Diese Arbeit wurde vom NIH DK080351 zu PC unterstützt.

Experimentelles Protokoll Der erste Schritt in diese experimentelle Protokoll ist eine große Anzahl von Maus-Hypophyse zu sammeln. Der zweite Schritt ist, um die Drüsen zu homogenisieren, um ein Protein Extrakt herzustellen. Der dritte Schritt besteht darin, diese Proteine ​​mit einer öligen Mischung zu emulgieren. Schritt 1: Sammlung und Lagerung von Maus Hypophyse Die Maus Hypophyse sitzt an der Basis des Schädels in einer Vertiefung des Keilbeins genannt der Sella turcica, seitlich durch den Trigeminus-Nerven und nach vorn durch das Chiasma umgeben. Die Drüse ist von einer größeren Vorderlappen und kleineren hinteren und mittleren Lappen zusammengesetzt, und wiegt im Durchschnitt 1,9 mg. Hypophyse sind von Mäusen mit gemischten Stämme, Geschlechter und Altersgruppen, dazu bestimmt, durch die Tierstation eingeschläfert werden gesammelt. Zur Isolierung der Hypophyse wird die Maus getötet, die Haut über den Schädel seziert, und die Schädelknochen aufgeschnitten mit einer Schere. Das Gehirn wird angehoben, um die Hypophyse, die dann von den umgebenden Dura mater ist isoliert aussetzen, schöpfte aus dem Keilbein und in einem Plastikröhrchen auf Trockeneis. In der Regel verwenden wir 300 Maus Hypophyse für ein Protein Vorbereitung, eine Zahl, die von einer Person in 9 Stunden gesammelt werden können. Sobald die Sammlung abgeschlossen ist, wird das Rohr mit der Hypophyse bei -80 ° C bis bereit für den nächsten Schritt gespeichert. Schritt 2. Extraktion von Maus Hypophyse Proteine <ol><li> Fügen Sie die Homogenisierungspuffer um einen 15-ml-Falcon-Röhrchen (Katalog Nr. 352059) gehalten in einem Eisbad mit 250 ul Puffer für alle 100 Hypophyse gesammelt. Verwenden Sie einen Puffer der Ionenstärke, ähnlich dem, das physiologisch im Inneren des Zytoplasma der Zelle (0,2 M) und des physiologischen pH-Wert (7,4) ist. Wir verwenden Phosphat-gepufferter Kochsalzlösung (0,161 M, pH 7,4), ergänzt mit einem Protease-Inhibitor-Cocktail (Sigma-Aldrich, Katalog Nr. P8340-1 ml). </li><li> Fügen Sie die Hypophyse auf den Puffer kurz vor der Homogenisierung und legen Sie dann den Generator (5-mm Durchmesser) der Polytron-Homogenisator an der Unterseite des Falcon-Röhrchen. Homogenisieren bei maximaler Drehzahl für 30 Sekunden, sanft bewegten die Falcon-Röhrchen auf und ab, während man selbst in das Eisbad. Dann ruhen die Homogenat auf Eis für 1 min. Wiederholen Sie die Homogenisierung und Kühlung Schritte zwei weitere Male. Diese sanfte Homogenisierung brechen sollte der Zellen und Freisetzung cytosolische Proteine, ohne dass der Kerne. </li><li> Zum Entfernen Kerne, ungebrochen Gewebe und unlösliche Stoffe (z. B. Bindegewebe und denaturiertes Protein-Komplexe), Zentrifuge das Homogenat bei niedriger Geschwindigkeit (1000g) für 10 min bei 4 ° C. </li><li> Übertragen Sie den Überstand (jetzt Post-Nuclear Überstand, PNS), um eine neue Falcon-Röhrchen, wobei darauf zu verlassen, das Pellet ungestört, und speichern Sie die PNS auf Eis. </li><li> Das Pellet in das ursprüngliche Volumen des Homogenisierungspuffer (250 ul Puffer für jeweils 100 Hypophyse gesammelt), wie oben zu homogenisieren, zentrifugieren wie oben, und kombinieren Sie die zweite PNS mit dem ersten. </li><li> Aliquot der beiden gepoolten PNS Proben in Reaktionsgefäße und bei -80 ° C bis bereit für den nächsten Schritt. Dieses Protein Präparat kann bei -80 ° C gelagert werden, sondern allmählich verliert seine Wirksamkeit, so empfehlen wir es innerhalb von 6 Monaten der Vorbereitung nutzen. Verwenden Sie eine kleine Teilmenge der Protein-Ausbeute und Konzentration von der BCA-Assay (Pierce Katalog-Nr. 23225), und die Qualität der Proteine ​​mittels Gelelektrophorese zu bestimmen. In der Regel erhalten wir etwa 60 mg Protein von 300 Maus Hypophysen in 1,5 ml Homogenisierungspuffer, bei einer Konzentration um 40 mg / mL. </li></ol> Schritt 3. Herstellung der Emulsion für die Immunisierung <ol><li> Tauwetter schnell die gefrorenen Protein Aliquots für das Experiment benötigt und stellen Sie die Proteinkonzentration auf 20 mg / mL. Dieses Protein-Extrakt wird 1:1 mit komplettem Freund-Adjuvans (CFA, Sigma F-5881) mit 5 mg / ml Hitze abgetötet Mycobacterium tuberculosis (Becton Dickinson, 231141) emulgiert werden. Jede Maus wird 1 mg Hypophysenextrakt in 100 ul der Emulsion zu erhalten. </li><li> Das hier angegebene Beispiel ist für die Immunisierung von 10 Mäusen, in denen jeder Maus mit 100 ul der Emulsion mit 1 mg Hypophyse Proteine ​​injiziert werden. Lassen Sie zwei extra Mäuse für materielle Verluste während der Vorbereitung, so mit 12 mg des Hypophysen-Proteine. Aspire 600 ul der 20 mg / mL Hypophyse Proteinextrakt beschreiben oben mit einem 2,5 mL Hamilton gasdichte Spritze. Resuspendieren CFA durch Schütteln, und streben 600 ul des CFA in ein anderes 2,5 ml Hamilton-Spritze. Befestigen und sichern eine 22-Gauge-Mikro-Emulgieren Nadel auf die Spritze mit CFA gefüllt. Langsam schieben Sie den Kolben der Spritze CFA, so dass die Nadel mit CFA gefüllt ist, dann befestigen und sichern Sie das andere Endedie Nadel auf die Spritze mit Hypophysenextrakt gefüllt. </li><li> Drehen Sie dazu den Kolben der Spritze CFA für die Öl-Komponente in die wässrige Hypophysenextrakt, so dass nur eine kleine Menge gemischt wird, voranzutreiben. Dann drücken Sie den Kolben der Hypophysenextrakt Spritze in den CFA Spritze. Wiederholen Sie diesen Vorgang schrittweise Erhöhung der Menge an Material gemischt. Weiter, bis der gesamte Inhalt der beiden Spritzen gemischt werden. Sie erhalten eine weißliche und gleichmäßig verteilte Lösung, dass das Wasser-in-Öl-Emulsion darstellt. </li><li> Nachdem die Emulsion entsteht, wiederholen Sie die Hin-und Rückwärtsbewegung mindestens 500 Mal, damit die Emulsion gründlich gemischt. Nach dem letzten Mischzyklus, ist die 1:1-Wasser-in-Öl-Emulsion gebrauchsfertig und enthält eine Hypophysen-Protein-Konzentration von 10 mg / mL. Die Emulsion wird injiziert selben Tag subkutan in narkotisierten Mäusen SJL werden, wie in der Begleiter JoVE Artikel beschrieben. </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>

Trotz der Tatsache, dass der erste Patient mit Autoimmun-Hypophysitis in 1962 4 wurde berichtet, bleiben die pathogenen Hypophyse Autoantigene identifiziert 5 sein. Tiermodelle können sehr nützlich sein, um diese Autoantigene zu identifizieren, Beihilfe zur Entdeckung von Biomarkern, die übersetzbar Patientenversorgung leisten. Dieser Artikel hat eine einfache Verfahrensweise zur Herstellung von Hypophysen-Proteine ​​in einer Form, die geeignet für Induktion autoimmune Hypophysitis bei Vers...

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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>Complete Freund's Adjuvant (CFA)</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

Autoimmune Hypophysitis ist eine chronische Entzündung der Hypophyse verursacht oder begleitet von Autoimmunität 1. Es ist traditionell eine seltene Erkrankung, aber die Berichterstattung hat sich in den letzten Jahren zugenommen. Hypophysitis in der Tat entwickelt sich nicht selten als &quot;Nebenwirkung&quot; bei Krebspatienten mit Antikörpern, die hemmenden Rezeptoren auf T-Lymphozyten, wie CTLA-4 2 und PD-1-Rezeptoren exprimiert Block behandelt. Autoimmune Hypophysitis kann experimentell durch die Injektion von Mäusen mit Hypophysen-Proteine ​​mit einem Adjuvans 3 gemischte induziert werden. In diesem Video-Artikel werden wir zeigen, wie Proteine ​​aus Maus Hypophyse und wie man sie in eine geeignete Form für die Induktion autoimmune Hypophysitis in SJL Mäusen vorbereiten zu extrahieren.

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

Autoimmune Hypophysitis kann in Mäusen durch Injektion ein Auszug aus Maus Hypophyse Proteine ​​reproduziert werden.

Vorbereitung der Maus Pituitary Immunogen für die Induktion der experimentellen autoimmunen Hypophysitis

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

preparation-mouse-pituitary-immunogen-for-induction-experimental

Research

JoVE Journal

Immunology and Infection

14.5K Aufrufe.  The Johns Hopkins University. Autoimmune Hypophysitis kann in Mäusen durch Injektion ein Auszug aus Maus Hypophyse Proteine ​​reproduziert werden.

Serie: 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.

Induktion der experimentellen autoimmunen Hypophysitis in SJL Mäuse

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

Forschung

Immunologie und Infektion

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.

Die Überwindung der nicht mehr reagiert, in der experimentellen autoimmunen Enzephalomyelitis (EAE) Beständig Mausstämmen durch adoptiven Transfer-und Antigen-Challenge

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.

Myelin Oligodendrozyten Glykoprotein (MOG<sub&gt; 35-55</sub&gt;) Induzierten experimentellen autoimmunen Enzephalomyelitis (EAE) in C57BL / 6 Mäuse

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.

Isolation der infiltrierenden Leukozyten aus Mäusehaut unter Verwendung enzymatischer Digest und Gradiententrennung

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.

Die Induktion der experimentellen autoimmunen Enzephalomyelitis bei Mäusen und die Auswertung des Krankheitsabhängige Verteilung von Immunzellen in verschiedenen Geweben

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.

Einfache und effiziente Herstellung und Reinigung von Maus-Myelin-Oligodendrozyten-Glykoprotein für experimentellen autoimmunen Enzephalomyelitis Studies

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.

Induktion der medikamenteninduzierten Autoimmunhepatitis bei BALB/c-Mäusen zur Untersuchung ihrer pathogenen Mechanismen

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;

Beeinträchtigung der Endothelzellen und glialen Barrieren des Referats neurovaskuläre während experimentelle Autoimmune Enzephalomyelitis In Vivo Visualisierung

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.

Flow Cytometric Analyse der Lymphozyteninfiltration im Zentralnervensystem während der experimentellen Autoimmunenzephalomyelitis

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.

Vorbereitung der Maus Pituitary Immunogen für die Induktion der experimentellen autoimmunen Hypophysitis

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