Name: intravenous endotoxin challenge in healthy humans: an experimental platform to investigate and modulate systemic inflammation
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The authors would like to thank Dr. Anthony Suffredini (Critical Care Medicine Department, National Institutes for Health Clinical Center, Bethesda, USA) for his careful review of the manuscript and many thoughtful suggestions. We would also like to thank the National Institutes of Health for making freely available the Clinical Center Reference Endotoxin employed within this study.
JNF and DWG are funded by the Wellcome Trust, which supported the generation of the described data and the publication of this article.

伦敦大学学院REC（UK）批准（参考5060/001）生成的代表结果部分显示的数据的研究。 伦理批准: 大多数使用IV内毒素挑战的研究在健康受试者已进行。作为这项研究没有任何健康益处的研究目标的重要性应该清楚地超过任何固有的风险。考虑相互作用时与新药用于修改炎症和具有夸大内毒素（ 例如，发热，在血压的变化，症状）宿主反应的潜力，这是相关的。 此前采用IV内毒素任何研究的开始，核准必须寻求并获得适当的研究伦理委员会（REC）/机构审查委员会（IRB）获得。四，内毒素政府一直采用了四十多年取得重要机甲nistic见解人类生物学，无严重或持久的不利影响，我们或其他作者的知识19。鉴于该模型的潜力，获得关于那些死亡和发病的主要原因多种炎症性疾病的重要信息，我们认为这在道德上是可以接受的健康志愿者和定义人口或临床组暴露于内毒素，提供了适当的风险最小化的策略到位。这可能需要由有资格的专家，或试验性研究独立的外部审查采用与内毒素引起的炎症新药及其相互作用特别是当以保证调查的安全通行。 制备 1.确定参与者的人口统计学和临床​​特征 注:大多数用人IV内毒素的挑战招募健康年轻男性志愿者&lt;30岁的研究。饰钉IES，额外承诺在健康妇女23，老年志愿者24,25和患者亚组26取决于实验的问题。老年参与者（ 即 ，大于60岁）可以具有更大的和更持久的响应（高发热，血压更大的下降），并与任何共病态状况或药物对内毒素诱导的炎症的相互作用时必须考虑这些研究的设计。请参阅在参与者选择进一步的考虑讨论。 2.征集参与者<ol><li>根据需要安排适当的广告。 注:参加者的时间和不方便经济补偿通常提供。这必须招募之前建立并通过REC / IRB批准。 </li><li>发出REC / IRB核准参与者资料单张（PIL）解释研究的性质，的概述协议，预计副作用，并与四内毒素相关的受访者符合入选标准的风险。 注:此应事先同意允许参加一个明智的决定，作出的对于任何问题或疑虑发给由研究人员来解决。 </li></ol> 3.获取正式知情同意书<ol><li>在进行任何研究相关程序，包括健康检查之前获得所有参与者的书面同意。 </li><li>求参与者保留和在PIL的信息的理解的口头确认。 </li><li>讨论IV内毒素管理的副作用。 注:在高剂量（2 - 4纳克/千克），这些严酷包括（寒战），头痛，畏光，肌痛，关节痛，恶心，而且很少，呕吐。峰症状强度发生约1 - 2小时后注入，由6之后缓和到基线 - 8小时。无严重或持续的不良EFFE已经报道了CTS继发于这些剂量的内毒素。少数情况下，志愿者可能会发现症状的响应高峰期间无法接受的程度。症状可以用对乙酰氨基酚/扑热息痛或非甾体药物的给药经口或静脉而改善（ 例如 ，阿司匹林，布洛芬）。这种试剂可改变炎症反应和它们的使用应记录。 </li><li>重新确认参加者在任何时候退出研究没有作出解释的能力。 </li></ol> 4.承诺在潜在参与了"健康网" 注:这是为了确保没有说把他们从IV内毒素伤害的风险更大未披露的医疗条件。作为次要目标信息相关的实验的问题可能被识别。 <ol><li>确定合适的临床环境和适当训练的医务人员进行健康屏幕。&lt;/ LI&gt; <li>以一个完整的病史，包括过去的医疗和社会历史，过去的和正在进行的药物治疗/治疗，新的健康食品或柜台药品，过敏状态，并进行调查的目前的症状，可能会提出新的或近期的疾病的存在。 </li><li>执行的正式临床检查，作为最低限度，心血管和呼吸系统。 </li><li>安排和审查的核心调查，包括常规观测（体重，心脏加快，血压升高，呼吸频率，血氧饱和度，温度），血液检查（全血细胞计数，肾，肝和凝血功能测试），12导联心电图。 注意:附加试验可以根据实验方案是必需的。 </li><li>与不起眼的病史，检查和调查参与者进行IV内毒素的挑战，取决于REC / IRB批准的包含/排除的标准。 注:我为新检测到的症状，体征或试验异常最佳实践中报告，并由参与者的例行医师研究。这些人应被排除在外，直到任何关注已经妥善解决。 </li></ol> 5.与会者通报出发至IV内毒素挑战; <ol><li>通知任何新的症状或疾病的调查。 注意:这将妨碍正常的进入研究。 </li><li>要求参与者在预先规定的时间和地点到达。建议学员穿着舒适的衣服，并把娱乐。 </li><li>要求参与者从午夜到快（透明液体允许的）。 </li><li>要求参与者从酒精和咖啡因不要24小时前注射和/或一个预定义的时间后（可选的协议而定）。 </li></ol>程序 6.内毒素挑战（在此之前施用内毒素） <ol><li>在45°准备用头一张床。 注意:确保这是坐落在一个适当的位置来获得临床援助和复苏设备。例如，临床研究设施，麻醉室或重症监护病房。 </li><li>确保所需的临床和实验设备访问和功能。 注意:这包括血压计，体温计，脉搏血氧仪，如果被雇用，3导心电监护。验证本地复苏设备和氧气的无障碍完整性。 </li><li>重建临床中心内毒素参考<ol><li>加入5毫升无菌注射用水，USP（SWI），以使用全无菌非触摸技术（ANTT）贯穿建业的先前未重构小瓶中。小瓶含有在一个白色，冻干粉末形式万内毒素单位（EU）（约1微克）。 </li><li>将小瓶Ø加入5毫升的SWI˚F建业在涡流摇动1小时，以确保内毒素附着在小瓶的玻璃表面被完全溶解。 注:建业不进入溶液容易，尽管他们看上去已经完全溶解。 </li><li>得出正确的体重调整容积内毒素（现为2000 EU / ml或大约200毫微克/毫升）到1毫升注射器。 注:使用玻璃或聚丙烯注射器内毒素粘附（和损失）最小化的设备。 </li></ol></li><li>参加者<ol><li>确认同意继续进行IV内毒素管理。查询有关新的疾病，症状和遵守协议的具体说明。 注意:如果有医疗关心或不遵守毒素，延缓管理。 </li><li>请参与者躺在床上准备，确认他们是舒适。确保他们有作废膀胱和肠道。 </li></ol></li><li>获得血管通路。 <ol&gt; <li>插入ANTT下静脉插管内毒素的管理，如果包括在协议中，静脉输液。 </li><li>地方持续抽血，避免反复静脉穿刺第二血管行。静脉内插入此并连接到一个3路抽头（具有或不具有扩展名），以促进血液抽取和冲​​洗。 注意:可替换地，用于连续血压监测和样品采集，部位的动脉线。这需要用于插入更多的专门知识和有局部并发症的风险增加。 <ol><li>对于持续使用静脉插管抽血，使用18号或更大口径套管与选址沿肘窝，避免溶血和凝血。静脉输液将有助于保持通畅。 注:血管内导管插入式与本站的选择应根据个人的实践者和参与者P拍摄地点参考。动脉线在传统上是在桡动脉或股动脉局部麻醉前滴入（如 1％利多卡因）。 </li></ol></li><li>抽取血液，用于在第一或第二线路的插入基线测试。丢弃前5毫升血液（在套管内占据的死空间和连接器）之前获得的血液进行采样。冲洗用10毫升0.9％的氯化钠抽血后的行。 注:血的体积绘制和样品的处理将通过实验方案和当地实验室程序来确定。 </li><li>附加临床监测适当的（ 例如 ，3导心电监护通常与高剂量使用挑战2 - 4纳克/千克）。 </li><li>记录基线常规临床观察在4.4适当的生命体征图表说明。主观症状评分（ 例如 ，使用头痛，肌肉痛视觉模拟评分，等）应该附加地执行监视参与者的经验。 </li></ol></li><li>内毒素管理<ol><li>冲洗用0.9％氯化钠的静脉插管以确保插管被正确地安放和专利。 </li><li>通过相同的静脉插管管理的预先准备，重量调整剂量重组建业的。注入丸药（&lt;2分钟）的剂量为在IV线的轮毂3路抽头。 </li><li>冲洗，用10ml 0.9％氯化钠，以确保所有建业进入循环。 注意:要施用的连续输注注入内毒素到稀释剂的已知体积， 如 100毫升0.9％的氯化钠以达到预先确定的浓度，注入以设定的速度（体积/时间）将所得的溶液。 </li></ol></li></ol> 7.监测，观测和采样抽奖<ol><li>监视参与者经历IV内毒素推后至少6小时的做唱或输注的持续时间。有一个合格的，有经验的临床医师执行此步骤。 </li><li>记录和审查生命体征和至少一次/ hr的任何临床观察。连续监测，如3导心脏监测可另外进行。 注:研究者应注意，在较高剂量推注（2 - 4纳克/千克）血管迷走神经反应，可能会引起心脏暂停。这些注射后最常见的为30分钟至2小时，不是恶性的（见进一步的细节讨论）。 </li><li>在研究者决定进行主观症状得分。 </li><li>辖2 - 3升晶体的（例如0.9％氯化钠或哈特曼溶液）6 - 4纳克/千克 - 2大丸剂施用后8小时。 注意:不存在任何可以接受的标准，上 ​​述流体管理。参与者的安全是至关重要的。这提供日常保养液和体积代表lacement（期间由嘴里不停地零和增加，由于提高体温和呼吸频率昏迷损失）。它们也可以对抗心律失常（见讨论）的风险。 </li><li>获得解答的实验问题需要预先确定的样品。 注:这些传统上包括但不限于，血液和尿液。采样的定时，数目和量应以获得准确的数据所要求的最小和已通过拍摄/ IRB。更频繁的采样可能在一定的时间点（ 例如，每30分钟）被要求确保峰（或波谷）不漏掉的值。取血量的计算必须考虑血死角撤离过程中丢弃。 </li><li>按要求处理样品。 </li></ol> 8.过程结束<ol><li>有主治医师确保参与者的症状已经解决，他们的观测到的趋势正常（所有修改的参数， 例如，提高的心脏速率和温度，显示出朝向基线值一致减少）制裁的观测和随后的放电结束之前。 注:2推注后- 4纳克/千克建业症状通常完全减弱了6 - 8小时。个人意见后续重叠，但离散时间课程。 12小时 - 这些都通常由10返回到基线。连续输注要求的观察作为症状和生命体征不会立即消退后施用内毒素的停止一个较长时期。 </li><li>删除所有监控设备和线路血管止血保障。 </li><li>确认参与者很高兴回家，并具有调查员的联系方式中的任何关注的情况下。 注:最佳实践是联系参与者第二天来检查自己的健康状况，并监控任何后遗症。 </li></OL&gt;

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The authors have nothing to disclose.

由于这里展示并在最近的评论文章充分说明，人类IV内毒素的挑战是提供洞察背后广阔的人类疾病的炎症通路独特的实验平台。允许全身性炎症的控制，可重现感应，该模型允许访问炎症级联的初始阶段，消除潜在的混杂因素。 参与者的响应可以相对于严重程度和持续时间，以及在他们的表型上介绍的基因组影响评价进行调制。此外，该模型可以用作试验台为治疗剂，不仅那些针对LPS配体本身，但LPS诱导的信号通路和两个炎症的急性和分辨率相介质。这可能包括针对内毒素耐受，免疫系统的低反应状态具有相似性，以重大疾病，我的恢复代理EP3受体激动剂诱导免疫麻痹33,34能够在不仅复制急性16和慢性炎症27的关键特征的生理/功能级别，而且一个转录一次35，IV内毒素攻击看似具有基本科学突破的翻译来发挥关键作用临床实践。当连接到中能够更加紧密地复制特定疾病（ 如鼠类盲肠结扎穿孔和败血症）的病理生理反应适当的动物模型直接调查这一研究性模式可能会特别强大。 在任何健康人的志愿者模型参与者的安全是至关重要的。 IV内毒素攻击已在2较高剂量范围施用- 4纳克/千克至数千人用，我们和其他作者的知识，没有严重的或永久性的不良事件（个人通信，安东尼苏弗雷迪尼博士）19。不Ë不过严重的孤立迷走神经反应的帐户已被报道36,37。其病因大概代表着高品质的休息迷走神经张力，禁食一夜后血容量不足和儿茶酚胺的释放与发热，寒战症状的发作，以夸张的Bezold-Jarisch反射达到高潮。这种风险可以通过排除与先前血管迷走神经晕厥或正倾斜测试，并与前和内毒素攻击37中的静脉注射液容积负荷个体得到改善。采用高剂量的内毒素的挑战（尤其是4纳克/千克）的研究人员应切实心动过缓和/或心脏停顿的风险，警告他们罕见的，但潜在发生的参与者，采用适当的监控，并有复苏设备一应俱全。其它排除标准，除了那些在协议中所述，可能需要依赖于实验问题。例如，而谨慎的做法是要排除那些有PArticipated在其他临床试验或经验丰富的手术/创伤，在过去三个月中，先前已在IV内毒素挑战试验可能也尝过条目（或使用一个平行组不是一个交叉设计的干预性研究）对这些限制内毒素耐受持续的时间在体内 38,39未知长度:如果免疫应答是待评估是必需的。 数限制是与模型显而易见。它在健康年轻男性志愿者联合国代表临床人口的传统进行。任何干预都需要施用内毒素的表型的后果来解决。纯化的LPS注射提供暴露于只有一个单一的TLR配体，而不是几个免疫原性部分上的活病原体。通过道德的必要性只有相对温和的炎性反应可引起。 DISE的每一个病理生理特征的模仿ASE没有实现40。因此，"任何期望该模型完全复制严重，局部或全身革兰氏阴性感染的临床状况是无许可的"18。 然而，我们会认为该模型的优势在于没有不适当的外推到临床上，但在询问的生理，血液学，免疫学，神经内分泌和一键炎症刺激感受态细胞和器官系统的代谢反应。瞬态变化的解释周到内毒素管理挑起并通过药理挑战自己调制一直是，而且将继续是，在设计新的治疗方法和预测其疗效翔实。

不同的病因，严重程度和持续时间的炎症在多种疾病的病理形成一个核心组成部分。从临界疾病状态，如败血症或外伤，其中严重的，相对短寿命炎症占优势，对慢性疾病，包括2型糖尿病，动脉粥样硬化和阿尔茨海默氏病，其中持续低水平炎症音被认为是病理因素，失调的炎症途径是不等全球发病率和死亡率1,2的关键驱动因素。 通过组织损伤3-5释放通常胞内组分-炎症是由病原体相关分子模式（PAMP）或危险相关分子模式（D-AMPS）的存在下引发。这些保守分子基序结合模式识别受体（的PRRs）如上皮细胞上发现的Toll样受体（TLRs），内皮细胞和那些先天免疫SYS的统，以触发共同的下游细胞内信号通路6。核转录因子核因子κB的活化，除其他外，导致编码建立炎症级联7促炎性细胞因子基因的转录。 炎症引起旨在隔离和删除初次损伤局部和全身的影响。当地改变包括上调附着力标记，趋化因子的释放，血管扩张，血管通透性增加，促进先天免疫细胞的轮回。补体和凝血系统也被激活。系统性，多器官特异性的生理变化观察，通过神经内分泌的网络策划。在体液调解显著上升出现和循环细胞轮廓从根本上改变。炎症的故障停止和内环境稳定，以重新建立，可能是由于在起始刺激持续性或专用分辨率的失败途径8。这些有力的机制，往往是脂类介质协调本身可能致病9,10。 在临床人群的炎症反应和这些关键调节步骤的调查是具有挑战性的。人口（年龄，性别，种族），时间（发病时间，暴露时间）和临床方差（启动刺激的类型，侮辱的严重程度，合并症负担，治疗干预）妨碍访问关键的生物学通路。传统的解决方案是使用动物模型。尽管，得到多重优势，其基本相似人类病理生理学11,12和输出的相关性，已受到越来越多的关注13-15。另一种方法是采用还原人体模型。 在20世纪60年代末开发，静脉注射（IV）施用内毒素对人提供了一个重要平台，通过它发现，划定和潜在的药物炎症通路16-19。内毒素（本文中脂多糖[LPS]同义使用）是一种强效的TLR4激动剂触发炎症级联以剂量依赖性。注入无论是作为大丸剂或输注它可因此被用于既低级别和不同持续时间的中度全身性炎症模型。所得短命炎症反应允许细菌感染期间，开发了高度复杂的宿主 - 病原体相互作用的单个组件的评估。而不是休克模型，IV内毒素挑战引发出现复制感染的早期阶段的反应。消除种间障碍，翻译，准确地模仿临床表型，如果定性不能定量，它可以提供炎症的影响，后果和机会来测试干预的实时解读。内毒素也可以被施用endobronchiallŸ20，21皮内或与LPS IV组合22，探索在不同车厢局部炎症反应。少广泛采用的替代方法包括近侧促炎细胞因子如肿瘤坏死因子α（TNFα），白细胞介素（IL）6 19的输注。这些将不进一步讨论。

<table><tbody><tr><td>Clinical Center Reference Endotoxin (CCRE, &lt;em&gt;Escherichia coli&lt;/em&gt; O:113:H10:K negative)</td><td>National Institutes for Health</td><td>n/a</td><td>GMP Manufactured Endotoxin</td></tr><tr><td>Syringes (1&amp;nbsp;ml, 5&amp;nbsp;ml)</td><td></td><td></td><td></td></tr><tr><td>Water for injection</td><td></td><td></td><td></td></tr><tr><td>0.9% sodium chloide (sterile, clinical grade)</td><td></td><td></td><td></td></tr><tr><td>Intravenous cannulae</td><td></td><td></td><td>Miminum 20 G French</td></tr><tr><td>Sphygmomanometer</td><td></td><td></td><td>If intraarterial line placed, transduction equipment for invasive blood pressure monitoring</td></tr><tr><td>Pulse oximeter</td><td></td><td></td><td></td></tr><tr><td>Thermometer</td><td></td><td></td><td>Typmanic, oral or core (rectal, oropharyngeal)</td></tr><tr><td>Electrocardiograph machine</td><td></td><td></td><td></td></tr><tr><td>Sample collection tubes</td><td></td><td></td><td>As utilized by local clinical laboratory</td></tr></tbody></table>

intravenous endotoxin challenge in healthy humans: an experimental platform to investigate and modulate systemic inflammation

炎症途径的激活代表在多种疾病状态的急性和慢性的中央机构。无论是通过病原体或组织损伤相关的分子图案为契机，共同的生化途径导致保守又变的生理和免疫学变化。解剖和决定因素和响应表型变异的潜在机制圈定预计产量新的治疗进展。 静脉内（IV）的内毒素（革兰氏阴性细菌脂多糖），一个特定的Toll样受体4激动剂，施用表示全身性炎症的在人的体内模型。国立卫生研究院临床中心参考内毒素（建业， 大肠杆菌 ○:113:H10:K负）被用来可靠地和可重复地生成血管，血液，内分泌，免疫学和器官特异性功能效应平行，在不同程度上那些在病理状态的早期阶段观察。剂量的变化（0.06 - 4纳克/千克）和时间尺度曝光的（大丸剂对输注）允许急性或慢性炎症和各种严重程度的复制被引出，与较高剂量（2 - 4纳克/千克）经常被用来建立一个"败血症样"状态。建立和新颖的药用化合物可额外给予前或后内毒素暴露欣赏他们对炎症级联效应。尽管在范围和普遍性的限制，人类IV内毒素的挑战提供了一个独特的平台来获得机械见解诱导的生理反应和炎症通路。合理使用它可能有助于这方面的知识转化为治疗的创新。

四，内毒素管理的多元化，多系统的后果最近已全面回顾，并不会全部在这里进行讨论（有关详情请参阅17-19,27）。代替的范围和模型的适用性的简要概述将沿着关于经典丸药-给药方案（2纳克/千克）主数据来提供。在图1中提供了示出了这样一个实验的时间线的示意图。 <img alt="图1" src="/files/ftp_upload/53913/53913fig1.jpg" /> 图 1:丸IV内毒素挑战议定书代表性示意图到来后，参与者的同意进行应与他们的健康状况和协议一致性（空腹，饮酒弃权等 ）一起确定。下面的血管插入线，attachme监测和基线临床观察和样品，体重调整剂量重新构成建业的集合的核苷酸施用。最少6临床监测 - 8小时是强制性的。样品，最常见的血液，可在预先选择的时间点服用。晶体静脉输液通常给出，在这种情况下，1升超过2小时，然后1升超过6小时。 <a href="https://www.jove.com/files/ftp_upload/53913/53913fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> IV内毒素的效果取决于所选择的剂量和给药方法（丸与输注）。 2推注- 4纳克/千克引起可靠与提高核心温度，心脏率和白细胞（WBC）的全身炎症反应综合征的特征计数16。这是伴随着炎症介导的体液，包括亲和蚂蚁的存在的i-炎性细胞因子，急性期蛋白（如C-反应性蛋白）（参见图2和图 3）和下丘脑-垂体轴28和两个亲混凝和纤溶系统29的活化。代谢的中枢和外周组织中被改变和多器官特异性功能的改变引起的19,30。参与者体验各种&#39;流感样"症状，在大约1的强度峰值- 2小时，6小时，在很大程度上减弱（参见图 4）。丸"大剂量"协议诱导志愿者31定性一致，但定量变量的反应。 <img alt="图2" src="/files/ftp_upload/53913/53913fig2.jpg" /> 图2:临床观察资料更改生命体征推注施用后临床 ​​中心参考的内毒素2 N。克/千克（以中位数IQR，N = 10）。呼吸频率（RR，每分钟呼吸，A），脉搏（每分钟节拍，B），收缩压（SBP，毫米汞柱，C）和温度（℃，通过鼓膜温度计，D）被显示。收缩压不降，由于静脉输液，小升可能反映增加交感神经的管理。时间显示在x轴（内毒素后人力资源管理）。 <a href="https://www.jove.com/files/ftp_upload/53913/53913fig2large.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/53913/53913fig3.jpg" /> 图 3: 细胞和体液免疫反应中总白细胞计数一致改变，包括瞬态中性粒细胞，观察到（A）中沿与inflammatio常用临床指标n个这样的C-反应蛋白（B）。四，内毒素后，循环血浆细胞因子浓度定量多种多样的，但定性的保守变化发生。 TNFα（C）和IL-6（D）显示为范例。临床中心参考内毒素2纳克/千克的所有数据后推注（以中位数IQR，N = 10，细胞因子浓度为2技术重复在每个时间点/个体平均值）。 <a href="https://www.jove.com/files/ftp_upload/53913/53913fig3large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图4" src="/files/ftp_upload/53913/53913fig4.jpg" /> 图4:症状档案累计症状积分记录在临床中心内毒素参考2纳克/千克的预先确定的时间点推后的管理（中位数与IQR，N = 10）。参与者被要求ŤØ得分头痛，寒战，肌肉酸痛，恶心的严重程度视觉模拟评分（0 - 10，最高分40） <a href="https://www.jove.com/files/ftp_upload/53913/53913fig4large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 相反"低剂量"内毒素对个人更加多样的效果，并且可以用来探索在炎症反应31个体间的差异。 0.06管理-无论是通过推注或输液0.2纳克/千克诱导2 - ​​血浆中的细胞因子与那些在慢性低度炎症性疾病32相称看到10倍的增长。这样的协议可以具有在探索炎症的贡献了关键作用，它调节到多个复杂心脏代谢条件27的通路。

Watch this Scientific Journal Video about Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation at JoVE.com

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

静脉注射内毒素挑战人类健康的实验平台，研究并调制全身炎症

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or ...

intravenous-endotoxin-challenge-healthy-humans-an-experimental

Research

JoVE Journal

Medicine

11.5K 次观看.  University College London. Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

视频: Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

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 ...

科研

免疫与感染

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

生物工程

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.
To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

注射脂多糖对小鼠肠道屏障破坏后微生物源产物的模拟进入

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

危重病人全血内毒素血症检测的内毒素活性测定

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

医学

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

分离来自癌细胞系的低内毒素含量细胞外囊泡

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and ...

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ranges between 20 and 30%. Novel treatment approaches in order to reduce sepsis-related multiorgan failure and death are urgently needed. Robust animal models allow for one or multiple treatment approaches as well as for testing their effect on physiological and molecular parameters. In this article, a simple animal model is presented.
First, general anesthesia is induced in animals either with the use of volatile or by intraperitoneal anesthesia. After placement of an intravenous catheter (tail vein), tracheostomy, and insertion of an intraarterial catheter (tail artery), mechanical ventilation is started. Baseline values of mean arterial blood pressure, arterial blood oxygen saturation, and heart rate are recorded.
The injection of lipopolysaccharides (1 milligram/kilogram body weight) dissolved in phosphate-buffered saline induces a strong and reproducible inflammatory response via the toll-like receptor 4. Fluid corrections as well as the application of norepinephrine are performed based on well-established protocols.
The animal model presented in this article is easy to learn and strongly oriented towards clinical sepsis treatment in an intensive care unit with sedation, mechanical ventilation, continuous blood pressure monitoring, and repetitive blood sampling. Also, the model is reliable, allowing for reproducible data with a limited number of animals in accordance with the 3R (reduce, replace, refine) principles of animal research. While animal experiments in sepsis research cannot easily replaced, repetitive measurements allow for a reduction of animals and keeping septic animals anesthetized diminishes suffering.

Here, we present a reproducible intensive care unit-oriented endotoxin model in rats.

一种可重复的重症监护病房导向内毒素模型在大鼠中的应用

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ...

通过 LPS 输注诱导猪内毒素血症的剂量依赖性

Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model

Sepsis and septic shock are frequently encountered in patients treated in intensive care units (ICUs) and are among the leading causes of death in these patients. It is caused by a dysregulated immune response to an infection. Even with optimized treatment, mortality rates remain high, which makes further insights into the pathophysiology and new treatment options necessary. Lipopolysaccharide (LPS) is a component of the cell membrane of gram-negative bacteria, which are often responsible for infections causing sepsis and septic shock. 
The severity and high mortality of sepsis and septic shock make standardized experimental studies in humans impossible. Thus, an animal model is needed for further studies. The pig is especially well suited for this purpose as it closely resembles humans in anatomy, physiology, and size. 
This protocol provides an experimental model for endotoxemic shock in pigs by LPS infusion. We were able to reliably induce changes frequently observed in septic shock patients, including hemodynamic instability, respiratory failure, and acidosis. This will allow researchers to gain valuable insight into this highly relevant condition and evaluate new therapeutic approaches in an experimental setting.

作者聚焦:猪实验性内毒素休克的诱导，用于研究血流动力学和呼吸衰竭

We provide a protocol for an experimental endotoxemic shock model in pigs by infusion of lipopolysaccharide.

脂多糖输注作为猪内毒素血症休克模型

Sepsis and septic shock are frequently encountered in patients treated in intensive care units (ICUs) and are among the leading causes of death in these ...

Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs

Source: Neun, B. W. et al., <a href="https://www.jove.com/v/58830">Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays.</a> J. Vis. Exp. (2019)
This video describes the turbidimetric format of the Limulus amoebocyte lysate, or LAL, assay to estimate endotoxin contamination in PEGylated liposomal doxorubicin nano-formulations. The turbidity of the solution following the reaction of the endotoxin with the LAL reagent helps to estimate the endotoxin in the sample.

比浊法鲎酰变形细胞裂解物测定:一种检测和定量纳米制剂药物中内毒素污染的技术

Source: Neun, B. W. et al., Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays. J. Vis. Exp. (2019)
This video ...

静脉注射内毒素挑战人类健康的实验平台，研究并调制全身炎症

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IL-8瞬时转化的小鼠模型

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

注射脂多糖对小鼠肠道屏障破坏后微生物源产物的模拟进入

危重病人全血内毒素血症检测的内毒素活性测定

分离来自癌细胞系的低内毒素含量细胞外囊泡

一种可重复的重症监护病房导向内毒素模型在大鼠中的应用

脂多糖输注作为猪内毒素血症休克模型

脂多糖输注作为猪内毒素血症休克模型

脂多糖输注作为猪内毒素血症休克模型

比浊法鲎酰变形细胞裂解物测定:一种检测和定量纳米制剂药物中内毒素污染的技术