这项工作是由美国国立卫生研究院（NIH）AI091759和克隆氏病和结肠炎基金会美国的威廉·谢尔比参数及家庭基金会研究奖（MN）的支持。 NIAID的血吸虫资源中心（NIAID的合同N01，A130026）提供的血吸虫感染小鼠。

1。净化血吸虫卵<ol><li>瑞士韦伯斯特小鼠感染血吸虫尾蚴，感染血吸虫生命周期8阶段。另外，获得NIAID的血吸虫病资源中心（血吸虫感染小鼠<a href="http://www.schisto-resource.org/" target="_blank">http://www.schisto-resource.org/~~V</a> ）。离开小鼠感染后6-7周，时间点，鸡蛋是在肝脏中，可以恢复如下所述（ 见图1）。 </li><li>安乐死与CO 2的小鼠，并用70％乙醇湿。将鼠标背面。使用钝钳和剪刀切去皮肤和基础腹膜。海关肝脏，胸腔和膈肌下方。 </li><li>精细肉末，肝及添加20毫升，每消化混合物肝，胶原酶/ Dispase酶（0.5毫克/毫升），青霉素（100 U / ml的）和链霉素（100微克/毫升）在PBS组成。把mixt余吕杏茜在50毫升猎鹰管，并在37℃摇床孵育过夜。 </li><li>离心混合物在400 XG / 3分钟/ 20℃轻轻倒出多余的液体，用PBS填充管。离心再次重复PBS洗涤。继去年离心，重悬沉淀在25毫升PBS。 </li><li>通过一个标准的厨房金属过滤器（使用50毫升注射器柱塞，通过把一个玻璃烧杯捣碎肝片），其次是通过细筛的混合物（用10毫升注射器推混合物通过细筛，应变再悬浮颗粒进入第二个玻璃烧杯）。 </li><li>紧张的混合物放入50毫升猎鹰管。离心机在400 XG / 5分钟/ 20°C。小心地倒出球不失上清。于3 mL PBS重悬沉淀。 </li><li>叠加混合到20％的Percoll轻轻，40毫升的Percoll 8毫升和32毫升0.25M蔗糖（FW = 342.3所以8.56Ğsucrose/100毫升H 2 0）梯度。 </li><li>离心机10分钟/ 800 XG / 20°C。吸取了1ð丢弃肝细胞的凝胶状的顶层。用移液管底部取出卵子颗粒和投入15毫升猎鹰管的。 </li><li>洗颗粒3X在10毫升的PBS / 1毫米EDTA / 1 mM的EGTA使用离心分离机设置：3分/ 30 XG / 20°C。在500μL的PBS重悬。 </li><li>重叠组合一个新的25％的Percoll，10毫升梯度（2.5毫升的Percoll和7.5毫升0.25M蔗糖）使用离心机设置：10分钟/ 800 XG / 20°C。 </li><li> 3X：在10毫升，用离心机设置PBS洗3分钟/ 30 XG / 20°C。 </li><li>在过去的10毫升洗，取出100μL，算鸡蛋。鸡蛋定居很快，所以猎鹰管必须混合，然后才取出样品。稀释900μLPBS 100μL样品。应该算作100μL混合使用解剖显微镜（1:10稀释）在显微镜幻灯片液滴。应使用大口径的枪头。 </li><li> 50,000蛋/毫升的PBS重悬卵。鸡蛋可以保存几个月，在-80℃，解冻一次或TWICE使用。使用前，检查在显微镜下，以确保鸡蛋仍然完好无损（ 见图1）。 </li></ol> 2。腹腔致敏与S。血吸虫卵：0天<ol><li>准备在5000 eggs/100微升PBS鸡蛋在5的毫升snapcap管。注射，估计超过所需的50％以上的鸡蛋。 </li><li>装入23 3/4inch针规，并与100μL鼠标悬浮鸡蛋每1毫升注射器。解决鸡蛋，所以鸡蛋立即使用前负荷针。 </li><li>岩石注射器来回混合鸡蛋，每次注射前，注入腹腔注射100μL每鼠标悬浮。 </li></ol> 3。眼窝静脉挑战与 S 血吸虫卵：14天<ol><li>在5000 eggs/100微升上述准备鸡蛋。 </li><li>与异氟醚（4％），或与甲苯噻嗪（10毫克/公斤）和氯胺酮（120毫克/千克）腹腔麻醉的小鼠在专门室。对ANEST深度hesia确定捏的脚垫，并确保没有生理反应。 </li><li>装载1毫升注射器和23计3/4inch针与鸡蛋，用钳子在面临成角的斜面弯曲90°角进针。确保无气泡存在。 </li><li>放在一方鼠标。收回的皮肤，眼睛会突出，眼球后面的船只注入100μL，在45°角的鼻子。 </li><li>撤出针，适用于轻的压力，以控制出血。 </li></ol>所有这些程序都必须小心，必须监测小鼠，直到他们从麻醉中恢复。这些协议被批准的由宾夕法尼亚机构动物照顾及使用委员会（IACUC）的大学。 4。实验的收获：第22天<ol><li>安乐死与CO 2的小鼠。 </li><li> 70％的乙醇，回地方的湿鼠标。 </li><li>用生硬的强迫EPS，抓住老鼠腹部，并在皮肤的小切口。用剪刀小心地取下的皮肤和公开的肋骨和腹膜。 </li><li>切开腹膜。切断腹主动脉，用巴斯德吸管恢复血液。存储在冰上。 </li><li>移动肝脏暴露隔膜。使用罚款剪刀剪下隔膜。仔细切开暴露肺组织的左肋。 </li><li>恢复parathymic淋巴结肿大，肺排出罚款钳。这些左肋下夹着，对胸腺9两旁。在1ml无菌媒体商店（DMEM培养基辅以10％热灭活小牛血清，100 U / mL青霉素，100μg/ mL链霉素，2毫米L-谷氨酰胺，2 - 巯基乙醇50微米，都可以从Invitrogen公司）在冰上。 </li><li>恢复灌洗，膨胀的肺部组织学分析，必须进行气管插管。由唾液腺移动到一边暴露气管，placi的吴细镊子下气管。用细剪刀，剪了一个洞，在气管中，并插入肺部油管。确保手术线程占用的油管。 </li><li>附加PBS 1 mL注射器充满油管和充气用1 mL PBS肺部。仔细检索用注射器灌洗洗。存储在冰上。 </li><li>附加4％多聚甲醛（PFA）在PBS填充1毫升注射器油管和膨胀的肺部。 </li><li>拆下油管，并收紧手术线，​​以防止泄漏的煤灰。 </li><li>仔细解剖出5毫升4％PFA在50毫升猎鹰管和肺组织的地方。 </li></ol> 5。回收组织的制备<ol><li> 血清：以下存储在冰上30分钟到2Ĥ血液凝固，血清离心恢复从血液（13000 XG / 10分钟/ 4℃）。血清储存于-20°C，可以用于细胞因子酶联免疫吸附或 S 血吸虫卵抗原特异性IgG亚型酶联免疫吸附<sup&gt; 6，10。 </li><li> 淋巴结肿大：准备单细胞悬液，用于评估细胞计数的免疫反应的严重程度。检查Th2细胞极化，细胞重新刺激与 S 血吸虫虫卵抗原72小时。可以用流式细胞仪检测细胞内细胞因子染色，上清可以回收和ELISA Th2细胞因子（IL-4，IL-5，IL-13）6，储存在-20°C。 </li><li> 灌洗：细胞颗粒（400 XG / 5分钟/ 4℃）。 BAL液被回收并储存在-20°C用ELISA Th2细胞因子的分析。 BAL细胞计数作为气道炎症的读数。 10,000-100,000细胞（500转/ 5分钟/ 4℃）cytocentrifuge准备，随后Diffquik染色将使枚举炎症渗透​​4，6。此外，BAL细胞可以检查，流式细胞仪6。 </li><li> 肺组织肺组织是STOR版一夜之间在4°C。固定组织，石蜡包埋，切片，组织学和免疫荧光分析的幻灯片上。 </li></ol> 6。代表结果该协议的细节必要（i）拟定纯化血吸虫卵在体内使用（ 图1），（二）敏感和挑战，这些鸡蛋的小鼠及（iii）检查肺部炎症反应中恢复器官的所有步骤。这在体内模型再现驱动Th2细胞肺癌的一个强有力的炎症反应。这是由呼吸道炎症和嗜酸性粒细胞浸润灌洗细胞计数和cytocentrifuge准备（ 图2）可视化。可以检查鸡蛋引起的肉芽肿（ 图3a），H＆E -染肺段，肺切片免疫荧光染色允许或者激活的巨噬细胞和Th2细胞因子诱导的基因，如电阻的可视化锡类分子（RELM）α（ 图3b）。通过酶联免疫吸附的抗原特异性CD4 Th2细胞因子的响应研究IL-5，IL-4和IL-13的抗原刺激parathymic淋巴结细胞（ 图4）。 <img alt="图1" src="/files/ftp_upload/3905/3905fig1.jpg" /> 图1显示血吸虫卵注入模型代表图片肉芽肿肝脏特点高S.mansoni鸡蛋负担，和S.mansoni鸡蛋- 。平均尺寸（L）120（宽）50微米微米。 <img alt="图2" src="/files/ftp_upload/3905/3905fig2.jpg" /> 图2。S。血吸虫卵注射液驱动气道炎症。答：灌洗细胞数（X10 5）从幼稚（N）或S.mansoni（SM）鸡蛋注入小鼠体内。 * P &lt;0.05;二BAL细胞Cytocentrifuge的准备。 Mac上，巨噬细胞; EO，嗜酸性粒细胞，淋巴结，淋巴细胞。酒吧，10微米。 <img alt="图3" src="/files/ftp_upload/3905/3905fig3.jpg" /> 图3。肺肉芽肿或者激活巨噬细胞周围的S.mansoni的鸡蛋的可视化。答：在H＆E -染肺部分肺肉芽肿周围的SM蛋（黑色箭头）可视化。 B.免疫荧光染色RELMα（绿色），甘露糖受体（红色）和DAPI（蓝色），揭示了周围的肉芽肿钐萤光蛋（黑色箭头）或者激活巨噬细胞（白色箭头）。酒吧，50微米。 <img alt="图4" src="/files/ftp_upload/3905/3905fig4.jpg" /> 图4。S.mansoni虫卵抗原特定的Th2细胞因子的响应。排水parathymic淋巴节点从天真（N）或SM鸡蛋注入小鼠细胞， 钐刺激&lt;/ EM&gt; 72小时的虫卵抗原，IL-4，IL-5和IL-13上清酶联免疫吸附试验。规模，毫微克/毫升。* P &lt;0.05;

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis:+report+of+a+WHO+expert+committee.">Prevention and control of schistosomiasis and soil-transmitted helminthiasis: report of a WHO expert committee.</a> WHO Technical Report Series 912. , (2002).</li><li>Pearce, E. J., MacDonald, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunobiology+of+schistosomiasis.">The immunobiology of schistosomiasis.</a> Nat. Rev. Immunol. 2, 499-511 (2002).</li><li>Sandler, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+profiles+during+acute+pathogen-induced+pulmonary+inflammation+reveal+divergent+roles+for+Th1+and+th2+responses+in+tissue+repair.">Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and th2 responses in tissue repair.</a> J. Immunol. 171, 3655-3667 (2003).</li><li>Perrigoue, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-31-IL-31R+interactions+negatively+regulate+type+2+inflammation+in+the+lung.">IL-31-IL-31R interactions negatively regulate type 2 inflammation in the lung.</a> J. Exp. Med. 204, 481-487 (2007).</li><li>Sandor, M., Weinstock, J. V., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulomas+in+schistosome+and+mycobacterial+infections:+a+model+of+local+immune+responses.">Granulomas in schistosome and mycobacterial infections: a model of local immune responses.</a> Trends Immunol. 24, 44-52 (2003).</li><li>Nair, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatively+activated+macrophage-derived+RELM-{alpha}+is+a+negative+regulator+of+type+2+inflammation+in+the+lung.">Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung.</a> J. Exp. Med. 206, 937-952 (2009).</li><li>Dalton, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+schistosome+eggs+and+miracidia+free+of+contaminating+host+tissues.">A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.</a> Parasitology. 115, 29-32 (1997).</li><li>Lewis, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+laboratory+maintenance+of+Schistosoma+mansoni,+with+observations+on+three+schistosome/snail+host+combinations.">Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations.</a> J. Parasitol. 72, 813-829 (1986).</li><li>Tilney, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+lymphatic+drainage+in+the+adult+laboratory+rat.">Patterns of lymphatic drainage in the adult laboratory rat.</a> J. Anat. 109 (Pt. 3), 369-383 (1971).</li><li>Lewis, F., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Current protocols in immunology. Chapter 19, Unit 19 (2001).</li><li>Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th2+cells+are+required+for+the+Schistosoma+mansoni+egg-induced+granulomatous+response.">Th2 cells are required for the Schistosoma mansoni egg-induced granulomatous response.</a> J. Immunol. 160, 1850-1856 (1998).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrotic+disease+and+the+T(H)1/T(H)2+paradigm.">Fibrotic disease and the T(H)1/T(H)2 paradigm.</a> Nat. Rev. Immunol. 4, 583-594 (2004).</li><li>Cheever, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+hepatic+fibrosis+and+granuloma+size+among+mouse+strains+infected+with+Schistosoma+mansoni.">Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni.</a> Am. J. Trop. Med. Hyg. 37, 85-97 (1987).</li></ol>

没有利益冲突的声明。

在这里，介绍一个模型，采用血吸虫卵诱导2型肺部发炎。这种模式的典型特征包括强有力的Th2型免疫反应，气道炎症和肺肉芽肿形成。由于这些参数依赖CD4 Th2细胞的反应11，这是一个有用的模型，探讨Th2细胞反应的特定蛋白质或特定谱系缺失的影响。这里的读数将包括抗原特异性Th2细胞因子的量化分析，呼吸道细胞浸润，肺肉芽肿的大小。一个额外的参数，可衡量的是鸡蛋引起的?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 尺寸/描述 </td><td> 公司 </td><td> 产品编号 </td></tr><tr><td>胶原酶/ Dispase酶</td><td> 500毫克</td><td>罗氏应用科学部</td><td> 11 097 113 001 </td></tr><tr><td>变化快客赫马3染色</td><td>收拾</td><td>尔科技有限公司有限责任公司</td><td> 122-911 </td></tr><tr><td> DMEM培养液</td><td>液体</td><td> Gibco公司</td><td> 11965 </td></tr><tr><td>猎鹰50毫升</td><td>收拾</td><td>鹘</td><td> 352070 </td></tr><tr><td> intramedic聚乙烯管</td><td> 0.58毫米</td><td>碧迪公司</td><td> 427410 </td></tr><tr><td>异氟醚</td><td> 250毫升</td><td>住持动物健康</td><td> 52600405 </td></tr><tr><td>氯胺酮</td><td> 100毫克/毫升</td><td>道奇堡，动物健康</TD&gt; <td> N / A </td></tr><tr><td>甘露糖受体抗体</td><td>素</td><td> ABD Serotec公司</td><td> MCA2235B </td></tr><tr><td>金属过滤器</td><td>标准厨房过滤器</td><td>目标</td><td>劳伦蒂斯</td></tr><tr><td>多聚甲醛</td><td> 16％ </td><td>电子Microsc。科学</td><td> 15710 </td></tr><tr><td>青霉素/ </td><td> 100X </td><td> Gibco公司</td><td> 15070 </td></tr><tr><td> Percoll密度</td><td> 1公升</td><td> GE医疗生物科学</td><td> 17 0891 01 </td></tr><tr><td> RELMα抗体</td><td> 50微克</td><td> PeproTech公司</td><td> 500-P214 </td></tr><tr><td>手术主题</td><td> 2.0美国药典100坪</td><td>公司外科专业</td><td> SP118 </td></tr><tr><td>针头</td><td> 23克，3/4英寸</td><td> BD真空采血管实验室。 MED。 </td><td>305143 </td></tr><tr><td>美国标准试验筛</td><td> 150微米，0.0059“ </td><td> WS泰勒公司</td><td> ASTME-11，规格。第100号</td></tr><tr><td>甲苯噻嗪</td><td> 20毫克/毫升</td><td>男管家</td><td> N / A </td></tr></tbody></table>

using eggs from schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

血吸虫寄生虫血吸虫感染估计有200万人，全世界1。在血吸虫 ，严重的病理，包括肝纤维化和脾肿大，慢性感染引起的免疫反应，而不是寄生虫本身2寄生虫卵。寄生虫卵诱导Th2反应特点是生产的IL-4，IL-5和IL-13，替代激活的巨噬细胞和嗜酸性粒细胞的招聘。在这里，我们描述了一个模型来研究特定的寄生虫在Th2细胞因子反应和肺淋巴结，形成肺肉芽肿周围的蛋，呼吸道炎症注射血吸虫卵。 腹腔宣传和静脉的挑战， 与血吸虫卵被运送到肺，通过他们被困在肺实质的肺动脉由淋巴细胞，嗜酸性粒细胞和另外激活的巨噬细胞3-6组成的肉芽肿。与肉芽肿形成，在炎症的支气管肺泡空间关联的，扩大的淋巴结和CD4 T细胞活化可以观察到。这里我们详细介绍了协议隔离（从7修改）感染的肝血吸虫卵，敏感和具有挑战性的老鼠，和恢复分析机关（支气管肺泡灌洗（BAL），肺和淋巴结）。我们也有代表性的组织学和免疫学数据和其他免疫分析的建议。 总体而言，这种方法提供了在体内模型，以探讨在肺寄生虫引起的免疫反应，这是广泛适用于Th2细胞的炎症性疾病，包括寄生虫感染，纤维化疾病，过敏性炎症和哮喘的研究。这种模式的优势，为典型研究E 2的肺部发炎，包括一个强有力的Th2细胞在肺部的炎症反应和淋巴结的重复性，便于鸡蛋周围的肉芽肿病理检查炎症的评估，和潜在的长期存储的寄生虫鸡蛋。

Watch this Scientific Journal Video about Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation at JoVE.com

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

血吸虫卵是强有力的刺激T辅助2型（Th2细胞）的免疫反应，寄生虫感染的特点，哮喘和过敏性炎症。该协议利用S。血吸虫卵注射产生的CD4 Th2细胞因子诱导的炎症反应在肺，肺肉芽肿形成的特点，各地的鸡蛋，嗜酸性粒细胞和巨噬细胞替代激活。

从使用鸡蛋<em&gt;血吸虫</em&gt;作为<em&gt;在体内</em&gt;蠕虫引起的肺部炎症模型

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

using-eggs-from-schistosoma-mansoni-as-an-vivo-model-helminth-induced

Research

JoVE Journal

Immunology and Infection

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

13.7K 次观看.  University of Pennsylvania. 血吸虫卵是强有力的刺激T辅助2型（Th2细胞）的免疫反应，寄生虫感染的特点，哮喘和过敏性炎症。该协议利用S。血吸虫卵注射产生的CD4 Th2细胞因子诱导的炎症反应在肺，肺肉芽肿形成的特点，各地的鸡蛋，嗜酸性粒细胞和巨噬细胞替代激活。

视频: Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

激光内在人肺组织中的新型细菌特异探针的光学筛选

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

成年日本血吸虫细胞外囊泡的分离与鉴定

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...