The authors acknowledge the help from the staff of Laboratory Animal Medicine, University of Nevada, Reno. MM received funding from Nevada INBRE.

下文概述的议定书（第00682号）由内华达里诺大学的机构动物护理和使用委员会（IACUC）批准，并遵循内华达大学，雷诺动物研究伦理委员会的指导方针。简言之，将小鼠用异氟醚麻醉，并使用鼻锥来维持连续的异氟醚水平20分钟。使用兽医软膏防止麻醉下眼睛的干燥。使用脚趾钳来确定麻醉水平，并且在整个程序中测量呼吸频率。将小鼠保存在单独的笼中并进行监测，直到意识恢复。小鼠仅使用一次蜱喂养，并在蜱回收后进行安乐死。安乐死由动物医学实验室人员进行。 CO 2和颈脱位用于安乐死动物。 注意:打勾时需要使用完整的个人防护等级uipment。为了计数未成熟的蜱，请佩戴白色实验室外套，长的丁腈手套，以覆盖用橡皮筋覆盖的实验室外套的袖子开口，以及封闭式脚趾鞋。对于用蜱虫感染小鼠，请使用头发网，一次性工作服，袖口上的长袖手套和脚套。使用白色或浅色的工作服来检测方向蜱。定期检查手套和袖子的蜱虫。 动物屋准备室<ol><li>指定一个单独的房间，以容纳被蜱虫感染的小鼠。在门外部和内部放置粘垫或双面地毯胶带，以防止意外剔除。 </li></ol> 2.计数小鼠感染蜱虫<ol><li>将双面胶带放在7"x 5"x 14"塑料容器的顶部边缘上，并充满水至约2厘米。 </li><li>将另一个小盒子或培养皿放在容器的中心，并加满水至1厘米，＃34;护城河"围绕这个容器</li><li>将含有幼虫或若虫的小瓶存放在培养皿内。 </li><li>使用精细的油漆刷从小瓶中取出幼虫或若虫，并在显微镜下计数。将50只幼虫或25只若虫计数到独立的闪烁瓶中。立即用尼龙网或有机布屏幕盖上小瓶，并用橡皮筋关闭。 </li></ol> 3.用不成熟的蜱感染小鼠<ol><li>使用白色或浅色工作台，并在工作区周边贴上双面胶带。 </li><li>用异氟烷麻醉老鼠通过脚趾夹紧检查麻醉水平。一旦麻醉，将鼠标转移到用纸巾覆盖的加热垫上，并连接到鼻锥上以继续供应异氟烷。 </li><li>使用基于油膏的眼膏软膏以避免干燥。注意鼠标的呼吸模式来调节异氟烷水平（每分钟80 - 230次呼吸是正常的，如果发生异烟灰，减少异氟烷水平呕吐率不超过每分钟80次呼吸以避免杀死动物）。 </li><li>取一瓶50只幼虫或25只若虫，并用油漆刷将耳朵上的毛皮放在耳朵之间的头皮上（ 图1 ）。在放置后将鼠标置于麻醉下20分钟，以给足够的时间附上蜱。 </li><li>将鼠标移动到带有静电盖和白色床上用品的标准塑料鞋盒型一次性鼠标笼。按照正常的老鼠护理， 随意提供玩具，水和食物。 </li><li>将鼠标笼放置在较大的大鼠或沙鼠笼内，双侧粘胶带围绕顶部边缘。用3厘米的水填充外笼（ 图2 ）。 </li></ol> 4.从小鼠收集蜱<ol><li>饲养3至6天之间不成熟的蜱分离。在第3天之后，每天检查笼子和水壕沟。 </li><li>在第4天和第6天之间从笼子收集分离的蜱。使用痛苦t刷或软镊子拾起蜷缩的蜱，并存放在用橡皮筋固定的尼龙网布上的干净的闪烁瓶中。 </li><li>麻醉和检查鼠标在第7天任何剩余附加的蜱。 </li><li>在第7天检查床上用品，食物槽和水瓶，以进行加注。按照上述步骤对小鼠进行安乐死。高压灭菌一次性笼子，床上用品，水瓶和食物槽，以避免任何逃逸的屁股。 </li></ol>美联储仓储<ol><li>保持湿度为90％的潮湿蜱 °C和12:12光:在湿度和温度控制的培养箱中的黑暗循环，直到发生蜕皮。这可能需要大约12到18周的时间才能发生。 </li><li>当高湿度促进蜱生存时，它也使蜱容易发生真菌过度生长。每星期至少检查一次显微镜下的喂食蜱，以进行模具。如果检测到，将70％乙醇中的幸存者洗涤5次分钟，在水冲洗，转移至滤纸干燥，并转移到新的，干净的小瓶中。 </li></ol>

<ol>
	<li>Gleim, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+associated+with+tick+bites+and+pathogen+prevalence+in+ticks+parasitizing+humans+in+Georgia,+USA.">Factors associated with tick bites and pathogen prevalence in ticks parasitizing humans in Georgia, USA.</a> Parasites &amp;amp; Vectors. 9 (125), 1-13 (2016).</li><li>Krober, T., Guerin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+feeding+assays+for+hard+ticks.">In vitro feeding assays for hard ticks.</a> Trends Parasitol. 23 (9), 445-449 (2007).</li><li>Kuhnert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+of+Hard+Ticks+In+Vitro:+New+Perspectives+for+Rearing+and+for+the+Identification+of+Systemic+Acaricides.">Feeding of Hard Ticks In Vitro: New Perspectives for Rearing and for the Identification of Systemic Acaricides.</a> ALTEX. 13 (2), 76-87 (1996).</li><li>Voigt, W. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+feeding+of+instars+of+the+ixodid+tick+Amblyomma+variegaturn+on+skin+membranes+and+its+application+to+the+transmission+of+Theileria+mutans+and+Cowdria+ruminantium.">In vitro feeding of instars of the ixodid tick Amblyomma variegaturn on skin membranes and its application to the transmission of Theileria mutans and Cowdria ruminantium.</a> Parasitol. 107, 257-263 (1993).</li><li>Gregson, J. D., Smith, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ticks.">Ticks.</a> Insect Colonization and Mass Production. , 49-72 (1966).</li><li>Sonenshine, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Ticks. 2, (1993).</li><li>Bouchard, K. R., Wikel, S. K., Marquaedt, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Care,+maintenance,+and+experimental+infestation+of+ticks+in+the+laboratory+setting.">Care, maintenance, and experimental infestation of ticks in the laboratory setting.</a> Biology of Disease Vectors. , 705-711 (2005).</li><li>Schumaker, T. S., Barros, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+Ornithodoros+(Alectorobius)+talaje.+(Acari:Argasidae)+in+laboratory.">Life cycle of Ornithodoros (Alectorobius) talaje. (Acari:Argasidae) in laboratory.</a> J Med Entomol. 32, 249-254 (1995).</li><li>Banks, C. W., Oliver, J. H., Hopla, C. E., Dotson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+life+cycle+of+Ixodes+woodi+(Acari:Ixodidae).">Laboratory life cycle of Ixodes woodi (Acari:Ixodidae).</a> J. Med. Entomol. 35, 177-179 (1998).</li><li>James, A. M., Oliver, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+and+host+preference+of+immature+Ixodes+dammini,I.scapularis,and+I.pacificus.(Acari:Ixodidae).">Feeding and host preference of immature Ixodes dammini,I.scapularis,and I.pacificus.(Acari:Ixodidae).</a> J. Med. Entomol. 27, 324-330 (1990).</li></ol>

The authors have nothing to disclose.

议定书内的关键步骤在饲养蜱时要有几个级别的安全措施，以避免意外逃跑。使用粘性胶带和水沟护理对于确保安全至关重要。将麻醉的鼠标保持在加热垫上以保持体温恒定是很重要的。我们还发现，剃须鼠标不会为蜱附着提供任何额外的好处。单个鼠标可以在指定的"蜱室"中保存在同一个笼子中一周，这进一步限制了蜱虫与人员的联系。 修改和故障排除<...

蜱是脊椎动物的专性吸血体外寄生虫和分布在世界各地。在美国，至少有11种蜱的是公众健康的重要性1病原体的载体。 肩突硬蜱负责若干病原体传播诸如莱姆病（ 布氏疏螺旋体 ）回归热（B. miyamotoi），人粒细胞无形体（ 无形体吞噬细胞 ），和巴贝虫病的病原体（巴贝虫属）。尽管肩突硬蜱的重要性作为一种疾病矢量，野生在实验室研究中收集大量这些蛛形纲动物并不总是可行的。因此，生产健康实验室饲养的蜱是靠赊账生物学的研究必不可少的，蜱病原体相互作用。 所有硬蜱（家庭硬蜱科），包括肩突硬蜱的生命周期，包括鸡蛋和三个ACTIV的e阶段:幼虫，若虫和成人。每个活动阶段饲养脊椎动物宿主。在几天的附着和喂养之间发生的蜱虫与其宿主之间发生的复杂相互作用几乎不可能使用人造饲料进行复制，并且不太可能提供足够数量的喂食蜱进行大规模饲养2,3,4 。因此，活体小鼠和兔子最常用作饲养未成熟（幼虫和若虫）和成熟阶段（成虫）蜱的宿主。在每个发育阶段，多个主机对供血的要求使得蜱饲养复杂化，并且是时间和成本密集的5,6,7 。大多数蜱饲养方案需要将小鼠保持在悬挂的网格地板笼7,8或圆柱形ca这样的尺寸，该动物不能自由地移动和新郎本身6，9，10的GE。 这些圆筒形保持架以后转移到鞋盒笼线栅。充血，独立蜱然后从下面的水收集。然而，在曝光馈此方法将导致蜱到污染的尿和粪便，可以增加真菌生长和蜱死亡率9的水。另外，它增加了从水槽蜱逃逸，以及引起应力小鼠的可能性。为了避免这些问题，我们在这里展示的塑料鞋盒型一次性笼中老鼠幼蜱喂养。此方法允许小鼠，充血蜱恢复增加的正常行为，并且降低由于污染蜱的死亡率。

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rearing ixodes scapularis, the black-legged tick: feeding immature stages on mice

肩突硬蜱 ，莱姆病的载体，是在东部和中西部的美国最重要的疾病载体之一。本种是三大主机剔需要从脊椎动物宿主的每个发展阶段血粉，而成年女性需要再现血粉。幼虫蜱附着其主机3 - 5天，喂食和脱落的主机时，完全充血。在几个不同的主机，这种依赖性和较长的附属时间怒张在实验室环境中蜱饲养复杂。然而，了解蜱生物学和蜱病原体相互作用，生产健康，实验室饲养的蜱是必不可少的。在这里，我们表现出不成熟蜱对小鼠喂食一种简单，经济高效的协议。我们修改了现有协议用于对小鼠应力减小和增大蜱通过使用一次性笼而不网格塔底供给成功和生存，以避免蜱无线的接触水被小鼠尿液和粪便污染。

我们修改现有蜱饲养协议6，10可以改善喂养效率和鼠标主机上减小的应力。结果表明，标准鞋盒风格鼠笼非常适合蜱饲养。白色的床上用品提供了美联储的刻度容易收集形成鲜明的对比。大多数蜱爬上容器壁后喂养，并很容易收集。此外，一次性的塑料盒，紧配合的盖子（常规地用于小鼠UNR住房）防止从盒剔逃逸。此外，我们?...

Watch this Scientific Journal Video about Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice at JoVE.com

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

健康实验室饲养蜱的生产对蜱生物学和蜱虫病原体相互作用的研究至关重要。在这里，我们展示了一种简单的方案，用于不成熟的蜱喂养，其成本效益和对小鼠的压力较小。

饲养<em&gt;肩突硬蜱，</em&gt;黑腿蜱:饲养对小鼠未成熟阶段

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

rearing-ixodes-scapularis-black-legged-tick-feeding-immature-stages

Research

JoVE Journal

Immunology and Infection

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

11.9K 次观看.  University of Nevada, Reno. 健康实验室饲养蜱的生产对蜱生物学和蜱虫病原体相互作用的研究至关重要。在这里，我们展示了一种简单的方案，用于不成熟的蜱喂养，其成本效益和对小鼠的压力较小。 

视频: Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

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 ...

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

饲养和注射<em&gt;烟草天蛾</em&gt;幼虫评估细菌的毒力

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

小鼠量度本地过敏反应

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

不成熟，成熟和耐受性树突状细胞的生成与代谢不同表型

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.