Name: development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fcεri
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The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

細胞株の1）準備： <ol><li>馬のFcεRIαを表現するRBL-2H3.1細胞株の開発： <ol><li>単層細胞株についての基本的な組織培養技術を用いて、pEE6プラスミドを使用して、親RBL-2H3.1細胞をトランスフェクト、ウマFcεRIα遺伝子（GenBank：Y18204.1）を有する28。 1.2×10 7細胞ml -1の濃度で細胞の0.8ミリリットルにプラスミドDNA2μgのμlの-1を追加します。その後すぐに氷上で10分間インキュベート0.4cmのelectrocuvetteを使用して、250V、960μFで細胞をエレクトロポ。 </li><li>その後、蛍光IgE抗体とそれらをタグ付けすることによって、FACSを通して残りの生きた細胞をソートし、ジェネティシンG418硫酸0.4グラムを含む培地を使用して形質転換細胞を選択します。捜査2、3のための馬FcεRIα細胞株を発現している結果としてRBL-2H3.1を使用してください。 </li></ol></li><li>プレ検定抗体感作性： <ol><li&gt;を収穫コンフルエントペトリ皿から馬FcεRIα細胞に発現RBL-2H3.1。 5×10 5細胞mlの細胞密度に培養培地中に再懸濁細胞を、その後洗浄する-1。 </li><li> 1 ngのml -1の最終的な濃度に懸濁した細胞への関心のIgEを追加した列1-6で96ウェルプレートに100μlの細胞をプレートし、37℃+ 5％CO 2 + 90％でインキュベート16時間、相対湿度。インキュベーション時間の後、および放出アッセイを行う前に、ウェルの集密度及び細胞接着のために顕微鏡下で、ウェルをチェックしてください。 </li></ol></li></ol> 2）放出アッセイ： <ol><li>細胞を洗浄する。 <ol><li>穏やかな細胞洗浄を可能にするために、37℃の温放出緩衝液（25mMのPIPES、120mMの塩化ナトリウム、5mMの塩化カリウム、0.04 mMの塩化マグネシウム、および1mM塩化カルシウム）。 </li><li> 、細胞培地を除去するためにプレートをフリックと暖かい100μlを加えることにより、細胞を洗浄37℃で、放出緩衝液。二回繰り返します。 </li></ol></li><li>抗原チャレンジ： <ol><li> 0.1、0 ngのmlと-1の抗原の段階希釈（NIP-HSAまたはDNP-HSA）を調製NGミリリットル-1、1 ngのmlで-1、10 ngのmlを-1、100 ngのmlで-1、千ngのミリリットル-1 10,000 ngのリリース·バッファ内ミリリットル-1および37℃で温めて。 </li><li>第二の細胞の洗浄後、メディアを破棄し、抗原溶液100μlで置き換え。同じ行のウェル（例えばA1-6）が同じ抗原濃度がそれらに追加されていることを確認してください。 </li><li> 0 ngのml -1の抗原を添加することにより、行Aにおける負の制御を設定します。陽性対照として使用する細胞を溶解するために、行H細胞におけるトリトン緩衝液（5％トリトンX-100）に続く行（BG）ダウン増大抗原濃度を追加する。細胞がそのメディエーターを放出できるように20分間37℃でインキュベートする。 </li></ol></li><li>個々のウェルコントロールの設定： <ol><li>インキュベーション後、プレートのもう半分（井戸A1-6井戸A7-12へ、等）への細胞上清50μlを移す。上清の残りを50μlを破棄して、列1-6のセル内の全メディエーターの割合として列7-12に各ウェルにリリースメディエーターの量の測定を可能にするためにトリトン-X緩衝液50μlと交換。 </li></ol></li><li>酵素基質： </li><li> 50（クエン酸緩衝液に添加することにより2mMのにまで希釈されたDMSO中で調製した50 mMの4-ニトロフェニルN-アセチルβ-Dグルコサミニド0.2 Mクエン酸、0.2 M酢酸ナトリウム、pH 4.5）βヘキソサミニダーゼ基質のμlを添加するすべてのウェルにβヘキソサミニダーゼ酵素による4-ニトロフェノールへの基質の変換を容易にします。 2時間、37℃でプレートをインキュベートする。 </li></ol><img alt="図2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" widt時間= &quot;400&quot; /&gt; <ol start="5"><li>反応を停止する。 <ol><li>各追加150μlのトリス緩衝液（1 Mトリス-HCl、pHを9）により反応を停止ならびに緩衝液の高いpHは、反応を停止させ、黄色に4-ニトロフェノールをオン。 </li></ol></li><li>結果を読み込むと分析する： <ol><li>黄色の吸光度を測定するために405nmでプレート分光光度計を用いてプレートをお読みください。以下の式を用いて放出βヘキソサミニダーゼの割合を算出した。 </li></ol></li></ol><img alt="図2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2"><li>平均値を行ごとに取得された後、各ウェルにこの数式を適用します。 A1およびA7が96ウェルプレートのウェルの位置を表す。 agai（総メディエーター放出に相当）βヘキソサミニダーゼ放出の割合のグラフをプロットします抗原濃度2,3 NST。 </li></ol>

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The authors declare that they have no competing financial interests in this paper.

要約すると、この調査の結果は、馬FcεRIαを表現するRBL-2H3.1細胞は馬のIgEで感作し、抗原によって挑戦されたとき、彼らは内部のメディエーターの合計量の4.88パーセント±36.68パーセントのピークメディエーター放出を与えることを示した馬FcεRIαを発現していないRBL-2H3.1親細胞に比べて細胞、。 したがって、このアッセイは、in vitroでの馬のアレルギー反応を調査...

アレルギーは、数千年前から知られている。喘息治療はエーベルス·パピルス（〜1550 BCE）として知られている古代エジプトの医学のテキストで説明して7を治療するための薬草療法を検討した。 今日アレルギーはTヘルパー細胞2型（TH 2）免疫系のアームはアレルゲンと呼ばれる、環境抗原に応答して、免疫グロブリンE（IgE）抗体の産生を操縦するI型過敏性反応、に分類される。 IgEの合成を高めるタバコの煙やディーゼル排気微粒子中の粒子がそうであるように、これらは9、インターロイキン-4およびインターロイキン13 8を含む一般的に免疫系の細胞と相互作用し、炎症誘発性サイトカインの合成および分泌を刺激する多様な物質である10。 過去50年間で先進国におけるアレルギー症状の上昇はの効果の組み合わせに起因している「衛生仮説」11によって提案されたように、TH 2サイトカインによって支配プロファイルに対する免疫応答をシフトするために組み合わせる環境汚染物質、より衛生的な環境へのトレンド、。 上述したように、人間がアレルギーに悩まさ唯一の哺乳類ではありません。注目すべき馬と犬はまた、古典的なアレルギー反応を開発することができますし、12の調査によると、人間のように、馬アレルギーは遺伝的要因と環境的要因に起因する、ことを示している。結果として、これらの動物は、一度臨床症状がで設定したアレルギーの遺伝的および環境的な原因、病気への感作からの進行、および可能な介入戦略との間の相互作用を研究するための優れたモデルを提示 1887年に、Stömmerは、馬の心血管系に対するヒスタミンの効果がある、人間と馬の喘息13の間の類似性を記述した最初の人だった人間14のものと非常に類似している。馬はまた、年間15米1150億ドルの賭け売上高は720億ドル、米国の価値がある競馬産業の礎である。 ほとんどの現代的な競走馬以降1878からレディアン·ブラントによって繁殖アラビア馬の数が少ないの子孫である。現代の競走馬は、一般的にパフォーマンス能力を選択するために近交系されている。彼らは、アレルギー反応をマウントするそれらの感受性であるそのうちの一つ、遺伝性疾患になりやすいです。彼らはまたも、最も深刻なアレルギー人間16よりも1000倍高い血清IgEレベルを持っている。馬アレルギー反応は、通常、虫刺され過敏症（IBH）17、18として明示されている。IBH結果は皮膚炎に起因刺されに属ヌカカで昆虫を形成する。ウマのアレルギー性​​疾患の別の形態は、これは肺と気道で明らかにされて、再発性気道閉塞（RAO）である。それは、喘鳴やラボによって特徴づけられるOR演算呼吸。 RAOは、一般的に胞子を成形することに応答して発生し、別の調査がこの20を確認していないが、高アレルゲン特異的IgEレベルは、一つの研究19にRAOに罹患している馬に記録された。 ウマアレルギーに関する研究の試みモニタリングにおける、および抗ウマのIgEモノクローナル抗体（mAb）21,22を開発することによって、馬のIgEを中和することを中心に展開し、さらに23による研究では、馬の高親和性Fc受容体のαの細胞外ドメインの生産を議論ウマ血清IgEを検出し、定量する試みで、チェーン（FcεRIα）受容体。 Ledin 24による関連研究では、自己/非自己免疫原を使用して免疫系をプライミングすることにより、血清IgEを中和することを目的とした新しいアプローチについて説明します。すべてのこれらの研究は、しかし、それらのプロトコルの安全性と有効性を試験するための効果的なアッセイを欠いていた。この記事では、我々は今そのようなアッセイsysを提示βヘキソサミニダーゼ放出馬システム、関連する診断および治療戦略の研究に適用可能なTEMは、細胞メディエーターの脱顆粒の指標として、ウマFcεRIαを表現するRBL-2H3.1細胞で評価した。このプロトコルは、異なる種由来のIgEに対する高親和性受容体のIgE結合ドメインをコードする遺伝子でトランスフェクトしたRBL細胞の工学を記述する先の刊行物25、4、5、2、3に基づいている。プロトコルは、結果は、三連の実験の平均値±標準偏差として提示されているβヘキソサミニダーゼ放出アッセイを実行する方法について説明します。 放出アッセイは、最初Siraganianによって開発され、人間のアレルギーを研究するために25をフックした。博士ルーベンSiraganian率いる研究室グループは、RBL細胞株を開発した。これらのRBL細胞は、ヒトFcεRIαを表現するために開発され、プロトコルが4によって出版された。最後のピースアッセイのハプテン4-ヒドロキシ-3-を標的IgEの可変領域をマウス遺伝子の下流に、その重鎖遺伝子をクローニングすることにより、IgE抗体の産生を記載しノイバーガー26の論文でのpSVプラスミドの開発に付属 - ニトロフェナセチル（NP）、得られたキメラ抗体は、完全に機能した。また、RBL細胞の表面上のその受容体をクローニングしながら、同じハプテンを対象とした全てのIgEを開発する能力は、好塩基球細胞の脱顆粒を測定するための有用なプロトコル作るアッセイの標準化をもたらした。 アッセイは、長所と短所を持っています。このアッセイの利点は、任意の哺乳動物系で使用される適応性である、我々の研究室は、ヒト、イヌおよびウマのシステムにおいて脱顆粒をテストするためにそれをこのように使用されており、これは、生物のIgEの合成およびその受容体をクローニングすることによって、単純に達成可能であるRBL細胞の表面上に。 一方、アッセイの短所は、RBL細胞は、それらを同じアッセイ内の脱顆粒のレベルの変化を与えること、熱的、機械とPH変化に非常に敏感であるということです。それは、このように強くアッセイは常に三重に繰り返された後、平均がそれらから取られることをお勧めします。また、RBL細胞は、彼らのメンテナンスが煩雑になっ、延長時間（&gt; 10週間）27組織培養で残っている場合、非放出表現型へシフトする傾向がある。彼らはまた、光学顕微鏡からは見えませんし、細胞の形態を変化させないマイコプラズマ細菌による感染症になりやすいですが、劇的に彼らの脱顆粒レベルを変更します。このように定期的なマイコプラズマ試験が必要とされている。

<table><tbody><tr><td>RBL-2H3.1 Expressing Equine Fc&amp;epsilon;RI&amp;alpha;</td><td>-</td><td>-</td><td>Produced in the lab</td></tr><tr><td>Equine IgE anti NIP-HSA</td><td>-</td><td>-</td><td>Produced in the lab</td></tr><tr><td>96 Well Plate</td><td>Sigma</td><td>CLS3595</td><td>-</td></tr><tr><td>Multi Channel Pipette</td><td>Anachem</td><td>-</td><td>-</td></tr><tr><td>Incubator</td><td>Galaxy R</td><td>-</td><td>-</td></tr><tr><td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td><td>Cambridge Research Biochemicals</td><td>N-1070-1</td><td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td></tr><tr><td>Dinitrophenyl Conjugated to Human Serum Albumin</td><td>Sigma</td><td>A6661</td><td>Abbreviated DNP-HSA</td></tr><tr><td>Plate Spectrophotometer</td><td>Anthos Labtec HT2</td><td>-</td><td>-</td></tr><tr><td>Pipes</td><td>Sigma</td><td>P1851</td><td>-</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S7653</td><td>-</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P9333</td><td>-</td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M2670</td><td>-</td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>C1016</td><td>-</td></tr><tr><td>Triton x100</td><td>Sigma</td><td>X100</td><td>-</td></tr><tr><td>4-nitrophenyl N-acetyl-&amp;beta;-D-glucosaminide</td><td>Sigma</td><td>N9376</td><td>Stock solution called &amp;beta;-hexosaminidase substrate was 50mM prepared in DMSO</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma</td><td>D2650</td><td>-</td></tr><tr><td>Citric Acid</td><td>Sigma</td><td>251275</td><td>-</td></tr><tr><td>Sodium Acetate</td><td>Sigma</td><td>S7670</td><td>-</td></tr><tr><td>Tris</td><td>Sigma</td><td>T5941</td><td>-</td></tr></tbody></table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

親RBL-2H3.1細胞およびウマFcεRIα受容体遺伝子でトランスフェクトされたものが最初にマウスIgE抗DNP-HSAで感作し、DNP-HSA抗原でチャレンジした。マウスIgEは、両方の細胞株における内因性ラット受容体に結合し、したがってメディエーター（ 図1 A）を放出するために、両方の細胞株の放出生存度を試験するための対照として働く。これは重要な検査であり、細胞培養における延長（...

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÃŽÂµRI at JoVE.com

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

開発<em&gt; in vitroで</emとの相互作用を研究するための&gt;モデル系<em&gt;エクウス</em&gt;<em&gt; caballus</emその高親和性受容体FcεRIとした&gt;のIgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

development-an-vitro-model-system-for-studying-interaction-equus

Research

JoVE Journal

Immunology and Infection

12.4K ビュー.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

ビデオ: Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

アレルゲン誘発性喘息のマウスモデル

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

免疫・感染学

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 &#197;. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

This antibody homology modeling prediction protocol is followed by antibody-receptor Pyrx docking and molecular dynamic simulation. These three primary methods are used to visualize the accurate antibody-receptor binding areas and the binding stability of the final structure.

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The ...

遺伝学

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

真のIgE発現するマウスB系統細胞の検出

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

牛にエフェクターおよびメモリーT細胞応答を評価するための長期培養インターフェロンγ酵素結合免疫アッセイの応用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

インフルエンザ血球凝集素抗体による Fc を介したエフェクター機能を評価する方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

治療抗体の存在下におけるFcγRIIIa関与によるヒトナチュラルキラー細胞事象の評価

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus

This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization.

ニュージーランド白ウサギにおける母体免疫の研究における鼻腔内免疫とミルクコレクション (オリクトラグス・キュニクルス)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of ...

3Dウシ回腸エンテロイド培養からの2D単分子膜の確立

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

著者スポットライト:宿主と病原体の相互作用を研究するための高度なエンテロイドモデル 

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

トランスレーショナルバイオメディカル研究への応用を目指したウシ一次エンテロイド由来2次元単層培養系の作製

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...

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トランスレーショナルバイオメディカル研究への応用を目指したウシ一次エンテロイド由来2次元単層培養系の作製

トランスレーショナルバイオメディカル研究への応用を目指したウシ一次エンテロイド由来2次元単層培養系の作製