从HIV-1阳性者的血浆分离外来体的
Summary
Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.
Abstract
外来体是小囊泡从从它们的多种细胞类型的释放都组成型和刺激后30纳米至100纳米不等的大小。他们发现，在一些生物体液的和已知携带多种蛋白质，脂质和核酸分子。原本以为要多一点水库细胞碎片，外来体调节生物过程和疾病中的作用也越来越赞赏。
几种方法已经被描述用于分离从细胞培养基和生物流体的外来体。由于它们的小尺寸和低的密度，差分超速离心和/或超滤是最常用的技术为外来体隔离。然而，HIV-1感染的个体的血浆中含有两个外来体和HIV病毒颗粒，其在尺寸和密度相似。因此，从人血浆中的HIV病毒颗粒的外来体的有效的分离已一个挑战。
为了解决此限制，我们开发了从Cantin 等改性的过程。 人，2008为来自HIV颗粒在人血浆中的外来体的纯化。碘克沙醇的速度梯度被用于外来体来自HIV-1颗粒的HIV-1阳性个体的血浆中分离。病毒颗粒确定P24 ELISA。外来体被确定的外来体标记物的乙酰胆碱酯酶的基础（乙酰胆碱酯酶）和CD9，CD63和CD45抗原上。我们的渐变过程产生游离病毒颗粒的外来体的准备。从人血浆外来体的高效净化使我们能够检查血浆衍生的外来体的含量，并调查它们的免疫调节潜力和其他生物学功能。
Introduction
在HIV-1流行继续在世界各地都有一个显著的影响。截至2013年，全球约有3500万人感染艾滋病毒，而这些210万新感染的个体1。预防战略和增加获得抗逆转录病毒疗法已在减少整体收购艾滋病毒有帮助。然而，个别人群仍面临上升收购HIV 1。因此，有必要继续努力来解决这一流行病。
其中一个HIV疾病进展的最强预测的是慢性免疫激活（CIA）2-10。持续高检测水平的细胞因子和T淋巴细胞的表面上表达升高的标记限定，CIA已被归因于：i）连续的树突状细胞产生I型IFN 11的类型; （二）直接免疫激活HIV蛋白达，NEF和gp120的12驱动; （三）易位相关的肠道免疫细胞6细菌蛋白质。然而，确切的机制（S）潜在慢性，在HIV感染的全身免疫激活仍有待完全阐明。
我们的研究小组和其他人证明外来体的艾滋病发病15-18的作用。我们小组已确定的Nef蛋白从感染的细胞中分泌的外来体15，和外来体的Nef（exNef）存在的HIV感染的个体中的纳克水平18在血浆中。我们已经表明，旁观者的CD4 + T细胞暴露于exNef导致活化诱导的细胞死亡取决于CXCR4途径19，20，另外，单核细胞/巨噬细胞难治exNef诱导的细胞凋亡，但表现出改变的细胞功能和细胞因子的表达。最近，我们集团已经显示出外来体从HIV感染者的血浆中分离出含有多种促炎细胞因子。福rther，诱导CD38表达对天真和中央记忆CD4 +和CD8 + T细胞的HIV感染患者暴露于血浆来源的外来天真的外周血单核细胞。这可能有助于全身性炎症，并通过旁观者细胞活化21病毒的繁殖，并表明外来体发挥在HIV发病机理中显著作用。
在调查外来体在HIV发病机理中的作用，一个挑战是发展技术以来自HIV颗粒，同时保持外体含量有效地分离外泌体，以及它们的功能免疫调节能力。几种方法已经被描述用于从细胞培养物和生物流体22,23分离外泌体。因为它们的小尺寸和低的密度（外来体漂浮在1.15的密度- 1.19克/毫升），差分超速离心和/或超滤是最常用的技术为外来体隔离23。但是，艾滋病毒感染细胞培养上清和病人的血浆同时包含外来体和HIV-1病毒颗粒。外来体和HIV-1的颗粒在尺寸和密度非常相似。另外，以独特的外体标记，如CD63，CD45，CD81和表达的优势，外来体一直在使用免疫亲和捕获方法23隔离。这个过程可以从外来体分离病毒。然而，这种技术的缺点是紧密附接抗体与纯化的外来体，这可能干扰在培养外来体的免疫调节的潜能的评估。
为了解决这些局限性，我们开发了用于使用碘克沙醇的速度梯度来自HIV颗粒在人血浆中由Cantin改性和同事22外来体的纯化过程。外来体被发现在低浓度偏析碘克沙醇梯度/上层级分，而病毒颗粒分离在高密度/较低的分数。病毒颗粒通过P24 ELISA检测和使用外来体标记的乙酰胆碱酯酶，CD9，CD63和CD45被确定外来体。收集上层低密度部分包含这是不含有HIV-1 P24污染外来体。艾滋病毒颗粒在人体血浆中外来体的高效纯化和分离方法可以从人血浆以及他们的免疫调节潜力的调查和外来体在HIV-1发病机制中的诊断和预后价值来源的外来内容的准确检查。
Protocol
Representative Results
Discussion
慢性免疫激活（CIA）和CD4 + T细胞耗竭是HIV-1感染的两个重要特征。他们已经被确立为预测的发病机制，与美国中央情报局是最好的预测。然而，驾驶慢性全身免疫激活和CD4 + T细胞衰退的作用机制还没有完全阐明。我们和其他的实验室已经开发出坚定的证据表明，外来体从HIV-1感染细胞分泌起到两个标志的作用。
在两种组合物和细胞外囊泡的功能继续关心导致了各种方法外来?…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank the following people: Jane Chu, Cameron Tran, James Lillard, Mafuz Khan, Masebonang Albert, Ken Rogers, and Syed A. Ali. Kateena Addae-Konadu was supported by UNCF/Merck Graduate Research Fellowship, American Medical Association Foundation, CRECD Grant 8R25MD007589-10, and NIH NIGMS MBRS Grant R25 GM058268. This work was supported by NIMHD grants 8G12MD007602, and 8U54MD007588, NIAID grant 1R21AI095150-01A1, Georgia Research Alliance grant GRA.VAC08.W, and Emory CFAR grant P30 A1050409.
Materials
| BD Vacutainer EDTA tubes (10ml) | Becton Dickinson | 368589 | pink top tubes |
| Lymphoprep Ficoll reagent | Cosmo Bio | AXS-1114545 | |
| Optiprep iodixanol reagent | Sigma | D1556 | |
| 14ml ultracentrifuge tubes | Beckman Coulter | 344060 | ultraclear tubes |
| Gradient Former Model 485 | BIO-RAD | 165-4120 | |
| Acetylthiocholine iodide | Sigma | 1480 | |
| Benzoic Acid | Sigma | D8130 | |
| Sodium Bicarbonate | Sigma | S5761 | |
| Acetyl Cholinesterase | Sigma | C3389 | |
| 96-well clear microtiter plate | Medical Supply Partners | TR5003 | |
| SpectraMax 190 microplate reader | Molecular Devices | 190 | Fluorescent plate reader |
| Criterion Gel Electrophoresis Cell | BIO-RAD | 165-6001 | |
| Transblot Gel | BIO-RAD | 170-3910 | |
| Transfer Cell | |||
| Tris-HCl Criterion precast gels | BIO-RAD | 567-1093 | |
| Anti-CD45 antibody | Abcam | Ab10558 | |
| CD63 Antibody (H-193) | Santa Cruz Biotech, Inc. | SC-15363 | |
| CD9 Antibody (H-110) | Santa Cruz Biotech, Inc. | SC-9148 | |
| Rabbit pAb p24 HIV-1 | ImmunoDX, LLC | 1303 | |
| Nitrocellulose membrane | BIO-RAD | G1472430 | |
| Tris Buffered Saline | BIO-RAD | 170-6435 | |
| HRP-conjugated IgG (H+L) secondary antibody | Thermo Scientific | 31460 | Goat-Anti-Rabbit |
| HRP-conjugated IgG (H+L) secondary antibody | Thermo Scientific | 31430 | Goat-Anti-Mouse |
| Western Blotting Luminol Reagent | Santa Cruz Biotech, Inc. | SC-2048 | |
| GE LAS-4010 Imager | GE Healthcare | LAS-4010 | |
| Human Procarta Cytokine Immunoassay Kit | Affymetrix | N/A | Custom immunoassay panel |
| Bio-Plex 200 Immunobead Reader | BIO-RAD | 171-000201 | |
| Coulter Z2 Particle Counter | Beckman Coulter | 383552 | Cell counter |
| Alexa Fluor 700-labeled anti-CD3 | BD Bioscience (UCHT1) | 300424 | |
| APC/Cy7-labeled anti-CD4 | Biolegend (OKT4) | 317418 | |
| PerCP-labeled anti-CD4 | BD Bioscience (RPA-T8) | 550631 | |
| V450-labeled anti-CD8 | BD Bioscience (RPA-T8) | 560347 | |
| Biotin-labeled anti-CD45RA | BD Bioscience (HI100) | 555487 | |
| PE/Cy7-labeled anti-CD62L | Biolegend (DREG-56) | 304822 | |
| PE/Cy5-labeled anti-CD38 | Biolegend (HIT2) | 303508 | |
| APC/Cy7-labeled anti-HLADR | Biolegend (L243) | 307618 | |
| PE-Texas Red-labeled anti-streptavidin | BD Bioscience | 551487 | |
| PE/Cy5-labeled mouse IgG1K | Biolegend (MOPC-21) | 400116 | |
| APC/Cy7-labeled mouse IgG2aK | Biolegend (MOPC-173) | 400229 |
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