Ce travail a été soutenu par NIH DK080351 au PC.

Protocole expérimental La première étape de ce protocole expérimental est de collecter un grand nombre de glandes pituitaires de souris. La deuxième étape consiste à homogénéiser les glandes de préparer un extrait de protéine. La troisième étape consiste à émulsifier ces protéines avec un mélange d&#39;hydrocarbures. Étape 1: Collecte et stockage des glandes pituitaires de souris La glande pituitaire de souris se trouve à la base du crâne dans une dépression de l&#39;os sphénoïde appelée la selle turcique, entouré latéralement par les nerfs trijumeau et en avant par le chiasma optique. La glande est composée d&#39;un grand lobe antérieur et les petits lobes postérieurs et intermédiaires, et pèse en moyenne 1,9 mg. Glandes hypophysaires sont collectées à partir de souches de souris mixtes, les sexes et les âges, destinée à être euthanasié par l&#39;établissement de soins pour animaux. Pour isoler la glande pituitaire, la souris est euthanasié, la peau au-dessus du crâne disséqué, et l&#39;os du crâne à ciel ouvert avec des ciseaux. Le cerveau est levé pour exposer la glande pituitaire, qui est alors isolé de la dure entourant mère, évidé de l&#39;os sphénoïde et stocké dans un tube en plastique sur la glace sèche. En général, nous utilisons 300 glandes pituitaires de souris pour une préparation de protéines, un nombre qui peut être recueilli par une personne en 9 heures. Une fois la collecte terminée, le tube contenant l&#39;hypophyse est stocké à -80 ° C jusqu&#39;au moment de l&#39;étape suivante. Etape 2. Extraction de la souris protéines pituitaires <ol><li> Ajouter le tampon d&#39;homogénéisation d&#39;un tube de 15 ml Falcon (no 352059) conservés dans un bain de glace, en utilisant 250 uL de tampon pour chaque 100 hypophyses collectées. Utilisez un tampon de force ionique semblable à celui qui est physiologiquement présents à l&#39;intérieur du cytoplasme de la cellule (0,2 M) et du pH physiologique (7,4). Nous utilisons tampon phosphate salin (0,161 M, pH 7,4), complété par un cocktail inhibiteur de protéase (Sigma-Aldrich, no de catalogue P8340-1ml). </li><li> Ajouter les glandes pituitaires pour le tampon juste avant l&#39;homogénéisation, puis placer le générateur (5 mm de diamètre) de l&#39;homogénéisateur Polytron au fond du tube Falcon. Homogénéiser au régime maximum pendant 30 secondes, doucement déplacer le tube Falcon de haut en bas tout en le gardant dans le bain de glace. Ensuite, repos de l&#39;homogénat sur la glace pendant 1 min. Répétez l&#39;homogénéisation et le refroidissement étapes deux fois plus. Cette homogénéisation douce devrait casser les cellules et la libération des protéines cytosoliques, sans perturber les noyaux. </li><li> Pour enlever les noyaux, les tissus et les matières insolubles ininterrompue (comme le tissu conjonctif et des complexes protéine dénaturée), Centrifuger l&#39;homogénat à basse vitesse (1000g) pendant 10 min à 4 ° C. </li><li> Transférer le surnageant (maintenant appelée post-nucléaire surnageant, SNP) dans un nouveau tube Falcon, en prenant soin de laisser intact le culot, et de stocker les SNP sur la glace. </li><li> Reprendre le culot dans le premier volume de tampon d&#39;homogénéisation (250 uL de tampon pour chaque 100 hypophyses collectées), homogénéiser comme ci-dessus, centrifuger comme ci-dessus, puis combiner les SNP second avec le premier. </li><li> Aliquoter les deux échantillons groupés de SNP dans des tubes à centrifuger et conserver à -80 ° C jusqu&#39;à ce que prêt pour l&#39;étape suivante. Cette préparation de protéines peuvent être conservés à -80 ° C mais perd progressivement de sa puissance, nous vous recommandons de l&#39;utiliser dans les 6 mois à partir de la préparation. Utilisez une petite aliquote de déterminer le rendement en protéines et la concentration par le dosage BCA (Pierce catalogue non. 23225), et la qualité des protéines par électrophorèse sur gel. En général, on obtient environ 60 mg de protéine de 300 hypophyses de souris dans 1,5 ml de tampon d&#39;homogénéisation, à une concentration d&#39;environ 40 mg / mL. </li></ol> Étape 3. Préparation de l&#39;émulsion pour la vaccination <ol><li> Dégel rapidement les protéines aliquotes congelés nécessaire pour l&#39;expérience et d&#39;ajuster la concentration en protéines à 20 mg / mL. Cet extrait protéique sera émulsionnée 01h01 avec l&#39;adjuvant complet de Freund (CFA, Sigma F-5881) contenant 5 mg / ml de chaleur a tué Mycobacterium tuberculosis (Becton Dickinson, 231141). Chaque souris reçoit 1 mg d&#39;extrait pituitaire dans 100 ul d&#39;émulsion. </li><li> L&#39;exemple donné ici est pour immuniser des souris 10, où chaque souris seront injectés avec 100 pi de l&#39;émulsion contenant 1 mg de protéines pituitaire. Autoriser deux souris supplémentaire pour pertes matérielles lors de la préparation, donc à utiliser 12 mg de protéines pituitaire. Aspire 600 uL des 20 mg / ml d&#39;extrait de protéines pituitaires décrit ci-dessus en utilisant un 2,5 mL Hamilton étanche seringue. Reprendre le CFA en secouant, et aspirer 600 pi de CFA dans une autre seringue de 2,5 mL de Hamilton. Attacher et sécuriser un calibre 22 micro-émulsification aiguille à la seringue remplie de CFA. Poussez lentement le piston de la seringue CFA tels que l&#39;aiguille est rempli avec le CFA, puis attacher et sécuriser l&#39;autre extrémité dul&#39;aiguille à la seringue remplie avec de l&#39;extrait pituitaire. </li><li> Lentement avancer le piston de la seringue CFA pour pousser la composante de l&#39;huile dans l&#39;extrait aqueuse hypophyse, de telle sorte que seule une petite quantité est mélangée. Puis repousser le piston de la seringue extrait pituitaire dans la seringue CFA. Répétez cette action en augmentant progressivement la quantité de matériaux mixtes. Continuez jusqu&#39;à ce que tout le contenu des deux seringues sont mélangés. Vous obtiendrez une solution blanchâtre et uniformément répartie, qui représente l&#39;émulsion eau-dans-huile. </li><li> Une fois l&#39;émulsion est formée, répétez le mouvement de va-et-vient au moins 500 fois pour assurer l&#39;émulsion est bien mélangé. Après le dernier cycle de mélange, l&#39;eau-01h01 dans-huile est prête à utiliser et contient une concentration en protéine hypophysaire de 10 mg / mL. L&#39;émulsion sera injecté le même jour sous-cutanée dans des souris SJL anesthésiés, comme détaillé dans l&#39;article JoVE compagnon. </li></ol>

<ol>
	<li>Caturegli, P., Newschaffer, C., Olivi, A., Pomper, M. G., Burger, P. C., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Autoimmune Hypophysitis Endocr Rev. 26, 599-614 (2005).</li><li>Blansfield, J. A., Beck, K. E., Tran, K., Yang, J. C., Hughes, M. S., Kammula, U. S., Royal, R. E., Topalian, S. L., Haworth, L. R., Levy, C., Rosenberg, S. A., Sherry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T-lymphocyte-associated+antigen-4+blockage+can+induce+autoimmune+hypophysitis+in+patients+with+metastatic+melanoma+and+renal+cancer.">Cytotoxic T-lymphocyte-associated antigen-4 blockage can induce autoimmune hypophysitis in patients with metastatic melanoma and renal cancer.</a> J Immunother. 28, 593-598 (2005).</li><li>Tzou, S. C., Lupi, I., Landek, M., Gutenberg, A., Tzou, Y. M., Kimura, H., Pinna, G., Rose, N. R., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+Hypophysitis+of+SJL+mice:+Clinical+Insights+from+a+New+Animal+Model.">Autoimmune Hypophysitis of SJL mice: Clinical Insights from a New Animal Model.</a> Endocrinology. 149, 3461-3469 (2008).</li><li>Goudie, R. B., Pinkerton, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+hypophysitis+and+Hashimoto's+disease+in+a+woman.">Anterior hypophysitis and Hashimoto's disease in a woman.</a> J Pathol Bacteriol. 83, 584-585 (1962).</li><li>Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+hypophysitis:+an+underestimated+disease+in+search+of+its+autoantigen(s).">Autoimmune hypophysitis: an underestimated disease in search of its autoantigen(s).</a> J Clin Endocrinol Metab. 92, 2038-2040 (2007).</li></ol>

Malgré le fait que le premier patient avec hypophysite auto-immune a été signalé en 1962 4, les auto-antigènes pathogènes hypophysaires restent à identifier 5. Les modèles animaux peut être très utile d&#39;identifier ces auto-antigènes, facilitant la découverte de biomarqueurs qui sont traduisibles les soins aux patients. Cet article a démontré une procédure simple pour la préparation de protéines pituitaire dans une forme qui est suffisante pour induire hypophysite auto-immunes c...

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<td>15-ml Falcon tubes</td>
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<td>homogenization buffer</td>
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<td>phosphate buffered saline</td>
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<td>protease inhibitor cocktail</td>
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<td>Polytron homogenizer</td>
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<td>BCA assay</td>
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<td>23225</td>
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<td>22-gauge micro-emulsifying needle</td>
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preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis

Hypophysite auto-immune est une inflammation chronique de la glande pituitaire provoqué ou accompagné d&#39;une auto-immunité. Il a traditionnellement été considérée comme une maladie rare, mais des rapports a nettement augmenté ces dernières années. Hypophysite, en fait, il n&#39;est pas rare que se développe un «effet secondaire» chez les patients cancéreux traités avec des anticorps qui bloquent les récepteurs inhibiteurs exprimés sur les lymphocytes T, tels que CTLA-4 2 et PD-1 récepteurs. Hypophysite auto-immunes peuvent être induite expérimentalement en injectant des souris avec des protéines pituitaires mélangé avec un adjuvant 3. Dans cet article, nous montrons comment la vidéo pour en extraire les protéines de souris glandes pituitaire et comment les préparer sous une forme appropriée pour induire hypophysite auto-immunes chez la souris SJL.

Watch this Scientific Journal Video about Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis at JoVE.com

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

Hypophysite auto-immunes peuvent être reproduits chez la souris par injection d&#39;un extrait de protéines de l&#39;hypophyse de souris.

Préparation de la souris Immunogène hypophysaires pour l&#39;induction de hypophysite auto-immune expérimentale

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

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JoVE Journal

Immunology and Infection

14.5K vues.  The Johns Hopkins University. Hypophysite auto-immunes peuvent être reproduits chez la souris par injection d'un extrait de protéines de l'hypophyse de souris.

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

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.

L&#39;induction de hypophysite auto-immune expérimentale chez la souris SJL

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

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Immunologie et infection

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.

Isolement de leucocytes infiltrant de la peau de souris Utilisation de digestion enzymatique et séparation par gradient

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.

Induction of Experimental encéphalomyélite allergique chez la souris et l&#39;évaluation de la distribution des maladies dépendant des cellules immunitaires dans divers tissus

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.

Production et purification de souris myéline oligodendrocyte glycoprotéine simple et efficace pour auto-immune expérimentale encéphalomyélite études

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

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Induction d’une hépatite auto-immune induite par des médicaments chez des souris BALB/c pour l’étude de ses mécanismes pathogènes

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

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;

Visualiser l’altération des barrières endothéliales et gliales de l’unité neurovasculaire au cours de l’encéphalomyélite allergique expérimentale In Vivo

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

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.

Uvéite auto-immune expérimentale : un modèle murin inflammatoire intraoculaire

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

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.

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.

Procédé d’homogénéisation rapide, simple et normalisé pour préparer des émulsions antigènes/adjuvants pour induire une encéphalomyélite auto-immune expérimentale

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

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

Génération d’un modèle de thyroïdite auto-immune spontanée chez la souris

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

Amélioration de l’immunogénicité par Mycobacterium paratuberculosis dans l’encéphalomyélite auto-immune

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.

Pleins feux sur l’auteur : Activité adjuvante de Mycobacterium paratuberculosis dans l’amélioration de l’immunogénicité des auto-antigènes au cours de l’encéphalomyélite auto-immune expérimentale  

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.

Activité adjuvante de Mycobacterium paratuberculosis dans l’amélioration de l’immunogénicité des autoantigènes au cours de l’encéphalomyélite auto-immune expérimentale

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