The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services (DHHS) or of the institutions and companies affiliated with the authors. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. M.R.H., K.J., D.P., L.B., and J.W. performed this work as employees of Battelle Memorial Institute. Subcontractors to Battelle Memorial Institute who performed this work are: R.B., an employee of Charles River Laboratories - Insourcing Solutions; L.K. and M.R.L., employees of MEDRelief Staffing Inc.; M.G.L. as an employee of Lovelace Respiratory Research Institute, Inc.; and J.H.K. as an employee of Tunnell Government Services, Inc.

该协议符合下列动物护理指南。动物被安置在由该协会的评估和实验动物国际评审认可的设施。所有的实验过程由国家过敏和传染病研究所，临床研究部，动物护理和使用委员会批准，均符合动物福利法的规定，公共卫生服务政策和指南的保养和使用实验动物的建议。 1.准备CT扫描（在"冷端"） <ol><li>条件的X射线管<ol><li>确认没有工作人员在扫描室和主题表是空的。 </li><li>按照扫描仪制造商的规格运行管调理过程。 </li></ol></li><li>执行空气校准<ol><li>确保没有对象在扫场。 </li&gt; <li>按照扫描仪制造商的说明进行空气校准程序。在许多平台上，这种校准自动或半自动的。 </li></ol></li><li>实施质量控制扫描（在"热面"） <ol><li>完成ABSL-4实验室西装入境手续（参考文献5中详细介绍），增加一个额外的步骤。如果程序涉及接触放射性物质，夹防护服下一个剂量计的磨砂。在转剂量计每2个月的辐射暴露的计算。 </li><li>放置在CT头部和身体幻象，它是由选自空气，水和聚四氟乙烯，在对扫描表中的相应保持器。 </li></ol></li><li>实施质量控制扫描（在"冷端"） <ol><li>从冷侧，推进幻象进入孔6的中心。 </li><li>使用实验室标准化的质量控制（QC）参数，以获得ENS扫描URE扫描仪制造商的规范之内。 </li><li>执行图像的质量控制计算机分析以集中于根据制造商的协议每种材料类型（空气，水或特氟隆）感兴趣区域内计算信号强度的平均值和标准偏差。 </li><li>通知CM人员的CT扫描仪就可以使用了。 </li><li>验证动物信息（识别号码，重量，出生日期）与CM人员。 </li></ol></li><li>注册专题信息到患者数据库。 <ol><li>在CT扫描仪控制台（在"冷端"），访问扫描仪的放射信息系统（RIS）。检查RIS填充与扫描时间表的详细信息。 </li><li>选择当前的主题和人口统计数据，如身份证号码，年龄，出生日期，和考试日期;那么，考试类型应自动填充。 </li><li>手动输入主题的重量。 </li><li>选择正确的苏在CT龙门bject方向（ 例如 ，头部先，脚先，俯卧，仰卧）。 </li></ol></li></ol> 2.准备工作区在ABSL-4实验室套装<ol><li>准备II级生物安全柜（BSC）或通气台在动物操作室。打开BSC（或通气台）至少10分钟后再使用。 <ol><li>清洁和使用经批准的消毒剂，如5％的双季铵消毒BSC或下降气流表的顶部表面的内表面（正烷基二甲基苄基氯化铵，正 - 烷基二甲基乙基苄基氯化铵）消毒剂溶液。喷在BSC或下降气流表的顶部表面的内表面和擦拭表面后的接触时间10分钟的干。喷雾和擦拭用70％的乙醇的表面，以除去残留的消毒剂。 </li><li>设备到位并在BSC或下降气流表所需物资，包括麻醉诱导框和动物处理手套。 </li></<html> OL&gt; </li><li>准备CT扫描仪床。 <ol><li>设置于CT床动物保持装置。 </li><li>设置的生命体征监测以检测心脏速率和氧饱和度。如果扫描会话长于10分钟，监测温度。 </li></ol></li><li>准备麻醉机。 <ol><li>检查蒸发器异氟醚量，如果需要添加更多的异氟醚。 </li><li>用氧气加压麻醉呼吸回路，检查逃脱气，视觉检查麻醉机是否有泄漏1执行泄漏测试。 <ol><li>如果检测到泄漏，确定源，纠正问题，并进行后续泄漏测试，以验证更正是正确实施。 </li></ol></li><li>称量一次性清除罐捕获垃圾麻醉气体。如果该罐是≥50克在罐的初始重量，更换罐7。 </li></ol></li></ol>标题"（热侧"&gt; 3。动物从动物手术室和麻醉诱导的制备在CT扫描仪对房间）运输" <ol><li>从其中的啮齿动物饲养室毗邻成像套件转移凯奇房屋豚鼠进入II级BSC。 <ol><li>验证动物ID。 </li><li>取下笼卡，并保持与动物。 </li></ol></li><li>打开盖子笼子和唐在正压力服手套保护动物的皮手套。轻轻地拿起豚鼠，将动物在麻醉诱导箱，并盖上盖子的框。 </li><li>从II级BSC删除包含动物的麻醉诱导箱，将其放在一个运输小车。 </li><li>使用车，取动物的CT扫描仪的房间。 </li><li>在CT扫描仪的房间，立即连接清除罐和麻醉机感应框。打开氧气到感应框，并设置汽化器至delivER 4％异氟醚麻醉8的初步归纳。 </li><li>监测麻醉诱导过程中对动物的麻醉深度足够（ 如反应迟钝对外界刺激，肌张力，呼吸平稳，心脏率）8。 </li><li>麻醉关闭到感应框时豚鼠被完全麻醉。 </li></ol> 4.排列在在CT扫描成像床主题（在"热面"） <ol><li>从感应盒中取出麻醉的豚鼠和成像床的地方。 <ol><li>放置在保持坐垫的豚鼠在俯卧位。 </li><li>双眼申请眼药膏保护角膜上皮干燥。 </li></ol></li><li>管理维护异氟醚麻醉。 <ol><li>广场上的豚鼠鼻锥，打开煤气，提供1升/分钟的氧，以鼻锥8。 </li><li>设置蒸发器，以提供2-3％的异氟醚通过鼻锥的豚鼠。 </li><li>一旦受试者已达到麻醉的所需平面（没有运动，〜每分钟60次呼吸），降低蒸发器设置为递送异氟烷1.0-1.5％为维持麻醉。 </li><li>监测生命体征，包括体温，心脏率和呼吸频率。如果呼吸频率分别开始9加速或减速，增加或减少异氟醚的百分比。 </li></ol></li><li>提供额外的热源来维持豚鼠体温37和39℃之间，如果需要的话，这取决于数据采集与麻醉深度的预期长度维持8,9。 </li><li>固定一个塑料盖放在夹持垫的顶部。 </li><li>推进成像床到容器管。 </li></ol> 5.设置视图的像场<ol><li>激活激光系统（成像人员）对CT SC的"冷端"anner空间位置CT检查豚鼠。使用表"中"和"out"按钮，以覆盖激光十字线下所关注的解剖结构。 </li><li>设置的视场，使用激光按钮设置在感兴趣的解剖结构的起点。 </li><li>在"偏热"跟CM的工作人员将自己置于和CT扫描仪之间的铅屏蔽。 </li></ol> 6.获取图像<ol><li>获得用于切片放置用于根据制造商的协议的CT研究图像调查扫描。 </li><li>规定对图像的调查研究CT片图像的位置。 </li><li>用"热侧"人员坐标如果注射对比剂是使用。 </li><li>根据制造商的协议获得研究扫描。 </li><li>根据制造商的协议在扫描器控制台重建CT图像。 </li><li>将图像发送到图像存档和通信系统。执行进一步的定性和QUAntitative从根据制造商的协议，该归档系统的分析。 </li></ol> 7.扫描后恢复<ol><li>从扫描仪床上转移豚鼠到一个干净的microisolator笼食物和零食。 </li><li>使用购物车，与动物运输笼回其住房面积。 </li><li>监控动物，直到从麻醉中完全恢复。 </li><li>一旦完全康复，回到笼子的高效率微粒空气（HEPA）-filtered通风架。 </li></ol> 8.扫描仪湾和消毒设备<ol><li>消毒扫描器托架区域（ 例如 ，这是在与动物，扫描器床，扫描器房间地板，门把手直接接触的表面）用5％的双季铵盐溶液为10分钟的接触时间。 </li><li>漂洗表面用10分钟暴露于双季铵盐溶液后70％的乙醇溶液中。 </li><li>擦拭不能直接喷项具有双季铵盐溶液（敏感的电子设备）具有双季铵饱和布后跟乙醇饱和布。 </li></ol>

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The authors have nothing to disclose.

在本系列前面的文章中都强调了广泛的培训，注重细节，安全程序，并以最高防护实验室12,13安全工作所需的额外工程控制。执行安全工作就是在这些实验室的最高优先级。这一理念更是用活的动物时重要，因为额外的危害，例如潜在的受感染的动物造成咬伤或抓伤或产生气溶胶7。这些程序强调动物的安全装卸和运输从保持房间成像室。麻醉期间动物操作和监测表明，得到观看者...

国家过敏和传染病研究所（NIAID）在德特里克堡弗雷德里克MD（IRF-弗雷德里克）综合研究机构的使命是执行新发传染性疾病的研究，以了解与严重程度相关的临床疾病过程微生物引起的疾病。该IRF-弗雷德里克必须在ABSL-4实验室1执行的后果严重病原体的动物模型医学成像的独特能力。提供给研究者的成像模态包括:计算机断层扫描（CT），磁共振成像（MRI），正电子发射断层扫描（PET），单光子计算机断层摄影（SPECT），超声波，X射线，和透视。研究人员利用现有的成像能力，监测病情进展和评价干预措施，例如药物治疗和接种疫苗的疗效，在纵向研究。 在IRF-弗雷德里克的成像方式是专门旨在保持设备的核心部件高围堵2,3之外，为维护和修理访问。这种设计成像套件为"热"（含病原体）和分离"冷端"。实现这种分离，专门设计的管构造成高度封闭空间延伸到每一个成像模态（ 图1）的孔中。除了提供生物围堵，这些管保护用于净化高封闭实验室气体和化学品的影像设备。成像的科学家和技术专家从"冷端"操作扫描仪，而比较医学（CM）的工作人员把手，显示器上的"偏热"的动物。由于CM的工作人员必须用影像的科学家紧密合作，协调这些实验中，这种分离可能造成通信的挑战。 评估可供选择之后，CM工作人员出来配有蓝牙耳枚发射短波长的超高频无线电波用来调用成像员工围堵之外的手机。由于设施的设计，无线接入点必须被安装在每个房间克服造成之间的"热"水泥和钢的各层和"冷侧"的信号的干扰。因此，CM的工作人员穿着嘈杂的正压力服和成像人员高度封闭外之间的通信现在是可靠的。摄像机也已安装上的成像室偏热成像人员看到了"偏热"的活动。随着摄像头，成像人员可以指导CM技师动物定位或在最后一分钟改变成像协议。 在IRF-弗雷德里克ABSL-4实验室套装的所有工作人员需要佩戴正压封装服4。穿着这些衣服减少流动性，和重晚点¯x手套附西装加手套多达三个附加层妥协灵巧。其结果是，过程需要更长的时间才能完成，需要精细运动技能任务要困难得多。作为生物安全水平的提高，动物处理和操纵变得更具挑战性和耗时的，特别是与小动物。在ABSL-4实验室程序最多可能需要2-3倍的时间比ABSL-2实验室。 本文的目的是为了在视觉上展示与成像动物模型中使用豚鼠，例如CT扫描过程的ABSL-4环境相关的挑战。

<table border="0" cellpadding="0" cellspacing="0" width="916">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Micro-Chem Plus</td>
			<td style="width:237px;">National Chemical Laboratories</td>
			<td style="width:139px;">255</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CT scanner</td>
			<td>Philips Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CT phantom</td>
			<td>Philips Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isovue-300 (CT contrast reagent)</td>
			<td>Bracco Diagnostics</td>
			<td>NDC 0270-1315-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ventilated rack</td>
			<td>Lab Products</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-isolator cage</td>
			<td>Lab Products</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biosafety cabinet</td>
			<td>Nuaire</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia machine</td>
			<td>SurgiVet</td>
			<td>WWV9000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia induction box</td>
			<td>VetEquip</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia mask</td>
			<td>Henry Schein</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Henry Schein</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waste gas scavenging canister</td>
			<td>Fisher</td>
			<td>F/AIR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Holding cushion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ophthalmic ointment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vital signs monitor</td>
			<td>Bionet</td>
			<td>BM3Vet</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mobile phone</td>
			<td>Spectralink</td>
			<td>8440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blue Tooth ear piece</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wireless access points</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sperian positive-pressure suit</td>
			<td>Honeywell Safety Products</td>
			<td>BSL 4-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Outer suit gloves (latex, Ansell Canners and Handlers)</td>
			<td>Fisher</td>
			<td style="width:139px;">19-019-601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Outer suit gloves (nitrile/rubber, MAPA)</td>
			<td>Fisher</td>
			<td>2MYU1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scrubs</td>
			<td>Cintas</td>
			<td>60975/60976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Socks</td>
			<td>Cintas</td>
			<td>944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Duct tape</td>
			<td>Pack-N-Tape</td>
			<td>51131069695</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Towels</td>
			<td>Cintas</td>
			<td>2720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zip lube</td>
			<td>Amazon</td>
			<td>B000GKBEJA</td>
			<td></td>
		</tr>
	</tbody>
</table>

safety precautions and operating procedures in an (a)bsl-4 laboratory: 4. medical imaging procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

严格遵守所有安全程序和标准作业程序，动物处理是在ABSL-4实验室安全工作至关重要。从动物操作室到成像套件感应箱内转印感染动物减少公共走廊污染的风险。按照规定的程序，没有实验室获得性感染或动物对象的交叉污染已记录，而在IRF-弗雷德里克导电ABSL-4的研究。 ABSL-4病毒病原体可诱导肺相关疾病10,11，和CT是...

Watch this Scientific Journal Video about Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures at JoVE.com

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

安全注意事项和操作步骤在（A）BSL-4实验室:4.医疗成像过程

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

safety-precautions-operating-procedures-an-bsl-4-laboratory-4-medical

Research

JoVE Journal

Immunology and Infection

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

10.8K 次观看.  National Institutes of Health (NIH). Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

视频: Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

科研

免疫与感染

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

安全注意事项和操作步骤在（A）BSL-4实验室:1.生物安全4级实验室西服套房出入境手续

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

成像免疫突触动力学内皮平面细胞模型

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

安全注意事项和操作步骤在（A）BSL-4实验室:2.常规做法

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

安全注意事项和操作步骤在（A）BSL-4实验室:3，大气生物学

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

毛里小肠内皮淋巴细胞的内质成像

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

小鼠中人类胸膜皮质瘤的PET/CT的植入与监测

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

小鼠骨髓移植程序研究克隆造血症

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...