这个项目是由美国凯斯西储大学启动资金提供给大肠杆菌乔利。感染性钉螺，血吸虫资源中心，国立卫生研究院NIAID的（NIAID的合同号HHSN272201000009I）。我们也感谢克里斯国王和罗纳德布兰顿确保感染血吸虫钉螺的支持和有益的讨论。

安全措施： 血吸虫尾蚴可直接穿透皮肤没有削减或毛孔需要，造成慢性疾病血吸虫病。S。血吸虫是一个生物安全2级的有机体，因此，适当的个人防护设备和感染尾蚴的工作时，必须遵循适当的安全措施。大多数必要的设备，成长和改造血吸虫是标准的，表中所指出的，该协议的结束与几个关键例外。所有的cercarial工作应该做在一个孤立的房间。 Schistosomules应培养在无菌的，经批准的生物安全罩。应佩戴两层腈或乳胶手套，实验室外套，一双防水的鞋子，裤子没有洞或泪水，并为您的手腕和手臂防水袖保护。在一个偶然泄漏的情况下，立即喷用70％乙醇或10％漂白粉的受污染的地区，揉大力如果你的人。这将是足以禁用的寄生虫，但所有可能感染的，应当报和重视。虽然schistosomules是不是天生感染，机构准则各不相同，应征询，以确定是否BSL2的预防措施，在非感染性的人生阶段所需的。来与任何潜在的感染性物质接触后，实验是在经批准的生物危害容器中进行丢弃废物的所有表面消毒。 注：盖蜗牛主机箱1〜2天前脱落和保护由轻 1。收获尾蚴从蜗牛主机<ol><li>填写一个干净，洗涤剂中途平衡至室温，用蒸馏水500ml烧杯。如果在不同的日期与毛蚴感染主机蜗牛的正常工作，使用感染日期为每一个单独的烧杯中（如果蜗牛将会为未来的实验中重用，这一点尤为重要）。烧杯的大小也可根据用户体验。 </li><li>收集你想摆脱仔细存款，他们使用一个标准的观赏鱼的网和通用钳需要烧杯中的受感染的蜗牛主机。 </li><li>泄漏应急放在烧杯托盘白炽灯光源下约8至12英寸。让我们站在为1-2小时，在灯光下。尾蚴离开蜗牛主机，当暴露在明亮的光线。 </li><li>仔细筛选出从尾蚴混合物蜗牛。采取额外的安全防范措施，作为混合物具有高度传染性。记住，尾蚴可直接穿透人体的皮肤！ </li><li>更换光源下约15分钟的混合物，使任何微粒废物问题解决。 </li><li>转移到一个干净的锥形烧瓶中，用吸管的尾蚴，避免在烧杯底部的碎片。烧瓶放置在约45 ° 45在黑暗中的冰角 - 60分钟，允许尾蚴融入一个角落。 </li><li>使用50 mL的吸管，去除75％至90％的上清液和存款中含10％漂白粉或70％乙醇的烧杯中，小心不要让水滴含有尾蚴从吸管滴。 </li><li>摇动三角瓶中含有尾蚴轻轻悬浮在溶液中的尾蚴。使用吸管样品转移到无菌50 ml锥形管。放在冰管，再次为在黑暗中20分钟。尾蚴将解决对试管底部，在黑暗中。 </li></ol> 2。手册转型的尾蚴schistosomules 开始之前：RPMI 1640完全“（5％胎牛血清的RPMI，1X笔/链球菌）和温暖至37 ° C。 （这是至关重要的，已孵化灭活胎牛血清使用56℃，1小时可以作为血吸虫敏感的补充）。 <ol><li>在生物危害罩，取出上清下降到5至10毫升每50 ml锥形解决方案，使用移液器或真空设备。 </li><li> 22计双端连接，露儿乐乳化针，以适当大小的注射器。 </li><li>慢慢地拉入注射器的混合物。收集来自所有50毫升注射器管的尾蚴。滴水周到。 </li><li>小心地翻过来，这样，独立的针尾注射器。将另一注射器乳化针的这一边。 </li><li>穿过针的混合物的40倍（20倍来回），改造尾蚴。 </li><li>位置垂直等混合物包含在底部注射器和顶部的注射器是空的注射器。卸下顶部的注射器和70％乙醇消毒。 </li><li>存款下降混合物放入一个干净的高壁百毫米x 50毫米耐热盘或结晶盘。 70％乙醇消毒针头和注射器。 </li><li>添加37℃的RPMI 1640完整的媒体，如培养皿的底部完全覆盖，仿佛一个卢里亚肉汤板浇筑。 </li><li>涡流轻轻收集在培养皿的中心schistosomules。避免飞溅。 注意：它可能是有用的这一步关闭罩灯，以更充分的可视化中心schistosomules的。 </li><li>立即存入一个新的12孔细胞培养板使用无菌滴管集中schistosomules。 </li><li>直到schistosomules菜（约10倍）的中心不再重复2.9和2.10。定金随后每一个新鲜以及的集合。 </li><li>填写半途而废（〜1.5毫升）预热的RPMI完整的媒体。 </li><li>倒置显微镜，可视化，以验证schistosomules和尾蚴的情况下是否存在。 </li><li>成长中的CO 2培养箱schistosomules设置为37 ° C和5％的CO 2。 </li></ol> 3。代表性的成果： 待转让schistosomules转化成文化传媒，下一个倒置的明场显微镜（见设备表为例）可​​视化应该产生一个类似于图1，其中只有转化schistosomules以及在目前的图像。潜在的污染物可能包括完整的尾蚴，切断cercarial尾巴，或其他杂物。如果没有倒置显微镜，一个标准的显微镜，甚至清扫范围，将有足够的schistosomules可视化。注意，此过程并不总是变换尾蚴的100％。 <img src="/files/ftp_upload/3191/3191fig1.jpg" alt="图1"/> 图1：转化Schistosomulum <img src="/files/ftp_upload/3191/3191fig2.jpg" alt="图2"/> 图2：尾蚴和痛苦的尾巴

<ol>
	<li>Ross, A. G., Bartley, P. B., Sleigh, A. C., Olds, G. R., Li, Y., Williams, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> N Engl J Med. 346, 1212-1220 (2002).</li><li>Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis+and+water+resources+development:+systematic+review,+meta-analysis,+and+estimates+of+people+at+risk.">Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk.</a> Lancet Infect Dis. 6, 411-425 (2006).</li><li>Savioli, L., Albonico, M., Engels, D., Montresor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis.">Progress in the prevention and control of schistosomiasis and soil-transmitted helminthiasis.</a> Parasitol Int. 53, 103-113 (2004).</li><li>King, C. H., Dickman, K., Tisch, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reassessment+of+the+cost+of+chronic+helmintic+infection:+a+meta-analysis+of+disability-related+outcomes+in+endemic+schistosomiasis.">Reassessment of the cost of chronic helmintic infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis.</a> Lancet. 365, 1561-159 (2005).</li><li>Haas, W., Grabe, K., Geis, C., Pach, T., Stoll, K., Fuchs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+and+invasion+of+human+skin+by+Schistosoma+mansoni+cercariae:+the+key-role+of+L-arginine.+Parasitology.">Recognition and invasion of human skin by Schistosoma mansoni cercariae: the key-role of L-arginine. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+cercarial+responses+to+irradiance+changes.">Schistosoma mansoni: cercarial responses to irradiance changes.</a> J Parasitol. 68, 120-124 (1982).</li><li>Chai, M., McManus, D. P., McInnes, R., Moertel, L., Tran, M., Loukas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+lung+schistosomula,+in+vitro+cultured+schistosomula+and+adult+Schistosoma+japonicum.">Transcriptome profiling of lung schistosomula, in vitro cultured schistosomula and adult Schistosoma japonicum.</a> Cell Mol Life Sci. 63, 919-929 (2006).</li><li>Gobert, G. N., Tran, M. H., Moertel, L., Mulvenna, J., Jones, M. K., McManus, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+changes+in+Schistosoma+mansoni+during+early+schistosomula+development+and+in+the+presence+of+erythrocytes.">Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes.</a> PLoS Negl Trop Dis. 4, (2011).</li><li>Basch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+Schistosoma+mansoni+in+vitro.+I.+Establishment+of+cultures+from+cercariae+and+development+until+pairing.">Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing.</a> J Parasitol. 67, 179-185 (1981).</li><li>Yoshino, T. P. &. a. m. p. ;. a. m. p., Laursen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Schistosoma+mansoni+daughter+sporocysts+from+mother+sporocysts+maintained+in+synxenic+culture+with+Biomphalaria+glabrata+embryonic+(Bge)+cells.">Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells.</a> J Parasitol. 81, 714-722 (1995).</li><li>Colley, D. G., Wikel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+simplified+method+for+the+production+of+schistosomules.">Schistosoma mansoni: simplified method for the production of schistosomules.</a> Exp Parasitol. 35, 44-51 (1974).</li><li>Lazdins, J. K., Stein, M. J., David, J. R., Sher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+rapid+isolation+and+purification+of+schistosomula+of+different+developmental+stages+by+centrifugation+on+discontinuous+density+gradients+of+Percoll.">Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll.</a> Exp Parasitol. 53, 39-44 (1982).</li><li>Cousin, C. E., Stirewalt, M. A., Dorsey, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+ultrastructure+of+early+transformation+of+skin-+and+shear-pressure-derived+schistosomules.">Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules.</a> Exp Parasitol. 51, 341-365 (1981).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. Chapter 19, 1-1 (2001).</li></ol>

没有利益冲突的声明。

在改造过程中可能出现的几个潜在的问题;然而，这个协议是为了避免这些问题大部分。一个潜在的危险是乳化针，这通常会出现由于在步骤1.3中的收获遗留下来的碎片堵塞。一定要允许所有的颗粒物质，解决在步骤1.5和转让步骤1.6尾蚴仔细，以避免这个问题。此外，如前所述，内生物安全柜工作，个人防护装备的使用（甚至面罩），避免过度的压力注射器最好。 分离cercarial尾巴可以在媒体上，这是贫困分离在步骤2.9可视化。尾巴表现出痉挛般的议案，并有不同的形状（见图2），而正是如此很容易分辨的schistosomules。可去除清洗多余的RPMI schistosomules完成schistosomules解决的尾巴。尾巴将保持浮动，并可以清理。 如果完整的尾蚴是目前在媒体上，在改造步骤2.5无法被正确执行。一个完整的尾蚴的形象描绘在图3b中，以供参考。如果发生此问题，确保正在使用的乳化针是适当的大小。此外，可以增加cercarial混合物是通过针通过倍，40倍，虽然通常比足够接近100％剪切cercarial尾巴。 尾蚴转化成schistosomules科利和Wikel（13）于1974年首次描述。从那时起，schistosomules准备已经完成在本议定书中，或使用一个振荡的方法和percoll梯度分离（14，15）或纷飞在这纸证明，使用针头和注射器剪切战略。这两种技术准备schistosomules精美的弗雷德刘易斯（16）和进一步培养长期增长schistosomules的信息，可在NIAID的血吸虫资源<a href="http://www.schisto-resource.org/">中心（</a> www.schisto resource.org ）。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> 22克x 2-7/8“乳化针</td><td> Fisher Scientific则</td><td> 14 - 825 - 17G </td></tr><tr><td> RPMI 1640培养液1X（500毫升） </td><td> Invitrogen公司</td><td> 11835030 </td></tr><tr><td>胎牛血清（FBS），热灭活</td><td> Invitrogen公司</td><td> 10082139 </td></tr><tr><td>青霉素/链霉素（100X） </td><td> Invitrogen公司</td><td> 15070063 </td></tr><tr><td>结晶菜（100毫米） </td><td> Fisher Scientific则</td><td> 08 - 741D </td></tr><tr><td>通用镊子</td><td> Fisher Scientific则</td><td> 10 - 316C </td></tr><tr><td> PETCO水族馆净盐水虾</td><td> PETCO </td><td>商品编号：1190954 </td></tr></tbody></table>

cercarial transformation and in vitro cultivation of schistosoma mansoni schistosomules

血吸虫寄生虫病，血吸虫病，长期衰弱的疾病，影响全球超过200万人次，跻身在公众健康和社会经济的影响（1-4）方面寄生虫病的第二个疟疾的病原体。血吸虫寄生虫吸虫蠕虫，软体动物和哺乳动物的主机之间的寄生生活，一个复杂的生命周期转乘干预自由游动阶段。简言之，自由游动的尾蚴感染哺乳动物的主机，通过穿透皮肤分泌的蛋白酶的援助，在这段时间内尾蚴失去它们的尾巴，schistosomules改造。现在schistosomules必须逃避宿主的免疫系统，开发出红血细胞的消化肠道，并迁移虽然肺部，他们在肝门系统的最终目的地途中门静脉循环和最终肠系膜静脉（曼氏血吸虫）其中男性和女性对蠕虫和队友，每天生产数百个鸡蛋。有些是从体内排出的鸡蛋，鸡蛋孵化成自由游动的毛蚴（5-10）到淡水。毛蚴感染特定的蜗牛种，并转化为母亲和女儿sporocysts，这反过来，产生感染尾蚴，完成生命周期。不幸的是，整个生命周期血吸虫不能在体外培养，但可以转化成schistosomules感染尾蚴，schistosomules可周的培养，在体外或微阵列分析分析血吸虫发展。在这个协议中，我们提供了一个可视化描述cercarial改造， 并在体外培养的schistosomules 。我们从蜗牛主机Biomphalaria光滑棚感染尾蚴和手动变换成schistosomules使用乳化针双端分离它们​​的尾巴。 体外 cercarial的改造和schistosomules养殖技术的描述，避免使用哺乳动物的主机，从而简化了血吸虫的可视化和便于实验分析收集的寄生虫。体外转化和血吸虫培养技术多年来一直做的（11， 12），但没有视觉的协议已经开发了整个社会。

Watch this Scientific Journal Video about Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules at JoVE.com

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

我们描述了一个<em&gt;在体外</em&gt;扁虫的寄生虫血吸虫童虫培养方法<em&gt;曼氏血吸虫</em&gt;，通过收获和改造感染尾蚴的淡水蜗牛中间宿主Biomphalaria光滑。

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

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

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Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

17.9K 次观看.  Case Western Reserve University. 我们描述了一个在体外扁虫的寄生虫血吸虫童虫培养方法曼氏血吸虫，通过收获和改造感染尾蚴的淡水蜗牛中间宿主Biomphalaria光滑。

视频: Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

科研

免疫与感染

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

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

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

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

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

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

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

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

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

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

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

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 ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

体外方法比较标的结合和CDC诱导治疗性抗体之间：应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

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

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

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

成年日本血吸虫细胞外囊泡的分离与鉴定

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

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

内生菌与人类肠道微生物群使用体外浴发酵系统的相互作用分析

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

具有体外转录mRNA的初级巨噬细胞的高效转染

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...