We would like to thank Ms. Amy Iverson in the Department of Infectious Diseases at St. Jude Children's Research Hospital for technical training on influenza virus culture and infections and Dr. Stacie Woolard in the Department of Flow Cytometry at St. Jude Children's Research Hospital for help in setting up the 14 color antibody cocktail for analysis. We would also like to thank Arthur Covington and Shelby Patrick of St. Jude for animal husbandry and care. These studies were supported by the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, under contract number HHSN26620070005C, Centers of Excellence in Influenza Research and Surveillance (CEIRS), N 01 7005C (JAM) and Le Bonheur Junior Faculty Grant (Samarasinghe).

Ethics Statement. All experiments were approved by the Institutional Animal Use and Care Committee at St. Jude Children&#39;s Research Hospital.
Note: All described experiments in this protocol must be conducted under BSL-2 and ABSL-2 conditions since Aspergillus fumigatus and influenza A virus are BSL-2 agents.
1. Modeling Allergic Asthma in Mice (Figure 1)
<ol>
	<li>Fungal sensitization
	<ol>
		<li>Re-constitute A. fumigatus antigen in sterile PBS at 1 mg protein/mL. Aliquot and store at -20&#176;C.</li>
		<li>House animals in a micro-isolator cages with autoclaved paper bedding under specific pathogen-free conditions with ad libitum food and water in a 12 hour light:dark cycle. Obtain C57BL/6J female mice at six weeks of age and acclimatize for one week. Start mice on the asthma protocol at seven weeks of age.</li>
		<li>Dilute A. fumigatus antigen to 20 &#181;g/mL in sterile PBS and add dropwise to an equal volume of Alum while stirring. Allow antigen to be adsorbed for 30 mins at room temperature with constant stirring.</li>
		<li>Fill 1 mL luer-lock syringes with the mixture and deliver 0.1 mL intraperitoneally and 0.1 mL subcutaneously between the ears to each mouse. Administer PBS and Alum as a mock-sensitization to mice that serve as na&#239;ve controls.</li>
		<li>Two weeks after the initial sensitization, dilute A. fumigatus antigen to a concentration of 200 &#181;g/mL in sterile PBS and deliver 20 &#181;L intranasally to each mouse. Repeat once weekly for a total of three intranasal administrations. Administer PBS as a mock-sensitization to mice that serve as na&#239;ve controls.</li>
	</ol>
	</li>
	<li>Fungal challenge
	<ol>
		<li>Prepare Sabouraud dextrose agar at 65 g/L per manufacturer&#39;s guidelines and sterilize in an autoclave at 121&#176;C for 15 mins. Place in a water bath set at 50&#176;C. to temper.</li>
		<li>Once tempered, aseptically add 10 mL to 25 cm2 canted neck, vented cap cell culture flasks. Lay the flasks flat and allow agar to set. Once set, recap flasks and store at 4&#176;C until use.</li>
		<li>Re-suspend the A. fumigatus culture aseptically by adding 5 mL of sterile PBS. Store overnight at 4&#176;C. Prepare 60 &#181;L aliquots for use and store at 4&#176;C.</li>
		<li>Eight days before the final intranasal administration of A. fumigatus antigen, culture A. fumigatus on Sabouraud dextrose agar for the fungal challenge. Add one well vortexed 60 &#181;L aliquot of fungus in each agar flask and spread using a disposable inoculating loop. Incubate flasks at 37&#176;C for 8 days.</li>
		<li>Set up the inhalation challenge apparatus in a class II biological safety cabinet as described in detail by Schuh and Hoselton.7 Create holes in the fungal culture flasks, one on the rear top and one on the bottom of the flask immediately above the culture by using a cork-borer.</li>
		<li>Attach the fungal culture flask to the regulated medical grade air supply through the bottom hole and to the inhalation chamber through the top hole. Cover the inhalation ports with laboratory tape.</li>
		<li>Run medical grade air over the culture at 2 psi for 10 mins to coat the chamber. Prepare sterile anesthetic cocktail with 75 mg/kg ketamine and 25 mg/kg xylazine.</li>
		<li>Inject the drug cocktail to three mice intraperitoneally and determine anesthesia by toe pinch reflex and lack of movement. Apply a drop of lubricating ointment in each eye to prevent drying out.</li>
		<li>Turn off the air, and replace the fungal flask with a new one and open the ports of the inhalation chamber.</li>
		<li>Place the anesthetized mice supine with their noses in the ports. Turn on the air over the culture and allow the animals to inhale the liberated fungal conidia for 10 mins.</li>
		<li>Place each animal supine in a new cage and place the cage on a heating pad. Monitor animals for recovery from anesthesia marked by turning over and movement around the cage.</li>
		<li>Repeat procedure after 14 days. Anesthetize animals that serve as na&#239;ve controls and place in a new cage.</li>
	</ol>
	</li>
</ol>
2. Modeling Influenza in Mice
NOTE: Adjust the age, gender, and strain of mice and the dose of the virus according to the experimental goals.
<ol>
	<li>Obtain female C57BL/6J mice at 14 or 17 weeks of age and acclimatize to the facility in micro-isolator cages with autoclaved paper bedding and ad libitum food and water in a 12 hour light:dark cycle. Number animals in each cage by tail mark or ear notch. One week later, weigh each mouse and record weight with date and time.</li>
	<li>Thaw a stock of A/CA/04/2009 virus and dilute in sterile PBS at log104.305 TCID50/mL in a glass vial. Anesthetize mice with 2.5% isoflurane with oxygen in an anesthesia chamber within the class II biological safety cabinet.</li>
	<li>Deliver 50 &#181;L of virus intranasally to each mouse for a final dose of 1000 TCID50 pH1N1 per mouse. Hold mouse supine until conscious and place in new micro-isolator cage. Administer PBS under anesthesia to na&#239;ve controls.</li>
	<li>Record animal weights every 24 hours until experimental endpoints are reached. 
	NOTE: It is good practice to use the same stock virus tube(s) to infect all animals in the study and not use previously thawed tubes as the viral titer may decrease during repeated freeze-thaw cycles.</li>
</ol>
3. Modeling Asthma and Influenza Co-morbidity in Mice (Figures 2 and 3)
<ol>
	<li>To mimic acute asthma and influenza, infect animals one week after the second fungal challenge with pH1N1 as noted in section 2. To mimic chronic asthma and influenza, infect animals 4 weeks after the second fungal challenge.</li>
</ol>
4. Pulmonary function test
<ol>
	<li>Weigh and record each mouse weight.</li>
	<li>Perform pulmonary function test on each animal. Note: Of the various equipment available to measure pulmonary function in rodents, we have opted to use the flexiVent FX1 module and flexiware software as previously described.22
	<ol>
		<li>Open the software and go to &#34;Study Definition and Planning.&#34; Click on &#34;Create a new study&#34; to launch the Study Definition Wizard.</li>
		<li>Follow the Wizard to input details of the study: Title and Status, Objective and Hypothesis; Protocol; Subject (animal) Groups.</li>
		<li>Create subjects by clicking on the mouse icon and add all necessary information on the animal including the Group Membership which will assign subjects to Subject Groups.</li>
		<li>Verify all information and confirm to close the wizard.</li>
	</ol>
	</li>
	<li>After concluding the &#34;Study Definition and Planning&#34; session, open the &#34;Experimentation Session&#34; in the software. Follow the steps of the pop-up wizard to set-up the instrument.
	<ol>
		<li>Select the Study (see 4.2.1) and the Experiment Template which depends on the instrument.</li>
		<li>Add details as deemed necessary to Session Properties. The instrument will then be connected to the software.</li>
		<li>Select the instrument module attached to the software when promoted and click OK.</li>
		<li>Select a subject in the Study to Site 1 and enter the current weight of the animal when prompted.</li>
		<li>Add 0.5 mL of saline to the finemist nebulizer and click OK when prompted to Prime the nebulizer (the generated mist will be visible). Attach the nebulizer to the instrument adaptor when complete.</li>
		<li>Perform Channel Calibrations when prompted. Calibrate the Cylinder Pressure and Airway Opening Pressure following the software wizard using a manometer to apply known pressures.</li>
		<li>At the conclusion of Channel Calibrations, the Tube Calibration Wizard will be initiated; attach an 18 g cannula to the Y-tubing adaptor and select the desired perturbations. Perform closed and open calibrations and verify that Calibration Results are void of errors.</li>
		<li>Once the calibrations are complete, select whether to start recording continuous data when prompted. Click &#34;Yes&#34; to start the default ventilation pattern. The instrument is ready to gather data.</li>
	</ol>
	</li>
	<li>Prepare a stock acetyl-&#946;-methylcholine (A&#946;M) at 125 mg/mL in sterile saline; freeze aliquots at -20&#176;C until use. Dilute the frozen stock of A&#946;M to the desired doses in sterile saline (3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, and 25 mg/mL).</li>
	<li>Prepare anesthetics in sterile PBS before procedure. Administer 10 mg/kg body weight xylazine intraperitoneally and place mouse its own cage to rest for 5 min. Administer 70 mg/kg body weight pentobarbital intraperitoneally and allow mouse to rest for 5 min.</li>
	<li>Intubate mouse as previously described.23
	<ol>
		<li>Briefly, place the anesthetized mouse supine on a slanted surgical board and fix in place with plastic tape over the forelimbs and tail.</li>
		<li>Make a small incision between the upper thorax and lower jaw. Separate the salivary glands and thyroid gland to expose the trachea. Gently pass curved surgical forceps underneath the trachea and pull a piece of thread underneath.</li>
		<li>Perform tracheostomy between two tracheal rings with sharp scissors taking care not to cut through the trachea.</li>
		<li>Insert the 18 g cannula that was used to calibrate the instrument. Fasten the cannula to the trachea with thread and attach the mouse to the Y-tubing of the instrument adaptor with the cannula.</li>
	</ol>
	</li>
	<li>Perform a deep inflation to recruit the airways and help the animal succumb to the ventilator. Select the &#34;mouse inhaled dose response v7.0&#34; script with default ventilation at 150 breaths/minute.</li>
	<li>Add saline followed by each incremental dose of A&#946;M to the nebulizer when prompted to perform a dose response curve.</li>
	<li>At the conclusion of the data acquisition after the highest A&#946;M dose, disconnect the mouse from the ventilator and humanely euthanize the animal by performing a cervical dislocation. Proceed to tissue harvest.</li>
	<li>Add saline to the nebulizer and activate the nebulizer to remove any remnant high dose A&#946;M. Change the subject in the software program and proceed with next mouse beginning with a tube calibration (step 4.1-4.3).</li>
</ol>
5. Tissue harvest
<ol>
	<li>Spray euthanized mouse with 70% ethanol and open the thoracic cavity. Open diaphragm and perform bronchoalveolar lavage (BAL) with 1 mL of sterile PBS injected through the 18 g cannula and gently draw out repeating once for a total of 2 mL.24 Place the sample in ice.</li>
	<li>Harvest the cardiac and diaphragmatic lobes of the right lung for RNA work, and azygous and apical lobes for protein work, place in separate 5 mL snap-cap tubes and snap freeze in liquid nitrogen.</li>
	<li>Harvest the left lobe using blunt forceps and scissors taking care not to damage/slice through the lobe. Fix the lung ex vivo with 10% normal buffered formalin with a 30 g needle. Store in 10% normal buffered formalin until histological analyses. 
	NOTE: When animals are taken through the model for determination of viral burden in the lungs, harvest whole lungs after euthanasia and snap freeze in liquid nitrogen.</li>
</ol>
6. Tissue processing for Flow Cytometry
<ol>
	<li>Centrifuge the BAL contents at 600 g for 10 mins at 4&#176;C and save the BAL fluid for protein assays at -80&#176;C until use.</li>
	<li>Perform red blood cell lysis if necessary noting that cells are more susceptible to death after viral infection. Perform red cell lysis by adding 8 parts cold distilled water and pulse-vortexing for 30 seconds followed by a one-minute incubation at room temperature. Add one part 10 PBS, mix to neutralize the water and centrifuge as above.</li>
	<li>Re-suspend the BAL cells in 200 &#181;L of staining media (sterile PBS with 5% FBS) and enumerate cells by trypan blue dye exclusion assay to determine the cell concentration.</li>
	<li>Cytospin 50,000 cells as follows:
	<ol>
		<li>Transfer the calculated volume of cells into a tube and bring volume up to 100 &#181;L with sterile PBS. Assemble the labeled slide and cytofunnel in the metal clip and place in the centrifuge.</li>
		<li>Add the diluted cells into the cytofunnel and spin at 55 g for 5 minutes to affix the cells to the slides.</li>
		<li>Perform differential staining by sequentially dipping the slides 20 times each in methanol, xanthene, and thiazine followed by distilled water and air-drying.</li>
	</ol>
	</li>
	<li>Add 1 &#181;L of human gamma globulin for every 10,000 cells in the cell suspension in step 6.3 and incubate in ice for 30 mins. Adjust volume to 1 mL with staining media and centrifuge at 600 g for 10 mins at 4&#176;C and aspirate supernatant. Re-suspend cells in 100 &#181;L of staining media and prepare four pooled samples with 10 &#181;L from each sample. Adjust volumes to 200 &#181;L and centrifuge as above.</li>
	<li>Re-suspend cells in 50 &#181;L of antibody cocktail containing fluorochrome-conjugated antibodies diluted in staining media (see table of materials). Re-suspend one of the pooled samples in 50 &#181;L of staining media, one pooled sample in 50 &#181;L of isotype control antibodies, and the remaining pooled samples with diluted antibodies individually. Incubate samples in ice protected from light for 30 mins. 
	NOTE: The use of antibodies will be based on research interests and should be optimized previously for the experiment. Each antibody used should be used individually in a pooled sample for instrument set up. The use of bone marrow cells or splenocytes for single color controls may be necessary if the cell yield is low and is acceptable if fluorochrome-conjugated antibodies are made against an abundant marker such as B220.</li>
	<li>Add 450 &#181;L of staining media and centrifuge at 600 g for 10 mins at 4&#176;C. Aspirate supernatant and repeat.</li>
	<li>Aspirate supernatant and re-suspend cells in 50 &#181;L of freshly prepared stabilizing fixative by gentle pipetting. Add 200 &#181;L more of stabilizing fixative and strain through a 40 &#181;m nylon mesh into a polystyrene FACS tube for analysis by flow cytometry. 
	NOTE: Whole lungs may be digested to make single cell suspensions and used to analyze cell populations by flow cytometry.</li>
</ol>
7. Tissue Processing for RNA Analysis
<ol>
	<li>Place frozen lung lobes in ice within a fume hood, and perform standard Trizol extraction of RNA according to manufacturer&#39;s recommended protocol. After DNase treatment and purification of RNA, convert 1 &#181;g of RNA to cDNA.</li>
	<li>Using RNA-specific validated primer sets, measure changes in gene expression with quantitative real-time PCR and analyze data using the 2-&#916;&#916;Ct method normalized to the internal housekeeping gene25 validated for lungs.</li>
</ol>
8. Tissue processing for Viral Titration
<ol>
	<li>Place frozen lungs in ice and add 1 mL of sterile PBS and mechanically homogenize tissue until entire tissue is pulsed. Centrifuge samples at 600 g for 10 mins at 4&#176;C and store supernatants in aliquots at -80&#176;C until use.</li>
	<li>Quantify virus using standard tissue culture infectious dose 50 (TCID50) method26 with MDCK.2 cells.</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+RSV+Infection+in+Asthma+Initiation+and+Progression:+Findings+in+a+Mouse+Model.">The Role of RSV Infection in Asthma Initiation and Progression: Findings in a Mouse Model.</a> Pulm Med. 2011, 748038 (2011).</li><li>Hsu, H. C., 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=Age-related+thymic+involution+in+C57BL/6J+x+DBA/2J+recombinant-inbred+mice+maps+to+mouse+chromosomes+9+and+10.">Age-related thymic involution in C57BL/6J x DBA/2J recombinant-inbred mice maps to mouse chromosomes 9 and 10.</a> Genes Immun. 4, 402-410 (2003).</li><li>Chang, Y. 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=Influenza+infection+in+suckling+mice+expands+an+NKT+cell+subset+that+protects+against+airway+hyperreactivity.">Influenza infection in suckling mice expands an NKT cell subset that protects against airway hyperreactivity.</a> J Clin Invest. 121, 57-69 (2011).</li><li>Wohlleben, 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=Influenza+A+virus+infection+inhibits+the+efficient+recruitment+of+Th2+cells+into+the+airways+and+the+development+of+airway+eosinophilia.">Influenza A virus infection inhibits the efficient recruitment of Th2 cells into the airways and the development of airway eosinophilia.</a> J Immunol. 170, 4601-4611 (2003).</li><li>Mori, H., 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=Differences+in+respiratory+syncytial+virus+and+influenza+infection+in+a+house-dust-mite-induced+asthma+mouse+model:+consequences+for+steroid+sensitivity.">Differences in respiratory syncytial virus and influenza infection in a house-dust-mite-induced asthma mouse model: consequences for steroid sensitivity.</a> Clin Sci (Lond). 125, 565-574 (2013).</li><li>Myles, P. 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=Predictors+of+clinical+outcome+in+a+national+hospitalised+cohort+across+both+waves+of+the+influenza+A/H1N1+pandemic+2009-2010+in+the+UK.">Predictors of clinical outcome in a national hospitalised cohort across both waves of the influenza A/H1N1 pandemic 2009-2010 in the UK.</a> Thorax. 67, 709-717 (2012).</li><li>van der Zalm, M. 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=Highly+frequent+infections+with+human+rhinovirus+in+healthy+young+children:+a+longitudinal+cohort+study.">Highly frequent infections with human rhinovirus in healthy young children: a longitudinal cohort study.</a> J Clin Virol. 52, 317-320 (2011).</li></ol>

The authors have nothing to disclose.

The high incidences of allergic asthma and influenza increase the likelihood that these two diseases would occur simultaneously in the same individual. While some viruses are clearly demonstrated to be causative agents initiating the pathogenesis of asthma,28,29 the impact of influenza virus infections on either the development or exacerbation of asthma is unclear. The association and the importance of studying influenza viruses in the context of allergic disease became evident when asthma was identified as a ...

Commonly referred to as a disease, asthma is a syndrome1 that affects over 235 million of the world's population.2 Allergic asthma is the most prevalent type of asthma and results from exposures to environmental factors such as pollen, house dust mite and cockroach antigens, and fungi.3 Fungal antigens are common sensitizing allergens affecting 25% of persistent asthmatics and can cause life threatening asthma attacks.4 The development of an accurate model of allergic asthma is challenging because mice do not naturally develop asthma and there are differences between mouse and human respiratory systems.5 The selection of the mouse asthma model system is largely dependent on the experimental question since the output may vary with the antigen used. A fungal asthma model using Aspergillus fumigatus conidia used in native form and natural route of entry into the airways of mice, described in detail elsewhere,6-8 has been shown to elicit all the hallmarks of human disease including airways hyperresponsiveness, eosinophilic inflammation, mucus hypersecretion, and airway wall remodeling events.9
Influenza affects millions worldwide every year. Genetic alterations that occur in influenza viruses by antigenic drifts and shifts, and re-assortments in common hosts can result in a novel virus with the potential to cause a pandemic. The influenza pandemic of 1918 (Spanish Flu) claimed over 50 million lives mostly of young adults.10 The first influenza pandemic of the 21st century occurred in 2009. Although the 2009 influenza pandemic was not as severe as that of 1918, approximately 90 million people were infected11 of which a little over 200,000 died.12 Hospitalized patients included those with underlying diseases such as obesity, cardiovascular and metabolic disease, asthma, COPD, and diabetes.13-16 Although asthma was identified as a risk factor for severe influenza morbidity,17,18 asthmatics were less likely to be admitted to the intensive care unit and die compared to patients without asthma.19,20 The reasons for these seemingly counterintuitive findings are unknown. Therefore, a reliable animal model system was necessary to study the intersection of these two diseases. 
Since other respiratory viruses such as rhinovirus and respiratory syncytial virus (RSV) have been shown to induce asthma, existing animal models of asthma and influenza explored whether influenza virus infections induces the development of asthma.21 The airways, lung parenchymal tissue, and the immune responses are different during acute allergic asthma exacerbations and chronic stable asthma. Therefore, models that can explore how influenza virus infections intersect allergic asthma in each situation are necessary to understand the underlying mechanisms of asthma and influenza comorbidities. This protocol describes a mouse model that can recapitulate these two scenarios.

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Aspergillus fumigatus antigen</td>
			<td style="width:124px;">Greer Labs</td>
			<td style="width:131px;">XPM3D3A25</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Aspergillus fumigatus</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:131px;">NIH 5233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Imject Alum</td>
			<td style="width:124px;">Pierce</td>
			<td style="width:131px;">77161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Difco Sabouraud dextrose agar</td>
			<td style="width:124px;">BD&#160;</td>
			<td style="width:131px;">210950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Spore-Klenz</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:131px;">04-324-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ketamine</td>
			<td style="width:124px;">Hospira</td>
			<td style="width:131px;">0409-2051-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Xylazine</td>
			<td style="width:124px;">Akorn Inc.</td>
			<td style="width:131px;">59399-110-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Isothesia (Isoflurane)</td>
			<td style="width:124px;">Butler Schein</td>
			<td>05260-04-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Euthasol</td>
			<td style="width:124px;">Virbac</td>
			<td style="width:131px;">710101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Acetyl-&#946;-methylcholine</td>
			<td style="width:124px;">Sigma Aldrich</td>
			<td style="width:131px;">A2251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">flexiVent FX1</td>
			<td style="width:124px;">SCIREQ</td>
			<td style="width:131px;">FV-FXM1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Nebulizer (standard mist)</td>
			<td style="width:124px;">SCIREQ</td>
			<td style="width:131px;">ANP-1100</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:227px;">flexiWare Software</td>
			<td style="width:124px;">SCIREQ</td>
			<td style="width:131px;">version 7.0.1 rel B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:227px;">Quik-Dip 3 Part Differential Stain</td>
			<td style="width:124px;">Mercedes Medical</td>
			<td style="width:131px;">MER 1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Human gamma globulin</td>
			<td style="width:124px;">Sigma Aldrich</td>
			<td style="width:131px;">G4386</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Rat anti-mouse CD3e-PE-Cy7</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">552774</td>
			<td>Clone: 145-2C11; Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Hamster anti-mouse CD8&#945;-FITC</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">553031</td>
			<td>Clone: 53-6.7, Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">FITC Rat IgG2a</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">553929</td>
			<td>Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">PE-Cy7 Armenian Hamster IgG</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">400922</td>
			<td>Clone: HTK888; Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Stabilizing fixative</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">338036</td>
			<td>Freshly prepare&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">MDCK.2</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:131px;">CRL-2936</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">TRIzol Reagent</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:131px;">15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Chloroform</td>
			<td style="width:124px;">IBI Scientific</td>
			<td style="width:131px;">IB05040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">iScript</td>
			<td style="width:124px;">Bio-Rad</td>
			<td style="width:131px;">170-8891</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">QuantiTect Primer Assays: HPRT1</td>
			<td style="width:124px;">Qiagen</td>
			<td style="width:131px;">QT00166768</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling asthma and influenza co-morbidity in c57bl/6j mice

Allergic asthma and influenza are both diseases of the pulmonary system that affect millions worldwide. During the 2009 influenza pandemic, asthma was identified as an underlying disease in hospitalized patients, although reasons for this increased susceptibility were unknown. Animal models are necessary to explore mechanisms of disease pathogenesis. However, models that could be used to study influenza virus infections in existing asthma are lacking. This protocol describes the development of mouse model systems of asthma and influenza comorbidity using an Aspergillus fumigatus-induced asthma model and the 2009 pandemic H1N1 influenza strain A/CA/04/2009. The host responses that occur in acute and chronic asthma can be explored by changing the timing of the influenza virus infection. These models suggest that the allergic host response to influenza virus depends on the state of the allergic airways and level of allergic inflammation.

The development and characterization of mouse models that recapitulated patient subsets with asthma and influenza is described. Such models are important in understanding host-pathogen interactions that occur during influenza virus infections in hosts that have underlying disease in the pulmonary system which alters the immune response to the invading virus.
Fungal asthma model
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Asthma and influenza are diseases affecting the pulmonary system that affects millions worldwide. The aim of this study was to develop a mouse model of asthma and influenza comorbidity to study the intersection of these two diseases in the same host.

Modeling Asthma and Influenza Co-morbidity in C57BL/6J Mice

Allergic asthma and influenza are both diseases of the pulmonary system that affect millions worldwide. During the 2009 influenza pandemic, asthma was ...

modeling-asthma-and-influenza-co-morbidity-in-c57bl6j-mice

Research

JoVE Journal

Biology

1.5K Views.  University of Tennessee Health Science Center. Asthma and influenza are diseases affecting the pulmonary system that affects millions worldwide. The aim of this study was to develop a mouse model of asthma and influenza comorbidity to study the intersection of these two diseases in the same host. 