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

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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。

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			<td style="width:147px;">33198</td>
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			<td>MT-15-040-CV</td>
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			<td>SH3003401</td>
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			<td>BP685-1</td>
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			<td>E6758</td>
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			<td>ICN15178101</td>
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			<td>PI-89811</td>
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			<td>10010-049</td>
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			<td>472301</td>
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			<td>V4629</td>
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			<td>93484</td>
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			<td>15-250-061</td>
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			<td>04-355-451</td>
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			<td style="width:203px;">Agilent</td>
			<td style="width:147px;">1260</td>
			<td>Multi-compenent</td>
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			<td style="width:203px;">AB Sciex</td>
			<td>4000</td>
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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 ...

Research

JoVE Journal

Immunology and Infection

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

In vitro Biofilm Formation in an 8-well Chamber Slide

In vitro Biofilm Formation in 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

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

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

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

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

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

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

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

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

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

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

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

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. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

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

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

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 ...