作者要感谢任孙（美国加州大学洛杉矶分校）和Seungmin黄某（华盛顿大学）的技术咨询和支持。这项工作是由巴克斯特基金会，国家卫生赠款研究院（R01 CA140964和R21 AI083841 C.梁）。

下面的协议，将描述研究的病毒滴度和病毒基因组的γHV68溶骨性和潜伏性感染周期，在小鼠中，理论上可以用于评估感染其他病毒，分享相​​似的生活方式γHV68负载。 1。扩增的γHV68 <ol><li>在37℃水浴解冻γHV68冷冻小瓶。 </li><li>将病毒接种到NIH3T12或3T3细胞培养（〜50％汇合）在10厘米的菜肴和文化感染的细胞在37℃，5％的CO 2。 </li><li>检查显微镜细胞病变效应（CPE）的文化和媒体时大于80％的细胞显示4〜5天，感染后CPE收集一次。为了最大限度地提高产量病毒，受感染的细胞培养可冻结和解冻两次释放所有细胞相关γHV68。 </li><li>在1000转速在室温（RT）5分钟离心收集媒体以去除细胞碎片。 </li><li>仔细上清转移到新管（高速离心管），而不触及底部的细胞碎片。 </li><li> 12,000 rpm的高速离心机（SA - 600 Sorvall）在4 ° C下至少1.5小时集中的病毒颗粒。 </li><li>到10γ漂白粉溶液倒掉上清，并仔细地重新悬浮颗粒（病毒和细胞碎片残留）用1ml无血清DMEM。转移到1.5 ml的微量离心管中的悬浮颗粒，拌匀由涡旋。 </li><li>离心机以最大速度为2分钟，以去除残留的细胞碎片和上清液（病毒存量）转移到一个新的管。这是病毒感染的最终股票。病毒的滴度是由斑块的检测（见下文）。 </li></ol> 2。滴鼻感染小鼠γHV68 <ol><li>感染〜6周龄的小鼠与1000〜5000γHV68每小鼠的菌斑形成单位（PFU）。 </li><li>麻醉小鼠腹腔注入氯胺酮/甲苯​​噻嗪（80毫克/毫升氯胺酮和12毫克/毫升甲苯噻嗪的混合物60μL/鼠标）。监测麻醉捏小鼠脚趾。它通常需要〜5-10分钟。如果老鼠是不充分麻醉的，他们会打喷嚏病毒。 </li><li>当鼠标完全麻醉，用吸管给小鼠接种〜30μLPBS中γHV68（15μL）在每个鼻孔下拉，缓慢但稳步下降。确保鼠标确实是吸入而试图形成气泡滴。 </li><li>让鼠标恢复为10-15分钟，监测，然后把他们送回笼的小鼠（如意识，呼吸等）的福利。动物疾病和充足的食物，水，和被褥条件，每天检查。如果疾病，疼痛或窘迫的临床体征的发展，将进行常规诊断，以确定病因。失去的体重超过20％的动物，将录得最大的减肥安乐死。最终决定执行安乐死是在临床兽医的自由裁量权，是与主要研究者协商后。 </li></ol> 3。斑块含量，以确定在急性感染肺病毒滴度<ol><li> Vero细胞单层斑块的形成是由病毒感染性滴度。 </li><li>斑块检测的前一天，在2.5 × 10 4细胞Vero细胞接种/ 12孔板。细胞应分布均匀，细胞密度应达到30 - 40％的斑块检测的一天。 </li><li>准备0.5％（W / V）甲基纤维素（MC）覆盖介质（见表1）。 </li><li>牺牲后5-7天γHV68感染小鼠滴鼻感染，以确定在肺部急性期病毒滴度。对于euthanization氯胺酮盐酸（80-100毫克/千克SC，IM IP）将给药的IM戊巴比妥钠（30-50毫克/千克的IP），静脉注射过量。这种方法是由T批准他在美国兽医医疗协会。 </li><li>收获的肺和1 × PBS冲洗两次，去除血细胞。收集1毫升冰冷的完整的DMEM培养基的一个侧面的肺部和保持在冰上。迅速冻结于-80 ° C，在肺部的qPCR（见下文）审查病毒DNA载量的肺部和存储的另一边。 </li><li>使用组织匀浆均质在肺组织的最高速度为1秒，冻结与解冻三次。洗净，在每一步改变样品时，用70％乙醇的匀浆。 </li><li>在10分钟3000转离心匀浆组织于4 ° C。收集上清，斑块检测转移到新的管。 </li><li>准备在1.5毫升微量离心管与普通的DMEM涡肺组织匀浆样品连续稀释（通常是从10 °到10 -8）。充液管的适当数量与540μLDMEM培养液稀释成60μL匀浆管头。传输我从第一管60μL没有下一个，依此类推。 </li><li>准备在12孔板VERO单层吸媒体和负载200μL/连续稀释匀浆含有传染性病毒，在相反的顺序（ 从 10-8到10 °） 。每次滴定的两口井（一式两份）。 </li><li>孵育Vero细胞病毒接种1小时37 ° C。轻轻摇动板每15分钟均匀地分布在单层病毒。 </li><li>取出接种2毫升/覆盖介质（见表1）替换它。一个星期板在37 ° C。 </li><li>删除覆盖介质（不需要是完整的），更换与修复/斑块检测的染色介质（见表1）和至少1小时在室温下孵育。 </li><li>当干板，隔离斑块的数量在每个稀释度计数。为了尽量减少错误，只包含在10和100斑块之间的水井也计算在内。 </li><li>计算病毒滴度使用follo翼公式：滴度（PFU / ml的）= [（斑块数/孔）/（接种/以及体积）] ×稀释倍数。 </li></ol> 4。传染病中心检测，以确定潜伏感染脾细胞中的病毒载量<ol><li>是由传染病中心（IC）检测单层Vero细胞病毒潜伏负载。 IC检测，种子Vero细胞在6孔板的前一天（1 - 2.5 × 10 5细胞/孔，我们通常使用脾样本的11％口井，10口井将用于连续稀释的脾细胞悬浮和一个用于测试冻融循环后自发激活病毒。 </li><li>牺牲后延迟（感染后12 - 14天）感染小鼠。收获的小鼠脾和游泳池到1毫升冷冻的DMEM。 </li><li>机械分离小鼠脾。新鲜分离的小鼠脾脏转移到一个10厘米的钢板，并添加10毫升含2％FBS的DMEM培养基所需。湿两种磨砂高端玻璃显微OPE幻灯片用冰冷的介质。将脾上一张幻灯片和尼克胶囊与其他幻灯片的磨砂结束边缘磨砂。机械游离于两个幻灯片之间的脾粉碎，直到所有的红色团块（去除脂肪颗粒如果存在的话）。 </li><li>用DMEM冲洗的幻灯片，幻灯片上都恢复剩余的细胞。 </li><li>通过细胞过滤器（40微米尼龙网）传输整个组织悬液，轻轻放入50毫升的锥形螺丝帽筒。 10 cm培养皿，同时过滤含有10毫升PBS和转让脾匀浆洗净。 </li><li> 375 XG离心机的单细胞悬液10分钟，弃上清。弗里克细胞沉淀，打破现有的细胞团块。加入5毫升的ACK缓冲液（见表1）裂解红细胞。孵育5分钟，再次离心375 X G. </li><li>弃上清，重新暂停颗粒，彻​​底，还轻轻在PBS，含2％FBS。保持细胞在4 ° C。使用吸管清除杂物，如果有的话。使用自动细胞计数器（BIO - RAD）的计数活细胞。 </li><li>离心4分钟XG 375的PBS重悬脾。弃上清，重悬细胞，在4毫升完全DMEM（2 × 1毫升，将用于集成电路检测原悬挂;串行五倍稀释在IC检测用0.6毫升0.4毫升病毒DNA的制备和定量PCR， 1毫升悬液，将用于评估三个冻融循环后自发激活潜伏的病毒） </li><li>准备串行的5倍稀释（即1 / 5，1 / 25，1 / 125，1 / 250）脾悬挂在重复。预填的15毫升2.4毫升的DMEM和第一管稀释成600μL脾悬挂拌匀锥形管。从以前的管传输到600μL，下管等，不要旋涡！ </li><li>从准备6井单层Vero细胞吸介质。种子1毫升到Vero细胞的连续稀释，每个稀释dupl脾icated（10口井将用于为0，1 / 5，1 / 25，1 / 125，1 / 250稀释）。 </li><li>板8-12小时在37 ° C和5％的CO 2。不要超过24小时。吸种子脾悬挂和加载4毫升覆盖媒体（见表1）每口井。孵育一个额外的6天。 </li><li>确定斑块检测显示，板块与0.2％（W ​​/ V）结晶紫染色脾​​传染病中心的水平。 </li></ol> 5。病毒基因组的量化<ol><li>从γHV68感染的组织，如肺，脾样本，在这个实验中提取总DNA。使用DNeasy血液和组织试剂盒（Qiagen），按照制造商的协议。 </li><li>确定每个样本的DNA的浓度，以确保DNA的定量PCR检测正在使用的相同数额。设计病毒的特异性引物（〜200 bp的扩增是一个定量PCR检测的首选）。我们使用γHV68ORF56特异引物（正向引物：5＆PRIME; - GTAACTCGAGACTGAAACCTCGCAGAGGTCC - 3&#39;，反向引物：5&#39; - CCGAAGCTTGCACGGTGCAATGTGTCACAG - 3&#39;），此法和β- actin引物（正向引物：5&#39; - cacccacactgtggcccatcat - 3&#39;反向引物：5&#39; - gtgaggatcttcatgaggtagtc - 3&#39;）控制的qPCR反应。 </li><li>混合DNA样本（100 - 500吴好）和适当的引物与2 × SYBR的master mix（Bio - Rad公司的iQ SYBR绿色Supermix）。使用Bio - Rad公司的实时PCR系统量化在受感染的组织的病毒载量。在下列条件下进行PCR：95℃15分钟和45周期95 ° C为30分钟，60 °彗星为30分钟，72 ° C下30分钟，其次是病毒和熔解曲线分析，肌动蛋白扩增产物。 </li><li>为了使病毒的基因组进行量化的标准曲线，一个γHV68与未受感染的细胞中分离出基因组DNA bacmid DNA连续稀释的已知金额。并行运行的qPCR的DNA与从感染检索TED组织使用同一组病毒特异性引物。 </li><li>目前病毒DNA每单位正常化后肌动蛋白的基因组DNA的拷贝数（例如100毫微克或500纳克）组织的病毒基因组负载。 </li></ol> 6。代表性的成果： 图1描绘了γHV68感染小鼠体内的测量实验的总体方案。代表性的成果，在γHV68斑块检测确定，急性感染期间肺部的病毒滴度，如图2a所示。一个含有γHV68非功能性的突变株病毒BCL - 2（vBcl 2）在肺部复制水平与野生型γHV68后7天滴鼻感染BALB / c小鼠（6 - 7％组小鼠）。肺病毒滴度检测两组差异无统计学意义。这一数据表明，vBcl - 2不是γHV68急性感染的关键因素小鼠。然而，28天postinfection，脾脏vBcl - 2变异病毒滴度下降6 - 10 - 倍的WT，传染病中心检测（图2B）来衡量，这表明vBcl - 2突变体γHV68病毒在感染后脾延迟的维修是有缺陷的。在协议减少传染病中心滴度，vBcl - 2突变病毒的病毒基因组负载严重减少相比，野生型病毒，在反复实验第28天，如在图2C所示。潜HV68vBcl - 2突变体病毒再激活体内潜伏的感染阶段的病毒基因组的负载和频率之间的密切的关系，突出了这种突变病毒感染的潜伏期缺陷。 <img alt="图1" src="/files/ftp_upload/3472/3472fig1.jpg" /> γHV68感染小鼠的斑块形成，传染病中心和定量PCR检测的测量示意图图 1。第<img alt="图2" src="/files/ftp_upload/3472/3472fig2.jpg" /> 图2。溶解的和潜在的WT和突变γHV68 在体内的病毒感染。在7天感染后DPI（）的BALB / c小鼠的肺部急性WT和突变vBcl - 2γHV68病毒的复制（一）是由斑块检测。 （B和C）测定脾传染病中心（B）和病毒基因组的负载（三）在感染小鼠脾脏的28 dpi的传染病中心检测和定量PCR，分别。红线，指示值的平均值。 NS，不显着。

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没有利益冲突的声明。

γHV68已被广泛用于作为一种模式来理解人类γ- HVS 2,4,5的发病机制。在这个协议中，我们描述了三个经常使用的方法，包括斑块传染性病毒滴度的检测，IC病毒潜伏负载检测，病毒基因组负载的qPCR，以评估后，在小鼠滴鼻接种γHV68急性和隐性感染者。 斑块的检测已广泛使用，以确定受感染的细胞或组织中的病毒滴度，但不同的病毒正在使用的最佳条件。这主要是因为?...

<table border="1"><tbody><tr><td> 材料名称 </td><td> 制备工艺 </td></tr><tr><td>甲基纤维素（MC）病毒斑块检测的覆盖介质</td><td><ol><li>热〜250毫升的蒸馏水在TC瓶&gt; 80 ° C（沸点为3分钟微波） </li><li>添加到热水逐步2.5克管委会粉，慢火搅拌直到溶解。 </li><li>高压灭菌立即液体周期为15分钟。 </li><li>搅拌4 ° C过夜的蒸压MC媒体。 </li><li>结合200毫升2 250毫升MC媒体x MEM（GIBCO - BRL）+ 50毫升FBS的500毫升的覆盖媒体。 </li></ol></td></tr><tr><td>修复/染色中等斑块检测</td><td> 0.2％（W ​​/ V）水晶紫在20％的乙醇。 </td></tr><tr><td> ACK裂解液</td><td> NH 4 CL 0.15米，KHCO 3 10MM，EDTA 0.1MM </td></tr><tr><td>完全DMEM </td><td>贝科的修改鹰的中期（DMEM），辅以10％小牛血清（FBS的; Invitrogen公司），2毫米L -谷氨酰胺，1％青霉素，链霉素（GIBCO - BRL）。 </td></tr></tbody></table>在本议定书中使用的特定媒体见表 1。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>氯胺酮</td><td> Sigma - Aldrich公司</td><td> K2753 </td></tr><tr><td>甲苯噻嗪</td><td> Sigma - Aldrich公司</td><td> X1251 </td></tr><tr><td>甲基</td><td> Sigma - Aldrich公司</td><td> M0512 - 250G </td></tr><tr><td> 2 x纪念</td><td> Life Technologies公司</td><td> 11935 </td></tr><tr><td>细胞过滤器</td><td>屋宇署Faclon </td><td> 352340 </td></tr><tr><td>全方位组织匀浆</td><td> OMNI国际</td><td> TH115 </td></tr><tr><td> DNeasy的血液和组织套件</td><td>洽根</td><td> 69504 </td></tr><tr><td> iQTM SYBRH绿色Supermix </td><td> BioRad公司</td><td> 170-8882 </td></tr><tr><td> CFX96实时PCR系统</td><td> Bio - Rad公司</td><td> 184-5072 </td></tr><tr><td>细胞计数</td><td> Bio - Rad公司</td><td> 145-0001 </td></tr><tr><td> Sorvall SA - 600 </td><td> Thermo Scientific的</td><td> 096-124022 </td></tr></tbody></table> 表2特定的试剂和设备 。

measurement of &#947;hv68 infection in mice

γ-疱疹病毒（γ- HVS）为显着的能力，建立潜伏感染的淋巴细胞1。人类γ- HVS，如EBV和KSHV的宿主范围狭窄，严重阻碍了详细的致病性研究。小鼠γ-疱疹病毒68（γHV68）股广泛的遗传和生物与人类的γ- HVS， 相似之处是2 murid啮齿动物的天然病原体。因此，γHV68感染小鼠近交系的评价，在病毒感染的不同阶段提供了一个认识过程中γ- HVS感染病毒的生命周期和发病机制的重要模式。 鼻内接种后，γHV68肺感染的急性血症结果，就是后来成隐性感染者脾细胞及其他细胞，这可能是整个生活的主机3,4恢复解决。在这个协议中，我们将描述如何使用斑块的检测，以评估在传染性病毒滴度肺组织匀浆后滴鼻感染（DPI）在早期阶段（5 - 7天）在Vero细胞单层。虽然急性感染主要是清除2 - 3周，隐性感染的γHV68各地建立了14 DPI，以后维持在小鼠的脾。隐性感染者通常会影响在受感染的组织细胞的一个非常小的人口，据此病毒保持休眠状态，并关闭其大部分基因的表达。潜伏感染的脾细胞自发激活后病毒组织培养，可以通过扼要传染病中心（IC）检测，以确定病毒潜伏负荷explanting。要进一步病毒基因组副本的数量估计在急性和/或潜伏感染的组织，实时定量PCR（定量PCR）用于其最大的灵敏度和准确性。的qPCR和斑块的检测和/或IC检测的结果结合起来分析，就会发现病毒的时空分布在体内的复制和传染性。

Watch this Scientific Journal Video about Measurement of Î³HV68 Infection in Mice at JoVE.com

Measurement of &amp;#947;HV68 Infection in Mice

γ-疱疹病毒（γ- HVS）在其主机建立终身续保。感染小鼠γ- HV68提供了一种基因听话<em&gt;在体内</em&gt;模型为表征的生命周期/γHVs发病机制。这个协议描述γHV68感染的检测和定量菌斑形成，传染病中心，qPCR实验感染后的急性和潜伏阶段。

γHV68感染小鼠的测量

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Research

JoVE Journal

Immunology and Infection

Measurement of &#947;HV68 Infection in Mice

13.5K 次观看.  University of Southern California, Los Angeles. γ-疱疹病毒（γ- HVS）在其主机建立终身续保。感染小鼠γ- HV68提供了一种基因听话在体内模型为表征的生命周期/γHVs发病机制。这个协议描述γHV68感染的检测和定量菌斑形成，传染病中心，qPCR实验感染后的急性和潜伏阶段。

视频: Measurement of &#947;HV68 Infection in Mice

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

标签胚胎小鼠血管的方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

科研

免疫与感染

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

胞内钙的测量 2 +</sup在隔离的收缩淋巴管

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Measurement of Chitinase Activity in Biological Samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

生物样品中赤子酶活性的测定

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

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 ...

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 ...