Este trabalho foi financiado pelo NIH conceder DK080351 para PC.

Protocolo Experimental O primeiro passo neste protocolo experimental é coletar um grande número de glândulas pituitária de rato. O segundo passo é homogeneizar as glândulas para preparar um extrato de proteína. A terceira etapa é para emulsionar estas proteínas com uma mistura de hidrocarbonetos. Passo 1: Recolha e armazenamento das glândulas pituitária de rato A glândula pituitária do mouse fica na base do crânio em uma depressão do osso esfenóide chamada sela túrcica, cercado lateralmente pelos nervos trigêmeo e anteriormente pelo quiasma óptico. A glândula é composta de um grande lobo anterior e menor lóbulos posterior e intermédio, e pesa em média 1,9 mg. Glândula pituitária são coletados de camundongos de linhagens mistas, sexos e idades, destinado a ser sacrificados pela facilidade de cuidado animal. Para isolar a glândula pituitária, o mouse é eutanásia, a pele acima do crânio dissecado, eo osso do crânio cortado com uma tesoura. O cérebro é levantada para expor a glândula pituitária, que é então isolado da dura-máter ao redor, escavou para fora do osso esfenóide e armazenadas em um tubo de plástico em gelo seco. Em geral, usamos 300 glândulas pituitária do mouse para uma preparação de proteínas, um número que pode ser recolhida por uma pessoa em nove horas. Depois que a coleção estiver completa, o tubo que contém as glândulas pituitária é armazenada a -80 ° C até que esteja pronto para a próxima etapa. Etapa 2. Extração de proteínas do mouse pituitária <ol><li> Adicione o tampão de homogeneização para um tubo Falcon de 15 ml (catálogo não 352.059) mantidos em um banho de gelo, com 250 mL de tampão para cada 100 glândulas pituitárias coletados. Use um buffer de força iônica semelhante à que é fisiologicamente presente no interior do citoplasma da célula (0,2 M) e de pH fisiológico (7,4). Nós usamos tampão fosfato (0,161 M, pH 7,4), complementado com um coquetel inibidor de protease (Sigma-Aldrich, o catálogo não-P8340 1 ml). </li><li> Adicione a glândula pituitária para o buffer antes da homogeneização, e em seguida, coloque o gerador (5 mm de diâmetro) do homogeneizador Polytron no fundo do tubo Falcon. Homogeneizar em rpm máxima por 30 segundos, movendo-se delicadamente o tubo Falcon cima e para baixo, mantendo-o em banho de gelo. Então, o resto homogeneizado em gelo por 1 min. Repita a homogeneização e resfriamento passos mais duas vezes. Esta homogeneização suave deve quebrar as células e liberação de proteínas citosólicas sem interromper os núcleos. </li><li> Para remover o tecido núcleos, ininterrupta e materiais insolúveis (como tecido conjuntivo e complexos de proteína desnaturada), centrifugar o homogeneizado a baixa velocidade (1000g) por 10 min a 4 ° C. </li><li> Transferir o sobrenadante (agora chamado de Post-Nuclear sobrenadante, PNS) para um tubo Falcon nova, tendo o cuidado de deixar o pellet intacta, e armazenar o PNS no gelo. </li><li> Ressuspender o sedimento em volume original de tampão de homogeneização (250 mL de tampão para cada 100 glândulas pituitárias coletados), homogeneizar como acima, centrífuga como acima, e depois combinar o PNS segundo com o primeiro. </li><li> Alíquota as duas amostras agrupadas PNS em tubos de microcentrífuga e armazenar a -80 ° C até que esteja pronto para a próxima etapa. Esta preparação proteína podem ser armazenadas a -80 ° C, mas aos poucos perde sua potência, por isso recomendamos a usá-lo dentro de 6 meses a partir da preparação. Use uma pequena alíquota para determinar a produção de proteína e concentração pelo ensaio BCA (Pierce catálogo no. 23.225), ea qualidade da proteína por electroforese em gel. Em geral, obtém-se cerca de 60 mg de proteína de 300 hipófises de rato em 1,5 mL de tampão de homogeneização, a uma concentração em torno de 40 mg / mL. </li></ol> Etapa 3. Preparação da emulsão para a imunização <ol><li> Descongele rapidamente as alíquotas de proteína congelados necessário para a experiência e ajustar a concentração de proteína de 20 mg / mL. Este extrato de proteína será emulsificada com adjuvante Freund 01:01 completo (CFA, Sigma F-5881), contendo 5 mg / mL de calor matou Mycobacterium tuberculosis (Becton Dickinson, 231.141). Cada rato vai receber 1 mg de extrato de hipófise em 100 mL de emulsão. </li><li> O exemplo dado aqui é para imunizar 10 ratos, onde cada rato serão injetados com 100 mL da emulsão contendo 1 mg de proteínas pituitária. Permitir que dois ratos extra para perda de material durante a preparação, então use 12 mg de proteínas pituitária. Aspire mL 600 dos 20 extrato de proteína mg / mL pituitária descrever acima usando uma seringa de 2,5 ml Hamilton herméticos. Ressuspender o CFA por agitação, e aspiramos 600 mL de CFA em mais 2,5 mL seringa Hamilton. Anexar e garantir uma agulha de calibre 22 micro-emulsionantes para a seringa cheia com CFA. Empurre lentamente o êmbolo da seringa CFA de tal forma que a agulha é preenchido com CFA, em seguida, anexar e segura a outra extremidade doa agulha para a seringa cheia com extrato de pituitária. </li><li> Lentamente avançar o êmbolo da seringa CFA para empurrar o componente óleo no extrato de pituitária aquosa, de modo que apenas uma pequena quantidade é misto. Em seguida, empurre o êmbolo da seringa extrato de hipófise na seringa CFA. Repita esta acção gradualmente aumentando a quantidade de material misturado. Continue até que todo o conteúdo das duas seringas são misturados. Você vai obter uma solução esbranquiçadas e distribuídas uniformemente que representa a emulsão de água em óleo. </li><li> Uma vez que a emulsão é formada, repita o movimento de vai-e-vem, pelo menos, 500 vezes para garantir a emulsão é bem misturado. Após o ciclo final de mixagem, de 1:1 de água em óleo, emulsão está pronto para usar e contém uma concentração de proteína pituitária de 10 mg / mL. A emulsão será injetado no mesmo dia por via subcutânea em ratos anestesiados SJL, conforme detalhado no artigo JOVE companheiro. </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>

Não há conflitos de interesse declarados.

Apesar do fato de que o primeiro paciente com hipofisite auto-imune foi relatada em 1962 4, a auto-antígenos patogênicos pituitária continuam a ser identificados 5. Modelos animais podem ser muito úteis para identificar esses auto-antígenos, auxiliando na descoberta de biomarcadores que são traduzíveis para o atendimento ao paciente. Este artigo tem demonstrado um procedimento simples para preparação de proteínas pituitária de uma forma que é adequada para induzir hipofisite auto-imunes...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>15-ml Falcon tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>homogenization buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>phosphate buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Polytron homogenizer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BCA assay</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>23225</td>
<td>&nbsp;</td>
<tr>
<td>Complete Freund's Adjuvant (CFA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F-5881</td>
<td>&nbsp;</td>
<tr>
<td>Mycobacterium tuberculosis</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>231141</td>
<td>&nbsp;</td>
<tr>
<td>2.5 mL Hamilton gastight syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>22-gauge micro-emulsifying needle</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis

Hipofisite auto-imune é uma inflamação crônica da glândula pituitária causada ou acompanhada de auto-imunidade 1. Ela tem sido tradicionalmente considerada uma doença rara, mas relatórios tem aumentado significativamente nos últimos anos. Hipofisite, de fato, não raro se desenvolve como um &quot;efeito colateral&quot; em pacientes com câncer tratados com anticorpos que bloqueiam os receptores inibitórios expressa nos linfócitos T, como CTLA-4 2 e PD-1 receptores. Hipofisite auto-imune pode ser induzida experimentalmente pela injeção de ratos com proteínas pituitária misturado com um 3 adjuvante. Neste artigo de vídeo demonstramos como extrair proteínas de rato pituitária glândulas e como prepará-los de forma adequada para induzir hipofisite auto-imunes em camundongos 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

Hipofisite auto-imune pode ser reproduzido em ratos através da injeção de um extrato de proteínas do mouse pituitária.

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 ...

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

Research

JoVE Journal

Immunology and Infection

14.4K visualizações.  The Johns Hopkins University. Hipofisite auto-imune pode ser reproduzido em ratos através da injeção de um extrato de proteínas do mouse pituitária.

Vídeo: 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.

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 ...

Imunologia e Infecção

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 de leucócitos de infiltração de rato pele utilizando digestão enzimática e separação por gradiente

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.

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) ...

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.

Produção simples e eficiente e Purificação de rato Mielina Oligodendrocyte glicoproteína de encefalomielite autoimune experimental Estudos

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.

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 ...

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 ...

A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

Um método de homogeneização rápido, simples e padronizado para preparar emulsões de antígenos/adjuvantes para induzir encefalomielite autoimune experimental

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

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

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

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

Geração de um modelo de tireoidite autoimune espontânea em camundongos

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

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 ...