This work was supported by a grant from the Multiple Sclerosis Society of Canada. RWJ is the recipient of the Waugh Family MS Society of Canada Doctoral Studentship Award.

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The authors declare no competing financial interests.

ここでは、MOG タグタンパク質および方法MOG タグタンパク質から純粋MOG 1-125を生成するための製造のためのプロトコルを記載しています。このプロトコルは、両方の標準的なHisタグベースのタンパク質精製方法、ならびに古いMOGベースのタンパク質15の世代のために以前に記載されたプロトコルに基づいています。それはここで説明されていないが、MOG タ?...

MSは、ミエリンに向けられた自己免疫応答によって駆動されると考えられているCNSの慢性炎症および神経変性によって特徴付けられるヒト疾患です。時間の経過とともにミエリンおよび軸索の損失は、認知および運動機能1の緩やかな衰退につながります。 「実験的自己免疫性脳脊髄炎」は、CNSミエリンに向け自己免疫疾患の動物モデルのための包括的な用語です。ヒトMSと同様に、EAEは、典型的には、CNSの免疫細胞の浸潤を特徴とし、場合によっては、脱髄2れます。しかし、任意のEAEモデルは、部分的に人間のMSに似ている度合いは、使用される種または株に、基礎となる抗ミエリン自己免疫応答の複雑さによって異なります。 抗ミエリン自己免疫は実験的に、いくつかの方法で誘導することができるが、今日使用される最も一般的な方法は、免疫優性CD4を模倣するアミノ酸の短いペプチドでマウスを免疫化することです<sミエリンタンパク質の&gt; + T細胞エピトープアップ。これは、病原性応答を誘導するための最小要件を示しています。おそらく、これらの中で最も一般的には、C57BL / 6マウス3にEAEを誘導するために使用されるミエリンオリゴデンドロサイト糖タンパク質（MOG 35-55）に由来する21アミノ酸ペプチドです。しかし、いくつかの実験目的のためには、より大きなタンパク質抗原で免疫化することが望ましい、あるいは必要であり、実際にこれにはいくつかの利点が短いペプチドを用いた免疫化の上にあります。タンパク質全体または特定のドメインのいずれかを表す、より大きなタンパク質抗原は、異なる種であっても、複数の近交系マウスでのプレゼンテーションのために正常に処理されるか、またはすることができながら、まず、MHC制限のため、短いペプチドは、通常の株の非常に限られた範囲でのみ有効です4。第二に、より大きなタンパク質抗原は、抗原認識にリンパ球の多くの種類を組み込んだより複雑な免疫応答を誘導するのではなくlimitinすることが可能ですCD4 + T細胞へのG抗原認識。例えば、それらのB細胞受容体（BCR）を介したB細胞は、全体ではなく、処理タンパク質と直接相互作用します。我々と他の人は、MOG 35-55免疫によって活性化されたB細胞は、MOGタンパク質5を認識しないことが示されました。 B細胞は、最近、ヒトMS 6に病原性の役割を果たすことが実証されたので、自己免疫病理にB細胞を組み込むEAEモデルは、ますます重要です。 EAEを誘導するために、より大きなタンパク質抗原を使用することの利点にもかかわらず、そのようなタンパク質のいくつかの市販の供給源が残ります。 MOG 35-55のような短いペプチドは、非常に迅速かつ比較的安価に合成することができるが、実際に、MOGタンパク質を商業オプション購入に限定されず、コスト実質的です。それにもかかわらず、MOGの細胞外ドメイン（MOG 1-125に ）自分自身を生成するために、研究グループのために利用可能ないくつかの発現ベクターがあります。 HoweveR、我々は文献で同定した発現系の全てを、以降、より効率的な発現系7で置換されている古い技術に基づいています。また、大部分はラットまたはヒトMOG 8に基づいています。マウスにおける自己免疫のいくつかの研究では、マウスMOG自己抗原に基づいて抗原が好ましいです。最後に、コマーシャルまたは発現ベクターのいずれかと、我々が同定したすべてのMOG系タンパク質は、MOG 1-125ベースに追加のアミノ酸を含む融合タンパク質です。これらは、同様に私たちは識別できなかった機能を備えているの多くを精製するためのタグと通常は他の配列が含まれます。 これらの制限に対処するために、我々は、MOGタンパク質5の既知の不溶性に対処するためにチオレドキシンを含むタグに融合したマウスMOGの細胞外ドメインに基づく新規な融合タンパク質を生成しました。タグ配列はまた、精製およびTEVプロテアーゼCLE用の6xHis配列を含みます必要に応じて、すべてのタグ配列を完全に除去することが可能avageサイト。これは、我々が純粋なMOG 1-125蛋白質を生成するの知っている唯一の方法です。タンパク質の大量生産を容易にするために、MOG 1-125配列を細菌発現のためにコドン最適化し、MOG タグ融合タンパク質は、PET-32発現系に挿入しました。ここでは、最も免疫学研究所で使用可能な非専門的な装置を使用して、詳細にMOG タグタンパク質を生成し精製するプロトコル、及び純粋MOG 1-125を記述する。

<table><tbody><tr><td>BL21 &lt;em&gt;E. coli&lt;/em&gt;- pet32-MOG&lt;sub&gt;tag&lt;/sub&gt;</td><td>Kerfoot lab</td><td></td><td>These bacteria are required to make the MOG&lt;sub&gt;tag&lt;/sub&gt; protein. Glycerol stocks of these bacteria are available upon request.</td></tr><tr><td>LB broth miller</td><td>Bioshop</td><td>LBL407.1</td><td></td></tr><tr><td>Ampicillin</td><td>bio basic</td><td>AB0028</td><td>Reconsititute the powder into 50% ethanol/ 50% H&lt;sub&gt;2&lt;/sub&gt;O at 100 mg/ml. Store at -20 &amp;deg;C.</td></tr><tr><td>IPTG</td><td>Bioshop</td><td>IPT002.5</td><td>Reconsititute the powder into H&lt;sub&gt;2&lt;/sub&gt;O at 1M and store at -20 &amp;deg;C.</td></tr><tr><td>Chicken-egg lysozyme</td><td>Bioshop</td><td>LYS702.10</td><td>Reconstitute in H&lt;sub&gt;2&lt;/sub&gt;O at 50 mg/ml and store at -80 &amp;deg;C.</td></tr><tr><td>Triton-X100</td><td>Sigma</td><td>T-8532</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>life technologies</td><td>20012-027</td><td>Commercial phosphate buffered saline is not required, any standard lab made phosphate buffered saline is sufficient.</td></tr><tr><td>Sodium chloride</td><td>Bioshop</td><td>SOD004.1</td><td></td></tr><tr><td>Tris-HCl</td><td>Bioshop</td><td>TRS002.1</td><td></td></tr><tr><td>Imidazole</td><td>Bioshop</td><td>IMD508.100</td><td></td></tr><tr><td>Guanidine-HCl</td><td>Sigma</td><td>G3272</td><td>The quality must be greater than 98% purity.</td></tr><tr><td>0.5 M EDTA</td><td>bioshop</td><td>EDT111.500</td><td></td></tr><tr><td>Nickel (II) sulfate</td><td>Bioshop</td><td>NIC700.500</td><td></td></tr><tr><td>His bind resin</td><td>EMD Millipore</td><td>69670-3</td><td>Store in 20% ethanol 80% H&lt;sub&gt;2&lt;/sub&gt;O at 4 &amp;deg;C</td></tr><tr><td>Anhydrous ethanol</td><td>Commercial Alcohols</td><td>P016EAAN</td><td>Dilute with water as needed.</td></tr><tr><td>Glacial acetic acid</td><td>Bioshop</td><td>ACE222.1</td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>Bioshop</td><td>SAA305.500</td><td></td></tr><tr><td>bovine serum albumin standard</td><td>bio-rad</td><td>500-0206</td><td></td></tr><tr><td>Bio-rad protein assay dye reagent concentrate</td><td>bio-rad</td><td>500-0006</td><td></td></tr><tr><td>Ethylenediamine tetraacetic acid, disodium salt dihydrate</td><td>Fisher scientific</td><td>BP120-500</td><td></td></tr><tr><td>Tris-base</td><td>Bioshop</td><td>TRS001.1</td><td></td></tr><tr><td>7000 MW Snakeskin dialysis tubing</td><td>Thermoscientific</td><td>68700</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M3148-25ml</td><td>This reagent should not be handled outside of a fume hood.</td></tr><tr><td>AcTEV protease</td><td>lifetechnologies</td><td>12575-015</td><td>Producing your own TEV protease can be accomplished using (https://www.addgene.org/8827/) and purified as in reference 17</td></tr><tr><td>Polyethyleneglycol 3350</td><td>Bioshop</td><td>PEG335.1</td><td></td></tr><tr><td>polyethyleneglycol 8000</td><td>Bioshop</td><td>PEG800.1</td><td></td></tr><tr><td>Nunc MaxiSorp flat-bottom 96 well plate</td><td>ebioscience</td><td>44-2404-21</td><td></td></tr><tr><td>Sonicator</td><td>Fisher scientific</td><td>FB-120-110</td><td></td></tr><tr><td>Eon microplate spectrometer</td><td>Biotek</td><td>11-120-611</td><td>This equipment uses the Gen5 data analysis software.</td></tr><tr><td>Gen5 data analysis software</td><td>BioTek</td><td></td><td></td></tr><tr><td>sodium dodecyl sulphate</td><td>Bioshop</td><td>SDS001</td><td></td></tr><tr><td>bromophenol blue</td><td>Bioshop</td><td>BRO777</td><td></td></tr><tr><td>Glycerol</td><td>Bioshop</td><td>GLY001</td><td></td></tr><tr><td>Protein desalting columns</td><td>Thermoscientific</td><td>89849</td><td></td></tr><tr><td>Glycine</td><td>Bioshop</td><td>GLN001</td><td></td></tr><tr><td>precast 12% polyacrylamide gel</td><td>bio-rad</td><td>456-1045</td><td></td></tr><tr><td>Rapid stain reagent</td><td>EMD Millipore</td><td>553215</td><td></td></tr><tr><td>Gel dock EZ imager</td><td>bio-rad</td><td>1708270</td><td></td></tr><tr><td>White Light Sample Tray</td><td>bio-rad</td><td>1708272&amp;nbsp;</td><td>Used along with gel dock EZ imager for coomassie blue stains</td></tr><tr><td>Protein ladder</td><td>bio-rad</td><td>1610375</td><td></td></tr></tbody></table>

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.

精製が完了すると、サンプルがステップ1.4、2.1、3.4に回収し、ステップ6.4からの最終生成物は、タンパク質ゲル（ 図3A）上で実行する必要があります。 MOG タグは、最初のT O / Nのサンプルで31.86 kDaのバンドとして表示されますが、ないT 0、および最終的に純粋な生成物にのみバンドでなければならない必要があります。 MOG タ?...

Watch this Scientific Journal Video about Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies at JoVE.co

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

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.

実験的自己免疫性脳脊髄炎研究のためのシンプルで効率的な生産とマウスミエリンオリゴデンドロサイト糖タンパク質の精製

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

simple-efficient-production-purification-mouse-myelin-oligodendrocyte

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.9K ビュー.  Western University. 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.

ビデオ: Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

慢性EAEを有するマウスにおける神経幹/前駆細胞の全身注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

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 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) ...

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 ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

マウスの主なオリゴデンドロ サイトの特異的・迅速免疫分離

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

神経科学

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 ...

自己免疫性脱髄における体液性反応の調査

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

著者スポットライト:多発性硬化症研究におけるB細胞応答を研究するための新しいアッセイ

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

実験的自己免疫性脳脊髄炎に対するマウスにおける自己反応性抗体の定量化

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

自己免疫マウスからすべてのCNS常在細胞型を同時に単離

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

著者スポットライト:すべての主要なCNS常在細胞タイプの同時分離による神経炎症経路の研究 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

成体自己免疫性脳脊髄炎マウスからの主要な中枢神経系常在細胞型の同時単離

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

実験的自己免疫性脳脊髄炎研究のためのシンプルで効率的な生産とマウスミエリンオリゴデンドロサイト糖タンパク質の精製

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慢性EAEを有するマウスにおける神経幹/前駆細胞の全身注射

ミエリンオリゴデンドロサイト糖タンパク質（MOG

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

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

マウスの主なオリゴデンドロサイトの特異的・迅速免疫分離

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

実験的自己免疫性脳脊髄炎に対するマウスにおける自己反応性抗体の定量化

実験的自己免疫性脳脊髄炎に対するマウスにおける自己反応性抗体の定量化

実験的自己免疫性脳脊髄炎に対するマウスにおける自己反応性抗体の定量化

成体自己免疫性脳脊髄炎マウスからの主要な中枢神経系常在細胞型の同時単離

実験的自己免疫性脳脊髄炎研究のためのシンプルで効率的な生産とマウスミエリンオリゴデンドロサイト糖タンパク質の精製

要約

さらに動画を探す

この動画の章

慢性EAEを有するマウスにおける神経幹/前駆細胞の全身注射

ミエリンオリゴデンドロサイト糖タンパク質（MOG

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

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

マウスの主なオリゴデンドロ サイトの特異的・迅速免疫分離

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

実験的自己免疫性脳脊髄炎に対するマウスにおける自己反応性抗体の定量化

実験的自己免疫性脳脊髄炎に対するマウスにおける自己反応性抗体の定量化

実験的自己免疫性脳脊髄炎に対するマウスにおける自己反応性抗体の定量化

成体自己免疫性脳脊髄炎マウスからの主要な中枢神経系常在細胞型の同時単離

マウスの主なオリゴデンドロサイトの特異的・迅速免疫分離