作者要感谢研究参与者和他们的父母。我们还要感谢Réjean拉波因特博士（覃巴黎圣母院，蒙特利尔，加拿大）访问他的酶联免疫斑点读者，鲁波亚历山德罗夫进行统计分析博士和丹尼斯布莱，桑德拉·卡隆，Silvie瓦卢瓦和马丁卡蒂的专家技术援助。支持由乐全宗D&#39;操作倒莱PROJETS去RECHERCHE倩碧等科特迪瓦评价; DES技术（朱圣 - 和Justine）到HS和PO，由La基金会中心去cancérologie查尔斯 - 布鲁诺，并通过对白血病和淋巴瘤协会补助加拿大。 ISF是由来自LA基金会朱圣 - 和Justine和乐全宗德拉RECHERCHE魁北克 - 桑特（FRQS）奖学金支持。 AJG奖学金是从微生物学，传染病学与免疫学，蒙特利尔大学（加布里埃尔 - 侯爵奖学金），FRQS部的收件人，以及加拿大卫生研究院ŘESEARCH（CIHR）。 NM是由拉朱基金会圣 - 和Justine，科尔基金会和FRQS支持。

本研究方案经朱圣 - 和Justine，蒙特利尔，魁北克，加拿大，那里的研究进行的制度伦理审查委员会。知情同意书，并寻求从所有研究参与者，他们的父母或法定监护人获得。在第一天进行的所有程序和2必须是无菌的条件下（ 即在层流罩）下进行。标准的安全程序来处理人体血液必须严格遵守。 1，涂料的板<ol><li>通过加入到每个孔中20微升35％乙醇中1分钟，透化聚偏氟乙烯（PVDF）膜上，在96孔MultiScreen IP白色的ELISpot板的底部。膜应该成为稍透亮。 </li><li>使用MUL;立即用200μl1X磷酸盐缓冲盐水（137 mM氯化钠，2.7 mM的氯化钾，10毫米的Na 2 HPO 4，2mM的KH 2 PO 4，pH 7.4的PBS）洗涤各孔3次声道音吸管或类似的装置。由于乙醇的残基可以影响细胞的活力和捕获抗体结合这一步是至关重要的。酶联免疫斑点法板是脆弱的，必须小心处理:使用自动洗板机，不推荐。 </li><li>涂层的孔与10微升纯化的小鼠抗人IFN-γ捕获抗体稀释在1×PBS中的10微克/毫升的最终浓度。覆盖板与普通保鲜膜孵育过夜，4°C。 </li></ol> 2，挡板<ol><li>空井，窃听他们干，并用200微升的1×PBS洗板5次。 </li><li>填充孔用200μl的完全RPMI 1640培养基中孵育板2小时，在37℃（分块步骤）。 </li><li>洗涤板5倍用200μl的1×PBS中并保持在孔中充满的1×PBS中，直到细胞准备进行电镀。 </li></ol> 3，电池和电镀刺激<ol><li>准备peripheral血单核细胞（PBMC）从人的静脉血样离心上使用标准方法的Ficoll-Paque上的梯度。备选地，使用标准方法，解冻PBMC的等分试样在液氮下冷冻。在水套培养箱在5％CO 2气氛下后，最后离心后，重新悬浮细胞以2×10 6个细胞/ ml的终浓度，孵育过夜，在37℃。 </li><li>从培养箱中取出PBMC重悬他们于5ml RPMI 1640完全培养基中的终体积。 </li><li>在10微升从粘质沙雷氏菌 5分钟，在室温下遗传工程内切核酸酶的存在下培养PBMC。 </li><li>除去PBMC的一个50μl的等分试样，并用50μl的台盼蓝染料混合。计数细胞，并用血球和显微镜检查（染色排除法）估算％的生存能力。各种自动化方法进行细胞计数和细胞活力罐人评价所以可以使用。 PBMC应&gt; 95％存活。丢弃当样品生存能力低于95％。 </li><li>离心机的PBMC，在700×g离心5分钟，弃去上清，重悬细胞以2×10 6个细胞/ ml的终浓度添加2％（V / V）灭活的人血清（HIS）的AIM-V培养基。保持在37°C，直到刺激混合物加入到平板。 </li><li>加至孔中，1滴一次，加入100μl适当的刺激混合物。三刺激的混合物，应使用如下所述: <ol><li>用AIM-V培养基补充有2％（V / V）灭活的人血清（IHS）作为阴性对照。 </li><li>使用抗CD3单克隆抗体（克隆OKT3）稀释在AIM-V培养基补充有2％（V / V）的IHS至0.5微克/毫升作为阳性对照）的终浓度。 </li><li>使用VZV抗原，无论是γ-辐射的形式（10,000戈瑞; 30分钟的曝光时间）减毒活疫苗VZV（1,350斑块形成单位[PFU] /毫升）的稀释泰德1:200在AIM-V培养基补充有2％（V / V）IHS）或1微克67肽（15个氨基酸残基）的基础上，VZV的顺序立即早期（IE63）磷蛋白合成的等摩尔混合物的。通过监测缺乏的细胞病变效应明显迹象后PBMC培养4天控制照射疗效。 </li><li>准备一式两份的所有井。 </li></ol></li><li>加至各孔，一减少的时间，将100μl制备步骤5.4至适当的孔中的细胞。两个井应保持无细胞（阴性对照）。在水套培养箱在5％CO 2气氛下孵育20小时，在37℃。不要摇晃，移动或堆叠在另一个孵化过程中顶部的板块。 </li></ol> 4，景点开发与检测<ol><li>弃去细胞用1×PBS洗涤板10倍的补充有0.05％（V / V）吐温20（PBST）。吐温20有助于分离已经坚持了我的PVDF细胞mbrane以下孵育过夜。 </li><li>使用多通道移液管加入100微升的0.5毫克/毫升生物素标记的抗IFN-γ单克隆抗体（克隆4S.B3）稀释在1×PBS中含有0.5％（重量/体积）牛血清白蛋白（BSA）。孵育板2小时在室温下。 </li><li>洗涤各孔6倍用PBST和添加每孔加入100μl链霉亲和有机共轭的碱性磷酸酶稀释1:1,000在1x PBS中含有0.5％（重量/体积）BSA中。覆盖板并孵育1小时，在室温下进行。 </li><li>洗涤板3倍用PBST和3X用1×PBS。添加100μl的5 - 溴-4 - 氯-3 - 吲哚磷酸（BCIP）/硝基蓝四唑氯化物（NBT）底物溶液孵育5分钟，在室温下进行。 </li><li>下运行蒸馏水冲洗板。取出的ELISpot板的底部部分，并用蒸馏水洗膜的两侧。空气干燥该板。 </li><li>检查各孔，并用立体ð列举的斑点issection显微镜或使用自动点计数器扫描板。保持板避光，如果他们不能检查在同一天。 <ol><li>平均斑点在相应的复孔计数的数量和从斑点中的测试孔计数的数目减去斑点在阴性对照孔计数的数目（无细胞）。 </li><li>表达IFN-γ的生产为斑点形成单位的数量（SFU）每10 6个PBMC。 </li><li>认为试验样品是阳性如果SFU的数量为&gt; 50每10 6个PBMC和2个标准差（SD）以上的负对照（细胞+ AIM-V培养基中补充了2％（V / V）IHS）。 </li><li>使用每孔400 SFU的值时，斑点更是不胜枚举进行计数。 </li></ol></li><li>测试是否酶联免疫斑点法数据是正态分布的，或不使用Kolmogorov-Smirnov检验。当数据是正态分布，采用学生t TES进行统计比较T（2组）或单因素方差分析和Bonferroni法检测后（多重比较）。如果数据不是正态分布，用曼-惠特尼U检验（2组）或Kruskal-Wallis检验和邓恩的后测（多重比较）。 </li></ol>

作者宣称，他们有没有竞争的财务权益。

修改和故障排除:IFN-γ酶联免疫斑点测定法已被用于检测细胞介导的针对多种微生物病原体，包括人类免疫缺陷病毒1型（HIV-1）的24,25，丙型肝炎病毒（HCV）26免疫反应， 27，和结核杆菌 28,29，只是仅举几例。在这里，我们描述的IFN-γ酶联免疫斑点法的发展来衡量细胞对免疫，用在定义儿科复苏脐血移植受者的VZV特异性免疫重建相互关系的希望。 <p class="jov...

在1989年第一次演出，脐血移植是越来越多地用作各种肿瘤和非肿瘤性血液疾病的儿童1治疗的一部分。水痘是一种细胞病变人力alphaherpesvirus这会导致两种不同的疾病，水痘（原发感染后），带状疱疹（激活后）。继原发感染，水痘持续整个背根神经节的感觉神经内避风的主机的使用寿命。其中最有威胁的感染性并发症以下UCBT是与水痘2-4关联。在我们的临床中心，在没有水痘预防，水痘水痘病的发病在3年累积发病率postUCBT为46％2。在这些患者中， 从头感染或VZV的激活通常与内脏传播到中枢神经系统，肺和肝5-7相关联。因此，阿昔洛韦，伐昔洛韦或泛昔洛韦预防通常给药至UBCT收件人8,9。然而，这种治疗策略不考虑VZV特异性T淋巴细胞或VZV特异性T细胞反应重建动力学的保护电位。随着国家扩大利用长期抗疱疹预防相关的潜在问题包括:a）病人过度治疗;二）抗病毒抗药性10,11的发展;和c）的VZV特异性免疫重建12,13减值。因为检测的功能VZV特异性T淋巴细胞相关性。和VZV感染长期保护的存在，改善临床结果4,14,15，监测细胞介导的针对水痘的免疫反应在移植后的时期，可能会导致更合理地使用抗病毒药物通过治疗使医生来区分谁的病人将受益于水痘预防从那些免疫系统能够控制VZV复制4,13的。 的IFN-γ酶联免疫斑点测定法被广泛地用于在多种试验系统及临床情况的监测细胞介导的​​免疫反应。斑产生下列显色底物的裂解，产生一个可见的和稳定的沉淀物在反应的位点。每个单独的点，从而代表一个单独的细胞因子产生细胞的足迹。 IFN-γ酶联免疫斑点法不仅测量单个细胞在体外的产生IFN-γ响应于体外刺激同源抗原的能力，但它也提供了在一个给定的细胞群体16,17响应单元的频率的估计。除了其高灵敏度，IFN-γ酶联免疫斑点法是直接执行，使得它的使用可能在旨在指导开始抗病毒治疗或终止个性化临床治疗方案的上下文。该过程详述如下describES是专门设计由外周血单核细胞下列体外刺激用VZV的抗原检测和测量产生IFN-γ的ELISPOT测定。

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leucocep tube</td>
			<td style="width:101px;">VWR</td>
			<td style="width:143px;">89048-936/89048-932</td>
			<td style="width:397px;">12 ml or 50 ml tubes may be used depending on the volume of blood.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ficoll-Paque</td>
			<td style="width:101px;">GE Healthcare</td>
			<td style="width:143px;">17-1440-02</td>
			<td>Protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzonase nuclease</td>
			<td style="width:101px;">Novagen</td>
			<td style="width:143px;">70746-3</td>
			<td>Keep at -20 C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MultiScreenHTS-IP Filter Plate</td>
			<td style="width:101px;">Millipore</td>
			<td style="width:143px;">MSIPS4W10</td>
			<td>Sterile with pore size of 0.45 &#181;m.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse anti-human IFN-&#947; capture antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">551221</td>
			<td>NIB42 clone.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pepmix VZV IE63&#160;</td>
			<td style="width:101px;">JPT Peptide Technologies</td>
			<td style="width:143px;">PM-VZV-IE63</td>
			<td>Dissolve contents of one vial in 40 &#956;L of DMSO. Use within 6 months.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotin-conjugated anti-IFN-&#947; monoclonal antibody</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:143px;">554550</td>
			<td>4SB3 clone.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Streptavidin conjugated with alkaline phosphatase&#160;</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-3554</td>
			<td>Dilute for use on the same day.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BCIP/NBT</td>
			<td style="width:101px;">Bio-Rad Life Science</td>
			<td style="width:143px;">170-6432</td>
			<td>Protect from light.</td>
		</tr>
	</tbody>
</table>

development of an ifn-&#947; elispot assay to assess varicella-zoster virus-specific cell-mediated immunity following umbilical cord blood transplantation

水痘 - 带状疱疹病毒（VZV）是发病率和死亡率以下脐血移植（UCBT）的显著原因。出于这个原因，抗疱疹预防系统地施用给小儿脐血移植​​接受者，以防止水痘病毒感染相关的并发症，但没有强大的，基于证据的共识，它定义了最佳治疗时间。因为T细胞介导的​​免疫是负责水痘感染的控制，评估的VZV特异性T细胞反应的重建下列UCBT可以表示出对预防是否应保持或可停药。为此，一个特定的VZVγ干扰素（IFN-γ）酶联免疫斑点（ELISPOT）测定法的开发是由T淋巴细胞响应于体外刺激与照射减毒活水痘疫苗来表征的IFN-γ的产生。此法提供了水痘特定的C的快速，重复性好，灵敏的测量埃尔介导的免疫适于监测VZV特异性免疫的重构在临床环境和评估免疫反应对VZV抗原。

上面详述的IFN-γ酶联免疫斑点协议被开发，并在我们的实验室优化测量的幅度和针对VZV 4细胞介导的免疫应答的质量。可用于刺激步骤的VZV抗原的不同来源。这些措施包括:a）由水痘市售清洁剂灭活提取物感染的Vero细胞18;从具体的VZV编码的蛋白质，包括IE63 15和19的ORF4重叠合成肽二）池;三）减毒活水痘带状疱疹疫苗20;及d）VZV紫外线灭活抗原准备从破碎的VZV?...

Watch this Scientific Journal Video about Development of an IFN-Î³ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation at Jo

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

新世代的功能测定法如γ干扰素（IFN-γ）的酶联免疫斑点法，其检测细胞因子的产生在单细胞水平，并提供T细胞应答的定量和定性特征，可用于评估细胞介导的​​针对水痘 - 带状疱疹免疫应答病毒（VZV）。

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

development-an-ifn-elispot-assay-to-assess-varicella-zoster-virus

Research

JoVE Journal

Immunology and Infection

15.6K 次观看.  Université de Montréal. 新世代的功能测定法如γ干扰素（IFN-γ）的酶联免疫斑点法，其检测细胞因子的产生在单细胞水平，并提供T细胞应答的定量和定性特征，可用于评估细胞介导的​​针对水痘 - 带状疱疹免疫应答病毒（VZV）。 

视频: Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

酶联免疫斑点法（ELISPOT）:TH - 1细胞对微生物抗原的免疫应答的定量

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

科研

免疫与感染

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

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.

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 ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...