我们要感谢克斯廷Thim（阿斯利康，瑞典隆德）贝尼塔·达尔伯格和安德斯·埃克伦德博士（Karolinska研究所，斯德哥尔摩，瑞典）以及马丁Stampfli博士（麦克马斯特大学，汉密尔顿，ON，加拿大）的熟练援助和专家的意见。

动物研究批准了北斯德哥尔摩动物福利伦理委员会。在实验过程中符合瑞典法律进行的。 1.生成一个LPS气溶胶<ol><li>溶解0.5克纯化P.假单胞菌内毒素在50ml无菌盐水中以温和搅拌并验证溶解。稀释1毫升9毫升无菌盐水溶解的LPS，以1毫克/毫升的最终浓度。防止光用铝箔并储存在-20℃。 </li><li>解冻溶解的LPS在黑暗中在室温下以及在使用之前立即混合。 </li><li>在通风II级生物危害罩中，插入一个红色入口进入喷雾器，并通过由制造商（审查方案呈递的实验装置图1）所提供的管道连接到雾化器的加压室内空气。 注意：适当的个人防护装备，包括一个半面罩可重复使用的respirat或具有微粒过滤器，护目镜，手套和防护服应该曝光的过程中被使用。 </li></ol><img alt="图1" src="/files/ftp_upload/51470/51470fig1highres.jpg" /> 图1：用于产生气溶胶的实验装置的示意图介绍的喷雾器的入口连接到空气供给。雾化器的出口经由15.9毫米管和空气过滤器，和空气供给第一连接至流量计在2千巴的压力被调整为5.0升/米。出口是下一个连接到有机玻璃盒子装有可拆卸盖和5毫米的孔，​​以防止压力积聚。 <ol start="4"><li>经由空气过滤器的雾化器的出口连接到一个质量流量计。质量流量计连接到电源供应。 </li><li>调节空气供给到5升/分钟，压力保持在1.0-2.0巴。 </li><li>取下质量流量计和切断气源。 </li><li>雾化器的出口连接到15.9毫米的管道，它分叉并连接两个有机玻璃盒子的尺寸：150×163 X205毫米，配有可移动的盖子。每个框应该有一个5毫米的孔中的一侧相对的所述入口，以防止压力积聚。 </li><li>将多达5只小鼠中的每个有机玻璃盒，并关闭盖子。 </li><li>打开雾化器和填充插入至少4毫升LPS溶解在无菌生理盐水或车辆单独（无菌生理盐水，音量不得超过8毫升）。重新连接的入口空气供应。 </li><li>允许气雾剂流入封闭有机玻璃盒子10分钟。持续监测动物。确保气源仍紧紧地固定在雾化器的入口。 </li><li>切断气源。用盖子封闭，让动物保持在有机玻璃盒子2分钟。 </li><li>打开盖子，并允许气雾剂以分散，并返回动物吨他的笼子。如果动物出现湿的，放置在一个加热垫设置为低热量的笼，以防止体温过低。 </li><li>连续监测动物的第30分钟，此后每隔2小时，第6小时。动物应表现出中和雾化过程后正常呼吸模式和活性。 </li></ol> 2.支气管肺泡灌洗（BAL） <ol><li>在试验终点，深深地麻醉动物用异氟醚的效果建议您机构的兽医人员。捏后爪以检查撤退反射，以确保麻醉足够的深度来进行大手术。喷洒下来，用70％的乙醇的动物的毛皮。 </li><li>打开用剪刀腹部，并切断主动脉抽血动物。将一块组织在腹部吸收血液。 </li><li> Follwing安乐死，用剪刀单前后下调至暴露胸部。抬起左肋通过胸骨的前尖端和使用剪刀刺穿隔膜在最腹侧点，而不切入任何肺叶。通过在前后方向上的两个切口（会议下颚下方）打开胸腔。 </li><li>轻轻拉开使用镊子肋骨，切喉下面的气管。 </li><li>抬起用钳子气管和通过切割连接所述瓣到胸腔的韧带，以及由脂肪和心脏组织轻轻拉动取出肺。 </li><li>插入聚乙烯管（内径0.58毫米;外径：0.965毫米）进气管，并固定管与丝线的字符串。 </li><li>打结的multilobe（四裂片右肺）用丝线。 </li><li>插入23号针插入聚乙烯管，慢慢加入250μl冰冷的无菌PBS注入用1ml注射器的单叶。 </li><li>仔细挖掘肺30次，并收集通过注射器的液体。用200微升PBS重复上述步骤（约300微升PBS应该从450微升的总注射量的单瓣恢复）。 </li><li>置于冰上的支气管肺泡灌洗液（BALF），或立即列举BAL细胞。计数细胞与heamocytometer，使用特克溶液染色细胞11。通过细胞数与染色溶液的稀释因子和heamocytometer的计数字段内的体积乘以计算总细胞数。 </li><li>其他地方11所描述的细胞离心细胞准备差分细胞计数。 </li></ol>肺组织的3福尔马林固定的组织学评估<ol><li>取出multilobe和管理单元冻结干冰。储存在-80℃。 </li><li>装入60毫升注射器与柱塞上的金属支座除去。填用10％福尔马林到注射器20厘米的实验台以上，较20厘米的恒定压力的高度。 </li><li>连接23号针通过​​塑料管与阀来控制福尔马林的流量固定在气管60毫升注射器的。 </li><li>吹进所述肺叶用福尔马林进行5分钟。断开针并与来自气管的聚乙烯管中取出在一起，同时拉动丝线上以关闭气管与保留在肺中的压力。 </li><li>淹没在福尔马林的肺叶，并在4℃下固定24小时。 </li><li>洗固定的组织3次进行至少20分钟，用70％的乙醇，和脱水二甲苯通过以下方案（每1小时）： <ul><li> 3倍70％的乙醇</li><li> 3倍的95％乙醇</li><li> 3×100％的乙醇</li><li> 3倍二甲苯</li><li> 1X（液体）石蜡</li></ul></li><li>嵌入脱水组织中的石蜡，切成4-5微米的部分，和染色用苏木精和曙红，以允许进行组织学评估。</li>
</ol>

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亚伯拉罕鲁斯已收到来自勃林格殷格翰，阿斯利康公司研发和阿斯利康研发一个研究资助旅费补助讲师费。马格努斯北是阿斯利康公司研发的全职员工，并已获得研究经费来自阿斯利康研发。约翰·格鲁内瓦尔德和托芙伯格是由阿斯利康研发资金无关的研究项目的合作研究者。

雾化LPS产生的气道炎症反应，在上皮粘膜下层特征在于嗜中性粒细胞，围绕传导气道，以及肺泡空格的空格。这是，与增加的总蛋白含量BALF中，表示血浆渗漏，有代表性的急性肺损伤的病理在一起。作为脂多糖诱导无菌炎症，反应是独立的适应性免疫应答的，并且有限制的相关性细菌感染。该技术可能，但是，被用于通过排除适应性免疫应答来剖析炎症机制。 虽然该方法是?...

脂多糖（LPS）是革兰氏阴性细菌1细胞壁成分。挑战LPS是急性肺损伤，特点是急性嗜中性粒细胞炎症和水肿2综合征的证据充分的模式。此外，肺中性白细胞增多也是慢性阻塞性肺疾病（COPD）3，和在人类中LPS刺激的一个标志已被用于模拟COPD恶化4。因此，LPS暴露的实验模型在临床上有关和有价值的工具来理解人类的病理。 的肺递送这里描述雾化脂多糖的目标是产生在导通和呼吸道，一个中性的炎症反应，无全身参与。的LPS攻击的几种技术已如前所述。静脉内注射LPS是最常用的给药途径。虽然这种技术是方便，T他主损伤是对内皮，与肺上皮以下中性粒细胞迁移至肺二次破坏。静脉内给药也诱导全身性炎症2，它可以在动物模型复杂化的临床图像。全身性炎症是相不与气管内给药观察到。这种技术，但是，是劳动密集型的，并且需要麻醉剂以及相当训练5,6。此外，肺部沉积通过施用这条路线是依赖于呼吸7。因此，肺部沉积由所需的帧内气管施用和可变沉积在气道可以观察到麻醉深度的影响。与此相反，肺递送用气雾脂多糖需要最少的培训，并且可以容易地实现对大量的动物几乎没有或个人5,8之间不存在差异。最近的研究证实，气雾剂递送优于对于沉积的气管内的路线，并有更多的相关剂量的LPS诱导中性粒细胞炎症与这种模式8。 以前的研究已经表明，挑战气溶胶假单胞菌假单胞菌 LPS产生的气道内腔和肺实质，包括肺泡腔9,10的显着的炎症反应。炎症的特征在于嗜中性粒细胞和肺水肿的存在的优势，因此可以用来解决急性肺损伤的发病机制和增益有助于疾病病理机制的进一步了解。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Name of the material/equipment&#160;</td>
			<td style="width:244px;">Company</td>
			<td style="width:145px;">Catalog number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:307px;">Purified Pseudomonas aeruginosa LPS&#160;</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Harmful. Recomended purification. LPS purified from other bactria may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pari LC sprint star nebulizer</td>
			<td style="width:244px;">PARI Respiratory Equipment Inc.&#160;</td>
			<td style="width:145px;">023G1250</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">TSI mass flowmeter 4040</td>
			<td style="width:244px;">TSI</td>
			<td style="width:145px;">4040</td>
			<td style="width:235px;">Alternative product from supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Saint-Gobain 15.9 mm Tygon tube</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">Z685704</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Plexiglas boxes with removable lids</td>
			<td style="width:244px;">Custom built</td>
			<td style="width:145px;">N/A</td>
			<td style="width:235px;">150 x 163 x 205 mm (a 2 mm hole on the side).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Half Facepiece Reusable Respirator</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">7503</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Advanced Particulate Filters (P100)&#160;</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">2291</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sissors</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">233-1104</td>
			<td style="width:235px;">Preferred scissors may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Forceps&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">232-1313</td>
			<td style="width:235px;">Preferred forceps may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Intramedic PE50 polyethylene tube</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">427411</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethicon 2-0 Perma-hand silk tread&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">95056-992</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">26 &#189;&#160; gage needle&#160;</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">1 mL BD slip-tip syringe, non-sterile</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025">301025</a></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">60 mL BD Luer-Lok syringe, non-sterile, polypropolene&#160;</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">301035</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluka Hematoxylin-Eosin</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">3972</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T&#252;rk&#39;s solution</td>
			<td style="width:244px;">Merck Millipore</td>
			<td style="width:145px;">109277</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Table top centrifuge</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative manufacturers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytospin 4 cytocentrifuge</td>
			<td style="width:244px;">Thermo Scientific</td>
			<td style="width:145px;">A78300003</td>
			<td style="width:235px;">Alternative centrifuge can be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEMA-3 stat pack</td>
			<td style="width:244px;">Fisher Scientific</td>
			<td style="width:145px;">23-123-869</td>
			<td style="width:235px;">Alternative staining kits exists.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">HT501128&#160;</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
	</tbody>
</table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

急性肺损伤（ALI）是一种严重的疾病，特点是肺泡中性粒细胞，用有限的治疗方法，死亡率高。 ALI的实验模型是提高我们对疾病发病机制的认识键。脂多糖（LPS）的革兰氏阳性菌引起得出中性粒细胞炎症的气道和小鼠的肺实质。有效的肺输送诸如LPS化合物，然而，难以实现。在这里所描述的方法中，肺递送在小鼠中通过挑战气溶胶绿脓杆菌脂多糖实现。溶解的LPS气溶胶由连接到压缩空气喷雾器。小鼠暴露于LPS气溶胶在一个有机玻璃盒子的连续流动10分钟，随后2分钟调理气溶胶被中断之后。气管插管和随后的支气管肺泡灌洗，接着福尔马林灌注在下一步进行，这允许无菌的p表征ulmonary炎症。雾化脂多糖产生肺部炎症特征在于肺泡中性粒细胞，在支气管肺泡灌洗和组织学评估检测。这种技术可以被设置在一个小的费用少用具及需要最少的培训和专门知识。曝光系统因此可以常规进行在任何实验室，有潜力，以提高我们的肺病理学的理解。

挑战雾化P.假单胞菌内毒素通常会产生在气道管腔和肺泡空间，在早期和晚期时间点的特征在于，中性粒细胞为主的显着的炎症反应。 雾化LPS诱导肺中性粒细胞增多 C57BL / 6BY和BALB / c小鼠暴露于雾化P.铜绿假单胞菌 LPS或单独的车辆和中性粒细胞列举BALF。对C57BL暴露于与车辆产生的烟雾/ 6BY小鼠的BALF细胞总数仅是通常大约或低于200,000个细胞，并?...

Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

我们描述诱导中性粒细胞肺部炎症通过挑战脂多糖雾化通过雾化，建模急性肺损伤的方法。此外，基本的手术技术对于肺隔离，气管插管和支气管肺泡灌洗也有所说明。

采用雾化脂多糖一种产生肺中性粒细胞增多

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

a-method-for-generating-pulmonary-neutrophilia-using-aerosolized

Research

JoVE Journal

Immunology and Infection

11.6K 次观看.  Karolinska Institutet. 我们描述诱导中性粒细胞肺部炎症通过挑战脂多糖雾化通过雾化，建模急性肺损伤的方法。此外，基本的手术技术对于肺隔离，气管插管和支气管肺泡灌洗也有所说明。 

视频: A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

标签胚胎小鼠血管的方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

科研

免疫与感染

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Xpert山地车/ RIF测试与诊断肺结核

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

用 crispr/cas9 基因编辑生成重组禽流感病毒载体

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

免疫沉淀法研究受体与酶的功能相互作用

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

一种新的生理免疫原树突状细胞生成方法

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

使用全身吸入系统在小鼠中暴露香烟烟雾

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

通过显微注射在类 圆线虫 物种中产生转基因和敲除

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...