Name: cultivation of heligmosomoides polygyrus: an immunomodulatory nematode parasite and its secreted products
Uploaded: 2015-04-06T13:00:00.000+00:00
Duration: 12 min 40 s
Description: Watch this Scientific Journal Video about Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products at JoVE.com

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

Research

JoVE Journal

Immunology and Infection

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

18.2K 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

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

鞭虫感染muris：在肠道内的2型免疫和炎症模型

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

科研

免疫与感染

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont&#8211;free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock&#8217;s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media.

在体内和昆虫病原线虫体外培育（ 斯氏和异小 ）

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

生物工程

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

生物学

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

通过显微注射在类 圆线虫 物种中产生转基因和敲除

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

秀 丽隐杆线虫 感染模型中的遗传免疫研究

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

培养和遗传操纵昆虫致病线虫

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

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 ...

NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection

Parasites generally have a negative effect on the health of their host. They represent a huge health burden, as they globally affect the health of the infested human or animal in the long term and, thus, impact agricultural and socio-economic outcomes. However, parasite-driven immune-regulatory effects have been described, with potential therapeutic relevance for autoimmune diseases. While the metabolism in both the host and parasites contributes to their defense and is the basis for nematode survival in the intestine, it has remained largely understudied due to a lack of adequate technologies. We have developed and applied NAD(P)H fluorescence lifetime imaging to explanted murine intestinal tissue during infection with the natural nematode Heligmosomoides polygyrus to study the metabolic processes in both the host and parasites in a spatially resolved manner. The exploitation of the fluorescence lifetime of the co-enzymes nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), hereafter NAD(P)H, which are preserved across species, depends on their binding status and the binding site on the enzymes catalyzing metabolic processes. Focusing on the most abundantly expressed NAD(P)H-dependent enzymes, the metabolic pathways associated with anaerobic glycolysis, oxidative phosphorylation/aerobic glycolysis, and NOX-based oxidative burst, as a major defense mechanism, were distinguished, and the metabolic crosstalk between the host and parasite during infection was characterized.

NADPH Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection

The present protocol describes the NAD(P)H fluorescence lifetime imaging of an explanted murine intestine infected with the natural parasite Heligmosomoides polygyrus, which allows one to investigate metabolic processes both in host and parasite tissues in a spatially resolved manner.

Parasites generally have a negative effect on the health of their host. They represent a huge health burden, as they globally affect the health of the ...

<meta charset="utf8"></head><body>研究实验室小鼠的 Strongyloid ratti 感染

Experimental Infection of Mice with the Parasitic Nematode Strongyloides ratti

Strongyloides ratti is a parasitic nematode that naturally infects wild rats. However, most laboratory rat and mouse strains are fully susceptible to infection. Immunocompetent BALB/c and C57BL/6 mice terminate S. ratti infections within a month in the context of a canonical type 2 immune response and&#160;remain semi-resistant to a re-infection. The course of infection can be divided into three phases: (a) the tissue migration phase of the infective third-stage larvae during the first two days; (b) the early intestinal phase, including the molting to the adult parasites and embedding in the mucosa of the intestine in days 3 to 6 post-infection with reproduction starting by day 5 to 6 post-infection; (c) the later intestinal phase ending with the complete clearance of the parasites. Experimental infections of mice with S. ratti enable the precise study of host-parasite interactions throughout the whole life cycle at the different sites of infection, as well as immune evasion strategies employed by the parasite. The protocol presented here describes the maintenance of the parasite in Wistar rats, the infection of laboratory mice, and the detection and quantification of S. ratti parasites in the tissue migrating phase and during the intestinal phase.

<meta charset="utf8"></head><body>作者聚焦： Strongyloides ratti 感染中的性别依赖性肠道免疫和位点特异性免疫反应

Strongyloides ratti is a parasitic nematode that causes transient infections in laboratory mice, displaying tissue-migrating and intestinal life stages. Here, we present a protocol for the maintenance of the parasite cycle in rats and experimental infection of mice, including parasite quantification in the head, lung, and intestine.

小鼠用寄生线虫 Strongyloides ratti 感染的实验性

Strongyloides ratti is a parasitic nematode that naturally infects wild rats. However, most laboratory rat and mouse strains are fully susceptible to ...

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鞭虫感染muris：在肠道内的2型免疫和炎症模型

在体内和昆虫病原线虫体外培育（斯氏和异小）

使用荧光显微镜可视化中的细菌线虫

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

通过显微注射在类圆线虫物种中产生转基因和敲除

秀丽隐杆线虫感染模型中的遗传免疫研究

培养和遗传操纵昆虫致病线虫

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection

小鼠用寄生线虫 Strongyloides ratti 感染的实验性

种植

摘要

探索更多视频

此视频中的章节

鞭虫感染muris：在肠道内的2型免疫和炎症模型

在体内和昆虫病原线虫体外培育（ 斯氏和异小 ）

使用荧光显微镜可视化中的细菌线虫

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

通过显微注射在类 圆线虫 物种中产生转基因和敲除

秀 丽隐杆线虫 感染模型中的遗传免疫研究

培养和遗传操纵昆虫致病线虫

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection

小鼠用寄生线虫 Strongyloides ratti 感染的实验性

在体内和昆虫病原线虫体外培育（斯氏和异小）

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

通过显微注射在类圆线虫物种中产生转基因和敲除

秀丽隐杆线虫感染模型中的遗传免疫研究