This work was supported by "Spanish Ministry of Economy and Competitiveness" [grant number BIO2014-5258P], "European Commission" by the H2020 programs [grant numbers TBVAC2020 643381].

所有小鼠保持在受控条件下和对疾病的任何迹象观察。试点工作是在与欧洲和国家指令实验动物保护，并与主管地方伦理委员会批准同意进行。 1. BCG丹麦的量化甘油股票和结核 分枝杆菌的制备注:所有所述的协议进行BSL3条件下进行。 <ol><li> BCG丹麦或分枝杆菌H37Rv菌株的解冻冷冻甘油股票和在10ml 7H9介质10补充有吐温80 0.05％（体积/体积）和ADC（白蛋白，葡萄糖，过氧化氢 ​​酶）的10％（V / V）接种100微升。 </li><li>静态条件下孵育一周，在37℃，直到培养是在对数生长期。 </li><li>由10毫升的文化转移到200毫升的新鲜培养基扩大细菌培养。孵育另外20静态条件下天。 </li><li>转移至50毫升离心管中并离心15分钟，在2500×克</li><li>丢弃上清液，剩余体积重悬细菌沉淀。添加到每个管无菌3-毫米直径的玻璃珠（10 - 15每管）。 </li><li>涡大力以1分钟内要分离的细菌团块。重悬在10毫升PBS中的各粒料用Tween-80 0.05％（体积/体积）。 </li><li>离开管10分钟允许的最大细菌聚集和玻璃珠定居下来。 </li><li>从每片含小团块和单细菌和转让卷一个新鲜的50毫升离心管（最终恢复卷38ml）中的4管的恢复9.5毫升上清液。离心机在400×g下10分钟。 </li><li>在一个单一的新鲜的50毫升离心管中回收35毫升上清液（主要含有单一细菌），并添加15毫升的PBS与甘油50％（体积/体积），得到15％的最终甘油浓度（体积/体积）。 </li><li>轻轻混匀并在1毫升的等分试样散布在适当的管中冷冻。存储在-80°C（一阄提供大约50等分甘油）。 </li><li>量化甘油股票，解冻甘油天冷冻后，在含有900微升的PBS单独1.5毫升管执行七点十分倍连续稀释。 </li><li>板在7H10固体琼脂培养基10 100微升每种稀释液的补充有55毫米直径的皮氏培养皿中制备的ADC 10％（体积/体积）。 </li><li>小心加入3毫米直径的玻璃珠（约10每盘）并轻轻摇动板均等地分布体积随琼脂平板上。丢弃珠和密封琼脂平板用封口膜。 </li><li>孵育21天，在37℃，并通过在稀释那里单个菌落可以准确分辨计数菌落形成单位（CFU）测定细菌的浓度。 </li></ol> 2.鼠标疫苗接种<ol><li>解冻预先定量卡介苗甘油并在PBS中稀释至准备两个悬浮液。计算最终体积考虑每只动物的剂量是用于皮下施用100微升（每毫升10 7 CFU用于皮下接种）和40微升用于鼻内给药（2.5×10 7 CFU鼻内疫苗）。 </li><li>使用专门麻醉工作站啮齿动物用异氟烷和氧气的混合物麻醉小鼠。使用异氟醚的5％以诱导麻醉和2％，以保持麻醉室中的小鼠。 （其他公认的麻醉方法都可以使用）。 </li><li>用于皮下施用疫苗填充1毫升注射器用＆G针用1ml细菌悬浮液（10 7 CFU /毫升）。去除气泡。 </li><li>放置在层流罩内俯卧的平坦表面麻醉小鼠并接种皮下100μl的疫苗（10 6 CFU /剂量）在小鼠的一侧面背面。返回鼠标笼并监视它，以确保它propeRLY从麻醉中恢复。 </li><li>改变各小鼠施用之间的注射器针头。如上所述除去气泡。 </li><li>用于鼻内给药，将麻醉小鼠在引擎盖内仰卧位。 </li><li>用微量取20微升悬浮的2.5×10 7 CFU /毫升。将接种下降逐滴两鼻孔直到整个卷已沉积之间，留下滴之间的时间来让鼠标呼吸音量注:鼠标可以在此过程中因呼吸窘迫这么辛苦，他们应该留给同化管理下一个，以防止它尽可能事先每下降。 </li><li>重新填写与另一个20微升微量并重复该操作。如果鼠标开始从第一接种后麻醉醒来，将其放置在麻醉室中的第二个前。返回鼠标笼子，并确保其恢复正常从麻醉。 </li></ol> 3.鼠标鼻内挑战与应变分枝杆菌<ol><li>解冻预先定量分枝杆菌甘油和稀释在PBS准备每毫升2500 CFU的菌悬液。计算最终体积考虑每只动物的剂量是40微升。 </li><li>在2.5中所述分枝杆菌是鼻内接种 - 2.7。每只小鼠最后的挑战剂量为10 2 CFU / 40微升。 </li></ol> 4.肺引起的免疫响应分析<ol><li> 肺细胞悬浮液 注:在肺中疫苗诱导的免疫应答的评估需要单细胞悬浮液的生成。为了这个目的，我们使用的组织离解，如GentleMACS。 <ol><li>牺牲颈椎脱位（或其他公认的人性化的方法）鼠标和鼠标确保是死触摸眼球，以确认没有瞬目反射。 </li><li>内部层流罩，提取第lungs用无菌剪刀和镊子，将其放在一个干净的表面。干净的肺移除气管和结缔组织，以及肺用PBS转移到1.5毫升试管（保持在冰上，直到处理）。 </li><li>以产生细胞悬浮液，将肺在含有5ml的10mM HEPES-NaOH中pH为7.4，150 mM氯化钠，5mM的氯化钾，1mM的MgCl 2的1.8毫米氯化钙2器官离解（C-离解器管）的适当管缓冲。 </li><li>添加的胶原酶D 100毫克/毫升100微升（2毫克/毫升终浓度），和DNA酶I 20000 IU / ml的40微升（160单位/毫升的最终浓度。 注:胶原酶D或DNA酶I的储备溶液制备分别溶解在PBS中冻干酶或甘油50％（V / V），20毫摩尔Tris -盐酸pH为7.5ý的MgCl 2为1mM，。 </li><li>将管在组织离解和使用肺碎片预定义的程序m_lung_01。孵育在37℃下在管的水浴中30分钟。 </li><li>第广场在离解ë管，并使用完全同质化的肺​​成单细胞悬浮液预定义的程序m_lung_02。 </li><li>通过装配到一个50毫升的离心管70微米尼龙细胞过滤器通过细胞悬浮液。用10ml的RPMI 1640培养基中洗涤过滤器。 </li><li>离心细胞在400 XG 5分钟，弃上清。 </li><li>加入1ml红细胞裂解缓冲液中，涡旋混合并孵育在室温下1分钟。 </li><li>加入10 mL的RPMI 1640培养基中，离心细胞在400×g下5分钟。 </li><li>在0.5的完全RPMI 1640培养基中重悬细胞在补充有10％热灭活胎牛血清（FCS），2mM的L-谷氨酰胺，100微克/毫升链霉素，100国际单位/毫升青霉素和50μM2-巯基乙醇。 注:FCS的温育在56℃灭活30分钟。 </li><li>计数细胞并添加完全RPMI 1640培养基中，达到10 7个细胞/ ml的终细胞密度。 注:对于细胞计数，我们使用自动细胞计数器。 </li><li>分发100微升细胞悬浮液在U型底96孔无菌板（10 6个细胞/孔）。 </li><li>添加50μl/孔的完全RPMI 1640培养基中的15微克/毫升的商业H37Rv的纯蛋白衍生物（PPD）（5微克/毫升的最终浓度）。 </li></ol></li><li> 上清液收集通过ELISA的细胞因子测定 <ol><li>孵育板从4.1.14为在37℃下48小时和5％ 的 CO 2。 </li><li>离心机板在400 xg离心5分钟，回收100μl的上清液。 </li><li>存储在-80℃。 </li><li>分别上清液稀释1/10和1/2在分析缓冲液的IFNγ和IL17A的决心。确定与商业ELISA试剂盒细胞因子浓度，根据制造商的说明（ELISA开发工具包）。 </li></ol></li><li> 细胞内染色测定细胞因子-producing CD4 +细胞通过流式细胞仪<ol><li>孵育板从4.1.14为在37℃下18小时和5％ 的 CO 2。 </li><li>添加1.5微升的布雷菲德菌素A 1毫克/毫升（股票溶于DMSO）至每孔（10微克/毫升终浓度）孵育额外六个小时。 </li><li>从离心4.3.2板1分钟在3200 XG弃上清。 </li><li>在抗小鼠CD4-FITC的50微升的RPMI 1640稀释，用10％的FCS（1/500）和悬浮细胞孵育在4℃下10分钟。 </li><li>离心从4.3.4板1分钟3,200 xg离心，弃去上清液，加入100μl/孔的固定溶液。孵育在4℃下20分钟。 </li><li>用通透的解决方案150微升/洗洗细胞两次。 注:10倍浓缩储液稀释于去离子水中以制备1×工作溶液。 </li><li>在50μl抗小鼠IFNγ-APC和抗小鼠IL17A-APC.Cy7的悬浮细胞稀释在permeabil化溶液（1/250两种抗体）。 </li><li>在室温下孵育1小时。 </li><li>用通透的解决方案150微升/洗洗细胞两次。 </li><li>悬浮细胞在200μl的PBS中进行流式细胞采集。 </li></ol></li></ol> 5.免疫球蛋白支气管肺泡灌洗液分析（BAL） <ol><li> BAL样品的获得 <ol><li>牺牲颈椎脱位（或其他公认的人性化的方法）鼠标和鼠标确保是死触摸眼球，以确认没有瞬目反射。 </li><li>揭露使用适当的精度无菌镊子和剪刀气管并执行气管一小截，确保不完全切除它。小心不要引起出血可能污染BAL样本。 </li><li>引入进气管连接到一个无菌的1-ml注射器用800微升冰冷的PBS的套管。 </li><li>慢慢地接种PBS进入肺部，则RECOVER BAL体积，以避免肺塌陷拉回注射器的活塞速度非常缓慢。 注:在500到600微升的恢复是令人满意的。 </li><li>转移BAL卷成1.5毫升管中并确认BAL保持无色，表明没有被血液污染的（保持在冰上，直到处理）。 </li><li>离心机BAL样品在400 xg离心5分钟，并转移上清液至新管。 </li><li>商店在上清-80℃。 </li></ol></li><li> 的IgA在BAL上清测定 注意:使用的所有试剂是在室温下。 <ol><li>用100μl的PPD在PBS（10微克/毫升）稀释分枝涂层高蛋白结合聚苯乙烯平底96孔板结核病特异性IgA检测，或用100微升BAL上清液稀释10倍的PBS总IgA决心。 </li><li>在4℃孵育过夜。 </li><li>弃上清并点击纸巾到D盘RY。用200微升/ PBS的好洗。 </li><li>用200μl/孔的牛血清白蛋白（BSA）的1％（重量/体积）在PBS吐温80 0.05％（体积/体积）（封闭缓冲液）阻断。 </li><li>孵育在RT 1小时。 </li><li>用200μl/ PBS吐温80 0.05％（体积/体积）（洗涤缓冲液）的孔洗一次。 </li><li>加入100μl未稀释的BAL上清液中的PPD-包被的孔。在室温下孵育两小时。 注意:如果总IgA判定与PPD特异性的IgA并行完成，离开总IgA孔用100μl洗涤缓冲液这个温育过程。 </li><li>用洗涤buffer.0170004的200μl/孔洗涤三次</li><li>添加100μl的辣根过氧化物酶（HRP）的缀合抗小鼠IgA稀释在封闭缓冲液（1 / 10,000）。 </li><li>孵育1小时RT。 </li><li>用洗涤缓冲液200μl/孔洗涤五次。 </li><li>添加的3,3100μl/孔&#39;，5,5&#39;-四甲基联苯胺（TMB）。孵育20分钟，在T室温他暗。 </li><li>添加100μl/孔的H 2 SO 4 0.5N。读在分光光度计板在450nm的波长。 </li></ol></li></ol> 6.细菌量测定肺<ol><li> 龙同质化和电镀 <ol><li>在攻击后四个星期，牺牲通过颈脱位（或其它公认的人道方法）的小鼠，并确保小鼠死触摸眼球确认不存在眨眼反射的。 </li><li>鼠标放置在固定在层流罩内平坦并消毒表面仰卧位。 </li><li>做一个切口，并从胸部区域去除皮肤留下明显的胸区域。 </li><li>剪下的肋骨和收获用无菌剪刀和镊子的肺和心脏。将机关在清洁的表面。 </li><li>通过移除心脏，气管和结缔组织清理肺部和肺部用1毫升去离子水转移到1.5毫升管（冰上保持，直至处理）。 </li><li>使用无菌镊子对各小鼠的肺转移到用于器官均质的适当管（M - 离解器管），含有1毫升去离子水。 </li><li>放置管在组织离解器和使用预定义的程序RNA_1至均质化肺部。 </li><li>使万分之五倍连续稀释在1.5毫升管，开始用100μl肺匀浆在900微升稀释。 </li><li>板稀释为1.11-1.14描述。 </li></ol></li><li> BCG和分枝杆菌之间的差异，通过PCR 注:在从6.1.6对应于鼻内途径接种BCG组的板块，我们分析殖民地的代表人数由BCG和分枝杆菌之间的挑剔的PCR，以确保CFU认为，评估疫苗赋予的保护效果相当以独特的分枝杆菌。 RD9的这种PCR扩增目标，基因组区域中BCG和M.涂不同berculosis，得到2618碱基对杆菌H37Rv，和465碱基对的BCG 11的片段。 <ol><li>制备主混合物的库存为每个样品体积所指示所有的样品，然后在PCR管分配（9微升/样品）。 注:缓冲区5X:2微升。正向引物25μM（GTGTAGGTCAGCCCCATCC）:0.32微升，反向引物25μM（GCCCAACAGCTCGACATC）:0.32微升，Taq DNA聚合酶5单位/微升:0.04微升，去离子水2 O:6.32微升。 </li><li>触摸菌落用无菌牙签在含有预混PCR管样品浸泡牙签。 </li><li>用下面的程序运行的PCR:10分钟95℃（此步骤是触发细菌破坏），10分钟95℃（此步骤是触发细菌破坏），35个循环（在95℃下30秒，1分钟在58℃，并在72℃4分钟）。 </li><li>加载的PCR样品用溴化乙锭和运行的样品在1％琼脂糖凝胶，以可视化的片段。 </l我&gt; </ol></li></ol>

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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=Bacteriology+of+tuberculosis:+laboratory+methods.">Bacteriology of tuberculosis: laboratory methods.</a> Am J Public Health Nations Health. 48 (7), 844-853 (1958).</li><li>Brosch, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+evolutionary+scenario+for+the+Mycobacterium+tuberculosis+complex.">A new evolutionary scenario for the Mycobacterium tuberculosis complex.</a> Proc Natl Acad Sci U S A. 99 (6), 3684-3689 (2002).</li><li>Kaushal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+vaccination+with+attenuated+Mycobacterium+tuberculosis+induces+strong+central+memory+responses+and+protects+against+tuberculosis.">Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis.</a> Nat Commun. 6, 8533 (2015).</li><li>Lochhead, J. J., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+biologics+to+the+central+nervous+system.">Intranasal delivery of biologics to the central nervous system.</a> Adv Drug Deliv Rev. 64 (7), 614-628 (2012).</li><li>Lochhead, J. J., Wolak, D. J., Pizzo, M. E., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+transport+within+cerebral+perivascular+spaces+underlies+widespread+tracer+distribution+in+the+brain+after+intranasal+administration.">Rapid transport within cerebral perivascular spaces underlies widespread tracer distribution in the brain after intranasal administration.</a> J Cereb Blood Flow Metab. 35 (3), 371-381 (2015).</li><li>Griffiths, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholera+toxin+enhances+vaccine-induced+protection+against+Mycobacterium+tuberculosis+challenge+in+mice.">Cholera toxin enhances vaccine-induced protection against Mycobacterium tuberculosis challenge in mice.</a> PLoS One. 8 (10), e78312 (2013).</li><li>Hirota, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+Th17+cells+in+Peyer's+patches+is+responsible+for+the+induction+of+T+cell-dependent+IgA+responses.">Plasticity of Th17 cells in Peyer's patches is responsible for the induction of T cell-dependent IgA responses.</a> Nat Immunol. 14 (4), 372-379 (2013).</li><li>Jaffar, Z., Ferrini, M. E., Herritt, L. 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The authors have nothing to disclose.

Although current vaccine against tuberculosis, BCG, is the most widely administered vaccine in history, tuberculosis remains one of the leading causes of death and morbidity from infectious diseases worldwide. This paradox is explained by the lack of protection of this vaccine against pulmonary tuberculosis, the responsible form of transmission. New vaccination approaches effective against pulmonary forms of the disease are urgently needed, as they would have the greatest impact on disease transmission globally.
Our data clearly show that a change in the route of administration of BCG to mimic the natural route of infection could be a successful strategy to prevent pulmonary tuberculosis. Our results are in accordance with other authors showing equivalent data in different animal models, including guinea pigs and non-human primates (NHP) 6,9,12.
Remarkably, our unpublished data indicated that the volume of administration by the intranasal route is a critical step of the protocol. These results revealed that following intranasal delivery of 100 H37Rv bacteria resuspended in 10 &#956;l (instead of 40 &#956;l), we only recovered lung CFUs from around 20% of the animals (data not shown. 
Importantly, one of the possible limitations of intranasal administration as a vaccine delivery route in clinic could be its proximity with the central nervous system13,14. In this regard, aerosol immunization might be safer as delivery route for pulmonary tuberculosis vaccines in humans.
This protocol describes a standardized methodology that can be adapted to other studies, as comparison of pulmonary vaccines, or different routes of pulmonary immunization, including aerosol.
Our data suggest that the analysis in parallel of protective efficacy and immune response, using lung samples from the same animal, could be a useful tool to identify biomarkers of protection. In this regard, our results reveal that lung IL17, but not IFN&#947;, seems to correlate with a better vaccine protective efficacy. These data highlight a possible role of IL17 in the protective response induced by intranasal BCG, which is in accordance with data reported by other authors using mucosal subunit TB vaccines 7,15. Our data also indicate that presence of IgA (both total and MTB-specific) in BAL samples correlates with protection conferred by intranasal BCG. Importantly, we described previously, in agreement with other works, that IL17 contributes to traslocation of IgA to respiratory airways and gut lumen.9,16,17

结核病（TB）是全球领先的传染性疾病导致性比HIV相关的死亡在世界之一，耐多药菌株的增加上升相结合，使得TB惊人的全球性健康问题1。新的诊断工具，更有效和毒性较小的药物，以及新的安全和有效的结核病疫苗的迫切需要，特别是在发展中世界。 减毒活卡介苗（BCG）是目前预防结核病，已在皮内诞生以来全球20世纪70年代给予唯一的持牌疫苗。卡介苗被认为是有效地防止儿童疾病（脑膜炎和粟粒TB）的严重形式，但已经显示出对负责疾病变速 ​​器2的肺结核功效不一致。 肺接种疫苗，它模仿结核感染的自然路线，代表了当地引发宿主的免疫反应有吸引力的方法秒。在这点上，相比于皮下或皮内途径3-6在不同的有关结核的动物模型的各种临床前工作已经证明以下肺免疫更大疫苗效力。尽管如此，通过肺接种引发的保护机制尚不十分清楚。在过去的几年中，一些作品指着具体TB-粘膜免疫应答的重要因子IL17介导的反应，如在缺陷的IL17粘膜疫苗诱导的保护效力小鼠模型受损7,8。 最近，我们展示的第一次鼻内BCG管理保护的DBA / 2鼠标，鼠标应变特点是皮下BCG免疫后9缺乏保护的。这些结果表明，呼吸道结核疫苗可以减少在流行国家，皮内BCG被认为是对pulmon无效结核病率更有效元TB。

<table border="0" cellpadding="0" cellspacing="0" width="895">
	<tbody>
		<tr height="21">
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			<td style="width:119px;">038-138003</td>
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			<td height="21" style="height:21px;width:409px;">Middlebrook 7H10 Agar</td>
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			<td height="21" style="height:21px;width:409px;">1-ml syringe 26GA 0.45x10 mm</td>
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			<td style="width:119px;">130-093-236</td>
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			<td style="width:119px;">11088882001</td>
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			<td style="width:119px;">35050-061</td>
			<td>100X concentrated</td>
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			<td height="21" style="height:21px;width:409px;">Scepter Cell Counter Sensors, 40 &#181;m</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:119px;">PHCC40050</td>
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			<td height="42" style="height:42px;width:409px;">Mycobacterium Tuberculosis - Tuberculin PPD</td>
			<td style="width:176px;">Statens Serum Institut (SSI)</td>
			<td style="width:119px;">2390</td>
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			<td style="width:176px;">Mabtech</td>
			<td>3321-1H</td>
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			<td height="21" style="height:21px;width:409px;">Mouse IL17A ELISA development kit&#160;</td>
			<td style="width:176px;">Mabtech</td>
			<td>3521-1H</td>
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			<td style="width:119px;">B7651</td>
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			<td style="width:176px;">BD</td>
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			<td height="21" style="height:21px;width:409px;">APC Mouse Anti-mouse IFNg</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">554413</td>
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			<td height="21" style="height:21px;width:409px;">LACHRYMAL OLIVE LUER LOCK 0.60 x 30 mm. 23G x 1 1/4&#8221;</td>
			<td style="width:176px;">UNIMED</td>
			<td style="width:119px;">27.134</td>
			<td>Used as trachea cannula for BAL</td>
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			<td height="21" style="height:21px;">high-protein binding polystyrene flat-bottom 96-well plates MAXISORP</td>
			<td style="width:176px;">NUNC</td>
			<td style="width:119px;">430341</td>
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			<td height="21" style="height:21px;width:409px;">Albumin, from bovine serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">A4503</td>
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			<td height="21" style="height:21px;width:409px;">Goat Anti-Mouse IgA (&#945;-chain specific)&#8722;Peroxidase antibody</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">A4789</td>
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			<td height="21" style="height:21px;width:409px;">3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB)&#160;</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">T0440</td>
			<td></td>
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			<td height="21" style="height:21px;width:409px;">MyTaq DNA Polymerase</td>
			<td style="width:176px;">Bioline</td>
			<td style="width:119px;">BIO-21107</td>
			<td>The kit Includes Buffer 5x</td>
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	</tbody>
</table>

protective efficacy and pulmonary immune response following subcutaneous and intranasal bcg administration in mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

这部作品描述了BCG给药两种途径的比较:皮下和鼻内。皮下途径是可比的皮内，这是全世界的卡介苗目前临床路径。疫苗接种的鼻内途径旨在模仿M的感染的天然途径肺结核 ，目标是直接诱导免疫反应在肺，这种病原体的主要靶器官。 图1描述了工作流程遵循。八到十周龄的雌性DBA / 2小鼠用10 6卡介苗CFU丹麦通过施用皮下或鼻内途径接种。 8周后，一组老鼠被牺牲分析由疫苗接种引起的肺免疫反应。 BAL样本首先获得，然后我们收获的肺。为了研究疫苗赋予的保护效果，我们接种各色核苷酸组小鼠与M的低剂量鼻内攻击结核分枝杆菌H37Rv株。一个月后，我们牺牲动物和收获肺。在这种情况下，对于每个动物，我们使用左肺确定细菌负荷和右肺来评估疫苗诱导的免疫应答攻击后。的目标是产生为了研究保护的潜在相关因素在肺部每只动物的细菌负荷和免疫反应的数据。 如图2中所示，肺离解，以获得任一器官匀浆或细胞悬浮液。均质肺铺板固体琼脂培养基上，以确定细菌负荷4周攻击后。以下肺酶消化胶原酶D和DNA酶I获得细胞悬浮液，研究疫苗诱导的免疫反应。 我们的结果清楚地表明，相当tÓ皮下途径，疫苗接种的鼻内途径赋予挑战（图3A）后更大的保护效力在肺部四个星期。此外，我们证实，在从鼻内BCG疫苗组的肺部细菌负载对应杆菌H37Rv和不卡介苗。为此，我们分析菌落有代表性的若干从该基通过特异性PCR为RD9基因组区域，其放大在BCG和M.不同长度的片段结核病 （图3B）。图清楚地表明，所有的菌落分析了提供0.4千碱基对的片段，其对应于分枝杆菌H37Rv。 我们的数据显示滴鼻接种卡介苗，并在攻击前肺部疫苗诱导的免疫反应所赋予的保护效果之间的相关性。鼻内BCG显然引发更高的IL17和IFNγ生产的肺，用ELISA法测定（图图4A）。 （数据未显示），这些数据是通过细胞内染色（ICS）的确认和流式细胞术。此外，我们还发现BAL样品（图4B）在总的和PPD特异性的IgA浓度的高浓度，这表明肺BCG接种诱导产生的IgA和易位到呼吸道，。 最后，我们研究了在肺部卡介苗诱导的免疫应答攻击后（图5）。我们的数据显示IL17和IFNγ之间的差异;只鼻内BCG组检测IL17A生产CD4 +细胞，而IFNγ产生细胞在感染分枝杆菌疫苗接种无关各组中。对应于IL17产生细胞和细菌负荷每只动物的数据的表示法表明IL17的存在和肺部细菌负载降低之间的显著相关性，这不是在IFNγ的情况下观察到。 <img alt="图1" src="/files/ftp_upload/54440/54440fig1.jpg" /> 图1 的工作流程比较BCG接种的鼻内和皮下途径。八周后接种BCG，一组小鼠（6每个实验组）被用来收获肺和执行BAL，和分析由疫苗诱导的肺的免疫反应。另一组小鼠（6 /组）中，接种M的低剂量鼻内攻击结核分枝杆菌H37Rv株（100 CFU）。一个月后，处死动物和细菌负荷左肺和右肺PPD特异性免疫反应进行了分析。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54440/54440fig2.jpg" /> <str翁&gt;图2. 肺标本的处理。使用了组织离解处理肺部。在实验中，以确定细菌负荷，肺直接制版他们坚实的琼脂培养基之前均质。在需要肺细胞悬浮液的实验，这是发生在与胶原酶D和DNA酶I酶消化。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54440/54440fig3.jpg" /> 图3. 保护效果卡介苗免疫赋予的。6的DBA的组/ 2小鼠通过皮下（卡介苗SC），鼻内（卡介苗）的路线，或者未接种疫苗的（Unvacc）用BCG丹麦疫苗10 6 CFU接种。两个个月接种后，小鼠用低剂量的鼻内接种（100CFU）H37Rv的挑战，并在肺中一个月后细菌负荷进行了测定。的两个独立的有代表性的实验中示出（A）中的数据的图表表示为平均值±SD。进行与邦费罗尼事后分析单向ANOVA检验来计算统计学意义。（二）从BCG滴鼻组单菌落通过PCR特异性RD9地区（不同的卡介苗和分枝杆菌基因组）进行了分析有代表性的若干辨别BCG和分枝杆菌菌落。 （此前公布的9）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54440/54440fig4.jpg" /> 图4. 疫苗特异性肺免疫反应分析与前向分枝杆菌挑战。6 DBA / 2组小鼠通过皮下（卡介苗sc）或鼻内（卡介苗）的路线，或者未接种疫苗的（Unvacc）用BCG丹麦疫苗10 6 CFU（A）疫苗接种术后两月接种后，得到从收获的肺细胞悬浮液。如在方法部分和IL17A（左图）和IFNγ（右面板）产量通过ELISA分析所述细胞用PPD刺激。（B）中的总IgA和M.肺结核 （MTB）特异性的IgA用ELISA法从BAL样本进行分析。从两个独立实验汇总数据被示出。在图的数据被表示为平均值±SD。 （此前公布的9）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54440/54440fig5.jpg" /> 图5. 疫苗特异性与分枝杆菌H37Rv攻击前肺的免疫应答分析6的DBA的组/ 2小鼠通过皮下（卡介苗sc）或鼻内（卡介苗）的路线，或者未接种疫苗的（NV）用BCG丹麦疫苗10 6 CFU接种。未接种，未感染的小鼠对照组也被列入（NV / NI）。两个个月接种后，小鼠用低杆菌H37Rv剂量（100 CFU）鼻内攻击，及1个月后动物安乐死。左和从相同动物右肺用于测定细菌负荷和IL17A-（A）或IFNγ-（B）中的CD4 +细胞，分别。在左面板数据对应于通过流式细胞术测定的细胞因子产生细胞的百分比，并且表示为平均值±SD。右图表示从细菌载荷和细胞因子产生用于每个小鼠获得的CD4 +细胞的数据。线性回归计算，并在各情况下所得到的p值在IL17A的情况下示出。汇集从两个独立实验的数据显示在该图中。 （此前公布的9）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice at JoVE.com

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

protective-efficacy-pulmonary-immune-response-following-subcutaneous

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.5K 次观看.  University of Zaragoza. We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

视频: Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

多色流流式细胞仪分析细胞在恒河猴的免疫应答

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

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 ...

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

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

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

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

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