この作業は、PCへ、そしてテリーとクリスティンゴアの家族の寛大な贈り物がNIHの助成金DK080351によってサポートされていました。

実験的なプロトコル下垂体免疫原が（関連記事参照）準備されると、それはSJLマウス（ジャクソンラボラトリーズ、株式000686）に皮下注射される。第二同一の注入は、7日後に繰り返されます。マウスは、 生体と検死結果に収集するために使用されています。 ステップ1。 SJLマウスに下垂体の免疫原を皮下注射マウスは、20 mg / mlの2,2,2 - トリブロムエタノールを0.5ml（AVERTIN、TCIアメリカ、T - 1420）を腹腔内注入による最初の麻酔です。マウスは、コンパニオンJoveの2181の記事で示したように、下垂体エマルジョン100μlを皮下注射する、下垂体抽出物と完全フロイントアジュバントの0.25ミリグラム、1ミリグラムが含まれています。エマルジョンは、左背側後肢領域（（50μL）と右鼠径部（50μL）に注入されます。下垂体エマルジョンは反対側の部位（50μlの右背側後肢の地域の中に7日目に再び注入されており、左鼠径部に50μlの）マウスは最初の3日間、毎日モニターし、初回免疫後に一般的に28日間である犠牲、の日まで、毎週されています。 ステップ2。 in vivoでの結果でのコレクション：血アッセイマウスはまだ生きている間、最も一般的に実験結果を評価するために使用される組織は、血液です。血液は、日常的に14日間下垂体抗体やサイトカインやケモカインなどの他のマーカーの血清レベルを測定するために初回免疫後に描画されます。 血液は、次のように生きたマウスから収集されます。マウスは首の首筋によって安全に保管されており、顎骨のバックが位置しています。顎骨の後部が特定されると、4mmのランセットは、ピアスに顎下維管束を使用している。血した後、血腫を避けるために、アルコール綿を使用して領域上での場所の圧力を集めている。使用されるまで、採取した血液は20分、2,000 gで遠心分離し、-80℃で保存されます。 ステップ3。事後の成果の収集：下垂体組織病理学マウスは、通常、28日初回免疫後に屠殺されています。 EAHの診断を確立するために必要な枢機卿の機能は、造血単核細胞による下垂体の浸潤である。 マウスをCO 2で潅流されているプラスチック封じチャンバ内に配置することにより安楽死されています。マウスは8分の合計のためのチャンバー内に保持されます。安楽死の後、下垂体は、関連記事で説明されているように収集されます。病気の下垂体は腫れと、よりしっかりと周囲の硬膜に付着されます。シャープ鉗子は、下垂体の周囲の髄膜を緩めるために使用されます。一度ちょう形骨上に自由に下垂体の動き、下垂体の一端を鉗子で把持され、下垂体は慎重に持ち上げられる。腺は、ベックステッドの固定液5を含むマイクロ遠心チューブに配置されます。下垂体は、一晩固定レンズの紙に包まれ、そしてカセットに配置されます。腺は、次のプロトコルを使用して処理されます。 <table border="1"><tbody><tr><td>ステーション番号</td><td>試薬</td><td>分単位の時間</td><td>温度（摂氏） </td></tr><tr><td> 1 </td><td> 70％アルコール</td><td> 5 </td><td> 25 </td></tr><tr><td> 2 </td><td> 70％アルコール</td><td> 15 </td><td> 25 </td></tr><tr><td> 3 </td><td> 95％アルコール</td><td> 15 </td><td> 25 </td></tr><tr><td> 4 </td><td> 95％アルコール</td><td> 15 </td><td> 25 </td></tr><tr><td> 5 </td><td> 100％アルコール</td><td> 15 </td><td> 25 </td></tr><tr><td> 6 </td><td> 100％アルコール</td><td> 7.5 </td><td> 25 </td></tr><tr><td> 7 </td><td> 100％アルコール</td><td> 7.5 </td><td> 25 </td></tr><tr><td> 8 </td><td>キシレン</td><td> 20 </td><td> 25 </td></tr><tr><td> 9 </td><td>キシレン</td><td> 20 </td><td> 25 </td></tr><tr><td> 10 </td><td>キシレン</td><tD&gt; 20 </td><td> 25 </td></tr><tr><td> 11 </td><td>パラフィン</td><td> 30 </td><td> 58 </td></tr><tr><td> 12 </td><td>パラフィン</td><td> 5 </td><td> 58 </td></tr><tr><td> 13 </td><td>パラフィン</td><td> 5 </td><td> 58 </td></tr></tbody></table>処理後は、下垂体腺はパラフィンに埋め込まれているし、少なくとも5連続していないセクションは、（5 -μm厚）各腺からカットされます。切削後は、腺を、ヘマトキシリン​​およびエオシンで染色し、単核細胞浸潤を定量化するために分析されています。最も深刻な浸潤を伴うセクションがスコアリングのために選択されています。重大度は、免疫細胞による下垂体前葉の交換や破壊の程度の主観的な見積もりに基づいて採点さ：グレード0、無疾患、グレード1、2％-20％の関与、グレード2、20％-30％、グレード3、 30％〜50％、グレード4の、50％-90％、5年生、90％以上。すべてのセクションは、セクションのソースを知らされていない二人の個人によって採点されます。

<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>

利害の衝突は宣言されません。

4つの技法は、動物実験で下垂体炎を誘導することが報告されている。フロイントアジュバントと混合して下垂体蛋白質の注入であるこの記事で説明する手法は、1967年6にさかのぼる最古の一つである、そして最近では1に改善されました。他の技術は、1992年7の水溶性風疹ウイルスの糖タンパク質の注入、2002 8で下垂体に直接アデノウイルスの定位の注入、および2010年9別のラット系統の腎被膜未満のラット系統から下垂体腺の移植です。 。フロイントアジュバントアプローチの主な利点は、それが密接に人間相手に似た病気を誘発において、低侵襲、より一貫性と、上記のすべて、より正確であるということです。このビデオの記事で説明されているテクニックは、この魅惑的な病気の我々の理解を向上させるために研究者によって使用できる下垂体炎の再現可能なモデルを生み出し、。

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<td>SJL mice (The Jackson Laboratories)</td>
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induction of experimental autoimmune hypophysitis in sjl mice

自己免疫性下垂体炎は、感受性のマウス1内にアジュバントに混合した下垂体蛋白質の注入によって実験的に再現することができます。マウスモデルは、しばしば人間に発生する同じプロセスの正常な複製を提供し、私たちは病気の展開方法を学ぶことができます。一部の自己免疫疾患の場合は、タイプ1Aの糖尿病のように、自発的に人間のものと同様の疾患を発症モデル（NODマウス）があります。他の多くの自己免疫疾患の場合は、しかし、モデルは実験的に誘導する必要があります。この点で一般的なアプローチが検討されて臓器由来する支配的な抗原をマウスに注入することです。例えば、自己免疫性甲状腺炎に興味のある研究者は、サイログロブリン2でマウスを注入し、重症筋無力症に興味のある人は、アセチルコリン受容体3とそれらを注入する。特定の自己免疫疾患の自己抗原が知られていない場合は、捜査官は、自己免疫反応の標的臓器から粗タンパク質抽出液を注入する。自己免疫性下垂体炎の場合は、病原性の自己抗原（s）は4をまだ明らかにされないままであるため、原油下垂体タンパク質調製物が使用されます。このビデオの記事では、SJLマウスにおける実験的自己免疫性下垂体炎を誘導する方法を示します。

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

このビデオでは、SJLマウスとどのように病理組織学的にその重症度を評価するために自己免​​疫性下垂体炎を誘導する方法を示しています。

SJLマウスにおける実験的自己免疫性下垂体炎の誘導

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.4K ビュー.  The Johns Hopkins University. このビデオでは、SJLマウスとどのように病理組織学的にその重症度を評価するために自己免​​疫性下垂体炎を誘導する方法を示しています。

ビデオ: 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.

実験的自己免疫性下垂体炎の誘導のためのマウス下垂体免疫原の調製

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

免疫・感染学

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.

ミエリンオリゴデンドロサイト糖タンパク質（MOG<sub&gt; 35〜55</sub&gt;）C57BL / 6マウスにおける実験的自己免疫性脳脊髄炎（EAE）誘発

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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.

マウスおよび種々の組織における免疫細胞の疾患に依存する分布の評価における実験的自己免疫性脳脊髄炎の誘導

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.

マウスにおける実験的細菌性肺炎の誘導と表現型のための非侵襲的および技術的に非集中的な方法

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

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

マウスからの初代肝細胞培養における炎症反応の誘導

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

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.

BALB/cマウスにおける薬物誘発性自己免疫性肝炎の誘導による発症機序の研究

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;

生体内で実験的自己免疫性脳脊髄炎時単位の内皮細胞およびグリアの障壁の障害の可視化

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.

グルコース-6-リン酸イソメラーゼ(G6PI)誘発RAマウスにおけるiNKT細胞の導入免疫療法

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.

実験的自己免疫性脳脊髄炎における中枢神経系におけるリンパ球浸潤のフロー細胞量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...