Este trabalho foi financiado pelo NIH conceder DK080351 para PC, e por uma oferta generosa do Terry e família Kristine Gore.

Protocolo Experimental Uma vez que o imunógeno pituitária está preparada (ver artigo companheiro), é administrado por via subcutânea em camundongos SJL (The Jackson Laboratories, estoque 000.686). Uma segunda injeção idêntico é repetido sete dias depois. Ratos são então usados ​​para coletar in vivo e post-mortem resultados. Etapa 1. A injecção subcutânea de imunógeno em camundongos SJL pituitária Ratos são os primeiros anestesiados pela injeção de 0,5 ml de 20 mg / ml 2,2,2 tribromoetanol (Avertin, TCI América, T-1420) por via intraperitoneal. Camundongos são injetados por via subcutânea com 100 l da emulsão pituitária que, conforme indicado no companheiro JOVE 2181 artigo, contém 1 mg de extrato de pituitária e 0,25 mg de adjuvante de Freund completo. A emulsão é injetado na região dorsal posterior da perna esquerda ((50 mL) e na região inguinal direita (50 mL). A emulsão pituitária é injetado novamente no dia 7 para os sites oposto (50 ul na região dorsal perna direita traseiras e 50 ul na região inguinal esquerda). Camundongos são monitorados diariamente durante os primeiros 3 dias e depois semanalmente até o dia do sacrifício, que normalmente é 28 dias após a primeira imunização. Etapa 2. Coleção de resultados no vivo: ensaios de Sangue O tecido mais utilizado para avaliar os resultados experimentais, enquanto os ratos ainda estão vivos é sangue. O sangue é rotineiramente elaborado 14 dias após a primeira imunização para medir os níveis séricos de anticorpos pituitária ou outros marcadores como as citocinas e quimiocinas. O sangue é coletado de ratos vivem como se segue. Ratos são realizadas de forma segura pela nuca do pescoço e parte de trás do maxilar está localizado. Uma vez que a parte de trás do maxilar está localizado, 4 mm são lancetas usadas para perfurar o feixe vascular submandibular. Após coletar o sangue, a pressão sobre o lugar sobre a área usando um algodão embebido em álcool para evitar um hematoma. Sangue coletado é centrifugado a 2.000 g por 20 minutos, e armazenadas a -80 ° C até sua utilização. Etapa 3. Coleção de post-mortem resultados: Pituitária histopatologia Ratos são tipicamente sacrificado 28 dias após a primeira imunização. A característica fundamental necessário para estabelecer o diagnóstico de HAE é a infiltração da glândula pituitária por hematopoiéticas células mononucleares. Os ratos são sacrificados, colocando-os em uma câmara de plástico selado que é perfundido com CO 2. Camundongos são mantidos na câmara para um total de 8 minutos. Após a eutanásia, a glândula pituitária é coletado como descrito no artigo do companheiro. A glândula pituitária doente aparece inchada e mais firmemente aderente à dura-máter ao redor. Pinças afiadas são usadas para soltar as meninges ao redor da hipófise. Uma vez que o move livremente pituitária no osso esfenóide, uma extremidade da glândula pituitária é apreendido com uma pinça e da glândula pituitária é cuidadosamente levantada. A glândula é então colocada em um tubo de microcentrífuga contendo 5 fixador de Beckstead. A glândula pituitária é fixo durante a noite, embrulhado em papel de lente, e colocado em uma fita cassete. As glândulas são então processadas utilizando o protocolo a seguir: <table border="1"><tbody><tr><td> Número da Estação </td><td> Reagente </td><td> Tempo em minutos </td><td> Temperatura (Celsius) </td></tr><tr><td> 1 </td><td> Álcool a 70% </td><td> 5 </td><td> 25 </td></tr><tr><td> 2 </td><td> Álcool a 70% </td><td> 15 </td><td> 25 </td></tr><tr><td> 3 </td><td> Álcool a 95% </td><td> 15 </td><td> 25 </td></tr><tr><td> 4 </td><td> Álcool a 95% </td><td> 15 </td><td> 25 </td></tr><tr><td> 5 </td><td> Álcool 100% </td><td> 15 </td><td> 25 </td></tr><tr><td> 6 </td><td> Álcool 100% </td><td> 7,5 </td><td> 25 </td></tr><tr><td> 7 </td><td> Álcool 100% </td><td> 7,5 </td><td> 25 </td></tr><tr><td> 8 </td><td> xileno </td><td> 20 </td><td> 25 </td></tr><tr><td> 9 </td><td> xileno </td><td> 20 </td><td> 25 </td></tr><tr><td> 10 </td><td> xileno </td><td&gt; 20 </td><td> 25 </td></tr><tr><td> 11 </td><td> parafina </td><td> 30 </td><td> 58 </td></tr><tr><td> 12 </td><td> parafina </td><td> 5 </td><td> 58 </td></tr><tr><td> 13 </td><td> parafina </td><td> 5 </td><td> 58 </td></tr></tbody></table> Após o processamento, as glândulas pituitárias são embebidos em parafina, e pelo menos cinco seções não consecutivas (5 m de espessura) são cortadas a partir de cada glândula. Após a secção, as glândulas são corados com hematoxilina e eosina, e analisadas para quantificar a infiltração mononuclear. A seção com a infiltração mais grave é selecionado para marcar. A gravidade é marcado por base uma estimativa subjetiva do grau de substituição hipófise anterior ou destruição por células do sistema imunológico: grau 0, nenhuma doença; grau 1, 2% -20% envolvimento; grau 2, 20% -30%; grau 3, 30% -50%; grau 4, 50% -90%; grau 5, mais de 90%. Todas as seções são marcados por dois indivíduos cegos para a fonte das seções.

<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>

Não há conflitos de interesse declarados.

Quatro técnicas têm sido relatadas para induzir hipofisite em animais experimentais. A técnica descrita neste artigo, que é a injeção de proteínas pituitária misturado com adjuvante Freund s, é o mais antigo, que remonta a 1967 6, e foi melhorado mais recentemente 1. Outras técnicas são a injeção de glicoproteínas do vírus da rubéola solúveis em 1992 7, a injeção estereotáxica de um adenovírus diretamente na glândula pituitária, em 2002 8, eo transplante...

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<td>SJL mice (The Jackson Laboratories)</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

Hipofisite auto-imune pode ser reproduzida experimentalmente pela injeção de proteínas pituitária misturado com adjuvante em camundongos suscetíveis 1. Modelos de ratos nos permitem estudar a forma como se desenrolam as doenças, muitas vezes fornecendo uma boa réplica dos mesmos processos que ocorrem nos seres humanos. Para algumas doenças auto-imunes, como diabetes tipo 1A, existem modelos (o mouse NOD) que, espontaneamente, desenvolver uma doença semelhante à contraparte humana. Para muitas outras doenças auto-imunes, no entanto, o modelo precisa ser induzida experimentalmente. Uma abordagem comum a este respeito é injetar o mouse com um antígeno dominante derivado do órgão em estudo. Por exemplo, os investigadores interessados ​​em tireoidite auto-imune injetar camundongos com tireoglobulina 2, e os interessados ​​em miastenia gravis injetá-las com o receptor de acetilcolina 3. Se o autoantígeno para uma determinada doença auto-imune não é conhecido, os investigadores injetar um extrato de proteína bruta do órgão alvo da reação auto-imune. Para hipofisite auto-imunes, a auto-antígeno patogênicos (s) continuam a ser identificados 4, e, assim, uma preparação de proteína bruta pituitária é usado. Neste artigo de vídeo demonstramos como induzir hipofisite auto-imune experimental em camundongos 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

Este vídeo mostra como induzir hipofisite auto-imunes em camundongos SJL e como avaliar sua gravidade pela histopatologia.

Indução de hipofisite Auto-Imune Experimental em Ratos 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 visualizações.  The Johns Hopkins University. Este vídeo mostra como induzir hipofisite auto-imunes em camundongos SJL e como avaliar sua gravidade pela histopatologia.

Vídeo: 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.

Preparação de rato Imunogénio Pituitária para a indução de hipofisite Auto-Imune Experimental

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

Imunologia e Infecção

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 glicoproteína (MOG<sub&gt; 35-55</sub&gt;) Induzida Encefalomielite Auto-imune Experimental (EAE) em murganhos C57BL / 6

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.

Indução de encefalomielite autoimune experimental em camundongos e avaliação da distribuição da doença dependente de células imunes em vários tecidos

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.

Um método não-invasivo e tecnicamente não-intensivo para indução e fenotipagem of Experimental pneumonia bacteriana em ratos

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.

Indução de Hepatite Autoimune Induzida por Drogas em CAMUNDONGOS BALB/c para o Estudo de Seus Mecanismos Patogênicos

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;

Visualizando o comprometimento das barreiras endoteliais e Glial da unidade Neurovascular durante encefalomielite auto-imune Experimental 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.

Imunoterapia adotiva de células iNKT em Isomerase Isomerase Induzida de Glicose-6-Fosfato (G6PI)-Induzido RaMice

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.

Análise Citométrica de Fluxo de Infiltração linfócito no Sistema Nervoso Central durante Encefalomielite Autoimune Experimental

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.

Uveíte Autoimune Experimental: Um Modelo de Rato Inflamatório Intraocular

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

Aumento da imunogenicidade por Mycobacterium paratuberculosis na encefalomielite autoimune

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.

Autor em destaque: Atividade adjuvante de Mycobacterium paratuberculosis no aumento da imunogenicidade de autoantígenos durante a encefalomielite autoimune experimental  

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.

Atividade Adjuvante de Mycobacterium paratuberculosis no Aumento da Imunogenicidade de Autoantígenos Durante Encefalomielite Autoimune Experimental

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