这项工作是由雷诺烟草公司（RJRT）根据与维克森林大学医学院的合作研究协议投资。 GL Prasad是RJRT的全职员工。

注：书面知情同意做这项研究在当地的临床研究单位根据IRB批准获得，每良好的临床实践。血液处理中，在无菌条件下执行PBMC和其它细胞培养实验的隔离，使用微生物的无菌用品和试剂。 1，WS-CM准备<ol><li>产生的WS-CM如前所述12。 <ol><li>准备WS-CM由四个3R4F参考香烟通过罗斯威尔公园纪念研究所（RPMI）1640培养基无酚红用下面的吸烟方案通过烟：35-60-2，在毫升抽吸量，以秒为单位的时间间隔粉扑，粉扑和持续时间以秒为单位，分别为。每种制剂产生20ml的样品。 </li></ol></li><li>标签管（S）与日期，时间完成，香烟名称和编号。存储500微升等分在-80℃吸烟后立即完成。 </li><li>分析冷冻WS等分-CM确定尼古丁，烟草特异性亚硝胺，和多环芳香烃的含量如前所述12。 </li></ol> 2.分离外周血单个核细胞<ol><li>收集来自健康捐赠者（谁是非消费者对烟草制品）新鲜血液隔离从新鲜血液的外周血单个核细胞在维克森林浸礼会教友健康IRB 15一个独立的审批如下所述。 <ol><li>在此之前的血袋的到来，具有的500毫升瓶中，剪刀，隔离缓冲器，和Dulbecco氏磷酸盐缓冲盐水（DPBS）（RT）准备在生物安全级别2（BSL-2）的细胞培养罩。隔离缓冲器必须避光。 </li><li>握住血袋颠倒并削减略低于它已被夹住造成至少3厘米管的管中。 </li><li>取下一瓶500毫升的上限，按住瓶口上面管。拿起血袋并倒转，使血液从包成吨自由流动他直到瓶的袋子是空的。允许血液流到瓶与直下作为的内壁以避免产生气泡。 </li><li>倾隔离缓冲器入瓶以1：5的比例的隔离缓冲器来的血液。瓶盖瓶口紧紧地，轻轻地颠倒了，最终在年底，10倍。离开瓶在细胞培养罩孵育与灯关闭，1小时，在室温。 </li><li>光，稻草色层将上述血液中积聚。用25毫升血清吸管入50ml锥形管中取出这一层。所收集的量可以从50变化 - 300毫升，这取决于谁捐献的血液的主题。 </li><li>离心管中，在200×g离心在室温10分钟。 </li><li>吸出半透明的上清液，留下暗血色的沉淀。小球会松动，但粘性。 </li><li>吸取3毫升隔离缓冲分成15毫升锥形管。 </li><li>到重新悬浮颗粒，每50毫升浅黄色液体收集的Add20毫升的DPBSTEP 2.1.5。涡旋调匀，并整合来自多个管的悬浮液。该液体含有悬浮的血细胞。 </li><li>用移液管，在步骤2.1.8转移将5ml悬浮的血细胞置于3mL的隔离缓冲器。倾斜的15毫升锥形管，其中包含的细胞悬浮液缓慢并轻轻以创建两个单独的层。离心管中，在400 XG 40分钟在室温以最小的加速和不带刹车。 </li><li>使用移液管，以除去含有的PBMC所得到的混浊的中间层（血沉棕黄层）到50毫升锥形管中。避免绘图等层次分明下方。转移不超过25 ml接种入各50ml锥形管中。 </li><li>加25毫升冷的运行缓冲液（或多种填充50ml锥形管的整个剩余体积）洗细胞。离心细胞，在400×g离心在4℃下10分钟。 </li><li>重悬沉淀用运行缓冲液10毫升这包含外周血单个核细胞。计数细胞，并使用即时通讯的治疗中，或置于冰上冻结。 </li></ol></li></ol> 3.冷冻和解冻的外周血单个核细胞<ol><li>离心收集在步骤2.1.13，对于以400×g的10分钟的PBMC。 </li><li>填充冷冻集装箱，每个制造商的说明异丙醇。警告：异丙醇是易燃，剧毒。 </li><li>重悬的RPMI 1640培养基（4℃）含有20％胎牛血清（FBS）和10％的二甲亚砜（DMSO）中的沉淀。这是RPMI 1640冷冻介质。悬浮颗粒与冷冻介质，这将导致在具有约5×10 7个细胞/ ml的悬浮液的量。可用于冷冻细胞的数量将有所不同。 </li><li>分配1ml等分细胞悬浮于2ml冷冻管并放置冻存管在冷冻容器中。将冷冻容器冷冻管在冷冻在-80°C至存储O / N，然后取出冻存管，并转移到存储在-150℃的低温冷冻至-190℃。 </li><li>除去从低温储藏的冻存管，并在37℃下温和搅拌迅速解冻它在水浴。 </li><li>立即传送解冻的PBMC中冻存管入15ml锥形管中以含有10％FBS，1％青霉素/链霉素和1％L-谷氨酰胺10毫升的RPMI 1640完全培养基（4℃）。解冻冻存管的内容，应尽快转移，以获得最大的细胞活力15。 </li><li>离心的PBMC，在400×g离心10分钟。 </li><li>悬浮用5ml RPMI 1640完全培养基中的小球，并计数细胞。 </li><li>通过已建立的方法，如台盼蓝排除法测定细胞活力。通常用这种方法的细胞生存力为约90 - 95％。在外周血单个核细胞现在准备在实验中使用。 </li></ol> 4.细胞死亡测定注意：这里列出稀释该STU的目的DY。稀释液可以相应地改变。 <ol><li>用RPMI完全培养基至100μl的总体积稀释的WS-CM或尼古丁在一个96孔板/孔，如下所示。 </li><li>稀的WS-CM以下列浓度：0.1，0.5，1,1.5，2，2.5，3，4，和5微克/毫升的相等烟碱单元（基于WS-CM中的尼古丁含量）12。 </li><li>稀尼古丁在RPMI培养基中，以下列浓度：100，200，500，750，1000，2000和3000微克/毫升。注意：尼古丁是急性毒性和对环境有害。 </li><li>加入100微升的PBMC悬浮于RPMI完全培养基中至每个孔1×10 6个细胞/孔的浓度。细胞加的WS-CM或尼古丁的总体积为200微升/孔。 </li><li>本研究的目的，准备两套板如上。覆盖板并孵育一个板放置24小时和一个板3小时，在37℃和5％的CO 2。调整的时间点是必要的。 </li&gt; <li>通过在300×g离心离心3分钟，吸取上清，涡旋板以板覆盖的底部，最后用200微升冰冷的缓冲液重新悬浮细胞洗细胞在RT，并重复该洗涤步骤一次。 </li><li>添加95微升缓冲液，随后用5微升7氨基放线菌素D（7AAD）至每孔100微升的总体积/孔。孵育板在黑暗中在RT下15分钟。 </li><li>加入100微升缓冲液，以集群管。从板到集群管的每个孔中转移细胞悬液的整个体积并采集样品上流式细胞仪。 </li><li>确定使用流式细胞分析软件7AAD阳性细胞的百分比。 </li></ol> 5. EC 50测定<ol><li>对WS-CM和尼古丁的EC 50值由外周血单个核细胞的7AAD阳性染色确定。 </li><li>的EC 50值被定义为浓entration在其中细胞的50％在24小时测定法不再可行，并且这些值被表示为相等尼古丁单位/毫升微克。 </li><li>这项研究的目的，EC 50值确定为1.56微克/毫升和1650微克/毫升的WS-CM和尼古丁，分别。 </li></ol> 6.分泌的细胞因子注意：这里列出的稀释是本研究的目的。稀释液可以相应地调整。 <ol><li>用RPMI完全培养基中加入100μl/孔在的浓度的总体积稀释的WS-CM在一个96孔板0.3，1.56，图3和5微克/毫升的相等尼古丁单元。 </li><li>稀释烟碱至下列浓度：100，200，500，750，1000，2000和3000微克/毫升。注意：尼古丁是急性毒性和对环境有害。 </li><li>加入100微升的PBMC悬浮于RPMI完全培养基中至每个孔1×10 6个细胞/孔的浓度。细胞加的WS-CM或尼古丁的总体积为200微升/孔。 </li><li>覆盖板并孵育在37℃，3小时，5％的CO 2。 </li><li>通过在300×g离心离心3分钟，吸取上清，涡旋板以板覆盖的底部，最后悬浮细胞用运行缓冲液并重复洗涤步骤一次加入200μl冰冷的洗细胞在RT。 </li><li>添加200μl的RPMI完全培养基中，并重复该洗涤步骤一次。 </li><li>加入200μl10微克/毫升的LPS培养基到每个孔中。 </li><li>覆盖板并孵育4小时，24小时，48小时，或72小时，在37℃和5％的CO 2。调整保温时间是必要的。 </li><li>离心板在300×g下3分钟。 </li><li>取175微升上清液从每孔中并储存在冰箱中在-80℃进行测定的步骤7和8。 </li></ol> 7.式微球分析 &lt;OL&gt; <li>解冻步骤6.10和使用在CBA实验制备的细胞上清液。执行流式微球阵列（CBA）测定按照制造商的说明。 </li></ol> 8.的IL-8的ELISA <ol><li>从步骤6.10和使用中的IL-8的ELISA解冻的细胞上清液。执行ELISA测定按照制造商的说明。 </li></ol> 9.细胞内染色和流式细胞仪注意：这里列出的稀释是本研究的目的。稀释液可以相应地调整。 <ol><li>用RPMI完全培养基中加入100μl/孔在的浓度的总体积稀释的WS-CM在一个96孔板0.3，1.56，图3和5微克/毫升的相等尼古丁单元。 </li><li>在相同的板，稀释烟碱至下列浓度：2，10，50，100，500，2000和4000微克/毫升。注意：尼古丁是急性毒性和环境哈萨dous。 </li><li>加入100微升的PBMC悬浮于RPMI完全培养基中以1×10 6个细胞/孔的浓度。细胞加的WS-CM或尼古丁的总体积为200微升/孔。 </li><li>覆盖板孵育3小时，在37℃和5％的CO 2。 </li><li>通过在300×g离心3分钟，吸取上清，涡旋板以板覆盖的底部，最后用200微升冰冷的缓冲液重新悬浮细胞洗细胞在RT，并重复该洗涤步骤一次。 </li><li>添加200μl的RPMI完全培养基中至板，并重复洗涤步骤一次。 </li><li>制备的2微升/毫升GolgiPlug使用RPMI完全培养基中的工作浓度和10微克/毫升的LPS和添加200μl的各孔中。 </li><li>孵育板6小时，在37℃和5％的CO 2。 </li><li>在步骤9.8的端部，用运行缓冲液（4℃）和纺丝，在300洗涤细胞XG为3分钟，在4℃。 </li><li>添加100微升Cytofix的每个孔中，孵育20分钟，在4℃。 </li><li>如在步骤9.9，在300×g离心在4℃下描述，带有1 Permwash（4℃）下进行3分钟洗细胞3次。 </li><li>添加45微升1×Cytoperm向每个孔中，随后用5微升以下每个抗体中的一个，每孔：TNF-α的Alexa Fluor 488，IFN-γ的V500，MIP-1α的PE。孵育在4℃下进行30分钟。 </li><li>如步骤9.9中所述，带有1 Permwash（4℃）和一个时间与运行缓冲液（4℃）在300×g离心3分钟，在4℃下洗涤细胞两次。 </li><li>重悬的细胞用200μl的2％多聚甲醛（4℃）的。转移细胞到12×75毫米管，和血细胞计数分析样品上的流动。注意：多聚甲醛具有腐蚀性，剧毒和健康危害。 </li></ol> 10. K562杀灭试验注：K562细胞应培养在培养于37℃和5％的CO 2的RPMI完全培养基中直到他们达到在测定前80％汇合。 <ol><li>通过加入18微升的DMSO至小瓶制备5mM的羧基琥珀酰亚胺酯（CFSE）原液。 </li><li>用RPMI完全培养基至100μl的总体积/孔以实现所需的等尼古丁单位或尼古丁浓度为每孔稀释的WS-CM或尼古丁在96孔板中。注意：尼古丁是急性毒性和对环境有害。 </li><li>加入100微升的PBMC的进RPMI完全培养基中以1.5×10 6个细胞/孔的浓度。细胞与WS-CM或尼古丁的总体积为200微升/孔。 </li><li>覆盖板孵育3小时，在37℃和5％的CO 2。 </li><li>通过加入10ml的DPBS和离心机在400×g离心在RT 8分钟洗K562细胞。 </li><li>将细胞重悬于10毫升的DPBS和计数的K562细胞。 </li><li>准备CFSE worki纳克溶液中加入1微升的CFSE原液至1ml DPBS。 </li><li>加入1毫升的CFSE工作溶液到1ml含1所述的K562细胞悬浮液- 2×10 7个细胞。涡和孵化正是在室温2分钟。 </li><li>立即加入200μlFBS的。涡和孵化正是在室温2分钟。 </li><li>加入10 mL RPMI完全培养基中，离心反应管，在400×g离心在RT 8分钟。 </li><li>除去上清液的RPMI并打破颗粒和重悬细胞，用10ml RPMI完全培养基中，计数的CFSE标记的K562细胞。 </li><li>通过离心板在300×g离心3分钟，在RT下洗涤的PBMC。通过倾析将液体吸出上清液。重新装上盖子，涡旋盘的底部。添加200μl的RPMI完全培养基中，并重复洗涤步骤一次。 </li><li>添加CFSE标记的K562细胞以1:15的比率：向PBMC板和后续的i的每个样品井（100,000 K562s 1.5×10 6个PBMC中）ncubate 5小时，在37℃和5％的CO 2。 </li><li>通过在300×g离心离心3分钟，在RT下洗涤细胞混合。通过丢弃该液体吸出上清液。放置在盖和旋涡振荡板的底部。 </li><li>添加200μl的运行缓冲液，并重复在步骤10.14一次描述的洗涤步骤。 </li><li>添加95微升缓冲液中，然后用5微升7AAD每孔100微升的总体积/孔。孵育板在黑暗中在RT下15分钟。 </li><li>加入100微升缓冲液到每个孔中，并从板到集群管的每个孔中转移细胞悬浮液的整个体积，并获得对流动的样品仪。 </li><li>确定使用流式细胞分析软件7AAD阳性和CFSE阳性细胞的百分比。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+Regulatory+Science:+Research+to+Inform+Regulatory+Action+at+the+Food+and+Drug+Administration's+Center+for+Tobacco+Products.">Tobacco Regulatory Science: Research to Inform Regulatory Action at the Food and Drug Administration's Center for Tobacco Products.</a> Nicotine Tob. Res. , (2014).</li><li>Arimilli, S., Damratoski, B. E., Bombick, B., Borgerding, M. F., Prasad, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cytotoxicity+of+different+tobacco+product+preparations.">Evaluation of cytotoxicity of different tobacco product preparations.</a> Regul. Toxicol. Pharmacol. 64, 350-360 (2012).</li><li>Gao, H., Prasad, G. 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The authors declare that there are no conflicts of interest.

我们和其他人已经证实了外周血单个核细胞治疗用的TPP的抑制几种反应，包括表达和细胞因子和功能性的措施，如靶细胞杀死14的分泌。在以前的工作中描述的实验方法需要更长的潜伏期并且是温和的幅度14。鉴于这种诱人的体外模型的潜在应用基础研究和应用研究，我们调查是否有任何在这些多步生物分析测定的参数可以优化。在这里，我们提出了简化的方法来衡量使用TP...

知识点大幅身体的慢性吸烟的健康的不利影响，包括心血管疾病（CVD），慢性阻塞性肺疾病（COPD）和癌症1,2。慢性吸烟已经知道，引起炎症和免疫抑制，并且这些改变被报告给有助于微生物感染和癌症中的吸烟者3的危险性增加。 在体外和离体技术是有用在阐明的病理生理作用的分子基础香烟烟雾4-9（ 表1）和被认为是用于引导各种烟草制品10,11新兴调控的重要工具。 例如，我们已经表明，可燃烟草产品制剂（的TPP）如全烟雾条件培养基（WS-CM）和总颗粒物质（TPM）远远更多的细胞毒性和损害DNA比非易燃的TPP或尼古丁12,13。一贯以发表的作品，这是最近报道，可燃的TPP或尼古丁12,13。一贯以发表的作品，我们最近报道说，可燃的TPP造成显着的免疫抑制。这是证明了抑制长话样受体（TLR）-ligands，刺激细胞因子的分泌，并有针对性的细胞（K562）的体外模型14杀了外周血单个核细胞。鉴于炎症在香烟烟雾诱导的疾病过程的重要性，对测定条件进一步优化以评价香烟烟雾的免疫调节作用，提出本报告中。 体外测定法通常测量胞内和分泌的细胞因子，以及细胞毒性T细胞和NK细胞对K562细胞的细胞溶解作用杀伤测定法14。该试验涉及预孵化与WS-CM和尼古丁和PBMC的后续刺激s的TLR激动剂历时3天;最终读数是使用酶联免疫测定（ELISA）和/或流式细胞术进行。我们利用细菌脂多糖（LPS），其结合到TLR-4受体，并刺激的PBMC导致产生细胞内细胞因子和细胞因子的分泌。除了各测定步骤的优化，用于评估的TPP，我们还本方法分离的PBMC，细胞死亡测定法，和IL-8的量化的免疫调节作用。这些方法可应用于解决其他研究问题，并进一步细化，以评估烟草制品中的调节上下文。 表1.出版的体外报告和离体方法用于研究烟草产品制剂varilus病理生理作用的CS，香烟烟雾介质。 CSC，香烟烟雾冷凝水; CSE，香烟烟雾提取物; ELISA，酶联免疫吸附测定; GADPH，甘油醛-3-磷酸脱氢酶;定量PCR，定量聚合酶链反应; RT，实时定量聚合酶链式反应; TS，烟草烟雾。 <table><tbody><tr><td> 作者（研究年） </td><td> Laan 等人（2004） </td><td> 穆迪等人（2004） </td><td> 奥尔特曼斯等人（2005） </td><td> Vayssier（1998年） </td><td> Witherden 等人（2004） </td><td> 比勒尔等人（2008） </td></tr><tr><td> 使用电池 </td><td>人支气管内皮细胞（BEAS-2B），人中性粒细胞</td><td>人肺泡上皮细胞（A549） </td><td>人体气道平滑肌细胞（HASMC） </td><td>人类premonocytic U937细胞，人单核细胞</td><td> II型肺泡上皮细胞（ATII） </td><td>人单核细胞系（THP-1），人肺巨噬细胞</td></tr><tr><td> TPP使用 </td><td> CSE </td><td> CSC </td><td> CSE </td><td> TS </td><td> CSE </td><td> CS </td></tr><tr><td> 使用方法 </td><td> ELISA，定量PCR，迁移，电动汽车转变</td><td>免疫组织化学，电泳，Arrayscan试剂盒，RT-PCR法，酶联免疫吸附</td><td> ELISA法，RT-PCR，定量PCR，电泳</td><td>凝胶迁移率</td><td>光显微镜，电子显微镜，电泳，ELISA </td><td>定量PCR，ELISA，E-toxate试剂盒（Sigma），P65平板试验（TRANSAM），电泳，各种免疫试剂盒</td></tr><tr><td> 措施 </td><td> IL-8，GM-CF，AP-1，NF-κB，迁移</td><td>组蛋白乙酰转移酶，组蛋白脱乙酰酶，NF-κB，IL-8，等电点κB-α，GADPH </td><td> HO-1，GADPH，RANTES，IL-8，趋化因子</td><td>热休克/应激蛋白（HSP / HSP70），HF转录因子NF-κB，TNF-α的</td><td>表面活性蛋白（SP-A，SP-C），IL-8，MCP-1，GRO-α，TNF-α，IL-1β，IFN-γ的</td><td> IL-8，IL-1β，IL-6，TNF-α，MIP1-α，GRO-α，MAPK / JNK / ERK磷酸化，cJUN：DNA结合，谷胱甘肽，P65：DNA结合</td></tr><tr><td> 最终的结果 </td><td> CSE通过抑制AP-1的活化下调细胞因子的产生。 </td><td> 的 H 2 O 2与CSC提高组蛋白的乙酰化，减少组蛋白脱乙酰酶活性，差异调节促炎细胞因子的释放。 </td><td>香烟烟雾可能引起的IL-8从HASMC释放，增强由TNF-α，20％CSE更少的IL-8释放，趋化因子的抑制作用和RANTES由香烟烟雾。 </td><td> TS激活的HF的转录因子，其用的Hsp70过表达和抑制的NFkB的结合活性和TNF-α的释放相关联。 </td><td>减压ATII细胞衍生趋化因子水平的折衷肺泡拉尔修理，有助于香烟烟雾诱导的肺泡损伤和肺气肿。 </td><td>数据提供机械解释为什么吸烟者增加呼吸道感染。抑制先天应答的伴随增加的IL-8。 </td></tr></tbody></table>

<table border="0" cellpadding="0" cellspacing="0" width="712">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:223px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">12 X 75 tubes</td>
			<td style="width:148px;">BD Falcon</td>
			<td style="width:119px;">352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">15 ml conical tubes</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">2 mL Microtubes</td>
			<td style="width:148px;">Axygen</td>
			<td style="width:119px;">MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">3R4F reference cigarettes&#160;&#160;</td>
			<td style="width:148px;">Univ. of Kentucky, College of Agriculture</td>
			<td style="width:119px;">3R4F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">50 ml conical tubes</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">500 ml bottle</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">7AAD</td>
			<td style="width:148px;">BD Pharmingen</td>
			<td style="width:119px;">559925</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well flat bottom plate</td>
			<td>Termo Nunc</td>
			<td>439454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">96 well round bottom plates</td>
			<td style="width:148px;">BD Falcon</td>
			<td style="width:119px;">353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture hood</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:119px;">1300 Series A2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Centrifuge</td>
			<td style="width:148px;">Eppendorf</td>
			<td style="width:119px;">58110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CFSE</td>
			<td style="width:148px;">Molecular Probes Life Technologies</td>
			<td style="width:119px;">C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cluster tubes</td>
			<td style="width:148px;">Corning</td>
			<td>4401</td>
			<td>Harmful if swallowed, carcinogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cytofix/Cytoperm (Permwash)</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">555028</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMSO (Dimethyl sulfoxide )</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DPBS</td>
			<td style="width:148px;">Lonza</td>
			<td style="width:119px;">17-512F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FBS</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">F2442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FCAP Array</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">652099</td>
			<td>Software analyzes CBA data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Filter unit</td>
			<td style="width:148px;">Nalgene</td>
			<td style="width:119px;">156-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Flow Cytometer</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">FACS Canto II</td>
			<td>&#160;8 colors, at Ex 405 and Em785.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Flow Cytometer</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">FACS Calibur</td>
			<td>&#160;4 colors at Ex 495 and Em 785.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Flow cytometry analysis software</td>
			<td style="width:148px;">Tree Star</td>
			<td style="width:119px;">FlowJo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Freezing Container</td>
			<td style="width:148px;">Nalgene</td>
			<td>5100-0001</td>
			<td>Contains DMSO,&#160; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">GogliPlug</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">555029</td>
			<td>Carcinogen, Irritant, Corrosive&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:223px;">H2SO4</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">339741</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human Inflammatory Cytokine Kit</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">551811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">IFN-&#947; V-500 Antibody</td>
			<td style="width:148px;">BD Horizon</td>
			<td style="width:119px;">561980</td>
			<td>&#160;skin sensitizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">IL-8 ELISA Kit</td>
			<td style="width:148px;">R and D Systems</td>
			<td style="width:119px;">DY208</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Isolation Buffer</td>
			<td style="width:148px;">Isolymph, CTL Scientific Corp.</td>
			<td style="width:119px;">1114868</td>
			<td>Flammable liquid, Irritant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Isopropyl alcohol</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">L-Glutamine</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">LPS</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td>L2630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">MIP1-&#945; PE Antibody</td>
			<td style="width:148px;">BD Pharmingen</td>
			<td style="width:119px;">554730</td>
			<td>Acute toxicity, Oral</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Monensin</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">M5273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaCl</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotine</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">N3876</td>
			<td>Acute toxicity, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Parafilm</td>
			<td style="width:148px;">Bemis</td>
			<td style="width:119px;">&#8220;M&#8221;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Paraformaldehyde</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>Flammable, Skin irritation&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Pen/strep</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">RPMI 1640</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Running buffer</td>
			<td style="width:148px;">MACS Running Buffer, Miltenyi Biotech</td>
			<td style="width:119px;">130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Th1/Th2 CBA Kit</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">551809</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:223px;">TNF-&#945; Alexa Fluor 488 Antibody</td>
			<td style="width:148px;">BioLegend</td>
			<td style="width:119px;">502915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Transfer pipette</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tris Base</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to evaluate cytotoxicity and immunosuppression of combustible tobacco product preparations

除其他病理生理改变，长期暴露于香烟烟雾导致炎症和免疫抑制，它已与吸烟者微生物感染和肿瘤发生率的增加的易感性。 体外抑制人外周血单核细胞中的受体介导的免疫应答（PBMCs）中与烟气成分处理是研究的机制和评估暴露于烟草制品的可能的长期影响的有吸引力的方法。在这里，我们的优化方法来执行体外使用的PBMC通过细菌脂多糖，Toll样受体4配体刺激的测定。整体烟条件培养基（WS-CM）的影响，可燃烟草制品的制备（TPP），和尼古丁通过在体外测定法的PBMC研究对细胞因子分泌和靶细胞杀伤。我们表明，分泌的细胞因子IFN-γ，TNF，IL-10，IL-6和IL-8和细胞内细胞因子IFN-47 ;, TNF-α和MIP-1α被压制在WS-CM-暴露的PBMC。效应器的PBMC的细胞毒性作用，如通过对K562靶细胞的杀伤测定法测定也减少通过暴露于WS-CM;尼古丁是效果最差的，这些检测。总之，我们提出了一组改进的测定法来评估的TPP的在体外测定法的影响，并且这些方法可容易地适用于测试其他感兴趣的产品。

结果以均数±标准平均（4供体样品）的错误。使用Excel软件以及t-检验对比进行与其对应的控制各处理，进行处理和未处理的对照样品之间的学生氏t检验。的统计学显着性指示的：*，P &lt;0.05; **，P &lt;0.005; *** P &lt;0.0005。 来测量暴露于WS-CM和尼古丁的影响，外周血单个核细胞分别用不同浓度的WS-CM和尼古丁3小时或24小时。细胞死亡是由坚固和可靠的7AAD染色法测定，7AAD插层成双?...

Watch this Scientific Journal Video about Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations at JoVE.com

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

方法评估细胞毒性的可燃烟草产品的准备和免疫抑制

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

methods-to-evaluate-cytotoxicity-immunosuppression-combustible

Research

JoVE Journal

Immunology and Infection

11.3K 次观看.  Wake Forest University Health Sciences. Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure. 

视频: Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

脊髓灰质炎病毒识别粒子凝集法

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

科研

免疫与感染

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

两种异核体的形成方法，发现丙型肝炎病毒的制约因素

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

脓毒症诱导免疫抑制的切卡结扎和穿刺和鼻内感染双重模型的设计

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...