这项工作是从退伍军人事务部的优异审查授予部分经费，由美国国立卫生研究院拨款AI045540，AI067874，AI076233 - 01和AI080801（水电部），和AI29646（SMB）。这项工作是在CT和JG的经费由美国国立卫生研究院的T32 AI07511部分。

1。小动物转基因利什曼原虫感染 1。寄生虫线转基因利什曼原虫 SPP。寄生虫表达荧光素酶的生成使用episomal或作为一个整合载体。1 2克隆线是首选。两个要点是： （一）综合超过episomal荧光素酶的荧光素酶是首选，因为这些寄生虫线，在理论上应该更好地保留在药物的压力，即没有引入到哺乳动物的基因。然而，即使整合的转基因可能会失去在低利率，3，因此它是至关重要的，保持对股市文化的选择性药物压力。在实践中， 利什曼原虫属。往往保留更长的时间内， 甚至在体内的episomal元素，虽然在每个细胞的质粒拷贝数的变化。两种类型的转基因寄生虫，他们应该总是通过在体外一次到动物接种前，以避免潜在的药物作用。 （二） 利什曼原虫 SPP。寄生虫可以减肥期间在体外培养的毒力。在某些物种（如属婴儿利什曼原虫，chagasi）这方面的损失可能会发生迅速的文化周。在其他物种（如属重大，L.墨西哥 ）的毒力保留长远来说，几个月到几年。尽管如此，克隆转染，应进行筛选，找出那些保留完整的小动物致害。许多实验室将连续传递寄生虫多次通过老鼠或仓鼠（4个或更多），以增加毒力前在实验中使用。 2。制备亚循环感染阶段寄生虫感染 利什曼原虫类感染形式。是由沙蝇传播到哺乳动物的主机是亚循环promastigote。因此，这种形式的寄生虫的动物感染的首选自然感染模型。虽然沙蝇都难以维持，在体外生长的寄生虫，将方便进行metacyclogenesis。文化在metacyclics％将随通行证，因为从动物，文化传媒和寄生虫的种类和应变隔离。 Metacyclics可以纯化由利什曼原虫的种类和线的积极或消极的选择。在终端的变化或侧链碳水化合物丰富的表面lipophosphoglycan石油气（LPG）L的残留主要让凝集损失5,6种其他利什曼原虫或 L纯化的外源凝集素PNA（花生凝集素）。缺乏亚循环液化石油气的主要突变体，可以净化一个聚蔗糖一步梯度7 IVIS可以使用大容量非纯化的文化，但应该认识到，这些含有亚循环的传染性形式的混合物。 通常情况下,15 - 20％的大宗固定相L.一chagasi文化恢复使用已经3个星期内的隔离仓鼠或鼠标的寄生虫metacyclics。寄生虫是通过离心洗涤，暂停缓冲液中所需的浓度（带或不带的Ca 2 +和Mg 2 +），并保持在4 °彗星长达2小时前接种到小鼠的HBSS或PBS。 3。 利什曼原虫属的接种途径的选择。到主机人类利什曼病的临床疾病表现取决于种寄生虫和宿主因素。有相应的差异，在小鼠感染，导致调查，使用不同的模型和感染途径。例如，物种造成人类CL（如属重大，L. amazonensis）L.墨西哥，往往接种到小鼠皮下的脚垫或在耳后皮下。8,9种引起人类VL的往往是通过尾静脉注射推出10-12静脉或皮内注射在耳，13尾静脉感染已执行，也可以与仓鼠派生的无鞭毛体或promastigotes 。我们经常感染小鼠L. promastigote形式一chagasi，无论是皮内注射在耳后或跗外侧地区的14或静脉注射。 4。治疗前与麻醉小鼠可用于野生型，转基因或基因敲除小鼠。虽然没有正式的，量化的，它似乎最有可能的，裸体或BALB / c小鼠，如白化允许通过组织黝黑的皮肤和头发的色素，如C57BL / 6小鼠相比，透光。 如注射氯胺酮加甲苯噻嗪的混合物[80-100毫克/公斤+ 10毫克/公斤，分别intrap anesthestic代理eritoneally（IP）]生理盐水稀释是一个首选的方法，因为动物会轻轻麻醉单剂量至少10分钟。 100 - 200ul总量腹腔注射使用25-30号针头。动物是手动坚决克制其腹部背侧皮肤和头部朝下。针头斜面向上，略有角度应定位。针尖应稍稍穿透腹壁左下象限。 一种吸入性麻醉剂，异氟醚等可交替使用，但动物只会留在麻醉状态下，只要吸入麻醉剂。 5。 利什曼原虫属鼠的感染。寄生虫一旦动物轻轻麻醉，感染部位清洗干净，用70％乙醇或异丙醇。寄生虫的种类，剂量和感染途径是由研究者。皮（10μL）或肌肉注射（25-50μL）感染途径，以防止过多的组织损伤，应尽量减少注射量。允许在小鼠注射量可静脉（100-200微升），皮下（高达2毫升）或腹腔感染途径（高达2毫升）。 在我们的实验中所用的感染途径和卷包括皮在耳耳廓（10μL），在侧面皮下（100-200微升），腹腔内（100-200微升）或静脉（100-200μL）。寄生虫的剂量范围从10 2 10 7 promastigotes 。 2。 利什曼原虫生物发光成像使用的IVIS（ 活体成像系统） 1。制备D -荧光素在体内生物发光成像 D -荧光素（卡尺生命科学）是重组，以浓度15毫克/毫升贝科的PBS 镁带或不带+和Ca 2 +和注射器过滤（0.2微米）。等分被冻结在-80℃直到使用。注射前，荧光素被加热到37℃水浴中。 2。 D -荧光素注射注射部位清洗和荧光素是由15毫克/毫升荧光素在DPBS溶液腹腔注射，剂量为150毫克/公斤，引入意识的动物。荧光素也可以被注入麻醉剂治疗的动物，但生物发光动力学可能会略有不同。 一旦注射到动物，荧光素迅速流传。它是非常有用的经验确定的最佳时间，收购后，每一个实验模型，并为不同的小鼠的解剖位置注射荧光素发光的数据，由于荧光素分布的动力学可能会有所不同。一个动力学曲线是由获得一个复制的图像序列。 3。动物轻轻麻醉吸入或注射麻醉剂，可用于成像过程。异氟醚是简单的管理在这一阶段，因为大多数IVIS系统，有一个附加的麻醉室，麻醉鼻锥内成像室多方面的。麻醉是麻醉室和歧管内的成像室之间的分裂。 这些动物被放置在明确有机玻璃麻醉室轻轻2.5-3.5％异氟醚麻醉。动物视力监测，以确保有效的麻醉程度已引起的，和他们的呼吸不受阻碍地。核实撤出从痛苦的爪子压力缺乏足够程度的麻醉。后动物轻轻麻醉，异氟醚的数量可减少到1.5-2％。 4。发光法数据收集充分麻醉动物从麻醉室转移到成像室和定位，使鼻子歧管锥将提供一个连续的异氟醚和调节流量。 生活图像软件程序（卡尺生命科学版）打开IVIS初始化。对于我们的实验中，我们利用精诺真IVIS 200的系统（卡尺生命科学）。 CCD摄像头将自动冷却，保持在适当的温度后，初始化的IVIS。摄像系统的设置和图像采集参数设置在IVIS系统控制面板。采集的参数主要是基于对动物的数量和生物发光的强度。重要的参数包括：成像模式（荧光或荧光），曝光时间（快门打开的时间），分级（决定在CCD上的像素大小），重点和主体高度（焦平面），F /停止（光圈大小）和领域的视角（FOV）。预设的视野s的IVIS成像系统200系列（方形的图像区域）4，6.5，13，20和25厘米（视场的宽度）。 按收购或收购的IVIS系统控制“窗口中的连续照片，发起的IVIS上的图像和数据采集。采集时间范围可以从几秒钟到几分钟。对于我们的实验中，我们使用60秒的默认曝光设置。 特写图像（小视野）可提供更高的分辨率，但不一定提高灵敏度相比，图像使用一个更大的视野。 后的成像过程中，动物是从成像室，回到笼子和监测，直到他们从麻醉中恢复过来。 7。数据分析发光的数据分析使用的生活图像软件（精诺真）和对应的CCD相机检测到的光子数目表示光的强度。这些数据代表的伪彩色图像覆盖到动物的黑白照片。具体形象的地区，可通过创建感兴趣区域（ROI）分析。生活图像软件，并提供多种数据​​输出选项。分析数据的最简单的方法之一是选择一个利息率（ROI）的地区，并检测到的光子/秒的测量平均＃。然后这些数据可以被导出到一个试算表，如Microsoft Office Excel（微软公司）的。随后GraphPad棱镜（GraphPad软件）是用于生成这个手稿图。 3。代表性的成果摘要： IVIS技术使荧光素酶表达的利什曼原虫 SPP的可视化。生活在实时麻醉BALB / c小鼠的寄生虫。当底物荧光素的分布，通过组织，迅速氧化导致的D -荧光素的转基因寄生虫表达的荧光素酶的光发射。那些没有组织吸收光子检测动物的IVIS成像技术的CCD相机的表面。 1。造成人的内脏与人类皮肤利什曼病寄生虫的感染模型。 内脏利什曼病的小鼠模型，超过皮肤利什曼病模型的问题，因为感染的进度要求安乐死组小鼠在每次评估点。 IVIS的一个限制是光发射淬火有不同程度的影响，在不同的内脏组织，并与高血流量，如肝，脾组织可能超过皮肤问题。因此，IVIS可能无法检测的敏感的荧光素酶表达寄生在皮肤在肝脏和脾脏。另外还建议寄生虫的负载不应该组织的网站之间的比较。尽管如此，寄生虫的负载可以在同一组织之间一脉相承的年龄相匹配的感染小鼠进行比较。 荧光素酶的信号很容易在我们的实验中检测到内脏器官，促进动物之间感染的比较。一个5周的感染，引起人的内脏利什曼病寄生虫，李用荧光素酶表达的一个例子。 chagasi SSU/IR1SAT-LUC（一），如图1所示。 visceralized寄生虫感染5周后，可以检测小鼠1和2，在所有小鼠的肝脏和脾脏。图2显示了相同的寄生虫线，一个小时后引进的寄生虫的剂量​​滴定。例如，一个纵向感染与导致人类皮肤利什曼病，属的寄生虫墨西哥 SSU/IR1SAT-LUC（一），如图3所示。 2。动力学荧光素酶检测。 初步动力学研究是必不可少的，以最大限度地发射光子从发光寄生虫的检测。这一点很重要，以确定这一经验，作为最佳的成像时间可能会有所不同的寄生虫的数量和亮度分离，并在小鼠体内的发光微生物的解剖位置而定。动力学研究依赖的荧光素的分布在整个组织，窝藏荧光素酶表达利什曼原虫的小鼠组织，并在每个隔离寄生虫的荧光素酶的表达/酶活动的效率速度。被感染小鼠接种和荧光素如上所述，一个图像序列收集荧光素注射后，每2分钟。发出的光的幅度量化和图形来确定最大的检测点（见例如图1B）。一旦最大的光检测的时间是确定的，在实验中的所有图像利用荧光素接种后成像的同时，在图1A。我们的动力学研究与 L IV感染的小鼠肝脏证明峰值的荧光素酶信号一 IP chagasi 10至25分钟后，荧光素接种（图1B） 。 3。评估效率寄生虫接种。 IVIS可用于估计与荧光素酶表达的利什曼原虫 SPP的初次感染的一致性和效率。只要使用同样的路线是对每个动物，发出的光子可以被用来作为一个粗略的估计接种的疗效。一个例子是如图2所示，在BALB / c小鼠接种的亚循环L.表示数字一chagasi promastigotes，利什曼原虫一个visceralizing物种。一个小时后，感染小鼠接种与荧光素的IP，并采取了20分钟后图像。图中显示的黑白图像（图2A）和伪彩色发光（图2B）覆盖的图像。图2C显示选择的地区的利益，从鼠标图像（ROI），发射光子的数量。 在图2中的例子表明，感染剂量成正比，与越来越多的寄生虫发射光子的渐变。数据显示，类似到 L 主要和L 。 墨西哥 ，皮肤感染利什曼原虫属成像。非常敏感，允许少于10 4寄生虫明确可视。如这些数据可以用来量化感染强度。在图2b所示的例子中，有大约1光子/秒/寄生虫。 4。监测小鼠感染的过程。 皮肤利什曼病的小鼠模型的特征作为衡量纵向遵循感染病灶的大小。虽然病情恶化的一个很好的估计，病灶的大小是在组织中的寄生虫复制率炎症反应程度的影响。老鼠必须安乐死在实际测量寄生虫负荷实验结束。 IVIS可用于估计寄生虫负荷的进展， 纵向与利什曼原虫的物种，导致皮肤利什曼病感染小鼠的皮肤组织。图3表明，BALB / c小鼠接种0天皮下注射10 5 L.墨西哥表 ​​达荧光素酶和成像对感染后的顺序几周内。图3A展品在感染后10-31周代表性的图像。相同的数据显示在定量的形式在图3B。重要的是，在感染部位的寄生虫的数量由IVIS可以量化，前病变是通过其他方法检测。 随着时间的推移，逐步增加在生物发光对应寄生虫数字的增长。信号强度，即光子/寄生虫/秒，可因寄生虫号码的功能，组织的位置，生长阶段和在体内的寄生虫健康。因此重要的是假设生物发光是成正比寄生虫数字的校准前寄生虫和发光之间的关系。在L 的情况下主要的 15 或 L 墨西哥 ，16出现在无鞭毛体生物发光衰减比较小，在脚垫病变相对promastigotes 。初步数据表明，衰减可能会更深刻属chagasi infantum 17或为L。 braziliensis 15。 <img src="/files/ftp_upload/1980/1980fig1.jpg" alt="图1" /> 图1。动力学的荧光素酶检测荧光素分布的动力学分析是建立在肝脏成像利什曼原虫感染的最佳时间。小鼠感染IV 10 7 L。一chagasi pIR1SAT - LUC（一）。 a）四个感染小鼠成像伴随着一个未受感染的小鼠，感染后5个星期。荧光素被注入了所有五个小鼠和图像IP收集24分钟，每两分钟。被选定的投资回报率和发光测定和量化（二）。 <img src="/files/ftp_upload/1980/1980fig2.jpg" alt="图2" /> 图2。评估寄生虫接种的效率 。成像和量化发光利什曼原虫寄生虫感染与IVIS 。在较低的右侧注入皮内，与野生型的BALB / c小鼠的HBSS（模拟） 或转基因利什曼原虫一chagasi L。一chagasi pIR1SAT - LUC（一）。 IVIS成像技术接种后的一个小时，小鼠，然后进行分析。 A）的小鼠的黑白照片立即采取前荧光信号（光强）测定。红色箭头指向每个注射部位动物。 B）发光的伪彩色图像叠加在照片上。三）被选定的感兴趣区域（ROI）（红色圆圈）和发光在每个ROI量化。这些数据代表感染的投资回报率减去的投资回报率的模拟感染的净发光。 <img src="/files/ftp_upload/1980/1980fig3.jpg" alt="图3" /> 图3。长期与造成人类皮肤利什曼病寄生虫感染寄生在一个单一的动物注射发光利什曼原虫墨西哥pIR1SAT - LUC（一）负担的时间课程评价。野生型BALB / c小鼠注射皮下100,000固定相的转基因利什曼原虫墨西哥pIR1SAT - LUC（一）在飞节。 IVIS收集资料，从感染小鼠在感染后表示数周。一）发光的伪彩色图像叠加到相应时间点的黑白照片。二）净投资回报率是通过测量发光在受感染的跗关节和减去背景的投资回报率，从对侧未受感染的典当绘制决定。散点图代表在不同的时间点感染后的净投资回报率。

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metacyclogenesis+is+a+major+determinant+of+Leishmania+promastigote+virulence+and+attenuation.">Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation.</a> Infect Immun. 55, 2802-2806 (1987).</li><li>Spath, G. F., Beverley, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lipophosphoglycan-independent+method+for+isolation+of+infective+Leishmania+metacyclic+promastigotes+by+density+gradient+centrifugation.">A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.</a> Exp Parasitol. 99, 97-103 (2001).</li><li>Scott, P., Caspar, P., 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=Protection+against+Leishmania+major+in+BALB/c+mice+by+adoptive+transfer+of+a+T+cell+clone+recognizing+a+low+molecular+weight+antigen+released+by+promastigotes.">Protection against Leishmania major in BALB/c mice by adoptive transfer of a T cell clone recognizing a low molecular weight antigen released by promastigotes.</a> J Immunol. 144, 1075-1079 (1990).</li><li>Belkaid, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+interleukin+(IL)-10+in+the+persistence+of+Leishmania+major+in+the+skin+after+healing+and+the+therapeutic+potential+of+anti-IL-10+receptor+antibody+for+sterile+cure.">The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure.</a> J Exp Med. 194, 1497-1506 (2001).</li><li>Wilson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+suppression+of+IFN-gamma+in+hepatic+granulomas+correlates+with+tissue-specific+replication+of+Leishmania+chagasi.">Local suppression of IFN-gamma in hepatic granulomas correlates with tissue-specific replication of Leishmania chagasi.</a> J Immunol. 156, 2231-2239 (1996).</li><li>McElrath, M. J., Murray, H. W., Cohn, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+granuloma+formation+in+experimental+visceral+leishmaniasis.">The dynamics of granuloma formation in experimental visceral leishmaniasis.</a> J Exp Med. 167, 1927-1937 (1988).</li><li>Ato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+dendritic+cell+migration+and+impaired+resistance+to+Leishmania+donovani+infection+in+mice+deficient+in+CCL19+and+CCL21.">Loss of dendritic cell migration and impaired resistance to Leishmania donovani infection in mice deficient in CCL19 and CCL21.</a> J Immunol. 176, 5486-5493 (2006).</li><li>Ahmed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intradermal+infection+model+for+pathogenesis+and+vaccine+studies+of+murine+visceral+leishmaniasis.">Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis.</a> Infect Immun. 71, 401-410 (2003).</li><li>Kamala, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hock+immunization:+a+humane+alternative+to+mouse+footpad+injections.">Hock immunization: a humane alternative to mouse footpad injections.</a> J Immunol Methods. 328, 204-214 (2007).</li><li>Lang, T., Goyard, S., Lebastard, M., Milon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+Leishmania+expressing+luciferase+for+rapid+and+high+throughput+screening+of+drugs+acting+on+amastigote-harbouring+macrophages+and+for+quantitative+real-time+monitoring+of+parasitism+features+in+living+mice.">Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote-harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice.</a> Cell Microbiol. 7, 383-392 (2005).</li><li>Brittingham, A., Miller, M. A., Donelson, J. E., Wilson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+GP63+mRNA+stability+in+promastigotes+of+virulent+and+attenuated+Leishmania+chagasi.">Regulation of GP63 mRNA stability in promastigotes of virulent and attenuated Leishmania chagasi.</a> Mol Biochem Parasitol. 112, 51-59 (2001).</li><li>Roy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Episomal+and+stable+expression+of+the+luciferase+reporter+gene+for+quantifying+Leishmania+spp.+infections+in+macrophages+and+in+animal+models.">Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models.</a> Mol Biochem Parasitol. 110, 195-206 (2000).</li><li>Contag, 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=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol Microbiol. 18, 593-603 (1995).</li><li>Hardy, J., Margolis, J. J., Contag, 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=Induced+Biliary+Excretion+of+Listeria+monocytogenes.">Induced Biliary Excretion of Listeria monocytogenes.</a> Infect. Immun. 74, 1819-1827 (2006).</li><li>Lecoeur, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Topical+Therapy+for+Leishmania+major+Localized+Cutaneous+Leishmaniasis+Using+a+Reliable+C57BL/6+Model.">Optimization of Topical Therapy for Leishmania major Localized Cutaneous Leishmaniasis Using a Reliable C57BL/6 Model.</a> PLoS Negl Trop Dis. 1, e34-e34 (2007).</li><li>Lang, T., Lecoeur, H., Prina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Leishmania+development+in+their+host+cells.">Imaging Leishmania development in their host cells.</a> Trends Parasitol. 25, 464-473 (2009).</li></ol>

在活体成像系统（IVIS），为整个动物的成像或体内成像实验感染利什曼病的不同形式的模型的 方法。18,16利什曼原虫SPP。寄生虫，可设计，以表达萤火虫荧光素酶在体内 IVIS成像技术检测的水平。这种方法的主要优势之一是它允许利什曼原虫属非侵入性可视化。活的动物宿主内。该方法已应用于其他传染性疾病模型，传染性病原体可以表达的转基因?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>D-Luciferin Potassium Salt </td>
<td>Reagent</td>
<td>Caliper LifeSciences (Formerly Xenogen)</td>
<td>122796</td>
<td>&nbsp;</td>
<tr>
<td>IVIS Imaging System 200 Series</td>
<td>Equipment</td>
<td>Caliper LifeSciences</td>
<td>&nbsp;</td>
<td>Other IVIS models that can be used include: Lumina II, Lumina XR, Kinetic, and Spectrum. </td>
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in vivo imaging of transgenic leishmania parasites in a live host

独特品种<em&gt;利什曼原虫，</em&gt;一个家庭的原生动物寄生虫<em&gt; Trypanosomatidae</em&gt;，通常会导致不同的人类疾病的表现。疾病最常见的形式是内脏利什曼病（VL）和皮肤利什曼病（CL）。利什曼病的小鼠模型被广泛使用，但在小鼠疾病的寄生虫负担的量化要求的小鼠在感染后的不同时间安乐死。寄生负载，然后在显微镜下测量，限制稀释实验中，或寄生虫的DNA定量PCR扩增。 “<em&gt;在体内</em&gt;成像系统（IVIS）有一个集成的软件包，使细胞在生物体相关的发光信号的检测。既可以减少动物的使用和纵向遵循感染在体内成像模型在个人，<em&gt;利什曼原虫</em&gt; SPP。造成VL或CL成立。寄生虫设计，以表达荧光素酶，这些被引入到小鼠皮下或静脉注射。转基因荧光素酶驾驶的生物发光的定量测量<em&gt;利什曼原虫</em使用IVIS&gt;鼠标内的寄生虫。个人小鼠可成像多次在纵向研究中，让我们评估在最初的实验寄生虫接种的动物间的变化，并评估在小鼠组织中的寄生虫的繁殖。寄生虫检测灵敏度高，与皮肤的位置。虽然这是极有可能的信号（光子/秒/寄生虫）是在更深的内脏器官比皮肤低，但信号的定量比较肤浅与深刻的网站还没有完成。这是可能的部位的寄生虫之间的数字不能直接比较，但可以在同一组织中的寄生虫负载之间的小鼠相比。一个visceralizing物种的例子（<em&gt; L。 infantum chagasi</em&gt;）和一个物种造成皮肤利什曼病（<em&gt; L。墨西哥</em&gt;）所示。 IVIS程序可用于监测和分析各种各样的小动物模型<em&gt;利什曼原虫</em&gt;物种造成人类利什曼病的不同形式。

Watch this Scientific Journal Video about In vivo Imaging of Transgenic Leishmania Parasites in a Live Host at JoVE.com

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

一个<em&gt;在体内</em&gt;成像系统是用来生成的定量测量与Trypanosomatid原虫感染小鼠<em&gt;利什曼原虫</em&gt;。这是一个慢性的过程中整个检测在许多组织中表达的荧光素酶的寄生虫的非侵入性和非致命性的方法<em&gt;利什曼原虫</em&gt; SPP。感染。

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

in-vivo-imaging-of-transgenic-leishmania-parasites-in-a-live-host

Research

JoVE Journal

Immunology and Infection

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

15.8K 次观看.  University of Iowa, and the VA Medical Center. 一个在体内成像系统是用来生成的定量测量与Trypanosomatid原虫感染小鼠利什曼原虫。这是一个慢性的过程中整个检测在许多组织中表达的荧光素酶的寄生虫的非侵入性和非致命性的方法利什曼原虫 SPP。感染。

视频: In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

在 Vivo 感染利什曼病亚马孙病评估小鼠的寄生虫毒性

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

在伤口反应期间斑马鱼中性粒细胞中趋化因子受体的实时成像

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

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 ...