作者感谢的实验帮助发展中国家从虹膜埃德曼，芭芭拉·冯·博伊斯特，朱莉娅玛丽亚·马丁内斯·戈麦斯小鼠淋巴免疫的方法。还要感谢玛吉阿拉斯和尼古拉Cesarovic您让我们使用他们的手术剧场这个视频制作。

1。小鼠麻醉<ol><li>在缓冲液混合氯胺酮（分离性麻醉剂）和甲苯噻嗪（镇静，镇痛）准备麻醉剂。氯胺酮和甲苯噻嗪最终溶液中的浓度是12.5和2毫克/毫升。 </li><li>使用注射器用25-30号针头通过腹膜内给药注射麻醉​​剂将小鼠。使用0.1 ml/10克的机身重量。 </li><li>为了防止干燥出的角膜应用眼用软膏对小鼠的眼睛。 </li><li>确保鼠标充分捏它的脚或脚趾与镊子麻醉。如果鼠标不与反射的捏反应，继续手术。如果鼠标反应捏有反射或肌肉收缩，等待1-2分钟，然后重复捏测试。如果鼠标仍然反应，请更换鼠标采用了新的动物，并从步骤1.2开始重复步骤。 </li></ol> 2。我的手术鼠标的腹股沟区ncision <ol><li>把鼠标在它的后面。 </li><li>湿1腹股沟区域用消毒乙醇（70％）。为了提供更好的消毒，腹股沟区可被剃光，除去皮毛消毒前用乙醇。 </li><li>取后腿和弯曲髋关节，产生髋关节的一个大约90°的角度。 </li><li>使用弯曲microdissecting钳，取小鼠皮肤在腹股沟区，其中髋关节弯曲和拉湿皮肤略有上升。 </li><li>同时保持肌肤与镊子，通过使用手术剪皮肤切开一个小切口（&lt;5毫米）。 </li><li>将闭合的剪刀的尖端插入切口，并进一步通过打开的剪刀，同时保持它的切口内打开切口。这会导致皮肤撕裂，并应当产生一个直径小于10毫米。注：具有灵活的光导冷光源，可以用来提高知名度。在较老的小鼠，特别是男性，淋巴结的识别可能是困难的，由于在腹股沟区过多的脂肪组织。 </li></ol> 3。淋巴管内注射<ol><li>准备一个注射器（0.5毫升或更少）用28-30ģ皮下注射针，短斜面可能是优选的一长斜面。吸出10μl的疫苗被注射并保证该注射器是自由的空气。 </li><li>本地化与弯钳的帮助和闭合剪刀的尖端处的腹股沟淋巴结。淋巴结会出现灰内越发白的脂肪组织，并且可以通过传入和传出的毛细血管进入和离开淋巴结进行进一步鉴定。 </li><li>受弯钳的分支之间保持它固定的淋巴结。 </li><li>就拿注射器和针头插入朝上的斜面淋巴结;确保整个斜面进入淋巴结。 </li><li>注射了疫苗（10微升）。如果淋巴结点头e的肿胀（吹），注射可以假设成功。如果针头未放置足够深到淋巴结，部分或整个疫苗可能会泄漏出来，而不是进入淋巴结。如果针头插入过深，注射的疫苗将被释放皮下下面的淋巴结。在后一种情况下，重复注射或用新的未处理的小鼠代替鼠标。 </li></ol> 4。通过缝合关闭切口<ol><li>打开无菌手术缝合线的包。 </li><li>抓住的缝合针与针座的针体的远端部分。通过挤压它，直到第一棘轮渔获拧紧持针器。 </li><li>使用镊子抓住皮肤的切口的一侧（取决于外科医生的偏好，齿形或不带齿的钳或皮肤钩可以使用）。插入针约 2-3毫米的切口的边缘上，从皮肤的外部进入。 </li><li>洛斯烯保持与钳子的皮肤和把握切口用钳子的相对侧。插入针约 2-3毫米的切口的边缘上，从皮肤的内侧进入。 </li><li>松开持针器，把握针端与钳。拉带通过皮肤胎面针，而使胎面的方便长度的第一针插入点之外（如果几个小鼠是被处理，留下较短端5-10小鼠可被操作上与一个缝合针/踩）。 </li><li>根据标准协议操刀制作的首选结。一个线圈就足够了，如果切口不大于10mm。对于较大的切口，进行第二次缝合。 </li></ol> 5。运行后的治疗<ol><li>将鼠标放置在笼子里，用纸巾覆盖保暖。当处理多个老鼠，把它们接近对方，以保持温暖。或者，将鼠标在一个温暖的垫子，直到他们才如梦初醒起来。 </li><li>观察小鼠直到他们醒来。 </li><li>每天观察小鼠的问候伤口愈合以及其他临床症状，如伤口感染。伤口通常足以在两天内关闭，并在该过程七天痊愈。 </li></ol>

<ol>
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TMK被命名为专利涉及在人淋巴免疫疗法的发明者。 TMK一直是科学顾问，并已收到差旅费由ImVisioN有限公司，Cytos生物技术，Mannkind公司，并XBiotech美国公司PJ没有任何利益冲突披露。

淋巴内免疫接种和免疫治疗已被证明是合适的两个抗体应答和T细胞应答的刺激。这表现这个视频文章中，接种疫苗的淋巴管内的程序是一个快速简便的方法刺激强烈的免疫反应的小鼠。一个训练有素的外科医生可以在3-4分钟执行程序。这次会议也可两名外科医生，其中一个通常做麻醉和缝合，第二个外科医生在做切口和注射之间共享。该过程的效率，更取决于运营商以外的免疫方法，尤其是当外...

疫苗通常是皮下或肌内注射对免疫应答的刺激。这个程序的成功需要疫苗的有效引流淋巴结其中抗原呈递细胞可以与淋巴细胞相互作用用于生成的T-和B-细胞应答。此外，强度和诱导免疫反应的类型也取决于这种相互作用的密度或频率以及微环境本身，细胞因子，特别是内容。作为注入到外周组织的疫苗只有一小部分到达淋巴结，小鼠和人类通过直接注射该疫苗进入淋巴结的疫苗，该网站被产生的免疫反应，已被执行。在男人中，程序是通过超声波，一个程序也可以用于可视化和诊断在淋巴系统成像剂的给药指导。在小鼠体内，是侵入性检查。在这里，一个小的（5-10毫米）incision被制成在麻醉的动物1的腹股沟区域，淋巴结进行定位和固定钳，和10-20微升的疫苗的一定体积的视觉控制下注入; 10微升用于第一注射和在年轻小鼠用小淋巴结，而20微升可以注入更旧的淋巴结或已催芽的小鼠，其具有较大的淋巴结。切口可闭合使用外科缝合线的单个针迹。通过该方法，将小鼠已接种的质粒DNA 2,3，信使RNA 4，1,3,5,6肽，蛋白质7-10，颗粒11，细菌12以及辅助剂7,13，和免疫大幅改善对所有类型的疫苗反应已​​被观察到。接种淋巴方法特别适合在常规疫苗接种产生的免疫力不足或可用疫苗的量是有限的或版本的情况下Ÿ昂贵。在人类，免疫的淋巴管内的方法已被应用到过敏患者14,15或癌症患者16-21。虽然目前的看法是，在淋巴管内的方法比其它可注射的方法，如肌内和皮下注射更具侵入性的，疼痛感觉不大于静脉穿刺15后更高。预期淋巴内注射疫苗将成为一种替代或补充的预防和治疗尤其是疫苗的其它方法。本文详细阐述了如何淋巴管内接种疫苗的过程中小鼠进行。描述的所有程序，批准了苏黎世和州兽医局，并根据瑞士联邦指导方针以及用于科学目的的动物的保护指令执行。

<table border="0" cellpadding="0" cellspacing="0" width="653">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Ketamine (Ketasol-100)</td>
			<td style="width:135px;">Graeub AG, Switzerland</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Anesthetics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Xylazine (Rompun)</td>
			<td style="width:135px;">Bayer, Germany</td>
			<td style="width:119px;"></td>
			<td>Anesthetics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Viscotears Eye-Gel</td>
			<td style="width:135px;">Novartis, Switzerland</td>
			<td style="width:119px;"></td>
			<td>To keep eyes from drying out during anesthesia.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">BD Micro-Fine 0.5 ml</td>
			<td style="width:135px;">BD Medical, France</td>
			<td style="width:119px;"></td>
			<td>29 G Insulin syringes with permanently attached needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">6-0 Dermalon Monofilament nylon</td>
			<td style="width:135px;">Covidien, MA, USA</td>
			<td style="width:119px;"></td>
			<td>For sutures (0.7 metric, 18G, 45 cm, Blue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Curved forceps, 4.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>For incision and holding of lymph node</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Straight surgical scissors, 4.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>For incision</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Needle holder, 5.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>To close incision with suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

intralymphatic immunotherapy and vaccination in mice

疫苗一般是皮下或肌内注射对免疫应答的刺激。这样做的成功需要疫苗的有效引流淋巴结，其中抗原呈递细胞可以与淋巴细胞相互作用的一代想要的免疫反应。的强度和诱导免疫反应的类型也取决于相互作用的密度或频率以及微环境，细胞因子，特别是内容。作为外周注射疫苗仅一分钟级分到达淋巴结，小鼠和人类的疫苗通过直接注射疫苗被执行到腹股沟淋巴结， 即淋巴内注射。在男人中，程序是由超声引导。在小鼠中，小（5-10毫米）切口，在麻醉的动物的腹股沟区域，淋巴结进行定位和固定钳，和10-20微升的疫苗的一定体积的视觉控制下喷射。切口闭合使用手术缝合线单针。小鼠接种的质粒DNA，RNA，肽，蛋白质，颗粒，细菌以及佐剂，并观察到对所有类型的疫苗的免疫应答的强改善。接种淋巴方法特别适合在常规疫苗接种产生的免疫力不足或可用的疫苗量有限的情况下。

在小鼠淋巴内注射的方法，尽管在手术的性质，是直线前进和比较快的。一个训练有素的人能在3-4分钟执行程序。是与一针封闭切口通常在两天内愈合（ 图1） 淋巴内注射疫苗或免疫接种用的mRNA被执行时，质粒DNA，肽，蛋白质，病毒和细菌。 图2示出了免疫与蛋白质磷脂酶A2（PLA2），主要蜜蜂毒液变应原后的抗体产生。 CBA小鼠进行免疫在0天，14，和2...

Watch this Scientific Journal Video about Intralymphatic Immunotherapy and Vaccination in Mice at JoVE.com

Intralymphatic Immunotherapy and Vaccination in Mice

预防性和治疗性疫苗往往不能刺激强烈的免疫反应，由于疫苗的周向引流淋巴结细胞和免疫细胞的参与因而不佳。通过直接注射疫苗到淋巴结的，所谓的淋巴内注射，疫苗的有效性，可以大大地改善和疫苗的剂量可以减少。

淋巴免疫治疗和疫苗接种小鼠

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

intralymphatic-immunotherapy-and-vaccination-in-mice

Research

JoVE Journal

Immunology and Infection

23.0K 次观看.  University Hospital Zurich. 预防性和治疗性疫苗往往不能刺激强烈的免疫反应，由于疫苗的周向引流淋巴结细胞和免疫细胞的参与因而不佳。通过直接注射疫苗到淋巴结的，所谓的淋巴内注射，疫苗的有效性，可以大大地改善和疫苗的剂量可以减少。 

视频: Intralymphatic Immunotherapy and Vaccination in Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

实验转移和CTL过继转移免疫小鼠模型

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

科研

免疫与感染

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

协议RBD - SARS疫苗的重组蛋白的制备，动物疫苗接种和中检测

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

制备肿瘤抗原负载成熟树突状细胞免疫治疗

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

CAR性T细胞过继治疗的护理标准胶质母细胞瘤的上下文生成

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

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 ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

小印度蒙古狼的异苯抗酸模拟分析(赫皮斯特奥罗普塔图斯)塞拉,用作口服狂犬病疫苗接种生物标记

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Methods Article

淋巴免疫治疗和疫苗接种小鼠