eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Antigen preparation for subcutaneous injection
<ol>
	<li>Perform all procedures in this section inside a chemical fume hood to prevent inhalation or skin contact with the Mycobacterium tuberculosis, Mtb H37Ra powder. Handle according to your institutional policies for Complete Freund&#39;s Adjuvant (typically BSL-1). This includes using chemical-resistant gloves, safety glasses, and protective work clothing (lab coat).</li>
	<li>Use a good sterile technique to prevent contamination of reagents that will be introduced into experimental animals.</li>
	<li>Make the Mtb suspension in phosphate-buffered saline (PBS) by mixing 5 mg of lyophilized, heat-killed&#160;Mycobacterium tuberculosis&#160;H37Ra powder with 2.5 mL of cold PBS in a 5 mL microcentrifuge tube. Vortex once for 30 seconds and then place on ice.</li>
	<li>To generate a fine suspension of the H37Ra in PBS, sonicate the suspension on ice for 5 min.
	<ol>
		<li>Unclamp the body of the converter unit and clean the probe with a 70% (v/v) alcohol swab.</li>
		<li>Switch on the sonicator, adjust the power setting to 4 by turning the power control knob, and immerse the probe&#39;s tip into the PBS-containing mycobacterial powder. Ensure that the probe tip is immersed to at least half the depth of the sample and that the probe tip is not touching the wall of the microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Sonicate the mixture on ice for 30 seconds, pause for 30 seconds, and repeat for a total of 5 min to fully disperse the powder into an even suspension without heating the liquid.</li>
	<li>Add 2.5 mL of Freund&#39;s Incomplete Adjuvant to the mixture and repeat the sonication process on ice until the emulsion forms a toothpaste-like consistency.</li>
	<li>Set the power to 0 using the control knob and turn off the unit to end the sonication. Remove the tip from the suspension and wipe the probe with an alcohol swab.</li>
	<li>Store the antigen emulsion at 4 &#176;C. Making batches of the emulsion will help ensure consistency across experiments. The emulsion can be stored at 4 &#176;C for up to 3 months.</li>
</ol>
2. Subcutaneous injection
<ol>
	<li>Perform subcutaneous injection a week prior to the intravitreal injection (designated as day -7).</li>
	<li>Load a 1 mL syringe (no needle attached) with the mycobacterial emulsion. Due to the viscosity and opacity of the emulsion, difficult-to-see air bubbles can fill the syringe.
	<ol>
		<li>To prevent air bubbles in the syringe, after loading 0.2-0.3 mL of emulsion, invert the syringe (tip facing up) and gently tap the syringe repeatedly on the edge of a counter to bring the bubbles to the surface.</li>
	</ol>
	</li>
	<li>Expel the air from the syringe and continue filling the syringe. Invert and intermittently tap until filled.</li>
	<li>Place a 25 G needle on the syringe and advance the emulsion to fill the needle. Store the syringe on ice until used.</li>
	<li>To perform the subcutaneous injection safely, either anesthetize the mouse or utilize humane restraint methods that allow easy access to the animal&#39;s hindquarters.</li>
	<li>To anesthetize for subcutaneous injection, place the animal in an isoflurane induction chamber (3%-4% for induction and 1%-3% for maintenance). Once anesthetized, ensure that the mouse has a slow respiratory rate and exhibits no signs of respiratory distress.</li>
	<li>Place the subcutaneous injections on either the dorsal surface of the hips or on the ventral surface of the legs proximal to the region of the inguinal lymph nodes.
	<ol>
		<li>Carefully insert the needle to prevent injecting into the muscle. Inject 0.05 mL of the Mtb emulsion into the subcutaneous space. Do not remove the needle immediately in order to allow the thick emulsion to be fully injected.</li>
		<li>Repeat the injection on both left and right sides for a total of 0.1 mL per animal.</li>
	</ol>
	</li>
	<li>If anesthetized, place the mouse on a warm heating pad until complete recovery. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Return the mouse to its cage upon complete recovery and label the cage card with the date of subcutaneous injection.</li>
	<li>Provide analgesia with oral acetaminophen (200 mg/kg/day), but not Non-steroidal anti-inflammatory drugs (NSAIDs) as anti-inflammatory agents can impact the induction of uveitis.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Freund&#39;s Incomplete Adjuvant</td>
			<td>BD Difco, NJ, USA</td>
			<td>263910</td>
			<td></td>
		</tr>
		<tr>
			<td>H37Ra lyophilized Mycobacteria extract</td>
			<td>BD Difco, NJ, USA</td>
			<td>231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin needle</td>
			<td>Exel International, USA</td>
			<td>26029</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>RM2255</td>
			<td>Leica Biosystems, IL,USA</td>
			<td></td>
			<td>Tissue Sectioning</td>
		</tr>
		<tr>
			<td>TB Syringe</td>
			<td>Becton, Dickinson and Company, NJ, USA</td>
			<td>309602</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Tylenol</td>
			<td>Johnson &#38; Johnson Consumer Inc, PA, USA</td>
			<td>NDC 50580-614-01</td>
			<td>Acetaminophen</td>
		</tr>
	</tbody>
</table>

subcutaneous injection of bacterial antigen to induce systemic inflammation in a murine model

Source: John, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62925">Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.</a>&#160;J. Vis. Exp. (2021)
This video demonstrates the induction of systemic inflammation in an anesthetized mouse. The injection of mycobacterial antigen-adjuvant depots triggers cytokine release and localized inflammation. Lymphatic vessels open, enabling immune cell entry into lymph nodes and activation of T cells, which release pro-inflammatory cytokines, leading to widespread inflammation.

Subcutaneous Injection of Bacterial Antigen to Induce Systemic Inflammation in a Murine Model

Source: John, S. et al., Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.&#160;J. Vis. Exp. (2021)
This video demonstrates the ...

To induce systemic inflammation &#8212; a widespread inflammatory response, begin by positioning an anesthetized mouse with its dorsal side up. Take a syringe containing mycobacterial emulsion of mycobacterial antigen-adjuvant depots &#8212; a localized oil reservoir of the antigen-adjuvant mixture.
Pierce the mouse&#39;s epidermis and dermis skin layers and inject the antigen-adjuvant emulsion subcutaneously proximal to the inguinal lymph node. Post-injection, the depot continuously releases antigens into the surrounding tissue &#8212; providing prolonged exposure of the antigens to the immune system.
Antigen-presenting cells, or APCs, in the tissue, interact with injected mycobacterial antigens, internalize, and process them into peptides. These peptides are then presented on the surface of APCs using major histocompatibility complexes, triggering the release of pro-inflammatory cytokines and chemokines.
This process generates localized inflammation and triggers the opening of lymphatic vessels, enabling the entry of immune cells including antigen-carrying APCs into the lymphatic system and reaching the inguinal lymph node.
In lymph nodes, T cell receptors of helper T cells interact with antigens on the APCs&#39; surface and become activated. The activated T cells release pro-inflammatory cytokines, which travel through lymphatic vessels and get released into various other tissues.
The released pro-inflammatory cytokines attract the immune cells to these tissues, inducing systemic inflammation.

subcutaneous-injection-bacterial-antigen-to-induce-systemic

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

283 次观看. 来源:John， S. et al.， 引发分枝杆菌葡萄膜炎 （PMU） 作为感染后葡萄膜炎的模型。J. Vis. Exp. （2021 年） 该视频演示了麻醉小鼠全身炎症的诱导。注射分枝杆菌抗原佐剂贮库会触发细胞因子释放和局部炎症。淋巴管打开，使免疫细胞能够进入淋巴结并激活 T 细胞，从而释放促炎细胞因子，导致广泛的炎症。 

视频: 皮下注射细菌抗原在小鼠模型中诱导全身炎症 - 概念

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances.
Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a &#39;sepsis-like&#39; state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

静脉注射内毒素挑战人类健康的实验平台，研究并调制全身炎症

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or ...

科研

JoVE Journal

医学

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

免疫与感染

副结核分枝杆菌在自身免疫性脑脊髓炎中的免疫原性增强

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

作者聚焦: 副结核分枝杆菌在增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原性中的辅助活性  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...

Subcutaneous Injection of Bacterial Antigen to Induce Systemic Inflammation in a Murine Model

成績單

Subcutaneous Injection of Bacterial Antigen to Induce Systemic Inflammation in a Murine Model