碧迪技术提供了支持。在本文件中所表达的意见是作者的，不旨在反映美国政府的官方政策。符合动物福利法“和其他联邦法规和规章，有关涉及动物和实验动物和以实验动物护理和使用 ，国家研究理事会，1996年” 指南 “中所列的原则，坚持进行研究。本研究进行了设施是完全认可的实验室动物保健国际评估和认证协会。

1。 NALT收集和培养<ol><li>安乐死使用IACUC批准指导的小鼠。避免使用吸入麻醉药可能影响NALT。小鼠转移到无菌工作区或生物安全柜。删除鼠标的下颚和清洁用酒精和碘抹布上腭区。 </li><li>用11号手术刀片，手术刀柄仔细削减和消费上腭，通过鼠标门齿和臼齿的内侧轮廓。 </li><li>轻轻剥开腭钳，小心不要撕的口感。 </li><li>这一步是必要的文化，以减少污染和处理多种口味。地方口味，将在无菌48孔板250μL完全培养基预填充第一列的个别井（37℃）组成的RPMI 1640，辅以10％小牛血清，100μg/ mL链霉素，100 UI / mL青霉素，50微克/毫升庆大霉素1微克/毫升fungizone /两性霉素。媒体应该是缓冲碳酸盐如果口味都在5％的CO 2/95％的潮湿空气培养箱（37℃），或者使用10毫米肝素钠pH值7.4非CO 2文化，培养。 </li><li>要洗的口味，用钳子移动到每个连续行以及口味，仔细攻板之间洗涤，直到腭经历了共8个洗。 </li><li>转移到一个新的无菌48孔板含有250μL新鲜，完整的培养基中井（37℃）的口味。 </li><li>将板含到37℃培养箱培养研究期间的口味。 </li><li>收集无菌转移100μL培养基，从文化的井，每24小时Eppendorf管，更换100μL新鲜培养基，37℃的样品进行分析。 </li><li>离心10分钟，4℃，颗粒碎片380 XG培养基样品。 </li><li>从离心样品上清转移新鲜Eppendorf管和储存在-20°C，直到准备分析。 </li><li>抗原特异性IgG和IgM和IgA，或分泌细胞因子的计量标准酶联免疫吸附试验（ELISA）检测培养上清组织。 </li></ol> 2。外科消融的NALT <ol><li>雌性BALB / c小鼠，7至9周的年龄，是合适的对象，但也接受其他菌株。手术前三天提供凝胶体状湿食品鼠标来替代食物和水，将手术后管理，以适应新环境。 </li><li>管理进入口腔手术前一滴Metacam止痛药（1.5毫克/毫升或0.05毫克/下降）。 </li><li>使用IACUC批准的指导下，例如氯胺酮，乙酰丙嗪，甲苯噻嗪的混合物（KAX），麻醉小鼠和Puralube兽医软膏涂于眼睛，防止干燥。避免使用吸入麻醉药可能影响NALT。一nesthesia可以温柔捏到脚趾反射反应的情况下确认。 </li><li> 1 mL生理盐水（0.9％氯化钠注射液USP），皮下管理之间的肩膀刀片，使用1 22-28号针头和注射器，以防止手术后鼠标潜在的脱水。还管理有0.1 mL的Respiram中皮下使用期间和手术后1 22-28号针头和注射器，以促进健康呼吸之间的肩膀刀片。 </li><li>提供热支持鼠标，在随后的所有操作。 </li><li>麻醉鼠标放置在仰卧位，左右上下切牙手术缝合线的两个单独的循环使用，轻轻地撬开了嘴，露出上腭。 </li><li>用11号手术刀片下来的的NALT现场的上腭中线长度约3毫米的切口。 </li><li>切口插入0.5毫米microcurette和下边缘轻轻刮去，以扰乱了NALT。 </li><li>烧灼切口上直热烧灼单位的罚款循环停止出血。 </li><li>继续热鼠标的支持，同时恢复到全意识和活动。 </li><li>用生理盐水皮下注射，最多一天三次，每毫升注射肩胛骨之间，提供鼠标作为必要和口服止痛药，以防止脱水一次手术后3天的日子。确定通过皮肤帐篷测试，以检查皮肤膨脱水。帐篷，以便它掌握的肩膀刀片之间的皮肤和皮毛。如果皮肤迅速恢复到正常的，以前的位置，鼠标不脱水。如果皮肤持续升高，缓缓移动，鼠标是在脱水状态，需要盐水。手术后至少3天为湿的食物，以允许足够的时间恢复。 </li><li>在此之前的任何实验程序，观察跟踪体重增加为8-10天的小鼠。体重恢复不佳通常表示不完全腭裂修复，并进一步研究这些小鼠应撤回。 </li></ol> 3。准备组织学NALT和评估的NALT手术成功<ol><li> NALT手术的成功，必须是经组织学检查确定如下所述。完成后，所有的实验研究，准备颅集合小鼠安乐死使用批准IACUC指导。避免使用吸入麻醉药可能影响NALT。 </li><li>掌握的安乐死鼠标的脖子的后颈。通过双方的剪刀剪断髁过程，从其余的头骨的下颌。 </li><li>颈背开始，从头骨的皮肤和皮毛，对头盖骨的腹部慢慢地剥离和切割。 </li><li>小心取出鼻腔周围地区的皮肤剪掉尖吻皮肤完全消除。 </li><li>脱离头骨，椎骨的机智公顷的剪刀剪断。 </li><li>枕骨大孔插入剪刀和削减一半沿中线向下头盖骨允许渗透到福尔马林中固定的软组织。 </li><li>修复头盖骨在10％中性福尔马林（NBF）在室温下24小时。 </li><li>除垢，将样品和单独的磁带，在纱布包裹，以防止接触骨头，在与甲酸混合瓶Rexyn。在室温下12小时的孵育，然后自来水下冲洗约20分钟不断。 </li><li>修剪样品隔过去的眼眶，用刀片。 </li><li>样品和磁带可存储10％为短期NBF。 </li><li>放置到一个通宵处理组织的处理器的样品。这是从样品准备石蜡包埋水。 </li><li>删除处理的组织，在蜡块嵌入一个吻下来，并使其冷却。 </li><li>修剪ŕough 15微米块与自动切片机，接近NALT的大致位置，横截面，然后继续削减到玻片上安装在44.3℃水浴中5微米的部分。 </li><li>应采用苏木精伊红（H＆E）组织学染色评估NALT消融。 </li></ol> 4。从老鼠的生物样品的收集 4.1血清<ol><li>仔细热灯体温升高，血流量就会增加。外科刀片刻痕尾静脉采集血液。让血液滴成Microtainer血清分离器管自由。 </li><li>尼克用吸水材料，如纱布或毛巾，应用温和的压力，以阻止血液流动。 </li><li>允许在30分钟Microtainers的血液凝固，然后离心之间的6-15×1000Ğ为至少90秒。 </li><li>权]血清中分离到一个干净的Eppendorf管从Microtainer贮存于-80°C。 </li></ol> 4.2唾液<ol><li>持有麻醉鼠标垂直，同时吸取20-30μL无菌磷酸盐缓冲液（PBS）之间的脸颊和牙龈线。收集枪头指导脸颊和牙龈线之间，稀释唾液。 </li><li>转让稀释的唾液含有10μL的2倍蛋白酶抑制剂和存储于-20°C到一个新的管</li></ol> 4.3鼻腔分泌物<ol><li>安乐死之前收集鼻腔分泌物的鼠标，使用核准IACUC指导。避免使用吸入麻醉药可能影响NALT。 </li><li>垂直握着鼠标，认真吸取30μL无菌PBS在一个鼻孔，收集从其他鼻孔冲洗。 </li><li>转让冲洗含有10μL的2倍蛋白酶抑制剂和存储于-20°C到一个新的管</li></ol> 4.4阴道分泌物<ol><li>微量的尖端插入到日开幕é一个安乐死鼠标外阴，50μL无菌PBS冲洗阴道，并收集所有的液体微量。 </li><li>转让冲洗含有10μL的2倍蛋白酶抑制剂和存储于-20°C到一个新的管</li></ol> 5。抗原特异性抗体或细胞因子，在收集的样本，用ELISA或其他定量方法可以测量。 6。代表结果 图1提供了一个体外分析与处理的NALT含组织所涉及的步骤的一般原理。在图2（甲，乙），腭裂的大小，以及消融手术切口在（A）的位置，如虚线所示。 NALT位置，在区域对切除苏木染腭图2（c）并行的组织，臼齿的箭头表示。 图3提出的NALT中断手术的步骤，显示访问到NALT上腭（甲，乙），消融（三），并最终烧灼切口（四）曝光。一个典型的H＆E的术前鼻窦周围NALT面积截面显示在图3E，而NALT由microcurette的破坏，直接手术后的图像出现在图3F。允许有足够的时间从手术休养，应关闭切口，鼻腔缺乏NALT（ 图3G）。 通过使用这些技术获得了典型的实验结果如图4所示，从比较研究的金黄色葡萄球菌的亚单位疫苗（STEBVax）组织培养上清液和生物样品。小鼠滴鼻（IN）或腹腔内（IP）路由管理STEBVax。制定疫苗与佐剂激活的Toll样受体4通路3,1<html> 4，和控制，只有生理盐水或疫苗无佐剂。从实验组获得的培养NALT分泌到培养基中，是衡量用ELISA抗原特异性免疫球蛋白。在这个例子中（ 图4A），结果表明，IGA的最大数额从与辅助相结合的亚单位疫苗接种在小鼠实验中获得的NALT发布。 可以用来控制或NALT小鼠获得的生物样品（如血清，唾液，鼻腔分泌物，阴道分泌物） 在体内的免疫反应与组织培养的结果比较，以鼻抗原来分析。 图4b，IgA和IgG反应的疫苗接种，大大减少无功能NALT。抗原特异性IgA水平普遍高于中IgG免疫小鼠的黏膜分泌物（唾液，鼻腔冲洗）。 upload/3960/3960fig1.jpg“/&gt; 图1。NALT收集和体外培养的原理图。 <img alt="图2" src="/files/ftp_upload/3960/3960fig2.jpg" /> 图2可视鼠标腭表示NALT和手术切口的位置。由虚线表示（一）手术切口的上腭，上腭切除（B）或苏木（三）染色，以查看在上颚臼齿区（前侧腭染色暗紫色的并行NALT的大小和位置）。 NALT用箭头表示。 <img alt="图3" src="/files/ftp_upload/3960/3960fig3.jpg" /> 图3。手术NALT中断。的NALT中断手术：仰卧鼠标上腭手术前视图（A），上腭中线切口访问NALT（二）; microcurette探头通过正中切口插入扰​​乱NALT结构，关键阶段tegrity（C），（四）手术结束切口烧灼。鼻腔，手术前，H＆E染色（五），显微图像后立即手术（女）;并成功治愈，无NALT鼠标（G）的。 <img alt="图4" src="/files/ftp_upload/3960/3960fig4.jpg" /> 图4。疫苗的具体NALT培养，唾液，鼻腔分泌物和收集免疫小鼠的抗体。 NALT免费或正常对照组小鼠接种或IP与STEBVax，生物样品采集。一式三份样本的抗体水平，用ELISA法测定。 （一），NALT被删除从对照组（不手术操作）和培养检查抗体反应。疫苗的特异性IgA文化反应在接种疫苗的老鼠（学生t检验，P≤0.01，比无佐剂或没有疫苗）的统计，从不同的控制。 （二）NALT干扰大大降低了特异性抗体反应疫苗接种。有特异性抗体水平显着差异NALT和NALT组唾液IgG的结果（学生t检验，P≤0.05）以外的所有比较（无外科手术或控制）。

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Vaccine Immunol. 14, 1499-1504 (2007).</li></ol>

没有利益冲突的声明。

我们已经提出了发展动物模型的集体方法，取得生物样品，分析和研究的NALT相关免疫反应1-4。有其他因素考虑，在这些方法的性能。应遵循标准的无菌技术，手术和组织文化。隔离和文化，以及保持消毒仪器，工作区和消毒口味过程中使用的抗菌和抗真菌剂的组合将减少污染的风险。唾液，鼻洗涤和类似的粘膜分泌物标本应为潜在污染的血液检查，发现高浓度一般都在血清抗体。此外，粘膜分泌物应该只被轻微稀释分析，因为低浓度的抗体，这些样本中血清相比。 小鼠直接手术后必须保持温暖预发泄潜在的麻醉诱导低温。在其两侧交替休息小鼠手术后休养期间尽量减少不规则的呼吸。手术过程是三个人一起工作来完成这些任务更有效执行手术，协助举办着嘴，提供手术后护理的小鼠从麻醉中恢复。 这是需要使用颅截面H＆E染色，在所有实验程序或研究，以验证每个鼠标的手术成功结束。可能手术的结果是：完整的和双边的NALT消融，消融不全，或完整NALT。因为并非所有的手术将导致NALT，完全丧失残留或完整NALT动物可作为内部控制。另一个潜在的结果是，口感未能彻底治愈，留下了一个开放连接鼻腔和口腔。不完整腭愈合会导致低体重和未能茁壮成长，这些人应该从研究中删除。 检查疫苗的反应，先用小鼠模型，将有助于建立在预期的研究成果（抗体反应，生存等）为NALT的作用。手术切除的NALT局部和全身免疫促进鼻贡献的决心。这里所描述的手术方法是最直接的方法获得NALT缺乏的小鼠模型。选择敲除小鼠模型已报告缺乏NALT，但这些动物也有缺陷，在其他二级淋巴组织的发展至关重要的细胞因子或趋化因子，并可能含有额外的缺陷12,13。此外，这里所描述的方法进行检查鼻腔内的免疫反应的几个方面。使用整个上palat研究基于我们的实验结果E从组织文化的鼠标，虽然它是可能的，可用于部分。最后，培养的NALT模型是进行组织培养试验完全。

<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>不锈钢无菌手术的刀片，第11号</td><td> miltex </td><td> 4-311 </td><td></td></tr><tr><td>刀柄，第3号</td><td> miltex </td><td> 4-7 </td><td></td></tr><tr><td> 48孔细胞培养板</td><td> COSTAR </td><td> 3548 </td><td></td></tr><tr><td> RPMI 1640培养</td><td> Invitrogen公司</td><td> 11875-093 </td><td></td></tr><tr><td>胎牛血清，热灭活</td><td> GIBCO / Invitrogen公司</td><td> 16000-044 </td><td>终浓度：在培养基量的10％ </td></tr><tr><td>硫酸链霉素</td><td>西格玛</td><td> S9137 </td><td>终浓度：100微克/毫升</td></tr><tr><td>青霉素</td><td>西格玛</td><td> P7794 </td><td>终浓度：100 UI /毫升</td></tr><tr><td>庆大霉素</td><td> Sigma-Aldrich公司</td><td> G1397-10ML </td><td>终浓度：50微克/毫升</td></tr><tr><td> fungizone </td><td> GIBCO / Invitrogen公司</td><td> 15290-018 </td><td>终浓度：1微克/毫升</td></tr><tr><td>肝素钠</td><td> Sigma-Aldrich公司</td><td> H0887 </td><td>终浓度：10毫米</td></tr><tr><td>离心离心管</td><td> Eppendorf公司</td><td> 022364111 </td><td></td></tr><tr><td> nutra凝胶樱桃味鼠标湿食物</td><td>生物服务</td><td> S4798托盘</td><td></td></tr><tr><td> metacam </td><td>勃林格殷格翰</td><td> 601531000 </td><td>一滴1.5毫克/毫升口服混悬液</td></tr><tr><td>氯胺酮</td><td>辉瑞公司</td><td> 00856440301 </td><td>最后有限公司NC：6.06毫克/毫升</td></tr><tr><td>乙酰丙嗪</td><td> vedco </td><td> VEDC207 </td><td>终浓度：0.061毫克/毫升</td></tr><tr><td>甲苯噻嗪</td><td>劳埃德</td><td> 4811 </td><td>终浓度：0.667毫克/毫升</td></tr><tr><td> puralube兽医软膏</td><td> pharmaderm动物健康</td><td> 1621 </td><td></td></tr><tr><td> 0.9％氯化钠注射液的USP </td><td>巴克斯特</td><td> 2B1302 </td><td></td></tr><tr><td> 0.5毫米Microcurette </td><td> roboz </td><td> RS-6350 </td><td></td></tr><tr><td>热烧灼单位</td><td>盖格尔</td><td> 150-S/150A-S </td><td></td></tr><tr><td> TCU更换提示，直精细环路</td><td>盖格尔</td><td> 214 </td><td></td></tr><tr><td>手术剪</td><td></td><td></td><td></td></tr><tr><td> 10％中性福尔马林</td><td>西格玛</td><td> HT501128 </td><td></td></tr><tr><td>蚁酸</td><td>费舍尔</td><td> A119P-4 </td><td>混合甲酸426毫升到1047毫升的自来水，再加入45毫升Rexyn 101（高） </td></tr><tr><td> rexyn 101（高） </td><td>费舍尔</td><td> R231-500 </td><td></td></tr><tr><td>组织-Tek的贵宾E150/E300 </td><td>樱花</td><td></td><td></td></tr><tr><td>旋转式切片机</td><td>徕卡</td><td> RM2255 </td><td></td></tr><tr><td> Mayer的苏木</td><td>西格玛</td><td> MHS16-500ML </td><td></td></tr><tr><td>吉尔一号苏木</td><td>西格玛</td><td> GHS116 </td><td></td></tr><tr><td>曙红B </td><td>西格玛</td><td> 2853 </td><td></td></tr><tr><td> microtainer血清分离</td><td>屋宇署中号edical </td><td> 365956 </td><td></td></tr><tr><td>磷酸盐缓冲液</td><td></td><td></td><td> 100毫米的NaH 2 PO 4计 ，140 mM氯化钠，pH值7.4 </td></tr><tr><td>蛋白酶抑制剂，EDTA无</td><td> Thermo Scientific的</td><td> 78415 </td><td></td></tr></tbody></table>

examining the role of nasopharyngeal-associated lymphoreticular tissue (nalt) in mouse responses to vaccines

鼻咽相关淋巴组织（NALT）发现，在人类，啮齿动物和其他哺乳动物，有助于在1-3鼻窦的免疫力。 NALT是两个平行的钟形结构位于硬腭以上的鼻腔，通常被认为是粘膜相关淋巴系统4-6二次元件。位于NALT穿插离散B和T淋巴细胞，树突状细胞抗原提呈4,7,8车厢。这些细胞被包围的上皮细胞层间与M-细胞是负责从空中通道9,10粘膜表面的抗原检索。正准备通过NALT循环的幼稚淋巴细胞回应7呼吸道病原体的第一次接触。虽然NALT在人类消失两岁，韦氏环和类似结构的淋巴器官继续坚持日roughout生活6。相反，人类，小鼠保留整个生命NALT，从而提供了一个方便的动物模型，为11鼻窦内的免疫反应的研究。 NALT单细胞悬液的文化是不实际的，由于产量低的单核细胞。然而，NALT生物学可以完整的器官体外培养的研究，该方法具有额外的优势，保持自然的组织结构。 在体内研究，提出限制到NALT缺陷的基因剔除模型是目前没有可由于缺乏了解的发展途径。例如，而淋巴毒素α基因敲除小鼠已萎缩NALT，Peyer氏斑，周围淋巴结滤泡树突状细胞和其他淋巴组织也改变了这些基因操作的小鼠12,13。作为一种替代基因敲除小鼠，手术消融带够永远接受从鼻腔消除NALT，而不影响其他组织。产生的小鼠模型已被用于建立NALT和免疫反应的疫苗1,3之间的关系。串行收集血清，唾液，鼻腔冲洗和阴道分泌物的宿主反应的基础上建立的疫苗，而直接源于NALT的免疫反应，可以通过组织培养证实是必要的​​。以下步骤概述了手术，组织培养和样品收集必要的检查局部和全身体液鼻腔（IN）的疫苗接种的免疫反应。

Watch this Scientific Journal Video about Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines at JoVE.com

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

描述的方法来检查鼻咽相关淋巴组织（NALT）鼻内疫苗的小鼠鼻腔和全身免疫反应的贡献。我们展示了一个外科手术，以建立一个的NALT依赖鼠标模型，<em&gt;体外</em&gt;提取NALT的文化。

鼻咽癌相关淋巴组织（NALT）在鼠标的响应中的作用研究疫苗

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

examining-role-nasopharyngeal-associated-lymphoreticular-tissue-nalt

Research

JoVE Journal

Immunology and Infection

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

25.4K 次观看.  U.S. Army Medical Research Institute of Infectious Diseases. 描述的方法来检查鼻咽相关淋巴组织（NALT）鼻内疫苗的小鼠鼻腔和全身免疫反应的贡献。我们展示了一个外科手术，以建立一个的NALT依赖鼠标模型，体外提取NALT的文化。

视频: Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

科研

免疫与感染

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility provides X. fastidiosa a means for long-distance intra-plant movement and colonization, contributing toward pathogenicity in X. fastidiosa. The twitching motility of X. fastidiosa is operated by type IV pili. Type IV pili of Xylella fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon encoding proteins that are involved with signal transduction pathways. To elucidate the roles of pilG in the twitching motility of X. fastidiosa, a pilG-deficient mutant Xf&#916;pilG and its complementary strain Xf&#916;pilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in Xf&#916;pilG, Xf&#916;pilG-C and its wild type strain. Using this recording system, it permits long-term spatial and temporal observations of aggregation, migration of individual cells and populations of bacteria via twitching motility. X. fastidiosa wild type and complementary Xf&#916;pilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant Xf&#916;pliG exhibited the twitching deficient phenotype. This study demonstrates that pilG contributes to the twitching motility of X. fastidiosa. The microfluidic flow chamber is used as a means for observing twitching motility.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce&#39;s disease in grapevine.

抽动运动功能的可视化和表征的角色的<em&gt; PilG</em&gt;在<em&gt;叶缘焦枯病菌</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

成人斑马鱼对 DNA 疫苗临床前筛选的免疫研究

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

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 ...