The authors would like to acknowledge Vertex Pharmaceuticals and the Cystic Fibrosis Research Innovation Award for funding R. Quinn and the NIH/NIAID for funding grant 1 U01 AI124316-01, a systems biology approach to treatment of multi-drug resistant pathogens. We would also like to thank the Department of Mechanical and Aerospace Engineering at UCSD's undergraduate mechanical engineering senior design course for facilitating the collaboration with the engineering aspects of this work.

1. Preparation of Stocks for the Artificial Sputum Media
<ol>
	<li>Create a 5% mucin solution. Add 1.0 g of dehydrated pig stomach mucin to 20 mL of deionized water. Autoclave the resulting solution. 
	NOTE: The mucin sterilization will destroy its inherent structure; other methods to sterilize the mucin in its dry form include UV sterilization and irradiation. These methods have not been extensively used for the WinCF system however.</li>
	<li>Add 2.2 g of KCl to 50 mL of deionized water and allow for dissolution. Add 5.0 g of NaCl to 50 mL of deionized water and allow for dissolution. Autoclave these two solutions.</li>
	<li>Add 100 mg of salmon sperm DNA to 10 mL of sterile deionized water. Heat this solution to about 85 &#176;C in a water bath for a few hours to ensure dissolution.</li>
	<li>Add 5.0 mg of ferritin to 5.0 mL of sterile deionized water.</li>
</ol>
2. Preparation of the Artificial Sputum Medium
<ol>
	<li>Combine the following components: 16 mL of mucin stock solution, 2.0 mL of KCl stock solution, 2.0 mL of NaCl stock solution, 200 &#181;L of egg yolk emulsion, 5.6 mL of DNA stock solution, 120 &#181;L of ferritin stock solution, 5.78 mL of essential amino acid solution, 5.78 mL of non-essential amino acid solution, and 2.44 mL of sterile water.
	<ol>
		<li>If small amounts of sediment appear, shake gently to mix.</li>
		<li>Pipet 5.0 mL of media into eight sterile 15 mL centrifuge tubes. 
		NOTE: The chemical conditions of each tube can be manipulated as desired. For example, buffer solutions and pH indicators can be added to each tube for the purpose of comparing microbial behavior at distinct pH levels. A demonstration of this is shown in the representative results section with 8 distinct pH levels, from 5.0 to 8.5 in 0.5 pH increments.</li>
	</ol>
	</li>
	<li>Once the chemical conditions of the media are successfully manipulated, freeze for later use. The media will remain stable frozen at -20 &#176;C for several months. Vortex upon thawing.</li>
</ol>
3. Preparation of a Control Run of the Capillary Tubes
<ol>
	<li>In a sterile biohood, fill eight sterile microcentrifuge tubes with 250 &#181;L of media each.</li>
	<li>Acquire eight more sterile 15 mL centrifuge tubes, each tube corresponding to a microcentrifuge tube mentioned in step 3.1.
	<ol>
		<li>Sterilize a paper towel with 70% ethanol solution and allow to dry. Once dry, tear the towel into pieces about four square inches each, and crumple each piece in the bottom of a 15 mL centrifuge tube. For additional tubes, spray and dry additional paper towels as needed.</li>
		<li>With one clump of paper at the bottom of each centrifuge tube, slightly dampen each paper clump with about 1.0 mL of sterile water to create a humid environment.</li>
	</ol>
	</li>
	<li>Obtain three glass capillary tubes for every microcentrifuge tube prepared in step 3.1 and a block of capillary tube putty sealant.</li>
	<li>For each microcentrifuge tube, fill three capillary tubes with media to about 5 mm away from the blue marker near the top of the tube.
	<ol>
		<li>Fill by holding one end of the tube in the microcentrifuge tube and tilting towards horizontal orientation, allowing capillary action to guide the medium into the tube (See Figure 1).</li>
		<li>Stop the filling by gently placing a gloved finger over the open end of the tube, and then seal the other end of the tube by pressing it down into the sealant block.</li>
	</ol>
	</li>
	<li></li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55532/55532fig1.jpg" /> 
Figure 1: Example pH Gradient, Filling Capillary Tube with Artificial Sputum Medium. The medium is added by inserting one end of the tube into the liquid and tilting to facilitate capillary action. The medium coloration in this example is due to pH indicator added to help demonstrate potential changes in acidity after incubation. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Place each set of three capillary tubes into 15 mL centrifuge tubes filled with paper towels completely moistened with sterile water, putty-sealed side down. Cap the tube and label. These three capillary tubes are for replication of each control condition.</li>
	<li>When all 15 mL centrifuge tubes are filled with their designated capillary tubes, place the tubes in a holding rack. Place the rack such that the tubes will be incubated horizontally (to catch gas bubbles) (See Figure 2). Incubate the capillary tubes inside the centrifuge tubes (containing the moistened paper clumps) at 37 &#176;C for 48 h.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55532/55532fig2.jpg" /> 
Figure 2: Example pH Gradient, Capillary Tubes Ready for Incubation. Once three capillary tubes have been filled and sealed, they are placed in a centrifuge tube with damp paper towel at the bottom. This tube is then capped and put into a rack. The rack must be oriented sideways during incubation, as pictured here, so that gas production can be observed once incubation is complete. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Imaging of the Control Capillary Tubes after Incubation
<ol>
	<li>Remove the rack of centrifuge tubes from the incubator, making sure to keep the tubes horizontal. Carefully slide the capillary tubes out of the centrifuge tubes, keeping each set of three separate from other sets.</li>
	<li>Arrange the capillary tubes next to each other on a lightbox, all lined up so that the contents of the tubes are visible and illuminated. Leave a gap every three tubes to separate different chemical conditions.</li>
	<li>With the tubes aligned and lightbox turned on, photograph from directly above. (See Figure 3)</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55532/55532fig3.jpg" /> 
Figure 3: Example pH Gradient, Control Run, Pre-incubation, No Sputum Added. Artificial sputum medium after being added to capillary tubes in sets of three, increasing in pH from left to right. The combination of indicators added to the medium result in more acidic tubes appearing more yellow, while less acidic tubes become more purple. The tubes are arranged horizontally and are illuminated from below, photographed from above. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Dispose of the control materials in biohazardous waste.</li>
</ol>
5. Inoculating the WinCF Capillary Tubes with a Sputum Sample
<ol>
	<li>In a sterile biohood, fill eight sterile microcentrifuge tubes with 225 &#181;L of media each.</li>
	<li>Homogenize the sputum sample by withdrawing and ejecting the sputum repeatedly with a 3 mL syringe (a plastic syringe without the needle). Do this until the sputum is of smooth consistency.</li>
	<li>Add 25 &#181;L of homogenized sputum to each microcentrifuge tube (1/10 dilution in ASM media) prepared in step 5.1. Then vortex the tubes for 30 s to mix sufficiently.</li>
	<li>Add the media to capillary tubes following the same procedure as steps 3.2 through 3.5.</li>
</ol>
6. Imaging of Sample Capillary Tubes after Incubation
<ol>
	<li>After the 48-h incubation time, remove and image the capillary tubes following the same procedure as steps 4.1 through 4.3.</li>
	<li>If bubbles are present in the tubes, make sure the photographs taken clearly depict the delineation between the bubbles and media in the tubes. If biofilm is present, make sure the photographs can clearly depict its presence as well.</li>
</ol>
7. Removal of Media for Downstream Applications
<ol>
	<li>To facilitate downstream analysis, remove the media from the capillary tubes after imaging. Potential applications include culturing and DNA/RNA sequencing and metabolomic profiling. 
	Caution: The glass capillary tubes filled with pathogens are a significant biohazard, thus, these steps must be done very carefully with specific equipment. If capillary tubes break, dispose in proper biohazard sharps container.</li>
	<li>Use blunt ended needles of 25 gauge and 0.5 inch in length to remove the media. Insert the blunt ended needle into the plugged end of the tube to break the seal.</li>
	<li>After breaking the seal, turn the capillary tube upside down and the media will drip out from the top portion. If the media does not drip out easily, use a pipette with a 200 &#181;L tip and expel the media out of the tube by pressing down on the pipette plunger when inserted into the end of the capillary tube. Collect the expelled tube media in an appropriate container (1.5 mL centrifuge tube). 
	NOTE: For transcriptomic or other RNA analysis, the media can be expelled directly into RNA stabilizing buffers.</li>
</ol>
8. The WinCF FLUD System
NOTE: The WinCF Fluid Loading Utility Device (FLUD) System is an optional suite of complementary devices designed to optimize the throughput of the WinCF System. The WinCF FLUD System is comprised primarily of 3D printable materials. 3D printed manufacturing allows for quick and easy replacement of materials to ensure minimal downtime for researchers as well as minimal manufacturing requirements. Designs, stl files, 3D printing instructions and the WinCF FLUD manual are available in the online supplement.
<ol>
	<li>Preparing media for capillary tube loading
	<ol>
		<li>In a sterile biohood, fill eight sterile 2 mL microcentrifuge tubes with 900 &#181;L of medium each.</li>
		<li>Homogenize any sputum samples by withdrawing and ejecting the sputum repeatedly with a 3 mL syringe (a plastic syringe without the needle). Do this until the sputum is of smooth consistency.</li>
		<li>Add 100 &#181;L of homogenized sputum to each microcentrifuge tube (1/10 dilution in medium) prepared in step 8.1.1. Then vortex the tubes for 30 s to mix sufficiently.</li>
		<li>Slot the filled and open microcentrifuge tubes into the rotator tube holder oriented so that the tubes are vertical.</li>
	</ol>
	</li>
	<li>Placement of capillary tubes
	<ol>
		<li>Retrieve three capillary tubes for every microcentrifuge tube in step 8.1.</li>
		<li>Slot three tubes into the rubber cradle so that they are aligned with the microcentrifuge tubes at the other end of the apparatus. Make sure the marked ends of the tubes face away from the microcentrifuge tubes. Fit the three holes on the bottom of the cradle properly over the three studs on the cradle stand.</li>
		<li>With the placed capillary tubes resting in their guide channels on the apparatus, place the rubber tamp over their midsections securely to prevent shifting. (See Figure 4)</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/55532/55532fig4.jpg" /> 
Figure 4: The FLUD System Fully Loaded with Capillary Tubes Secured by the Rubber Tamp Over Their Midsections. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Loading media into the capillary tubes
	<ol>
		<li>Carefully use one hand to grasp the end of the apparatus where the capillary tubes are loaded, and use the other hand to hold the rotator rack in which the microcentrifuge tubes are loaded.</li>
		<li>Delicately rotate the microcentrifuge rack so that the microcentrifuge tubes are nearly horizontal and proceed to slowly push the rack towards the capillary tubes. (See Figure 5)</li>
		<li>When the ends of the capillary tubes make contact with the media in the microcentrifuge tubes, ensure that capillary action immediately begins filling the capillary tubes. To adjust fill rate and fill level, gently rotate the apparatus as a whole. While doing this, be careful not to spill media out of the microcentrifuge tubes. (See Figure 6)</li>
		<li>When the capillary tubes are filled to desired levels, place the apparatus level on a surface and carefully yet quickly pull the rack of microcentrifuge tubes off the ends of the capillary tubes to cease filling. The microcentrifuge tubes can now be retracted all the way back to vertical position and closed.</li>
		<li>Seal the protruding ends of the capillary tubes by pressing a sealant block onto each triplicate set, sealing one set at a time until all sets are sealed. To reduce risk of contamination, press a different part of the sealant block onto each triplicate set (See Figure 7).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/55532/55532fig5.jpg" /> 
Figure 5: The FLUD System with Medium Tubes Deployed to a Horizontal Orientation, Ready to Make Contact with Capillary Tubes. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55532/55532fig6.jpg" /> 
Figure 6: The FLUD System with Capillary Tubes Loading with Media via Capillary Action. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/55532/55532fig7.jpg" /> 
Figure 7: Sealing Filled Capillary Tubes on the FLUD System One Triplicate Set at a Time Using a Sealant Block. This sealant block had plastic along the edges that was cut off to prevent contact with neighboring triplicate sets during sealing. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Incubation
	<ol>
		<li>Remove the rubber tamp over the tube midsections and lift the rubber cradle off the main apparatus. This should take all the capillary tubes with it. Now set the cradle and the tubes being held onto the imaging rack. This rack has three stubs that fit into the cradle, as well as small guide channels to set the tubes into.</li>
		<li>Set the imaging rack as a whole into the clear plastic incubation box. Soak small amounts of sterile paper towels in sterile water and place along the two shorter sides of the box to provide humidity during incubation. (See Figure 8)</li>
		<li>Close the box completely and set inside a 37 &#176;C incubator, making sure to keep the tubes horizontal. Incubate for 48 h.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 8" src="/files/ftp_upload/55532/55532fig8.jpg" /> 
Figure 8: Capillary Tubes in Rubber Cradle Transferred from FLUD System to an Imaging Rack, Which has been Placed in a Clear Incubation Box Alongside Damp Paper Towels to Provide Humidity. <a href="http://cloudfront.jove.com/files/ftp_upload/55532/55532fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Imaging and extraction
	<ol>
		<li>Remove the imaging rack holding the tubes from the incubation box and set it on a lightbox, illuminated from below. With the tubes already in the imaging rack, the triplicate sets will be properly spaced and ready to image immediately.</li>
		<li>Focus the camera on the tubes such that all are visible in the field of view and light from the light box provides sufficient contrast and visualization of the color in the tube dyes. Photograph from directly above.</li>
		<li>To extract the contents of the tubes, remove the capillary tubes from the rubber cradle one set of triplicates at a time. Gently lift the tubes up and out of the cradle or slide them out.</li>
		<li>For each set of triplicates, follow the extraction procedure detailed in steps 7.1 through 7.3.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Quinn, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Winogradsky-based+culture+system+shows+an+association+between+microbial+fermentation+and+cystic+fibrosis+exacerbation.">A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation.</a> ISME J . 9, 1024-1038 (2015).</li><li>Quinton, P. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+lung+microbiome:+Opportunities+to+reconsider+management+of+airway+infection.">Cystic fibrosis lung microbiome: Opportunities to reconsider management of airway infection.</a> Pediatr pulmonol. 50, S31-S38 (2015).</li><li>Blainey, P. C., Milla, C. E., Cornfield, D. N., Quake, S. 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The authors have nothing to disclose.

The microbiological makeup of a lung with CF contains a great variety of organisms, but the conditions within the lung likely have a significant influence on what kinds of microbes can survive and thrive13,15. Specific mechanisms through which these conditions change and the exact effects they have on the lung microbiome are generally unclear at present. In this experimental method, we present an analysis of microbiological changes based upon manipulated chemical...

The method described in this manuscript is called the WinCF system1. The overall goal of WinCF is to provide an experimental setup capable of simulating the environment of a mucus-filled lung bronchiole. This will allow for a tractable system to study microbial pathogens of lung diseases with a mucus hypersecretion phenotype including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), asthma and others. The procedure was designed specifically for the study of CF, which is characterized by mutations that cause lung secretions to become thick and hard to clear, eventually filling bronchioles and other small passageways with mucus2. Such blockages in the lung inhibit gas exchange because inhaled air is no longer able to reach many alveoli and also provide a habitat for bacterial colonization3,4. The inability to prevent microbial growth in the excessive lung mucus eventually leads to the development of complex chronic infections of the airway. These communities contain a variety of organisms, including viruses, fungi, and bacteria like Pseudomonas aeruginosa, all interacting with one another5,6,7,8. The activity of the CF lung microbiome is believed to be involved in flares of symptoms called pulmonary exacerbations1,9,10,11. WinCF enables study of the microbial community behavior around these exacerbations and is now being expanded to act as a base experimental system to study lung microbial ecology. Traditionally, exacerbations have been studied through direct analysis of samples taken from the lung. Many confounding factors make direct analysis of microbial behavior in lungs challenging, with the WinCF system, many of these factors are removed and the behavior of the lung microbiome can be studied more directly, allowing for finer analysis of bacterial activity in a mucus-plugged bronchiole.
The WinCF system provides a method to grow and analyze bacteria in a manner that effectively mimics the lung environment. Traditional methods for growing lung bacteria often involved culturing samples on traditional agar plates. These methods leave the samples open to atmospheric oxygen, neglecting to account for the hypoxic and often anoxic conditions found in lung bronchioles plugged with mucus12,13. Culturing on agar under aerobic conditions is nothing like the environment of the CF lung and can mislead clinicians and researchers concerning the behavior of the pathogens that they are trying to treat. Additionally, the nutrients available to bacteria on agar plates are dissimilar to those available in actual sputum, which is accounted for in WinCF by utilizing artificial sputum media (ASM). As shown by the Pseudomonas cultures in Sriramulu et al.14, ASM includes a specific set of components that mimics the resources available to sputum microbes and also replicates the physical consistency of sputum. Because a diseased lung has a specific microbiome, the study of such microorganisms should ideally take place in the specific conditions of the lung as well.
The WinCF system enables rapid analysis and easy manipulation of the experimental conditions to observe microbial changes similar to how they would occur in an actual lung bronchiole. This technique allows for the inoculation of a myriad of related sample types including sputum, saliva, other body secretions and pure or mixed bacterial cultures. The nature of the experimental setup allows for immediate visual interpretation of the microbial community behavior and is designed to enable easy downstream application of a multitude of microbiological and omics procedures. Such studies are important because bacterial community composition changes based upon the physiochemical conditions of their environment. With WinCF the chemical conditions of the media can be manipulated to analyze the effects on bacterial activity. For example, the acidity of the media can be altered prior to inoculation with a sample. After incubation, the bacterial activity in each of these conditions can be directly compared, and conclusions can be drawn about how bacteria in those sputum samples behave in response to varying pH. Here, we outline the procedures for applying the WinCF system and examples of how the media chemistry can be manipulated to study the effects on the lung microbiome.

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Color-Coded Capillary Tubes</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">22-260943</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cha-seal Tube Sealing Compound</td>
			<td style="width:93px;">Kimble-Chase</td>
			<td align="right" style="width:119px;">43510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mucin from porcine stomach</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">M1778</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ferritin, cationized from horse spleen</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">F7879</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Salmon sperm DNA Sodium salt (sonified)</td>
			<td style="width:93px;">AppliChem Panreac</td>
			<td>A2159</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MEM Nonessential Amino Acids</td>
			<td style="width:93px;">Corning cellgro</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MEM Amino Acids</td>
			<td style="width:93px;">Cellgro</td>
			<td style="width:119px;">25-030-CI</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Egg Yolk Emulsion, 50%</td>
			<td style="width:93px;">Dalynn Biologicals</td>
			<td style="width:119px;">VE30-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Chloride</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">P2157500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Chloride</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">S271500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">15mL centriguge tubes with Printed Graduations and Flat Caps</td>
			<td style="width:93px;">VWR</td>
			<td style="width:119px;">89039-666</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50mL centrifuge tubes with Printed Graduations and Flat Caps</td>
			<td style="width:93px;">VWR</td>
			<td style="width:119px;">89039-656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5mL microcentrifuge tubes</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">MCT-150-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2.0mL microcentrifuge tubes</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">MCT-200-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

the wincf model - an inexpensive and tractable microcosm of a mucus plugged bronchiole to study the microbiology of lung infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

Microbiological growth across the various chemical conditions induced within the samples varied dramatically in some cases and more subtly in others. Many changes in activity were visual in nature, being readily apparent as soon as the incubation period ended. In the example of pH manipulation, the samples across the pH spectrum varied greatly as shown by multiple factors that became apparent after incubation. When no sputum samples were added to the media, the only change exhibited acros...

Watch this Scientific Journal Video about The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections at JoVE.com

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

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Immunology and Infection

Unit 1

Lower Respiratory Disorders

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9.0K Views.  University of California, San Diego. The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Video: The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

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

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

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

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

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

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...