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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Induction of Skin Inflammation in pIL1-DsRed Mice
<ol>
	<li>Anesthetize mouse by intraperitoneal (i.p.) injection of an anesthetic cocktail containing 100 mg/kg ketamine, 10 mg/kg xylazine, and 1 mg/kg acepromazine. Adequately anesthetized mice do not show hind paw withdrawal to toe pinch.</li>
	<li>Apply hair-removal cream to the dorsal surface of both ears of the mouse. Wait 30 sec to 1 min. Wipe ear surface with water-wetted cotton and allow to air-dry.</li>
	<li>Measure the ear thickness using a micrometer and replace the mouse in a separate cage. Observe until recovery from anesthesia (~ 15 - 30 min). To resolve skin inflammation induced by the hair removal product, allow the mouse to rest for 3 days before further experimentation is carried out.</li>
	<li>Prepare 1.25% (w/v) oxazolone (OX) by dissolving 16.7 mg of OX in 1 ml acetone and mixing 750 &#181;l of OX/acetone solution with 250 &#181;l of olive oil. Prepare vehicle solution by mixing 750 &#181;l of acetone with 250 &#181;l of olive oil.</li>
	<li>Re-anesthetize mouse by intraperitoneal (i.p.) injection of anesthetic cocktail (1.1). Measure the ear thickness as before.</li>
	<li>Apply 12.5 &#181;l of 1.25% OX onto each side of the right ear. Apply 12.5 &#181;l of acetone/olive oil vehicle solution onto each side of the left ear.</li>
	<li>Keep the mouse separate from cage-mates until fully recovered to prevent insult to its ears by other mice, then replace the mouse in its original cage and return it to the animal housing facility. 
	NOTE: Hair removal may result in minor skin inflammation following by change of ear thickness. This minor inflammation will be resolved 3 days later confirmed by normal ear thickness. After topical application for 24 hr, OX-treated ears show significant (p &#60; 0.01) swelling compared to vehicle solution alone-treated ears.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Hospira</td>
			<td>NDC 0409-2051-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>LLOYD Laboratory</td>
			<td>NADA #139-236</td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Boehringer Ingelheim</td>
			<td>ANADA 200-361</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair-removal cream</td>
			<td>Church &#38; Dwight</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A16P4</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxazolone</td>
			<td>Sigma-Aldrich</td>
			<td>E0753</td>
			<td></td>
		</tr>
	</tbody>
</table>

a technique to assess the immune response against an antigen by inducing skin inflammation

Source: Yao, Y., et al. <a href="https://app.jove.com/v/54070">Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice.</a> J. Vis. Exp. (2016).
This video demonstrates an in vivo technique to assess a test antigen by inducing skin inflammation in a murine model. The test antigen&#8212;a hapten in an oil/acetone solution&#8212;is topically applied to the skin of the mouse ear, and inflammation of the skin is assessed after an adequate period.

A Technique to Assess the Immune Response against an Antigen by Inducing Skin Inflammation

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take an anesthetized mouse with depilated ears, and apply the test antigen &#8212; a hapten mixed in a suitable vehicle solution &#8212; on the exposed skin. The vehicle solution facilitates hapten penetration through the outermost epidermis layer to reach the underlying dermis.
Hapten binds to skin proteins, forming an immunogen recognized by antigen-presenting cells or APCs. Upon engulfment, APCs process and display the immunogen on their surface.
APCs migrate to lymph nodes, presenting the immunogen to na&#239;ve T cells and activating them. Activated cells differentiate into primed memory T cells &#8212; a subset of which migrate to the skin.
Upon hapten re-exposure, APCs present the immunogen to the primed T cells, inducing pro-inflammatory cytokine release.
Cytokines trigger keratinocytes and mast cells to release chemokines, which bind to circulating neutrophils, facilitating their extravasation. The extravasated neutrophils reach the inflammation site.
Cytokines activate neutrophils to produce proteolytic enzymes and reactive oxygen species &#8212; causing tissue damage and amplifying the inflammation.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

77 Views. Source: Yao, Y., et al. Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice. J. Vis. Exp. (2016). This video demonstrates an in vivo technique to assess a test antigen by inducing skin inflammation in a murine model. The test antigen—a hapten in an oil/acetone solution—is topically applied to the skin of the mouse ear, and inflammation of the skin is assessed after an adequate period. 

Video: A Technique to Assess the Immune Response against an Antigen by Inducing Skin Inflammation - Concept

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrain&#160;the development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses ...

JoVE Journal

Immunology and Infection

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40&#160;conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40&#160;peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

Biology

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

A Technique to Assess the Immune Response against an Antigen by Inducing Skin Inflammation

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