains the neutrophils. The clear layer above this pellet contains the density gradient media. A thin layer between the density gradient media and the blood plasma layer on top contains PBMCs.</li>
	<li>Carefully collect the PBMCs with a Pasteur pipette and transfer cells to a new 50 ml tube. From now on keep the PBMCs on ice and use ice-cold PBS. Add ice-cold PBS to a volume of 50 ml and centrifuge at 400 x g&#160;for 10 min at 4 &#176;C (with the brake set at 9).</li>
	<li>Discard the supernatant by aspiration, resuspend the pellet in 50 ml of ice-cold PBS and centrifuge at 400 x g&#160;10 min at 4 &#176;C, repeat 2x.</li>
	<li>After discarding the supernatant, add 1 ml of ice-cold medium (RPMI + 1% P/S) and count cells using a counting chamber.</li>
	<li>Adjust the cell concentration to 5 x 106 PBMCs/ml.</li>
</ol>
5. Stimulation of PBMCs with Live HRSV
<ol>
	<li>Prepare a setup of the experiment; include experimental duplicates for each condition.</li>
	<li>Dilute all necessary stimuli in medium (RPMI + 1% P/S). Keep in mind that the final volume per well will be 200 &#956;l. A volume of 50 &#956;l of stimuli will be added so prepare 4x concentrated stimulation stocks.</li>
	<li>Add 100 &#956;l of the above mentioned cell suspension to each well of a round bottom 96-well&#160;plate&#160;(5 x 105 PBMC/well).</li>
	<li>Add 50 &#956;l of the diluted stimuli to each well. It is possible to add two different stimuli, which gives a volume of 200 &#956;l. If only one stimuli is needed adjust the volume to 200 &#956;l with medium.</li>
	<li>Incubate the 96-well plate for the required incubation period at 37 &#176;C in a 5% CO2 incubator. For most cytokines 24 hr of incubation will be sufficient.</li>
</ol>
6. Readout of Stimulation
<ol>
	<li>After the incubation period, centrifuge the 96-well&#160;plate for 5 min at room temperature at 450 x g&#160;to collect the cells at the bottom.</li>
	<li>Collect the supernatant (take a maximal volume of 180 &#956;l) and store for subsequent cytokine analysis (-20 &#176;C) or for virus production (-80 &#176;C).</li>
	<li>The stimulated PBMCs that are present in the pellet can either be analyzed by qPCR/Western blot by adding lysis buffer or alternatively, PBMCs can be stained for flow cytometry analysis.</li>
</ol>

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We have nothing to disclose.

In this study, we demonstrate that infection of PBMC with HRSV is a fast and reliable model system in which inflammatory pathways can be studied. In order to stimulate PBMCs an HRSV stock has to be prepared and quantified.
Multiple cell lines are susceptible to HRSV infection. Cell lines most used for HRSV culturing are HEp-2, HeLa,&#160;and Vero cells. However, we would not recommend the usage of Vero cells for culturing of HRSV because it has been shown that culturing of HRSV in Vero cells r...

Human Respiratory Syncytial Virus (HRSV) is the most common cause of lower respiratory tract infections in children. Each year, over 33 million children under the age of five are infected with HRSV, leading to over three million hospitalizations and almost 200,000 deaths1. A growing body of evidence suggests that HRSV also poses a significant threat to the elderly and adults with underlying chronic illnesses2.
The majority of HRSV infections in young children presents with mild symptoms, comparable to the common cold, and do not require clinical intervention. However, a small proportion of patients require hospitalization and mechanical ventilation due to severe bronchiolitis.
Part of the pathogenesis of HRSV disease is the host&#39;s overexuberant and inadequate immune response to infection3,4. This is illustrated by several observations. The period of maximal illness is often preceded by the peak of viral infection and coincides better with cellular infiltration of infected tissues and the release of inflammatory cytokines3. Another line of evidence comes from the formalin inactivated HRSV (FI-RSV) trials in the 1960s. Instead of inducing protection in young infants, the vaccine resulted in an exaggerated immune response resulting in enhanced respiratory disease and higher morbidity and mortality.
Several models have been developed to study the pathogenesis of HRSV infections. Continuous cell lines, like HEp-2 and A549, have been used extensively to study HRSV infection in vitro. Epithelial cells are the primary targets of HRSV infection5, therefore a lot of focus has been on these cell types. However, the in vivo situation is much more complex and not limited to one cell type. In order to examine these complex interactions, several animal infection models have been developed to study HRSV pathogenesis. Thus far, two different strategies have been employed, focusing either on heterologous (nonhuman) models as well as cognate host-pneumovirus models. Examples of heterologous models for HRSV include chimpanzees, sheep, cotton rats and mice. RSV infection in its natural host has been studied in cattle and in mice, using bovine RSV and pneumovirus, respectively.
Whilst each of these models has provided important insights into disease pathogenesis, HRSV is highly adapted to humans. Therefore, as an addition to the cell lines and animal models currently used, we propose to use human primary PBMCs as a model for stimulation with HRSV. Human PBMCs can be obtained from a range of individuals to address specific research questions, including young children6, immunocompromised individuals as well as healthy adults7 or elderly individuals8. PBMCs can be used to study innate aspects of infection as well as the adaptive response to the virus. Activated inflammatory pathways important for the pathogenesis of HRSV disease can be studied at the mRNA and protein level. Further, viral infection of (subsets of) immune cells can be determined by flow cytometry. We have used this model previously to identify the synergistic effects of HRSV infection together with the bacterial ligand muramyl dipeptide (MDP) on the induction of proinflammatory cytokines7. We propose to use HRSV stimulation of human primary PBMCs as a robust, easy and fast in vitro model to study inflammatory pathways involved in the pathogenesis of HRSV disease.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td>PBS without Ca2+ Mg2+ or phenol red</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025-100</td>
			<td>HBSS, Calcium, Magnesium, no Phenol Red</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966-047</td>
			<td>DMEM, High Glucose, GlutaMAX, Pyruvate</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Invitrogen</td>
			<td>72400054</td>
			<td>RPMI 1640 Medium, GlutaMAX, HEPES</td>
		</tr>
		<tr>
			<td>Reduced Serum Medium (Opti-MEM)</td>
			<td>Invitrogen</td>
			<td>51985-026</td>
			<td>Opti-MEM I Reduced Serum Medium, GlutaMAX</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Greiner Bio-one</td>
			<td></td>
			<td>FBS (Fetal Bovine Serum)</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7030</td>
			<td>Albumin from bovine serum</td>
		</tr>
		<tr>
			<td>P/S</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Penicillin-Streptomycin, liquid</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>0.05% Trypsin-EDTA (1x), Phenol Red</td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td>Sucrose BioXtra, &#8805;99.5%</td>
		</tr>
		<tr>
			<td>IgG2 anti-mouse-FITC</td>
			<td>BD Pharmingen</td>
			<td>555057</td>
			<td>Ms IgG2b K FITC isotype control</td>
		</tr>
		<tr>
			<td>Anti-NP-RSV-FITC</td>
			<td>Abcam</td>
			<td>ab25849</td>
			<td>Anti-Respiratory Syncytial Virus antibody [671] (FITC)</td>
		</tr>
		<tr>
			<td>Density gradient medium (Lymphoprep)</td>
			<td>Axis-Shield</td>
			<td>1114545</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA tubes</td>
			<td>BD Biosciences</td>
			<td>367525</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Merck Millipore</td>
			<td>1000141000</td>
			<td>Acetone for analysis</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material </td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td>Thinwall, Polyallomer, 38.5 ml, 25 mm x 89 mm</td>
		</tr>
		<tr>
			<td>SureSpin 630 Rotor with 36 ml buckets</td>
			<td>Sorvall</td>
			<td>79368</td>
			<td>Swinging bucket titanium rotor</td>
		</tr>
		<tr>
			<td>Sorvall WX80 Ultracentrifuge</td>
			<td>Sorvall</td>
			<td>46900</td>
			<td></td>
		</tr>
		<tr>
			<td>CB 150l CO2 Incubator</td>
			<td>Binder</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The HRSV A2 cultivation and purification method described here will give a yield between 2 x&#160;107 and 2 x&#160;108 FITC-50 quantified infectious viral particles/ml. Results of microscopic analysis, where FITC-stained infected cells were counted (results not shown), were compared to results of quantification by FACS (Figure 1). Both methods resulted in similar titers, although the FACS method had a much lower standard deviation between different experiments and was less laborious...

Watch this Scientific Journal Video about An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection at JoVE.com

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.

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 ...

an-vitro-model-to-study-immune-responses-human-peripheral-blood

Research

JoVE Journal

Immunology and Infection

7.8K Views.  Radboud university medical center. 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.

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

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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

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

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

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

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

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...