This work was supported by NIH Grant HL093196 (R.C.R.) and the Atlanta Research and Education Foundation (AREF).

I. Allergen Sensitization and Challenge (see Figure 1)
A. For Intratracheal Challenge
<ol>
	<li>For initial sensitization, inject male or female C57BL/6 or BALB/c mice (6-8 weeks old) intraperitoneally on day 0 and again on day 7 with 20 &mu;g of ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO) emulsified in 0.2 ml of sterile phosphate buffered saline (PBS) containing 2 mg of aluminum hydroxide (Sigma-Aldrich) or with 2 mg aluminum hydroxide in 0.2 ml of sterile PBS as control.</li>
	<li>Challenge with antigen as appropriate (e.g., on days 14, 16, 18, and 20). Challenge procedure follows.</li>
	<li>Anesthetize mouse with an intraperitoneal (i.p.) injection of a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg). Ensure the mouse is fully anesthetized for at least 10 min.</li>
	<li>Angle operation surface at 45 degrees or greater. Place mouse on this surface keeping ventral side upward and head at top.</li>
	<li>Hook thread under front incisors to hold head back. Level the paws with one another to ensure the trachea is straight and use label tape to hold legs. Soak surgical site with 70% EtOH and swab.</li>
	<li>Apply bupivicaine (0.1 to 0.2 ml of 0.25% solution) topically at the incision site.</li>
	<li>Nip skin on the throat with forceps and pull gently outward. Make a small vertical incision with surgical scissors. Minimize size of incision.</li>
	<li>Prepare a 1 ml syringe with 50 &mu;l of PBS or 0.1% OVA in PBS and insert it into a repetitive pipette (Tridak Stepper, Torrington, CT). Take the pipette in one hand and use the other to hold the tissue back with the tweezers and expose the trachea.</li>
	<li>Holding the syringe as parallel to the trachea as possible, insert needle through trachea wall and inject the solution.</li>
	<li>Maintain mouse in a vertical orientation following injection to allow time for solution to settle in the lungs.</li>
	<li>Gently and sterilely close wound area with tweezers and seal with suture.</li>
	<li>Place mouse sternum-down on a heating pad and allow it to recover until fully ambulatory. Following recovery, return the mouse to the animal care facility and monitor it daily for signs of seroma, inflammation or infection, and wound dehiscence until the wound is fully healed.</li>
</ol>
B. For Challenge by Nebulization
<ol>
	<li>Sensitize mice on day 0 by intraperitoneal injection of 20 &mu;g of OVA (Sigma-Aldrich) emulsified in 0.2 ml of sterile PBS containing 2 mg of aluminum hydroxide (Sigma-Aldrich) or with 2 mg aluminum hydroxide in 0.2 ml of sterile PBS as control.</li>
	<li>On day 14, boost sensitization by i.p. injection as described above.</li>
	<li>On the 21st, 22nd, 23rd, 24th and 25th day after initial sensitization, challenge mice by exposure for 30 min to nebulized 1% OVA or PBS alone delivered via an ultrasonic nebulizer (Buxco Research Systems, Wilmington, NC).</li>
	<li>Place mice in main chamber of WBP; acclimatize them within the plethysmography chamber for at least 10 min.</li>
	<li>Place 1 ml of 0.1% OVA in sterile PBS or sterile PBS alone, as described in steps 4-6 below, via a nebulizer cup. Nebulize for 30 min.</li>
	<li>Remove nebulizer cup and discard any remaining solution.</li>
	<li>Nebulization can be performed on all mice simultaneously by using a nebulization chamber.</li>
</ol>
II. Determination of Airway Hyperresponsiveness to Methacholine
A. Noninvasive Measurement of Airway Hyperresponsiveness by Whole-body Plethysmography (WBP; Buxco Research Systems, Wilmington, NC)
<ol>
	<li>Allow the powdered methacholine bottle to warm to room temperature before opening (methacholine is very hygroscopic and will form useless clumps if allowed to absorb water). Prepare a 200 mg/ml stock solution in sterile PBS, then make serial 2-fold dilutions (e.g., 100, 50, 25, 12.5 and 6.25 mg/ml). Keep solutions cold.</li>
	<li>Set up equipment as follows: connect main inlet of WBP to nebulizer, bias-flow inlet to air-pump, and WBP outlet to gas trap using tight-fitting rubber tubing. Attach pressure transducer to bridge the outlets of the main and reference chambers of the WBP. Connect pressure transducer to preamplifier with the cables provided, and connect preamplifier to PC using specific data-acquisition card.</li>
	<li>Calibrate the preamplifier using the software according to manufacturer's recommendations.</li>
	<li>Place mice in main chamber of WBP; acclimatize them within the plethysmography chamber for at least 10 min, then record baseline readings (Penhbase) for 3 min.</li>
	<li>Place 1 ml of sterile PBS in the nebulizer cup. Nebulize for 2 min and then monitor respiratory variables for an additional 6 min during the drying phase. Remove nebulizer cup and discard any remaining PBS.</li>
	<li>Place 1 ml of 6.25 mg/ml methacholine in the nebulizer cup and repeat nebulization for 2 min plus a 6-min monitoring cycle.</li>
	<li>Repeat measurement with 12.5, 25, 50 and 100 mg/ml methacholine, using the same 2-min nebulization period and 6-min monitoring cycle.</li>
	<li>Remove the mice from the chambers and return them to their cages.</li>
	<li>Refill nebulizer cup with 1 ml sterile PBS and run another sequence to flush the tubing.</li>
	<li>Shut down the air flows, disassemble, and wipe clean all chambers before running a second set of animals.</li>
</ol>
B. Invasive Measurement of Airway Responsiveness by Computer-controlled Ventilator (flexiVent; SCIREQ Inc., Montreal, Canada)
<ol>
	<li>Weigh the mouse and anesthetize by intraperitoneal (i.p.) injection of 60 mg per kg body weight pentobarbital sodium.</li>
	<li>Following adequate anesthesia, position the mice ventro-dorsally for tracheostomy.</li>
	<li>Disinfect the neck skin with 70% ethanol. Apply bupivicaine (0.1 to 0.2 ml of 0.25% solution) topically at the incision site. Incise and open the neck skin. Separate the neck muscles and expose the trachea.</li>
	<li>Make a 1- to 2-mm incision in the trachea with fine scissors (be certain not to sever the trachea) and insert the tracheal tube cautiously. Tie a suture around the trachea to prevent an air leak.</li>
	<li>Lay the mouse in the body plethysmograph chamber and connect the inserted tracheal tube to the ventilator.</li>
	<li>Start mechanical ventilation. Set appropriate respiratory rate and tidal/stroke volume (150 strokes/min and 200 &mu;l, respectively for a 20-g mouse). Be sure that the thorax is moving in synchrony with the ventilator. If the mouse is "fighting" with the ventilator (self-respiration), inject more anesthetic and wait for the synchronization.</li>
	<li>Following baseline measurements, maintain mice under baseline ventilation for another 3 min, and then take a 2nd set of impedance measurements. This 2nd set of baseline measurements is used to calculate the mean baseline values.</li>
	<li>Deliver PBS or methacholine (MCh) challenges (6.25, 12.5, 25, 50 and 100 mg/ml) by channeling inspiratory flow from the ventilator through an ultrasonic nebulizer.</li>
	<li>Following each challenge with MCh (6.25, 12.5, 25, 50 and 100 mg/ml), return the piston to delivering a Vt of 10 ml/kg at 120 breaths/min and take impedance measurements.</li>
</ol>
III. Measurement of Cellular Infiltration into the Airspace
A. Perform Bronchoalveolar Lavage (BAL)
<ol>
	<li>After measuring the AHR, euthanize mice with CO2, and position each mouse on its back on the surgical pad.</li>
	<li>Soak the area with 70% EtOH.</li>
	<li>Beginning at the lower abdomen, cut open the abdominal cavity and remove skin/upper muscle, moving upwards toward the ribs.</li>
	<li>Once the ribs are visible, use scissors to carefully puncture the diaphragm. Lungs should collapse away from the diaphragm. Be especially careful not to nick the lungs or heart.</li>
	<li>Cut away the ribcage to fully expose the lungs/heart (avoid cutting any major blood vessels to keep blood from filling the site).</li>
	<li>Using a 1 ml syringe with a 27 gauge needle (BD syringes, Franklin Lakes, NJ), puncture the heart ventricles and slowly and carefully pull back the syringe to collect the blood. Take care to avoid collapsing the heart.</li>
	<li>Collect the serum from this blood using standard protocol. Store at -70 &deg;C until use.</li>
	<li>Cut away skin and tissue from the throat until the trachea is revealed. Clear away sufficient tissue to work easily within the field (again, avoid cutting any major blood vessels).</li>
	<li>Using curved scissors, cut under the trachea to clear a path.</li>
	<li>Pass the point of a curved forceps under the trachea and grasp the end of a piece of suture. Draw the suture thread under the trachea.</li>
	<li>Tie a loose half-knot about the trachea, low in the throat.</li>
	<li>Carefully cut a notch, sufficient in size for the cannula, above the suture thread.</li>
	<li>Carefully insert the cannula into the hole and down the trachea past the point of the suture thread. Gently press forward until the cannula emerges just at the entrance to the lungs (too far: puncture lungs; too short: collapse trachea when attempting to recover BAL).</li>
	<li>Tighten suture thread and complete knot to seal trachea around cannula.</li>
	<li>Lock syringe (containing 1 ml PBS) on the cannula, and gently press the fluid into the lung. Lung lobes should individually inflate slowly. Do not over-fill. For a full grown mouse 0.9-1.0 ml is the absolute maximum. 0.8 ml may be safer. Lock syringe loosely to cannula, otherwise it is likely to cause damage when attempting to disengage.</li>
	<li>Withdraw fluid from lungs. If resistance is encountered (tissue sucked into cannula), press cannula slowly further into the lung and resume removing. Also try rotating the cannula in place. If all else fails, withdraw the cannula part way; the trachea is much more likely to collapse in this case.</li>
	<li>Detach syringe from cannula, deposit BAL fluid in container, and repeat 2 times with fresh PBS solution.</li>
	<li>Keep the BAL solution on ice until spun down.</li>
	<li>Use the BAL fluid and serum to measure the OVA specific IgE using commercially available mouse IgE ELISA kits (MD Bioproducts, St. Paul, MN).</li>
</ol>
B. Count Cells and Determine Differentials
<ol>
	<li>Centrifuge the BAL fluid 5 min at ~600 &times; g, 4 &deg;C.</li>
	<li>Resuspend the cell pellet gently in PBS and keep on ice.</li>
	<li>Load a standard Neubauer hemacytometer with the diluted cell suspension and count the cells.</li>
	<li>Remove aliquots of 2 &times; 104 cells in 10 to 40 &mu;l volume for cytospins. Dilute cells if necessary.</li>
	<li>For cytospins, mix 2 &times; 104 cells, 130 &mu;l PBS and 10 &mu;l FBS. Add entire cell mixture to double cytospin funnel and centrifuge 10 min at 700 rpm, using double cytoslides for duplicate samples.</li>
	<li>Allow the slides to dry at room temperature for 1 h prior to staining.</li>
	<li>Stain the slides by using Diff-Quick stain (Siemens, Newark, DE).</li>
</ol>
IV. Representative Results
Excessive airway constriction following provocative stimuli is a prominent feature of clinical asthma. We describe two methods for measuring such airway hyperresponsiveness to methacholine in OVA-sensitized and challenged mice: Whole-body plethysmography (Figure 2) and forced oscillation using the flexiVent system (Figure 3). Both methods demonstrate that OVA sensitization and challenge produces airway hyperresponsiveness in mice.
Eosinophil-rich airway inflammation is another prominent feature of both clinical asthma and allergic airway disease in mice. As shown in Figure 4, OVA sensitization and challenge greatly increases the total number of cells that can be recovered from the airways by BAL. The numbers of eosinophils and, to a lesser extent, neutrophils are especially increased.
Evidence indicates that allergic airway disease results from overproduction of IgE antibodies to sensitizing antigens. Sensitization and challenge with OVA using the protocols we describe increases IgE levels in both serum and BAL fluid of treated mice (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/3771/3771fig1.jpg" /> 
Figure 1. Experimental schema for OVA-induced allergic asthma. Mice were sensitized twice i.p. with 20 &mu;g of OVA emulsified in 2 mg of aluminum hydroxide in 0.2 ml of sterile PBS, or 2 mg of aluminum hydroxide in 0.2 ml of sterile PBS alone, followed at the indicated time points by i.t. challenge with 0.1% OVA or sterile PBS solution or by daily exposure for 30 minutes to nebulized 1% OVA in PBS or PBS alone delivered via an ultrasonic nebulizer (Buxco). Twenty-four hours after the final OVA exposure, airway responsiveness was determined. Subsequently, BAL fluid, blood samples, lung cells, and tissues were collected for further analysis.
<img alt="Figure 2" src="/files/ftp_upload/3771/3771fig2.jpg" /> 
Figure 2. Assessment of allergen-induced airway hyperresponsiveness by a noninvasive method. Mice (n=4/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, airway hyperresponsiveness to inhaled methacholine was determined using whole-body plethysmography as described in the protocol. Penh was determined and expressed as Penh ratio (average Penh over the 8-min time interval with methacholine divided by the average Penh over the 8-min interval with PBS). *, P &lt; 0.05 vs. PBS.
<img alt="Figure 3" src="/files/ftp_upload/3771/3771fig3.jpg" /> 
Figure 3. Assessment of allergen-induced airway hyperresponsiveness by an invasive method (forced oscillation). Mice (n=4/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, airway hyperresponsiveness to increasing concentrations of inhaled methacholine was determined by the forced oscillation (flexiVent) method as described in the protocol. A, B) Airway resistance; C) Lung elastance. *, P &lt; 0.05 vs. PBS.
 
<img alt="Figure 4" src="/files/ftp_upload/3771/3771fig4.jpg" /> 
Figure 4. BAL fluid cell count. Mice (n=4/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, (Top) BAL cells were collected and total cells were counted as described in the protocol. (Bottom) Cytospin slides were prepared and stained with Diff-Quick. Tot = total cells; Eos = eosinophils; Neu = neutrophils; Mac = macrophages; Lym = lymphocytes. *, P &lt; 0.05 vs. PBS.
<img alt="Figure 5" src="/files/ftp_upload/3771/3771fig5.jpg" /> 
Figure 5. OVA-specific IgE. Mice (n=4/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, IgE was measured in BAL fluid and in serum from the blood collected by cardiac puncture as described in the protocol. *, P &lt; 0.05 vs. PBS.

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Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Atlanta VAMC IACUC Committee under protocol #V010-10.

Animal models of allergic airway disease provide important tools for studies relevant to clinical asthma. A number of different models, employing varying species and antigens, have been developed. The mouse, an attractive and frequently used laboratory species, also offers a number of advantages for models of allergic airway disease.9,10 Although such models do not mimic asthma in every respect,11 with aspects of chronic disease being particularly difficult to reproduce,12,13 we confirm h...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ovalbumin</td>
			<td>Sigma-Aldrich 
			St. Louis, MO</td>
			<td>A5503</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aluminum hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>239186</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetyl-&beta;-methylcholine chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A2251</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Whole body plethysmography 
			(WBP) system</td>
			<td>Buxco Research Systems 
			Wilmington, NC</td>
			<td>&nbsp;</td>
			<td><a href="http://www.buxco.com" target="_blank">http://www.buxco.com</a></td>
		</tr>
		<tr>
			<td>FlexiVent</td>
			<td>SCIREQ, Inc. 
			Montreal, Canada</td>
			<td>&nbsp;</td>
			<td><a href="http://www.scireq.com" target="_blank">http://www.scireq.com</a></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Leica Microsystems, Inc. 
			Buffalo Grove, IL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cytospin 4</td>
			<td>Thermo Scientific 
			Asheville, NC</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Diff-Quick stain</td>
			<td>Siemens 
			Newark, DE</td>
			<td>B4132-1A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Repetitive pipette</td>
			<td>Tridak 
			Torrington, CT</td>
			<td>STP4001-0025</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

murine model of allergen induced asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

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Murine Model of Allergen Induced Asthma

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

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Immunology and Infection

Unit 1

Lower Respiratory Disorders

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39.8K Views.  Emory University and Atlanta VA Medical Center. Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Video: Murine Model of Allergen Induced Asthma

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...