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.

여기서는 MOG 태그 단백질 및 방법 MOG 태그 단백질 순수한 MOG 1-125을 생성하는 생산을위한 프로토콜을 기술 하였다. 이 프로토콜은 표준 그의 태그 기반의 단백질 정제 방법뿐만 아니라 이전 MOG 단백질 계 (15)의 생성을위한 전술 한 두 프로토콜에 기초한다. 그것은 여기에서 설명되지 않지만, MOG 태그 단백질의 주 사용은 단백질 항원을 면역 EAE를 유도하는 ?...

MS 만성 염증과 미엘린 향하는자가 면역 반응에 의해 구동되는 것으로 생각되는 CNS의 신경 퇴행을 특징으로하는 인간의 질병이다. 시간이 지남에 따라 수초와 축색 돌기의 손실은인지 및 모터 기능을 하나의 점진적 감소가 발생. &quot;실험자가 면역 뇌척수염은&quot;중추 신경계의 수초 지향자가 면역 질환의 동물 모델에 대한 포괄적 인 용어이다. 인간의 MS와 마찬가지로 EAE는 일반적으로 어떤 경우에는 CNS의 면역 세포 침윤을 특징으로하고,이 탈수 초화. 그러나, 임의의 주어진 EAE 모델은 부분적으로 인간의 MS와 유사한 과정에 사용되는 화학 종 또는 균주 및 하부 반 미엘린 면역 반응의 복잡성에 좌우된다. 안티 미엘린 실험적자가 면역은 여러 가지 방법으로 유도하지만, 현재 사용되는 가장 일반적인 방법은 면역 CD4 흉내 아미노산의 짧은 펩타이드 마우스 면역화 될 수있다 <s미엘린 단백질&gt; + T 세포 에피토프입니다. 이 병원성 반응을 유도 할 수있는 최소 요구 사항을 나타냅니다. 아마도 이들 중 가장 일반적인 C57BL / 6 마우스 3 EAE를 유도하기 위해 사용된다 (MOG 35-55) 미엘린 희소 돌기 아교 세포의 당 단백질에서 유래하는 21 개의 아미노산 펩타이드이다. 그러나 일부 실험 목적이 큰 단백질 항원 실제로 짧은 펩타이드와 예방 접종을 통해이 몇 가지 장점이와 예방 접종하는 것이 바람직 심지어 필요가있다. 전체 단백질 또는 특정 도메인의 어느 쪽인지를 표현하는 더 큰 단백질 항원이 다른 종에서도 여러 근친 마우스 변종에 프리젠 테이션을 위해 정상적으로 처리하거나 할 수 있지만 먼저, MHC의 제한 때문에, 짧은 펩티드는, 일반적으로 균주의 매우 제한된 범위에서만 유효하다 4. 둘째로, 더 큰 단백질 항원은 항원 인식 림프구 종 이상을 포함하는보다 복잡한 면역 반응을 유도하기보다는 limitin 할CD4 + T 세포에 항원 g 인식. 예를 들어, 그들의 B 세포 수용체 (BCR)을 통해 B 세포는 전체 단백질보다는 처리와 직접 상호 작용한다. 우리와 다른 MOG 단백질 (5)을 인식하지 못하는 MOG 35-55 예방 접종에 의해 활성화하는 B 세포를 보여 주었다. B 세포는 인간 최근 MS 6에서 병원성 역할을하는 것으로 입증 되었기 때문에,자가 면역 병리에서 B 세포를 포함 EAE 모델은 점점 중요하다. EAE를 유도하는 큰 단백질 항원을 사용하는 장점에도 불구하고, 단백질에 대한 몇 시판 소스가 남아있다. MOG 35-55 같은 짧은 펩티드가 매우 빠르고 상대적으로 낮은 비용으로 합성 할 수 있지만 실제로, MOG 단백질 상업 옵션을 구입 제한 및 비용 실질적으로 더 있습니다. 그럼에도 불구하고, MOG 세포 외 도메인을 생성하는 연구 그룹에 사용할 수있는 여러 가지 발현 벡터 (MOG 1-125) 자신이있다. HoweveR, 우리는 문헌에서 확인 하였다 발현 시스템의 모든 사람보다 효율적인 발현 시스템 (7)로 대체 한 이전 기술에 기초한다. 또한, 대부분의 쥐 또는 인간의 MOG 8을 기반으로합니다. 마우스에서자가 면역 반응의 일부 수사 마우스 MOG의자가 항원에 기초하여 항원이 바람직하다. 마지막으로, 상업적 또는 발현 벡터와 같은 하나 우리가 식별 된 모든 MOG 계의 단백질은 상기 MOG 1-125 기재에 추가적인 아미노산을 포함하는 융합 단백질이다. 다음은 우리가 식별 할 수 없습니다 많은 기능이있는뿐만 아니라 정화 및 일반적으로 다른 시퀀스에 대한 태그를 포함한다. 이러한 한계를 해결하기 위해, 우리는 MOG 단백질 5의 공지 불용성 싸우는 오레 독신을 포함하는 태그에 융합 된 마우스 MOG 외 도메인에 기초하여 신규 한 융합 단백질을 생성. 태그 시퀀스는 정화와 TEV 프로테아제 CLE에 대한 6xHis 시퀀스를 포함원하는 경우, 모든 태그 서열의 완전한 제거를 허용 avage 사이트. 이것은 우리가 순수 MOG 1-125 단백질을 생성 할 알고있는 유일한 방법입니다. 단백질의 대량 생산을 용이하게하기 위해, MOG 1-125 서열은 박테리아 발현 코돈 - 최적화하고, MOG 태그 융합 단백질 애완-32 발현 시스템 내로 삽입되었다. 여기서는 가장 면역학 연구실 사용할 비 특수 장비를 사용하여 생산 MOG 태그 단백질 순수 MOG 1-125를 정화 상세히 프로토콜을 설명한다.

<table border="0" cellpadding="0" cellspacing="0" width="1434">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">BL21 E.coli- pet32-MOGtag</td>
			<td style="width:139px;">Kerfoot lab</td>
			<td style="width:119px;"></td>
			<td style="width:803px;">These bacteria are required to make the MOGtag protein. Glycerol stocks of these bacteria are available upon request.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LB broth miller</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LBL407.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Ampicillin</td>
			<td style="width:139px;">bio basic</td>
			<td style="width:119px;">AB0028</td>
			<td>Reconsititute the powder into 50% ethanol/ 50% H2O at 100 mg/ml. Store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">IPTG</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IPT002.5</td>
			<td>Reconsititute the powder into H2O at 1M and store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Chicken-egg lysozyme</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LYS702.10</td>
			<td>Reconstitute in H2O at 50 mg/ml and store at -80 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Triton-X100</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">T-8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Phosphate buffered saline</td>
			<td style="width:139px;">life technologies</td>
			<td style="width:119px;">20012-027</td>
			<td>Commercial phosphate buffered saline is not required, any standard lab made phosphate buffered saline is sufficient.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium chloride</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SOD004.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-HCl</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS002.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Imidazole</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IMD508.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Guanidine-HCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">G3272</td>
			<td>The quality must be greater than 98% purity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">0.5M EDTA</td>
			<td style="width:139px;">bioshop</td>
			<td style="width:119px;">EDT111.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nickel (II) sulfate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">NIC700.500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">His bind resin</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">69670-3</td>
			<td>Store in 20% ethanol 80% H2O at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Anhydrous ethanol</td>
			<td style="width:139px;">Commercial Alcohols</td>
			<td style="width:119px;">P016EAAN</td>
			<td>Dilute with water as needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glacial acetic acid</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">ACE222.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium acetate trihydrate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SAA305.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bovine serum albumin standard</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Bio-rad protein assay dye reagent concentrate</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Ethylenediamine tetraacetic acid, disodium salt dihydrate</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-base</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">7000 MW Snakeskin dialysis tubing</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">68700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">2-mercaptoethanol</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">M3148-25ml</td>
			<td>This reagent should not be handled outside of a fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">AcTEV protease</td>
			<td style="width:139px;">lifetechnologies</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;width:375px;">Polyethyleneglycol 3350</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG335.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">polyethyleneglycol 8000</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG800.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nunc MaxiSorp flat-bottom 96 well plate</td>
			<td style="width:139px;">ebioscience</td>
			<td style="width:119px;">44-2404-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sonicator</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">FB-120-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Eon microplate spectrometer</td>
			<td style="width:139px;">Biotek</td>
			<td style="width:119px;">11-120-611</td>
			<td>This equipment uses the Gen5 data analysis software.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gen5 data analysis software</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">sodium dodecyl sulphate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SDS001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bromophenol blue</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">BRO777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycerol</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLY001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein desalting columns</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">89849</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycine</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLN001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">precast 12% polyacrylamide gel</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">456-1045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rapid stain reagent</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">553215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gel dock EZ imager</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">White Light Sample Tray</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708272&#160;</td>
			<td>Used along with gel dock EZ imager for coomassie blue stains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein ladder</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">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.

정화가 완료되면, 샘플 단계 1.4, 2.1, 3.4에서 수집하고, 단계 6.4의 최종 생성물은 단백질 겔 (도 3A)에서 실행되어야한다. MOG 태그 먼저 T O / N 샘플에서 31.86 kDa의 밴드로 나타날 수 있지만 0 T, 최종 순수한 생성물의 유일한 밴드되어야한다. 모그 태그 단백질이 올바르게 접혀 여부를 테스트하기 위해, MOG 태그 단백질 FACS?...

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.

실험자가 면역 뇌척수염 연구 간단하고 효율적인 생산 및 마우스 미엘린 희소 돌기 아교 세포 당 단백질의 정제

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

Research

JoVE Journal

Immunology and Infection

10.9K 조회수.  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.

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

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.

실험 면역 Hypophysitis의 유도를위한 마우스 뇌하수체 Immunogen의 준비

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

연구

면역학 및 감염병학

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.

SJL 마우스의 실험적 면역 Hypophysitis의 유도

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

입양 이전 및 Antigenic 챌린지에 의해 실험적 면역 Encephalomyelitis (EAE) 모성 마우스 변종에 응답하지 않는 문제를 극복

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

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.

수초 희소 돌기 아교 세포 당 단백질 (MOG<sub&gt; 35-55</sub&gt;) C57BL / 6 쥐에 실험자가 면역 뇌척수염 (EAE) 유도

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

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.

효소 다이제스트 및 그라데이션 분리를 사용하여 마우스 피부에서 침투 백혈구의 분리

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.

다양한 조직에서 면역 세포의 질병에 의존하는 유통의 마우스 및 평가에 실험자가 면역 뇌척수염의 유도

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;

Vivo에서 실험적인 자기 면역 뇌 중 Neurovascular 단위의 내 피와 폐해 장벽의 장애 시각화

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

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.

실험적인 자가면역 뇌척수염 중 중추신경계에서 림프구 침투의 흐름 세포 분석

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.

실험적 자가면역 포도막염: 안구내 염증성 마우스 모델

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

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

체외 및 생체 내 연구를 위한 마우스 불변 천연 킬러 T 세포의 정화 및 확장

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...