我们要感谢强生公司，结核病联盟，阿斯利康Zeneca，Rib-X和Trius治疗公司分别提供床上苯胺，PA-824（pretomanid），AZD5847，radezolid和tedizolid。 Brendan Prideaux，Matthew Zimmerman，Stephen Juzwin，Emma Rey-Jurado，Nancy Ruel，Leyan Li和Danielle Weiner为MALDI分析，生物分析方法，干酪替代品的制备，化学合成和兔子白蛋白的分离提供了支持。这项工作是由比尔和梅林达·盖茨基金会资助的，拨款为OPP1044966和OPP1024050的V. Dartois，NIH Shared Instrumentation Grant S10OD018072，以及Bill和Melinda Gates基金会以及Wellcome Trust for A卓越中心的联合资助。将发展中世界疾病的潜在优化用于P.Wyatt。

所有动物研究按照指南国立卫生研究院的实验动物护理和使用从NIAID的机构动物护理和使用委员会（NIH），马里兰州贝塞斯达的批准进行。所有涉及结核分枝杆菌的研究是在实验室中的生物安全防护水平3（BSL-3）执行。 1.兔感染模型和Caseum收藏<ol><li>使用鼻式气溶胶曝光系统如前所述感染新西兰白兔结核分枝杆菌 。 12，13允许感染为12-16周进行。稳重用35毫克/千克氯胺酮和5mg / kg的甲苯噻嗪肌内，兔子安乐死与0.22毫升/公斤戊巴比妥钠和苯妥英钠兔子静脉内并与尸检进行。 </li><li>使用镊子和手术刀，从胸部取出CA肺VITY。从每个肺叶，使用手术刀解剖单个腔和大的坏死性肉芽肿。仔细地从空腔和肉芽肿壁上刮掉面包。称量，记录并将样品储存在-20℃的2 mL螺旋管中，直至准备使用。 </li><li> Gamma在干冰上照射3 MegaRad上的感染性病例样本，使其在BSL-2实验室中不受感染和安全使用。 </li></ol> 2.来自THP-1细胞的表皮代谢产物的体外生成<ol><li>在T175细胞培养瓶（80 mL /瓶）中，在RPMI 1640培养基（2 mM L-谷氨酰胺和10％胎牛血清）中培养THP-1单核细胞。将烧瓶在5％CO 2气氛中于37℃孵育3-4天。 </li><li>将来自T175烧瓶的培养物以150×g离心5分钟的两个50mL锥形管中。弃去上清液并将沉淀悬浮在10mL的RPMI 1640培养基中。 </li><li>将5μL该培养液移至含有45μLt的1.5 mL试管中紫苏蓝通过移液彻底混合。将10μL转移到血细胞计数器，并使用光学显微镜（10倍放大）计数活的THP-1单核细胞数（未染色）。计算每毫升培养物的活细胞数。用RPMI培养基稀释至1.25×10 6个细胞/ mL的最终密度。 </li><li>在大细胞培养板（50×10 6个细胞/板）上加入40mL培养物。加入40μL100μMPMA（佛波醇12-肉豆蔻酸乙酯13-乙酸乙酯制备），并使细胞在培养箱中过夜。 注意：PMA的最终浓度为100nM。 </li><li>在乙醇中稀释纯油酸（OA）（0.89 g / mL）至浓度为0.1 M（ 即在968.3μL乙醇中的31.7μLOA）。将此溶液稀释至新鲜预热的RMPI培养基中，浓度为10 mM。将该OA悬浮液稀释至预温至37℃的RPMI培养基中的0.4mM（最终工作浓度）。 </li><li>删除现有媒体和非从细胞培养板中吸收细胞，并轻轻加入40 mL 0.4 mM OA至THP-1巨噬细胞（THP-M）。在培养箱中37℃孵育过夜。 </li><li>使用40倍放大的光学显微镜来目视确认每个THP-M中存在许多脂质体夹杂物。从细胞培养板中取出所有的RPMI培养基，并用50 mL血清移液管用磷酸盐缓冲盐水（PBS）轻轻洗涤贴壁细胞两次。 注意：脂质体在THP-M的细胞质中表现为小的，清晰的球形结构。 </li><li>在PBS中加入40mL 5mM乙二胺四乙酸（EDTA）的PBS。在37℃孵育15分钟。 </li><li>通过使用10 mL血清移液管重复吸取整个板的表面，分离泡沫巨噬细胞（FM）。将细胞悬浮液转移到50mL锥形管中，并以150×g离心5分钟。 </li><li>将细胞沉淀重悬于10mL PBS（第三次PBS洗涤）中并转移到预先称重的15-mL锥形管中。以150 xg再次旋转5分钟。使用血清移液管小心吸出上清液并弃去。 </li><li>将FM颗粒置于3个冻融循环中以裂解细胞并将其在75℃下孵育20-30分钟以使基质中的蛋白质变性。将颗粒储存在-20°C直到准备使用。 </li></ol>快速平衡透析（RED）测定<ol><li>在二甲基亚砜（DMSO）中制备所有测试化合物的10mM储备溶液。在每次测定之前，稀释至DMSO中的500μM工作溶液。 </li><li>称重含有小麦替代颗粒的管。减去空管的重量以得出单独的颗粒的重量。每管添加2-3个金属珠，并使用组织匀浆器以1200冲程/分钟1分钟，破碎干酪或PBS（1：9w / v）中的替代物基质，以达到每种基质的10倍稀释悬浮液。 </li><li> Spike 6.5μL的500试验化合物的μM溶液进匀浆643.5μL达到5μM（≤1％DMSO）和涡流的最终浓度。 </li><li>放置RED插入到基座板。添加药物掺入基质到每个RED插入物的供体腔室（红色环）和350μLPBS到每个接收器的腔室200μL。制备3插入各试验化合物（一式三份样品）。密封板用粘接剂密封板并孵育在37℃下在以200rpm（1×g）离心4小时的恒温。 </li><li>培育后，轻轻上下吹打2-3次的供体和受体室的内容混合。从供体室吸取出匀浆的20μL等分试样，并添加至PBS干净的20μL在1.5mL管（1：1）。类似地，从接收器腔室吸取出PBS样品的20μL的等分试样，并添加至20μL的清洁匀浆（矩阵匹配）。 8 注：矩阵匹配消除了NEED为2条独立的校准曲线（在匀浆和PBS）用于定量分析进行。供体腔的内容可能沉淀一段时间。轻轻由前中吸取吹打混合的内容。 </li></ol> 4. LC-MS定量和数据分析<ol><li>加1的160μL：1甲醇：乙腈含有500毫微克/毫升的双氯芬酸或10ng / mL的维拉帕米（内标），以每管和涡流以沉淀蛋白质。离心机以10,000×g离心5分钟以沉淀沉淀物和上清液转移到96孔用于液相色谱 - 质谱（LCMS）分析深孔板。 7 </li><li>从1-1,000纳米的各试验化合物建立校准曲线，同时保持相同的基质组合物作为样品的上方。量化测试化合物在从使用LC-MS方法的供体和受方腔室的样品的浓度。 </li><li>计算未结合的分数（f u）的 Ôf使用方程1稀释的基质中的药物。使用等式2计算未稀释基质中的f u （ D =稀释因子10）。 14 <img alt="公式1" src="/files/ftp_upload/55559/55559eq1.jpg" /> <img alt="公式2" src="/files/ftp_upload/55559/55559eq2.jpg" /></li><li>使用方程3检查每种化合物的恢复（质量平衡），以鉴定具有稳定性/代谢/非特异性结合问题的化合物。 注意：恢复通常在70％到130％之间。 15 <img alt="公式3" src="/files/ftp_upload/55559/55559eq3.jpg" /></li></ol>

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没有竞争的经济利益。

肺坏死病灶和空洞肺结核感染的患者含有细菌是顽抗到药物治疗的亚群。这些结构的干酪性芯是用于在细胞外环境中携带这些持留尤其负责。抗细菌剂为这些远程位置的16有利分布被认为是肺结核药物疗效的重要决定因素。在此之前协议的验证，没有体外技术，可以预测干酪样病变的药物发现的化合物的分布可用。其结果是，在这些关键病灶药物分布的评价在很大程度上?...

肺结核病的治疗需要的药物有效的分配到不同类型的病变。坏死性病变和腔包含窝藏毒品宽容或“老大难”细菌的亚群干酪中心。 1，2空洞病与劣质治愈率和预后较差。 3，4以前的研究表明，使用定量和成像技术，即穿透caseum的能力而变化显著从一个药物类到另一个。 5,6这些方法，但是，需要使用动物感染模型是缓慢而乏味的。测量药物结合到离体 caseum 在体外测定的目的。这种结合被发现成反比地干酪样肉芽肿药物渗透和相互关联的，因此，是作为预测工具。 7 平衡透析被认为是黄金标准的方法来血浆蛋白结合研究。快速平衡透析（RED）装置提供进行这种测定的一种快速，易于使用，可靠的系统。 8所述的装置由两个部件组成：单次使用的，由通过半透膜的垂直筒2个分隔室的一次性刀片;和可重复使用的基板，其可容纳在一个时间48点的插入物。透析膜具有8kDa的分子量截止（MWCO），是理想的药物 - 大分子结合研究。膜隔室的高表面积 - 体积比允许快速透析和平衡。两个插入件和底板已经验证为最小的非特异性结合。用生物分析技术RED设备的组合提供了药物在p中未结合的级分的精确估计lasma。 8,9 虽然最初设计用于测量血浆蛋白结合，该RED设备已在若干组织结合研究使用匀浆使用。 10，11在这个协议中，我们测量药物结合到caseum，坏死碎屑从坏死损害和结核病感染的兔的空腔切除。干酪性材料的所述无细胞和非血管性质可以很容易地均匀成均匀的悬浮液，其与测定兼容。 鉴于caseum是乏味的，以产生和来之不易，该协议也被验证为与从泡沫巨噬细胞制备的替代矩阵使用。 THP-1单核细胞衍生的巨噬细胞与油酸诱导的积累，让他们自己的“泡沫”外观的多个脂质体。这些脂质加载收获细胞和加工成产生一个矩阵，我们用作表壳的替代品。这项研究表明，与该替代基质结合的药物与结合蛋白质有很好的相关性，有效地模拟了妨碍药物在肉芽肿和空腔的核心中的渗透的体内过程。

<table border="0" cellpadding="0" cellspacing="0" width="960">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">New Zealand White Rabbits</td>
			<td style="width:203px;">Covance</td>
			<td style="width:147px;">-</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">HN878 Mycobacterium tuberculosis</td>
			<td style="width:203px;">BEI Resources</td>
			<td style="width:147px;">NR-13647&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ketathesia (Ketamine) 100mg/mL C3N</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">56344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Anased (Xylazine) 100mg/mL</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">33198</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Euthasol (pentobarbital sodium and phenytoin sodium) Solution&#160;</td>
			<td style="width:203px;">Virbac</td>
			<td style="width:147px;">710101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">THP-1 monocytic cell line</td>
			<td style="width:203px;">ATCC</td>
			<td style="width:147px;">ATCC TIB-202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">175cm&#178; TC-Treated Flask (T175)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-3400-175</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 media w/o glutamine</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>MT-15-040-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone Fetal Bovine Serum, Gamma irradiated&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3091003IR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone L-glutamine, 200mM</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3003401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellstar TC dish, 145x20mm, vented</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-2881-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phorbol 12-myristate13-acetate (PMA)&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>BP685-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:203px;">Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oleic acid&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>ICN15178101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Reusable Base Plate</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Inserts, 50/box&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED insert removal tool&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>89812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adhesive plate seal</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>08-408-240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH7.4, 10 X 500 mL (Gibco)&#160;</td>
			<td style="width:203px;">Life Technologies&#160;</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">DMSO</td>
			<td style="width:203px;">Sigma</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Acetonitrile</td>
			<td style="width:203px;">Sigma</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Methanol</td>
			<td style="width:203px;">Sigma</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Verapamil hydrochloride</td>
			<td style="width:203px;">Sigma</td>
			<td>V4629</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Diclofenac sodium salt</td>
			<td style="width:203px;">Sigma</td>
			<td>93484</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-250-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ethanol, 200 proof</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">2010 Geno/Grinder</td>
			<td style="width:203px;">SPEX SamplePrep</td>
			<td style="width:147px;">2010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Bead Mill Homogenizer Accessory, Metal Bulk Beads</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-340-158</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">484R Cobalt 60 Irradiator</td>
			<td style="width:203px;">JL Shepard</td>
			<td style="width:147px;">7810-484-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">INCYTO&#160;C-Chip Disposable Hemacytometers</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:147px;">22-600-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Upright Light Microscope</td>
			<td style="width:203px;">Leica</td>
			<td style="width:147px;">DM1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Binary Liquid Chromatography system</td>
			<td style="width:203px;">Agilent</td>
			<td style="width:147px;">1260</td>
			<td>Multi-compenent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Mass spectrometer</td>
			<td style="width:203px;">AB Sciex</td>
			<td>4000</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro caseum binding assay that predicts drug penetration in tuberculosis lesions

消除结核病需要能够穿透多层复杂肺部病变的药物治疗方案。空腔和病变的干酪核心中的药物分布尤其重要，因为它们具有耐药细菌的亚群，通常也称为持久性细菌。用于测量结核病变中药物渗透的现有方法涉及与生物分析或成像技术相结合的昂贵且耗时的体内药代动力学研究。提出了与大肠杆菌大分子结合的药物的体外测定作为这种技术的替代方法，因为这种结合阻碍了药物分子通过底物的被动扩散。快速平衡透析是一种用于进行血浆蛋白和组织结合研究的快速可靠的系统。在该方案中，我们使用快速平衡透析（RED）装置来测量药物与排泄物的匀浆的结合从结核病感染的兔子的病变和腔中。该方案还描述了如何从脂质负载的THP-1巨噬细胞产生代替基质来代替小麦蛋白。这种病例/替代结合试验是结核病药物发现的重要工具，可用于帮助研究由其他疾病引起的病变或脓肿中的药物分布。

使用这个协议，我们已经测试了数百种结核病药物开发化合物，以达到预期的穿透性白蛋白的效率。 图1显示了RED测定的基本概念。 RED插入物的透析膜允许未结合的小分子从供体孔扩散到接收器，最终实现两个室之间的平衡。与大分子如蛋白质或脂质结合的小分子被捕获在供体井中。来自两个室的样品的定量分析允许计算小分子或替代基质中每个小分子的未?...

Watch this Scientific Journal Video about An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions at JoVE.com

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

在这里，我们描述了一种快速平衡透析（RED）方法，用于测量肺结核病变和腔内药物与病例的结合。该方案也与泡沫巨噬细胞衍生基质一起使用，这是一种有效的替代试剂。

一个<em&gt;体外</em&gt; Caseum结合测定，预测药物在渗透结核病变

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

an-vitro-caseum-binding-assay-that-predicts-drug-penetration

Research

Education

JoVE Journal

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Pharmacokinetics and Pharmacodynamics

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Immunology and Infection

Unit 1

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Nonlinear Pharmacokinetics

Introduction to Respiratory System Drugs

SCIENTISTS IN ACTION

11.6K 次观看.  Rutgers. 在这里，我们描述了一种快速平衡透析（RED）方法，用于测量肺结核病变和腔内药物与病例的结合。该方案也与泡沫巨噬细胞衍生基质一起使用，这是一种有效的替代试剂。 

视频: An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

科研

免疫与感染

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

促进药物发现：斑马鱼在一个自动化的高含量检测炎症

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

参考微量稀释法达巴万星<em&gt;体外</em&gt;细菌敏感性测试生长有氧

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

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. ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

体外方法比较标的结合和CDC诱导治疗性抗体之间：应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...