这项研究得到了最大麦吉少年Diabetesat医学院的威斯康星州和威斯康星儿童研究所国家工程研究中心。出资者已经没有任何作用，在研究设计，数据收集和分析，或准备的手稿。

1。设立抑制法检测之前，需要对细胞的刺激和事后大衣tosylactivated珠抗人CD3（克隆UCHT1，终浓度为1μg/ml）检查是否有效地通过设立在体外增殖实验中使用人类的涂层珠T细胞<ol><li>以从原来的小瓶，在磁场的立场地点的M - 450 tosylactivated珠1毫升并按住，直到所有的珠子都坚持管方。删除缓冲区，而管仍然是在磁场的立场。以管的磁立场，并添加了缓存1毫升，放置​​在磁场的立场管，并删除缓存1，在磁场的立场，而管，重悬珠缓存1毫升，加入抗人CD340μl，激荡在37 ° C 15分钟，加0.1％W / V牛血清白蛋白，并继续为未来的16小时搅拌。 </li><li>在buffer2清洗珠两次5分钟;删除缓冲区buffer21毫升重悬珠，珠是在2-8℃，一旦5分钟buffer3在2-8 ° C使用磁性的立场，如上面所述4 × 8 / ml的终浓度和准备使用。 </li><li>分装50000和25000外周血单核细胞/孔在96孔板一式三份，并添加变量CD3涂层珠（4 × 8珠/毫升，CD3 +1μg/ml，例如1，2，3，4，5珠/细胞），以确定每个细胞珠的最佳数目。文化在72小时后，加1μCi[3H]胸腺嘧啶，继续孵育37 °彗星在未来16个小时。多屏收获板（Millipore公司）收获细胞，加入闪烁液，并读取每分钟计数（CPM）/使用最高级NXT（惠普，CT）。使用珠/ CPMS 5000以上时，细胞的比例，但小于15000的调节性T细胞，这可能会失去抑制功能，以避免过度刺激。通常情况下，3珠/细胞的比例，刺激响应者和Treg细胞在测试到目前为止1-4所有学科组。 </li></ol> 2。从全血PBMC的隔离从健康的捐赠者或从人类leukopacks或捉鬼外套（BC），通常采取从健康志愿者和从当地输血中心免费提供（图1） <ol><li>稀BC（〜50毫升），用PBS 1:6（添加250毫升）。现在有300毫升总量。慢慢地稀释公元前层25毫升上15毫升聚蔗糖帕确顶部加至50ml猎鹰管，互不干扰的图层。在800xg离心30分钟在4 ° C与刹车，（在一个水平转头SH - 3000 Sorvall离心机1400rpm）关闭。 </li><li>仔细收集外周血单个核细胞层（中间相），将它转移到一个新的50毫升猎鹰管。洗净PBMC的灌装管与DPBS至50ml。一管收集到2个细胞沉淀。在400xg离心10分钟，4 ° C。重复洗涤步骤两次，每次单管组合成2个细胞沉淀。合并成一个单一的50毫升管细胞沉淀。 </li><li>计数外周血单个核细胞用台盼蓝排斥试验。加入20μL的PBMC台盼蓝180μl染色台盼蓝在1:10稀释。拌匀，取20μL立即hemacytometer计数。位于两个见方的块，每个都含有16个小方格的所有不染，只有蓝染色细胞计数数字。这两个数字的平均值乘以10。这个数字除以100得到的外周血单核细胞/毫升。活菌百分比计算为[1 - （蓝色细胞/细胞总数）× 100]。如果所得的可行性是≥95％。 </li></ol> 3。 MACS预排序的CD4 T细胞<ol><li>出发前，转移到一个新的管的PBMC1毫升，添加媒体4毫升准备照射5000rad这些细胞将抗原提呈细胞（APC）。 </li><li>离心10min后PBS/2mM EDTA/0.5％BSA缓冲休息250xg细胞在4 ° C。 PBS/2mM EDTA/0.5％BSA缓冲4毫升倒掉上清，悬浮细胞。 </li><li> MACS抗CD4微珠添加200μL和孵育4℃20分钟的彗星。 </li><li> 10min后洗净加入250xg PBS/2mM EDTA/0.5％BSA缓冲液和离心机40毫升在4 ° C。脱气，室温PBS/2mM EDTA/0.5％BSA缓冲8毫升倒掉上清液和悬浮。 </li><li>过滤器分离的细胞悬液，采用预分离过滤器，再装上一个LS列。 </li><li>分割的细胞悬液，每超过校准的LS列（deggassed PBS/2mM EDTA/0.5％BSA缓冲3毫升）层4毫升。 LS列在MidiMACS分隔是固定的。细胞悬液之前用完，要么重新运行流穿或添加3毫升deggassed室温PBS/2mM EDTA/0.5％BSA缓冲，让缓冲区贯穿。添加更多的缓冲区，直到它明确的。 </li><li>移液器5毫升到LS列deggassed缓冲区，删除从MidiMACS分隔和地方到一个新的无菌15毫升收集管让〜1.0毫升贯穿LS列。放入列中的柱塞，慢慢推，其余量出来。 </li><li>同时LS列，并结合两个CD4 +分数。添加至50ml PBS和计数细胞。预期收益率高达5X10 8细胞。在400xg离心10分钟，悬浮细胞以及2毫升PBS/2mM EDTA/0.5％BSA缓冲。 </li></ol> 4。荧光激活细胞分选（FACS）隔离（图2） <ol><li>制作鸡尾酒抗体CD标记，以下面的细胞表面标志物（保持避光）：20μL抗人CD8 - FITC（克隆RPA - T8），20μL抗人CD14 - FITC（克隆M5E2;脂多糖受体），20μL抗人CD32 - FITC（克隆FLI8.26;FcγRⅡ型）和6μL抗人CD116 - FITC（克隆M5D12; GM -CSFRα链），另外，加入40μl反人类CD4 - APCCy7（克隆RPA - T4）。 </li><li>取5μL2毫升细胞悬液和染色染色的鸡尾酒2μL;这是一个确定的阈值（荧光色素减一，鱼类统营处）5管。 </li><li>加入50μL抗人CD25 - PE（克隆的M - A251; IL -2Rα）污渍的鸡尾酒，并添加鸡尾酒细胞悬液孵育，在4 ° C 30分钟。在PBS缓冲液，离心10分钟，至10 7 / ml的细胞浓度的悬浮细胞的细胞在400xg洗净。 </li><li>准备不染的细胞和细胞或单个荧光染色作为补偿控制细胞咏叹调流式细胞仪（BD公司，圣何塞，新泽西州）的排序中使用的珠。 </li><li>收购在鱼类统营处管的细胞，这将允许用户设置的门槛，为CD25 + T细胞（图2a）的排序。设置FITC阴性细胞周围的门，排除FITC阳性细胞，包括单核细胞，巨噬细胞和所有其他的CD4 +非T细胞（图2b）。 </li><li>在一个单独的情节，借鉴盖茨+ CD25 -，CD25low和CD25high T细胞的调节性T细胞（顶部细胞表达的CD25 +的最高数量的1％）（图2c）。细胞亚群通常表现出高纯度（图2D，2E和2F）。 </li><li> 10分钟400xg细胞离心收集管，并保持，直到镀冰。 </li></ol> 5。设置细胞培养在96孔板200μl/well（计划表连接） <ol><li> CD3涂层珠分装50μL（1微克/毫升）计算，3珠/应答者的细胞，以及完整的媒体悬浮汇集10％人AB血清U型底96孔板。 （例如，使CD3涂层2毫升媒体，采取7.5μl股票CD3涂层珠终浓度为4 × 8 /毫升，稀释媒体2毫升;每50μL将包含75,000 beads-3beads/cell）。 </li><li>照射的APC 5X10 5 / ml细胞浓度稀释，加入50μL到每口井（将包含2.5 × 10 4细胞）与以前增加的刺激，包括标示为“调节性T细胞只”井“，APC的唯一”和“媒体“表1。 </li><li>一式三份以下表1中的设计，添加2.5 × 10 4 / CD4CD25或CD4CD25low T细胞。 </li><li>添加Treg细胞联合培养的比例为1:10（2500 Treg细胞）（B行，表1）和“调节性T细胞只”标记的井，5％的CO 2，CO 2培养箱培养板在37 ° C ，在饱和湿度为72小时。 </li><li>脉冲1μCi[3H]胸腺嘧啶井，并继续在37℃，未来16小时的潜伏期。 </li></ol> 6。收获和点票<ol><li>多屏收获板收获细胞，使用惠普filtermate收割机或替代系统。 </li><li>加入闪烁液（Microscint 20），封面用透明塑料盖在最后一步准备收获板。 </li><li>阅读每分钟计数（CPM）/以及使用最高级NXT（惠普，CT）或替代系统。 </li></ol> 7。抑制计算的百分比<ol><li>细胞培养在一式三份，平均计算每个条件。如果变异系数&gt; 30％，离群被淘汰，只有两口井CPM的平均值。通过计算得到抑制百分比（SC）/ S] × 100％，其中s = CPM在单一的文化和C = CPM共培养。 </li><li>为幼稚（CD25 -）和效应T细胞在体内激活（CD25low）镀作为响应者T细胞，调节性T细胞的能力，以抑制这些子集的区别是捕获和使用作为一个潜在的功能预后指标的前提基础，活化细胞难以抑制。 </li></ol> 8。代表性的成果： 使用方法的变化很大，并在体外培养的人抑制实验所得的结果促使我们进行一个全面的研究条件 ，影响检测1。我们已经制定了一个试验，测试不仅Treg的功能，而且其纯度，考虑调节性T细胞之间的低比例：Teffs（1:10），我们确定前面6。此外，调节性T细胞与在日EIR的能力，成功地抑制天真，在体内激活T细胞，即使在健康受试者，在图3和我们以前的研究2,4，显得更为突出显示免疫平衡被破坏，在危险的科目发展T1D 。该检测工作非常出色，我们比较自然（nTregs），诱导（iTregs）， 并在体外培养扩增nTregs抑制功能的研究，使我们能够健康对照组之间比较它们的功能，最近发生的（RO）的T1D和长期（LS ）T1D科目。我们得出的结论是反渗透T1D科目有更好的能力，产生功能iTregs和扩大nTregs，比LS T1D和健康对照组7。因此，此法可作为承认的免疫失衡的早期和晚期的状态极好的工具。 计划在体外抑制实验所涉及的步骤1的原理演示<img src="/files/ftp_upload/3071/3071fig1.jpg" alt="图1"/> 图1的照片呈现在体外抑制实验的步骤 。 <img src="/files/ftp_upload/3071/3071fig2.jpg" alt="图2"/> 图2流式细胞仪细胞分离。浇注战略。一个）+ CD25 +阈值调整，根据荧光色素减一（FMO），b）细胞分别为FITC标记的负面门控，彗星）FITC标记的负面细胞被进一步门和CD + CD25 +，CD + CD25low和光盘+ CD25high收集（调节性T细胞）的百分比，D所示），流式细胞仪排序Tregs的排序后，E）流式细胞仪排序排序后的CD + CD25low，和f）流式细胞仪排序后排序的CD4 + CD25 - T细胞<img src="/files/ftp_upload/3071/3071fig3.jpg" alt="图3"/> 图3。正常人的代表 ）代表每分钟计数的结果，参与所有细胞亚群的单一文化的健康受试者（ 天真，CD25 +，在体内的活性CD25low，抗原提呈细胞- APC（CPM）和调节性T细胞，Treg细胞）以及应答器T细胞共培养（+ CD25 -或CD25low）和调节性T细胞，B）的百分比抑制每个CD25和CD25low响应T的调节性T细胞自体细胞，为健康对照组（ N = 4）。制止被计算为[（SC）/ S] × 100％，其中s = CPM在单一的文化和c = CPM共培养。虽然略有差异调节性T细胞的能力，抑制急救员T细胞被注意到，这是不显着（配对t检验，P = 0.08），C）是CPM的风险科目，每个单一的文化，包括CD25和CD25low作为响应者T细胞，以及APC和调节性T细胞，并联合培养，每个应答者的T细胞亚群调节性T细胞（CD25-/Tregs和CD25low/Tregs）D）每个CD25和CD25low响应者T细胞的抑制百分比接种自体调节性T细胞是风险科目（N = 4）。抑制CD25 -与CD25low响应者T细胞的调节性T细胞的能力差异有显着性（配对t检验，P = 0.04）。 表1。示意图设立在体外抑制实验<table border="1"><tbody><tr><td></td><td> 1-3 </td><td> 4-6 </td><td> 7-9 </td><td> 10-12 </td></tr><tr><td>一</td><td> CD4CD25 - </td><td> CD4CD25low </td><td>媒体只</td><td>媒体只</td></tr><tr><td>乙</td><td> CD4CD25 / - 调节性T细胞</td><td> CD4CD25low /调节性T细胞</td><td>调节性T细胞只</td><td> APC的唯一</td></tr><tr><td>彗星</td><td></td><td></td><td></td><td></td></tr><tr><td> ð </td><td></td><td></td><td></td><td></td></tr><tr><td> Ë </td><td></td><td></td><td></td><td></td></tr><tr><td> F </td><td></td><td></td><td></td><td></td></tr><tr><td>摹</td><td></td><td></td><td></td><td></td></tr><tr><td> H </td><td></td><td></td><td></td><td></td></tr></tbody></table>

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K., Kolodziej, A., Looby, R. J., Sun, W. -. C., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lysine+walk+to+high+relaxivity+collagen-targeted+MRI+contrast+agents.">A lysine walk to high relaxivity collagen-targeted MRI contrast agents.</a> Chem. Commun. , 430-432 (2009).</li><li>Le&#244;n-Rodr&#237;guez, L. M. D., Kovacs, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+and+chelation+chemistry+of+DOTA-peptide+conjugates.">The synthesis and chelation chemistry of DOTA-peptide conjugates.</a> Bioconjugate Chem. 19, 391-402 (2008).</li><li>Boswell, C. A., Eck, P. K., Regino, C. A. S., Bernardo, M., Wong, K. J., Milenic, D. E., Choyke, P. L., Brechbiel, M. 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作为唯一的独特功能，调节性T细胞，抑制功能应可靠，统一的测试在同一疾病发展的不同阶段和不同的研究主体之间。我们提供我们这个实验的标准化贡献在我们的实验室开发的抑制实验的细节。在我们广泛的优化研究，我们有决心，刺激T细胞与抗人CD3涂层珠（UCHT1克隆，在1μg/ml浓度组合（相对于市售5μg/ml）作为APC PBMC提供了天然的刺激激活测试科目1都响应者的T细胞和调节性T细胞的能...

<table border="1"><tbody><tr><th>试剂或仪器名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>聚蔗糖 - 帕确游</td><td> Amersham公司Pharmacia公司生物技术</td><td> 17-1440-03 </td><td></td></tr><tr><td> DPBS - 1X </td><td> Gibco公司</td><td> 14190-144 </td><td></td></tr><tr><td>台盼蓝</td><td> Invitrogen公司</td><td> 15250-061 </td><td></td></tr><tr><td>抗CD4 +微珠</td><td>美天旎</td><td> 130-045-101 </td><td></td></tr><tr><td>预分离过滤器</td><td>美天旎</td><td> 130-041-407 </td><td></td></tr><tr><td> LS列</td><td>美天旎</td><td> 130-042-401 </td><td></td></tr><tr><td> EDTA </td><td> Invitrogen公司</td><td> 15575-020 </td><td></td></tr><tr><td>牛血清白蛋白</td><td> Sigma - Aldrich公司</td><td> B4287 </td><td></td></tr><tr><td>抗人CD4 - APCCy7（克隆RPA - T4） </td><td> BD Pharmingen公司</td><td> 557852 </td><td></td></tr><tr><td>抗人CD25 - PE（克隆的M - A251; IL -2Rα） </td><td> BD Pharmingen公司</td><td> 555432 </td><td></td></tr><tr><td>抗人CD8 - FITC（克隆RPA - T8） </td><td> BD Pharmingen公司</td><td> 555366 </td><td></td></tr><tr><td>抗人CD14 - FITC（克隆M5E2;脂多糖受体） </td><td> BD Pharmingen公司</td><td> 555397 </td><td></td></tr><tr><td>抗人CD32 - FITC（克隆FLI8.26;FcγR- II型） </td><td> BD Pharmingen公司</td><td> 555448 </td><td></td></tr><tr><td>抗人CD116 - FITC（克隆M5D12; GM -CSFRα链） </td><td> BD Pharmingen公司</td><td> 554532 </td><td></td></tr><tr><td> Dynalbeads的M - 450 tosylactivated </td><td> Invitrogen公司</td><td> 140-13 </td><td></td></tr><tr><td>抗人CD3 </td><td> Ancell </td><td> 144-024 </td><td></td></tr><tr><td>缓存1 </td><td>自制</td><td></td><td> 0.1M娜2，B 4 O 7的 pH7.6 </td></tr><tr><td> Buffer2 </td><td>自制</td><td></td><td> PBS/2mM / EDTA的0.1％BSA的PH7.4 </td></tr><tr><td> Buffer3 </td><td>自制</td><td></td><td> 0.2M Tris/0.1％BSA的pH8.5 </td></tr><tr><td>完全RPMI媒体</td><td>自制</td><td></td><td>的RPMI 1640媒体2毫米L -谷氨酰胺5毫米的HEPES 100 U /微克/毫升peni /链霉0.5毫米丙酮酸钠</td></tr><tr><td> [3H]胸腺嘧啶</td><td> Perkin Elmer公司</td><td> NET027Z005MC </td><td></td></tr><tr><td>人类汇集AB血清</td><td>亚特兰大生物</td><td> S40110 </td><td></td></tr><tr><td>多屏收获板</td><td> Millipore公司</td><td> MAHFC1H60 </td><td></td></tr><tr><td> Microscint 20 </td><td> Perkin Elmer公司</td><td> 6013621 </td><td></td></tr></tbody></table>

human in vitro suppression as screening tool for the recognition of an early state of immune imbalance

调节性T细胞（Treg细胞）的自身抗原免疫耐受的关键调解员。此外，他们被感染后的免疫反应的关键调节因子。尽管努力找出独特的调节性T细胞表面标记，唯一的功能是抑制效应T细胞的增殖和功能的能力。虽然很明显，只有在体外试验 ，可用于评估人Treg功能，这将成为问题时，评估的结果，从横断面研究健康细胞和细胞与自身免疫性疾病（如1型糖尿病的T1D）需要的科目中分离进行比较。有一个响应者的T细胞，刺激的性质和强度，Treg细胞的数量和类型的实验室之间的变化很大：应答者比率和使用的数量和类型的抗原提呈细胞（APC）在人类体外抑制实验。这种差异使得比较难以测量Treg细胞功能的研究。 Treg的领域，需要一个标准化的抑制与正常人和自身免疫性疾病的检测工作。我们已经开发出在体外抑制实验表明很少批内变异在健康志愿者相比，与底层的胰腺β细胞的自身免疫性破坏的科目分离的T细胞的刺激。这件作品的主要目的是描述一个在人类体外抑制实验，允许不同的学科组之间的比较。此外，该法有可能划定nTreg功能的一个小的损失，并预计在未来的进一步丧失，从而确定谁可以受益于预防免疫疗法1的科目。下面，我们提供在此过程中所涉及的步骤的详细描述。我们希望用来衡量Treg细胞功能的体外抑制实验的标准化做出贡献。此外，我们提供了这个实验作为一种工具，认识到早期免疫失衡状态和T1D功能的一个潜在的生物标志物。

Watch this Scientific Journal Video about Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance at JoVE.com

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

调节性T细胞是免疫系统的强有力的抑制器。有一个独特的表面标志物来定义它们的缺乏，因此，调节性T细胞的定义主要功能。在这里，我们描述了一个优化的<em&gt;在体外</em&gt;检测能够识别风险科目的免疫失衡发展T1D。

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

human-vitro-suppression-as-screening-tool-for-recognition-an-early

Research

JoVE Journal

Immunology and Infection

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

14.4K 次观看.  Medical College of Wisconsin. 调节性T细胞是免疫系统的强有力的抑制器。有一个独特的表面标志物来定义它们的缺乏，因此，调节性T细胞的定义主要功能。在这里，我们描述了一个优化的在体外检测能够识别风险科目的免疫失衡发展T1D。

视频: Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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 ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

人类承认免疫失衡的早期国家禁止作为筛检工具