このプロジェクトは、E.ジョリーに提供されるケースウェスタンリザーブ大学のスタートアップ資金によって賄われていた。感染したカタツムリは住血吸虫リソースセンター、NIH - NIAID（NIAID契約番号HHSN272201000009I）により提供された。我々はまた、感染した住血吸虫カタツムリを確保し、有用な議論のためのサポートのためにクリスキングとロナルドブラントンに感謝。

安全対策： 住血吸虫セルカリアは直接カットや毛穴のニーズなく皮膚に浸透する、と慢性疾患の住血吸虫症を引き起こす可能性があります。S.マンソンは、バイオセーフティレベル2微生物であり、そのため感染のセルカリアで作業する場合、適切な個人用保護具と適切な安全対策に従う必要があります。住血吸虫の成長と変革するために必要な機材のほとんどは、プロトコルの末尾の表に記載されているいくつかの重要な例外を除いて、標準です。すべてのセルカリアの仕事は、隔離された部屋で行われるべきです。 Schistosomulesは、滅菌、承認されたバイオセーフティフードで培養する必要があります。実験用の上着、靴、穴や涙なしのパンツ、そして手首と腕用の防水スリーブプロテクタの防水ペアが必要として、ニトリルやラテックス手袋の二層は、着用してください。偶発的な流出の場合には、すぐにあなたの人になら、激しくこすり、70％エタノールまたは10％漂白剤で汚染されたエリアをスプレー。これは、寄生虫を無効にして十分ですが、すべての可能な感染症が報告され、真剣に取られるべきである。 schistosomulesが本質的に感染性はないが、制度的なガイドラインは変わる、とBSL2注意が非感染性の生命の段階で必要とされているかどうかを決定するために調べられるべきである。実験が行われ、承認バイオハザード容器に廃棄物を廃棄された後に感染性物質と接触するすべての表面を消毒する。 注記：カバーのカタツムリのホストのビン1〜2日前に捨て、光から保護するために 1。カタツムリのホストからセルカリアを収穫<ol><li>室温に蒸留水で半分フル洗剤の自由なきれいな、500ミリリットルビーカーを埋める。別の日にmiracidiumの複数形に感染したホストのカタツムリで作業する場合、（カタツムリが将来の実験のために再利用される場合、これは特に重要です）それぞれの感染の日付ごとに別々のビーカーを使用してください。ビーカーの大きさはまたユーザーの経験に応じて小さくすることができます。 </li><li>あなたが当てると慎重に、必要に応じて標準的な観賞魚ネットと汎用の鉗子を用いてビーカーにそれらを堆積する感染カタツムリのホストを収集する。 </li><li>緊急時の流出の上に置いてビーカーは白熱光源の下に約8〜12インチをトレイ。光の下で1-2時間放置する。明るい光にさらされるとセルカリアは、カタツムリのホストを終了します。 </li><li>慎重にセルカリアの混合物からカタツムリを除外。混合物は非常に感染であるため、余分な安全予防策を講じる。 忘れないで、セルカリアは直接人間の皮膚に浸透することができます！ </li><li>任意の粒子の廃棄物が解決できるように、約15分間の光源下で混合物を交換してください。 </li><li>ビーカーの底に破片を避け、ピペットを用いてきれいな三角フラスコにセルカリアを転送します。 60分、セルカリアが一角に定着できるようにすること - 45のための暗所で氷上で約45 °の角度でフラスコを置きます。 </li><li>上清の75〜90％を除去し、10％の漂白剤または70％エタノールを入れたビーカーにそれを預ける、水滴がピペットからセルカリアのドリップを含むように注意しながら、50 mLのピペットを使用してください。 </li><li>スワール三角フラスコは、溶液中のセルカリアを再懸濁させる優しくセルカリアを含む。滅菌50 mlコニカルチューブにサンプルを転送するためにピペットを使用してください。暗所で20分間氷上で再びチューブを置きます。セルカリアは暗闇の中でチューブの底に向かって沈殿します。 </li></ol> 2。 schistosomulesにセルカリアの手動変換 作業を始める前に：RPMI 1640で完了（RPMI、5％ウシ胎児血清、1 ×ペン/連鎖球菌）と37℃まで暖かくしてください（これは、住血吸虫は、補完するように注意を払う必要があるとして使用されるウシ胎児血清で1時間インキュベーション56℃で不活性化されていることが重要です）。 <ol><li>バイオハザードフードでは、ピペッターまたは真空装置を使用して、各50 mlコニカルにおけるソリューション5〜10mlにダウン上清を取り除く。 </li><li>適切なサイズのシリンジに22ゲージダブルエンド、ルアーロック乳化針を取り付けます。 </li><li>ゆっくりと注射器に混合物を描く。シリンジ内のすべての50 mlチューブからセルカリアを収集する。滴下の気配りである。 </li><li>慎重にアタッチされていない針の端が上になるように上の注射器を回します。乳化針のこの側面に別のシリンジを取り付けます。 </li><li>セルカリアを変換するために針を通して40倍（20倍前後に）の合計を混合して渡す。 </li><li>混合物が底の注射器に含まれており、トップシリンジが空であることを垂直にそのような注射器を置きます。トップシリンジを外し、70％エタノールで消毒。 </li><li>きれいな高い壁に囲まれた100ミリメートルx 50mmのパイレックスの皿または結晶皿の中にドロップによる預金の混合物をドロップ。 70％エタノールで針と注射器を消毒する。 </li><li> 37℃のRPMI 1640完全なメディアがそのようなこと追加ルリアブロスプレートを注ぐかのようにペトリ皿の底は完全に、覆われている。 </li><li>ペトリ皿の中央にschistosomulesを収集するために穏やかに渦巻く。水はねを避ける。 注：これは、より適切に中央にschistosomulesを視覚化するこのステップのためにボンネットのライトをオフにすると便利かもしれません。 </li><li>直ちに滅菌スポイトを使用しても新鮮な12ウェル細胞培養プレートに集中schistosomulesを入金。 </li><li> schistosomulesが皿の中心（約10倍）で、もはや存在しなくなるまで、2.9、2.10繰り返します。新鮮なだけでなく後続の各コレクションを入金。 </li><li>よく、最大半分（〜1.5 mL）に温めておいたRPMI完全培地でそれぞれを埋める。 </li><li> schistosomulesとセルカリアの不在の存在を確認するために倒立顕微鏡で視覚化する。 </li><li>成長するCO 2インキュベーターでschistosomulesは37に設定されて℃、5％CO 2。 </li></ol> 3。代表的な結果： 培養培地に変換schistosomulesの転送、保留中、反転明視野顕微鏡（例えば、機器の表を参照）の下で可視化は、変換schistosomulesがよく中に存在する1を、図に類似した画像を生成する必要があります。潜在的な汚染物質がそのままセルカリア、切断セルカリア尾、または他の破片を含めることができます。は倒立顕微鏡が利用できない場合は、標準的な顕微鏡あるいは解剖スコープはschistosomulesを可視化するために十分であろう。この手順は常にセルカリアの100％を変換しないことに注意してください。 <img src="/files/ftp_upload/3191/3191fig1.jpg" alt="図1"/> 図1：変換された幼住血吸虫<img src="/files/ftp_upload/3191/3191fig2.jpg" alt="図2"/> 図2：セルカリアと断絶された尾

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利害の衝突は宣言されません。

いくつかの潜在的な問題は、変換手順の実行中に発生する可能性がありますが、このプロトコルは、これらの問題のほとんどを回避するために設計されています。つの潜在的な危険は、通常、ステップ1.3の収穫から残っている破片のために生じた乳化針の目詰まりです。ステップ1.5で解決すると、ステップ1.6で慎重にセルカリアを転送するすべての粒子状物質は、この問題を回避できるようにしてください。また、のような生物学的安全キャビネット内部の作業、前に説明した、個人用保護具の使用（さえフェイスシールド）と注射器の過度の圧力の回避が望ましい。 切り離されたセルカリア尾は、ステップ2.9の間に貧困層の分離の指標である、メディアで視覚化することができます。尾は、痙攣のような動きを示すと（図2を参照）異なる形状を有し、そしてマンドリーバschistosomulesと簡単に区別できます。尾はschistosomulesが解決できるように完全RPMIを超えるschistosomulesを洗浄することによって除去することができます。尾はフローティングのままとなり、オフバキュームすることができます。 無傷のセルカリアがメディアに存在する場合、ステップ2.5での変換が正しく実行されませんでした。無傷のセルカリアの画像は参考のため図3bに示されている。この問題が発生した場合は、使用されている乳化剤針が適切なサイズであることを保証する。 40倍が一般的に十分なセルカリア尾の100％のせん断にアプローチするよりもはるかに多くのですがまた、セルカリア混合物が針を通過する回数は、増加することができます。 schistosomulesにセルカリアの転換は、最初のコリーとWikel（13）によって1974年に記述されていた。それ以来、schistosomulesの調製は、このプロトコルで説明されているように、針と注射器との併用でもせん断戦略を使用して、またはボルテックスアプローチを使用し、パーコールの勾配（14、15）で区切ったり、本論文で示すように渦巻いて行われてきました。準備schistosomulesのためのこれらの2つのテクニックは、美しくフレッドルイス（16）によって記述されており、長期的な成長のためのschistosomulesのさらなる培養のための詳細については、NIAID住血吸虫リソースセンター（で見つけることができます<a href="http://www.schisto-resource.org/">www.schisto - resource.org</a> ）。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td> 22グラムX 2-7/8&quot;乳化針</td><td>フィッシャーサイエンティフィック</td><td> 14から825 - 17G </td></tr><tr><td> RPMI 1640培地1X（500ミリリットル） </td><td>インビトロジェン</td><td> 11835030 </td></tr><tr><td>ウシ胎児血清（FBS）熱不活化</td><td>インビトロジェン</td><td> 10082139 </td></tr><tr><td>ペニシリン/ストレプトマイシン（100X） </td><td>インビトロジェン</td><td> 15070063 </td></tr><tr><td>結晶皿（100ミリメートル） </td><td>フィッシャーサイエンティフィック</td><td> 08 - 741D </td></tr><tr><td>汎用ピンセット</td><td>フィッシャーサイエンティフィック</td><td> 10 - 316C </td></tr><tr><td>ブラインシュリンプのためのペトコ水族館ネット</td><td>ペトコ</td><td> SKU：1190954 </td></tr></tbody></table>

cercarial transformation and in vitro cultivation of schistosoma mansoni schistosomules

住血吸虫寄生虫は住血吸虫症の原因物質、世界的に2億人以上の人々に影響を与え、公衆衛生と社会経済的インパクト（1-4）の点では寄生虫疾患の中でマラリアに次ぐ慢性的に衰弱させる病気です。住血吸虫寄生虫は自由遊泳性の段階が介在すると軟体動物と哺乳類宿主における寄生生活の間で交換する複雑な生活環を持つ吸虫ワームです。簡単に言えば、自由遊泳性のセルカリアはschistosomulesに変換、セルカリアは彼らの尾を失い、その間、分泌されるプロテアーゼの助けを借りて、皮膚を​​貫通することによって哺乳動物宿主に感染する。 schistosomulesは、宿主免疫システムを回避する赤血球の消化のための腸を開発し、移行する必要がありますしかし、肺および肝門脈系の最終的な宛先に向かう途中門脈循環および最終的に腸間膜静脈（S.マンソン用）ここで、男性と女性のワームのペアとチームメイト、毎日卵の何百もの生産。卵の一部は、自由遊泳miracidiumの複数形（5-10）に卵の孵化新鮮な水、体内から排泄される。 miracidiumの複数形は、特定のカタツムリの種に感染し、順番に、ライフサイクルを完了、感染のセルカリアを生み出す母と娘sporocysts、変身。残念ながら、全体の住血吸虫のライフサイクルは 、in vitro で培養することはできませんが、感染性のセルカリアはschistosomulesに変換することができる、とschistosomulesは、 インビトロまたはマイクロアレイ解析における住血吸虫開発の分析のために週間培養することができます。このプロトコルでは、我々はセルカリア変換とschistosomules の in vitro培養での視覚的な説明を提供します。私たちは、カタツムリのホストビオンファラリア属のglabrataから感染セルカリアを当てると手動で乳化両頭針を使用して尻尾を切り離して、schistosomulesに変換すれば良い。 体外セルカリア変換とschistosomules培養技術で 、住血吸虫の可視化を簡素化し、実験的な分析のために寄生虫の収集を容易に。vitroでの変換と住血吸虫の培養技術で年間行われている哺乳動物宿主、（11の使用を避ける説明12）が、コミュニティ全体で利用できる開発されている視覚的なプロトコル。

Watch this Scientific Journal Video about Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules at JoVE.com

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

我々は、説明<em&gt; in vitroで</em扁形動物寄生虫の培養schistosomulumの複数形のための&gt;方法<em&gt;マンソン住血吸虫</em&gt;、淡水カタツムリ中間のホストビオンファラリア属のglabrataから感染セルカリアの収穫とトランスフォーメーションによる。

セルカリア変換と in vitroで</emの>栽培マンソン住血吸虫 Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

cercarial-transformation-vitro-cultivation-schistosoma-mansoni

Research

JoVE Journal

Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

18.0K ビュー.  Case Western Reserve University. 我々は、説明 in vitroで方法マンソン住血吸虫、淡水カタツムリ中間のホストビオンファラリア属のglabrataから感染セルカリアの収穫とトランスフォーメーションによる。

ビデオ: Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

HIV - 1コアのin vitroでの脱殻で

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

免疫・感染学

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

から卵を使用して、<em&gt;マンソン住血吸虫</em&gt;として<emインビボ&gt;</em蠕虫によって誘発される肺の炎症の&gt;モデル

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

定量的な<em&gt;インビトロ</em静的な条件の下で活性化された初代ヒト微小血管内皮細胞への好中球の接着を測定するために&gt;アッセイ

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

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 ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

日和見菌病原体への蛍光タグ付き遺伝子のバイオリスティック変容<em&gt;クリプトコックス·ネオフォルマンス</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

マウスにおける強制経口投与に続いてプロバイオティクス酵母の形質転換および消化管免疫組織から彼らの回復

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

治療抗体間の標的結合およびCDC誘導を比較するためのインビトロ法：Biosimilarity Analysisにおける応用

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

インビトロバス発酵システムを用いて内生生物とヒト腸内細菌叢との相互作用の解析

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

インビトロ転写mRNAによる一次マクロファージの高効率トランスフェクション

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

セルカリア変換と in vitroで栽培マンソン住血吸虫 Schistosomules

セルカリア変換と in vitroで栽培マンソン住血吸虫* Schistosomules*