eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
CAUTION: Working with human blood samples and human malaria parasites requires precautionary measures. Always wear a lab coat, and 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 considerations should also be put in place for working with human immunodeficiency virus 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 an 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 ultraviolet (UV) for 1 hr following use. Note that each institution will have its 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&#160;P. falciparum&#160;malaria parasites exist. ITG was used for these experiments, but different strains can be used. Excellent instructions on freezing and thawing&#160;Plasmodium falciparum&#160;parasites are available at the MR4 website.
1. Making RPMI-A for Malaria Parasite Culture
<ol>
	<li>Make RPMI-0 by mixing 950 ml of double-distilled water (ddH2O), 1 packet of Roswell Park Memorial Institute (RPMI)-1640 powder, 6 g of N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (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 the serum contains particulate matter or is thick spin at 2,000 rpm and then filter the 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.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	Note: Parasite growth may vary in different donor serums. 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&#39;s ID and date of collection on the label.</li>
	<li>Store blood at 4&#160;oC until needed. Use blood for parasite cultures within 1 month.</li>
	<li>Wipe the top of the tube with 70% ethanol. Carefully remove the stopper and discard it. 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 an 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&#160;oC until needed.</li>
</ol>
3. Maintaining Parasite Cultures
<ol>
	<li>Pre-warm RPMI-A to 37&#160;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 the 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 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&#160;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 so 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 the slide to dry.</li>
	<li>Place 4 ml of fresh RPMI-A into the flask, gently mix, gas for 30 sec, and place in an incubator at 37 &#176;C.</li>
	<li>Stain the slide using the Hema3 staining kit according to the manufacturer&#39;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 Plasmodium falciparum RBC, 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 it with alanine. Only ring-stage parasites and uninfected RBCs 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 the alanine solution to 37&#160;oC.</li>
	<li>Spin down the parasite culture (5 min x 1,000 x g), and remove the medium.</li>
	<li>Resuspend pellet in 19 volumes of alanine solution (1 ml packed RBC to 19 ml alanine solution). Incubate for 15 minutes at room temperature (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&#160;oC.&#160;&#160;&#160;&#160;&#160;&#160; 
	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 RBCs per mL of parasite culture using a hemocytometer.&#160;&#160;&#160; 
	Number of PfRBC = % parasitemia x total RBC/ml x ml of culture. For example, 10 ml of culture at 10 x 106&#160;RBC/ml and 10% parasitemia is equal to 0.1 x 106/ml x 10 ml = 10 x 106&#160;PfRBC.</li>
	<li>Spin the parasite culture for 5 min at 1,000 x g (RT). Aspirate medium, and resuspend at 6 x 106&#160;PfRBC per ml in RPMI-S+ (500 ml RPMI-1640 supplemented with L-glutamine and HEPES, 10% heat inactivated fetal bovine serum (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 Ethylenediaminetetraacetic acid (EDTA) as an anti-coagulant as EDTA&#39;s calcium-chelating action affects cell function. On average expect between 5-10 x 106&#160;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 it 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 an equal volume of cold Dulbecco&#39;s phosphate-buffered saline (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 &#39;no brake&#39; setting is on.</li>
	<li>Once the cells have spun, look for the interface between the two phases. This is where the PBMCs are. Using a sterile plastic transfer pipette collect the PBMC from the interface (see&#160;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&#160;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 hemocytometer and count the living cells (cells that have not taken up the blue dye &#8211; all blue cells are dead). Use a hemocytometer to calculate cell number in a solution as follows.&#160;&#160;&#160; 
	Note: A hemocytometer has a grid of specified dimensions so that the area covered by the lines is known.
	<ol>
		<li>Make sure the hemocytometer 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 be filled by capillary action.</li>
		<li>Place the loaded hemocytometer under a microscope. The full grid of the hemocytometer contains 9 squares of 1 mm2&#160;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,&#160;e.g.,&#160;(500 cells/5 squares) x dilution factor of 20 x 10,000 cells/ml = 20 x 106&#160;cells total.</li>
	</ol>
	</li>
	<li>Adjust the medium to a final concentration of 10 x 106&#160;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&#160;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(-) samples.</li>
	<li>For the PfRBC wells, place 3 x 106&#160;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 positive controls to ensure cells are functional.</li>
	<li>Place the plate at 37&#160;oC in a 5% CO2&#160;incubator.</li>
	<li>Optional: use excess cells for phenotypic analysis using flow cytometry or store them 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 the 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 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>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alanine</td>
			<td>Sigma</td>
			<td>A7377</td>
			<td></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>Parasite Gas Mixture</td>
			<td>By special order</td>
			<td></td>
			<td>3% CO2, 1% O2, balance N2</td>
		</tr>
	</tbody>
</table>

generation of an in vitro cell culture model of malaria-hiv co-infection

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

<img alt="Figure 1" src="/files/ftp_upload/21717/21717_Figure1.jpg" /> 
Figure 1.&#160;Depiction of Ficoll gradient post spin demonstrating the position of the PBMCs.

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

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

Begin with a multi-well plate containing human peripheral blood mononuclear cells, or PBMCs derived from a human immunodeficiency virus, or HIV-infected donor.
PBMCs contain various immune cells, including HIV-infected CD4-positive T cells with integrated proviral DNA.
Seed the wells with a mixture of Plasmodium falciparum parasitized erythrocytes, or PfRBCs &#8212; expressing parasite-derived antigens, and uninfected RBCs.
During incubation, the PfRBCs&#39; antigens interact with receptors of immune cells, activating them and releasing cytokines.
These cytokines trigger downstream signaling in CD4 T cells, initiating transcription of HIV proviral DNA and synthesis of new viral RNAs.
The viral RNAs and HIV proteins form virus particles that bud off, infecting new CD4 T cells and spreading HIV infection.
Progressive HIV infection disrupts cytokine secretion, impairing the immune response against PfRBCs and leading to persistent malaria infection.
Over time, infected RBCs release parasites that invade new RBCs, proliferating malarial parasites and establishing the malaria-HIV co-infection cell culture model.

generation-an-vitro-cell-culture-model-malaria-hiv-co

Research

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

83 Views. Source: Finney, C. et al., An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection. J. Vis. Exp. (2015) This video demonstrates the generation of an in vitro malaria-HIV co-infection cell culture model. HIV-infected peripheral blood mononuclear cells co-cultured with Plasmodium falciparum parasitized erythrocytes potentially support each other's growth and provide insights into the immune responses and disease progression in HIV and mala...

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

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

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

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

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

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

JoVE Journal

Immunology and Infection

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

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

Transcript

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

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

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness