作者感谢云南，中华人民共和国和阿博维尔，科特迪瓦，他们参与上述研究的社区。我们非常感激的测试和执行的协议，协助当地的野队。我们特别感谢下列同事从瑞士热带和公共健康研究所:Endriss伊薇特提供在视频拍摄过程中的显微图像，Hanspeter实验室的支持和孙燕姿克劳思·马蒂和他的团队对她的帮助。最后，我们感谢两位匿名审稿进行了一系列有益的意见。

1。改良加藤技术<ol><li>将一个标准的改良加藤模板为41.7毫克（mg），大便显微镜载片上有一个洞。 </li><li>炒到2-3克（g）的新鲜粪便样品放在一张报纸或铝箔，然后按顶部的金属丝或塑料网筛它一块。 </li><li>使用一个小的塑料小铲，刮离网筛分的材料，并完全填充改良加藤模板的孔中。以除去过量的粪便物，平与刮刀的孔的含量。 </li><li>垂直删除，而不会干扰现在坚持粪便的显微镜幻灯片模板。模板和抹刀可以在水中用洗涤剂清洗，并重复使用。 </li><li>将一块玻璃纸，预浸泡在甘油孔雀石绿溶液在至少24小时（小时），在显微镜载玻片上的粪便样品。 </li><li>要传播的粪便成厚厚的涂抹，轻轻按下一个干净的微样品滑动的滑动范围，均匀分布的材料内的圆的直径略小于显微镜载玻片的广度。 </li><li>允许的幻灯片，以清除为30-60分钟（min），在此期间，幻灯片必须远离阳光直射的地方。然后，系统地研究了厚厚的涂抹在光学显微镜下（40到100倍的放大倍率）。数一数......鸡蛋和分层物种（即，A.lumbricoides，T.trichiura，和钩虫）。其他寄生虫卵（如血吸虫 ）值得注意的是，也可以通过该方法来检测，并应分开计数和记录。 </li><li>要得到感染的强度的一个标准化的估计，通常表示为每克粪便（EPG）的卵数，乘以蛋计数由24（24×41.7毫克≈1克）。 </li></ol> 2。贝尔曼技术<ol><li>建立一个独立，可容纳一个玻璃漏斗和连接橡胶管，关闭机智公顷剪辑，漏斗底部。 </li><li>形状象一个圆锥体的金属丝网筛的筛或一块放置在液漏斗和层与医用纱布一块。 </li><li>填补了漏斗用自来水，并确保在整个设置也不会漏出。 </li><li>舀到中间的医用纱布20-30克新鲜粪便，并确保它是完全浸没在水中。如果需要添加更多的水。医用纱布折叠过的样品。 </li><li>从下面，人造光照耀在2小时的漏斗。 </li><li>漏到离心管中的50毫升（ml）的下部，在漏斗中的水轻轻释放的橡胶管上的剪辑。 </li><li>在600×g离心5分钟（相对离心力）离心收集的水，并小心地倒出上清液，而不会干扰沉积物。保留最后几滴在管。 </li><li>重悬沉淀中加入2滴在显微镜载片上的解决方案和检查幻灯片在光学显微镜下确认的物种进行检测和100-400X的放大倍率（40倍放大）。第一阶段的幼虫（L1）的S。粪类圆线虫是活的，积极的姿态，在显微镜下。 </li></ol> 3。 20米折返跑测试<ol><li>测量一个20米的平直运行过程，并把它标记为锥。 </li><li>帮助测试参与者佩戴一个心脏监测仪上的手腕和相应的发射器周围的胸部18。此设备允许参与者的心脏速率试验之前和之后进行测量，从而监控该参与者是否已经产生了极大的努力。 </li><li>指导参与者在球场上来回运行，通过预先录制的声音信号，划定20米圈的步伐。从8.5千米每小时（公里/小时）的行驶速度，频率的信号指示一个相当于0.5公里/小时每分钟20米的间隔增加。 </li><li>确保日在参与者开始一个新的20米的间隔，每次一个信号响起，下一个信号到达之前完成。 </li><li>停止参与者，如果他/她没有跟随的步伐，连续两个20米的间隔。 </li><li>记录的最高运行速度参与者完成充分，沿着与该参与者的年龄和性别，最后的间隔表示。 </li><li>验证，在测试结束的心脏率达到是孩子的最大心脏率19。如果没有达到这个心脏率，孩子可能不会产生了最大的努力，因此，应重复测试。 </li><li>估计参与者的最大有氧能力（VO最多2个 ），使用Léger的和他的同事提出的一个方程:最大VO 2 = 31.025 + 3.238 *速度（公里/小时） - 3.248 *年龄（岁）+ 0.1536 *速度（公里/小时）*年龄（岁）。 </li></ol> 4。代表性的成果在运输及房屋局局长诊断与改良加藤技术，独特的特点，不同的寄生虫卵允许的识别大多数物种20。的蛋A.lumbricoides（约45-75微米（μm）长，宽和35-50微米， 图1），比T.trichiura（ 图2），钩虫（ 图3）的蛋具有较厚的壳壁。一个受精卵A.lumbricoides（ 图1）具有外胚乳的外衣，这使得它看起来呈金黄色，而一个未受精的卵子缺乏这方面的外层披毛和稍微细长，更大的整体。鸡蛋T.trichiura（约50μm的长，宽和22微米， 图2）中是细长的，并具有不同的极性的插头。钩虫鸡蛋（约64-76微米的长，宽和36-40微米， 图3），在另一方面，有一个非常薄的壁，随后由明确的环围绕的致密的细胞群在中心的蛋。 s.stercoralis·L-1幼虫（ 图4）是约180-380微米长，并有一个很短的口腔内。一个明显的生殖器原基（突出显示一个蓝色的箭头在图4）可以看出，和幼虫（突出显示一个红色箭头， 图4）的尾部有一个尖底。 作为真正的影响STH感染儿童的体能仍存在争议13,14,15，有代表性的结果，在这一点上他们的影响。可以用各种方式进行20米折返跑测试结果的解释。可以比较谁是相似的年龄，性别，其他可能相关的健康状况，社会经济地位和文化习惯的感染和非感染科，儿童的体能。这可以通过在一个横截面的调查13,14,15运输及房屋局局长感染状况及感染者和非感染儿童的身体素质进行评估在一个特定的时间点。证据的强度会更高时，以下的适龄儿童的体能评估之前和之后驱虫/安慰剂治疗的随机对照试验设计，使短期和长期的评估。可以进一步尝试找出一个尽可能小的剂量-反应关系，运输及房屋局局长感染的强度和体能与体能，并比较不同程度的multiparasitism 1。在工作邑和他的同事15，强身健体，最大VO 2所描述的，被认为是显着减少感染与T.trichiura的儿童相比，孩子们谁没有被感染与T.trichiura（ 图5） 。 <img alt="图1" src="/files/ftp_upload/3966/3966fig1.jpg" /> Figu<html>重1。显微图像的A.lumbricoides鸡蛋受精（A）和（B）500倍的放大倍率下未受精的。比例尺= 50微米。 <img alt="图2" src="/files/ftp_upload/3966/3966fig2.jpg" /> 图2。500倍的放大倍率下的T.trichiura蛋。比例尺=50μm的显微图像。 <img alt="图3" src="/files/ftp_upload/3966/3966fig3.jpg" /> 图3的钩虫鸡蛋500倍的放大倍率下的显微图像。比例尺= 50微米。 <img alt="图4" src="/files/ftp_upload/3966/3966fig4.jpg" /> 图4的S.stercoralis·L-1幼虫在250倍放大倍数下的显微图像。箭头指示的尾巴（红色）和生殖器原基（蓝色）。比例尺= 100微米。 d/3966/3966fig5.jpg"/&gt; 图表显示的体能相比，的儿童感染T.trichiura水平的体能水平的儿童没有感染T. 图5。 虫 。

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R., Rollinson, D., Marti, H., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+soil-transmitted+helminths+in+the+era+of+preventive+chemotherapy:+effect+of+multiple+stool+sampling+and+use+of+different+diagnostic+techniques.">Diagnosis of soil-transmitted helminths in the era of preventive chemotherapy: effect of multiple stool sampling and use of different diagnostic techniques.</a> PloS Neglected Tropical Diseases. 2, e331 (2008).</li></ol>

没有利益冲突的声明。

本文中所描述的三个协议已经过测试，并执行在西南部云南省的一部分，中华人民共和国各成员之间的布朗族少数民族21在不同地区的非洲6,13,14。 有几个模板，可用于改良加藤技术。每一个不同的孔尺寸将提供一个独特的粪便量。对于计算的EPG，因此，有必要知道单元的模板，以乘以鸡蛋的计数量的修正因子，以到达的EPG估计。此外，当在社区钩虫是本调查下，它是必不可少的制备后不久（结算后的时间为30分钟，但60分钟内的最大清除时间）读取幻灯片。主要的原因是，钩虫，在高温下的鸡蛋迅速瓦解22。粪便生产的研究面值之间的时间间隔ticipant和检查的样品也是重要的，因为3小时或以上，可导致改良加藤技术钩虫诊断23的灵敏度降低50％的时间间隔。最后，为了提高诊断的敏感性的改良加藤技术，尤其是在低强度STH感染，应该从参与者多的粪便样本，双或一式三份改良加藤厚涂片地区应做好每次大便样本7,24。 随着到贝尔曼技术方面，必须采取谨慎进行实验时，感染期幼虫可能存在，因此，必须戴手套。虽然贝尔曼技术示出了相当高的浓度比其他技术的灵敏度并且是不太费力的培养方法相比，有一些重要的缺点。例如，大量的新鲜粪便材料是必需的，这是一个问题，当患者提交小粪便样品。在另一方面，一个大的粪便样品可能会增加在低强度的感染9幼虫检测的机会。此外，存在这样的风险，其他线虫，，如钩虫或毛线虫属。幼虫，可能会对本，从而小心镜检是必要的。 有直接和间接的方式测量VO 2最大有氧体适能评估。直接协议要求跑步机，自行车测力计，和其他专业的和笨重的设备，它可以测量的量和呼出的空气的气体浓度。在资源制约农村发展中世界的设定，这种做法是不可行的。对于这样的设置，间接的方式，如20米折返跑测试，是首选方法。虽然间接的方法是从来没有直接的方法准确，Léger的和他的同事建立了由20米折返跑测试，表现出很高的再现性（R = 0.89）和效度y对最大摄氧量在跑步机上16。此外，研究者和参与者比低，有几位与会者可以同时进行测试，节省人力资源和时间。心脏速率监视器通常不用于此类测试13,16。然而，他们的允许调查的参与者是否产生了极大的努力，在测试中获得更多的信息。最后，最好是让参与者做了试运行的前几个间隔，使他们能够熟悉的运行速度和航向。 总之，我们认为，结合上述方法，按照标准的协议，允许STH感染的发生率和强度的适龄儿童在科学合理的，管理的和负担得起的方式和体能评估。

注意:在此列表中的材料是通用的，可以从不同的来源获得。 <table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>改良加藤试剂盒 [400带有一个孔的6毫米（mm）（直径）在1.5毫米厚的模板塑料模板400塑料抹刀; 80网目尺寸的尼龙筛20米的作用; 20米的作用的亲水性的玻璃纸，34微米厚。] </td><td>韦斯特弗兰德森</td><td></td><td>请注意的甘油孔雀石绿溶液不包含在该试剂盒中，但可以从任何化学品供应商购买。 </td></tr><tr><td>玻璃漏斗[约8厘米（cm），宽6厘米深] </td><td></td><td></td><td>为贝尔曼技术在玻璃漏斗应该是能够容纳约60毫升水。钍E尺寸的橡胶软管和纱布是依赖于所使用的玻璃漏斗的类型。 </td></tr><tr><td>生物显微镜</td><td>奥林巴斯</td><td> CX21LED/CX21 </td><td></td></tr><tr><td>离心分离</td><td>上海医疗器械集团</td><td> 80 - 2T </td><td></td></tr><tr><td>预先录制的信号</td><td> Bitworks设计</td><td>团队哔哔测试版1.3.1 </td><td> iPhone应用程序</td></tr><tr><td>心脏监测仪</td><td> POLAR </td><td> FT1 </td><td>手表和发射器</td></tr></tbody></table>

determining soil-transmitted helminth infection status and physical fitness of school-aged children

土壤传播的蠕虫（STH）感染是常见的。事实上，超过1亿人受到影响，主要是在发展中世界，那里的贫困盛行，卫生行为，供水和卫生设施往往缺乏1,2， 蛔虫 ， 鞭虫和钩虫种类， 十二指肠钩虫 ， 钩虫美洲 ，是最普遍的STHs 3。由于钩虫病，蛔虫病和鞭虫病的全球疾病负担的估计是22.1，10.5，和640万伤残调整寿命年（DALY），分别为4。此外，估计有30-100亿人感染粪类圆线虫 ，最被忽视的STH种具有全球意义，可以说是也造成相当大的公众健康的影响5,6。多物种感染（即不同的STHs窝藏在一个单一的个体）是常见的，已经感染林<html>糟透了降低生产率，从而促进经济的发展中国家的前景1,3。 的诊断的常见STHs的，世界卫生组织（WHO）建议的改良加藤技术7.8，这是一个相对简单的方法来确定这类感染的发生率和强度。它促进了检测，感染者通过在其粪便中的寄生虫卵。 随着的诊断S.stercoralis方面，目前还没有简单而准确的工具。的的贝尔曼技术是应用最广泛的方法，其诊断。的贝尔曼技术背后的原理是，积极S.stercoralis幼虫迁移出照明的新鲜粪便样品的幼虫趋光9。它需要不太复杂的实验材料和消耗更少的时间比文化和免疫学方法5。 T"发病率与运输及房屋局局长感染范围从急性，但共同的症状，如腹痛，腹泻，皮肤瘙痒，慢性症状，如贫血，不足和营养不良，和认知功能障碍10。一般都是非特异性的症状，微妙的，他们经常被忽视，被认为是一个正常的条件下受影响的个体，或者被处理作为可能会对多见于一个给定的设定的其他疾病的症状，因此，可以想象的是，真正的负担被低估了评估的STH感染工具依靠自我宣称的症状和体征通常情况下人口为基础的调查。 在20世纪80年代末和90年代初，Stephenson和他的同事们强调STH感染的可能性降低了体能的男孩6-12岁11,12。最近13,14,15这条线的科学调查获得了新的动力。 20米（m）折返跑测试开发ð验证莱热等。，是全球通用的衡量有氧健身的儿童17。测试是简单的规范和可以执行的地方有20米长，平坦的运行过程和音频源，使得它的使用有吸引力的资源限制设置13。为方便和规范的尝试，在评估是否STH感染有效果的适龄儿童的体能，我们提出的方法，诊断STH感染或措施的体能是简单的执行，提供精确和可重复的结果。这将有助于产生新的证据，STH感染对健康的影响。

Watch this Scientific Journal Video about Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children at JoVE.com

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

用土壤传播的的蠕虫（STHs）的慢性感染引起吸收不良，发育迟缓，浪费在成长中的孩子。因此，它是合理的，这些感染也可以减少儿童的体能。在这里，我们可视化技术的诊断STHs和20米折返跑测试，以评估儿童的身体健康。

确定土壤传播的蠕虫感染状况和体质的学龄儿童

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

determining-soil-transmitted-helminth-infection-status-physical

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

23.3K 次观看.  Swiss Tropical and Public Health Institute, Basel, Switzerland. 用土壤传播的的蠕虫（STHs）的慢性感染引起吸收不良，发育迟缓，浪费在成长中的孩子。因此，它是合理的，这些感染也可以减少儿童的体能。在这里，我们可视化技术的诊断STHs和20米折返跑测试，以评估儿童的身体健康。

视频: Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

确定临床抗体样品的吞噬活性

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

科研

免疫与感染

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

从使用鸡蛋<em&gt;血吸虫</em&gt;作为<em&gt;在体内</em&gt;蠕虫引起的肺部炎症模型

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

成人斑马鱼对 DNA 疫苗临床前筛选的免疫研究

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

确定土壤传播的蠕虫感染状况和体质的学龄儿童

摘要

探索更多视频

此视频中的章节

确定临床抗体样品的吞噬活性

从使用鸡蛋

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

总蛋白提取和二维凝胶电泳方法

体外共生分析评估病原体诱导中性粒细胞跨上皮迁移

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性

成人斑马鱼对 DNA 疫苗临床前筛选的免疫研究

确定土壤传播的蠕虫感染状况和体质的学龄儿童

摘要

探索更多视频

此视频中的章节

确定临床抗体样品的吞噬活性

从使用鸡蛋

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

总蛋白提取和二维凝胶电泳方法

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性

成人斑马鱼对 DNA 疫苗临床前筛选的免疫研究

体外共生分析评估病原体诱导中性粒细胞跨上皮迁移