这项研究得到了美国国立卫生研究院（NIH）国家过敏和传染病研究所（National Institute of Allergy and Infectious Diseases）校内研究计划的支持。我们感谢 Linden T. 胡、Sam R. Telford III、Kenneth Dardick、Carla Williams、Erin Chung 和 Christina Brandeburg 参与制定程序。

让志愿者暴露于蜱虫叮咬是一种实验方法，必须根据相关监管机构批准的临床研究方案进行。临床研究（NCT01143558、NCT02446626 和 NCT05036707）由各自的机构审查委员会批准，在美国食品和药物管理局授予的研究性器械豁免下进行，并根据良好临床实践指南进行。此外，这些研究已在 ClinicalTrials.gov 注册，并获得了所有参与者的书面知情同意书。
1. 密闭敷料准备
<ol><li>要创建测量模板，请沿测量线的一侧复印 3" x 3" 水胶体密封敷料（图 1A 和 材料表）。</li>
	<li>沿 1 英寸线切割复印模板。然后，沿着 2 英寸的线切割，创建一个 2 英寸的圆，中心为 1 英寸（图 1B）。</li>
	<li>使用此模板，切割非粘性泡沫敷料，创建一个 2 英寸的圆圈，中心为 1 英寸（图 2A、B 和 材料表）。</li>
	<li>使用相同的模板，在 4" x 4" 超薄水胶体敷料的中心切一个 2 英寸的圆圈（图 2C，D）。</li>
	<li>在 11/2" 线上切割 3" x 3" 密闭敷料的水胶体层。</li>
	<li>拉回安全壳敷料的水胶体层的纸衬垫，露出粘合剂。将切割的泡沫敷料放入粘合剂的 2 英寸圆内（图 3A）。这将确保泡沫敷料延伸到安全壳敷料的开口之外。将纸衬垫更换到敷料的水胶体层上（图3B）。 注:这些测量值用于涉及多达 10 个 幼虫 I. scapularis 蜱的研究。对于涉及多达 30 只 肩胛蜱 幼虫的研究，泡沫中心的模板在 1 英寸和 11/2 英寸标记之间切割，以留出更大的放置区域。密闭敷料的测量值可以根据研究设计进行调整。</li>
</ol>2. 刻度放置
<ol><li>即时报价存储<ol><li>将 肩胛蜱 幼虫储存在医疗级冰箱中，温度设置为 9-10 °C。</li>
		<li>将蜱虫存放在覆盖有网帽的小瓶中，以允许足够的气流。用轻轻湿润的海绵将一小瓶蜱虫包装在可密封的塑料袋中，以保持湿度并防止干燥。</li>
	</ol></li>
	<li>放置刻度<ol><li>从冰箱中取出蜱虫，让它们加热到室温。蜱虫可以在冰箱外短短 15 分钟内变得活跃。</li>
		<li>在放置区域周围放置一张白纸，以便轻松看到任何无意中掉落的刻度。</li>
		<li>用清水或生理盐水弄湿 4 英寸 x 4 英寸的纱布垫，然后用它来清洁放置部位的皮肤。该区域应拍干或让其风干。 注意:在选择放置蜱虫的身体部位时，请考虑不限制志愿者日常活动的区域，并且参与者可以轻松监测敷料的完整性。不要将蜱虫放置在可能因直接压力而被压碎的区域（例如背部）。评估参与者的体毛，如有必要，使用剪刀在粘合剂部位小心修剪过多的体毛，避免对皮肤造成伤害。避免使用剃须刀脱毛，因为它可能会增加皮肤受伤和感染的风险。</li>
		<li>一旦皮肤干燥，从准备好的敷料的水胶体侧取下粘性衬垫，并将其牢固地固定在放置部位的皮肤上，确保网状层保持开放。</li>
		<li>将木棉签（见 材料表）对半掰开，露出锯齿状的末端。</li>
		<li>小心地打开一小瓶蜱虫。</li>
		<li>使用木棉签的锯齿状端，从小瓶中转移活性蜱虫，并将它们放在收容敷料内的皮肤上（图4A）。让额外的团队成员监控蜱虫放置部位，以确保蜱虫留在收容敷料内。在此过程中，使用胶带捕获从小瓶中逸出的蜱虫。 注意: 打破木签并使用锯齿状的一端可以有效地操作和转移蜱虫，而不会对蜱虫造成损坏。带有浅色柔软刷毛的小刷子也可用于将蜱虫从容器转移到皮肤部位。</li>
		<li>放置研究的指定数量的刻度后，从密闭敷料的网状层上取下粘性衬垫，并将其封闭在开口处。</li>
		<li>在确保粘合剂牢固附着后，将准备好的 4" x 4" 超薄水胶体敷料涂在密闭敷料上以增加安全性（图 4B）。 注:有关无病原体肩胛胛蜱菌落和引用研究中使用的蜱暴露程序持续时间的更多信息，可在已发表研究19,22 的方法部分找到。</li>
	</ol></li>
</ol>3. 现场护理
<ol><li>为研究参与者提供防水罩和 2 英寸低过敏性胶带（参见 材料表）。</li>
	<li>提供护理蜱虫遏制部位的说明。<ol><li>定期检查敷料并监测边缘是否牢固地粘附在皮肤上。根据需要使用额外的胶带来加固敷料边缘。</li>
		<li>在研究期间，请勿刮擦敷料部位，并避免打开或取下敷料。</li>
		<li>避免洗澡或浸泡在水中。此外，避免热水淋浴并限制淋浴的时间。淋浴前，用提供的防水罩保护安全壳敷料，并在完成淋浴后将其取下。轻轻拍干敷料，并检查它以确保它保持牢固。</li>
		<li>避免可能导致大量出汗和损害敷料完整性的活动（例如，有氧运动、长时间徒步旅行）。</li>
	</ol></li>
</ol>4. 去除蜱虫
<ol><li>收集瓶制备<ol><li>在蜱虫收集瓶上贴上所需的标识符（例如，研究代码、日期）。</li>
		<li>如果需要保存活蜱虫，用20G针在小瓶盖上穿孔。使用一层尼龙或聚酯网以及瓶盖密封小瓶。将一小块湿海绵与一小瓶蜱虫一起放入可密封的袋子中以保持湿度。</li>
	</ol></li>
	<li>去除蜱虫<ol><li>在绷带部位周围铺上白色床单，并确保收集盆触手可及。</li>
		<li>使用酒精湿巾，小心地从皮肤上取下绷带。</li>
		<li>完全取下后，将绷带放入收集盆中，并检查是否存在脱落的蜱虫。</li>
		<li>评估放置地点是否有附着的蜱虫。已经补充的蜱虫将分离（图5A）。对于剩余的附着的蜱虫，使用细尖镊子去除（图5B）。</li>
		<li>用酒精湿巾或肥皂和水清洁皮肤。</li>
	</ol></li>
</ol>

人类接触幼虫蜱虫用于蜱传疾病研究

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Adriana Marques 博士拥有美国专利 8,926,989 B2;并且是全球莱姆病联盟和美国莱姆病基金会的无薪科学顾问。Siu Ping Turk 和 Aleah Eschman 没有可能构成利益冲突的关联。本出版物的内容并不一定反映美国卫生与公众服务部的观点或政策，提及商品名称、商业产品或组织也不意味着美国政府的认可。

虽然涉及暴露于蜱虫的动物研究 4,5,6,7,21 对于提高我们对宿主对蜱传疾病和蜱虫叮咬反应的理解非常宝贵，但这些模型在预测人类宿主反应的程度上存在局限性。该模型描述了以可控方式将人类暴露于蜱虫叮咬的方法，可以很容易地适应回答不同的研究问题，并扩展了在人?...

硬蜱（硬蜱科:Acari）是全球范围内发生的专性血液摄食性体外寄生虫，能够传播广泛的病原体，包括细菌、病毒和寄生虫，具有重要的医学和兽医意义。硬蜱蜱必须与宿主保持附着数天才能完成血粉，它们有能力保持附着在皮肤上，同时避免识别，防止局部血液凝固，并促进长期摄食1,2,3。动物研究表明，不宽容的宿主会通过反复接触蜱虫叮咬获得对蜱虫叮咬的抵抗力，这可能导致传播病原体的能力降低，而蜱虫可以反复寄生于宽容的宿主。获得性蜱耐药性取决于宿主免疫反应的性质 4,5,6,7。
蜱传疾病在美国是一个日益严重的威胁，2004 年至 2016 年间报告的病例数增加了一倍多 8,9。由于气候变化，不同蜱虫的地理范围继续扩大10,11。美国主要的蜱传疾病包括莱姆病、无形体病、埃立克体病、斑疹热立克次体病、巴贝虫病、土拉菌病和波瓦桑病毒病8。莱姆病由伯氏疏螺旋体感染引起，是美国和欧洲最常见的蜱传疾病12.美国每年约有 476,000 人被诊断患有莱姆病，这给个人和社会带来了公共卫生和经济负担 13,14,15。
肩胛硬蜱 （黑腿蜱或鹿蜱）是莱姆病的主要传播媒介，在美国也是无形体病、巴贝虫病、 宫本疏螺旋体 病和波瓦桑病毒病的主要传播媒介。在美国，其他具有医学重要性的蜱种包括 Amblyomma americanum （孤星蜱）、 Dermacentor variabilis （美国狗蜱）、 太平洋硬蜱 （西部黑腿蜱）、 Dermacentor andersni （落基山木蜱）、 Cookei硬蜱 （土拨鼠蜱）、 Dermacentor occidentalis （太平洋沿岸蜱）、 Rhipicephalus sanguineus （棕色狗蜱）和 Amblyomma maculatum （墨西哥湾沿岸蜱）16。
开发一种使研究志愿者暴露于蜱虫叮咬的方法支持使用自然载体寻找感染证据的研究，这一过程称为异种诊断 17,18,19,20,21，并更多地了解暴露于蜱虫引起的免疫力，这有助于发现抗蜱虫疫苗 5,6,7.本文描述的程序是在2014年发表的第一项人类研究中开发和使用的，该研究使用实验室饲养的肩胛硬蜱幼虫在抗生素治疗（NCT01143558）后对伯氏疏螺旋体感染进行异种诊断19。该系统已成功用于一项 2 期研究，该研究调查了阳性异种诊断是否与莱姆病 （NCT02446626） 抗生素治疗后症状的持续存在相关，以及一项探索宿主对蜱虫叮咬反应的研究 （NCT05036707）。
该程序协议描述了创建收容敷料、蜱虫放置程序和蜱虫清除程序的过程，以及维持收容敷料所需的现场护理。关于上述研究中使用的无病原体肩胛胛蜱菌落和蜱暴露程序的详细信息之前已经描述过19,22。这种方法提供了一种灵活的研究工具，可用于研究人类宿主对蜱虫叮咬的反应、蜱虫预防药物的有效性以及莱姆病和其他蜱传疾病的不同方面。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20 G needle</td>
			<td>Any brand</td>
			<td></td>
			<td>For puncturing the vial cap.</td>
		</tr>
		<tr>
			<td>3&#34; x 3&#34; containment dressing</td>
			<td>Monarch Labs Names</td>
			<td>LeFlap</td>
			<td>https://www.monarchlabs.com/ordering</td>
		</tr>
		<tr>
			<td>4&#34; x 4&#34; extra-thin hydrocolloid dressing</td>
			<td>ConvaTec</td>
			<td>DuoDerm</td>
			<td>https://www.convatec.com/products/advanced-wound-care/brand-names/pc-wound-duoderm-granluflex/duoderm-extra-thin-dressing/</td>
		</tr>
		<tr>
			<td>4&#34; x 4&#34; gauze</td>
			<td>Monarch Labs Names</td>
			<td></td>
			<td>For cleaning skin</td>
		</tr>
		<tr>
			<td>Clean water or saline</td>
			<td></td>
			<td></td>
			<td>For cleaning skin</td>
		</tr>
		<tr>
			<td>Moisture barrier (e.g. 7&#34; x 7&#34;)</td>
			<td>AquaGuard</td>
			<td>TIDI</td>
			<td>For showering, ttps://www.tidiproducts.com/product-listing/aquaguard-shower-cover-sheets&#160;</td>
		</tr>
		<tr>
			<td>Non-adhesive foam dressing</td>
			<td>Coloplast</td>
			<td>Biatain</td>
			<td>https://www.coloplast.us/biatain-non-adhesive-en-us.aspx</td>
		</tr>
		<tr>
			<td>Roll of 2&#34; hypoallergenic tape</td>
			<td>Monarch Labs Names</td>
			<td>Durapore</td>
			<td>For reinforcing containment dressing.</td>
		</tr>
		<tr>
			<td>Roll of adhesive tape</td>
			<td></td>
			<td></td>
			<td>For trapping ticks</td>
		</tr>
		<tr>
			<td>Vials for collection (e.g. cryovials)</td>
			<td>Ependorf</td>
			<td>ECC200</td>
			<td></td>
		</tr>
	</tbody>
</table>

a model for experimental exposure of humans to larval ixodes scapularis ticks

蜱传疾病是美国和全世界的一个重大公共卫生问题。蜱虫是专性吸血节肢动物;硬蜱必须保持附着在宿主的皮肤上，并完成其为期多天的摄食过程以获取其血粉。将动物暴露于蜱虫是研究宿主对蜱虫叮咬和蜱传疾病反应的常见做法。我们开发了该程序，进行了第一项人体研究，并发表了关于将人类志愿者暴露于未感染的 肩胛硬蜱 幼虫的研究结果。本文介绍了用于构建收容敷料的方法，如何将蜱虫应用并固定到宿主身上，如何维护敷料，以及如何从宿主中清除蜱虫。让志愿者暴露于蜱虫叮咬是一种实验程序，必须根据相关监管机构批准的临床研究方案进行。这种方法允许转化研究，以更好地了解人类对蜱虫叮咬的反应，并促进蜱传疾病的诊断、预防和治疗的发展。

该研究表明，该手术是安全且耐受性良好的，主要不良事件是叮咬部位的轻度瘙痒，在 58% 的手术中观察到。当使用干净的实验室饲养的肩 胛蜱幼 虫时，没有发生与该程序相关的严重不良事件19.在进行的 43 个程序中，与放置的蜱虫相比，附着的蜱虫的平均恢复百分比为 45% ± 27% （SD），中位数百分比为 40%（图 6）。
肩胛蜱</e...

Watch this Scientific Journal Video about A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks at JoVE.com

作者聚焦:用于蜱虫研究和莱姆病研究的受控人类暴露模型

本文介绍了将人类暴露于 肩胛硬蜱 幼虫以进行临床研究的方法。该技术比较简单，研究志愿者可以忍受，并且可以根据实验需要进行修改。此类涉及人类受试者的研究必须根据适当监管机构批准的临床研究方案进行。

人类接触 肩胛蜱 幼虫的实验暴露模型

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick ...

该协议为人类受控地暴露于蜱虫提供了一种有效的方法。对于研究，该协议可用于研究人类免疫反应蜱叮咬，以及开发针对蜱传疾病的诊断和预防措施。没有公布对人类研究志愿者进行蜱虫的程序。挑战在于开发一种系统，让蜱虫能够进食数天，同时又让参与者感到舒适。该模型包含喂养期间的蜱虫，并允许研究参与者继续进行日常活动，并对生活方式进行微小的调整。

a-model-for-experimental-exposure-humans-to-larval-ixodes-scapularis

Research

JoVE Journal

Immunology and Infection

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

394 次观看.  National Institutes of Health. 该协议为人类受控地暴露于蜱虫提供了一种有效的方法。对于研究，该协议可用于研究人类免疫反应蜱叮咬，以及开发针对蜱传疾病的诊断和预防措施。没有公布对人类研究志愿者进行蜱虫的程序。挑战在于开发一种系统，让蜱虫能够进食数天，同时又让参与者感到舒适。该模型包含喂养期间的蜱虫，并允许研究参与者继续进行日常活动，并对生活方式进行微小的调整?...

视频: 作者聚焦:用于蜱虫研究和莱姆病研究的受控人类暴露模型

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA干扰在蜱

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

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 ...

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 ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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

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 three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

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

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

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 ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

甲醛暴露对大鼠新生辣椒素致过敏性皮炎模型的影响

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

大鼠异位心脏和心肌细胞移植的免疫模型

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

人类接触 肩胛蜱 幼虫的实验暴露模型

人类接触肩胛蜱幼虫的实验暴露模型