Questo lavoro è stato sostenuto da NIH concedere DK080351 a PC, e da una generosa donazione della famiglia Terry e Kristine Gore.

Protocollo sperimentale Una volta che il immunogeno pituitaria è pronto (vedi articolo correlato), è iniettata per via sottocutanea in topi SJL (I Laboratori Jackson, fotografia di 000.686). Una seconda iniezione identico si ripete 7 giorni dopo. I topi sono poi utilizzati per raccogliere in vivo e post mortem risultati. Fase 1. L&#39;iniezione sottocutanea di immunogeno pituitaria in topi SJL I topi sono prima anestetizzati iniettando 0,5 ml di 20 mg / ml di 2,2,2-Tribromoethanol (Avertin, TCI America, T-1420) per via intraperitoneale. I topi sono poi iniettata per via sottocutanea con 100 microlitri della emulsione pituitaria che, come indicato nella compagno JOVE articolo 2181, contiene 1 mg di estratto di ipofisi e 0,25 mg di adiuvante completo di Freund. L&#39;emulsione viene iniettato nella regione dorsale sinistra zampa posteriore ((50 mL) e la regione inguinale destra (50 mL). L&#39;emulsione pituitaria viene iniettato di nuovo il giorno 7 nei siti opposto (50 ml nella regione dorsale destra zampa posteriore e 50 microlitri nella regione inguinale sinistra). topi sono monitorati giornalmente per i primi 3 giorni e poi ogni settimana fino al giorno del sacrificio, che in genere 28 giorni dopo la prima immunizzazione. Fase 2. Raccolta dei risultati in vivo: test del sangue Il tessuto più comunemente usato per valutare i risultati sperimentali, mentre i topi sono ancora in vita è il sangue. Il sangue è di routine redatto 14 giorni dopo la prima immunizzazione per misurare i livelli sierici di anticorpi anti-ipofisi o altri indicatori come citochine e chemochine. Il sangue viene raccolto da topi vivi come segue. I topi sono tenute saldamente per la collottola del collo e la parte posteriore del mascellare si trova. Una volta che la parte posteriore della mandibola si trova, 4 lancette mm utilizzato per forare il fascio vascolare sottomandibolare. Dopo il sangue viene raccolto, la pressione sul posto sulla zona con un batuffolo di cotone imbevuto di alcol per evitare un ematoma. Sangue raccolto viene centrifugato a 2.000 g per 20 minuti, e conservati a -80 ° C fino al momento. Fase 3. Raccolta di post-mortem risultati: ipofisari istopatologia I topi sono generalmente sacrificati 28 giorni dopo la prima immunizzazione. La funzione cardinale necessarie per stabilire la diagnosi di EAH è l&#39;infiltrazione della ghiandola pituitaria da ematopoietiche cellule mononucleate. I topi sono eutanasia mettendoli in una camera di plastica sigillata che viene perfuso con CO 2. I topi sono tenuti in camera per un totale di 8 minuti. Dopo l&#39;eutanasia, la ghiandola pituitaria è raccolto come descritto nell&#39;articolo compagno. Una ghiandola pituitaria malato appare gonfio e più saldamente aderente al mater circostante dura. Pinze taglienti sono utilizzati per allentare le meningi che circondano la ghiandola pituitaria. Una volta che l&#39;ipofisi si muove liberamente sul sfenoide, un capo della ghiandola pituitaria è afferrato con una pinza e la ghiandola pituitaria è alzato attentamente. La ghiandola viene poi posto in una provetta contenente 5 fissativo Beckstead è. La ghiandola pituitaria è fisso durante la notte, avvolto in carta per lenti, e posto in una cassetta. Le ghiandole vengono poi elaborati utilizzando il seguente protocollo: <table border="1"><tbody><tr><td> Numero stazione </td><td> Reagente </td><td> Tempo in minuti </td><td> Temperatura (Celsius) </td></tr><tr><td> 1 </td><td> 70% di alcol </td><td> 5 </td><td> 25 </td></tr><tr><td> 2 </td><td> 70% di alcol </td><td> 15 </td><td> 25 </td></tr><tr><td> 3 </td><td> 95% di alcol </td><td> 15 </td><td> 25 </td></tr><tr><td> 4 </td><td> 95% di alcol </td><td> 15 </td><td> 25 </td></tr><tr><td> 5 </td><td> 100% di alcol </td><td> 15 </td><td> 25 </td></tr><tr><td> 6 </td><td> 100% di alcol </td><td> 7,5 </td><td> 25 </td></tr><tr><td> 7 </td><td> 100% di alcol </td><td> 7,5 </td><td> 25 </td></tr><tr><td> 8 </td><td> xilene </td><td> 20 </td><td> 25 </td></tr><tr><td> 9 </td><td> xilene </td><td> 20 </td><td> 25 </td></tr><tr><td> 10 </td><td> xilene </td><td&gt; 20 </td><td> 25 </td></tr><tr><td> 11 </td><td> paraffina </td><td> 30 </td><td> 58 </td></tr><tr><td> 12 </td><td> paraffina </td><td> 5 </td><td> 58 </td></tr><tr><td> 13 </td><td> paraffina </td><td> 5 </td><td> 58 </td></tr></tbody></table> Dopo l&#39;elaborazione, ipofisi sono inclusi in paraffina e di almeno 5 sezioni non consecutivi (5-micron di spessore) sono tagliati da ogni ghiandola. Dopo sezionamento, le ghiandole sono colorate con ematossilina ed eosina, e analizzati per quantificare l&#39;infiltrazione mononucleare. La sezione con l&#39;infiltrazione più grave è stato selezionato per il punteggio. La gravità è segnato sulla base di una valutazione soggettiva del grado di sostituzione anteriore dell&#39;ipofisi o distruzione da parte delle cellule immunitarie: grado 0, nessuna malattia, di grado 1, 2% -20% coinvolgimento, grado 2, 20% -30%, grado 3, 30% -50%, di grado 4, il 50% -90%; grado 5, superiore al 90%. Tutte le sezioni sono segnati da due individui accecati alla fonte delle sezioni.

<ol>
	<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. , (2008).</li><li>Twarog, F. J., 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=The+production+of+thyroid+autoantibodies+in+mice.">The production of thyroid autoantibodies in mice.</a> J Immunol. 101, 242-2450 (1968).</li><li>Patrick, J., Lindstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+response+to+acetylcholine+receptor.">Autoimmune response to acetylcholine receptor.</a> Science. 180, 871-872 (1973).</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><li>Beckstead, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+preservation+of+fixation-sensitive+antigens+in+paraffin-embedded+tissues:+addendum.">A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues: addendum.</a> J Histochem Cytochem. 43, 345-345 (1995).</li><li>Levine, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergic+adenophypophysitis:+new+experimental+disease+of+the+pituitary+gland.">Allergic adenophypophysitis: new experimental disease of the pituitary gland.</a> Science. 158, 1190-1191 (1967).</li><li>Yoon, J. W., Choi, D. S., Liang, H. C., Baek, H. S., Ko, I. Y., Jun, H. S., Gillam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+an+organ-specific+autoimmune+disease,+lymphocytic+hypophysitis,+in+hamsters+by+recombinant+rubella+virus+glycoprotein+and+prevention+of+disease+by+neonatal+thymectomy.">Induction of an organ-specific autoimmune disease, lymphocytic hypophysitis, in hamsters by recombinant rubella virus glycoprotein and prevention of disease by neonatal thymectomy.</a> Journal of Virology. 66, 1210-1214 (1992).</li><li>Davis, J. R., McMahon, R. F., Lowenstein, P. R., Castro, M. G., Lincoln, G. A., McNeilly, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+in+the+ovine+pituitary+gland+is+associated+with+hypophysitis.">Adenovirus-mediated gene transfer in the ovine pituitary gland is associated with hypophysitis.</a> J Endocrinol. 173, 265-271 (2002).</li><li>Rotondo, F., Quintanar-Stephano, A., Asa, S. L., Lombardero, M., Berczi, I., Scheithauer, B. W., Horvath, E., Kovacs, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenohypophysitis+in+rat+pituitary+allografts.">Adenohypophysitis in rat pituitary allografts.</a> Int J Exp Pathol. , (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

Quattro tecniche sono stati segnalati per indurre ipofisite negli animali da esperimento. La tecnica descritta in questo articolo, che è l&#39;iniezione di proteine ​​pituitaria miscelati con adiuvante di Freund s, è il più antico, risalente al 1967 6, ed è stata migliorata più di recente 1. Altre tecniche sono l&#39;iniezione di glicoproteine ​​solubili virus della rosolia nel 1992 7, l&#39;iniezione stereotassica di un adenovirus direttamente nella ghiandola pituitaria nel...

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<td>SJL mice (The Jackson Laboratories)</td>
<td>&nbsp;</td>
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<td>stock 000686</td>
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<td>2,2,2-Tribromoethanol (Avertin)</td>
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<td>TCI America</td>
<td>T-1420</td>
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<td>Beckstead's fixative</td>
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induction of experimental autoimmune hypophysitis in sjl mice

Ipofisite autoimmune può essere riprodotto sperimentalmente mediante l&#39;iniezione di proteine ​​pituitaria mescolato con un adiuvante in topi suscettibili 1. Modelli murini ci permettono di studiare come le malattie si svolgono, spesso fornendo una buona replica degli stessi processi che si verificano negli esseri umani. Per alcune malattie autoimmuni, come il diabete di tipo 1A, ci sono modelli (il topo NOD) che sviluppano spontaneamente una malattia simile alla controparte umana. Per molte altre malattie autoimmuni, invece, il modello deve essere indotta sperimentalmente. Un approccio comune su questo punto è quello di iniettare il mouse con un antigene dominante deriva dal organo oggetto di studio. Per esempio, i ricercatori interessati alla tiroidite autoimmune iniettare topi con tireoglobulina 2, e coloro che sono interessati miastenia gravis loro iniettare con il recettore dell&#39;acetilcolina 3. Se l&#39;autoantigene per una particolare malattia autoimmune non è noto, gli investigatori iniettare un estratto grezzo di proteine ​​dal organo bersaglio della reazione autoimmune. Per ipofisite autoimmune, l&#39;autoantigene patogeni (s) devono ancora essere identificati 4, e quindi una preparazione in proteine ​​gregge pituitaria è utilizzato. In questo articolo video che dimostrano come indurre ipofisite autoimmune sperimentale nei topi SJL.

Watch this Scientific Journal Video about Induction of Experimental Autoimmune Hypophysitis in SJL Mice at JoVE.com

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Questo video mostra come per indurre ipofisite autoimmune in topi SJL e come valutare la sua gravità per via istopatologica.

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 ...

induction-of-experimental-autoimmune-hypophysitis-in-sjl-mice

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Immunology and Infection

11.4K Views.  The Johns Hopkins University. Questo video mostra come per indurre ipofisite autoimmune in topi SJL e come valutare la sua gravità per via istopatologica.

Video: Induction of Experimental Autoimmune Hypophysitis in SJL Mice

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

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

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

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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Immunologia e infezioni

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 ...

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) ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Un metodo non invasivo e tecnicamente non intensiva per l&#39;induzione e Fenotipizzazione Sperimentale polmonite batterica nei topi

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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 ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Immunoterapia adottiva delle cellule iNKT in isomerasi indotta da glucosio (G6PI)

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

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.

Uveite autoimmune sperimentale: un modello murino infiammatorio intraoculare

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

Potenziamento dell'immunogenicità da parte di Mycobacterium paratuberculosis nell'encefalomielite autoimmune

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.

Autore in primo piano: Attività adiuvante del Mycobacterium paratuberculosis nel migliorare l'immunogenicità degli autoantigeni durante l'encefalomielite autoimmune sperimentale  

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.

Attività adiuvante del Mycobacterium paratuberculosis nel migliorare l'immunogenicità degli autoantigeni durante l'encefalomielite autoimmune sperimentale

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