从国立卫生研究院的资助（以R01 NS37253和N01，NS055167）和国家多发性硬化症协会（的RG 3288 - A6）在部分支持。

1。准备终审法院乳化的抗原<ol><li>在磷酸盐缓冲液（PBS）的2毫克/毫升的浓度，溶解豚鼠MBP（根据的方法Swanborg1的准备，并在冻干的形式储存在4°C）。如果合成的MBP肽（MBP的60-80 SHHAARTTHYGSLPQKSQR）2，准备2毫克/毫升的解决方案。达成鲁尔乐的玻璃注射器吸取适量抗原溶液（0.1毫升每鼠标进行免疫接种）。 </li><li>画了一个完全弗氏佐剂H37Ra株（0.1wt％的）（终审法院，Difco公司实验室）等于另一个玻璃注射器到鲁尔乐配备的抗原溶液的体积。 </li><li>连接一个三通阀的两个玻璃注射器。混合来回推动活塞与终审法院的MBP的解决方案。保持运动，直到形成乳状液。 </li><li>如果要测试形成乳状液，推到一个注射器的混合物。断开空的注射器，拉ţ他柱塞后退了几步，排出残留的乳液在室温下的清洁水的烧杯下降。如果乳化降保持完好的水面上，形成乳状液。如果分散乳化降，重新连接注射器和继续搅拌议案。 </li><li>使用1毫升的塑料注射器的免疫程序，以确保提供一个准确的抗原剂量。转移抗原乳剂的塑料注射器，拉出柱塞和开放三通阀1.4断开以前的玻璃注射器插入空塑料注射器。 </li><li>填补了塑料注射器，玻璃注射器的柱塞推到边缘。插入背面的塑料注射器的柱塞和向后推，直到达到1.2毫升标记的塑料注射器多余的乳液。这个动作可确保无气泡的塑料注射器内被困。 </li><li>从三通阀中取出的塑料注射器。阿塔CH＃26号针头的塑料注射器。推柱塞让乳液填充空隙体积的针1毫升标记。 </li></ol> 2。供体小鼠的免疫注：通常是雌性C57BL / 6小鼠注射抗原乳液在四个网站在侧翼，麦克法林的3组，在国家卫生研究院原先设计的方法。对于一个有经验的研究者，一个人就可以完成整个任务。对于初学者来说，帮助可征求这样一个人持有鼠标和其他人注射。另外，鼠标就可以注射麻醉。 <ol><li>牢牢按住鼠标，颈部皮肤和尾巴之间的第 4和第 5 次手指，胸部和腹部等方便忍住。 </li><li> 1毫升的塑料注射器针头放置在略高于右腋下胸部网站（莫使用）和0.05毫升抗原乳化皮下注入。可以看出，通过面色苍白，皮肤稍隆起的白色终审法院。重复相同的网站在略高于左腋下胸部。 </li><li>松开尾巴，并采取举行的右脚（鼠标），打开它，并保持它之间的第 4和第 5 次手指。定位略低于肋骨的情况下，在右侧的网站，并注入抗原乳剂0.05毫升。 </li><li>松开右脚，转其他方式和鼠标之间的第 4和第 5 次手指按住左脚（鼠标）。找到下面的肋骨的情况下只是在左翼的网站，并注入抗原乳剂0.05毫升。 </li><li>改变到一个新的针头，注射后10只小鼠，或是否变得迟钝针。 </li></ol> 3。接种后7至10天组织文化的淋​​巴结细胞的分离<ol><li>接种后7至10天，牺牲老鼠清除淋巴结。安乐死的首选方法是通过CO 2吸入。牺牲后，在夹层板打下鼠标与四肢伸出，并与针板固定在它的后面。小鼠70％的乙醇喷雾润湿。 </li><li>纵向切缝在皮肤中的下巴，用无菌剪刀和镊子腹部的下端中间。 </li><li>从腹股沟区切下来每条腿腹部皮肤。剥离背部和牵制的皮肤，暴露腹股沟淋巴结肿大。 </li><li>削减的皮肤，使皮肤可以去皮背部和牵制暴露腋窝淋巴结附近的腋下沿前肢（左，右）。 </li><li>两个双钳共同努力，拉远覆盖的结缔组织和周围的腹股沟淋巴结。当结缔组织清除，放置一对钳下的淋巴结和拉远离身体。放置淋巴结在35毫米培养皿用无菌PBS填充。 </li><li>拉远覆盖两个腋窝淋巴结肿大，使用两对镊子的结缔组织。孤立淋巴结和放置在培养皿中。必须非常小心，不要打破过路的面积出血使得很难确定淋巴结的血管。 </li><li>所有的淋巴结收集后，将培养皿生物安全罩。打破和挑逗的淋巴结除了两对钳子，研磨反对。此外，淋巴结，可以打破柱塞3毫升的塑料注射器对培养皿底部的淋巴结按使用平头。 </li><li>通过一个尼龙网戏弄淋巴结细胞悬液去除膜和杂物。 </li><li>弥补的淋巴结细胞悬液50毫升，用无菌PBS。在1000 rpm离心10分钟。培养液中悬浮颗粒。做一个可行的çELL计数，调整浓度4×10 6细胞/ ml。辅以10％小牛血清，2毫米L-谷氨酰胺，1毫米的MEM丙酮酸钠，0.1毫米的MEM非必需氨基酸，100 U / mL青霉素，100μg/ mL链霉素，10毫米的HEPES缓冲和5×UseRPMI 1640 10 -5 m 2-ME公司。预温的文化之前，重新悬浮细胞。 </li><li>培养的细胞于24孔培养板，每孔2 mL体积（最终细胞浓度为8×10 6细胞/孔）。在终浓度为100微克/毫升，每孔加入抗原。放置在37℃培养箱培养板，用5％的CO 2。孵育5天的细胞。 </li></ol> 4。收获后5天启动文化培养淋巴结细胞<ol><li>在每口井的细胞混合巴斯德玻璃吸管或小的体积移液器吸取了上下。收获细胞，以50 mL离心管中。 </li><li> 10分钟1000转离心细胞。 resus挂起细胞沉淀成小体积的PBS。 </li><li>计数细胞，调整可行的细胞浓度为2.5×10 8细胞/毫升无菌PBS。到装有26号针头注射器吸取细胞。 </li><li>在受体小鼠的笼子里，以帮助扩张尾静脉放置热灯。 </li><li>使用鼠标持有人的持有人通过的缝隙，延长鼠标的尾部。拉扩大尾巴。找到静脉。 </li><li>注入细胞悬液0.2毫升/鼠标，相当于5×10 7细胞。 </li></ol> 5。受体小鼠的抗原挑战20天细胞转移和疾病症状的发展<ol><li>挑战受体小鼠抗原与疾病的征兆。作为“供体小鼠免疫”第2相同的程序。 </li><li>观察发展麻痹病抗原的挑战后，7至10天。 <ol><li>疾病开始在尾部的弱点。疾病被评定为1当尾巴变得松弛。当鼠标在一个后肢瘫痪的疾病被评定为2。疾病等级为3时，两后肢瘫痪和鼠标拖动两个后肢向前爬行。疾病等级为4时，鼠标失去使用两个前肢，使鼠标完全动弹不得。疾病等级为5时，鼠标是奄奄一息，身体蜷缩起来。死亡，疾病年级6 4拿下。 </li></ol></li><li> MBP特定脑脊髓炎诱导C57BL / 6小鼠后，此协议是单相。小鼠可能从疾病中恢复过来，但不进行自发的复发。 </li></ol> 6。代表结果老鼠收到引物，并在体外扩大捐助淋巴结细胞的发展并不EAE的疾病症状，反映他们的抵抗疾病的感应。然而，严重的疾病后抗原的挑战（ 见表一 ），表明抗原挑战的能力逆转耐药机制。抗原挑战，指出两个特殊功能的抗原剂量和挑战，诱发疾病的动力学。需要非常低剂量的抗原，如表三所示。令人惊讶的是，小鼠获得供体细胞较上年同期仍可能发展为严重疾病与挑战抗原（ 表四 ）。这两种意见强烈建议“长寿”的T细胞的参与，在维护EAE的阻力。该协议是适用于许多EAE的抗小鼠品系。 <img alt="图1。" src="/files/ftp_upload/3778/3778fig1.jpg" /> 图1。在著名的抗小鼠品系对EAE诱导的实验设计流程图。 <table border="1"><tbody><tr><td></td><td></td><td colspan="3"> 临床脑脊髓炎 继转移后 </td><td colspan="3"><stroNG&gt;临床脑脊髓炎 经过抗原的挑战</td></tr><tr><td> 应变 </td><td> 抗原 </td><td> 发生率 </td><td> AV。病级 （范围） </td><td> AV。发病日 （范围） </td><td> 发生率 </td><td> AV。病级 （范围） </td><td> AV。发病日 （范围） </td></tr><tr><td> SJL </td><td> MBP的</td><td> 21/21 </td><td> 4.0（3-5） </td><td> 8.0（6-10） </td><td> ND折价</td><td> ND折价</td><td> ND折价</td></tr><tr><td> C57BL / 6小鼠</td><td> MBP的</td><td> 0/10 </td><td> - </td><td> - </td><td> 7/7 </td><td> 3.85（3-5） </td><td> 9.28（9-10） </td></tr><tr><td> BALB / C </td><td> MBP的</td><td> 0/7 </td><td> - </td><td> - </td><td> 5/5 </td><td> 3.2（3-4） </td><td> 7.0（7） </td></tr><tr><td> C3H/HeJ小鼠</td&gt; <td> MBP的</td><td> 0/7 </td><td> - </td><td> - </td><td> 4/4 </td><td> 3.2（3-4） </td><td> 8.0（8） </td></tr><tr><td> C57BL / 6小鼠</td><td> MBP的60-80 </td><td> 0/4 </td><td> - </td><td> - </td><td> 4/4 </td><td> 2.75（2-4） </td><td> 10.2（9-11） </td></tr></tbody></table> 表一，在C57BL / 6小鼠的MBP-介导的EAE的诱导。在C57BL / 6小鼠EAE的引物和体外扩大供体细胞中没有引起过继转移。抗原的挑战后细胞转移呈现易患疾病诱导小鼠。 AV。，平均。钕，没有这样做。 <table border="1"><tbody><tr><td></td><td></td><td colspan="3"> 抗原的挑战后的临床脑脊髓炎 </td></tr><tr><td> 吸抗原 </td><td> 挑战抗原 </td><td> 发生率 </td><td> AV。病级 （范围）</td><td> AV。发病日 （范围） </td></tr><tr><td> MBP-终审法院</td><td>终审法院仅</td><td> 0/3 </td><td> - </td><td> - </td></tr><tr><td> MBP-终审法院</td><td>卵，终审法院</td><td> 0/3 </td><td> - </td><td> - </td></tr><tr><td> MBP-终审法院</td><td> MBP-终审法院</td><td> 3/3 </td><td> 4.0（4.0） </td><td> 7.7（7-9） </td></tr></tbody></table> 表二。特异性抗原的挑战。收件人小鼠吸抗原以及其他无关抗原的挑战，。只吸抗原诱发严重的疾病。卵：鸡卵清蛋白。 AV。，平均。 <table border="1"><tbody&gt; <tr><td></td><td></td><td colspan="3"> 抗原的挑战后的临床脑脊髓炎 </td></tr><tr><td> 挑战抗原剂量（微克/鼠标） </td><td> 发生率 </td><td> AV。病级 （范围） </td><td> AV。发病日 （范围） </td></tr><tr><td> 200 </td><td> 4/4 </td><td> 3.75（3-4） </td><td> 8.25（7-9） </td></tr><tr><td> 100 </td><td> 4/4 </td><td> 3.67（3-4） </td><td> 7.5（7-8） </td></tr><tr><td> 50 </td><td> 4/4 </td><td> 3.25（3-4） </td><td> 8.25（7-10） </td></tr><tr><td> 25 </td><td> 4/4 </td><td> 3.0（3.0） </td><td> 8.25（8-9） </td></tr><tr><td> 10 </td><td> 4/4 </td><td> 3.0（3.0） </td><td> 8.76（8-9） </td></tr><tr><td> 5 </td><td> 4/4 </td><td> 2.5（2-3） </td><td> 9.75（9-10） </td></tr><tr><td> 1 </td><td> 2/4 </td><td> 1.0（1.0） </td><td&gt; 16（14-18） </td></tr><tr><td> 0 </td><td> 0/4 </td><td> - </td><td> - </td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table> 表三。挑战抗原剂量需要诱导疾病。用于各种挑战受体小鼠的MBP浓度进行了测试。 AV。，平均。 <table border="1"><tbody><tr><td></td><td></td><td colspan="3"> 抗原的挑战后的临床脑脊髓炎 </td></tr><tr><td> 挑战抗原 </td><td> 天的挑战 </td><td> 发生率 </td><td> AV。病级 （范围） </td><td> AV。发病日 （范围） </td></tr><tr><td> MBP-终审法院</td><td> 5 </td><td> 3/4 </td><td> 1.33（1-2） </td><td> 9.67（9-10） </td></tr><tr><td> MBP-终审法院</td><td> 10 </TD&gt; <td> 4/4 </td><td> 3.0（3.0） </td><td> 8.25（7-9） </td></tr><tr><td> MBP-终审法院</td><td> 15 </td><td> 4/4 </td><td> 3.75（3-4） </td><td> 7.25（7-8） </td></tr><tr><td> MBP-终审法院</td><td> 25 </td><td> 4/4 </td><td> 4.0（4.0） </td><td> 8.0（7-9） </td></tr><tr><td> MBP-终审法院</td><td> 35 </td><td> 4/4 </td><td> 3.5（3-4） </td><td> 7.5（7-8） </td></tr><tr><td> MBP-终审法院</td><td> 60 </td><td> 4/4 </td><td> 3.75（3-4） </td><td> 9.0（8-10） </td></tr><tr><td colspan="5"> -------------------------- </td></tr><tr><td> MBP-终审法院</td><td> 343 </td><td> 3/3 </td><td> 3.0（3.0） </td><td> 10.33（10-11） </td></tr><tr><td> MBP-终审法院</td><td> 445 </td><td> 3/3 </td><td> 3.0（3.0） </td><td> 7.0（7.0） </td></tr></tbody></table> 表四。动力学抗原挑战。受体小鼠在不同的时间点后的挑战采取IVE细胞转移。 AV。，平均。

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+proteolipid+protein-specific+CD4+CD25++regulatory+cells+mediate+genetic+resistance+to+experimental+autoimmune+encephalomyelitis.">Myelin proteolipid protein-specific CD4+CD25+ regulatory cells mediate genetic resistance to experimental autoimmune encephalomyelitis.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 15434-15439 (2004).</li><li>Korn, T., Reddy, J., Gao, W., Bettelli, E., Awasthi, A., Petersen, T. R., Backstrom, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin-+specific+regulatory+T+cells+accumulate+in+the+CNS+but+fail+to+control+autoimmune+inflammation.">Myelin- specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation.</a> Nature Med. 13, 423-431 (2007).</li><li>Shaw, M. K., Li, J., Ho, P. P., Hao, H., Lisak, R. P., Tse, H. Y., Broglie, P. V. <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+myelin+basic+protein-specific+adoptive+experimental+autoimmune+encephalomyelitis+in+C57BL/6+mice.+Longevity+of+donor+T+cells+and+lack+of+disease+relapses.">Induction of myelin basic protein-specific adoptive experimental autoimmune encephalomyelitis in C57BL/6 mice. Longevity of donor T cells and lack of disease relapses.</a> Neuroimmunology Research Focus. , 175-191 (2007).</li></ol>

没有财务披露。

在小鼠EAE的研究往往利用的neuroantigens，髓鞘碱性蛋白（MBP），蛋白脂蛋白（PLP）有或髓鞘少突胶质细胞蛋白（MOG）。早先的研究中大多采用的MBP。工党刺激SJL小鼠的强烈和一致的反应。最近，MOG是共同neuroantigen使用，因为很容易受到疾病与此抗原诱导B6小鼠。许多有针对性的小鼠品系的基因B6的遗传背景。有趣的是，B6小鼠容易与MOG诱导EAE，但抵抗疾病的MBP诱导。 许多以前的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>豚鼠脊髓</td><td>罗克兰免疫化学品公司 gilbertsville，扩音19525 <a href="http://www.rockland-inc.com" target="_blank">www.rockland-inc.com</a> </td><td> GP-T065 </td><td>冻结</td></tr><tr><td>弗氏佐剂，完整H37Ra株</td><td> DIFCO实验室 密歇根州底特律</td><td> 231131 </td><td></td></tr><tr><td> MULTIFIT交换能够注射器</td><td>屋宇署 <a href="http://www.bd.com/us" target="_blank">www.bd.com /我们</a> </td><td> 512133 </td><td></td></tr><tr><td> RPMI 1640培养</td><td>敏达公司 弗吉尼亚州马纳萨斯 <a href="http://www.cellgro.com" target="_blank">www.cellgro.com</a> </td><td> 10-040-CM </td><td></td></tr><tr><td>卵</td><td> Sigma-Aldrich公司 <a href="http://www.sigmaaldrich.com" target="_blank">www.sigmaaldrich.com</a> </td><td> A5503 </td><td></td></tr><tr><td>小鼠</td><td>杰克逊实验室 缅因州巴尔港， </td><td> B6小鼠：0000664 </td><td></td></tr></tbody></table>

overcoming unresponsiveness in experimental autoimmune encephalomyelitis (eae) resistant mouse strains by adoptive transfer and antigenic challenge

实验性自身免疫性脑脊髓炎（EAE）是一种中枢神经系统（CNS）炎症性疾病，并已作为研究人类脱髓鞘疾病，多发性硬化症（MS）的动物模型。脑脊髓炎的特点是进入中枢神经系统的病理单核细胞浸润和麻痹性疾病的临床表现。类似于MS，EAE的是在某些小鼠品系遗传控制下也很容易受到疾病的诱导，而其他有抗药性。通常情况下，免疫C57BL / 6小鼠（H-B）与髓鞘碱性蛋白（MBP）的失败发展麻痹的迹象。此反应迟钝是肯定不会由于抗原加工缺陷或抗原的MBP介绍，这里描述的一个实验性协议已用于诱导C57BL / 6小鼠以及其他知名的抗小鼠品系严重脑脊髓炎。此外，脑炎C57BL / 6和BALB / c小鼠，以MBP的反应性T细胞克隆成功完全孤立和传播。 实验协议涉及使用蜂窝收养传输系统的MBP-底漆（200微克/鼠标）C57BL / 6捐助淋巴结细胞分离和培养五天与扩大池的MBP-特异性T细胞的抗原。在文化时期的结束，50万个活细胞通过尾静脉转移到天真同源收件人。通常这样对待的受体小鼠不发展脑脊髓炎，从而重申了他们的耐药现状，他们可以无限期地保持正常。十天后细胞移植受体小鼠挑战与完全弗氏佐剂（CFA）乳化四个网站在侧翼的MBP。严重的EAE的开始发展，在这些小鼠十至十四天后的挑战。结果表明，诱导疾病的抗原具体作为与不相干抗原挑战没有引起疾病的临床症状。值得注意的是，抗原剂量滴定挑战受体小鼠表明，它可能是为5微克/鼠标。此外，抗原挑战的时机动力学研究表明，早在5天抗原的挑战，如超过445天的抗原挑战，诱发疾病的挑战是有效的。这些数据有力地指向保持反应迟钝“长寿”的T细胞人口的参与。在这个系统中的调节性T细胞（Treg细胞）的参与，没有被定义。

Watch this Scientific Journal Video about Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge at JoVE.com

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

某些鼠标菌株能够抵抗与髓鞘碱性蛋白诱导实验性自身免疫性脑脊髓炎（EAE）。这里描述的是一个简单的免疫协议，扭转反应迟钝和麻痹性疾病引起几个典型的EAE的耐鼠标污渍。

在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝

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

overcoming-unresponsiveness-experimental-autoimmune-encephalomyelitis

Research

JoVE Journal

Immunology and Infection

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

20.2K 次观看.  St. John-Providence Health System. 某些鼠标菌株能够抵抗与髓鞘碱性蛋白诱导实验性自身免疫性脑脊髓炎（EAE）。这里描述的是一个简单的免疫协议，扭转反应迟钝和麻痹性疾病引起几个典型的EAE的耐鼠标污渍。

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

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

实验转移和CTL过继转移免疫小鼠模型

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

科研

免疫与感染

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.

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

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.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

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

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

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

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;

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

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.

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

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.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

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

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.

实验性自身免疫性葡萄膜炎：眼内炎症小鼠模型

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

副结核分枝杆菌在自身免疫性脑脊髓炎中的免疫原性增强

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

作者聚焦： 副结核分枝杆菌在增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原性中的辅助活性  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

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