Questo lavoro è stato sostenuto da NIH concedere DK080351 al PC.

Protocollo sperimentale Il primo passo in questo protocollo sperimentale è quello di raccogliere un gran numero di topo ghiandola pituitaria. Il secondo passo è quello di omogeneizzare le ghiandole a preparare un estratto di proteine. Il terzo passo è quello di emulsionare queste proteine ​​con una miscela di idrocarburi. Fase 1: Raccolta e conservazione di mouse ghiandola pituitaria La ghiandola pituitaria del mouse si trova alla base del cranio in una depressione dello sfenoide detta sella turcica, circondato lateralmente dai nervi trigemino e anteriormente dal chiasma ottico. La ghiandola è composta da un lobo anteriore più ampia e più piccoli lobi posteriori e intermedi, e pesa in media 1,9 mg. Ghiandola pituitaria sono raccolti dai topi di ceppi misti, i sessi e di età, destinato ad essere l&#39;eutanasia dalla struttura di cura degli animali. Per isolare la ghiandola pituitaria, il mouse è eutanasia, la pelle sopra il cranio sezionato, e l&#39;osso del cranio taglio aperto con le forbici. Il cervello viene sollevato per esporre la ghiandola pituitaria, che viene poi isolato dalla dura mater circostante, scavato dello sfenoide e conservati in un tubo di plastica in ghiaccio secco. In generale usiamo 300 ghiandola pituitaria del mouse per una preparazione di proteine, un numero che può essere raccolta da una sola persona in 9 ore. Una volta che la collezione è completata, il tubo contenente la ghiandola pituitaria è conservato a -80 ° C fino al momento per il passo successivo. Fase 2. Estrazione di proteine ​​del mouse pituitaria <ol><li> Aggiungere la soluzione tampone di omogeneizzazione ad un tubo da 15 ml Falcon (catalogo n. 352059) conservati in un bagno di ghiaccio, con 250 microlitri di tampone per ogni 100 ghiandola pituitaria raccolti. Utilizzare un tampone di forza ionica simile a quello che è fisiologicamente presente all&#39;interno del citoplasma della cellula (0,2 M) e del pH fisiologico (7,4). Noi usiamo tampone fosfato (0,161 M, pH 7,4), integrato con un cocktail di inibitori delle proteasi (Sigma-Aldrich, catalogo n. P8340-1 ml). </li><li> Aggiungere la ghiandola pituitaria al buffer poco prima omogeneizzazione, quindi posizionare il generatore (5-mm di diametro) del omogeneizzatore Polytron sul fondo della provetta Falcon. Omogeneizzare a giri massimo per 30 secondi, spostare delicatamente il tubo Falcon su e giù mantenendolo nel bagno di ghiaccio. Poi, il resto l&#39;omogenato in ghiaccio per 1 min. Ripetere l&#39;omogeneizzazione e raffreddamento passi due volte. Questa omogeneizzazione dolce deve rompere le cellule e il rilascio delle proteine ​​citosoliche senza interrompere i nuclei. </li><li> Per rimuovere i nuclei, tessuto ininterrotto e materiali insolubili (come il tessuto connettivo e complessi di proteine ​​denaturate), centrifugare la omogenato a bassa velocità (1000 g) per 10 minuti a 4 ° C. </li><li> Trasferire il surnatante (ora si chiama post-nucleare supernatante, PNS) in una nuova provetta Falcon, avendo cura di lasciare indisturbato il pellet, e memorizzare il PNS sul ghiaccio. </li><li> Risospendere il pellet nel volume originale di buffer di omogeneizzazione (250 ml di buffer per ogni 100 ghiandola pituitaria raccolti), omogeneizzare come sopra, centrifuga come sopra, e poi unire il PNS secondo con il primo. </li><li> Aliquota i due campioni raccolti in provette da microcentrifuga PNS e conservare a -80 ° C fino al momento per il passo successivo. Questa preparazione di proteine ​​possono essere conservati a -80 ° C ma perde gradualmente la sua potenza, per cui si consiglia di utilizzarlo entro 6 mesi dalla preparazione. Utilizzare una piccola aliquota per determinare la resa di proteine ​​e la concentrazione dal saggio BCA (Pierce catalogo no. 23.225), e la qualità delle proteine ​​mediante elettroforesi su gel. In generale, si ottengono circa 60 mg di proteina da 300 pituitaries mouse in 1,5 mL di buffer di omogeneizzazione, ad una concentrazione di circa 40 mg / mL. </li></ol> Fase 3. Preparazione della emulsione per l&#39;immunizzazione <ol><li> Scongelare rapidamente le aliquote proteina congelato necessari per l&#39;esperimento e regolare la concentrazione di proteine ​​a 20 mg / mL. Questo estratto proteico sarà emulsionato 1:1 con adiuvante completo di Freund (CFA, Sigma F-5881) contenente 5 mg / mL di calore ucciso Mycobacterium tuberculosis (Becton Dickinson, 231.141). Ogni topo riceverà 1 mg di estratto pituitario in 100 ml di emulsione. </li><li> L&#39;esempio dato ecco per 10 immunizzando topi, dove ogni mouse saranno iniettati con 100 l di emulsione contenente 1 mg di proteine ​​pituitaria. Consentono a due topi in più per la perdita di materiale durante la preparazione, in modo da utilizzare al massimo 12 mg di proteine ​​pituitaria. Aspire 600 microlitri della mg / ml 20 estratto proteico pituitario descritto sopra utilizzando una siringa da 2,5 ml di Hamilton a tenuta di gas. Risospendere il CFA agitando, e aspira 600 ml di CFA in un&#39;altra siringa da 2,5 ml Hamilton. Attaccare e garantire un 22-gauge micro-emulsione ago alla siringa piena di CFA. Spingere lentamente lo stantuffo della siringa CFA in modo che l&#39;ago è pieno di CFA, quindi collegare e fissare l&#39;altra estremità dell&#39;ago della siringa riempita con l&#39;estratto di ipofisi. </li><li> Lentamente avanzare lo stantuffo della siringa CFA per spingere la componente del petrolio estratto acquoso nella pituitaria, in modo tale che solo una piccola quantità è misto. Poi spingere indietro lo stantuffo della siringa estratto pituitario nella siringa CFA. Ripetete questa azione aumentando gradualmente la quantità di materiale misto. Continuare fino a quando l&#39;intero contenuto delle due siringhe sono mescolati. Otterrete una soluzione biancastra e uniformemente distribuito che rappresenta l&#39;acqua in emulsione di olio. </li><li> Una volta che l&#39;emulsione è formata, ripetere la parte posteriore e indietro movimento almeno 500 volte a garantire l&#39;emulsione è accuratamente miscelati. Dopo il ciclo finale di miscelazione, il rapporto 1:1 acqua in emulsione olio è pronto all&#39;uso e contiene una concentrazione pituitaria proteina di 10 mg / ml. L&#39;emulsione verrà iniettato lo stesso giorno per via sottocutanea in topi anestetizzati SJL, come dettagliato nell&#39;articolo JOVE compagno. </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>

Nessun conflitto di interessi dichiarati.

Nonostante il fatto che il primo paziente con ipofisite autoimmune è stato segnalato nel 1962 4, l&#39;ipofisi autoantigeni patogeni devono essere ancora identificate 5. I modelli animali possono essere molto utili per identificare questi autoantigeni, favorendo la scoperta di biomarcatori che sono traducibili alla cura del paziente. Questo articolo ha dimostrato una procedura semplice per la preparazione di proteine ​​pituitaria in una forma che è sufficiente per indurre ipofisite autoimmuni...

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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>Complete Freund's Adjuvant (CFA)</td>
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<td>Mycobacterium tuberculosis</td>
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<td>Becton Dickinson</td>
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<td>2.5 mL Hamilton gastight syringe</td>
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preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis

Ipofisite autoimmune è una infiammazione cronica della ghiandola pituitaria causata o accompagnati da autoimmunità 1. E &#39;stata tradizionalmente considerata una malattia rara ma segnalazione è aumentato notevolmente negli ultimi anni. Ipofisite, infatti, non si sviluppa raramente come un &quot;effetto collaterale&quot; nei pazienti con tumore trattati con anticorpi che bloccano i recettori inibitori espressi sui linfociti T, come CTLA-4 2 e PD-1 recettori. Ipofisite autoimmune può essere indotta sperimentalmente iniettando nei topi con le proteine ​​pituitaria mescolato con un adiuvante 3. In questo articolo video che dimostrano come estrarre proteine ​​dal topo ghiandola pituitaria e su come prepararli in una forma adatta per indurre ipofisite autoimmune in topi 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

Ipofisite autoimmune può essere riprodotta nei topi iniettando un estratto di proteine ​​del mouse pituitaria.

Preparazione di Immunogeno mouse ipofisari per l&#39;induzione di ipofisite autoimmune sperimentale

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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Immunology and Infection

14.5K Views.  The Johns Hopkins University. Ipofisite autoimmune può essere riprodotta nei topi iniettando un estratto di proteine ​​del mouse pituitaria.

Video: 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.

Induzione di ipofisite autoimmune sperimentale nei topi 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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Immunologia e infezioni

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.

Superare blocco nel encefalomielite autoimmune sperimentale (EAE) ceppi di topi resistenti dal trasferimento adottivo e uno stimolo antigenico

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.

Mielina Oligodendrocyte glicoproteina (MOG<sub&gt; 35-55</sub&gt;) Indotta encefalomielite autoimmune sperimentale (EAE) in C57BL / 6 Mice

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.

Isolamento di leucociti infiltranti da mouse pelle utilizzando enzimatica Digest e Separazione 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.

Induzione di encefalomielite autoimmune sperimentale nei topi e valutazione della distribuzione delle malattie-dipendente delle cellule immunitarie in vari tessuti

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.

Produzione semplici ed efficaci e Purificazione di topo mielina Oligodendrocyte glicoproteina di encefalomielite autoimmune sperimentale Studi

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.

Induzione dell'epatite autoimmune indotta da farmaci in topi BALB / c per lo studio dei suoi meccanismi patogenetici

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;

Visualizzazione di danno delle barriere endoteliali e gliali dell'unità neurovascolare durante l'encefalomielite Autoimmune Sperimentale In Vivo

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.

Analisi citometrica del flusso dell'infiltrazione di linfociti nel sistema nervoso centrale durante l'encefalomielite autoimmune sperimentale

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.