这项工作是由个人从威康信托Beate Kampmann（056608，GRO77273）的奖学金。

1。卡介苗勒克斯记者分枝杆菌股票的制备<ol><li>成长卡介苗LUX（牛分枝杆菌卡介苗蒙特利尔应变穿梭质粒pSMT1的转化）用颤 ​​抖的Middlebrook 7H9肉汤含0.2％甘油，0.05％Tween 80和10％的ADC浓缩在200rpm中旬对数生长期。 </li><li>在光度计试管一式两份（900μlPBS +100μL卡介苗勒克斯文化），准备在无菌PBS 1:10稀释的卡介苗勒克斯文化1毫升。 </li><li>装入管中的N -癸醛（荧光素酶底物）到本机的背面，并确保盖子担保和管到位。 </li><li>总理通过基板的黄金节目，注入100 UCL × 3每3个空吸管放在光度计，光度计喷油。 </li><li>装入2管光度计和采取的程序，在每个管注入100 UCL基板的读数，并在1秒的间隔为20秒读。 </li><li>当股价已达到1 × 10 8 RLU /毫升，以2X10 8 RLU /毫升（约3-4天的生长需要），加等体积的无菌30％甘油在50毫升猎鹰管文化，轻轻混匀。 </li><li> 1.5毫升卷分装成标记的螺丝帽2毫升微管和储存在-80 ° C。 </li></ol> 2。确定股票RLU / CFU相关等分内容<ol><li> Middlebrook 7H9培养基添加10％的ADC补充一个发泄的第一个200毫升锥形瓶中15毫升。 </li><li>潮霉素和20％吐温30μl加入15μl。 </li><li>在安全内阁在室温（RT）的冷冻和解冻取出一小瓶卡介苗勒克斯 。小瓶的内容添加到中等，并拧紧瓶盖。 </li><li>孵育晃动在200rpm在37 ° 4天。 </li><li>在每一天，弥补了串行的10倍稀释（1:10，1:100，1:1000，1:10,000和1:100 000）CFU决心。 </li><li>在平行设置的相同的稀释和重复，以确定发光（1.2-1.6） 。 </li><li>准备Middlebrook 7H11琼脂（1升含0.5％甘油，10％OADC补充，20％吐温潮霉素和1毫升1毫升的液体介质）的两个3车厢板。 </li><li>盘开出100μL每个稀释到每个板，使用单独的传播者段，同样分布在个别室的液体，。 </li><li>用封口膜密封每个板块，在无菌塑料袋的地方，与反应釜胶带密封，并在37 ° 2周，眼皮朝下彗星。 </li><li>定期检查，直到CFUs出现（2-3周）。 </li><li>要计数的殖民地，从孵化器和地方上的菌落计数器删除板块。计算平均每个稀释重复板。 </li><li>使用相当于每个稀释度RLU和CFU计数计算RLU / CFU的比例。的比例应为3至5 RLU / CFU。 </li></ol> 3。 勒克斯文化卡介苗接种到全血制备<ol><li> Middlebrook 7H9培养基添加10％的ADC补充一个发泄的第一个200毫升锥形瓶中15毫升。 </li><li>潮霉素和20％吐温30μl加入15μl。 </li><li>在安全内阁在室温下冷冻和解冻取出一小瓶卡介苗勒克斯 。小瓶的内容添加到中等，并拧紧瓶盖。 </li><li>孵育晃动在200rpm在37 ° 2到4天的彗星。 </li><li>在光度计试管一式两份（900μlPBS +100μL卡介苗勒克斯文化），准备在无菌PBS 1:10稀释的卡介苗勒克斯文化1毫升。 </li><li>与基总理光度计（如上所述），轻轻涡管到光度计负载，并采取读数（如上所述）。 </li><li>使用此读来稀释在PBS的文化，给予相当于 7x10 6 RLU（这将使约2 × 10 5 CFU / ml的血液和卡介苗的比例约为1:1的单核细胞接种，假设一个单核细胞计数的约2 × 10 5 4 × 5单核细胞每毫升血液）。 </li></ol> 4。全血制备<ol><li>取35毫升静脉血液中含不含防腐剂肝素管。 </li><li>血液转移至50ml猎鹰管和平等的含有量的谷氨酰胺和25mm HEPES（没有笔/链球菌）的RPMI 1640稀释。 </li><li>分装到每个时间点的不育茹管（共6管为0hrs：3和3为96小时，额外的时间点设置额外的管，如果有必要的900μl稀释血液，一式三份。 </li><li>分装900μl10％的ADC补充+50μg/ml潮霉素（在同一介质中，用于文化卡介苗勒克斯）Middlebrooks 7H9培养基生长控制的重复。 </li><li>添加稀释卡介苗勒克斯 100μL每管血，2增长控制管。 </li><li>搅拌均匀，放置96小时的时间点和2生长控制方在摇摆孵化器在37 3 bijous ° C，20转/分（CO 2的不需要） 。 </li></ol> 5。测量发光（0hr和96小时） <ol><li>在2000克离心10分钟3 bijous。 </li><li>小心地取出，而不会干扰2毫升螺丝帽微管沉淀和冻结在-80300μL上清℃，随后的细胞因子分析。 </li><li>加入300μL的PBS每个茹，以取代删除上清量。 </li><li>成相应的50毫升猎鹰管吸每个茹的内容，并添加8毫升蒸馏水，以每管。开始了10分钟的计时器。 注：这是一个时间敏感的一步，这个孵化不能有任何超过10分钟，从开始时，细胞首先接触到水 。 </li><li>冲洗，蒸馏水和5秒的旋涡2毫升相应的猎鹰管引爆之前，每个茹。 </li><li>在结束了10分钟的孵育，离心2000克猎鹰管10分钟。 </li><li>倒出上清液进入Surfanios消毒剂。 </li><li>添加一些玻璃珠，每个颗粒和涡。 </li><li>添加无菌PBS 1ml到每个管和涡。 </li><li>在光度计管中，每个样品一式两份，用PBS（900μlPBS +100μL样品），并在96小时的时间点1:10稀释。每个生长控制的相同稀释。 </li><li>轻轻涡管和负载到的光度计，并采取读数（如上面所述） 注：同一样品一式三份的读数应在彼此的15 ％。 </li></ol> 6。代表性的成果： 重要的是要使用全血勒克斯检测的增长对数生长期的细菌，即接种样品前增长超过48-72小时，代谢活动是最理想的，如果任直出的冰柜或固定相。前增长应为48或72小时的实验系列，以避免变异标准化。图1显示了一个代表性的文化卡介苗勒克斯的生长曲线。倍增时间约24小时，直到达到固定相。 <img alt="图1" src="/files/ftp_upload/3332/3332fig1.jpg" /> 图1。代表BCG的生长曲线勒克司超过96小时7H9培养基和相关RLU和CFU 。 虽然RLU值总是与CFU关联，可以改变单一的CFU数量相应的RLU取决于股票，这就是为什么它是很好的做法，整个一系列的实验中使用相同的冻结的股票，以保证一个一致的多重感染（ MOI）。 表1显示了一个原始日期的接种时间（T 0），并在96小时的例子。增长比率计算公式T 96 / T 0，但其他的间隔时间，当然也可以测量。它是，但是，最好不要使用超过96小时的文化，如发生显着的细胞死亡。 <img alt="表1" src="/files/ftp_upload/3332/3332table1.jpg" /> 表1的接种时间（T0），并在96小时（T96）和3个成人捐助者计算增长比率的原始数据的例子。 根据现有的抗原特异性记忆反应和中性粒细胞计数也可能，增长比率因人而异如图所示，在图2与儿童的血液中，这里没有感染艾滋病毒。平均，幼儿比成年人有较大幅度的增长比率，结核菌素皮肤试验（TST）+已经个人有较低的增长比率比尖沙咀- VE个人，和艾滋病毒感染者由于缺乏症的CD4 T细胞人口的高增长比率，分枝杆菌的细胞免疫反应的关键调解人之一。 <img alt="图2" src="/files/ftp_upload/3332/3332fig2.jpg" /> 图2捐助者间的变异 ：增长的比率为一组患者的T 0与96小时，取决于底层的艾滋病毒状况。 过了一段时间的增长比率的可重复性是在图3所示，它总结了64流血超过12个月内，从一个单一的捐助者为对照实验，反复流血的两次捐助者的结果。变异的潜在原因可能是，在分枝杆菌致敏或在宿主细胞因子水平的变化的变化，许多生物测定。 <img alt="图3" src="/files/ftp_upload/3332/3332fig3.jpg" /> 图3。 （a）单一捐助12次超过12个月。（二）多（64）2次捐助者超过12米onths。

<ol>
	<li>Cheng, S. H., Walker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+increased+anti-mycobacterial+activity+in+peripheral+blood+monocytes+after+BCG+vaccination+in+British+school+children.">Demonstration of increased anti-mycobacterial activity in peripheral blood monocytes after BCG vaccination in British school children.</a> Clin Exp Immunol. 74, 20-205 (1988).</li><li>Cheng, S. H., Walker, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+antimycobacterial+activity+before+and+after+Mycobacterium+bovis+BCG+vaccination+in+Chingleput,+India,+and+London,+United+Kingdom.">Monocyte antimycobacterial activity before and after Mycobacterium bovis BCG vaccination in Chingleput, India, and London, United Kingdom.</a> Infect Immun. 61, 4501-4503 (1993).</li><li>Silver, R. 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没有利益冲突的声明。

在结核病疫苗研究中的杀菌检测以及建立动物模型，它们应用到屏幕上可能有前途的候选疫苗。作为主要的免疫原性读出各个器官功能的细菌负荷减少。 人类杀菌实验的第一代设计单独使用巨噬细胞，感染与 M. 结核病 。 CFU担任分枝杆菌生存的主要读出后最短的时间为3周。依靠这些检测外周血单个核细胞，贴壁的单核细胞/巨噬细胞和裂解准备在特定时间点<...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 猫没有 </td></tr><tr><td>卡介苗勒克斯 </td><td>各种</td><td> N / A </td></tr><tr><td> Middlebrook 7H11琼脂</td><td>碧迪</td><td> 283810 </td></tr><tr><td> Middlebrook 7H9肉汤</td><td> BD公司</td><td> 271310（500克） </td></tr><tr><td> Middlebrook ADC的浓缩</td><td> BD公司</td><td> 212352 </td></tr><tr><td> Middlebrook OADC的补充</td><td>碧迪</td><td> 212240 </td></tr><tr><td>吐温80 </td><td>西格玛爱秩序</td><td> 274364 </td></tr><tr><td>甘油</td><td>西格玛爱秩序</td><td> G5150 </td></tr><tr><td>潮霉素B </td><td>罗氏公司</td><td> 10843555001（20毫升） </td></tr><tr><td> N -癸醛</td><td>西格玛</td><td> D7384 - 100G </td></tr><tr><td>乙醇（&gt; 99.7％） </td><td> VWR </td><td> 101077 </td></tr><tr><td>无菌PBS </td><td>众议院</td><td> N / A </td></tr><tr><td> RPMI - 1640 L -谷氨酰胺，25MM的HEPES </td><td>西格玛</td><td> R0883 </td></tr><tr><td>无菌蒸馏水</td><td>众议院</td><td> N / A </td></tr><tr><td colspan="3"> 设备 </td></tr><tr><td>管光度计（强制注射端口） </td><td>贝特霍尔德AutoLumat LB953或单管光度计天狼星</td><td></td></tr><tr><td>光度计管，12 × 75毫米，耐热无框</td><td>康宁从VWR </td><td> 99445-12 </td></tr><tr><td>通风帽的锥形瓶，无菌聚碳酸酯250毫升</td><td>康宁通过费舍尔科学</td><td> 2150329 </td></tr><tr><td> BD真空采血管血液收集管（不含防腐剂肝素） </td><td>屋宇署</td><td> 367676 </td></tr><tr><td> 50ml隼管中，圆锥形</td><td>屋宇署</td><td> 352077 </td></tr><tr><td> 7毫升Sterilin标本聚丙烯茹管</td><td> VWR </td><td> 99445-12 </td></tr><tr><td> Sterilin环球货柜（30毫升） </td><td>实验室分析有限公司</td><td> 128C </td></tr><tr><td>玻璃珠直径2mm </td><td> SigmaAldrich </td><td> Z143928 </td></tr><tr><td> 10毫升移液器和移液器男孩</td><td>任何</td><td></td></tr><tr><td> 1000μl和200μL的移液器和过滤嘴</td><td>任何</td><td></td></tr><tr><td colspan="3"> 通用实验室设备： </td></tr><tr><td> II级安全柜柜</td><td></td><td></td></tr><tr><td>冰箱（4℃） </td><td></td><td></td></tr><tr><td>离心分离</td><td></td><td></td></tr><tr><td>混合管的摇摆振动筛平台（180 0 ） </td><td></td><td></td></tr><tr><td>长大细菌培养轨道摇床培养箱在37 ° C </td><td></td><td></td></tr></tbody></table>

a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)

功能测定早已起到了关键作用的一个给定的疫苗的免疫原性的测量。这是常规表现为血清杀菌滴度。儿童疫苗的血清杀菌滴度的研究使我们能够开发和验证切断保护性免疫反应的水平和削减权衡，在日常使用。没有这样的检测已采取迈进的诱导主要是细胞免疫效应T细胞反应的形式，如结核病疫苗，疫苗的例行评估。表现一个给定的候选疫苗在动物模型，定期评估标准化杀菌检测，目前所有的新型结核病疫苗候选人受到这一步在他们的评价，第1阶段的人体试验之前。理想的免疫原性，因此，新型抗结核病疫苗的保护效力的可能性的评估应在人体模型进行类似的分步进行的评价，现在，包括杀菌实验的测量。 杀菌检测在结核病疫苗研究方面已经建立的动物模型，它们应用到屏幕上可能有前途的候选疫苗。作为主要的免疫原性读出各个器官功能的细菌负荷减少。然而，没有这样的检测已被纳入临床试验新型抗结核病疫苗的最新。 虽然仍存在不确定性的确切机制，导致人类巨噬细胞内结核分枝杆菌杀，分枝杆菌与巨噬细胞和T细胞的相互作用是明确要求。本文中所描述的实验代表的新型杀菌检测等关键的细胞成分的研究，使全血中目前没有作出有关其相对独立的贡献的假设与所有其他细胞和体液因素。本集团所描述的检测使用全血的小卷，并已受聘在成人和儿童的研究结核病流行的设置。我们已经表明BCG疫苗的免疫原性，增加分枝杆菌的增长，在HIV阳性患者，以及抗逆转录病毒疗法和维生素 D分枝杆菌在体外生存的影响。在这里，我们总结的方法，目前我们的重复性数据，使用这个相对简单，低成本和友好的领域模型。 注：定义/缩写卡介苗勒克斯= M 牛卡介苗，蒙特利尔应变，转化与穿梭质粒进行哈维氏弧菌的luxAB基因pSMT1，分枝杆菌GroEL的（HSP60）启动子的控制下。 CFU =集落形成单位（结核分枝杆菌的可行性措施）。

Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

我们描述了一个替代的方法来列举分枝杆菌<em&gt;在体外</em&gt;，它使用的记者基因标记，而不是细胞集落形成单位（CFU）的分枝杆菌。 “生存”的生物体，以及宿主反应的标记，同时测量，提供一个低成本，灵活和功能系统在结核病中的主机/病原体相互作用的研究。

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

a-functional-whole-blood-assay-to-measure-viability-mycobacteria

Research

JoVE Journal

Immunology and Infection

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

13.8K 次观看.  Imperial College London. 我们描述了一个替代的方法来列举分枝杆菌在体外，它使用的记者基因标记，而不是细胞集落形成单位（CFU）的分枝杆菌。 “生存”的生物体，以及宿主反应的标记，同时测量，提供一个低成本，灵活和功能系统在结核病中的主机/病原体相互作用的研究。

视频: A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

非遗传表面修饰的素-生物素系统在牛分枝杆菌卡介苗中的外源抗原表达

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

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, ...