この作品は、カナダ衛生研究所（MSH - 95368、MOP - 89773とCZのMOP - 106623）とイノベーションのためのカナダ財団によってサポートされていました。 SCMは、消化器ポスドクのCIHR /カナダの協会の受信者です。 CZはCIHR新しい研究者でもある。

1。伝播するネズミ鞭虫の卵<ol><li> （または遺伝的に感受性のあるマウスネズミ鞭虫の卵の新しいバッチを生成するには、20〜30免疫不全マウスに感染（）例：NOD.Cg - Prkdc SCID Il2rg tm1Wjl / SzJ（NSG）または129S6/SvEvTac-Rag2 tm1Fwa（RAG2 - - /）例：。経口胃管栄養法により約300ネズミ鞭虫の卵を6〜8週間経過しているAKR / J）。 </li><li> 32から35日後にCO 2窒息によりマウスを犠牲にする。 </li><li>マウスの腹側を露出させ、70％エタノールで腹部を濡らす。 </li><li>鉗子を使用すると、腹部の皮膚をつかんで、はさみを使って小さな切開を行います。腹部の内容を公開するために皮膚を切り取ります。 </li><li>盲腸と近位大腸を識別し、それらを削除します。 </li><li> 500 U / mlペニシリンおよび500μg/ mlのストレプトマイシン（P / S）とダルベッコ改変イーグル培地（DMEM）を含む10cmペトリ皿に盲腸と近位大腸に置きます。ルーメンとワームを公開するオープンな盲腸を切る。リン酸のビーカーに鉗子の場所の盲腸を使用して、緩衝食塩水（PBS）と糞を除去するために振とうする。 </li><li>ペトリ皿に盲腸を返し、ゆっくりとワームを引き出し、DMEM + P / Sの5mlのを含む6ウェルプレートのウェルに配置滑らかな曲面鉗子（Roboz RS - 5047）を使用して</li><li>マウスのすべてに対してこのプロセスを繰り返します。 </li><li> 37℃湿度とインキュベートを維持するために湿らせたペーパータオルで4時間のためのCでタッパーウェアの容器で6ウェルプレートを置く。 </li><li> 4時間は滑らかな曲面鉗子を使用してワームを削除し、5mLのDMEM + P / Sを含む6ウェルプレートの2つの新しい井戸に分割して後15ミリリットルファルコンチューブにオリジナルも、場所の内容を取得する。 4時間の抗原（典型的なタンパク質の収量が20匹あたり10から20ミリグラムであるPBS透析、後の細胞の再刺激のために使用することができる）を再懸濁しますとして3000rpmで取り外しおよび予備清でチューブを回転させて卵を含むオートクレーブした水道水でペレットを。 </li><li> 37℃で一晩戻し、6ウェルプレートを置く。取り外して、ワームを処分。ファルコンチューブ、5分間3000rpmでスピンに井戸の内容を取得する。一晩抗原として上清と予備を削除する（典型的なタンパク質の収量が20匹あたり10から20ミリグラムであるPBS透析、後鞭のAg特異的抗体のアイソタイプELISA法に使用することができる）と懸濁しますのオートクレーブ水道水でペレットを卵を含む。 </li><li> 4時間と一晩卵を結合し、残ったワームを削除するには、70μmストレーナーを通して卵をフィルタリングするには、75 cm 2のフラスコに移す。室温（RT）で6週間のための箔で覆われた暗い場所でフラスコをしてください。 </li><li>オートクレーブした水道水に卵を洗うと50μlの50現実的な卵で懸濁します。これを行うには、〜50ミリリットル滅菌水道水で5分間懸濁し、3000 rpmで卵を遠心する。スライドガラス上に50μlの卵と場所を削除します。 40倍の倍率を使用すると、50μlの実行可能な孵化卵の数を数えます。 50μlの50実行可能な卵を与えるために卵を希釈する。実行可能な卵のイメージのためにコッパーとマンスフィールド16でスタディを参照してください。 </li></ol> 2。 ネズミ鞭虫卵（200卵/マウス）と実験用マウスの急性感染<ol><li>徹底的に再懸卵とは、マウスの数を治療するために十分なボリュームを削除してX 2（例えば10匹に対して200μlのX線写真を撮る10匹× 2 = 4卵のMLS）と14mlのスナップキャップチューブに配置。 </li><li>ミックスに14mlのスナップキャップチューブを反転。適切なサイズの餌針で1mlのシリンジ（25グラムのマウスを、私たちは18ゲージ1 1 / 2を使用する&quot;針）に卵液200μlを立ち上げます。片方の手の首筋には、マウスや他の手を使っての強制経口投与による卵の管理。 </li><li> 21日待ってください。 </li></ol> 3。実験的な収穫<ol><li> 21日目にCO 2を使用してマウスを生け贄に捧げる。 </li><li>心臓穿刺によりマウスから血液を収集し、非ヘパリンシリンジで、そして4℃で保存します。次の日は、-20℃で血清と凍結を分離血清は、ELISAによって鞭特異IgGをおよびIgEの検出に用いることができる。 </li><li>マウスの腹側を露出させ、70％エタノールで腹部を濡らす。 </li><li>鉗子を使用すると、腹部の皮膚をつかんで、はさみを使って小さな切開を行います。皮膚や腹部の内容を公開するために、基礎となる膜を切り取ります。 </li><li>小腸、盲腸および結腸を識別します。ピンセットで盲腸を取得し、腹腔から引き出します。鉗子の別のペアで腸間膜リンパ節（mLNs）を公開し、右に小腸を押してください。メディアの2 MLSを含む15 50mlファルコンの腸と場所にカットしないように注意しながらmLNsを削除します。これらは、必要なら、ELISAおよび細胞内のfでサイトカインの解析のための72時間処理し、in vitro（1μg/ mlのαCD3/CD28付きまたは50μg/ml4H 鞭銀の24ウェルプレートに1 mlの4 × 10 6個 / ml）で再刺激することができますローサイトメトリー。 </li><li>盲腸と大腸を切り取る。 </li><li>鉗子を使用して、2 mlのAxygenチューブの遠位結腸と場所から糞を押し出す。 -20℃で凍結糞は、レジスチン様分子-β（RELM -β）の発現のためのウェスタンブロットによって分析することができます。 </li><li>盲腸（〜0.5 cm）の先端を取り外します。糞便を削除するにはピンセットで盲腸の先端を保持し、PBSはペトリ皿に3 mlのシリンジと22ゲージの針を使用して洗い流してください。 5ミリリットルファルコンのスナップキャップチューブに新鮮な4％パラホルムアルデヒドで場所。 4℃で保存します。これらはパラフィン包埋、切片と組織学的分析のためのスライド上にマウントすることができます。 </li><li> RNAのための近位結腸の一部（〜1 cm）を取り外します。 500μlのRNAlater中で2ml Axygenチューブにサンプルを置く。 4℃で保存します。これらのサンプルは、定量PCRによるmRNAの発現について分析することができる。 </li><li> -20℃でラベルされたペトリ皿や凍結で盲腸の残りの部分を置き</li></ol> 4。盲腸のネズミ鞭虫ワームの列挙<ol><li> 〜5 mlの水を含むペトリ皿で​​冷凍し、場所から盲腸を削除します。 </li><li>はさみを使用して、内腔を公開するオープンな盲腸を切る。 </li><li>鉗子で盲腸を持ち、精力的に糞便の大半を除去するために盲腸を振る。 </li><li>水を含むと優しくIECSの基盤となる組織を掻き滑らかな曲面鉗子を使用して別のシャーレに盲腸を転送する。 </li><li>水を含む新しいペトリ皿に盲腸を移し、精力的に小さい部分にティッシュをリッピング、ピンセットで残りの組織を削る。 </li><li>シャーレのすべての3でワームのすべてをカウントする解剖顕微鏡を使用してください。このワームは、同じワームを確実にするために皿からそれを削除見つけるの倍カウントされません。ワームが損傷している場合（すなわち半分に壊れて）正確なカウントを取得する唯一の厚いまたは薄い端を数える。 </li></ol> 5。代表的な結果 ネズミ鞭虫の感染モデルでは、研究者が寄生虫の負担と全身免疫応答だけでなく、組織学的に調べるために遠位の盲腸内の炎症反応を定量化することができます。収穫盲腸の内腔が露出されると、 鞭のワームは、解剖顕微鏡（図1A - B）を使用して列挙することができます。 鞭への免疫のパラメーターにも影響を受けやすい動物に抵抗性動物のIgG1の（Th2型の免疫に関連する）、およびIgG2a（Th1型の免疫に関連する）に切り替える抗体のアイソタイプ（図1C）が含まれています。杯細胞特異的タンパク質レジスチン様分子-β（RELM -β）の発現は、 鞭のクリアランスに関連付けられており、ウェスタンブロット解析（図2）で検出可能です。盲腸の先端部分は、杯細胞の応答と感染症（図3）に対応して腸の炎症の程度を評価するために過ヨウ素酸シッフ（PAS）で染色することができます。 <img src="/files/ftp_upload/2774/2774fig1.jpg" alt="図1"/> 図1。 ネズミ鞭虫への耐性の定量化 。 （A）抵抗性（B6）マウスは、21日後に感染により鞭のムリスを追放。これとは対照的に、影響を受けやすい（AKR / J）マウスは、寄生虫の確立で、その結果、 鞭で慢性的に感染する。各バーは平均±グループごとに4-8のSEMは、動物を表しています。 （B）ワームは、解剖顕微鏡を使ってカウントされます。トップパネルが単独でワームを示し、下部パネルには、IECSと糞の存在下でワームを示しています。 （C） 鞭 -特異的IgG2a力価は、排泄の鞭の抗原（O / N銀、5μgの/でコーティングしたプレートに希釈した血清のELISA（1:2希釈、1:20希釈から始まると1:2560で終わる）によって決定されますml）を。感受性のAKR / Jマウスは抵抗性B6マウスよりも鞭特異的血清IgG2aの特に高いレベルを持っている。 <img src="/files/ftp_upload/2774/2774fig2.jpg" alt="図2"/> 図2。 ネズミ鞭虫のクリアランスは、杯細胞特異的タンパク質のRELM -β7の発現に関連付けられています 。 RELMは、-βの生産は、ウェスタンブロット分析によって鞭感染抵抗性（B6）ではなく、影響を受けやすい（AKR / J）マウス由来の糞から検出することができます。各レーンは、（N）=ナイーブ、（TM）= 21日間の鞭感染したマウスの単一動物の代表です。 <img src="/files/ftp_upload/2774/2774fig3.jpg" alt="図3"/> 図3。遠位盲腸以下の鞭の感染症の組織学的検査 。遠位盲腸5-7μmのセクションでは、PASで染色。杯細胞の過形成と粘液産生は、耐性（B6）で明らかですが鞭の感染後のマウスを（AKR / J）影響を受けることはありません。

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動物実験は、ブリティッシュコロンビア大学で定められたガイドラインおよび規制に準拠して行った。

このプロトコルは、詳細調査員の必要に応じて変更できます標準の高用量急性ネズミ鞭虫の感染を。例えば、マウスを屠殺することができ、組織は別の日に収穫。マウスが正常に完全なワームの負担が確立されていることを決定するために、それらはすべてマウスでは約200ワームの重荷を運ぶ必要のある時点で、14日目に犠牲にすることができます。マウスはまた、検出されたワーム?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>動物給餌針（18 × 1 ½&quot;） </td><td>ポッパー</td><td> 7912 </td><td></td></tr><tr><td>滑らかな曲面鉗子</td><td> Roboz </td><td> RS - 5047 </td><td></td></tr><tr><td> DMEM </td><td>ギブコ</td><td> 11965 </td><td></td></tr><tr><td> NOD.Cg - Prkdc SCID Il2r gtm1Wjl / SzJ（NSG） </td><td>ジャクソン研究所</td><td> 005557 </td><td>これらは我々が使用したマウスは、しかし、いかなる免疫不全マウスや感受性株は動作するはずです。 </td></tr><tr><td> RNAlaterは</td><td>キアゲン</td><td> 76104 </td><td></td></tr><tr><td> 2 mlのチューブ</td><td> Axygen </td><td> MCT - 200 - C </td><td></td></tr><tr><td> 15 mlチューブ</td><td>ファルコン</td><td> 352096 </td><td></td></tr><tr><td> 6ウェルプレート</td><td>ファルコン</td><td> 353046 </td><td></td></tr><tr><td>パラホルムアルデヒド</td><td>電子顕微鏡の科学</td><td> 15710 </td><td></td></tr><tr><td> α-RELMβ抗体</td><td>ぺプロテック株式会社</td><td> 0694270Rb </td><td></td></tr></tbody></table>

trichuris muris infection: a model of type 2 immunity and inflammation in the gut

ネズミ鞭虫はネズミの天敵病原体であり、ヒトや家畜1を感染させる鞭の種の生物学的および抗原的に類似しています。感染卵を経口胃管栄養法、遠位小腸で孵化によって与えられる、環境1に線が盲腸の陰窩と近位結腸そのと成熟時に腸上皮細胞（IECS）ワームのリリースの卵に侵入する。このモデルは、コントロールのCD4 + Tヘルパー（Th）細胞の活性化だけでなく、腸上皮の変化その要因を調べるために強力なツールです。このようなC57BL / 6およびBALB / cのような耐近交系、で発生する免疫応答が、Th2の偏サイトカイン（IL - 4、IL - 5およびIL - 13）とTh1細胞関連サイトカイン間（ILワームの追放によって特徴付けられる-12、IL - 18、IFN -γ）遺伝的に感受性のAKR / Jマウス2-6の慢性感染を促進する。 Th2サイトカインが急速IECSの売上高、杯細胞の分化、募集およびワームの追放7-15に関与しているすべてが上皮透過性と平滑筋収縮の変化、を含む腸内微小環境における生理学的変化を促進する。ここでは詳細にその後の実験で使用することができるネズミ鞭虫の卵を伝播するためのプロトコルを。我々はまた、感染後の分析のための提案をサンプル実験的な収穫を提供しています。全体的に、このプロトコルは番目の消化管における傾向と同様にIECSの免疫エフェクター機能に関係する質問に対処するために使用することができるネズミ鞭虫のマウス感染モデルを実行するための基本的なツールを研究者に提供します。

Watch this Scientific Journal Video about Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut at JoVE.com

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

ネズミ鞭虫の感染は、抵抗性のマウスは、保護のTh2応答を生成し、影響を受けやすいマウスは、病理学的Th1応答を生成するTh2の免疫の腸のモデルです。

ネズミ鞭虫感染症：腸内のタイプ2免疫と炎症のモデル

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

trichuris-muris-infection-model-type-2-immunity-inflammation

Research

JoVE Journal

Immunology and Infection

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

16.2K ビュー.  University of British Columbia. ネズミ鞭虫の感染は、抵抗性のマウスは、保護のTh2応答を生成し、影響を受けやすいマウスは、病理学的Th1応答を生成するTh2の免疫の腸のモデルです。

ビデオ: Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

の莢膜血清型<em&gt;肺炎球菌</em&gt;ラテックス凝集による

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

免疫・感染学

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

単離、特徴付けおよび歯肉免疫細胞ネットワークの機能検討

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

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 ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

によってエフェクター転効率を検査するための蛍光共鳴エネルギー移動（FRET）を適用<em&gt; Q熱リケッチア</em&gt;のsiRNAサイレンシング中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

マウスにおける実験的細菌性肺炎の誘導と表現型のための非侵襲的および技術的に非集中的な方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密カットライブ肺樹状細胞を可視化するために、マウスの肺スライスを

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

蛍光ベースのノイラミニダーゼ阻害アッセイ抗ウイルスのノイラミニダーゼ阻害剤クラスにインフルエンザウイルスの感受性を評価します

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

シグナリング蛋白質の廃止、炎症性腸病気のマウスモデルで腸の炎症に味の効果

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

ウイルス感染とキイロショウジョウバエのホスト ウイルスの相互作用の解析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

播種性カンジダ症のハチミツガWaxworm 感染症モデル

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...