This work was supported by a grant from the Multiple Sclerosis Society of Canada. RWJ is the recipient of the Waugh Family MS Society of Canada Doctoral Studentship Award.

<ol>
	<li>Compston, A., Coles, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis.">Multiple sclerosis.</a> Lancet. 372 (9648), 1502-1517 (2008).</li><li>Baxter, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+application+of+experimental+autoimmune+encephalomyelitis.">The origin and application of experimental autoimmune encephalomyelitis.</a> Nat Rev Immunol. 7 (11), 904-912 (2007).</li><li>Bittner, S., Afzali, A. M., Wiendl, H., Meuth, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+oligodendrocyte+glycoprotein+(MOG35-55)+induced+experimental+autoimmune+encephalomyelitis+(EAE)+in+C57BL/6+mice.">Myelin oligodendrocyte glycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice.</a> J Vis Exp. (86), (2014).</li><li>Shetty, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodominant+T-cell+epitopes+of+MOG+reside+in+its+transmembrane+and+cytoplasmic+domains+in+EAE.">Immunodominant T-cell epitopes of MOG reside in its transmembrane and cytoplasmic domains in EAE.</a> Neurol Neuroimmunol Neuroinflamm. 1 (2), 22 (2014).</li><li>Dang, A. K., Jain, R. W., Craig, H. C., Kerfoot, S. 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+cell+recognition+of+myelin+oligodendrocyte+glycoprotein+autoantigen+depends+on+immunization+with+protein+rather+than+short+peptide,+while+B+cell+invasion+of+the+CNS+in+autoimmunity+does+not.">B cell recognition of myelin oligodendrocyte glycoprotein autoantigen depends on immunization with protein rather than short peptide, while B cell invasion of the CNS in autoimmunity does not.</a> J Neuroimmunol. 278, 73-84 (2015).</li><li>Barun, B., Bar-Or, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+multiple+sclerosis+with+anti-CD20+antibodies.">Treatment of multiple sclerosis with anti-CD20 antibodies.</a> Clin Immunol. 142 (1), 31-37 (2012).</li><li>Bettadapura, J., Menon, K. K., Moritz, S., Liu, J., Bernard, C. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+and+human+myelin+oligodendrocyte+glycoproteins+induce+experimental+autoimmune+encephalomyelitis+by+different+mechanisms+in+C57BL/6+mice.">Rat and human myelin oligodendrocyte glycoproteins induce experimental autoimmune encephalomyelitis by different mechanisms in C57BL/6 mice.</a> J Immunol. 171 (1), 462-468 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning.">JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> J Vis Exp. , (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JoVE+Science+Education+Database.+Basic+Methods+in+Cellular+and+Molecular+Biology.+Restriction+Enzyme+Digests.">JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Restriction Enzyme Digests.</a> J Vis Exp. , (2016).</li><li>Hamada, H., Arakawa, T., Shiraki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+additives+on+protein+aggregation.">Effect of additives on protein aggregation.</a> Curr Pharm Biotechnol. 10 (4), 400-407 (2009).</li><li>Dang, A. K., Tesfagiorgis, Y., Jain, R. W., Craig, H. C., Kerfoot, S. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thioredoxin+gene+fusion+expression+system+that+circumvents+inclusion+body+formation+in+the+E.+coli+cytoplasm.">A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm.</a> Biotechnology (N Y). 11 (2), 187-193 (1993).</li><li>Raines, R. T., McCormick, M., Van Oosbree, T. R., Mierendorf, R. 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Y., 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=Separation+and+purification+of+Escherichia+coli-expressed+human+thymosin-alpha1+using+affinity+chromatography+and+high-performance+liquid+chromatography.">Separation and purification of Escherichia coli-expressed human thymosin-alpha1 using affinity chromatography and high-performance liquid chromatography.</a> Protein Expr Purif. 77 (2), 140-145 (2011).</li><li>Tegel, H., Ottosson, J., Hober, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+protein+production+levels+in+Escherichia+coli+with+a+strong+promoter.">Enhancing the protein production levels in Escherichia coli with a strong promoter.</a> FEBS J. 278 (5), 729-739 (2011).</li><li>Akirav, E. M., Bergman, C. M., Hill, M., Ruddle, N. 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The authors declare no competing financial interests.

Here, we have described a protocol for the production of MOGtag protein and how to generate pure MOG1-125 from the MOGtag protein. This protocol is based both on standard His-tag based protein purification methods, as well as a previously described protocol for the generation of an older MOG-based protein15. Although it is not described here, the primary usage of the MOGtag protein is to induce EAE through immunization with protein antigen. A protocol describing how ...

MS is a human disease characterized by chronic inflammation and neurodegeneration of the CNS which is thought to be driven by an autoimmune response directed towards myelin. The loss of myelin and axons over time result in the gradual decline of cognitive and motor function1. &#34;Experimental Autoimmune Encephalomyelitis&#34; is an umbrella term for animal models of autoimmune disease directed towards CNS myelin. Like human MS, EAE is typically characterized by immune cell infiltration of the CNS and, in some cases, demyelination2. However, the degree to which any given EAE model resembles human MS in part depends on the species or strain used and on the complexity of the underlying anti-myelin autoimmune response.
Anti-myelin autoimmunity can be experimentally induced in several ways, but the most common method used today is to immunize mice with a short peptide of amino acids mimicking the immunodominant CD4+ T cell epitope of a myelin protein. This represents the minimum requirement to induce a pathogenic response. Perhaps the most common of these is a 21 amino acid peptide derived from myelin oligodendrocyte glycoprotein (MOG35-55), which is used to induce EAE in C57Bl/6 mice3. However, for some experimental purposes it is desirable or even necessary to immunize with larger protein antigens and indeed there are several advantages to this over immunization with short peptide. First, because of MHC restriction, short peptides are usually only effective in a very limited range of strains, while larger protein antigens representing either the whole protein or a specific domain can be processed normally for presentation in multiple inbred mouse strains or even in different species4. Second, a larger protein antigen is capable of inducing a more complex immune response incorporating more types of lymphocytes in antigen recognition, rather than limiting antigen recognition to CD4+ T cells. For example, B cells via their B cell receptor (BCR) interact directly with whole rather than processed protein. We and others have shown that B cells activated by MOG35-55 immunization do not recognize MOG protein5. Since B cells were recently demonstrated to play a pathogenic role in human MS6, EAE models that incorporate B cells in autoimmune pathology are increasingly important.
Despite the advantages of using larger protein antigens to induce EAE, there remain few commercially available sources for such proteins. Indeed, while short peptides like MOG35-55 can be synthesized very quickly and at a relatively low cost, the commercial options for MOG protein are limited and cost substantially more to purchase. Nonetheless, there are several expression vectors available for research groups to generate MOG extracellular domain (MOG1-125) themselves. However, all of the expression systems that we have identified in the literature are based on older technologies that have since been replaced with more efficient expression systems7. Further, most are based on rat or human MOG8. For some investigations of autoimmunity in mice, an antigen based on the mouse MOG autoantigen is preferable. Finally, all MOG-based proteins that we have identified, either commercial or as expression vectors, are fusion proteins containing additional amino acids to the MOG1-125 base. These include a tag for purification and usually other sequences as well, many of which with a function we were unable to identify.
To address these limitations, we generated a novel fusion protein based on the mouse MOG extracellular domain fused to a tag containing thioredoxin to combat the known insolubility of MOG protein5. The tag sequence also contains a 6xHis sequence for purification and a TEV protease cleavage site that allows for the complete removal of all tag sequences, if desired. This is the only method that we are aware of to generate pure MOG1-125 protein. To facilitate production of large amounts of protein, the MOG1-125 sequence was codon-optimized for bacterial expression and the MOGtag fusion protein was inserted into the pET-32 expression system. Here, we describe in detail the protocol to produce and purify MOGtag protein, and pure MOG1-125, using non-specialized equipment available to most immunology laboratories.

<table border="0" cellpadding="0" cellspacing="0" width="1434">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">BL21 E.coli- pet32-MOGtag</td>
			<td style="width:139px;">Kerfoot lab</td>
			<td style="width:119px;"></td>
			<td style="width:803px;">These bacteria are required to make the MOGtag protein. Glycerol stocks of these bacteria are available upon request.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LB broth miller</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LBL407.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Ampicillin</td>
			<td style="width:139px;">bio basic</td>
			<td style="width:119px;">AB0028</td>
			<td>Reconsititute the powder into 50% ethanol/ 50% H2O at 100 mg/ml. Store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">IPTG</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IPT002.5</td>
			<td>Reconsititute the powder into H2O at 1M and store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Chicken-egg lysozyme</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LYS702.10</td>
			<td>Reconstitute in H2O at 50 mg/ml and store at -80 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Triton-X100</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">T-8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Phosphate buffered saline</td>
			<td style="width:139px;">life technologies</td>
			<td style="width:119px;">20012-027</td>
			<td>Commercial phosphate buffered saline is not required, any standard lab made phosphate buffered saline is sufficient.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium chloride</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SOD004.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-HCl</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS002.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Imidazole</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IMD508.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Guanidine-HCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">G3272</td>
			<td>The quality must be greater than 98% purity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">0.5M EDTA</td>
			<td style="width:139px;">bioshop</td>
			<td style="width:119px;">EDT111.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nickel (II) sulfate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">NIC700.500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">His bind resin</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">69670-3</td>
			<td>Store in 20% ethanol 80% H2O at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Anhydrous ethanol</td>
			<td style="width:139px;">Commercial Alcohols</td>
			<td style="width:119px;">P016EAAN</td>
			<td>Dilute with water as needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glacial acetic acid</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">ACE222.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium acetate trihydrate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SAA305.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bovine serum albumin standard</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Bio-rad protein assay dye reagent concentrate</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Ethylenediamine tetraacetic acid, disodium salt dihydrate</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-base</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">7000 MW Snakeskin dialysis tubing</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">68700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">2-mercaptoethanol</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">M3148-25ml</td>
			<td>This reagent should not be handled outside of a fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">AcTEV protease</td>
			<td style="width:139px;">lifetechnologies</td>
			<td style="width:119px;">12575-015</td>
			<td>Producing your own TEV protease can be accomplished using (https://www.addgene.org/8827/) and purified as in reference 17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Polyethyleneglycol 3350</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG335.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">polyethyleneglycol 8000</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG800.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nunc MaxiSorp flat-bottom 96 well plate</td>
			<td style="width:139px;">ebioscience</td>
			<td style="width:119px;">44-2404-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sonicator</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">FB-120-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Eon microplate spectrometer</td>
			<td style="width:139px;">Biotek</td>
			<td style="width:119px;">11-120-611</td>
			<td>This equipment uses the Gen5 data analysis software.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gen5 data analysis software</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">sodium dodecyl sulphate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SDS001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bromophenol blue</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">BRO777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycerol</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLY001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein desalting columns</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">89849</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycine</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLN001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">precast 12% polyacrylamide gel</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">456-1045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rapid stain reagent</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">553215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gel dock EZ imager</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">White Light Sample Tray</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708272&#160;</td>
			<td>Used along with gel dock EZ imager for coomassie blue stains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein ladder</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1610375</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Once the purification is complete, samples collected in steps 1.4, 2.1, 3.4, and the final product from step 6.4 should be run on a protein gel (Figure 3A). MOGtag should first appear as a 31.86 kDa band in the TO/N sample, but not T0, and should be the only band in the final pure product. To test whether the MOGtag protein has correctly folded, the MOGtag protein can be used to label MOG-protein specific B cells by F...

Watch this Scientific Journal Video about Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies at JoVE.co

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

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 ...

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Research

JoVE Journal

Immunology and Infection

10.9K Views.  Western University. 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.

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

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 ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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) ...

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 ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...