This work was supported by NIH grants to T.W. (R01AI072060 and R01AI099123). G. Xie was supported by a Sealy Center for Vaccine Development Predoctoral Fellowship. We thank Dr. Richard Flavell (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven) and Dr. Shizuo Akira (Osaka University, Japan) for providing the MyD88&#8211;/&#8211; mice and Dr. Y Cong (UTMB, Galveston) for providing OTII transgenic mice.

全ての動物実験は、テキサスメディカル支店大学の動物実験委員会によって承認された。 非感染およびWNV感染マウスからのDCの単離<ol><li>年齢および性別が一致6-10週齢の野生型C57BL / 6（B6）とのMyD88 - / -マウス。腹腔内（IP）WNV NS4B- P38G変異体の500プラーク形成単位（P​​FU）で接種する。 - / - CO 2を用いたマウス感染後3日目に、B6マウス及びMyD88-を安楽死させる。コントロールとして、両グループの非感染マウスを使用してください。各グループから3匹のマウスを使用してください。 </li><li> 70％エタノールを使用して屠殺したマウスの左側にウェット毛皮と、マウスの左側に沿って前後脚の間の約半分の毛皮を切り取る。体腔を切り開い。鉗子を使用して脾臓を削除します。 RPMIでペトリ皿に脾臓を置きます。 </li><li>に従って抗CD11c磁気ビーズを使用して、脾臓DCを単離する製造元の説明書。 </li></ol> 2.精製とOTIIトランスジェニックマウスのT細胞の標識<ol><li>ナイーブOTIIマウスから収穫1脾臓は、上記のステップ1.2で説明したと2スライドのすりガラス端部の間に脾臓を均質化するように。 14ミリリットルにRPMI培地を加えるを15mlコニカルチューブに細胞懸濁液を移す。サスペンションは5分間座って、新しい15ミリリットルコニカルチューブにそれの13ミリリットルを転送してみましょう。 </li><li>製造業者の説明書に従ってCD4 + T細胞単離キットを使用して、脾臓CD4 + T細胞を単離する。 </li><li>を50mlコニカルチューブに転送細胞は、PBSで2回洗浄する。再懸 ​​濁し、1ml当たり1×10 6細胞を、0.5％ウシ血清アルブミン（BSA）を有するPBS中の細胞とCFSEの0.5モル/ mlに加える。光から保護し、10分間、37℃でインキュベートする。 </li><li> 10％の熱不活化ウシ胎児血清を5ミリリットルの冷完全培地（RPMI-1640を追加し、100分の1巻1000×1000の2-メルカプトエタノール、抗生物質/抗真菌剤、1/100体積L-グルタミンおよび1 / 1,000倍容量）。 </li><li>氷上で5分間インキュベートします。 PBSで細胞を1回洗浄し、2％PFAで0.2×10 6ラベルのCD4 + T細胞を固定し、フローサイトメーターで取得する。 CFSEの基礎レベルを決定するために、このサンプルを使用します。完全培地中の標識細胞の残りを再懸濁。 </li></ol> WNV感染マウスのDCと3。共培養OTII T細胞<ol><li>培養2×10 OTIIマウスの5精製CD4 + T細胞単独で、または野生型もしくはMyD88の精製されたDCと- / -またはOVA残基323-339無し24ウェルプレート中のマウス（2×10 4）（1グラムウェルあたり1mlの完全培地中/ ml）を。 </li><li> 5日間、37℃で細胞をインキュベート。収穫細胞は、（1％FBSを含むPBS）、FACS緩衝液中で2回洗浄すると0.25mlの2％PFAで固定する。ボルテックスすぐに。 </li></ol> インビトロの4。フローサイトメトリー分析&lt;/ em&gt;のT細胞プライミング<ol><li>買収のために、フローサイトメトリーソフトウェアのアイコンをダブルクリックする。 対 SSC-AのFSC-Aと200,000 0上の線形SSC-A、チャンネルを選択0 200,000リニアFSC-Aの密度プロットのために。その後、生細胞にゲートを作成（P2; 図1A参照 ）。 </li><li> CFSEの蛍光強度を分析するために、FL1チャンネルのヒストグラムを開き、ゲーテッド集団20,000のイベントを取得する。単独で培養し（ 図1A）OTII細胞のための試料上のマーカーを設定します。 </li><li>野生型のDC（ 図1B）またはMyD88のと共培養OTII細胞のためのサンプルを取得する- / -樹状細胞（ 図1C）と同様の方法で。 </li></ol> 5.絶縁およびサイトカインアッセイ用の脾細胞の刺激<ol><li> steのと同様の手順を用いて、WNV NS4B-P38G変異を有するマウス- / - B6およびMyD88に感染するP 1.1。日、30日8と21ポストのプライマリWNV感染または二次感染後4日目に、野生型WNV株IPの2000 PFUで感染後、再チャレンジ生存マウスでは、脾臓の収集のためにCO 2でマウスを安楽死させる。 注：我々は、各時点についてグループごとに2〜3匹のマウスを使用していました。 </li><li> ステップ1.2＆ステップ2.2で上記のように脾臓細胞の単一細胞懸濁液を作成します。血球計数器を用いて細胞をカウントし、10mlの完全培地中に再懸濁これら。 </li><li> 2.5×10 6細胞/ mlの完全培地で細胞を希釈する。 1.5ミリリットルのマイクロ遠心管に脾細胞の1ミリリットルを追加します。 </li><li> DMSO中1mg / mlのWNV特異NS4B及びEペプチド（SSVWNATTAとIALTFLAV）を、CD8 + T細胞をシミュレー希釈する。 CD4 + T細胞の刺激のために、DMSO中1mg / mlのWNV特異的NS3およびEペプチド（RRWCFDGPRTNTILEとPVGRLVTVNPFVSVA）を希釈する。 followe細胞へのペプチドの10μlのを追加します。ブレフェルジンの1μlの溶液によるD。コントロールとしてのペプチド処理なしに細胞を使用してください。細胞を混ぜる。 </li><li>チューブのキャップで18 G針で2穴パンチ。 5時間、37℃で細胞をインキュベート。 </li></ol> 6.細胞内サイトカイン染色<ol><li>新しいマイクロ遠心チューブに細胞を移す。 300〜400×gで5分間細胞をスピンし、上清を捨てる。 120μlのFACS緩衝液でピペッティングにより1mlのFACS緩衝液を追加し、300〜400×gで5分間スピンし、再懸濁細胞。 </li><li> 2μlのFcの遮断を追加し、10分間室温で細胞をインキュベートする。各チューブに1ミリリットルFACSバッファーを追加します。 400×G - 300でスピン5分。 </li><li>再懸 ​​濁300μlのFACS緩衝液で細胞をし、3本のチューブ（チューブ当たり約0.8×10 6細胞）にそれらを分割する。 表1に示すように、ラットのIgG-PE染色のために、各培養条件の第一の管を予約する。第二の管の場合、CD4ペプチド処理細胞への抗CD4 APCの3μlを添加、3μペプチド処理なしの細胞に対するCD8ペプチド処理細胞または双方の抗体に対する抗CD8 FITCのリットル。補償染色するための第3のチューブを使用してください。各培養条件については、APC結合CD4、FITC結合CD8またはFL1、FL2及びFL4チャネルの補償コントロールなどの単独のPE-ラベルCD3抗体（チューブあたり3μL）で染色した細胞の3本のチューブを使用しています。 </li><li>簡単にボルテックス細胞。氷上で20分間のままにして、光から保護する。 1.4ミリリットルFACS緩衝液を追加します。 300〜400×gで5分間スピンし、上清を捨てる。 </li><li>細胞ペレット（または簡潔に渦を）破壊すること攪拌する。 250μlの固定/透過化solutionbyのペッティングで細胞ペレットを再懸濁。 20分間室温でインキュベートし、光から保護する。 </li><li> 1.2ミリリットルFACS緩衝液を追加します。 300μlのFACS緩衝液中で300〜400×gで再懸濁細胞でのスピン5分。 注：この時点で、細胞は、さらなる処理のためにBL2ラボに転送することができるか、冷蔵庫に保存し、uのために光から保護3日のp。 </li><li> 500μlのFACSバッファーを追加します。スピン1X透過化/洗浄緩衝液500μl中で300〜400×gで再懸濁細胞で5分。スピン300~400×gで5分間。 </li><li>以前に予約済みチューブのステップ6.3および3.5μlのラットのIgG-PEでCD4のための抗体および/ ​​またはCD8 T細胞マーカーを付加したチューブに3.5μlの抗IFNγ-PEを追加し、100μlの1×パーマ/洗浄で細胞を再懸濁氷上で25分間のIgGコントロールのために、光から保護する。 1X透過化/洗浄緩衝液1.4ミリリットルを追加することによって、細胞を洗浄。スピン300~400×gで5分間。 1.4ミリリットル1X透過化/洗浄バッファーを追加することによって、洗浄工程を繰り返します。 </li><li> 300〜400×gで5分間スピンし、上清をデカント。 FACS緩衝液1.4ミリリットルと、最終的な獲得のためのFACS緩衝液400μlの再懸濁を追加することによって、細胞を洗浄。 </li></ol>細胞内サイトカイン染色の7フローサイトメトリー分析<ol><li> ステップ4.1と同じ取得設定を使用します。クレア生細胞上のゲートTE（P2を、 図2Aを参照）。各グループの補償管を使用して電圧を調整します。 </li><li>二つの新しいドットプロット、 対 FL1のために収集したデータを表示するための1を開きます。 FL2（CD8 対 。IFN-γ）、他方は対 FL4のために収集されたデータを表示することである。 FL2（CD4 VS。IFN-γ）。各サンプルのゲートされ、人口5万のイベントを取得します。 </li></ol>

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著者らは、開示することは何もない。

WNVは、BL3の病原体である。起因する安全規制に、WNVに感染したサンプルを用いた免疫学的アッセイは、多くの場合、BL3施設または実行するために、より長くて退屈な時に機器の利用可能性によって制限されている。最近の研究では、WNV感染の5中の細胞性免疫応答を研究するための2つのフローサイトメトリーに基づく方法を使用する。両方のアッセイでは、WNV感染細胞を直接またはPF...

西ナイルウイルス（WNV）、神経向性、プラス感知されたフラビウイルスは、新興の公衆衛生上の脅威である。現在、ワクチンは、ヒトでの使用1のために承認されていない。非構造（NS）（b）のタンパク質でP38G置換を有する弱毒WNV株は、野生型WNV NY99株の2以上のマウスには、マウスにおける致死性が、より高い先天性サイトカインおよびT細胞応答を誘導しないことが知られている。 NS4B-P38G変異体で免疫したマウスは、すべての致命的な野生型WNVと二次攻撃から保護された。これは、NS4B-P38G変異体は理想的なワクチン候補に適した特徴を有することを示唆している。 NS4B-P38G変異体は、高い防御適応免疫を誘導する機構は明らかにまだ理解されていない。病原体関連分子パターンを認識するToll様受容体（TLR）は、ウイルス感染に対する自然免疫の開始に重要な役割を果たしている。コアTLRシグナル伝達経路は、骨髄分化一次応答geと利用プライマリアダプタ3,4としてNE 88（MyD88に）。最近の研究では、MyD88のシグナル伝達は、マウス5におけるWNV NS4B- P38G変異感染中の細胞性免疫の発達に重要な役割を果たすことが示された。樹状細胞（DC）は、ウイルス感染の6,7の間に、一次T細胞応答を開始するためのユニークな能力を示す最も重要な抗原提示細胞の一つである。 CD4 +およびCD8 + T細胞は、長期間持続する防御免疫に寄与し、野生型WNV感染の8,9次宿主の生存のために重要である両方。二つの免疫学的アッセイは、NS4B-P38G変異体に感染したマウスにおけるこれらの細胞の機能を評価するために、本研究に使用した。 - / -マウスで最初に、 インビトロでの T細胞プライミングアッセイは、WNV感染野生型マウス及びMyD88-のDCの抗原提示能力を比較するために利用された。アッセイ、ナイーブCD4の感度を高めるために、&lt;商標&gt; + T細胞は、323、ニワトリオボアルブミン（OVA）ペプチドをVα2/Vβ5TCR特異的に発現OTIIトランスジェニックマウスから単離した- WNV感染したマウスの339 DCは、カルボキシフルオレセインスクシンイミイースターで精製し、共培養した（CFSE OVAペプチドの存在下で）標識CD4 + T細胞。共培養の5日後、細胞を採取し、パラホルムアルデヒド（PFA）で固定し、フローサイトメトリーによって分析した。増殖アッセイは伝統的に、5-ブロモ-2&#39;-デオキシウリジン（BrdU）の取り込みまたはトリチウム化チミンデオキシリボシド（3 HTdr）10を介して行われている。それにもかかわらず、これらのアッセイは、放射性および/またはバイオセーフティーレベル（BL）WNVの研究が行われている3施設、での特別な設備を必要としてのいずれかである。生体染色色素の蛍光強度CFSEのシリアル半減によるリンパ球増殖のフローサイトメトリー分析は、より一般的に染料をより安定的であるように、免疫学的アッセイにおいて使用されるようになっている均等に、細胞内に取り込まれ、フローサイトメトリーによって容易に検出され、11非放射性である。アッセイはまた、細胞分裂の数を評価する能力を有する。 WNVの研究において、このアッセイを使用する1つの大きな利点は、1〜2％PFAによる感染細胞の固定はBL2実験において、フローサイトメーターでサンプル採取を可能にするWNV 12を不活性化する可能性があることである。 次に、変更された細胞内サイトカイン染色（ICS）手順がNS4B-P38G変異体感染マウスにおけるWNV特異的T細胞応答の調節におけるシグナル伝達のMyD88の役割を研究するために使用された。このアッセイでは、感染したマウスから単離した脾細胞を、WNV特異的なペプチドを用いてインビトロで処理した。ブレフェルジンAは、細胞内サイトカインを保持するために添加した。 5時間のインキュベーション後、細胞を回収し、洗浄し、T細胞サブセットを染色した。次いで、細胞を（IFN）-γおよびフローサイトメトリーによって分析し、インターフェロンのために透過処理、染色、PFAで固定した。細胞は、PFAを含む固定および透過化緩衝液で処理されると、他のフローサイトメトリーに基づくアッセイと同様に、感染したサンプルは、さらなる処理および分析のためBL2室に移すことができる。いくつかの公表された研究では、我々は、WNV感染マウス13,14におけるT細胞エフェクター機能を測定するためにICSを使用している。それは十分に確立さですが、このアッセイの一つの主要な欠点​​は、手順が非常に長いですし、BL3施設内で行わより多くの時間がかかることができることです。ここで、微小遠心管ベースのICS法はより実現可能であることが示された、進行しやすくとBL3研究所内で行うより少ない時間がかかる。

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in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection

マウスにおける野生型WNVより生得サイトカインおよびT細胞応答を誘導した弱毒西ナイルウイルス（WNV）、非構造（NS）4B-P38G変異体。最近、骨髄分化因子88（MyD88に）シグナリングがWNV NS4B-P38G変異体感染の間に、初期T細胞プライミングおよびメモリーT細胞の発達に重要であることが示された。本研究では、2つのフローサイトメトリーに基づく方法-アッセイおよび細胞内サイトカイン染色（ICS）をプライミングin vitroで T細胞は-樹状細胞（DC）およびT細胞の機能を評価するために利用された。 OTIIトランスジェニックマウスのCD4 + T細胞を標識-アッセイプライミングT細胞では、細胞増殖は、カルボキシフルオレセインスクシンイミジルエステル（CFSE）でのマウスの両群からのDCの共培養の後にフローサイトメトリーによって分析した。このアプローチは、伝統よりも有意に改善全体的な感度で増殖するCD4 + T細胞の割合の正確な決定を提供放射性試薬を用いたアルアッセイ。マイクロ遠心チューブシステムは、ICSプロトコルの両方の細胞培養およびサイトカイン染色法に使用した。従来の組織培養プレートベースのシステムと比較して、この修正された手順は、バイオセーフティーレベル（BL）3施設で実行することが容易であった。また、WNV-感染細胞BL3施設外さらなる分析を可能に両方のアッセイにおいて、パラホルムアルデヒドで処理した。全体として、これらのin vitro免疫学的ア ​​ッセイは、効率的にWNV感染の間に細胞性免疫応答を評価するために使用することができる。

- / - OVAペプチドの存在下または非存在下でマウスT細胞プライミングアッセイでは、CFSE標識CD4 + T細胞は、NS4B-P38G変異感染野生型マウス及びMyD88-から精製されたDCと培養した。 5日間またはOVAなしで単独で培養した標識​​されたT細胞は、陰性対照として使用した。 図1Aに示すように、総T細胞は、FL1チャネルで蛍光強度を分析するためにゲートした。マーカーはDC?...

Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

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 ...

in-vitro-analysis-myd88-mediated-cellular-immune-response-to-west

Research

JoVE Journal

Immunology and Infection

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

8.1K ビュー.  The University of Texas Medical Branch. Two flow cytometry-based methods – 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.

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

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

アカゲザルの細胞性免疫応答のマルチカラーフローサイトメトリー解析

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

免疫・感染学

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

の寄生蜂の紹介<em&gt;ショウジョウバエ</em&gt;と抗寄生虫免疫応答

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

感染を防ぐための新しいアプローチとして、PRP：準備と<em&gt;インビトロ</emPRPの&gt;抗菌特性

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

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 ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

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. ...

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

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

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

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

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

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

突然変異体ワクシニアウイルスベクターを構築するためのシンプルで効率的なアプローチ

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

マウスのモデルを評価する生得の免疫反応黄色ブドウ球菌感染症

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...