我们感谢惠淑哦，乔安妮黑樱桃和阿兰Collmer，植物病理学和植物 - 微生物生物学系，康奈尔大学，有价值的讨论。我们感谢Munkvold文章评论杰西。是由美国国家科学基金会植物基因组计划提供资金，奖DBI - 0605059（GBM）的。

第1部分：植物的生长和维护 烟草benthamiana在试验中使用的植物应约7周之久。他们应修剪前至少有4-5天检测，以除去所有腋的分支机构和花卉。这是一个好主意，去除腋窝分行后，很快便出现在为了使植物更便于管理。 第2部分：细菌培养第一天： <ol><li>在检测中使用的所有株板或文化都在同一时间启动。对于假单胞菌 。其中包括2诱导和3株挑战到KBM板，条纹细菌含冷冻甘油股票（见表2）适当的抗生素。板中心应少量的细菌传播。约18-24小时，在30 ° C孵育板。 农杆菌介导 ，启动于2 mL LB液体文化，从一个全新的板块和成长在250转过夜，30 ° C 。 </li></ol>第2天： <ol start="2"><li>对绿脓杆菌 ，加入150-200μl液体KBM的细菌，它们遍布整个板使用无菌玻璃吊具。板背面在孵化器，并让它为20-24小时，增长。应该有一个细菌草坪上板，第二天，显示良好的增长势头。 农杆菌介导 ，启动约20微米的乙酰丁香酮5-10毫升LB次要的文化，让它过夜，以20小时增长250 RPM，30 ° C 。 </li></ol>第3部分：准备检测植物第2天： <ol><li>检测前一天的植​​物应该被移到一个房间在22-24℃，约35-40％RH和恒定的光。 </li><li>马克叶片上，至少有直径1.5厘米，用厚厚的黑色标记的圆圈。可以标明每叶二至四个圆圈。界应该得到很好的间距，最好不要跨大静脉。选择良好的扩展叶。避免坚韧或较厚的触摸和避免标志着各界对叶下部老叶或树叶。 </li></ol>标记各界实验前一天是没有必要的协议。然而，节省了时间，如果被检测的植物有大量，使得它更容易实现之间的公共交通交汇处的感应和挑战接种的精确定时。 第4部分：电磁炉的公共交通交汇处第3天： <ol><li>收获从板的细胞在10毫米MgCl 2的 荧光为 P 和 P 恶臭假单胞菌 。 农杆菌介导 ，离心机的文化，收集细胞，悬浮10MM 氯化镁 2 + 10毫米MES的pH值5.6 。重复离心和悬浮在MgCl 2的 MES解决方案。测量细胞在600 nm的光密度（OD）。消耗臭氧层物质调整到最后的表2中所述的氯化镁假单胞菌应终于在10毫米悬浮氯化镁2和农杆菌 2 - MES解决方案所需的值。一个25毫升的总量是足够的接种约40-50点。 </li><li>渗入叶使用1毫升的无针注射器的标界的交汇处诱导。写下在植物渗透的秩序和渗透开始时的确切时间。 </li></ol>第5部分：挑战接种<ol><li>准备P。丁香 PV。 番茄 DC3000，DC3000 的PstΔhopQ1 - 1和PS PV。 粉虱的部分4.1和表2中所述的挑战。 </li><li>七小时后，诱导渗透，执行相同的顺序在植物的挑战渗透，作为诱导。一般来说，第一次接种圈的边缘上的点可以用来作为第二次接种圈的中心。如果有一片叶子上的多点，然后确保不重叠渗透。 </li><li> KBM或含有适当抗生素的LB板的检测，以确定确切的CFU数量的所有文化板块连续稀释。 </li></ol>第6部分：交汇处分项评分第5天： <ol><li>看看，由于到ETI细胞死亡的外观，在用Pst DC3000挑战点。渗透重叠区域内的细胞死亡表示交汇处击穿，应被视为一个积极的表型中取得的。 </li></ol>第7天： <ol start="2"><li>寻找点用 Pst DC3000ΔhopQ1 - 1或 PS光伏挑战由于疾病的细胞死亡的外观。粉虱</em&gt;。分数如6.1部分所述的表型为以同样的方式积极。 </li></ol>当控制植物（不应该损害交汇处）开始出现在重叠区域的渗透细胞死亡，停止任何进一步的意见。 第7部分：代表结果图1显示的检测结果时， 体育被用作荧光诱导和面临的挑战PST DC3000。 <img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="图1" border="0" /> 图1。 体育荧光是渗透到N 。 benthamiana叶（黑圈），以促使公共交通交汇处和7个小时后，当场被用 Pst DC3000（白色圆圈）的挑战。 FLS2沉默植株所在地区体育的公共交通交汇处击穿荧光是渗透（一），细胞死亡。不沉默的控制，植物没有显示在重叠区，由于公共运输交汇处（二）诱​​导细胞死亡。红色箭头表示缺乏或细胞死亡存在重叠区域的渗透。照片是2天之后的渗透。请<a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">点击这里</a>看到图1的放大版本。 <table border="1"><tbody><tr><td> 公共交通交汇处诱导 </td><td> 细胞死亡，从而引发的挑战</td><td> 细胞死亡的性质</td><td> 守则 </td></tr><tr><td> 荧光假单胞菌 55 </td><td> 假单胞菌丁香光伏番茄（PST）。DC3000 6 </td><td> ETI </td><td> PF / DC </td></tr><tr><td> P.恶臭假单胞菌 KT2440 </td><td> PST DC3000 </td><td> ETI </td><td> PP / DC </td></tr><tr><td> P.荧光 55 </td><td> PST DC3000ΔhopQ1 - 1 6 </td><td>疾病</td><td> Pf/Q1-1 </td></tr><tr><td> 农杆菌 GV2260 </td><td> P.丁香 PV。粉虱11528R </td><td>疾病</td><td>农业/ PTAB </td></tr></tbody></table> 表1：公共交通交汇处的诱导和细胞死亡引出的公共交通交汇处的细胞死亡的实验中使用的微生物组合。 <table border="1"><tbody><tr><td> 菌株 </td><td> 选择培养基 </td><td> 最后外径在实验中使用 </td><td> 相应的CFU /毫升</td></tr><tr><td> P.荧光 55 </td><td> KBM氨苄青霉素（100微克/毫升） </td><td> 0.5 </td><td> 1 × 10 9 </td></tr><tr><td> P.恶臭假单胞菌 KT2440 </td><td> KBM氨苄青霉素（100微克/毫升） </td><td> 0.5 </td><td> 1 × 10 8 </td></tr><tr><td> A.农杆菌 GV2260 </td><td> LB利福平（100微克/毫升） </td><td> 0.5 </td><td> 5 × 10 8 </td></tr><tr><td> PST DC3000 </td><td> KBM利福平（100微克/毫升） </td><td> 0.02 </td><td> 2 × 10 7 </td></tr><tr><td> PST DC3000ΔhopQ1 - 1 </td><td> KBM利福平（100微克/毫升） </td><td> 100倍稀释液0.1 </td><td> 1 × 10 6 </td></tr><tr><td> P.丁香 PV。粉虱11528R </td><td> KBM利福平（100微克/毫升） </td><td> 100倍稀释液0.1 </td><td> 1 × 10 6 </td></tr></tbody></table> 表2：文化条件，并在实验中使用的接种水平。分光光度计之间的不同品牌的OD和相应的CFU水平可能会有所不同。

<ol>
	<li>Abramovitch, R. B., Anderson, J. C., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+elicitation+and+evasion+of+plant+innate+immunity.">Bacterial elicitation and evasion of plant innate immunity.</a> Nat Rev Mol Cell Biol. 7 (8), 601-60 (2006).</li><li>Jones, J. D., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+immune+system.">The plant immune system.</a> Nature. 444 (7117), 323-323 (2006).</li><li>Zipfel, C., Robatzek, S., Navarro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+disease+resistance+in+Arabidopsis+through+flagellin+perception.">Bacterial disease resistance in Arabidopsis through flagellin perception.</a> Nature. 428 (6984), 764-764 (2004).</li><li>Oh, H. S., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+resistance+against+bacteria+in+Nicotiana+benthamiana+leaves+is+accompanied+by+reduced+vascular+staining+and+suppressed+by+multiple+Pseudomonas+syringae+type+III+secretion+system+effector+proteins.">Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.</a> Plant J. 44 (2), 348-348 (2005).</li><li>Goodin, M. M., Zaitlin, D., Naidu, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotiana+benthamiana:+its+history+and+future+as+a+model+for+plant-pathogen+interactions.">Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.</a> Mol Plant Microbe Interact. 21 (8), 1015-1015 (2008).</li><li>Wei, C. F., Kvitko, B. H., Shimizu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pseudomonas+syringae+pv.+tomato+DC3000+mutant+lacking+the+type+III+effector+HopQ1-1+is+able+to+cause+disease+in+the+model+plant+Nicotiana+benthamiana.">A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.</a> Plant J. 51 (1), 32-32 (2007).</li></ol>

可用于细胞死亡为基础的检测，以确定参与基因在公共交通交汇处。例如，一个感兴趣的基因丧失功能的突变体，可以使用这个实验测试。诱导和表1中给出的挑战接种的4种组合，这是可能的，只有一个组合可能会导致在您的工厂背景的表型。我们观察到植物FLS2沉默PF / DC和Pf/Q1-1诱导和挑战接种（见表1）组合在一个公共交通交汇处击穿。 它是必不可少的，检测的环境条件尽可能接近3.1部分所述。湿度低，导致细胞死亡进行太快，使检测难以得分。我们的协议中所描述的条件，如果不工作在自己的实验室，然后尝试改变诱导或挑战接种水平。另一个参数是可以修改的诱导和挑战之间的时间差距，虽然我们已经发现，较短的时间差距造成的检测，以打破在控制植物太快。 我们建议，至少4-5植物在实验测试和积极的表型应至少在3-4个独立实验重复。

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<td>MES</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
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<td>Life Science UV/Vis Spectrophotometer</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
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assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants

察觉到他们的环境中潜在的病原体，植物使用其细胞膜上的模式识别受体（PRRS）。 PRRS承认保守的微生物称为病原体相关分子模式（PAMPS）的功能，这种检测PAMP触发的免疫力（PTI），从而有效地防止非致病菌 1,2的定植植物组织。在公共交通交汇处的最深入研究的系统是FLS2 依赖途径3。 FLS2识别PAMP flg22是细菌鞭毛蛋白的一个组成部分。 成功的病原体具有致病因素或效应，可以抑制公共运输交汇处和允许的病原体引起的疾病 1 。反过来，有些植物具有耐药基因检测的效应或他们的活动，从而导致效应触发免疫“（ETI）2 。 我们描述了一个公共交通交汇处的细胞死亡为基础的检测OH和Collmer 4修改。该实验是在 N标准化benthamiana，正在越来越多地用来作为一个模型系统研究植物病原体的相互作用5。 PTI是诱导渗透到叶的非致病细菌菌株。七个小时后，一个细菌菌株引起的疾病或激活ETI是渗透到原来的渗透区重叠地区。第一浸润引起PTI是能够延缓或阻止细胞死亡的第二个挑战浸润的外观。相反，在接种重叠区的出现表明细胞死亡的一个公共交通交汇处的击穿。 4个公共交通交汇处和挑战接种诱导的不同组合进行了规范（见表1）。检测测试对非沉默北路benthamiana植物，作为控制和FLS2沉默的植物进行了预测，在他们的能力，发展公共交通交汇处受到损害。

Watch this Scientific Journal Video about Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants at JoVE.com

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

一个细胞死亡为基础的公共交通交汇处的检测在烟草benthamiana植物描述。

含量为病原体相关分子模式（PAMP）触发免疫功能的植物（公共交通交汇处）

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

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Immunology and Infection

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

27.0K 次观看.  Boyce Thompson Institute for Plant Research. 一个细胞死亡为基础的公共交通交汇处的检测在烟草benthamiana植物描述。

视频: Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

科研

免疫与感染

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

幽门螺杆菌感染与胃病理的小鼠模型

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;