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 border="0" cellpadding="0" cellspacing="0" width="1183">
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			<td height="22" style="height:22px;width:209px;">Material Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:143px;">Catalogue Number</td>
			<td style="width:157px;">Comments</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">Amicon concentrator</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:143px;">5112</td>
			<td style="width:157px;">Model 8050 50 ml 
			http://www.emdmillipore.com/INTL/en/product/Stirred-Cell-Model-8050,-50%C2%A0mL,MM 
			_NF-5122</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Hanks&#8217; buffered salt solution</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;">H9394</td>
			<td style="width:157px;">500 ml 
			http://www.sigmaaldrich.com/catalog/product/sigma/h9394?lang=en&#38;region=GB</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Penicillin streptomycin</td>
			<td style="width:83px;">Invitrogen</td>
			<td style="width:143px;">15070-063</td>
			<td style="width:157px;">100 ml 
			http://www.lifetechnologies.com/order/catalog/product/15070063</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">L-glutamine</td>
			<td style="width:83px;">Invitrogen</td>
			<td style="width:143px;">25030-081</td>
			<td style="width:157px;">200 MM 100ml 
			https://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-25030081</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">Activated charcoal</td>
			<td style="width:83px;">Merck</td>
			<td style="width:143px;">1.09624.0500</td>
			<td style="width:157px;">18-35 mesh 
			http://www.merckmillipore.com/GB/en/product/Charcoal-activated,MDA_CHEM-109624</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Glucose solution</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;">G8769</td>
			<td style="width:157px;">100 ml 45% solution 
			http://www.sigmaaldrich.com/catalog/product/sigma/g8769?lang=en&#38;region=GB</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Falcon tubes</td>
			<td style="width:83px;">Sarstedt</td>
			<td style="width:143px;">62.547.254</td>
			<td style="width:157px;">50 ml 
			http://www.scribd.com/doc/124320105/Sarstedt-Falcon</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">T25 Flasks</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;">CLS430639&#160;</td>
			<td style="width:157px;">25cm vented flask 
			http://www.sigmaaldrich.com/catalog/product/sigma/cls430639?lang=en&#38;region=GB</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">Chromogenic LAL assay</td>
			<td style="width:83px;">Lonza</td>
			<td style="width:143px;">50-647U</td>
			<td style="width:157px;">QCL-1000 assay 
			http://www.lonza.com/products-services/pharma-biotech/endotoxin-detection/endotoxin-detection-assays/endpoint-chromogenic-lal-assay.aspx</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Gentamicin</td>
			<td style="width:83px;">Gibco</td>
			<td style="width:143px;">15710-049</td>
			<td style="width:157px;">100 ml 
			http://www.lifetechnologies.com/order/catalog/product/15710072</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">Roswell Park Memorial Institute (RPMI) 1640</td>
			<td style="width:83px;">Gibco</td>
			<td align="right">11875093</td>
			<td style="width:157px;">500 ml; without L-glutamine 
			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

18.0K 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

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

科研

免疫与感染

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

人骨髓间充质干细胞（干细胞）的免疫调节性能评估

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

通过“Acufection”和它的皮肤研究中的应用的经济型DNA递送系统的开发

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

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 ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

针对天然产物的单克隆抗体的产生

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

在伊蚊中传播微孢子寄生虫Edhazardia aedis

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...