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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RBL-2H3.1 Expressing Equine Fc&#949;RI&#945;</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-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>Stock solution called &#946;-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 FcÎµRI 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

Universidad de Cartagena UDEC

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

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

のための負の選択マーカーの開発赤痢アメーバ</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

免疫・感染学

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

小説<em&gt;インビトロ</em&gt;ヒト全血と内皮の間の相互作用を研究するためのモデル

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

アン<em&gt;インビトロ</em&gt; HIV重感染のコンテキストでのマラリアに対する免疫応答を測定するためのモデル

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

微生物群集を学びます<em&gt;インビボ</em&gt;：ホスト間の媒介の相互作用のモデル<em&gt;カンジダ</em&gt;と<em&gt;緑膿菌</em&gt;航空で

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

アン<em&gt;インビトロ</em&gt;インピーダンス分光法を用いた血液脳関門のモデル：T細胞 - 内皮細胞の相互作用に焦点を当て

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

「Acufection」と肌の研究への応用により、経済的なDNAデリバリーシステムの開発

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

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微生物群集を学びます

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チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

「Acufection」と肌の研究への応用により、経済的なDNAデリバリーシステムの開発

使用してリンパ球血管外漏出の分析