从人类母乳中分离白细胞，用于HIV靶点抗体依赖性细胞噬菌体测定
Summary
母乳传播人体免疫缺陷病毒（HIV），尽管只有15%的感染艾滋病毒的母亲母乳喂养的婴儿被感染。母乳喂养的婴儿每天摄入+105+108母性白细胞，尽管这些细胞研究不足。在这里，我们描述了乳腺癌白细胞的分离，并分析了它们的噬菌体能力。
Abstract
即使在没有抗逆转录病毒药物的情况下，只有15%的感染艾滋病毒的母亲母乳喂养的婴儿受到感染，这表明母乳（BM）具有很强的保护作用。除非获得清洁水和适当的婴儿配方奶粉是可靠的，否则世卫组织不建议感染艾滋病毒的母亲停止母乳喂养。许多因素可能协同作用，以减少 BM 传输。母乳喂养的婴儿每天摄入105~108个母性白细胞，但基本上仍不清楚这些细胞对BM抗病毒特性的贡献。 目前，我们的目标是从人类BM中分离细胞，以测量抗体依赖性细胞噬菌体（ADCP），BM噬菌体对HIV靶点最基本和普遍的先天免疫反应之一。从在哺乳不同阶段获得的5个人类BM样本中分离出细胞。分离是通过温和的离心进行，然后仔细去除乳脂和反复清洗细胞颗粒。涂有HIV包络（Env）表位的荧光珠子被用作ADCP分析的目标。细胞被染色与CD45表面标记，以识别白细胞。结果发现，ADCP活性明显高于对照实验，使用HIV特异性抗体830A可重复可测量。
Introduction
母乳 （BM） 由母细胞组成，这些细胞的存活率为 >90%1。细胞组成受到哺乳阶段、母婴健康状况和个人变异的强烈影响，对1、2、3、4仍然知之甚少。鉴于BM含有+103+105白细胞/mL，可以估计母乳喂养的婴儿每天摄入+105~108母性白细胞。各种体内研究表明，母性白细胞对婴儿提供关键免疫，其功能远远超过最初摄入5、6、7、8这些部位。 ，9，10，11.婴儿摄入的所有母性衍生细胞都有可能与婴儿一起发挥免疫功能或补偿婴儿自己的白细胞12。
在资源有限的国家，人体免疫缺陷病毒（艾滋病毒）的母婴传播仍然是一个危机。由于腹泻和呼吸道疾病是造成资源有限国家婴儿死亡率的显著原因，而且通过纯母乳喂养，这些疾病大大减少，这对感染艾滋病毒的母亲有利。母乳喂养远远超过风险13，14，15。除非获得清洁水和适当的婴儿配方奶粉是可靠的，否则世卫组织不建议感染艾滋病毒的母亲停止母乳喂养。每年通过 BM 进行约 100，000 次 MTCT;然而，只有15%的婴儿由其感染艾滋病毒的母亲母乳喂养感染，这表明BM17，18，19，20，21的强烈保护作用。许多因素可能协同作用，以防止传输。重要的是，BM中艾滋病毒特异性抗体（Abs）与MTCT的减少和/或减少婴儿死于艾滋病毒感染有关22，23。在很大程度上，仍不清楚的是BM细胞部分对其抗病毒特性的贡献。
许多Abs促进各种抗病毒活动，由免疫球蛋白分子的”恒定”区域，可结晶片段（Fc），通过与Fc受体（FcRs）的相互作用，发现几乎所有的先天免疫细胞，几乎所有发现在人类BM24。抗体依赖性细胞噬菌体（ADCP）已被证明是清除病毒感染所必需的，在预防艾滋病毒MTCT的情况下，已经研究不足。28,29.鉴于对BM噬菌体活性对预防艾滋病毒MTCT的潜在贡献缺乏了解，我们旨在开发一种严格的方法，将细胞从人类BM中分离，以便对由细胞介导的ADCP进行研究。BM在哺乳的不同阶段获得。
Protocol
本研究的每个参与者都是根据道德和机构审查委员会 （IRB） 的批准招募和面试的，并经过西奈山人类主体保护计划 （PPHS） 的指导和授权，使用 IRB 批准的获取母乳样本的协议。 1. 目标微球制备 选择相关靶抗原。注：在此示例中，使用了重组蛋白V1V2-2F5K，它旨在模仿原生HIV包络30的三分位。 根据制造商的协议，?…
Representative Results
牛奶可在室温或冷却器下保存（但不冷冻）;然而，鉴于我们观察到当牛奶保持非常寒冷时，其生存能力降低（数据未显示），并且在环境温度下进行短暂收集、短暂储存和运输更简单，因此建议不要冷藏样品，以减少样本到样本的可变性。在产后7-183天获得的牛奶中，由自动细胞计数器测定的细胞浓度为16，083~222，857细胞/mL。图 1说明了消除双精度?…
Discussion
本文描述的用于测量ADCP活性的流式细胞学技术于2011年首次被描述30，并自此被证明为稳健型，并在80多项研究中被引用。此处描述的协议首次将该技术用于初级 BM 单元。以前对BM细胞的Fc介导功能的研究主要限于使用从对口乳分离的细胞（出生后0⁄4天）测量氧化性爆裂或基于组织学的噬菌体测定。几乎没有研究对超过隆利阶段的人类BM细胞进行过检查。使用共体细胞的研究通常?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢西奈山伊坎医学院医学系和微生物学系的苏珊·佐拉-帕斯纳博士的手稿评论。NIH/NICHD为该项目提供了资助，赠款号为R21 HD095772-01A1。此外，R. Powell还得到西奈山伊坎医学院传染病司医学部的资金支持。
Materials
| 1 µm FluoSpheres NeutrAvidin-Labeled Microspheres | Thermo Fisher | F8776 | |
| BD Pharmingen PE Mouse Anti-Human CD45 | BD | 560975 | |
| Bovine serum Albumin | MP Biomedicals | 8810025 | |
| Corning V-bottom polystyrene 96-well plate | Corning | 3894 | |
| Cytochalasin D | Sigma | 22144-77-0 | |
| EZ-Link NHS-LC-LC-Biotin kit | Thermo Fisher | 21338 | |
| Falcon 15mL Conical Centrifuge Tubes | Corning | 352196 | |
| Falcon 50mL Conical Centrifuge Tubes | Corning | 352070 | |
| Fixable Viability Stain 450 | BD | 562247 | |
| Formaldehyde solution | Sigma | 252549 | |
| HBSS without Calcium, Magnesium or Phenol Red | Life Technologies | 14175-095 | |
| Human BD Fc Block | BD | 564219 | |
| Human Serum Albumin | MP Biomedicals | 2191349 | |
| Kimtech Science Kimwipes Delicate Task Wipers | Kimberly-Clark Professional | 34120 | |
| PBS 1x pH 7.4 | Thermo Fisher | 10010023 | |
| Polystyrene 10mL Serological Pipettes | Corning | 4488 | |
| Protein Concentrators PES, 3K MWCO, 0.5 mL | Pierce | 88512 |
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Cite This Article
Powell, R. L., Fox, A. Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets. J. Vis. Exp. (151), e60149, doi:10.3791/60149 (2019).