著者は、アイリスエルトマン、バーバラ·フォン·ボイスト、ジュリア·マリア·マルティネス·ゴメスからマウスではリンパ管内免疫化の方法を開発する上での実験の助けに感謝しています。私たちは、このビデオ制作のために彼らの手術室を使用させるためにもマギーアラスとニコラCesarovicに感謝します。

1。マウスの麻酔<ol><li>緩衝生理食塩水でケタミン（解離性麻酔薬）およびキシラジン（鎮静および鎮痛）を混合することにより、麻酔薬を準備します。最終溶液中のケタミン及びキシラジンの濃度は、それぞれ、12.5および2 mg / mlのです。 </li><li> 25〜30gの針で注射器を用いて腹腔内投与によりマウスに麻酔薬を注入する。体重10gあたり0.1mLを使用してください。 </li><li>角膜の乾燥を防ぐために、マウスの目に眼軟膏を適用します。 </li><li>マウスが十分にピンセットでその足やつま先をつまんで麻酔されていることを保証する。マウスがピンチに反射神経と反応しない場合は、手術に進みます。マウスは反射神経や筋肉の収縮とピンチに反応する場合は、1〜2分待ってからピンチテストを繰り返します。それでもマウスが反応した場合は、新しい動物とマウスを交換し、以降の手順1.2から手順を繰り返してください。 </li></ol> 2。私は外科マウスの鼠径部のncision <ol><li>仰向けにマウスを置きます。 </li><li>消毒エタノール（70％）で1鼠径部を濡らす。より良好な消毒を提供するため、鼠径部をエタノールで消毒前のファーを除去するために剃られてもよい。 </li><li>後ろ足を取り、股関節の約90°の角度を生成する股関節を曲げる。 </li><li>湾曲したmicrodissecting鉗子を使用して、股関節を曲げた鼠径部でのマウス皮膚を取り、少し湿らせた肌を引き上げます。 </li><li>鉗子で皮膚を保持しながら、外科はさみを使って皮膚を介して小切開（&lt;5 mm）をカット。 </li><li>切開部に閉じたハサミの先端を配置し、さらに、切開の中にそれを保持しながら、はさみを開くことによって、切開を開きます。これは、皮膚が破れの原因になり、10mm未満の直径を生成しなければならない。注：可撓性光ガイドを有する冷光源は、可視性を改善するために使用することができる。古い中マウス、特に男性、リンパ節の同定は、鼠径部における多くの脂肪組織に困難である。 </li></ol> 3。リンパ内注射<ol><li> 28〜30 Gの皮下注射針と注射器（0.5ミリリットル以下）を調製、短い斜面は長くベベルすることが好ましい場合がある。ワクチンの吸引除去10μlを注入し、注射器は、空気を含まないことを保証する。 </li><li>湾曲した鉗子と、閉じたハサミの先端の助けを借りて、鼠径リンパ節を局所化。リンパ節は、より白っぽい脂肪組織内で灰色表示され、さらに入力し、リンパ節を残して求心性と遠心性毛細血管を介して同定することができる。 </li><li>湾曲鉗子の枝の間に保持することにより、リンパ節を固定する。 </li><li>シリンジを取り、上に向けベベルとリンパ節に針を挿入し、全体のかさがリンパ節に入ることを確認すること。 </li><li>ワクチン（10μl）を注入する。もしリンパNODeは（爆破）腫れている、注入が成功したと仮定することができます。針がリンパ節に深く十分に配置されていない場合、一部または全体のワクチンが漏れやリンパ節に入らないことがあります。針が深すぎる挿入されている場合は、注入されたワクチンは、皮下リンパ節の下にリリースされます。後者の例では、注入を繰り返したり、新しい未処理のマウスでマウスを交換します。 </li></ol> 4。縫合することによって切開を閉じる<ol><li>無菌手術用縫合糸のパックを開きます。 </li><li>針本体の先端部に針ホルダとの縫合針をつかむ。第1ラチェットキャッチまでそれを絞ることによって針ホルダを締めます。 </li><li> （歯又は非歯付き鉗子または皮膚のフックを使用することができる、外科医の好みに応じて）鉗子を用いて切開部の片側に皮膚のホールドを取る。針CAを挿入2-3ミリ切開部の端から、皮膚の外部から侵入。 </li><li>ロース鉗子で皮膚のホールドENとピンセットで切開の反対側を把握する。針CAを挿入2-3ミリ切開部の端から、肌の内側から侵入。 </li><li>針ホルダを緩め、ピンセットで針先を把握する。数匹のマウスを治療する場合は、短い方の端を残して（最初の針挿入点の外にトレッドの便利な長さを残して、皮膚を​​通してトレッド針を引いて、5月10日のマウスは1縫合針を操作でき/）トレッド。 </li><li>標準外科医プロトコルによる好ましい結び目を作る。切開部が10mm以下である場合に一針で十分である。大きな切開のために、第二のステッチを作る。 </li></ol> 5。ポストの運用トリートメント<ol><li>ケージにマウスを置き、暖かい保つために組織にカバーしています。複数のマウスを治療する場合、暖かく保つために互いに近接して配置します。彼らは目覚めまで、または、暖かいパッドの上にマウスを置くアップ。 </li><li>彼らが目を覚ますまで、マウスを観察します。 </li><li>癒しだけでなく、創傷感染症などの他の臨床症状を巻かに関しては毎日マウスを観察します。傷は通常、十分に2日以内に閉鎖し、手順の7日以内に癒されます。 </li></ol>

<ol>
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特定目的会社は、ヒトにおけるリンパ管内免疫療法を扱う特許の発明者として名前が付けられています。特定目的会社は、科学顧問されており、PJは開示する競合の関心を持っていないImVisioN社、CYTOSバイオテクノロジー、MannKind株式会社、およびXBiotech USA社から旅費を受け取った。

リンパ管内免疫化および免疫療法は、抗体応答およびT細胞応答の両方を刺激するため適切であることが示されている。このビデオの記事で示されているように、ワクチン接種のリンパ管内の手順は、マウスにおいて強力な免疫応答を刺激するための迅速かつ簡単な方法です。訓練された外科医は、3〜4分の間に手続きを行うことができます。セッションも1は、一般的に麻酔や縫合をしている...

ワクチンは、典型的には、免疫応答の刺激のために、皮下または筋肉内に注射する。この方法の成功は、抗原提示細胞は、T-およびB細胞応答の生成のためにリンパ球と相互作用することができるリンパ節へのワクチンの効率的な排水を必要とする。また、強​​度と誘導される免疫応答のタイプも密度や周波数などの相互作用ならびに微小環境自体、サイトカイン、特に内容によって異なります。末梢組織に注入されたワクチンのごく一部は、リンパ節に到達すると、リンパ節へのワクチンの直接注射によりマウスおよびヒトのワクチン接種は、免疫応答部位が生成された、行われている。ヒトにおいて、手順は、超音波、また、リンパ系における可視化および診断のための造影剤の投与のために使用した手順によって案内される。マウスでは、手順が侵襲的である。ここでは、小さな（5〜10 mm）のincisioN麻酔した動物1の鼠径部で行われ、リンパ節はローカライズやピンセットで固定化して、ワクチンの10〜20μLの容量は、視覚的な制御の下で注入されて、10μlを最初の注射のために使用され、若いマウスでは小リンパ節、20μlの大きなリンパ節を有するより古いか、すでにプライミングしたマウスのリンパ節に注入することができる一方。切開は、外科用縫合糸を使用して、シングルステッチで閉鎖することができる。この方法によって、マウスをプラスミドDNA 2,3、メッセンジャーRNA 4、1,3,5,6ペプチド、タンパク質7-10、11粒子 、細菌12ならびにアジュバント7,13、および免疫の強力な改善でワクチン接種されているワクチンは、すべての種類に対する応答が観察されている。ワクチン接種のリンパ管内方法は、従来のワクチン接種が不十分な免疫を生産するか、利用可能なワクチンの量が限られていたり、バージョンである状況では特に適しています高価なY。人間では、予防接種のリンパ管内の方法は、アレルギー患者14,15にまたは癌16〜21の患者に適用されている。現在の概念はリンパ管内の方法は、筋肉内および皮下注射などの他の注射方法より侵襲的であるということですが、痛みの知覚は、静脈穿刺15後よりも高いではありません。これは、リンパ内ワクチン接種は、予防および特に治療予防接種の他の方法に代わる、または補完になることが期待されている。この記事では、リンパ管内ワクチン接種の手順は、マウスで実行する方法を詳細に説明します。記載されているすべての手順は、チューリッヒ州立獣医庁によって承認され、スイス連邦のガイドラインおよび科学的目的のために使用される動物の保護に関する指令を実施した。

<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

ワクチンは、典型的には、免疫応答の刺激のために、皮下または筋肉内に注射する。これが成功するかどうかは、抗原提示細胞が欲しかった免疫応答を生成するためのリンパ球と対話することができ、リンパ節へのワクチンの効率的な排液を必要とします。強さと誘導される免疫応答のタイプは、密度や周波数の相互作用ならびに微小環境、サイトカインの、特に内容によって異なります。末梢注入されたワクチンの分画のみがリンパ節に到達すると、マウスおよびヒトのワクチン接種、 すなわちリンパ内注射、鼠径リンパ節へのワクチンの直接注射により実施した。男性では、手順は、超音波によって導かれる。マウスでは、小さな（5〜10 mm）の切開が、リンパ節の局在化及び鉗子で固定化されているが、麻酔した動物の鼠径部に形成されており、ワクチンの10〜50μlの容量を視覚的制御下に注入される。切開部を外科用縫合糸を使用して、シングルステッチで閉じられる。マウスを、プラスミドDNA、RNA、ペプチド、タンパク質、粒子、および細菌ならびにアジュバントでワクチン接種し、ワクチンのすべてのタイプに対する免疫応答の強力な改善が観察された。ワクチン接種のリンパ管内方法は、従来のワクチン接種が不十分な免疫または利用可能な場合、ワクチンの量が限られているが生成状況では特に適しています。

マウスではリンパ管内注射の手順は、外科的な性質にもかかわらず、まっすぐ進むと、比較的高速である。訓練を受けた人は3〜4分で手続きを行うことができます。一針で閉じられ切開は、典型的には2日以内に治癒する（ 図1） リンパ内ワクチン接種またはimmunizationがmRNAと行ったプラスミドDNA、ペプチドタンパク質ウイルスおよび細菌2タンパク質</stro...

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

Intralymphatic Immunotherapy and Vaccination in Mice

予防的および治療的ワクチン接種は、多くの場合、週のリンパ節へのワクチンの排水および免疫細胞の関与の結果として、乏しいによる強力な免疫応答を刺激することができない。リンパ節へのワクチンの直接注射することによって、いわゆるリンパ内注射は、ワクチンの有効性を強く改善することができ、ワクチン用量を低減することができる。

マウスのリンパ管内免疫療法やワクチン

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 ビュー.  University Hospital Zurich. 予防的および治療的ワクチン接種は、多くの場合、週のリンパ節へのワクチンの排水および免疫細胞の関与の結果として、乏しいによる強力な免疫応答を刺激することができない。リンパ節へのワクチンの直接注射することによって、いわゆるリンパ内注射は、ワクチンの有効性を強く改善することができ、ワクチン用量を低減することができる。 

ビデオ: 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.

実験的転移とCTLの養子移入免疫療法のマウスモデル

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

免疫・感染学

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.

タンパク質調製物、動物の予防接種と中和の検出：遺伝子組換えRBDベースのSARSのワクチンのためのプロトコル

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.

免疫療法のための腫瘍抗原にロードされた成熟樹状細胞の調製

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.

RNAウイルスの広域スペクトルの化学抑制剤のためのハイスループットスクリーニング

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.

ケアの膠芽腫の標準のコンテキストにおける養子療法のためのCAR T細胞の生成

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.

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

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.

まれな抗原特異的 B 細胞血液の循環から弁別に対するモノクローナル抗体の生成

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.

同所性同種人間神経膠腫異種移植片のマウスモデルの細胞傷害性免疫細胞の定位の養子転送

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.

ワクチン接種後のモルモット モデル メジャー T 細胞反応に最適化されたインターフェロン-γ しなさいアッセイ

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.

小さなインドのマングースにおけるイオフェノキ酸類似体の分析 (ヘルペステス・オーロプンクタトゥス)経口狂犬病ワクチン接種生物学的マーカーとしての使用のためのセラ

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

マウスのリンパ管内免疫療法やワクチン