Name: bioluminescence imaging to detect late stage infection of african trypanosomiasis
Uploaded: 2016-05-18T14:00:00.000+00:00
Duration: 7 min 59 s
Description: Watch this Scientific Journal Video about Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis at JoVE.com

我们感谢约翰·凯利和马丁·泰勒（伦敦卫生与热带医学院）提供T.湾布氏 GVR35-VSL-2和Andrea Zelmer博士（LSHTM），用于体内成像的建议。这项工作是由比尔和梅林达·盖茨基金会全球健康项目（批准号OPPGH5337）的支持。

伦理 所有工作被英国内政部动物的批准（科学程序）1986年法令和卫生与热带医学院动物福利伦理审查委员会的伦敦经济学院下进行。 ARRIVE准则得到遵守本报告。 1.在生物发光布氏锥虫布氏的体内通道<ol><li>删除T的冷冻股票（称为stabilate） 湾布氏应变GVR35-VSL-2（可从约翰·凯利教授在卫生和热带医学伦敦学校通过MTA）从液氮9，并允许平衡至室温。稀释stabilate至2×10 4锥虫/ ml的无菌磷酸盐缓冲的葡萄糖盐水（PBS-G）的pH为8.0（1L PBS + 15克葡萄糖）10。 注:T。湾布氏 GVR35-VSL-2是非感染到人类和要求的SAPO授权设施来进行实验。 </li><li>注射等一20-25g的雌性CD1小鼠经腹腔途径（IP）稀释锥虫0.2毫升一个地下25针。这是"供体鼠"。将受感染的鼠标变成无特定病原（SPF）-cage和饲料自由采食 。 注:CD1小鼠是远交应变和T.湾布氏 GVR35感染充分表征在该菌株。然而，该协议也适用于近交或遗传修饰的菌株，但要注意，皮毛颜色可以在成像6,9的灵敏度产生影响是重要的。 </li><li>监测采血外围原虫。鼠标放置到塑料限制器和取5微升的血液从尾部通过与21g的针或放血静脉穿刺和用氯化钠0.85％无菌铵混合1/10以裂解红血细胞，并允许更容易计算。 </li><li>算在Neubauer血球稀释血液。 <ol><li>加载稀释血液到这两个腔室。计数整个中央GR一个腔室的ID，以得到N锥虫。在血液= 正 ×10 4锥虫/ ml的锥虫的浓度，调整为任何稀释因子，并计算两个腔室的平均值。 注意:在约5-7天感染后，外周血原虫应充分上升以2×10 4个锥虫/ ml的浓度来进行感染。 </li></ol></li><li>当锥虫水平足够高的所需的小鼠的数量，继续前进到步骤1.6。在一个供体小鼠的6×10 4个锥虫/ ml的原虫水平足以为10只小鼠的感染。 注意:对于统计学显著组大小中，n = 6对于每个治疗组的小鼠的需要8个 。感染了其他模型，功率计算将需要用于确定适当的组大小。 </li><li>地方的小鼠成热箱（在28-30℃下进行约5-10分钟），以允许尾静脉扩张。为了诱导终端麻醉，辖20毫克/公斤戊巴比妥静脉与地下25针。确认鼠标轻轻按下脚垫，以确保没有撤退反射麻醉。 </li><li>通过等分在5000 USP单位/毫升5 ml肝素成珠宝管和进出附连到1ml注射器针头的抽吸两次Heparinize一个21g的针头。 </li><li>取肝素21g的针和1ml注射器，并通过集中插入针左肋下执行对麻醉通道小鼠心脏穿刺（血液珠在针的底部池）并轻轻向后拉柱塞以免倒塌心脏的腔。收集至少0.7毫升的血液。 </li><li>稀释受感染的血液，以产生的在PBS-G的pH 8.0的2×10 4个锥虫/ ml的如上步骤1.1接种物。 注意:在这一点上，剩余的血液可以冷冻保存由用8％的甘油，20％热灭活胎钙混合以产生stabilateLF血清（HI-FCS）和72％感染者的血液。使用慢速冷冻冷冻集装箱达到-1℃/ min的冷却速度，转移到后液氮之前在-80℃下慢慢地冻结。 </li></ol> 2.实验小鼠感染<ol><li>将CD1雌性小鼠（21-25〜G）进入加热箱轻轻地暖扩张静脉。放置温热小鼠到塑料约束和一个地下25针入尾静脉，相当于每只小鼠4000锥虫静脉内注射0.2毫升稀释接种物。将压在注射部位事后阻止出血。 注意:腹膜内感染是病毒感染的有效途径，并在以前的研究中10已被使用，但我们已发现这比静脉内途径少重复的。 </li><li>随机感染小鼠和保持架在SPF-通风笼3组。作为对照，注射CD1雌性小鼠与野生型非生物发光寄生虫，T。湾布氏 GVR35（N =3）。 </li><li>监测感染通过血液电影显微镜<ol><li>从静脉穿刺或放血点5微升血液到载玻片并执行血涂片（使用第二载玻片轻轻地推血滴横跨滑动，以产生一个薄膜）。允许幻灯片空气干燥。 </li><li>修复血涂片用100％的甲醇和染色用姬姆萨10分钟与卫生署2 O冲洗前，晾干。下100X客观计数锥虫中的视场（FOV）10的字段的数目。 注意:血拍戏被整一起非侵入性动物的成像早一天后感染进行。当处理一批动物，锥虫定量此方法优于血球计数作为样品可以储存在室温和更高计数。 </li></ol></li></ol> 3.生物发光成像跟踪感染注意:要监视的感染，整个动画人非侵入性成像可以使用。 <ol><li>制备30毫克/毫升D-萤光素，萤火虫荧光素酶底物，在Dulbecco氏磷酸盐缓冲盐水（DPBS）中的储备溶液。过滤消毒，并在-20℃下以等分试样冷冻。 注意:D-荧光素对光敏感，因此，通过在箔包裹等份避光。不要反复冻融萤光素，因为它会影响活动11。 </li><li>暖D-荧光素的室温的等分试样。除去从笼（包括感染nonbioluminescent野生型寄生虫菌株的对照小鼠）各小鼠和注射150毫克/公斤的温热D-荧光素（在DPBS稀释的）的腹膜内与地下25针。将小鼠成一个异氟醚室大约5分钟，在2.5升/分，诱导用2％异氟烷/ O 2的混合麻醉。 注:当他们麻醉状态下的小鼠变得动弹不得。对于这里使用的成像器，3只小鼠可在同一时间内进行处理。如果小鼠anesthet源化的时间超过30分钟，眼用人工泪液软膏可应用于防止小鼠的眼睛干燥。 </li><li>对于此处所描述的成像器，打开软件并使用该软件按照制造商的说明初始化机器。采集图像，一旦摄像头，加热板达到的温度。 注:本仪器通常不会关闭，从而使机器能够在一夜之间执行后台校准。在它需要重新启动的情况下，它必须首先运行校准检查。 </li><li>放置在麻醉小鼠放入成像仪（见3.5）。使用小鼠之间大黑分离器，以减少通过任何光亮生物发光信号的流血。使用一组曝光时间图像的小鼠; 1，3，10，30，60和180秒，具有中等分级，1 F /停止和开口的过滤器，并实地查看电子商务（12.5×12.5 平方厘米）。 注:感染野生型nonbioluminescent GVR35寄生虫的对照小鼠提供background生物发光值。从萤光素动力学实验（ 图6）与该模型中，我们已发现，在10分钟时的生物发光信号峰值给药后和成像需要大约5分钟为3只小鼠。麻醉和成像的小鼠时，把这个时间尺度考虑。 </li><li>为了确保小鼠的彻底成像，旋转获得腹，背和横向视图。保持方向为顺序动物之间的成像顺序的一致性。 </li><li>成像后，让小鼠从麻醉状态下观察返回SPF-笼前恢复。 </li><li>继续成像小鼠每7天到21天感染后。应该有生物发光信号在整个动物稳步增长。 </li><li>在D21，图像和血膜，如上所述，和剂量的小鼠用40毫克/千克diminazene aceturate腹膜内小鼠。这种药物会清除外围原虫但不会穿过血braiN阻挡，因此，能使中枢神经系统感染的更好的可视化。 注:Diminazene aceturate是用来治疗早期动物锥虫病一套行之有效的药物。 </li><li>在D28 D35和成像继续和监督。 </li></ol> 4.确认感染中枢神经系统<ol><li>在D35，上面详细图像小鼠（步骤3.1-3.4）。 </li><li>成像后，允许按照步骤1.6-1.8详细说明，以供进一步分析采血小鼠从麻醉中和抽血小鼠恢复。 </li><li>以下心脏穿刺，灌注用20ml PBS小鼠（可选的:加入3毫克/毫升的荧光素灌注溶液）经由心脏从器官清除血液和重新引入可能已经从大脑区域上的时间间隔清除萤光素标签从步骤4.1。 </li><li>轻轻通过使用弯曲的剪刀，从围绕颅骨的圆周基部切割，并剥离所述颅骨顶部部分，以允许大脑取出大脑，用弯钳轻轻提起。 </li><li>切除脑放置到黑色塑料和吸管50微升的15毫克/毫升的荧光素在成像前的器官的截面，以确保有足够的萤光素已被添加到该切除大脑。图像使用上述的设置。这将证明感染的定位到大脑半球。 注意:在切下脑寄生虫血症的下游量化可以通过qPCR如前所述8进行。其他替代下游应用可能包括组织学或免疫识别以下固定脑组织的寄生虫。 </li></ol> 5.生物发光成像的定量注:生物发光可使用的与成像软件感兴趣区域（ROI）的区域进行量化，并作为背景生物发光校正。 <ol><li>使用ROI工具，创建整个动物定量一个矩形的投资回报率第二它定位在鼠标图像覆盖鼻尖鼠标的尾部基地。使用同样大小的盒子，每动物和每一个时间点。当在ROI为大脑看，使用一个圆形的ROI的相同方式，将其定位在图像中的小鼠头部区域。 </li><li>如果需要的话，调整图像在相同的频谱出现感染的视觉表示。 注意:此处所用的软件为每个图像的彩色地图为最小和最大光子计数自动调整。以使一系列的图像之间的视觉比较，色标必须设置为所有图像的相同的值。 <ol><li>使用最高的生物发光并在工具选项板的"图像调整"选项卡中的图片，选择一个对数标度和适当的颜色比例的最小值和最大值（凭经验确定）。这个规模应用到整个实验过程中的所有图像。 </li></ol></li></ol>

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The authors have nothing to disclose.

生物发光T的发展湾布氏 GVR35应变允许锥虫感染从早期到晚期的可视化。以前感染模型是无法检测到后期，当寄生虫在大脑中，在从血液膜显微镜实时，和所需的大脑的剔除和除去从被感染的小鼠，以确定寄生虫负荷12。生物发光降低小鼠间变异性为单个鼠标可以在整个感染的全部，并且可以在任何阶段被观察感染的快速定性评估进行跟踪。生物发光成像的高度敏感的方法使?...

Human African trypanosomiasis (HAT), or sleeping sickness, is caused by the vector-borne protozoan parasites of the Trypanosoma brucei spp1. Estimated numbers of current cases is fewer than 7 thousand every year with almost 70 million people exposed to the risk of the parasite infection within the African continent. The disease, which is most often lethal if left untreated, comprises an early hemolymphatic stage where parasites are present in the blood, progressing to the late stage when parasites invade the central nervous system (CNS) and are no longer susceptible to treatment by early stage trypanosomal drugs2. The current drug therapies for late-stage HAT have both complex, prolonged, treatment regimens and severe adverse effects as well as reported resistance, therefore research into new drug therapies is imperative3,4. 
The study of late-stage human African trypanosomiasis (HAT) within traditional mouse models is lengthy and complex, with the removal of brain tissue being required to monitor parasitic burden5. The animal infective strain T. b. brucei is used as the study model of trypanosomiasis with the late stage appearing 21 days post infection (dpi). To monitor the wild type nonbioluminescent parasite infection in the mouse model, peripheral blood films or quantitative PCR are the only methods to determine parasite burden. For parasite burden in the brain, the mouse needs to be culled, brain excised and qPCR carried out on tissues, making it impossible to track parasites through multiple time points in the late stage infection. This results in the inability to follow real-time infections within the central nervous system (CNS). 
In vivo bioluminescence imaging (BLI) can provide highly sensitive, non-invasive detection of parasite dissemination and disease progression in a mouse model that can be followed in a single animal for the entirety of the experiment6. BLI is based on the emission of light in the visible spectrum produced by a luciferase-catalyzed reaction. The emitted photons are then detected by a charge coupled device (CCD) camera7. For this purpose, the pathogen is genetically modified to express a luciferase protein and the substrate, luciferin, is introduced at time points of interest by injection. The main advantage of this method is the ability to carry out longitudinal studies, in which the same animal can be imaged several times with minimal adverse effects. The acquired bioluminescence signal can be quantified, thus indicating the pathogen burden.
The optimization and validation of a red-shifted bioluminescent T. b. brucei has enabled the investigation of the late stage infection through non-invasive procedures, detecting parasites earlier than blood film microscopy and greatly reducing the time, cost and numbers of animals needed to study CNS infection and drug screening in late-stage trypanosomiasis8,9. In this protocol we demonstrate infection of mice with bioluminescent trypanosomes and how to then visualize the parasites in vivo for quantification of disease progression and CNS penetration.

<table><tbody><tr><td>PBS</td><td>Sigma, UK</td><td>P4417</td><td>tablets pH 7.4</td></tr><tr><td>Glucose</td><td>Sigma, UK</td><td>G8270</td><td>99.5% (molecular) grade</td></tr><tr><td>Ammonium chloride</td><td>Sigma, UK</td><td>A9434</td><td>99.5% (molecular) grade</td></tr><tr><td>Heparin (lithium salt)</td><td>Sigma, UK</td><td>H0878</td><td></td></tr><tr><td>Hi-FCS</td><td>Gibco, Life Technologies, UK</td><td>10500-064</td><td>500 ml</td></tr><tr><td>DPBS</td><td>Sigma, UK</td><td>D4031</td><td>Sterile filtered</td></tr><tr><td>Mr. Frosty</td><td>Nalgene, UK</td><td></td><td></td></tr><tr><td>Giemsa</td><td>Sigma, UK</td><td>G5637</td><td></td></tr><tr><td>D-Luciferin</td><td>Perkin Elmer, UK</td><td></td><td></td></tr><tr><td>Sigma, UK</td><td>115144-35-9</td><td></td><td></td></tr><tr><td>Diminazene aceturate</td><td>Sigma, UK</td><td>D7770</td><td>Analytical grade</td></tr><tr><td>IVIS Lumina II</td><td>Perkin Elmer, UK</td><td></td><td>other bioimagers available &lt;em&gt;e.g.&lt;/em&gt; from Bruker, Kodak</td></tr><tr><td>Living Image v. 4.2</td><td>Perkin Elmer, UK</td><td></td><td>proprietary software for Perkin Elmer IVIS instruments; other instruments may have their own</td></tr><tr><td>1 ml Syringe</td><td>Fisher Scientific, UK</td><td>10142104</td><td></td></tr><tr><td>20 ml Syringe</td><td>Fisher Scientific, UK</td><td>10743785</td><td></td></tr><tr><td>25 G Needles</td><td>Greiner Bio-one</td><td>N2516</td><td></td></tr><tr><td>21 G Needles</td><td>Greiner Bio-one</td><td>N2138</td><td></td></tr><tr><td>Twin-frosted microscope slide</td><td>VWR, UK</td><td>631-0117</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tube</td><td>StarLab, UK</td><td>I1415-1000</td><td></td></tr><tr><td>7 ml Bijou tube</td><td>StarLab, UK</td><td>E1412-0710</td><td></td></tr><tr><td>Mouse restrainer</td><td>Sigma, UK</td><td>Z756903</td><td>our restrainer was made in-house, this is a similar model</td></tr></tbody></table>

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.

此协议演示了如何遵循以下小鼠感染T.疾病进展湾布氏 ，为非洲人类锥虫病的模型。 图1显示了实验方案的时间表，这表明对于治疗和成像的步骤的时间表; 图2演示了用于定量外围原虫固定吉姆萨染色的血涂片的视图的典型字段，用锥虫和红血细胞存在。后面可以通过测量生物发光如在图3，其显示了与在每个时间点拍摄?...

Watch this Scientific Journal Video about Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis at JoVE.com

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

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 ...

bioluminescence-imaging-to-detect-late-stage-infection-african

Research

JoVE Journal

Immunology and Infection

7.7K 次观看.  London School of Hygiene & Tropical Diseases. 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.

视频: Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

艾滋病毒/艾滋病的综合成本有效的实验室监测：非洲的角色模型

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

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 ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

用于锥虫病检测和分类的 AI 程序

Superior Auto-Identification of Trypanosome Parasites by Using a Hybrid Deep-Learning Model

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of hotspot areas under active surveillance is a fundamental procedure for controlling disease transmission. Microscopic examination is a commonly used diagnostic method. It is, nevertheless, primarily reliant on skilled and experienced personnel. To address this issue, an artificial intelligence (AI) program was introduced that makes use of a hybrid deep learning technique of object identification and object classification neural network backbones on the in-house low-code AI platform (CiRA CORE). The program can identify and classify the protozoan trypanosome species, namely Trypanosoma cruzi, T. brucei, and T. evansi, from oil-immersion microscopic images. The AI program utilizes pattern recognition to observe and analyze multiple protozoa within a single blood sample and highlights the nucleus and kinetoplast of each parasite as specific characteristic features using an attention map.
To assess the AI program&#39;s performance, two unique modules are created that provide a variety of statistical measures such as accuracy, recall, specificity, precision, F1 score, misclassification rate, receiver operating characteristics (ROC) curves, and precision versus recall (PR) curves. The assessment findings show that the AI algorithm is effective at identifying and categorizing parasites. By delivering a speedy, automated, and accurate screening tool, this technology has the potential to transform disease surveillance and control. It could also assist local officials in making more informed decisions on disease transmission-blocking strategies.

作者聚焦:从显微图像中检测 AI 驱动的锥虫物种 

Worldwide medical blood parasites were automatically screened using simple steps on a low-code AI platform. The prospective diagnosis of blood films was improved by using an object detection and classification method in a hybrid deep learning model. The collaboration of active monitoring and well-trained models helps to identify hotspots of trypanosome transmission.

使用混合深度学习模型对锥虫寄生虫进行卓越的自动识别

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of ...

生物工程

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

肿瘤进展和血管生成的双生物发光成像

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

生物发光成像检测到非洲锥虫病的晚期感染

摘要

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此视频中的章节

在Live主机转利什曼原虫寄生虫在体内成像

艾滋病毒/艾滋病的综合成本有效的实验室监测：非洲的角色模型

生物发光成像的NADPH氧化酶活性在不同的动物模型

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

利用荧光蛋白监视非洲锥虫Glycosome动态

高通量基因定位在

使用混合深度学习模型对锥虫寄生虫进行卓越的自动识别

使用混合深度学习模型对锥虫寄生虫进行卓越的自动识别

使用混合深度学习模型对锥虫寄生虫进行卓越的自动识别

肿瘤进展和血管生成的双生物发光成像