The authors are funded by the Wellcome Trust through the Edinburgh Clinical Academic Track (C.J.C.J.) and Programme Grant (Y.H., and R.M.M.) and Fellowship (J.R.G) funding, the BBSRC (G.C.), the American Asthma Foundation (E.R., H.J.McS. and R.M.M.), Asthma UK (H.J.McS.) and the Rainin Foundation (D.J.S. and R.M.M.).

注:在本协议中的所有程序都是按照载列由英国内政部和爱丁堡兽医服务的大学指导进行。 1.感染小鼠灌胃<ol><li>商店Heligmosomoides polygyrus L3幼虫在蒸馏水中长达六个月，在4℃。 </li><li>在使用前，洗L3幼虫三次蒸馏水:离心机在300×g离心10分钟（与制动器），除去的水几乎500微升（以避免扰乱丸状L3幼虫）和每一次重悬沉淀。 </li><li>对于第三次洗涤，用200μl的尖端切割加水到一个确切的体积（通常为40毫升）和抽吸20微升，以扩大其孔径。放置两个20微升样品60毫米培养皿的表面上并计算幼虫的L3（通常是移动的，并且下50X放大率最佳效果与解剖显微镜）。悬浮在蒸馏水中最终沉淀到concentration个每毫升2000 L3幼虫。 </li><li>对于生命周期的生产，感染8周岁的F1（C57BL / 6xCBA）小鼠400 H. polygyrus L3幼虫在200μl蒸馏水中通过口服管饲法（由颈背抑制小鼠在直立位置并轻轻穿过口腔和食道的钝灌胃针进入胃）。彻底搅动之前，每个感染（幼虫尽快解决水）和吸200微升以1毫升注射器;使用专用的管饲针以一圆形端。对实验性感染年轻小鼠（6-8周龄），或其它近交系（ 例如 C57BL / 6或BALBC），感染小鼠200的L3幼虫。 </li></ol> 2.传播和H.保养polygyrus <ol><li>放置木炭在一个大的塑料桶的中心，并允许冷自来水以运行在它为至少30分钟（未洗的活性炭是有毒至L3幼虫）。从桶排出水，并放置在吨的炭WO层的吸水纸，离开它暴露在室内空气直至完全干燥。 </li><li>如果蛋是必需的，刮屎先用钳子结肠（和剪刀如有必要）。如果需要大量的L3幼虫，将小鼠上的线栅和持续数天收集粪便颗粒。 </li><li>混合与造粒木炭粪便以至少1:1的比例，以实现一致性刚好足够潮湿坚持滤纸。涂抹一薄层上有些挫伤滤纸的中心在培养皿并将其放置这在潮湿箱在黑暗中（加一些湿纸巾和水皿）为12-14天。 </li><li>从第7天起除去L3幼虫，并收集它们在至少两个场合纸张被丢弃之前。幼虫形成围绕滤纸的边缘环;提起滤纸出培养皿和冲洗被留在板的（用移液管和5ml每板用无菌水）到50ml管中的幼虫。 </li><li>电梯断滤纸和收获剩余幼虫残留在板上用蒸馏水至50ml管中。离心在300×g离心流出物溶液10分钟。三次，用蒸馏水洗涤幼虫，然后在4℃下在高达50毫升蒸馏水储存直到需要。 注:幼虫保持存活和感染性为至少6个月。 </li></ol> 3.收集成人的H. polygyrus蠕虫<ol><li>制备改性贝尔曼装置提前， 如图1。 </li><li>卡尔小鼠感染后十四日。 </li><li>洗用70％乙醇的腹部。切开皮肤在腹部和拉回来，露出前腹壁。使中线切口进入腹腔。 </li><li>删除整个小肠和大肠（从近端十二指肠远端直肠）。放入干燥的培养皿中。 </li><li>拉直沿其整个长度的肠;切除含粪便冒号;把这个成蛋的准备后，一个单独的菜。 </li><li>切除近端20厘米×小肠含有由于内部管腔蠕虫由比较厚的墙体十二指肠和经常红色外观的修正 - 重新鉴定成虫。放置成（直径100mm）的培养皿用5ml的Hanks&#39;溶液，加热到37℃（每皿2样品）。 </li><li>用剪刀（圆头剪刀是最适合这个）纵向打开蠕虫填充近端肠道部分，并且向下刮肠里面有两个载玻片衬删除蠕虫。然后丢弃干净的肠壁。 </li><li>尖端蠕虫成小棉布袋，钉封闭，并与周围的玻璃漏斗（ 图1）的边缘纸夹固定。 </li><li>填写漏斗与汉克斯的解决方案，并添加蠕虫大约4培养皿到每一个漏斗。 </li><li>在37℃培养箱地方设备1-2小时，通过轻轻搅拌中途赶走碎片从肠道准备，可能会堵塞细布过滤器。请注意，以避免棉布袋外碎片溢出 - 这将导致最终HES制剂的污染。成虫应该通过细棉布缓慢迁移和在玻璃试管的底部结算。仔细分离从连接橡胶软管在水槽的试管（注意避免在这一点上失去蠕虫）。 </li><li>使用塑料移液管，转移蠕虫到50ml管中并洗涤六次用Hanks&#39;溶液（允许蠕虫解决与重力，除去介质用stripette，添加40毫升汉克斯&#39;解并重复5次）。 注意:蠕虫的文化必须保持无菌从这时开始。 <ol><li>移动到层流罩和在无菌Hanks液补充有100U / ml青霉素和100μg/ ml链霉素，准备用于体外培养洗另一六次。 </li></ol></li><li>算通过采取两个样本，以扩大其光圈拍摄了黄色针尖削减20微升恢复成虫;期望接种幼虫的数量的约50％。 </li></ol> 4.设置了文化HES:中等准备，洗成虫<ol><li>泡从3.11蠕虫在大约10毫升的RPMI的补充有10％庆大霉素20分钟，而使管搁在一个角度，以确保蜗杆完全覆盖。 </li><li>执行此在一个层流罩:用Hanks&#39;溶液再次洗涤六次（补充有5 U / ml的青霉素和5微克/ ml链霉素）。 </li><li>准备H. polygrus媒体。 </li><li>保持无菌的层流罩，至500ml的RPMI1640，加11.1毫升45％葡萄糖（终浓度为1.2％的RPMI1640作为含有0.2％葡萄糖），将5ml 100×Peniclllin - 链霉素（终浓度5U / ml青霉素的， 5微克/ ml链霉素），将5ml L-谷氨酰胺（最终浓度为2mM），并5毫升庆大霉素（终浓度为1％）。不添加FCS。 </li><li>分装成蠕虫通风T25瓶，约1000蠕虫在15ml H。每瓶polygyrus介质，并置于直立在37℃培养箱（5％CO 2）3周。 </li></ol> 5.准备的HES <ol><li>收集HES-含有培养物的培养基中以不超过两次，每周较长的时间间隔，在培养24小时后，搁置所述第一集合（由于高含量的LPS污染及宿主蛋白质的 - 可单独处理或丢弃）。保持每个单独收集，并明确标有日期和批号。更换H.等体积polygyrus媒体每次。 </li><li>离心机HES-含有介质，在400×g下5分钟。然后过滤通过0.2微米的低蛋白结合过滤器消毒到50ml管中。储存在-20°C冰箱标明蠕虫收获日期HES的c和日期ollection。 </li><li>经过从文化21天HES收集，丢弃蠕虫。 </li><li>池500-1000毫升HES上清液（通常从冷冻的储存，并且不包括第一24小时收集），并在氮气压力下浓缩过的超滤装置中的3000 MWCO过滤器。 注意:要非常小心，不要让过滤器运行干燥。 <ol><li>设置的过滤器装置中，首先在1升的烧杯中，用蒸馏水向下洗3 kDa的膜光泽面为3×20分钟，同时搅拌。组装超滤装置按制造商的使用滤光膜光泽面向上的指令。发生在内阁在4℃，并开始集中汇集HES前一次的50毫升蒸馏水通过。 </li><li>加入HES的每个管进入过滤装置所要求（每天100-140毫升），直到体积浓缩至2-5毫升。 </li></ol></li><li>为了从含有HES培养基除去污染物，加50毫升吡的ogen无PBS的过滤装置和，然后浓缩降至大约2毫升重复此步骤两次（150毫升PBS中的总数）。转移HES入15ml管中，过滤消毒（用0.2微米过滤器）在层流罩和使用分光光度计（E 280 = 10）或通过Bradford测定法测定蛋白质浓度。 </li><li>等分试样，标记与在-80℃下批号和日期，并冷冻。 </li><li>根据制造商对每一个批次的HES的使用前的协议执行生色LAL测定。如果LPS水平高于每1微克蛋白1 U LPS，考虑不使用此批体内实验和体外培养。 </li><li>处理分别以相同的方式收集在24小时的HES;它将包含LPS和一些宿主蛋白质，虽然不适合功能实验，它是可以由单克隆抗体亲和纯化来分离单个分子的有用来源。 </li></ol&gt;

The authors have no conflicts of interest.

H的生命周期polygyrus收益的可靠一致的方式。下面自然摄入或第0天L3幼虫灌胃，囊肿开始形成下十二指肠浆膜第5天，发展到幼虫蜕皮，然后逐渐成为成虫为10天肠腔，鸡蛋可以从粪便中可以看出14天及肉芽肿从21天以上所述（并总结在图6中 ）的协议的十二指肠浆膜表面可见允许高通量生产的H. polygyrus排泄-分泌产物（HES），除了可行的L3幼虫可靠地恢复为未来的实验和生命周期的感染。 H. polygyrus感染已被证明是保护性哮喘依赖OVA或明镜第1页（屋尘螨变应原）14的模型。此外，抑制气道炎症可以转移与CD4 + CD25 + </suP&gt;调节性T细胞14或CD19 + CD23 喜调节性B细胞24非致敏，H. polygyrus感染的小鼠。在自身免疫，H.车型polygyrus感染已被证明是抑制在实验性自身免疫性脑脊髓炎（EAE）模型多发性硬化9和抑制可传送与任CD4 + T细胞或CD19 + B细胞从感染小鼠24。 Heligmosomoides polygyrus排泄-分泌产物（HES）调节宿主免疫应答和抑制Th2细胞介导的炎症通过许多机制（ 图7中所概述），包括:1）抑制树突细胞应答的刺激25，二）诱导的CD4 + Foxp3 +调节性T细胞18。和c）IL-33生产20封锁。 HES已被证明是保护性给药时一吨致敏或质疑哮喘19的OVA，明矾模型，并与交链孢提取物过敏原20滴鼻给药时，通过早期抑制IL-33的释放。 HES的LPS污染避税往往是未来的免疫实验成功的关键。在这里介绍的协议，在实现此的关键步骤是确保包含在贝尔曼设备不会进入最终的HES制剂的棉布袋碎屑（见2.10），并留出的ES溶液从培养的第24小时（见5.1）。 近年来，HES已经彻底表征的蛋白质水平，并确认了26,27超过370不同的蛋白质。此外，ES从第四阶段幼虫已进行蛋白质组学分析28。正在进行的工作包括HES特征的主要成分多糖，已知免疫原性强3，和分泌微RN作为被封装在50-100纳米囊泡或外来体（Buck 等 ，提交出版）。与建立可重复的协议为ES的集合从这个高度免疫调节寄生虫，并具有广泛的蛋白质组数据标识的HES的分子成分，所述阶段是现在用于鉴定和从H.单个分子的治疗性测试设置polygyrus可以介导宿主免疫系统的关键作用。

Heligmosomoides polygyrus是一种天然鼠胃肠寄生虫是密切相关的高度流行人类线虫寄生虫1。与其他线虫模型，如巴西日圆线虫，H. polygyrus始终建立慢性感染的小鼠作为多个强大的免疫调节机制的直接结果它采用抑制宿主的免疫应答2。 H. polygyrus有直接的生命周期:感染性幼虫L3由经办口传播摄入（或通过在实验室设置口服管饲法给药），于是它们到十二指肠和包囊的浆 ​​膜层迁移返回到肠腔如前成虫初次感染后大约八天。交配和产蛋量发生了10天，这是可能的收获成虫对从DA排泄 - 分泌产物培养和收集Ÿ14日起3。H. polygyrus也感染易感小鼠4-6，和H.水平增加相互作用与共生菌群，具有增加的乳杆菌存在polygyrus感染小鼠之后暴露于乳酸菌6。 活动性感染与H. polygyrus已经显示保护免受免疫病理学中自身免疫7-10的许多动物模型中，结肠炎11,12和过敏13-16。目前已因此一直在排泄分泌分子从这种寄生虫的潜力极大的兴趣（"HES"），在体内17,18向下调节的病理。实际上，保护作用被视为治疗后小鼠的HES制品19，通过它现在正在查明18,20通路。在这里，我们描述了一个协议的可靠的生产Heligmosomoides polygyrus的分泌其产品和恢复可进一步用于多种功能的生化和免疫学调查。

<table><tbody><tr><td>Amicon concentrator</td><td>Millipore</td><td>5112</td><td>Model 8050 50 ml&lt;br/&gt; http://www.emdmillipore.com/INTL/en/product/Stirred-Cell-Model-8050,-50%C2%A0mL,MM&lt;br/&gt; _NF-5122</td></tr><tr><td>Hanks&amp;rsquo; buffered salt solution</td><td>Sigma</td><td>H9394</td><td>500 ml&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/h9394?lang=en&amp;amp;region=GB</td></tr><tr><td>Penicillin streptomycin</td><td>Invitrogen</td><td>15070-063</td><td>100 ml&lt;br/&gt; http://www.lifetechnologies.com/order/catalog/product/15070063</td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>25030-081</td><td>200 MM 100ml&lt;br/&gt; https://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-25030081</td></tr><tr><td>Activated charcoal</td><td>Merck</td><td>1.09624.0500</td><td>18-35 mesh&lt;br/&gt; http://www.merckmillipore.com/GB/en/product/Charcoal-activated,MDA_CHEM-109624</td></tr><tr><td>Glucose solution</td><td>Sigma</td><td>G8769</td><td>100 ml 45% solution&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/g8769?lang=en&amp;amp;region=GB</td></tr><tr><td>Falcon tubes</td><td>Sarstedt</td><td>62.547.254</td><td>50 ml&lt;br/&gt; http://www.scribd.com/doc/124320105/Sarstedt-Falcon</td></tr><tr><td>T25 Flasks</td><td>Sigma</td><td>CLS430639&amp;nbsp;</td><td>25cm vented flask&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/cls430639?lang=en&amp;amp;region=GB</td></tr><tr><td>Chromogenic LAL assay</td><td>Lonza</td><td>50-647U</td><td>QCL-1000 assay&lt;br/&gt; http://www.lonza.com/products-services/pharma-biotech/endotoxin-detection/endotoxin-detection-assays/endpoint-chromogenic-lal-assay.aspx</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-049</td><td>100 ml&lt;br/&gt; http://www.lifetechnologies.com/order/catalog/product/15710072</td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640</td><td>Gibco</td><td>11875093</td><td>500 ml; without L-glutamine&lt;br/&gt; http://www.lifetechnologies.com/order/catalog/product/11875093</td></tr></tbody></table>

cultivation of heligmosomoides polygyrus: an immunomodulatory nematode parasite and its secreted products

Heligmosomoides polygyrus（原名Nematospiroides蝗，并且还通过一些作为H. bakeri简称）是一种胃肠道寄生虫，它采用多种免疫调节机制来建立慢性感染小鼠和近似于人类流行的蠕虫感染。H. polygyrus已被广泛研究中的寄生虫衍生的免疫调节领域，已经发现有力地抑制（都是具有活性的感染的和孤立的分泌产物）变态反应和自身免疫的实验模型。本文描述的协议，概述了H.管理polygyrus生命周期一致的生产L3幼虫，成虫寄生的恢复，收集他们的排泄分泌产品（HES）的。

对感染的易感性与H. polygyrus被控制在很大程度上由鼠标菌株（ 表1）的遗传背景; C57BL / 6小鼠CBA是高度敏感21,22。为了维护寄生虫生命周期的，这两个品系之间的杂交F1代已被选定为它能够承受更高的蠕虫负担无发病率（肠道中过多的上皮损伤）相比，无论是父母的压力。 400的L3幼虫口服管饲法被用来维持生命周期中的F1小鼠（导致图2中所示成虫负担），而在200的L3幼虫的剂量通常用于在纯合近交系的实验（ 例如 ，C57BL / 6或BALBC）。然而，这种剂量可能需要根据环境共同因素，可能动物设施，如变型中肠道菌群之间相差减小。 HES的批次已被证明可重复艾菲cacy在功能测定法和蛋白质组合物;而且，当从培养的每个连续一周的上清液进行分析，最多到总共4周，该蛋白分布被发现是非常相似的（ 图3），当 HES浓度通过Bradford测定法测定（见5.5），总蛋白质通常是约1毫克/毫升（ 图4）。 浓缩的HES的另一种方法是用离心浓缩器（如的Vivaspin 3-kDa截止的膜），在这种样品在一个开式转子离心机离心在高达4000克，除去缓冲的盐和低分子量组分。离心浓度是最适合于小处理量（1-10毫升）和被限制在约30倍的最大浓度的因素。 当收集的HES，避免污染是非常关键的。为了避免污染与主体分子，我们放弃HES-CONT从成虫收获从小鼠肠后的第一个24小时癌宁培养基。我们还定量的LPS污染水平每批HES的具有显色LAL测定（见5.7）。 LPS的1 U相当于〜100皮克LPS和低于这个水平被认为是可以忽略不计23。在我们的手中，HES大部分批次都显著低于这个限度，LPS的HES的平均浓度为0.23 U /微克（ 图5）。 LPS在体内模型病理的影响（如哮喘反应的抑制）至少需要10纳克LPS 23。因此， 在 5微克HES从一批次100皮克LPS /微克的HES 体内施用将包括500皮克脂多糖，远低于有效浓度，其中脂多糖成为问题。 <img alt="图1" src="/files/ftp_upload/52412/52412fig1highres.jpg" /> 图1:贝尔曼仪器贝尔曼仪设置为成人收藏的H. polygyrus（如在第2描述的）。 <img alt="图2" src="/files/ftp_upload/52412/52412fig2highres.jpg" /> 图2:感染400 L3幼虫后14天平均虫负担14天感染400 L3幼虫平均虫后的负担。显示的数据点是从19个独立轮C57BL / 6xCBA小鼠的感染。平均值±SEM表示。 <img alt="图3" src="/files/ftp_upload/52412/52412fig3highres.jpg" /> 图3:HES的蛋白图谱从收集在连续几周的文化HES蛋白质文化 SDS-PAGE曲线连续周 。 <img alt="图4" src="/files/ftp_upload/52412/52412fig4highres.jpg" /> 图4: </s仲&gt; HES从500毫升ES-含从约500ml培养上清液中得到11个不同批次的HES蛋白质的产量媒体最终数量 。平均值±SEM表示。 <img alt="图5" src="/files/ftp_upload/52412/52412fig5highres.jpg" /> 图5:LPS污染的HES的41批次的鲎ameobocyte法测定HES水平的LPS污染 。平均LPS浓度= 86üHES每毫克。 <img alt="图6" src="/files/ftp_upload/52412/52412fig6highres.jpg" /> 图6:H的动画示意图关键生命周期阶段，从L3幼虫灌胃，通过回收幼虫和成虫以HES隔离polygyrus生命周期摘要。 图7:HES其功能包含在HES可溶性介质和外来体的主要免疫调节作用的动画示意图 。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> 基因型（和背景株） </td><td> 原发感染表型 </td><td>参考</td></tr><tr><td></td><td></td><td></td></tr><tr><td>近交系</td><td></td><td></td></tr><tr><td>的A / J，CBA，C3H </td><td>高感</td><td> 22,29 </td></tr><tr><td>的C57BL / 6和C57BL / 10 </td><td>易感</td><td> 22,30 </td></tr><tr><td> BALB / c，DBA / 2，129 / J </td><td>中间</td><td> 22,31 </td></tr><tr><td>美国国立卫生研究院，SJL，SWR </TD&gt; <td>低易感性</td><td> 22,32 </td></tr><tr><td></td><td></td><td></td></tr><tr><td colspan="2"> 转基因的细胞因子或细胞因子受体 </td><td></td></tr><tr><td> IL-1β - / - </td><td>更容易</td><td> 33 </td></tr><tr><td rowspan="2"> IL-1R - / - </td><td rowspan="2">较不敏感（一）;在敏感性没有变化，但增加了肉芽肿（二） </td><td> （一）33 </td></tr><tr><td> （二）34 </td></tr><tr><td> IL-2Rβ转基因（C57BL / 6） </td><td>耐</td><td> 35 </td></tr><tr><td> IL-4 - / - </td><td>高繁殖力</td><td> 36 </td></tr><tr><td> IL-4Rα - / - （C57BL / 6或BALB / c小鼠） </td><td>高感</td><td> 22 </td></tr><tr><td>的IL-6 - / - （BALB / c或C57BL / 6） </td><td>高抗</td><td> 37 </td></tr><tr><td> IL-9的转基因（FVB） </td><td>耐</td><td> 38 </td></tr><tr><td> IL-21R - / - （C57BL / 6） </td><td> Th2细胞缺乏，降低肉芽肿gormation </td><td> 39,40 </td></tr><tr><td> IL-25 - / - （BALB / c小鼠） </td><td>更容易</td><td> 33 </td></tr><tr><td> IL-33R（T1 / ST2） - / - （BALB / c小鼠） </td><td>在敏感性无变化</td><td> 33 </td></tr><tr><td> TGFβRIIdn（C57BL / 6） </td><td>高Th1细胞，增加易感性</td><td> 41,42 </td></tr><tr><td></td><td></td><td></td></tr><tr><td colspan="2"> 转基因的T细胞标记物 </td><td></td></tr><tr><td> CD28 - / - （BALB / c小鼠） </td><td>小幅高繁殖力</td><td> 43 </td></tr><tr><td> CD86（B7-2） - / - （BALB / c小鼠） </td><td>高繁殖力</td><td> 44 </td></tr><tr><td> OX40L - / - （BALB / c小鼠） </td><td>高繁殖力和＃160; </td><td> 45 </td></tr><tr><td></td><td></td><td></td></tr><tr><td colspan="2"> 转基因先天免疫基因位点 </td><td></td></tr><tr><td> 1型干扰素受体（IFNAR） - / - （C57BL / 6） </td><td>高繁殖力，更肉芽肿</td><td> 34 </td></tr><tr><td>的MyD88 - / - （C57BL / 6） </td><td>更耐磨，更肉芽肿</td><td> 34 </td></tr><tr><td> C-KitW / Wv的（WBB6） </td><td>更容易</td><td> 46,47 </td></tr></tbody></table> 表1:H.主要感染polygyrus在遗传上不同的和基因针对性小鼠品系。

Watch this Scientific Journal Video about Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products at JoVE.com

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus是鼠类线虫具有强大的免疫调节功能，非常类似于那些高度流行的人类寄生虫感染。在这里，我们描述了一个协议，用于长期维持H的polygyrus生命周期。

种植<em&gt; Heligmosomoides Polygyrus:</em&gt;免疫调节线虫寄生虫及其分泌产物

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

cultivation-heligmosomoides-polygyrus-an-immunomodulatory-nematode

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

18.3K 次观看.  University of Edinburgh.  Heligmosomoides polygyrus是鼠类线虫具有强大的免疫调节功能，非常类似于那些高度流行的人类寄生虫感染。在这里，我们描述了一个协议，用于长期维持H的polygyrus生命周期。 

视频: Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

从小鼠的补丁中提取体液免疫的关键参与者: 先进和优化的淋巴细胞分离协议

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

科研

癌症研究

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

免疫与感染

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

人性化的 nod/scid/il2γ空 (hu-nsg) 小鼠 hiv 复制和延迟研究模型

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

在 Vivo 增强的古特-霍姆调节 T 细胞诱导

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

种植

摘要

探索更多视频

此视频中的章节

从小鼠的补丁中提取体液免疫的关键参与者: 先进和优化的淋巴细胞分离协议

新工具展开调节性T细胞的HIV-1感染的个体

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

人性化的 nod/scid/il2γ空 (hu-nsg) 小鼠 hiv 复制和延迟研究模型

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

在 Vivo 增强的古特-霍姆调节 T 细胞诱导

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

种植

摘要

探索更多视频

此视频中的章节

从小鼠的补丁中提取体液免疫的关键参与者: 先进和优化的淋巴细胞分离协议

新工具展开调节性T细胞的HIV-1感染的个体

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

人性化的 nod/scid/il2γ空 (hu-nsg) 小鼠 hiv 复制和延迟研究模型

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

在 Vivo 增强的古特-霍姆调节 T 细胞诱导

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响