The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

1）制备细胞系: <ol><li>发展表达马了FcεRIα的RBL-2H3.1细胞系: <ol><li>使用基本的组织培养技术的单层细胞系，转用pEE6质粒父母RBL-2H3.1细胞，承载着马FcεRIα基因（GenBank:Y18204.1）28。加入2微克微升-1的质粒DNA，以0.8毫升细胞以1.2×10 7个细胞毫升-1的密度。使用0.4厘米electrocuvette然后立即在冰上孵育10分钟，电穿孔的细胞在250伏960μF。 </li><li>用含有0.4克遗传霉素G418硫酸盐媒体选择转化的细胞，然后通过用荧光的IgE抗体标记它们通过FACS剩余的活细胞进行排序。使用所得的RBL-2H3.1表达马FcεRIα细胞系为调查2,3。 </li></ol></li><li>预测试抗体致敏: <ol><l我&gt;收获的RBL-2H3.1表示从融合培养皿马FcεRIα细胞。洗，然后重悬浮细胞培养基中，以5×10 5细胞毫升-1的细胞密度。 </li><li>的兴趣增加了IgE与悬浮细胞，以1毫微克毫升-1的终浓度，然后板100微升的细胞在96孔板中在列1-6和孵育在37℃+ 5％CO 2 + 90％的相对湿度为16小时。经过培养时间，并进行释放试验前，检查井显微镜的良好融合和细胞粘附下。 </li></ol></li></ol> 2）释放试验: <ol><li>洗涤细胞: <ol><li>暖释放缓冲液（25mM PIPES，120毫摩尔氯化钠，5mM的氯化钾，0.04毫摩尔氯化镁和1mM氯化钙）在37℃下，以允许细胞轻柔洗涤。 </li><li>通过轻弹板以除去细胞培养基，并加入100μl的温暖洗涤细胞，3个7°C，释放缓冲区。重复两次。 </li></ol></li><li>抗原挑战: <ol><li>准备一个连续稀释的抗原0纳克毫升-1，0.1纳克毫升-1，1纳克毫升-1，10纳克毫升-1，100纳克毫升-1（NIP-HSA或DNP-HSA），1000纳克毫升-1，万纳克毫升-1释放缓冲区，并在37℃的温水。 </li><li>第二小区洗涤后，弃去培养基，并用100μl抗原溶液代替。确保在同一行中的孔中（A1-6例如）具有相同的抗原浓度加入到其中。 </li><li>加入0纳克毫升-1抗原设立阴性对照A行。添加渐增的抗原浓度下后跟曲拉通-X缓冲液（5％的Triton X-100）中排H细胞以裂解细胞中的行（BG）被用作阳性对照。孵育在37℃下20分钟以使细胞释放其调停。 </li></ol></li><li>建立单井控制: <ol><li>培养后，转移50μl的细胞上清液，以在板的另一半（井A1-6至A7-12孔等）。丢弃剩余的50微升上清液，并用50μl的曲拉通-X缓冲替换，以允许在每个释放介质以及在列7-12的数量的测量作为细胞内的总介体在柱1-6的百分比。 </li></ol></li><li>酶底物: </li><li>添加50μl的β氨基己糖苷酶底物（50mM的4-硝基苯基N-乙酰基β-D-葡糖苷加入到柠檬酸盐缓冲液0.2M的柠檬酸和0.2M的醋酸钠制备在DMSO中稀释，以2毫米，pH值4.5）的到所有的孔中，以促进基片的转换，以4-硝基苯酚的β氨基己糖苷酶。孵育所述板在37℃下搅拌2小时。 </li></ol><img alt="图2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" widtH ="400"/&gt; <ol start="5"><li>终止反应: <ol><li>停止通过加入150微升的Tris缓冲液（1M的Tris-盐酸，pH为9），向每个孔中作为缓冲液的高pH值，反应停止反应并接通4-硝基苯酚的成黄色。 </li></ol></li><li>阅读和分析结果如下: <ol><li>使用平板分光光度计在405nm测定的黄颜色的吸光度读板。计算使用下面的公式释放β氨基己糖苷酶的百分比: </li></ol></li></ol><img alt="图2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2"><li>应用此式向每个孔中，在这之后的平均值取为每一行。 A1和A7代表各孔中的96孔培养板的位置。积的β氨基己糖苷酶的释放（其对应于总的介质释放）的百分比的曲线图agaiNST抗原浓度2,3。 </li></ol>

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The authors declare that they have no competing financial interests in this paper.

在总结本研究的结果表明，当表达马FcεRIα的RBL-2H3.1细胞通过抗原致敏马IgE和挑战，他们得到的36.68％的峰值介质释放±介体内部的总量的4.88％细胞相比，所述的RBL-2H3.1亲本细胞不表达马FcεRIα。 因此该测定提供了用于调查研究马过敏反应体外的有用工具。其允许介由肥大细胞/嗜碱粒细胞谱系细胞释放的量的确定，从而可以预期到在马过敏推进研究，评估是否过敏致病剂，或研究的IgE /受体阻断剂。 此法进行了修改，研究从用它来 ​​研究人类和犬类过敏症4529为同一个目的而以前的出版物马过敏。该工程化细胞系所表达的人类和犬FcεRIα被用来研究过敏阻断剂的疗效和安全性，例如靶向的IgE /受体相互作用30的抑制的疫苗。调查表明免疫原，其包含整个Cε3域，其还建议其他人作为疫苗，用于衰减IgE介导的过敏性反应的狗24的anaphylactogenic副作用。根据我们的调查，对于这种潜在的严重并发症的分子基础，可以通过含有靶向于Cε3表位和抗IgE抗体，抗IgE抗体的免疫血清进行合理化识别中IgE的抗体的表位的基础上时，不会遮挡IgE的络合与其受体。这一观察结果不能已在没有该检测系统，是鲜明的对比调查了24，谁也研究了基于体内 immuni抗过敏的干预策略矩阵特殊积与Cε3域，但是没有讨论该策略的安全性，由于没有这样的测定的。 该协议的关键步骤，是确保广大RBL-2H3.1细胞是分泌型的;具有该细胞的培养大于10周使它们开始朝 ​​着非分泌型27漂移不管它们是否表示感兴趣的克隆FcεRIα与否。因此，它总是必不可少运行小鼠IgE阳性对照旁边的实验（ 图1），并维持分泌细胞的大量冷冻的储存。 此外，所有缓冲区的pH值必须准确，否则风险提供虚假阴性结果;延伸缓冲液的货架寿命会改变其pH值，并因此任何缓冲液2个月以上需要被丢弃。 该技术示出了一种化学品与RBL-2H3.1策的相互作用LLS，以及它如何促进，或者抑制，其介质释放体外，这是体内可能发生的事情一个很好的迹象，因此，该协议是一个重要的步骤研究潜在的抗过敏治疗策略的时候。该技术没有，但是，表明该抗体/受体复合体的目的分子与之交互的哪一部分。 其他的研究工作，如20表明，马饶是不相关的IgE的调解，这就是为什么有些差距可以看出，与其他出版商，如使用莫兰总马血清进行测试，以钙时等 31 2+潮饶患马用非特异性reaginic抗体，而不同的抗体将负责细胞钙离子内流。 该测定是特定的IgE介导的超敏反应，其也可以适用于其他生物，如人体和狗29，并且具体用于测试的抗过敏干预策略，如合成/嵌合抗过敏抗体30的安全性。

过敏已经知道了几千年。哮喘的治疗中被称为埃伯斯纸草（〜1550 BCE）的古埃及医书被描述和讨论草药治疗呢7。 今天过敏被分类为I型超敏性反应，其中，所述辅助性T细胞2型（TH 2）对免疫系统的臂牵引其生产免疫球蛋白E（IgE）的抗体响应于环境抗原称为变应原。这些是多种多样的物质，这些物质通常与细胞的相互作用在免疫系统，并刺激了合成和促炎性细胞因子的分泌，包括白细胞介素4和白细胞介素13 8,9作为别在香烟烟雾或柴油机排气颗粒颗粒增强IgE合成10。 在过敏表现上升工业化国家在过去50年来一直归因于效果的组合环境污染物和一种趋势，更加消毒环境中，它们组合转向由TH 2细胞因子占优势的轮廓的免疫应答，所建议的"卫生假说"11。 如上所述，人类不是由过敏折磨的唯一哺乳动物。特别是马和狗也可以开发经典过敏反应和研究12表明，在人类，马过敏是由于遗传和环境因素。其结果是，这些动物呈现良好的模型用于研究过敏的遗传和环境的原因，从致敏到疾病的进展，和可能的干预策略，一旦临床表现中已经设置之间的相互作用 1887年，Stömmer是第一个来描述人类和马哮喘13之间的相似性，组胺的马心血管系统的效果非常接近人类14。马也都是赛马业的基石，这是值得72美元十亿与115美元十亿年15投注额。 最现代的赛马都是小数量从1878年起，培育夫人安妮·布朗特阿拉伯马的后代。现代赛马通常近交系选择性能的能力。它们容易遗传性疾病，其中之一是其易安装过敏反应。它们还具有高1000倍的血清IgE水平甚至比最严重的过敏性人16。马过敏反应通常表现为昆虫叮咬过敏（IBH）17，18。IBH导致皮炎由于叮咬形成虫属中的库蠓 。马过敏性疾病的另一种形式是经常性的气道梗阻（RAO），这是表现在肺和呼吸道。它的特点是气喘和实验室或运算呼吸。饶通常发生在响应于模具孢子和高变应原特异性IgE水平已经记录在马从饶在一项研究中19痛苦虽然另一个调查还没有证实这20。 研究马过敏围绕在监控中和马的IgE通过开发抗马的IgE单克隆抗体（mAbs）21，22的企图。此外，研究了23讨论了生产的马的高亲和力Fc受体的α的胞外结构域的链（FcεRIα）受体，以试图检测和定量的马血清IgE。一项相关研究Ledin 24讨论了一种新方法，旨在中和血清IgE通过使用自体/非自吸免疫的免疫系统。所有这些研究中，但是，缺乏有效的测定法，以测试它们的协议的安全性和有效性。在这篇文章中，我们现在提出这样一个实验SYS统适用于相关的马系统，其中β氨基己糖苷酶的释放，作为细胞介脱粒的指标，进行了评估上表达马FcεRIα的RBL-2H3.1细胞诊断和治疗策略的研究。这个协议是基于以前的出版物25，4，5，2，3，描述用编码的高亲和力受体的IgE来自不同物种的IgE结合结构域的基因的RBL细胞的工程。该协议说明如何执行一个β氨基己糖苷酶释放测定，其结果表示为平均值±的一式三份实验的标准偏差。 释放法最早是由Siraganian开发和钩25来研究人类过敏。为首的鲁本Siraganian博士的实验室小组还开发出了RBL细胞系。这些RBL细胞开发了表达人FcεRIα和协议出版了4。最后一块的测定附带的PSV质粒在纸张其中所述的制造的IgE抗体的克隆及其小鼠基因的下游重链基因为靶向的半抗原4-羟基-3-一个的IgE可变区的发展由Neuberger的26硝基 - 苯乙（NP），将得到的嵌合抗体是全功能的。能力开发任何的IgE靶向相同​​的半抗原，同时也克隆及其受体导致使其成为一个有用的协议来测量嗜碱性粒细胞的脱颗粒测定的标准化的RBL细胞的表面上。 该法确实有优点和缺点。该测定法的优点是它的适应性，在任何哺乳动物系统中使用，我们的实验室已因而用它来测试在人，犬和马系统的脱粒，并且这是可以实现简单地通过合成的生物体的IgE和克隆受体到RBL细胞的表面上。 另一方面，该测定法的缺点是，RBL细胞对热，机械和PH值的变化非常敏感，使得它们得到脱粒水平的相同的测定范围内的变化。因此，这是强烈建议的分析总是重复一式三份，然后平均取自他们。此外，RBL细胞趋向于朝向非剥离性表型转变，如果它们被留在组织培养延长的时间（&gt; 10周）27，使得其维修很麻烦。他们还容易感染支原体的细菌，这是不是从一个光学显微镜可见的，不改变细胞的形态，但会极大地改变他们的脱颗粒的水平。因此，需要定期检查支原体。

<table><tbody><tr><td>RBL-2H3.1 Expressing Equine Fc&amp;epsilon;RI&amp;alpha;</td><td>-</td><td>-</td><td>Produced in the lab</td></tr><tr><td>Equine IgE anti NIP-HSA</td><td>-</td><td>-</td><td>Produced in the lab</td></tr><tr><td>96 Well Plate</td><td>Sigma</td><td>CLS3595</td><td>-</td></tr><tr><td>Multi Channel Pipette</td><td>Anachem</td><td>-</td><td>-</td></tr><tr><td>Incubator</td><td>Galaxy R</td><td>-</td><td>-</td></tr><tr><td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td><td>Cambridge Research Biochemicals</td><td>N-1070-1</td><td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td></tr><tr><td>Dinitrophenyl Conjugated to Human Serum Albumin</td><td>Sigma</td><td>A6661</td><td>Abbreviated DNP-HSA</td></tr><tr><td>Plate Spectrophotometer</td><td>Anthos Labtec HT2</td><td>-</td><td>-</td></tr><tr><td>Pipes</td><td>Sigma</td><td>P1851</td><td>-</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S7653</td><td>-</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P9333</td><td>-</td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M2670</td><td>-</td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>C1016</td><td>-</td></tr><tr><td>Triton x100</td><td>Sigma</td><td>X100</td><td>-</td></tr><tr><td>4-nitrophenyl N-acetyl-&amp;beta;-D-glucosaminide</td><td>Sigma</td><td>N9376</td><td>Stock solution called &amp;beta;-hexosaminidase substrate was 50mM prepared in DMSO</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma</td><td>D2650</td><td>-</td></tr><tr><td>Citric Acid</td><td>Sigma</td><td>251275</td><td>-</td></tr><tr><td>Sodium Acetate</td><td>Sigma</td><td>S7670</td><td>-</td></tr><tr><td>Tris</td><td>Sigma</td><td>T5941</td><td>-</td></tr></tbody></table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

亲本的RBL-2H3.1细胞和那些转染了马FcεRIα受体基因进行了首次致敏的小鼠的IgE抗DNP-HSA和攻击用的DNP-HSA的抗原。小鼠IgE结合在两种细胞系中内源性大鼠受体并因此充当控制来测试这两种细胞系的生存力释放，释放介质（ 图1中的A）。这是一个重要的检查和常规自应进行后将延长（&gt; ​​10周），通过在细胞培养物，RBL-2H3.1细胞漂移朝向非分泌型27。亲本细胞支持的51.54％的峰值介质释放±4.79％，同时表示马FcεRIα受体细胞有45.99％的峰值介质释放±5.76％。这些相同的细胞系，然后在那里与致敏马的IgE抗NIP-HSA和挑战与NIP-HSA抗原（ 图1B）。亲本细胞，并没有经过介质的释放，由于马IgE的不绑定到内源性受体的老鼠。另一方面，正如所料，表达马FcεRIα受体的RBL-2H3.1细胞发生介体释放时用马的IgE抗NIP-HSA致敏和激发与NIP-HSA的抗原，给人的36.68％的峰值释放±4.88％ 。 这些结果证实了此版本的分析方案的介质释放的通过马IgE受体调查的效率。它表明，该细胞系是在寻求驴新的治疗干预策略在马过敏体外减少了对动物实验的有用的诊断工具。相同的方案也适用于转染的人类和犬FcεRIα受体的RBL-2H3.1细胞。 该协议也被用于测试的一个潜在的免疫策略，试图以中和犬血清IgE抗体30的安全性。从结果中（ 图2）可以判断，该免疫策略是不安全的。抗犬的血清IgE的成功结合到犬科动物的IgE作为最初打算以中和血清IgE水平，并防止其结合到它的受体。但此结合是特异性，因此抗IgE血清交联在细胞的表面上的受体，从而导致介质的释放，从而可能引起过敏性休克，如果此免疫策略被用来作为抗变态反应疫苗。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> RBL-2H3.1表达马FcεRIα </td><td> - </td><td> - </td><td>产于实验室</td></tr><tr><td>马IgE的抗NIP-HSA </td><td> - </td><td> - </td><td>产于实验室</td></tr><tr><td> 96孔板</td><td>西格玛</td><td> CLS3595 </td><td> - </td></tr><tr><td>多道移液器</td><td> Anachem </td><td> - </td&gt; <td> - </td></tr><tr><td>恒温箱</td><td>银河ř </td><td> - </td><td> - </td></tr><tr><td> 4-羟基-5-碘代-3-硝基苯基乙酸</td><td>剑桥研究生物化学</td><td> N-二1070-1 </td><td> NIP-OH的缀合与人血清白蛋白，使NIP-HSA在实验室</td></tr><tr><td>共轭二硝基苯与人血清白蛋白</td><td>西格玛</td><td> A6661 </td><td>缩DNP-HSA </td></tr><tr><td>酶标仪</td><td> ANTHOS蓝铂HT2 </td><td> - </td><td> - </td></tr><tr><td>管</td><td>西格玛</td><td> P1851 </td><td> - </td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S7653 </td><td> - </td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P9333 </td><td> - </td></tr><tr><td>氯化镁</td><td>西格玛</td><td> M2670 </td><td> - </td></tr><tr><td>氯化钙</td><td>西格玛</tD&gt; <td> C1016 </td><td> - </td></tr><tr><td> TRITON X100 </td><td>西格玛</td><td> X100 </td><td> - </td></tr><tr><td> 4-硝基苯基N-乙酰基β-D-葡糖苷</td><td>西格玛</td><td> N9376 </td><td>原液称为β氨基己糖苷酶底物的50mM在DMSO中制备</td></tr><tr><td>二甲基亚砜</td><td>西格玛</td><td> D2650 </td><td> - </td></tr><tr><td>柠檬酸</td><td>西格玛</td><td> 251275 </td><td> - </td></tr><tr><td>醋酸钠</td><td>西格玛</td><td> S7670 </td><td> - </td></tr><tr><td>三</td><td>西格玛</td><td> T5941 </td><td> - </td></tr></tbody></table> 表1:表材料和设备: <img alt="图1" fo:content-width="5.5in" src="/files/ftp_upload/52222/52222fig1highres.jpg" width="700" /> 图1: </str翁&gt;发布检测结果:图A显示了预制的RBL-2H3.1细胞不表达马FcεRIα，并表示使用小鼠IgE抗DNP-HSA受体的释放试验。的小鼠IgE结合导致介体释放的内源性大鼠受体当与DNP-HSA抗原攻击，从而证实这两种细胞系是可行的，并支持IgE介导的抗原诱导的介质释放2,3。图B示出了预成形的释放测定对RBL-2H3.1亲细胞不表达马FcεRIα和那些表达马FcεRIα，用马的IgE抗NIP-HSA对细胞致敏。该马的IgE结合到马受体，但不是内源性大鼠的受体，导致介质的释放，这是由于亲代细胞证实没有释放介体介体，而在细胞中表达的马受体支撑马IgE介导的，抗原诱导的，介质释放2，3。 <img alt="图2" fo:content-width="6.5in" src="/files/ftp_upload/52222/52222fig2highres.jpg" width="650" /> 图2:释放免疫检测结果:本图显示了预制的表达犬FcεRIαRBL-2H3.1细胞释放试验，用犬的IgE抗NIP-HSA对细胞致敏，并免疫大鼠抗犬，血清IgE水平的抗原。所用的对照物的预免疫的血清，阳性对照是NIP-HSA的抗原。犬的IgE结合到犬科动物的受体。此外，抗IgE血清结合到受体结合的犬IgE和交叉链接的，并且该受体，在细胞的表面造成介质的释放。此，实际上，实践证明这特定的免疫策略有可能引起过敏性休克反应的潜力，并因此被认为是不安全30。

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÎµRI at JoVE.com

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

的发展<em&gt;在体外</em&gt;模型研究的交互系统<em&gt;雅科仕</em&gt;<em&gt; caballus</em&gt;以其高亲和力受体了FcεRI的IgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

development-an-vitro-model-system-for-studying-interaction-equus

Nanyang Technological University

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Immunology and Infection

12.4K 次观看.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

视频: Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

科研

神经科学

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

免疫与感染

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

从 3D 牛回肠类结肠培养物中建立 2D 单层

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

作者聚焦：用于研究宿主-病原体相互作用的高级类肠模型 

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

生成用于转化生物医学研究的牛原代肠源二维单层培养系统

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

生物学

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

雌激素原代牛颗粒细胞的组织培养模型

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

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此视频中的章节

视网膜色素上皮的生理与病理生理学研究的实验模型

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

生成用于转化生物医学研究的牛原代肠源二维单层培养系统

生成用于转化生物医学研究的牛原代肠源二维单层培养系统

生成用于转化生物医学研究的牛原代肠源二维单层培养系统

糖皮质激素粪分析:在马属动物无创监测肾上腺

雌激素原代牛颗粒细胞的组织培养模型