The author would like to acknowledge the animal housing units at Lund University, Camilla Bj&#246;rkl&#246;v and Agnieszka Czopek, for their support, and Richard Williams, Kennedy Institute of Rheumatology, University of Oxford, UK, for constructive criticism and linguistic support producing this manuscript.

All animal procedures were performed according to the practices of the Swedish Board of Animal Research and were approved by the Animal Ethics Committee, Lund-Malm&#246;, Sweden (Permit number: M126-16).
NOTE: A schematic flow of the method is described in Figure 1.
1. Material preparation
NOTE: Prepare all the reagents aseptically in a sterile hood, and aliquot and store at the indicated temperature. The reagents can be stored up to 2 years without losing their effect.
<ol>
	<li>Prepare CFA containing 20 mg/mL&#160;Mycobacterium tuberculosis as described below.
	<ol>
		<li>Add 100 mg of freeze-dried M. tuberculosis H37RA (see Table of Materials) and five 3.2 mm steel beads to a 7 mL tube (see Table of Materials). Shake the tube in a homogenizer (see Table of Materials) for 60 s at the highest speed setting (5,000 rpm).</li>
		<li>Add 5 mL of incomplete Freund&#39;s adjuvant (IFA) to the tube and shake again for 60 s at the highest speed. Transfer the slurry of homogenized M. tuberculosis in IFA (now named CFA) to a fresh tube with a pipette, leaving the beads behind, and store at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Add ultrapure water to the freeze-dried powder of MOG35-55 peptide (see Table of Materials) to obtain a final peptide concentration of 10 mg/mL. Sterilize the solution by passing it through a 0.2 &#181;m filter. Prepare 60 &#181;L aliquots and store at -20 &#176;C until use. 
	NOTE: The MOG35-55 peptide used for this experiment is of ImmunoGrade purity (~70%), is TFA-cleaved, dissolves easily in water, and contains a C-terminal amide (-NH2) to increase in vivo stability14. Peptide aliquots were never thawed and re-frozen more than twice, and were stored at -20 &#176;C until use.</li>
	<li>Prepare 100 mL of pertussis toxin buffer: Dissolve 2.9 g of NaCl in 80 mL of 1x Ca2+/Mg2+ free PBS and add 100 &#181;L of 10% Triton X-100. Adjust the volume to 100 mL with PBS and sterilize the solution by passing it through a 0.2 &#181;m filter. Prepare 10 mL aliquots and store at 4 &#176;C until use.</li>
</ol>
2. Preparation of CFA/peptide emulsions
<ol>
	<li>Calculate the amount of emulsion needed for immunization. In this standard protocol, each mouse receives 50 &#181;g of MOG35-55 peptide and 300 &#181;g of M. tuberculosis dispersed in Freund&#39;s adjuvant (CFA). The amount of each reagent (MOG35-55 peptide, PBS, CFA, and IFA) required for preparing the emulsions can be calculated using Supplemental File 1. An additional 20% of all reagents, to compensate for the small loss of emulsion, is included in the calculation. 
	NOTE: The commercial emulsion kit (see Table of Materials) used in this study comprises a tube, screw cap, and a plunger in a sterilized pouch. Each tube of the emulsion kit holds a maximum of 9.6 mL, making it possible to prepare 8 x 1 mL syringes sufficient for immunizing 40 mice (or 80 mice if using 100 &#181;L of emulsion per animal).</li>
	<li>Place the screw capped tube (from the commercial emulsion kit) and reagents to be used on ice.</li>
	<li>Add the emulsion components in the following order in the volumes calculated in step 2.1: PBS, then the peptide, then M. tuberculosis in CFA, and finally IFA.</li>
	<li>Close the tube with the cap firmly by tightening and loosening it several times. Shake the tube vigorously for 5-10 s by hand to pre-mix the reagents.</li>
	<li>Place the tube in the shaking homogenizer and secure it with the rod. Set the speed to the highest setting and the time to 60 s. Once the run is finished, place the tube on ice for 3 min. Repeat the run one or two more times with the same settings.</li>
	<li>Centrifuge the tube at 300 x g for 1 min to remove trapped air and compact the emulsion.</li>
	<li>Remove the cap from the tube, insert the plunger into the tube, and push it down slowly until it reaches the top of the emulsion. Remove the snap off closure at the bottom of the tube by twisting it.</li>
	<li>Remove the plunger from an injection syringe (1 mL; see Table of Materials). Add a needle (preferably 25-27 G) to the injection syringe.</li>
	<li>Attach the back end of the injection syringe to the dedicated lock at the bottom of the tube and lock it with a short twist.</li>
	<li>Transfer the emulsion from the tube to the injection syringe by pushing the plunger gently. Stop when the emulsion reaches the 0.15 mL graduation of the injection syringe.</li>
	<li>Separate the injection syringe from the tube and insert the plunger carefully, taking care that no air enters the syringe. Push the plunger until the emulsion comes out of the needle. 
	NOTE: At this point, some of the emulsion can be used for quality control (see step 3).</li>
	<li>Repeat steps 2.8-2.11 for the rest of the emulsion present in the tube. 
	&#8203;NOTE: To minimize the waste of expensive CFA/antigen emulsions, 1 mL syringes should be used. Other syringes that fit the dedicated lock at the bottom of the tube may be used; however, a larger volume of emulsion may need to be prepared. The CFA/MOG35-55 peptide emulsion can be stored at 4 &#176;C for up to 15 weeks without losing its EAE-inducing capacity.</li>
</ol>
3. Quality control of emulsion
<ol>
	<li>Drop-test: Add a small drop of the emulsion into a sterile 50 mL conical tube filled with 20 mL of cold water. Close the tube and shake it by hand for a couple of seconds. The appearance of tiny distinct droplets confirms the formation of a water-in-oil emulsion.</li>
	<li>Examine the emulsion using phase contrast microscopy.
	<ol>
		<li>To analyze the size of the water-in-oil particles, add a tiny drop (2-3 &#181;L) of the emulsion to a microscope slide, smear it out with a cover slip, and then push hard in a circular motion to flatten out the emulsion.</li>
		<li>Examine the smeared emulsion under a phase contrast microscope (see Table of Materials) with 400x magnification and focus on a field with a monolayer of the emulsion. Ensure that small uniform grey/white particles are visible (Figure 2A).</li>
	</ol>
	</li>
	<li>Analyze the emulsion particle size using laser diffraction. 
	NOTE: Laser diffraction measures particle size distributions using a laser beam. This is the ultimate quality control test for emulsions. A representative result of particle size distributions using the method described here is shown in Figure 2B (for a detailed description, see Topping et al.6). Nevertheless, the drop test and microscope examination are sufficient to validate whether a satisfactory emulsion has been formed.
	<ol>
		<li>Set the refractive indices for both red and blue lasers for the dispersant (see Table of Materials) to 1.456 and for the sample to 1.33 on the particle size analyzer (see Table of Materials). Set the absorbance of the refractive index to 0.02.</li>
		<li>Add one drop from the syringe containing the peptide emulsion to the dispersion unit containing light mineral oil. Start the data acquisition instantly after dispersion of the emulsion. 
		&#8203;NOTE: A general-purpose model was applied, and spherical particles were assumed, for the estimation of particle size distribution.</li>
	</ol>
	</li>
</ol>
4. EAE induction using the MOG&#160;35-55 peptide emulsion in C57BL6/J mice
NOTE: In all experiments, five 8-12-week-old female C57BL6/J mice were used.
<ol>
	<li>Prior to immunization, anesthetize the mice using 2.5% vaporized isoflurane and confirm using a firm toe pinch.</li>
	<li>Inject each mouse subcutaneously using 1 mL syringes into two different sites, on the right and left sides of the hind flank with 200 &#181;L of emulsion containing 50 &#181;g of MOG35-55 peptide in a 1:1 (v/v) ratio of peptide/CFA.</li>
	<li>At least 1.5 h after the immunization with the emulsion, and then again after 24 h, administer a total of 80 ng of Bordetella pertussis toxin in 200 &#181;L of pertussis toxin buffer to each mouse, through intraperitoneal injections (100 &#181;L on the left and 100 &#181;L on the right site of the belly). 
	NOTE: Precise localization of intraperitoneal injection with pertussis toxin has been shown to be essential for the activation of T cells in the draining lymph node15.</li>
	<li>Optional: Inject MOG35-55 peptide again on day 7 (as used in some protocols16) by repeating step 4.2. 
	NOTE: Most importantly, make sure that the syringes and needles used for immunization containing MOG/CFA or pertussis toxin are adequately disposed of in biohazard bags/containers.</li>
	<li>Evaluate the clinical score daily, using a scale from 0-6 as previously described6.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+and+T+cells+driving+multiple+sclerosis:+identity,+mechanisms+and+potential+triggers.">B and T cells driving multiple sclerosis: identity, mechanisms and potential triggers.</a> Frontiers in Immunology. 11, 760 (2020).</li><li>Sambucci, M., Gargano, F., Guerrera, G., Battistini, L., Borsellino, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One,+No+One,+and+One+Hundred+Thousand:+T+Regulatory+Cells'+Multiple+Identities+in+Neuroimmunity.">One, No One, and One Hundred Thousand: T Regulatory Cells' Multiple Identities in Neuroimmunity.</a> Frontiers in Immunology. 10, 2947 (2019).</li><li>Mix, E., Meyer-Rienecker, H., Hartung, H. P., Zettl, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+multiple+sclerosis--potentials+and+limitations.">Animal models of multiple sclerosis--potentials and limitations.</a> Progress in Neurobiology. 92 (3), 386-404 (2010).</li><li>Terry, R. L., Ifergan, I., Miller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Autoimmune+Encephalomyelitis+in+Mice.">Experimental Autoimmune Encephalomyelitis in Mice.</a> Methods in Molecular Biology. 1304, 145-160 (2016).</li><li>Topping, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+antigen-emulsion+preparations+for+the+induction+of+autoimmune+disease+models.">Standardization of antigen-emulsion preparations for the induction of autoimmune disease models.</a> Frontiers in Immunology. 13, 892251 (2022).</li><li>Flies, D. B., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+vortex+method+for+preparing+antigen/adjuvant+emulsions+for+immunization.">A simple and rapid vortex method for preparing antigen/adjuvant emulsions for immunization.</a> Journal of Immunological Methods. 276 (1-2), 239-242 (2003).</li><li>M&#228;&#228;tt&#228;, J. A., Er&#228;linna, J. P., R&#246;ytt&#228;, M., Salmi, A. A., Hinkkanen, A. 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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+modifications+inhibit+proteolytic+degradation+of+an+immunogenic+MART-1(27-35)+peptide:+implications+for+peptide+vaccines.">Terminal modifications inhibit proteolytic degradation of an immunogenic MART-1(27-35) peptide: implications for peptide vaccines.</a> International Journal of Cancer. 83 (3), 326-334 (1999).</li><li>Kool, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alum+adjuvant+boosts+adaptive+immunity+by+inducing+uric+acid+and+activating+inflammatory+dendritic+cells.">Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells.</a> The Journal of Experimental Medicine. 205 (4), 869-882 (2008).</li><li>Hasselmann, J. P. C., Karim, H., Khalaj, A. 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BTB Emulsions AB has submitted a patent application for the use of a device and method for preparing emulsions for immunization and animals and humans (European patent application number: EP3836884A1). BTB is the CEO and founder of the company, and a shareholder in BTB Emulsions. Bertin-Instruments is distributing the emulsion kits used in this publication world-wide.

Water-in-oil emulsions, such as antigen/Freund&#39;s adjuvant, have been used for more than half a century to induce EAE17. There is currently no standardized method to prepare antigen emulsions that is independent of human influence. Manual mixing using syringes is standard for most laboratories, however this method is time consuming, often results in an excessive loss of material, and the quality differs depending on the scientist preparing it.
The method presented in...

A breakdown of immunological tolerance can result in the generation of autoimmune disorders, such as multiple sclerosis (MS). It is estimated that 2.8 million people are living with MS worldwide1. Although the exact cause of MS is still largely unknown, dysregulation of autoreactive T and B cells, as well as defects in Treg function, play important roles in the pathogenesis of the disease2,3.
Animal models of autoimmune diseases are essential tools to investigate potential therapeutic modalities. The experimental autoimmune encephalomyelitis (EAE) model has been used for almost a century by researchers interested in MS4. In early experiments, the incidence of the disease was relatively low. The introduction of complete Freund&#39;s adjuvant (CFA), containing Mycobacterium and pertussis toxin, enabled the consistent induction of EAE in mice4. Most importantly, it is necessary to mix CFA with a central nervous system (CNS)-specific antigen to generate a homogenous water-in-oil emulsion for inducing EAE. The most common currently available EAE models are based on the active immunization of mice with encephalitogenic peptides. The genetic background of the mice plays an important role in disease susceptibility, with myelin oligodendrocyte glycoprotein (MOG35-55) and myelin proteolipid protein (PLP139-151) peptides used to induce EAE in C57BL/6J and SJL mice, respectively5. However, other mouse strains and CNS-derived peptides can also be used.
The quality of the CFA/peptide emulsion is a critical factor that determines disease penetrance in the active immunization EAE model6. A homogeneous water-in-oil emulsion must be prepared by mixing the encephalitogenic peptides dissolved in aqueous buffer with CFA, otherwise animals will not develop the disease. Numerous protocols have been published on the preparation of CFA/peptide emulsions. Examples include the use of a vortex7, sonication8, syringes and a three-way T connector9, or one syringe only5. However, all these methods are difficult to standardize and are often associated with lengthy and complicated protocols.
Compared to all the above methods, the simple method described here for emulsion preparation offers the advantages of having no person-to-person differences and being relatively fast. The emulsion is generated by a homogenizer shaking the reagents with a set speed, time, and temperature, ensuring fast and consistent results. In addition to inducing disease in the EAE model, this method can also be used to study other autoimmune disease models such as collagen-induced arthritis (CIA) and antigen induced arthritis (AIA)6. Therefore, it is anticipated that this method can be used to consistently induce disease in other animal models that depend on water-in-oil emulsions with autoantigens, such as experimental autoimmune neuritis (EAN)10, experimental autoimmune thyroiditis (EAT)11, autoimmune uveitis (EAU)12, and myasthenia gravis (MG)13. This method also induces general immune responses such as delayed-type hypersensitivity (DTH) consistently6, and could therefore be used for delivering cancer and malaria vaccines (see discussion).
Thus, a rapid (total preparation time ~30 min), simple (all reagents can be prepared in advance and stored), and standardized (the emulsion is accomplished using a shaking homogenizer) method has been developed and is presented here. The CFA/antigen emulsions prepared using this protocol consistently induce disease in autoimmune animal models.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Injection syringe</td>
			<td>B. Braun</td>
			<td>9166017V&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Injection syringe</td>
			<td>Sigma-Aldrich</td>
			<td>Z683531</td>
			<td></td>
		</tr>
		<tr>
			<td>7 ml empty tubes with caps</td>
			<td>Bertin-Instruments</td>
			<td>P000944LYSK0A.0</td>
			<td>7 mL tube</td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tube&#160;</td>
			<td>Fisher Scientific</td>
			<td>10788561</td>
			<td>50 mL tube</td>
		</tr>
		<tr>
			<td>Bordetella pertussis toxin</td>
			<td>Sigma-Aldrich</td>
			<td>P2980</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Dispersant, light mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Emulsion kit</td>
			<td>Bertin-Instruments</td>
			<td>D34200.10 ea</td>
			<td>Containing a tube, cap, and plunger</td>
		</tr>
		<tr>
			<td>Incomplete Freund&#39;s Adjuvant</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;F5506</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis, H37RA</td>
			<td>Fisher Scientific</td>
			<td>DF3114-33-8</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Mastersizer 2000&#160;</td>
			<td>Malvern Panalytical</td>
			<td>N/A</td>
			<td>Particle size analyzer</td>
		</tr>
		<tr>
			<td>Minilys-Personal homogenizer</td>
			<td>Bertin-Instruments</td>
			<td>P000673-MLYS0-A</td>
			<td>Shaking homogenizer</td>
		</tr>
		<tr>
			<td>MOG 35-55 Peptide&#160;&#160;&#160;</td>
			<td>Innovagen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Montanide ISA 51 VG</td>
			<td>Seppic</td>
			<td>36362Z</td>
			<td>FDA-approved oil adjuvant</td>
		</tr>
		<tr>
			<td>Pall Acrodisc Syringe Filters 0.2 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>17124381</td>
			<td>Sterlie filter</td>
		</tr>
		<tr>
			<td>PBS, Ca2+/Mg2+ free</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Phase-Constrast Microscope</td>
			<td>Olympus</td>
			<td>BX40-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel Beads 3.2 mm</td>
			<td>Fisher Scientific</td>
			<td>NC0445832</td>
			<td>Autoclave and store at RT</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>648463</td>
			<td>Store at RT</td>
		</tr>
	</tbody>
</table>

a rapid, simple, and standardized homogenization method to prepare antigen/adjuvant emulsions for inducing experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

The rapid, simple, and standardized protocol for the preparation of CFA/MOG emulsions is depicted in Figure 1. This method has recently been described elsewhere6. The CFA/MOG emulsions can also be prepared with other methods, such as the traditional syringe method or by vortexing. These methods were compared here by assessing the quality of the emulsions. All the methods produced water-in-oil emulsions; the homogeneity and quality of these emulsions were assessed by a...

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A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely ...

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Research

JoVE Journal

Immunology and Infection

3.1K Views.  Lund University. To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

Video: A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Immunogenicity Enhancement by Mycobacterium paratuberculosis in Autoimmune Encephalomyelitis

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.

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

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.

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

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...