这项工作是由NIH RO1 AI079253和退伍军人事务部。

1。动物模型<ol><li>鼠标:使用P47 PHOX-/ -小鼠与年龄和性别匹配的C57BL6/DBA小鼠。获得的实验，从实验动物护理和使用委员会的批准。 </li><li>麻醉:使用连续的异氟醚麻醉诱导系统。汽化器的系统（VetEquip）填充用异氟醚（2-3％）。确认，老鼠是完全麻醉，观察呼吸，运动及角膜反射，在对外部刺激的反应。 </li><li>外科手术:磨砂手术区（台式），用70％的乙醇。戴无菌手套和干净的手术衣和面具。准备小鼠经削波的头发和应用聚乙烯吡咯酮碘，用70％乙醇，将切口的地方交替。无菌的表面上，并使用无菌器械进行手术。监视器小鼠手术后，直到清醒和自由​​移动。 </li><li>气管酵母管理<ol><li>管理麻醉如在1.2中描述。 </li><li>聚维酮碘和70％乙醇消毒手术区（颈部），暴露气管的手术剥离。 </li><li>皮尔斯气管用27号针头注射酵母多糖（圣路易斯，MO）的剂量为1微克/克，采用0.5微克/微升的溶液（总体积为50微升的25克鼠标）溶解于无菌PBS中。 </li><li>在无菌条件下，用5-0无菌丝线缝合关闭小鼠颈部伤口。 </li><li> 4小时和24小时后的图像。 </li></ol></li><li>盲肠结扎穿刺<ol><li>管理麻醉，如在1.2中描述。 </li><li>剪辑的头发在手术区（腹部），并与聚乙烯吡咯酮碘和70％乙醇消毒。 </li><li>执行中线剖腹探查术，并确定盲肠。 </li><li>用4-0丝线缝合穿刺盲肠末端的21号针，结扎一次暴露盲肠结扎远端的50％。 </li><li>用4-0无菌丝线缝合关闭切口。 </li><li>图片4小时后，24小时。 </li></ol></li><li>口腔四氯化碳（CCL 4）管理<ol><li>管理麻醉，如在1.2中描述。 </li><li>管理CCl 4中 （2微克/克，圣路易斯，MO）溶解在玉米油中，通过口腔管饲法。 IBC / EHS政策，四氯化碳管理的程序应在指定的区域内进行。 </li><li> 4小时和24小时后的图像。 </li></ol></li></ol> 2。获取生物发光图像<ol><li>初始化IVIS 200（精诺真公司，加利福尼亚州Alameda）成像系统，设置发光模式，电荷耦合器件（CCD）的温度来锁定和等待。 </li><li>选择曝光时间，分箱（中）和F / STOP（8）设置。 </li><li>将小鼠仰卧位成像室在加热阶段，以维持正常的体温。管理麻醉在经鼻锥1.2，而老鼠是在IVIS成像室。 </li><li>管理L-012<html> （20微克/克）溶解于无菌PBS（10微克/微升）静脉内通过在每个时间点选择为成像眶注射。 </li><li> L-012注射开始后2分钟拍摄图像。我们通常使用40秒曝光。 </li></ol> 3。数据分析使用的感兴趣区域<ol><li>利用活体成像软件4.2版（精诺真公司，加利福尼亚州Alameda）进行数据分析。 </li><li>打开图像，选择测量的地区利益的工具。 </li><li>选择地区利益的形状和大小的胸部和/或腹部的鼠标在一个摄影图像重叠后的伪彩色数字图像（即光子探测）。 </li><li>量化信号强度（光子通量）从本地区的利益。 </li></ol> 4。统计<ol><li>使用统计分析软件包进行双向方差分析与Bonferroni测试后在各个时间点。 </li></ol>TLE"&gt; 5代表性的成果。 酵母多糖是一种促炎症酵母细胞壁产品和强有力的激活NADPH氧化酶3。我们以前发现，气管酵母多糖诱导肺部炎症和更强大的嗜中性粒细胞的促炎性细胞因子产生的P47 PHOX / -比野生型小鼠1。在这里，我们的目标是比较ROS生成的野生型P47 PHOX-/ -小鼠的肺部后，酵母多糖的挑战。 4小时后气管内的酵母管理，我们观察到了显着的增加光子发射的胸部在野生型小鼠相比，基线以及增加光子发射在野生型相比，P47 PHOX-/ -小鼠在4小时和24小时。相比之下，生物发光信号的p47 PHOX-/ -小鼠在4小时后，酵母多糖处理（ 图1 AB）保持在基准水平。因此会出现，NADPH氧化酶为bË产生的ROS在肺部酵母管理的主要来源。 接下来，我们评估ROS生产的CLP诱导的脓毒症模型。败血症是一种危及生命的症状与终末器官损伤，包括急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）4，5。 ROS介导的损伤一直被认为是一个重要的驱动因素败血症引起的多器官功能障碍。我们发现，ROS显着增加了胸部（4小时），并在腹部（24小时）后，中电在野生型小鼠相比，基线。然而，活性氧（ROS）的p47 PHOX-/ -小鼠的水平基线水平相似，在两个时间点（ 图2交流 ）。这些结果表明，对CLP-诱导的败血症小鼠模型中的ROS的产生NADPH氧化酶依赖性。 最后，我们利用生物发光成像检测ROS的产生，在四氯化碳诱导的肝损伤模式升。 CCl 4中引起肝细胞坏死，并已被用于建模急性肝损伤和肝纤维化6。在先前描述的模型的炎症和损伤相反，我们发现，在腹部的ROS产生温和（约35％）增加基线相比的p47 PHOX-/ -小鼠在CCl 4中给药后4小时。然而，比在野生型小鼠中的p47 PHOX-/ -小鼠（ 图A，B）的CCl 4诱导的ROS的产生显着的幅度更大。这些数据表明，P47 PHOX含NADPH氧化酶依赖和独立的ROS生成，发生在4诱导的急性四氯化碳肝损伤。 <img alt="图1" src="/files/ftp_upload/3925/3925fig1.jpg" /> 图1。ROS生产在肺部后酵母的治疗。 A）代表bioluminescenCE成像ROS的产生。需要注意的是，除了胸部，荧光检测在野生型和P47 PHOX-/ -小鼠腹部发光是不变的酵母多糖治 ​​疗腹部。 B）光子计数从胸部的野生型P47 PHOX / -小鼠气管内一次性注射酵母多糖（IT）。生物发光成像在基线，4小时，24小时。胸部以上从感兴趣的区域中的光发射被确定使用所指示的伪彩色规模。结果以平均值±SE，n = 6-9每个组，* P &lt;0.05。 <a href="http://www.jove.com/files/ftp_upload/3925/3925fig1large.jpg" target="_blank">点击查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3925/3925fig2.jpg" /> 图2。ROS检测CLP。 A）ROS的产生，B代表生物发光成像）光子计数的胸部，C）光子计数野生型和P47 PHOX-/ -小鼠的腹部在基线，4小时，24小时后电。结果以平均值±SE N = 4％至5组，* P &lt;0.05。 <a href="http://www.jove.com/files/ftp_upload/3925/3925fig2large.jpg" target="_blank">点击查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/3925/3925fig3.jpg" /> 图3。ROS生产在CCL腹部后4诱导的肝损伤。 A）代表的生物发光成像ROS的产生与B）光子计数野生型和P47 PHOX-/ -小鼠的腹部在基线，4小时和24小时后口服CCl4处理。结果以平均值±SE，n = 4％至5组，P &lt;0.05。 <a href="http://www.jove.com/files/ftp_upload/3925/3925fig3large.jpg" target="_blank">点击查看大图</a> </A&gt;。

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没有利益冲突的声明。

"实时"测量的活性氧（ROS）可以通过使用荧光和化学发光探针在生活的动物。虽然荧光探针患有具有弱的信号-噪声比12，成像技术描述的是更敏感的检测发光基于鲁米诺-底物L-012的化学反应的活性氧。像所有的生物发光的成像技术，这种方法是有限的器官和组织的依赖于波长的光的吸收和散射。这些实验都集中在建立适当的测量ROS在野生型P47 PHOX-/ -小鼠的实验条件。这些转基因小鼠的使用，使我们能区分所产生的活性氧的p47的PHOX含NADPH氧化酶的活性氧生成的其他途径。 虽然功能NADPH氧化酶P47 PHOX的活性氧的主要来源，以及其他的NADPH氧化酶的异构体存在，和额外的ROS产生系统，包括黄嘌呤氧化酶和线粒体呼吸，可以产生活性氧。 NADPH氧化酶和活性氧产生的途径有重要的影响和互动抗菌宿主防御和下游信号通路的调节炎症和损伤7-10。利用生物发光成像，我们能够测量的动力学体内 ROS的产生，并划定NADPH氧化酶的活性氧水平不同炎症模型的贡献。 这种方法的一个限制涉及到生物发光的空间分辨率。虽然我们可以测量发光在特定的解剖车厢（ 例如 ，胸部，腹部），它是很难定位的解剖部位的发光器官水平。在我们的实验模型中，我们选择了生物发光测量的3个时间点:基线，4小时和24小时。我们不知道，如果生物发光成像在ROS的产生可以识别的微小差异，在较短的时间间隔的分析，还是在体内活性氧的产生和生物发光之间的关系是线性的。这些问题需要进一步研究。在这些研究中使用的模型中，我们认为，吞噬细胞，主要负责招募中性粒细胞和巨噬细胞的吞噬细胞NADPH氧化酶系统的活性氧的产生通过。未来的研究中，被耗尽，或者骨嵌合体生成特定的细胞类型可以提供更多的知识，从不同的细胞群相对水平的ROS的产生。使用L-012的研究是另一个潜在的限制，其他的自由基（除了ROS）的反应可能与此荧光探针，减少在某些情况下，ROS检测的特异性。 继酵母多糖注射，缺乏中ROS产生的p47 PHOX-/ -小鼠表明，NADPH氧化酶是在这个模型肺部炎症的ROS的主要来源。 CLP是败血症11和生物发光成像的最常见的动物模型之一显露在肺部和腹部，NADPH氧化酶依赖增加ROS信号。对比度的酵母多糖和CLP模型，活性氧（ROS），增加了两个野生型和P47 PHOX-/ -小鼠，与基线相比，在腹部，但是在野生型小鼠在更大程度上，表明CCl 4中诱导ROS产生的p47 PHOX -含有NADPH氧化酶和其他机制。 总之，我们的数据表明，鲁米诺为基础的生物发光成像法检测ROS 在体内的活性氧生产的监管和后果的研究是很有价值的工具。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> L-012 </td><td>光化工美国公司</td><td> 120-04891 </td><td></td></tr><tr><td>酵母多糖</td><td> Sigma公司，圣路易斯，密苏里州</td><td> Z4250 </td><td></td></tr><tr><td>四氯化碳</td><td> Sigma公司，圣路易斯，密苏里州</td><td> 289116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

NADPH氧化酶是一种关键的酶介导的抗菌和抗真菌的宿主防御。 NADPH氧化酶除了其抗菌宿主防御中的作用，具有关键的信令功能，调节炎症反应1。因此，发展的方法来衡量"实时"的动力学NADPH氧化酶衍生的活性氧的产生是一个有价值的研究工具，了解相关的宿主防御，炎症和损伤的机制。 慢性肉芽肿病（CGD）是一种遗传性疾病的特点是严重感染和过度炎症反应的NADPH氧化酶。吞噬细胞的NADPH氧化酶的激活需要易位其胞浆亚基（PHOX，P67 PHOX的p47和p40的PHOX）和Rac的膜- ，绑定flavocytochrome（由一个gp91 phox和22页PHOX异二聚体）。损失在任何这些NADPH氧化酶成分结果在CGD的功能的突变。相似患者CGD，gp91 PHOX-缺陷小鼠和的p47 PHOX-缺陷小鼠有缺陷的吞噬细胞的NADPH氧化酶的活性和受损的宿主防御13，14。除了的吞噬细胞，其中含有上述成分的NADPH氧化酶，各种其他类型的细胞表达的NADPH氧化酶的不同的亚型。 在这里，我们描述了一种方法来量化ROS生产的活老鼠，并划定的贡献，NADPH氧化酶的活性氧的产生的炎症和损伤模型中的。这种方法是基于对ROS与L-012（鲁米诺类似物）反应，以发射由一个电荷耦合器件（CCD），被记录的发光。在原始描述的L-012探针，L-012-依赖化学发光完全废除了超氧化物歧化酶，表明在该反应中检测到的主要活性氧是超级超氧阴离子15。随后的研究表明，L-012，可以检测到的其他自由基，包括活性氮物种16，17。 kielland等17表明，局部应用佛波醇肉豆蔻酸乙酸酯，一种强力的NADPH氧化酶的活化剂，导致NADPH氧化酶-依赖性ROS生成使用发光探头L-012的小鼠中可检测到的。在这个模型中，他们表明:，P47 PHOX缺失的小鼠L-012-依赖的发光被取消。 我们比较了野生型小鼠和NADPH氧化酶缺乏P47 PHOX-/ -小鼠在以下三种模式:1）气管内给药的酵母，真菌细胞的促炎症墙衍生产品，可以激活NADPH氧化酶的活性氧的产生; 2）盲肠结扎穿刺（CLP），继发性急性肺部炎症和损伤腹内​​脓毒症模型; 3）口服四氯化碳（CCL），ROS依赖的肝损伤模型。这些模型是专门选定在非感染性炎症，多菌性败血症，毒素诱导的脏器损伤的情况下，分别评估NADPH氧化酶 - 依赖性ROS生成。在野生型小鼠比较，生物发光P47 PHOX-/ -小鼠，使我们能够描出P47 PHOX含NADPH氧化酶的生物发光信号在这些模型中所产生的ROS的具体贡献。 生物发光成像的结果表明增加细胞内ROS水平在野生型小鼠相比，P47 PHOX-/ -小鼠NADPH氧化酶产生的ROS的主要来源是炎症刺激。这种方法提供了一种微创的方法为"实时"监控产生的ROS在体内的炎症。

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH氧化酶在巨噬细胞的活性氧（ROS）的主要来源。由于ROS的短暂性的，它是难以衡量和监测中生活的动物的细胞内ROS水平。 ROS在活小鼠的串行量化的一种微创的方法。

生物发光成像的NADPH氧化酶活性在不同的动物模型

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

bioluminescence-imaging-nadph-oxidase-activity-different-animal

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

16.2K 次观看.  Vanderbilt University School of Medicine. NADPH氧化酶在巨噬细胞的活性氧（ROS）的主要来源。由于ROS的短暂性的，它是难以衡量和监测中生活的动物的细胞内ROS水平。 ROS在活小鼠的串行量化的一种微创的方法。

视频: Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Ecto和实验室大鼠和小鼠体内寄生虫的诊断

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

科研

免疫与感染

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

生物发光成像检测到非洲锥虫病的晚期感染

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

在抗菌蓝光治疗的体内研究的多药耐药鲍曼不动杆菌感染烧伤利用生物发光成像

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

颅内 Subarachnoidal 感染对猪链球菌生物膜在小鼠感染模型中作用的研究

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

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. ...