笔者想承认的贡献AM万巴斯柯克我们的理解的发展DTH反应，移植受者的监管。这项工作是支持的NIH拨款PO1AI084853和R01AI066219-06，由欧盟资助的一个研究。

1。淋巴细胞的制备<ol><li>收集血液进入ACD（酸性柠檬酸葡萄糖）管。 </li><li>按照标准方法，用淋巴细胞分离液从新鲜人外周血的PBMC隔离。 </li><li>外周血单个核细胞用PBS三次洗净，去除污染的血小板。血小板被发现干扰反式被检体内的DTH试验。最大允许PBMC制备血小板污染是≤1×10 7 /注塑。 </li><li>如果有明显的红血细胞污染，执行第一次洗涤后，使用ACK裂解缓冲液裂解红细胞。将ACK缓冲液，用PBS洗涤2次。 </li></ol> 2。制备特异性抗原<ol><li>使用上面所示的过程从供体外周血的PBMC隔离。 </li><li>于PBS中的浓度为120x10 6细胞/ ml（4×10 6个细胞/ 30微升）重悬供体的PBMC。 </li><li>加入1μM的苯甲基磺酰氟的混合物，以防止蛋白质退化。 </li><li>超声处理的细胞悬浮液，用2毫米的探头超声波破碎仪使用7个1秒的脉冲。 （注意:保持材料的冷，并避免过多的气泡，如果出现泡沫，让细胞悬液，坐冰2-3分钟）。 </li><li>验证使用血球细胞&gt; 90％的中断。 </li><li>在14,000 rpm离心该混合物在4℃下冷​​藏20分钟的离心。 </li><li>上清转移到一个新的2.0毫升安全锁管，并确定蛋白浓度。 </li></ol> 3。细胞制备的注射<ol><li>为每次注射等分7×10 6到2.0毫升安全锁管PBMC。 </li><li>离心一分钟6000转离心，去除上清。 </li><li>带或不带抗原在PBS中重悬细胞。调整进样体积为35微升，用PBS。下面的计划是用于注射: 阴性对照:外周血单个核细胞+ PBS 阳性对照:PBMC + TT / DT（25亩克/注射液） 实验抗原特异性反应:外周血单个核细胞+测试AG（4-8微克/注塑） 实验抗原特异性调节:PBMC +测试的Ag + TT / DT </li></ol> 4。测量前，注射，测量后<ol><li>异氟醚麻醉CB17 SCID小鼠。使用弹簧式卡尺测量后脚掌厚度。认厚度足垫的中心，一个边缘上行走的脚的焊垫接触，以提供一个基准，以保持测量部位一致。脚垫的厚度记录时，压力表读数稳定。 </li><li>慢慢细胞悬液注入皮下的小鼠使用半CC胰岛素注射器，28号针头的脚垫。针指向脚趾，斜面朝上，执行注入。 （注意:确保没有任何泄漏）。 </li><li>注射后18-24小时，麻醉鼠标垫肿胀与异氟醚和重复测量。 </li><li> Subtr行事每个足垫的厚度，在注射之前，从后喷射值取得足垫的膨胀值，在10 -4英寸为单位表示的数据。计算的抗原特异性的净肿胀:控制垫肿胀（外周血单个核细胞+的PBS）中减去从足垫肿胀的处理获得的值（外周血单个核细胞+供体同种抗原，TT / DT，或供体同种抗原+ TT / DT）。阳性对照响应召回抗原TT / DT≥25×10 -4英寸以上背景响应PBS所需的测试被认为是有效的。 </li><li>确定供者抗原的存在下进行比较，使用下列公式计算各喷射净肿胀抑制召回响应: <img alt="公式1" fo:src="/files/ftp_upload/4454/4454eq1highres.jpg" src="/files/ftp_upload/4454/4454eq1.jpg" /></li></ol>

<ol>
	<li>Carrodeguas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans+vivo+analysis+of+human+delayed-type+hypersensitivity+reactivity.">Trans vivo analysis of human delayed-type hypersensitivity reactivity.</a> Hum. Immunol. 60, 640-651 (1999).</li><li>VanBuskirk, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+allograft+acceptance+is+associated+with+immune+regulation.">Human allograft acceptance is associated with immune regulation.</a> J. Clin. Invest. 106, 145-155 (2000).</li><li>Burlingham, W. J., Jankowska-Gan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+and+injection+site+are+crucial+for+detecting+linked+suppression+in+transplant+recipients+by+trans-vivo+DTH+assay.">Mouse strain and injection site are crucial for detecting linked suppression in transplant recipients by trans-vivo DTH assay.</a> Am. J. Transplant. 7, 466-470 (2007).</li><li>Burlingham, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+tolerance+to+a+maternal+kidney+transplant+is+selective+for+HLA+class+II:+Evidence+from+trans-vivo+DTH+and+alloantibody+analysis.">Loss of tolerance to a maternal kidney transplant is selective for HLA class II: Evidence from trans-vivo DTH and alloantibody analysis.</a> Human Immunology. 61, 1395-1402 (2000).</li><li>Geissler, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+liver+allograft+acceptance+and+the+'tolerance+assay':+In+vitro+anti-donor+T+cell+assays+show+hyporeactivity+to+donor+cells+but,+unlike+DTH,+fail+to+detect+linked+suppression.">Human liver allograft acceptance and the 'tolerance assay': In vitro anti-donor T cell assays show hyporeactivity to donor cells but, unlike DTH, fail to detect linked suppression.</a> Transplantation. 72, 571-580 (2001).</li><li>Jankowska-Gan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+liver+allograft+acceptance+and+the+'tolerance+assay'.+II.+donor+HLA-A,+-B+but+not+DR+antigens+are+able+to+trigger+regulation+of+DTH.">Human liver allograft acceptance and the 'tolerance assay'. II. donor HLA-A, -B but not DR antigens are able to trigger regulation of DTH.</a> Hum. Immunol. 63, 862 (2002).</li><li>Cai, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minor+H+Antigen+HA-1-specific+Regulator+and+Effector+CD8++T+Cells,+and+HA-1+Microchimerism,+in+Allograft+Tolerance.">Minor H Antigen HA-1-specific Regulator and Effector CD8+ T Cells, and HA-1 Microchimerism, in Allograft Tolerance.</a> J. Exp. Med. 199, 1017-1023 (2004).</li><li>Rodriguez, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+and+graft+survival+in+kidney+transplant+recipients+are+both+enhanced+by+human+leukocyte+antigen+matching.">Immune regulation and graft survival in kidney transplant recipients are both enhanced by human leukocyte antigen matching.</a> Am. J. Transplant. 4, 537-543 (2004).</li><li>Xu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD4+CD25low+adaptive+T+regulatory+cells+suppress+delayed-type+hypersensitivity+during+transplant+tolerance.">Human CD4+CD25low adaptive T regulatory cells suppress delayed-type hypersensitivity during transplant tolerance.</a> J. Immunol. 178, 3983-3995 (2007).</li><li>Derks, R. A., Jankowska-Gan, E., Xu, Q., Burlingham, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+type+determines+the+mechanism+of+bystander+suppression+by+adaptive+T+regulatory+cells+specific+for+the+minor+antigen+HA-1.">Dendritic cell type determines the mechanism of bystander suppression by adaptive T regulatory cells specific for the minor antigen HA-1.</a> J. Immunol. 179, 3443-3451 (2007).</li><li>Jankowska-Gan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+reduction+of+immunosuppression+in+older+renal+transplant+recipients+who+exhibit+donor-specific+regulation.">Successful reduction of immunosuppression in older renal transplant recipients who exhibit donor-specific regulation.</a> Transplantation. 88, 533-541 (2009).</li><li>Jankowska-Gan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretransplant+immune+regulation+predicts+allograft+outcome:+bidirectional+regulation+correlates+with+excellent+renal+transplant+function+in+living-related+donor-recipient+pairs.">Pretransplant immune regulation predicts allograft outcome: bidirectional regulation correlates with excellent renal transplant function in living-related donor-recipient pairs.</a> Transplantation. 93, 283-290 (2012).</li><li>Knechtle, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+and+limited+use+of+tacrolimus+to+avoid+rejection+in+an+alemtuzumab+and+sirolimus+regimen+for+kidney+transplantation:+clinical+results+and+immune+monitoring.">Early and limited use of tacrolimus to avoid rejection in an alemtuzumab and sirolimus regimen for kidney transplantation: clinical results and immune monitoring.</a> Am. J. Transplant. 9, 1087-1098 (2009).</li><li>Haynes, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-specific+indirect+pathway+analysis+reveals+a+B-cell-independent+signature+which+reflects+outcomes+in+kidney+transplant+recipients.">Donor-specific indirect pathway analysis reveals a B-cell-independent signature which reflects outcomes in kidney transplant recipients.</a> Am. J. Transplant. 12, 640-648 (2012).</li><li>Burlingham, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17-dependent+cellular+immunity+to+collagen+type+V+predisposes+to+obliterative+bronchiolitis+in+human+lung+transplants.">IL-17-dependent cellular immunity to collagen type V predisposes to obliterative bronchiolitis in human lung transplants.</a> J. Clin. Invest. 117, 3498-3506 (2007).</li><li>Bobadilla, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TH-17,+Monokines,+Collagen+Type+V,+and+Primary+Graft+Dysfunction+in+Lung+Transplantation.">TH-17, Monokines, Collagen Type V, and Primary Graft Dysfunction in Lung Transplantation.</a> Am. J. Respir. Crit. Care Med. 177 (6), (2008).</li><li>Olson, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+prostate+tumor+antigen-specific+CD8++regulatory+T+cells+are+inhibited+by+CTLA-4+or+IL-35+blockade.">Human prostate tumor antigen-specific CD8+ regulatory T cells are inhibited by CTLA-4 or IL-35 blockade.</a> J. Immunol. 189, 5590-5601 (2012).</li></ol>

这份手稿的作者有没有利益冲突的披露。

跨体内的DTH检测是一种新型的诊断测试与评估细胞介导的​​反应，移植，癌症和自身免疫性病人中的一个潜在的临床应用。它是有价值的，因为它不仅是有用的监测召回T效应反应，而且还可以检测调节性T反应。一种可靠的方法来检测人类DTH调控可能预测免疫抑制戒断安全的患者中单药治疗或耐受性试验的候选人是谁。 DTH电视的间接通路的MHC allorecognition的检测是非常敏感?...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>ACD tube for blood collection</td>
			<td>BD</td>
			<td>02-684-26</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lymphocyte Separation Medium</td>
			<td>Cellgro</td>
			<td>25-072-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate-Buffered Saline</td>
			<td>Cellgro</td>
			<td>21-031-CM</td>
			<td>Without calcium &amp; magnesium</td>
		</tr>
		<tr>
			<td>ACK Lysis Buffer</td>
			<td>BioWhittaker</td>
			<td>10-548E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TT/DT or EBV</td>
			<td>Sanofi Pasteur Inc./ Meridian Life Science, Inc.</td>
			<td>&nbsp;</td>
			<td>TT/DT 25 &mu;g/injection EBV 8 &mu;g/injection</td>
		</tr>
		<tr>
			<td>Protease inhibitor PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>78830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Eosine for cell count</td>
			<td>Sigma-Aldrich</td>
			<td>E-6003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alloantigen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Purified HLA antigens, synthetic allopeptides can be used instead of donor cell-free lysates</td>
		</tr>
		<tr>
			<td>50 ml sterile centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>06-443-18</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml pipettes and pipettor</td>
			<td>BD Falcon</td>
			<td>13-675-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 ml safe-lock tubes</td>
			<td>Costar</td>
			<td>3213</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1000 &mu;l, 100 &mu;l , 10 &mu;l pipettes with sterile tips</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Full size centrifuge and microfuge</td>
			<td>Beckman Coulter/Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1/2cc or 1cc insulin syringes</td>
			<td>Becton Dickinson</td>
			<td>14-826-79</td>
			<td>28 gauge</td>
		</tr>
		<tr>
			<td>Vibracell sonicator</td>
			<td>Divtech Equipment Co. Sonocs&amp; Materials Inc</td>
			<td>&nbsp;</td>
			<td>2 mm probe</td>
		</tr>
		<tr>
			<td>Dial thickness gauge</td>
			<td>Mitutoyo, Japan</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SCID mice</td>
			<td>Harlan</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>&nbsp;</td>
			<td>Inhalant anesthesia</td>
		</tr>
	</tbody>
</table>

trans-vivo delayed type hypersensitivity assay for antigen specific regulation

体内 T细胞依赖的免疫应答，宿主的免疫系统在最近的过去经历了一个外来抗原（Ag）的表现形式是一种快速的迟发型超敏反应（DTH）。 DTH反应通常分为致敏阶段，指的是最初的抗原经验，挑战阶段，通常如下致敏后数天。往往被视为无能的证据缺乏的迟发型超敏反应皮肤测试证明召回银。传统的DTH试验已有效地用于诊断许多微生物感染。 尽管共享相似的免疫功能，如淋巴细胞浸润，水肿，组织坏死，直接DTH直喷导致致敏供者抗原与移植物丢失的可能性，因为不是一个可行的诊断技术在移植患者。为了避免这个问题，哼哼开发一个鼠标"体内反"DTH检测1,2。此测试本质上是一个传输的DTH试验，人外周血单核细胞（PBMCs）和特异性抗原皮下注射到羽片或一个天真的的鼠标和DTH像肿胀足垫3 18-24小时后测量。人类抗原提呈细胞如巨噬细胞或区议会高度血管小鼠组织中的T细胞的抗原提呈触发炎症级联反应，并吸引了小鼠的免疫细胞，导致肿胀反应。该反应是抗原特异性的，并需要事先致敏抗原。一个积极的捐助者反应在电视DTH检测反映的DTH反应，移植的病人已经发展向移植同种抗原的亲炎症免疫处置。 在此检测中最重要的特征是，它也可以被用于检测调节性T细胞，从而引起旁观者抑制。一个旁观者抑制供体抗原的存在是的DTH召回响应特征与接受同种异体移植受者2,4-14。同种异体反应性和监管的TV-DTH移植术后监测可能确定谁可以受益于减少免疫抑制的患者没有拒绝或肾功能恶化的风险升高的一个子集。 一个有前途的领域是电视DTH检测监测自身免疫15,16和17在肿瘤免疫学的应用。

1。评估肾移植供体抗原特异性反应使用电视DTH检测为了测试我们的捐助者反应在肾移植术后细胞免疫注射PBMC从这些患者与供者抗原单独或与召回抗原破伤风类毒素（TT）。仅与TT作为阳性对照细胞注射。我们观察到三个主要模式的迟发型超敏反应移植受者（ 图1）。所有患者反应强烈，召回AG（TT）。患者＃62所示的调节柄，其特征在于由一个弱响应于供体抗原和一?...

Watch this Scientific Journal Video about Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation at JoVE.com

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

我们描述了一个有价值的诊断方法，有可能被用来决定撤出未经移植排斥反应的风险升高，移植后的免疫抑制。该法采用迟发型超敏反应的原则，并提供准确的评估，既供者特异性效应和调节免疫应答安装收件人。

跨体内迟发型变态反应检测方法的抗原特异性调节

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

trans-vivo-delayed-type-hypersensitivity-assay-for-antigen-specific

Research

JoVE Journal

Immunology and Infection

16.0K 次观看.  University of Wisconsin-Madison, School of Medicine and Public Health. 我们描述了一个有价值的诊断方法，有可能被用来决定撤出未经移植排斥反应的风险升高，移植后的免疫抑制。该法采用迟发型超敏反应的原则，并提供准确的评估，既供者特异性效应和调节免疫应答安装收件人。

视频: Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

科研

免疫与感染

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

嗜碱粒细胞激活测试IgE介导的药物过敏机制的调查

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...