1。 Endoparasite考试（见表1） 1。肛周胶带测试（见第5节;这些通常是在同一时间执行） <ol><li>从自动售货机中取出一个明确的长度，不磨砂，透明胶带。胶带应足够长的时间来处理，而不触及中间（约5厘米）一端。它可能会更容易免除一次几个长度，连接到一个干净的工作表面的边缘，并使用需要。 </li><li>电梯从一只老鼠笼和笼盖的地方，它的尾巴。层流柜或生物安全柜中，如果​​动物的健康状况要求它执行此操作。 </li><li>抑制鼠标的尾巴，从笼子里解除它的后腿。抓住拇指和食指之间的磁带结束，然后应用磁带中坚决鼠标会阴，包括肛周面积几次。头发，应看作是附着到磁带的检测，被认为是成功的。 </li><li>将在笼子里的鼠标背部。 </li><li>放在一个标记洁净的载玻片一滴矿物油，适用于磁带的幻灯片，然后又一滴矿物油。盖上玻璃盖滑。 </li><li>阅读显微镜幻灯片，使用光学显微镜上的10倍和40倍的目标。肛周胶带测试是最好的检测Syphacia鸡蛋 ，虽然有时会发现其他寄生虫卵。 </li></ol> 2。粪便浮选<ol><li>组装浮选解决方案，平底瓶（丸小瓶或Ovatector如粪便浮选设备），培养皿，盖玻片，显微镜幻灯片，并撒施/搅拌棒。浮选解决方案，如Fecasol，应该有一个比重1.20-1.30具体，可制成各种钠盐，糖，硫酸锌，或购买商业。 （见表2） </li><li>收集到的浮选室2-5从笼子里的粪粒或新鲜的动物（S）。如果粪便是极其干燥的，无论是由于年龄或物种产生的排泄物，润燥用0.9％生理盐水500μL的粪便可能是有益的。 </li><li>放置在培养皿中的小瓶保护工作表面溶解粪便溢出。添加浮选剂和糊状物的体积小，彻底搅拌。没有大块的材料应维持不变。继续添加到小瓶的边缘上方的半月板形式介质浮选。 </li><li>将盖玻片上的半月板，在室温下孵育15分钟。寄生虫卵和一些原虫的卵囊将上升到顶部，坚持以盖玻片。 </li><li>孵育后，升降机及反转盖玻片。将盖玻片上的玻璃显微镜幻灯片。 </li><li>检查的幻灯片，使用光学显微镜下的10倍和40倍的目标。 </li><li>虽然技术含量低，易于操作，一般来说，这种技术是不建议对小鼠或大鼠。作为一项规则，它是更合适的动物粪便产生量较大的。 </li></ol> 3。粪便中的浓度和离心<ol><li>组装浮选解决方案，离心管中，盖玻片，显微镜幻灯片，和撒施/搅拌棒和管帽。浮选解决方案应该有一个比重1.18-1.30具体，可制成各种钠盐，糖，硫酸锌，或购买商业。 （见表2） </li><li>一个收集管收集到2-10从笼子里的粪便颗粒或新鲜的动物（S）。如果粪便极为干燥，无论是由于年龄或物种产生的排泄物，粪便润燥500微升0.9％生理盐水或用浮选溶液您使用的可能是有益的的。 </li><li>玻璃离心管内混合在一个合适的浮选解决方案样品。可用于与vortexer机械搅拌混合样品。如果使用一个vortexer，弹簧帽应放在管的顶部，以防止溢出和交叉污染。常规样品准备在1.18比重硫酸锌，但是其他的解决方案可用于除了在一个单独的准备管或替代。 </li><li>每管添加额外的浮选解决方案，以形成积极在每个管有轻微的半月板。应用塑料盖玻片，每管，确保管唇接触。放置管（S）进入离心机。 </li><li>在约616-760离心力离心10分钟。一个新的盖玻片，如果盖玻片在离心过程中丢失或破，可放在样品管和管可以轻轻放倒，使半月板接触到新的盖玻片。没有额外的离心是必要的。 </li><li>删除一个标记，干净的玻璃显微镜玻片上覆盖离心管中的支路和地点。如果有多个离心解决方案被用来评价一个粪便样本，两个盖玻片可能被放在同一张幻灯片上。 </li><li>与碘染色的幻灯片。这使得电子asier鉴定囊肿。 </li><li>检查幻灯片，使用光学显微镜上的10倍和40倍的目标。 </li></ol> 4。直接检查肠道寄生虫和原生动物<ol><li>将安乐死鼠标在背斜卧在一个干净的夹层板或类似的工作表面或大鼠胴体。 </li><li>使用镊子，电梯在生殖器部位的腹壁。用剪刀，小心地切开腹腹壁从生殖器区域除去皮肤和肌肉和肋骨露出的肠子基地。取出肠子，开始在十二指肠（小肠开始在胃的出口段）和继续降结肠（段结束在肛门和通常包含形成粪便的肠道）。 </li><li>在100毫升的培养皿放置的肠子。收集从安乐死的动物和解剖板盲肠和十二指肠的一部分。段纵向切割每个肠道黏膜暴露。 </li><li>用剪刀，剪切成小部分，其余的肠子。加入足够的自来水几乎淹没收集组织的菜。 </li><li>在35-40℃在实验室烘箱或至少10分钟的孵化器，孵育样品的混合物。这将解放和揭露管腔寄生虫。而样本是孵化，进行以下步骤。 </li><li>将两个下降0.9％生理盐水侧方的单个标记的幻灯片，允许从两个肠段（十二指肠和盲肠）的样品制备。 </li><li>热杀菌和冷却接种环或同等学历。 </li><li>刮十二指肠粘膜，并放置在左侧的幻灯片（最接近磨砂边侧）刮出。 </li><li>刮盲肠黏膜和地点从右侧的幻灯片刮出。 </li><li>顶部与盖玻片刮出。检查准备幻灯片，使用相衬显微镜下的40倍，目标）;需要识别的增加放大。如果发现寄生虫，确定根据形态。 </li><li>这道菜的内容将准备检查这一点。在解剖显微镜下检查的菜内容。使用作为探针或涂药棒内移动盘的内容，完成了全面检查。如果存在蛲虫，它们将出现小，白色，毛发状的蠕虫。如果绦虫存在，他们将出现分割，平面蠕虫（大于线程像蛲虫）。 </li><li>如果蠕虫被发现或怀疑，用一个小镊子对收集标本。 </li><li>安装在一个标记，石蜡或矿物油下降洁净的载玻片标本，标本上放置盖玻片。检查幻灯片，使用光学显微镜上的10倍和40倍的目标。 </li></ol> 2。体外寄生虫检查（表3） 5。毛皮动物内脏外寄生虫检查（胶带测试） <ol><li>从自动售货机中取出一个明确的长度，不磨砂，透明胶带。胶带应足够长的时间来处理，而不触及中间（约5厘米）一端。它可能会更容易免除一次几个长度，连接到一个干净的工作表面的边缘，并使用需要。 </li><li>电梯从鼠标或老鼠笼和笼盖的地方，它的尾巴。层流柜或生物安全柜中，如果​​动物的健康状况要求它执行此操作。 </li><li>抑制小鼠或大鼠。抓住皮草与止血和鼠标的肩胛区，腹侧颈部，腋窝区域，腹股沟区，背臀部，轻轻地摘下从毛皮。放置在磁带上的毛皮。头发，应看作是附着到磁带的检测，被认为是成功的。将它在笼中的小鼠或大鼠背部。 </li><li>放置一个标记洁净的载玻片上滴的矿物油，适用于磁带，然后又一滴矿物油。盖上玻璃盖滑。 </li><li>阅读使用光显微镜下的10倍和40倍的目标的显微镜幻灯片。 </li><li>这种检查是最好的检测，如Radfordia，Myobia和Myocoptes皮毛螨。 </li></ol> 6。皮肤刮<ol><li>汇编下列材料：动物进行测试，矿物油，显微镜载玻片，盖玻片，手术刀，剪刀。如果这个测试是在活的动物，它们应该被麻醉，然后再开始。 </li><li>样品背部的尾巴和头部颞区附近的基地。另外，皮肤损伤和/或其他网站可能会被刮掉。 </li><li>深用手术刀刀片刮皮肤，毛发的生长方向相反，削弱表皮。修剪毛发刮前，可能会提高筛查的灵敏度，减少视觉上阻塞（多余的毛发）幻灯片。 </li><li>在幻灯片上放置一个石油下降。应用范例滴油抹刀片表面上的幻灯片（与样本附后）。如果有必要的幻灯片，并与盖玻片上添加额外的石油。 </li><li>阅读使用光显微镜下的10倍和40倍的目标的显微镜幻灯片。 </li><li>皮肤刮一般是用来检测的蠕形螨（和dermatophytic真菌） 。 </li></ol> 7。直接检查的​​皮毛<ol><li>将安乐死鼠标或大鼠解剖显微镜的阶段。 </li><li>检查的皮约10倍的头发，使用涂药棒或类似的文书部分的头发和观察基地的头发轴。 </li><li>检查颅地区，眼睛和羽片之间的羽片，肩胛之间，下颌骨，以及腹股沟和腋窝地区之间。另外，整个胴体进行审查。 </li><li>收集任何体外寄生虫或使用小镊子对可疑物质看到。体外寄生虫往往可能看起来像头皮屑或黄色的头发轴的基地，或直接在皮肤上的蜡质堆积。 </li><li>山石蜡或矿物油的下降，在洁净的载玻片上的标本，标本上放置盖玻片。检查的幻灯片，使用光学显微镜下的10倍和40倍的目标。 </li></ol> 3。代表性的成果： 参见确定下面的寄生虫附加文件：（注：这些程序会检测到任何鸡蛋，蠕虫，或囊肿目前，在粪便中，或在皮肤上和皮毛;其中只有少数是下面列出） 体内寄生虫： <table border="0"><tbody><tr><td> Syphacia muris（鸡蛋，蠕虫病毒） </td><td> Chilomastix bettencourti </td></tr><tr><td> Syphacia obvelata（鸡蛋，蠕虫病毒） </td><td> Hexamastix muris </td></tr><tr><td> Aspiculuris tetraptera（鸡蛋，蠕虫病毒） </td><td> Retortamonas SP。 </td></tr><tr><td> Rodentolepis娜娜 （鸡蛋，蠕虫病毒） </td><td> 贾第虫菌。 </td></tr><tr><td> Tritrichomonas muris </td><td> Spironucleus muris </td></tr><tr><td> 阿米巴muris </td><td></td></tr></tbody></table>体外寄生虫： <table border="0"><tbody><tr><td> Myocoptes musculinis </td><td> Radfordia affinis </td></tr><tr><td> Myocoptes musculinis </td><td> Radforida ensifera </td></tr><tr><td> Myobia musculi </td><td></td></tr></tbody></table><table border="1"><tbody><tr><td>&nbsp;</td><td> 胶带测试 </td><td> 粪便浮选 </td><td> 联邦通讯委员会 </td><td> 直接考试1 </td><td> PCR扩增 </td></tr><tr><td> 原生动物 </td><td> - </td><td> + </td><td> + </td><td> + + </td><td> + + / NA 2 </td></tr><tr><td> 后生动物 </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 蛲虫 </td><td> + / - 3 </td><td> + / - 4 </td><td> + 4个 </td><td> + + </td><td> + + </td></tr><tr><td> 绦虫5 </td><td> - </td><td> + </td><td> + </td><td> + + </td><td>不适用</td></tr><tr><td> 5其他蛔虫 </td><td> - </td><td> + </td><td> + + </td><td> + + </td><td>不适用</td></tr></tbody></table> 1，这种方法需要的动物实施安乐死。 2，有没有PCR检测方法，目前每原生动物。 3，这种方法是最合适的检测Syphacia SPP。 4，此方法将会更容易检测Aspiculuris，并不太可能检测Syphacia。 5，绦虫和蛔虫，蛲虫等，是非常罕见的，在现代化的实验室小鼠和大鼠。 表1。类的endoparasite和适当的检测方法。有些方法会要求安乐死的动物。 NA表示方法目前没有可用于这些寄生虫+表示寄生虫问题的检测方法的适用性， - 表明，该方法是不建议，寄生虫。 <table border="1"><tbody><tr><td> 解决方案 </td><td> 比重 </td><td> 成份每公升H 2 O </td></tr><tr><td>氯化钠</td><td> 1.20 </td><td> 311克氯化钠</td></tr><tr><td>硝酸钠</td><td> 1.20 </td><td> 338克硝酸钠</td></tr><tr><td>硝酸钠</td><td> 1.30 </td><td> 616克硝酸钠</td></tr><tr><td>糖</td><td> 1.20 </td><td> 1170克， 蔗糖 1 </td></tr><tr><td> Sheather的糖</td><td> 1.27-1.30 </td><td> 1563年克蔗糖1 </td></tr><tr><td>硫酸锌</td><td> 1.18 </td><td> 493克硫酸锌</td></tr></tbody></table> 1，这些解决方案需要冷藏或加入9毫升苯酚作为防腐剂。 表2。粪便浮选解决方案（Smith 等人。） <table border="1"><tbody><tr><td></td><td> 毛皮动物内脏（磁带测试） </td><td> 皮肤刮1 </td><td> 直接考试1 </td><td> PCR扩增 </td></tr><tr><td> 虱子 </td><td> - </td><td> - </td><td> + </td><td>不适用</td></tr><tr><td> 螨 </td><td> + </td><td> + </td><td> + + </td><td> + + + / NA 3 </td></tr><tr><td> 跳蚤4 </td><td> - </td><td> - </td><td> + </td><td>不适用</td></tr><tr><td> 蜱4 </td><td> - </td><td> - </td><td> + </td><td>不适用</td></tr></tbody></table> 1。这种方法需要麻醉，如果它是一个活的动物进行。 2，这种方法需要的动物实施安乐死。 3，有没有目前螨的每一个物种的PCR检测方法。 4，跳蚤和扁虱是极其罕见的，在现代化的实验动物设施。 表3类外寄生物和适当的检测方法。有些方法会要求安乐死的动物，和其他方法将需要麻醉执行他们在活的动物。 NA表示方法目前没有这些寄生虫。 NA表示方法目前没有可用于这些寄生虫+表示寄生虫问题的检测方法的适用性， - 表明，该方法是不建议，寄生虫。

<ol>
	<li>Baker, D. G., Fox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+23.">Chapter 23.</a> The Mouse in Biomedical Research: Diseases. Diseases Vol. 2, (2007).</li><li>Baker, D. G., Baker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11.">Chapter 11.</a> Flynn's Parasites of Laboratory Animals. , 303-398 (2007).</li><li>Owen, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Parasites of Laboratory Animals. 12, (1992).</li><li>Pritchett, K. R., Fox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+22.">Chapter 22.</a> The Mouse in Biomedical Research: Diseases. Diseases Vol. 2, (2007).</li><li>Smith, P. H., Wiles, S. E., Malone, J. B., Monahan, C. M., Baker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.">Chapter 1.</a> Flynn's Parasites of Laboratory Animals. , 1-13 (2007).</li><li>Wasson, K., Fox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+21.">Chapter 21.</a> The Mouse in Biomedical Research: Diseases. Vol. 2, 517-550 (2007).</li></ol>

作者是查尔斯河，诊断测试和试剂的供应商，包括上文所述的测试的所有员工。

在实验室工作时，安全应始终关注的问题。记得穿适当的防护设备，与动物的工作时，并用消毒剂清洁之前和之后您的工作站。这些方法主要是设计中的位置找到任何啮齿类实验动物寄生虫检查，也就是说，他们可以检测外来的或极其罕见的寄生虫以及更常见的蛲虫和毛皮螨。虽然它们同样适用于其他物种，野生啮齿类动物可能有额外寄生虫，如肝，皮下和脑的位置，而不是通过上述方法进行评估。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>菜板</td><td>热电公司</td><td> CAT＃36114 </td><td></td></tr><tr><td>小剪刀</td><td> ROBOZ </td><td> RS - 5910，G204 </td><td> 23毫米刀片，3.5“长，直</td></tr><tr><td>中等剪刀</td><td> ROBOZ </td><td> RS - 6808，G207 </td><td> 5“ </td></tr><tr><td>钳弯</td><td> ROBOZ </td><td> RS - 8254（M1/21004） </td><td> 4.5英寸，锯齿，轻微的曲线</td></tr><tr><td>镊子，Microdissecting </td><td> ROBOZ </td><td> RS - 5238 </td><td>哈德森（埃瓦尔德） </td></tr><tr><td>钳，组织钳</td><td> ROBOZ </td><td> RS - 8160 </td><td>鼠齿</td></tr><tr><td>金属探针</td><td> VWR </td><td> CAT＃25778-000 </td><td></td></tr><tr><td>止血</td><td>华帝</td><td> V97 - 48 </td><td></td></tr><tr><td>涂药棒</td><td>清教徒</td><td> 6英寸的喷头，楼盘＃807 </td><td></td></tr><tr><td>解剖显微镜</td><td>奥林巴斯</td><td> SZ51，肖特，ACE1 </td><td></td></tr><tr><td>培养皿</td><td> VWR </td><td>为100mm，猫＃3401PDNL </td><td></td></tr><tr><td>盖玻片</td><td> VWR </td><td>猫＃48366-067微玻璃盖， </td><td></td></tr><tr><td>幻灯片</td><td> VWR </td><td> VistaVision显微镜载玻片猫＃16004-368 </td><td></td></tr><tr><td>接种环</td><td> VWR </td><td> CAT＃50815-040 </td><td></td></tr><tr><td>光镜下</td><td>奥林巴斯</td><td>型号＃BX41TF </td><td></td></tr><tr><td>实验室烘箱</td><td>昆西实验室公司</td><td> 10型实验室的烤箱</td><td></td></tr><tr><td>离心分离</td><td> Beckman Coulter公司</td><td> Allegra的X - 12R </td><td></td></tr><tr><td> Vortexer </td><td> VWR </td><td>迷你- Vortexer </td><td></td></tr><tr><td>试管中</td><td>金布尔大通</td><td> 15毫升的一次性离心管中，猫＃73790-15 </td><td></td></tr><tr><td>盖玻片</td><td> VWR </td><td>塑料显微镜的盖玻片上，22毫米，猫＃48376-049 </td><td></td></tr><tr><td>白色鸭舌帽</td><td> VWR </td><td> CAT＃60869-089 </td><td></td></tr><tr><td>碘</td><td>罗利生化研究所</td><td>猫＃SO - 364 </td><td></td></tr><tr><td>硫酸锌</td><td>西格玛爱秩序</td><td>猫＃1000917519，Z4750 - 500G </td><td></td></tr><tr><td>蔗糖</td><td> Mallinckrodt化学品</td><td> CAT＃8360-06 </td><td></td></tr><tr><td>苯酚</td><td>电解二氧化锰</td><td> CAT＃PX0510 - 1 </td><td></td></tr><tr><td>玻璃纸胶带</td><td>斯台普斯</td><td>隐形胶带，猫＃504712 </td><td></td></tr><tr><td>矿物油</td><td> Mallickrodt </td><td> CAT＃6358 </td><td></td></tr></tbody></table>

diagnosis of ecto- and endoparasites in laboratory rats and mice

内部和外部的寄生虫仍然是一个显著的关注，在实验室灭鼠设施和许多科研设施海港一些寄生的动物。开始对动物进行寄生虫检查前，应考虑两件事情。一：有什么用，将所收集的信息，和两个：这是最合适的测试。得知是寄生的动物，可能是设施接受的东西，但往往有一个需要治疗的动物，然后才能确定治疗的疗效。发现动物体内的寄生虫可能通过各种技术，包括从居住或实施安乐死的动物抽取样本。从历史上看，最大的诊断敏感性测试所需的动物实施安乐死，虽然PCR允许几种类型的寄生虫的高灵敏度的测试。本文演示程序的检测内，并在小鼠和大鼠体外寄生虫。同样的程序适用于其他啮齿类动物，虽然发现寄生虫的种类会有所不同。

Watch this Scientific Journal Video about Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice at JoVE.com

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

本文介绍了筛选大鼠和小鼠的各种程序，以检测内或ectoparasitism。几种诊断方法将被证明，那些适合使用活的动物和那些使用后的动物实施安乐死。将包括照片，以帮助确定大鼠和小鼠的寄生虫。

Ecto和实验室大鼠和小鼠体内寄生虫的诊断

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

diagnosis-of-ecto-and-endoparasites-in-laboratory-rats-and-mice

Research

JoVE Journal

Immunology and Infection

41.0K 次观看.  Charles River. 本文介绍了筛选大鼠和小鼠的各种程序，以检测内或ectoparasitism。几种诊断方法将被证明，那些适合使用活的动物和那些使用后的动物实施安乐死。将包括照片，以帮助确定大鼠和小鼠的寄生虫。

视频: Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

原位大鼠后肢移植

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

嗜碱粒细胞激活测试IgE介导的药物过敏机制的调查

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

在暴露于臭氧的小鼠的弹性周期中的肺微 rna 分析

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

小鼠的共性共性隔离和识别

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...