这项工作是由奥地利科学基金会（FWF）授予P18820 - B13。 伦理声明: 这项研究是由伦理委员会批准（PLUS_Ethik_090514）在萨尔茨堡大学，涉及人类和/或动物实验与修订于1983年，所有患者参加了他们的书面知情同意遵守赫尔辛基Declaraction。

1。药物结合物的制备<ol><li>溶解于1.5 ml反应管1毫升DH 2 O 10毫克的DF。在进一步的程序，使用本DF解决方案的95μL。共轭聚丙烯人血清白蛋白溶于500μL0.3 M氢氧化钠（NaOH）的30.4毫克PP衍生工具（304克/摩尔）。 </li><li>新增430.6μL生署2 O和100μL0.5 M的2 - （N -吗啉）乙磺酸（MES）的缓冲液pH值6.5至95μL溶解的DF。加入33.1μL生署2 O和140μL2米MES缓冲液pH值2.9的聚丙烯样品。 </li><li>加入57.5μL新鲜制备的N -乙基- N&#39; - - （3 - dimethylaminopropyl）碳二亚胺盐酸盐（EDC），卫生署2 O溶解原液浓度为100毫克/毫升的DF样品，或10μLEDC解决方案PP的样本。涡1分钟的样品。 </li><li>用118毫克/毫升的浓度，每个样品添加的HSA溶液16.9μL。 </li><li>孵育样品在室温下2小时，而在300转/分的晃动。 </li><li> 2磷酸盐缓冲液（PBS）pH值7.4升，每几个小时，透析对DF和PP样品的3倍。执行所有在4 ° C透析的步骤</li><li> 14000 XG离心样品10分钟，收集上清液含DF或聚丙烯共轭与HSA。 </li><li>检查执行的SDS - PAGE蛋白质的上清液，用12％的凝胶。装入一个凝胶槽约12微克HSA的共轭。 </li></ol> 2。确定药物的共轭耦合率（图2） <ol><li>对于DF和PP共轭耦合率的HPSEC分析，使用100毫米磷酸钠缓冲液的pH值6.5包含150 mM氯化钠和0.05％的叠氮化钠。使用一个7.8 × 300毫米TSK - GEL - G2000的SWXL列。 </li><li>执行通过使用一个在线耦合RI和紫外检测器系统的信号检测。 </li><li>准备一个HSA的标准样品，用1毫克/毫升的浓度，卫生署2 O探测器的校准注入50μL血清白蛋白标准溶液。 </li><li>计算RI常数k RI和常数k UV紫外线探测器。使用的公式面积RI = K RI * [（DN / DC） 人血清白蛋白 （HSA）的]和地区紫外线 = K 紫外线 * [（DA / DC）， 白蛋白 （HSA）]与* C（DN / DC） 人血清白蛋白 = 0.185和（DA / DC） 人血清白蛋白 = 0.518 5。 </li><li>准备DF和聚丙烯衍生HPSEC，5，10和30 nmol金额两种标准可以很容易地注入标准。 </li><li>计算平均值（DN / DC） 的药物和（DA / DC）两种标准的药物。对于东风，（DN / DC）的0.235 ± 0.010和（DA / DC）为39.000 ± 0.493决心。对于PP，（DN / DC）和0.328 ± 0.016（DA / DC）53.155 ± 2.464决心。 </li><li>上HPSEC注入药物的结合物，并确定他们的RI和UV峰面积。 </li><li>解决这两个方程面积RI = K RI * [（DN / DC） 药物 * C（药）+（DN / DC） 人血清白蛋白 * C（HSA）]和地区紫外线计算DF，聚丙烯衍生物，与HSA的浓度= K * [ 紫外线 （DA / DC） 药物 * C（药）+（DA / DC） 人血清白蛋白 * C（HSA）的未知数。 </li></ol> 3。英美烟草公司使用药物结合物<ol><li>准备7流式细胞仪样品瓶和标签取决于其内容:未染色的控制，阴性对照，阳性对照组，和药物的结合物。 </li><li>移液器50μL刺激的B - CCR - STB缓冲区中保留未染色和阴性对照的小瓶（Flow2科协，Bühlmann实验室，Schönenbuch，CH）。作为阳性对照，用50μLFlow2科协套件提供了一个反FcεRI解决方案。共轭溶液中加入适当的金额，创造了从0.02 -20微克/毫升DF共轭和PP共轭（终浓度的检测量在200μL）1:10稀释系列药物共轭保留小瓶。必须执行这些滴定实验，以确定最佳嗜碱性粒细胞活化为每一个病人样本的浓度。 </li><li>每管加入100μL刺激缓冲区和50μL患者的EDTA全血。仔细混合样品。 </li><li>移液器20μLFlow2科协染色试剂含有藻红蛋白（PE）和异硫氰酸荧光素（FITC）标记，以每管的抗CD63抗体标记的抗CCR3抗体，再次组合。不要到不染样品的染色试剂移液器！ </li><li>在37℃水浴中孵育45分钟的样品。 </li><li> 5分钟，停止对冰的反应。 </li><li>加入2.0毫升Flow2科协裂解试剂，每小瓶和旋涡轻轻裂解红细胞。在黑暗中孵育样品在室温下10分钟。 </li><li>离心5分钟，小心倒出上清液500 XG样本。 </li><li>重悬于500μLFlow2科协洗涤缓冲液和冰，直到流式细胞仪存储的细胞。 </li><li>调整流式细胞仪的电压Settings前部和侧散射（FSC，SSC），同时收购未染色样品。 </li><li>从SSC - FSC阴谋的SSC - PE情节淋巴细胞门细胞预测。嗜碱性粒细胞识别CCR3 您好 SSC LO和门到FITC标记的PE图的PE标记的抗CCR3抗体。用FITC标记的抗CD63抗体检测激活嗜碱性粒细胞。 </li><li>设置截止到最大的嗜碱性粒细胞CD63的在喜阴性对照的2.5％ 。 A样品嗜碱粒细胞的活化被认为是积极的，如果&gt; 5％嗜碱性粒细胞CD63 的喜和除以阴性对照组的MFI样品平均荧光指数（MFI）超过2（图3） 。 </li></ol> 4。代表性的成果: 这个实验评估作为一个有益的工具来检测IgE依赖的药物过敏的英美烟草公司。用于嗜碱性粒细胞激活蛋白结合物的非甾体抗炎药。通过HPSEC RI和紫外检测聚集状态，耦合度，并有效的结合物产量的决定。 如图4所示，43.5％的DF共轭保持了5.0 DF / HSA的耦合度的单体。对于总量的9.5耦合度决定的。 propyphenazone共轭显示，68.5％与21.2耦合度单体组成。聚集的耦合度为27.9。 DF的共轭确定的耦合度，共计7.6 DF和PP的结合物，为23.2。 英美烟草公司与结合物进行优化条件（偶联物的浓度，刺激时间）在允许调查甾体抗炎药过敏的IgE依赖的反应。正如图5所示，只有PP的结合，能够触发嗜碱性粒细胞激活可视化中的上调CD63的表面表达:20.6％，嗜碱性粒细胞被激活，在荧光强度的日志移特点。的MFI增加至386（阴性对照），到1893年的4.9倍。相比之下，使用DF共轭只有3.1％的嗜碱性粒细胞被激活，低于5％的切断。小额信贷机构只增加了从197到210（阴性对照）的一个因素1.1（限2.0）。含量的有效性是由（i）&lt;5％嗜碱粒细胞的活化，阴性对照（二）日志移一个嗜碱粒细胞亚群的荧光强度，及（iii）激活嗜碱性粒细胞&gt; 50％（阳性对照）。 <img alt="图1" src="/files/ftp_upload/3263/3263fig1.jpg" /> 图1。DF和PP衍生物的耦合与HSA 。溶解后的药品，水，MES缓冲液，和EDC（偶联剂）。样品振荡1分钟前HSA是吸管的药物解决方案。在室温下摇晃2小时内的药物共价键结合血清白蛋白。单体以及聚合可能的结果。 <img alt="图2" src="/files/ftp_upload/3263/3263fig2.jpg" /> 图2。耦合度的计算。首先，k RI和K 紫外线的常量确定。因此，HSA与已知浓度的标准是注入HPSEC系统。 （DN / DC）和（DA / DC）对HSA给定值0.185和0.518，分别为5。峰面积计算的常数。其次，每种药物注入三个标准，以确定药物浓度的特定的RI和UV领域。以来的K RI和K 紫外线常数从上面的步骤，可以计算出特定药物（DN / DC）和（DA / DC）衍生物。第三，药物人血清白蛋白结合物注入HPSEC。由此产生的峰值区是用于解决两个未知变量的两个方程来计算药物和HSA的浓度每峰。 <img alt="图3" src="/files/ftp_upload/3263/3263fig3.jpg" /> 图3。嗜碱粒细胞浇注。预测的细胞如淋巴细胞与前向散射的闸门侧散射（SSC - FSC图）。嗜碱性粒细胞是从门控淋巴细胞（SSC - CCR3 - PE图）认定为CCR3 您好 SSC LO。嗜碱性粒细胞都激活（CD63 您好 ）CD63 - FITC - CCR3 - PE情节分析。阴性对照是用来设置最高2.5％，其余CD63您好嗜碱性粒细胞切断。 <img alt="图4" src="/files/ftp_upload/3263/3263fig4.jpg" /> 图4。药物共轭确定的耦合度。 RI和UV信号显示两峰代表聚集（低保留体积洗脱）和单体（高保留体积洗脱）的药物 - 人血清白蛋白的结合物。单体和聚合的百分比（RI信号来确定），其耦合度描述（N = 1）。 <img alt="图5" src="/files/ftp_upload/3263/3263fig5.jpg" /> 嗜碱粒细胞activati ​​图5。在测试。共轭激活药物样品，阴性对照，阳性对照结果显示CD63 - FITC - CCR3 - PE图（N = 1）。

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	<li>Himly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgE-mediated+immediate-type+hypersensitivity+to+the+pyrazolone+drug+propyphenazone.">IgE-mediated immediate-type hypersensitivity to the pyrazolone drug propyphenazone.</a> J Allergy Clin Immunol. 111, 882-888 (2003).</li><li>Demoly, P., Pichler, W., Pirmohamed, M., Romano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+questions+in+Allergy:+1--drug+allergy/hypersensitivity.">Important questions in Allergy: 1--drug allergy/hypersensitivity.</a> Allergy. 63, 616-619 (2008).</li><li>Ebo, D. G., Hagendorens, M. M., Bridts, C. H., Schuerwegh, A. J., Clerck, L. S., Stevens, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+in+vitro+activated+basophils,+specific+IgE+and+skin+tests+in+the+diagnosis+of+pollen-associated+food+allergy.">Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy.</a> Cytometry Part B (Clinical Cytometry). 64B, (2005).</li><li>Harrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diclofenac+hypersensitivity:+antibody+responses+to+the+parent+drug+and+relevant+metabolites.">Diclofenac hypersensitivity: antibody responses to the parent drug and relevant metabolites.</a> PLoS One. 5, e13707-e13707 (2010).</li><li>Wen, J., Arakawa, T., Philo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-exclusion+chromatography+with+on-line+light-scattering,+absorbance,+and+refractive+index+detectors+for+studying+proteins+and+their+interactions.">Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.</a> Anal Biochem. 240, 155-166 (1996).</li><li>De Weck, A. L., Sanz, M. L., Gamboa, P. M., Aberer, W., Bienvenu, J., Blanca, M., Demoly, P., Ebo, D. G., Mayorga, L., Monneret, G., Sainte Laudy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tests+based+on+human+basophils:+more+potentials+and+perspectives+than+pitfalls.+II.+Technical+issues.">Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. Technical issues.</a> J Investig Allergol Clin Immunol. 18, 143-155 (2008).</li><li>Ebo, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-assisted+allergy+diagnosis:+current+applications+and+future+perspectives.">Flow-assisted allergy diagnosis: current applications and future perspectives.</a> Allergy. 61, 1028-1039 (2006).</li><li>Poulsen, L. K., Quan, S., Kragh, C., Platzer, M. H., Skov, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basophil+granulocyte+in+allergic+reactions:+experimental+models+and+their+use+for+the+identification+of+drugs+with+effects+or+side+effects+on+basophils.">The basophil granulocyte in allergic reactions: experimental models and their use for the identification of drugs with effects or side effects on basophils.</a> Curr Med Chem - Anti-inflammatory &#38; Anti-Allergy Agents. 3, 167-180 (2004).</li><li>Sanz, M. L., Gamboa, P., de Weck, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+combined+test+with+flowcytometric+basophil+activation+and+determination+of+sulfidoleukotrienes+is+useful+for+in+vitro+diagnosis+of+hypersensitivity+to+aspirin+and+other+nonsteroidal+anti-inflammatory+drugs.">A new combined test with flowcytometric basophil activation and determination of sulfidoleukotrienes is useful for in vitro diagnosis of hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs.</a> Int Arch Allergy Immunol. 136, 58-72 (2005).</li></ol>

没有利益冲突的声明。

英美烟草公司是一个良好的成立虽然尚未用于常规诊断IgE介导的过敏性疾病的6,7的方法。然而，药物过敏，其适用性受到损害为低分子化合物不能交联的IgE受体，嗜碱性粒细胞活化8的一个先决条件。因此，根据调查的药物，需要合适的载体蛋白质（如白蛋白）共价结合。重要的是，耦合度（ 即每载体蛋白的药物分子的数量），需要加以控制，以保证免疫活性的结合物 （即IgE受体交联）。从理论上讲，载体分子，每两个半抗原应足以IgE受体交联，只有百分之五的人血清白蛋白的DF分子已被证明在基于单元的检测 4触发介质释放。两者结合物，PP - HSA和DF - HSA的，已被确定为显示足够高的耦合度（HPSEC），使他们在BAT合适的试剂，免疫积极。另一个问题可能是药物代谢物可能会起到一定的作用，而不是母体药物的代谢产物可能会引起过敏反应。在东风的情况下，这种可能性已经在以前使用的五个主要阶段我的代谢产物和联动变种4的详细评估。掌握所描述的技术重要因素包括血液样本（&lt;12小时后采血，检测嗜碱性粒细胞数&gt; 500）的质量和优化的刺激条件（时间，剂量）。此外，所谓的非应答，谁也不确定阳性对照（抗体IgE受体的指示）反应。因此，验证标准，包括抗FcεRI抗体为阳性对照。一个重要的优势是使用药物共轭的事实，他们出现在对比纯净的药品，作为DF - HSA的共轭抗FcεRI 英美烟草 4抗体协同刺激无毒。相比之下，纯东风，显示在浓度为1.25毫克/毫升的细胞毒作用，导致英美烟草 9解释性的问题。一般情况下，潜在的细胞毒性测试，强烈建议每一个新产生的药物共轭。 在这里，我们已经表明PP - HSA的结合物的潜力，在英美烟草公司用来揭示IgE介导的超敏反应。与此相反，DF过敏是不是与IgE的表现在缺乏嗜碱粒细胞的活化响应DF - HSA的共轭。重要的是，关于IgE介导的机制的不确定性的情况下，结合物的免疫活性，在体外细胞为基础的检测系统进行评估，已为东风4所示。 使用的描述设置合适的蛋白载体共价键的共轭低分子量药物分子对包括抗生素药，非甾体抗炎药，造影媒体，肌肉松弛剂，麻醉剂的过敏反应等，可进行调查的IgE介导的参与机制。因此，英美烟草公司可能会作为一个除了现有的方法进行诊断（皮试，口服激发试验）。

<table border="1"><tbody><tr><th>试剂/设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> 2 - （N -吗啉）乙磺酸</td><td>西格玛</td><td> M3671 </td><td> - </td></tr><tr><td> 7.8 × 300毫米TSK凝胶 - G2000SWXL列</td><td>东曹生命科学</td><td> 08540 </td><td> - </td></tr><tr><td> BD FACSCanto二</td><td>碧迪</td><td> 338962 </td><td> - </td></tr><tr><td>台式离心机</td><td>西格玛</td><td> - </td><td> - </td></tr><tr><td>双氯芬酸钠盐</td><td>西格玛</td><td> D6899 </td><td> - </td></tr><tr><td> EDTA的真空采血管</td><td>碧迪</td><td> 368589 </td><td> - </td></tr><tr><td> Flow2科协套件</td><td> Bühlmann实验室</td><td> FK - CCR </td><td>自2011年6月14日，Flow2 CAST套件现在流量科协</td></tr><tr><td> HP1100型高效液相色谱系统</td><td>惠普</td><td> - </td><td> - </td></tr><tr><td>人血清白蛋白</td><td>西格玛</td><td> A9511 </td><td> - </td></tr><tr><td>实验室离心机</td><td> Eppendorf公司</td><td> - </td><td> - </td></tr><tr><td> N -乙基- N&#39; - （3 - dimethylaminopropyl）碳二亚胺盐酸盐（EDC） </td><td>西格玛</td><td> E6383 </td><td> - </td></tr><tr><td> PBS片</td><td> AppliChem </td><td> A9199 </td><td> - </td></tr><tr><td> Propyphenazone衍生</td><td>奥地利萨尔茨堡大学分子生物学系</td><td> - </td><td> - </td></tr><tr><td>沙克尔</td><td> Eppendorf公司</td><td> - </td><td> - </td></tr><tr><td>叠氮化钠</td><td>西格玛</td><td> S8032 </td><td> - </td></tr><tr><td>氯化钠（NaCl） </td><td>西格玛</td><td> S1679 </td><td> - </td></tr><tr><td>碳酸氢钠</td><td>西格玛</td><td> 90421C </td><td> - </td></tr><tr><td>氢氧化钠</td><td>西格玛</td><td> S5881 </td><td> - </td></tr><tr><td>磷酸钠</td><td>西格玛</td><td> 342483 </td><td> - </td></tr><tr><td>三探测器阵列</td><td>的Viscotek </td><td> TDA302 </td><td> - </td></tr><tr><td>个人BioVortex的V - 1加</td><td> Peqlab </td><td> 90 - V - 1 </td><td> - </td></tr><tr><td>水洗澡1012 </td><td> GFL </td><td> 1012 </td><td> - </td></tr></tbody></table>

basophil activation test for investigation of ige-mediated mechanisms in drug hypersensitivity

对像propyphenazone（PP）和双氯芬酸（DF）的非甾体抗炎药（NSAIDs）的过敏反应可表现为I型样过敏反应 1 。在临床实践中，对药物过敏的诊断主要是由患者的病史，皮肤测试是不可靠的和口头挑衅测试熊生命危险， 为病人2的。因此，一个潜在的IgE介导的病理机制证据是很难获得。 在这里，我们目前的基础上，使用药物过敏的患者，可以模仿在体内的效应反应过敏的人类嗜碱性粒细胞的体外培养方法。由于药物过敏的患者嗜碱性粒细胞进行特定的IgE分子的罪魁祸首药物，他们被激活后IgE受体交联，释放过敏效应分子。嗜碱性粒细胞的活化，可以监视通过上调CD63的表面表达用流式细胞仪检测3测定。 在低分子量药物的情况下，共轭旨在使嗜碱性粒细胞上的IgE受体的交联。正如在图1中 ，非甾体抗炎药的两名代表所示，PP和DF，共价结合，通过与主赖氨酸残基的氨基酸组的羧基反应组的人血清白蛋白（ HSA）的。东风带有一种内在的羧基，因此，可以直接使用 4，而含羧基衍生的PP organochemically合成前研究 1 。 耦合度低分子量蛋白质载体分子化合物及其空间分布，重要的是要保证两个IgE受体分子的交联。这里所描述的协议，适用于高性能尺寸排阻色谱（HPSEC）配备一个连续的折射率（RI）和紫外线（UV）检测系统的耦合度的决心。 由于描述的方法可应用于其他药物，嗜碱性粒细胞活化试验（BAT）熊潜力的IgE介导的机制的药物过敏的决心。在这里，我们确定IgE介导和DF英美烟草公司非IgE抗体介导的过敏PP的超敏反应。

Watch this Scientific Journal Video about Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity at JoVE.com

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

嗜碱性粒细胞活化试验是一种IgE依赖的过敏检测的有力工具<em&gt;在体外</em&gt;。在这里，嗜碱性粒细胞活化试验的优化协议是用来研究药物过敏。一种高效的生产药物蛋白的共价结合物及其理化性质的方法是描述。

嗜碱粒细胞激活测试IgE介导的药物过敏机制的调查

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

basophil-activation-test-for-investigation-ige-mediated-mechanisms

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Unit 1

Adverse Drug Effects and Chemical Toxicity

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33.4K 次观看.  University of Salzburg. 嗜碱性粒细胞活化试验是一种IgE依赖的过敏检测的有力工具在体外。在这里，嗜碱性粒细胞活化试验的优化协议是用来研究药物过敏。一种高效的生产药物蛋白的共价结合物及其理化性质的方法是描述。

视频: Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

促进药物发现:斑马鱼在一个自动化的高含量检测炎症

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

跨体内迟发型变态反应检测方法的抗原特异性调节

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.