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

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

げっ歯類における腸運動と腸壁の炎症の機能評価：腸管操作の標準化されたモデルで分析

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

医学

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

ザ<em&gt;シトロrodentium</em&gt;マウスモデル：感染性大腸炎に勉強病原体とホスト貢献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

免疫・感染学

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

の栽培<em&gt; Heligmosomoides Polygyrus：</em&gt;免疫調節虫寄生虫とその分泌製品

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

T細胞への抗原提示細胞による抗原の提示を研究するための抗原駆動性大腸炎モデルの開発

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the entrance of bacteria or bacteria-derived products into the host, accessing the host circulation and inner organs leading to the uncontrolled inflammation as observed in patients with inflammatory bowel disease (IBD), that are characterized by an increased intestinal epithelial permeability.
To mimic the entrance of bacterial-derived compounds into the host, an endotoxemia model has been adopted in which lipopolysaccharide (LPS), a component of the outer cell wall of Gram-negative bacteria, were injected into mice. In this study, a sublethal dose of LPS was intraperitoneally injected and the mice were subsequently monitored for 8 h using a disease score. Furthermore, the expression levels of the inflammatory cytokines Il6, Il1b, and Tnfa were analyzed in the spleen, liver and colon by qPCR at different time points post LPS injection. This model could be useful for the studies involving investigation of immune responses after the invasion of microorganisms or bacterial-derived products caused by a barrier breach of body surfaces.

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

リポ多糖の腸粘膜バリアー違反の後微生物由来製品の入り口を模倣するマウスに注射

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

IBDのDSS誘発モデルにおける腸炎の調査

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

生物学

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

慢性サルモネラ感染誘発性腸線維症

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

マウスの遠位人工肛門、転用 c57bl/6 マウス大腸炎の新たなモデル

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

免疫学的洞察のための強化された腸内チッププラットフォーム

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system&#39;s response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.

著者スポットライト:感染症研究のための免疫適格臓器チップモデルの開発

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

腸粘膜免疫応答を解析するための免疫適格腸内チップモデル

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent ...