この研究は、カナダ血液サービス（CBS）と健康科学（CIHR）研究パートナーシップ基金のカナダの協会によって資金を供給された。作者は彼らの研究施設の使用のために血の研究のためにUBCのセンターでLMB高分子ハブに感謝します。インフラ施設は、イノベーションのためのカナダの基礎（CFI）と健康研究のためのマイケル·スミス財団（MSFHR）によってサポートされています。 R. Chapanianは輸血科学MSFHR研究研修生ポスドクフェローシップのレシピエントにおける（CIHR / CBS）のポスドクの受賞者です。 JN KizhakkedathuはMSFHRキャリア調査官Scholarの賞の受賞者です。

A.ハイパーブランチポリグリセリンの変更（SS-HPG） <ol><li>場所は、丸底フラスコ中の60 kDaの（0.5グラム、0.0083 mmol）をHPG凍結乾燥させ、90℃で真空下で一晩乾かし℃に</li><li>室温にフラスコを冷蔵、無水ピリジン（3ml）中の乾燥HPGを溶かす。 </li><li>カルボキシル基とHPGで約8個のヒドロキシル基を官能基化するには、HPGの溶液に触媒量のジメチルアミノピリジン（ピリジン中5 mg / mlの溶液を1滴）を追加します。この混合物に、10分かけて0.5ミリリットルを滴下ピリジンに溶解し、無水コハク酸、（0.0067グラム、0.0664 mmol）を追加します。アルゴン下、室温で一晩混合物をかき混ぜる。 </li><li> 50 mlの遠心チューブに冷アセトン（4℃）40mlに混合物を沈殿させ、15分間27000×gでベックマンJ2-MCの遠心分離器を用いて遠心分離する。上清をデカントし、室温でアルゴンで洗浄することによって残留アセトンを除去する。 </li><li>カルボキシをアクティブにするにはスクシンイミジルスクシネート（SS）のグループとL基は、無水DMF（3ml）にカルボキシル官能HPGを溶かす。 HPG液にN-ヒドロキシスクシンイミド（0.0077グラム、0.0664 mmol）およびN、N&#39;-ジイソプロピルカルボジイミド（0.0084グラム、0.0664 mmol）を追加し、アルゴン下、室温で一晩混合物をかき混ぜる。 </li><li>冷アセトンで沈殿させることにより、SS-HPGを精製する。 </li><li>アルゴンで洗浄することによって残留アセトンを削除します。 </li><li> NMR分析-変更HPGの純度とプロトン（1 H）によるカルボキシルおよびSS官能化度を決定します。 </li></ol> B.全血採取し、赤血球の分離（赤血球） <ol><li>クエン酸バキュテイナチューブに健康なヒトのドナーを承諾から全血（40％ヘマトクリット、3ミリリットル）を採取します。 </li><li> 4分間1,000×gでクエン酸塩チューブを遠心します。 </li><li>血漿や血小板を含む上清を取り出して、白血球やplateleを含む中間軟膜パスツールピペットを用いてTS。 </li><li> 12ミリリットルファルコンの遠心管に濃厚赤血球（80％ヘマトクリット、1.2ミリリットル）を転送し、赤血球を洗浄するために、PBSバッファー（8ミリリットル液、pH = 8.0）を追加します。 </li><li>均一な細胞分布を得るために反転することによって赤血球を混ぜる。 </li><li> 4分間1,000×gでファルコンチューブを遠心し、上清を除去する。 </li><li>ステップ5と6を繰り返して、PBSで2回以上で赤血球を洗浄します。 </li><li> 1.6ミリリットルエッペンドルフチュー​​ブに入れ、パック-赤血球（100μl）を、20％ヘマトクリット赤血球を取得するためにPBS緩衝液（300μlの液、pH = 8.0）を追加します。 </li></ol> C. HPGは赤血球へのグラフト<ol><li>直ちにステップA6のアセトン中の変性HPGの降水量、ガラスバイアル（1 DRAM）で行われ、SS-HPG（150 mg）を加え、PBS緩衝液（300μlの液、pH = 8.0）でそれを溶解した後。 </li><li> 20％ヘマトクリットにSS-HPGポリマー溶液を加えると、最終的に400μlの体積および0.5mM〜3mmの範囲で、SS-HPG濃度を得るために、赤血球を洗浄した。例えば0.5mMのSS-で処理赤血球を準備するHPG、洗浄赤血球からのPBS 36μlを削除するとSS-HPGのポリマー溶液36μlを置く。 </li><li>渦優しくサスペンション、1時間室温でオービタルシェーカー上のエッペンドルフチュー​​ブを配置します。 </li><li> 4分間1,000×gでエッペンドルフチュー​​ブを遠心分離し、上澄みをピペットで。 </li><li> 、赤血球を洗浄し遠心分離し、上清を除去するために1mlのPBSを加える。 </li><li> 1 mlの生理食塩水を追加し、2倍のステップ5で赤血球を洗浄してください。 </li><li> 20％ヘマトクリットの最終濃度に濃厚赤血球液100μl（80％ヘマトクリット）に生理食塩水を300μlを加える。 </li></ol>変性赤血球をHPG】D.クタリゼーション I.補体媒介性溶解<ol><li>健康なヒトのドナーからBDバキュテイナガラス血清分離管に全血の約8ミリリットルを採取し、30分間室温で血液凝固を可能にします。 </li><li> 10分間2000 xgでチューブを遠心分離し、血清の約3ミリリットルを収集します。 </li><li>出せるの1ミリリットルmは、熱不活性化血清を準備するために30分間60℃の水浴中に入れた。 </li><li> 20％ヘマトクリットHPG修正赤血球または変更されていない赤血球の60μlを含むエッペンドルフチュー​​ブに新鮮な血清または熱不活性化血清60μlを添加する。 </li><li> 37℃で1時間、赤血球をインキュベート℃に</li><li>場所96ウェルプレートに三重に変更された赤血球、あるいは非改変細胞をHPG 20％のヘマトクリット値の5.9μlの細胞懸濁液中のヘモグロビンの量を定量する。 Drabkin試薬（spectrophotomericallyヘモグロビンの量を定量するために使用される試薬）294μlを添加する。 inとoutペッティングによりDrabkinの試薬で細胞を混ぜる。 SPECTRA MAX 190プレートリーダーを用いて540nmでヘモグロビンcyanoderivati​​veの吸光度を測定します。 </li><li>上清中のヘモグロビンの量を定量化するために、1分間13000×gで細胞を遠心分離します。 96ウェルプレートで三重に上清50μlを置きます。 Drabkinの試薬250μlを添加する。 Drabkinの試薬で細胞を混ぜるinとoutピペッティングで。 SPECTRA MAX 190プレートリーダーを用いて540nmでヘモグロビンcyanoderivati​​veの吸光度を測定します。 </li><li>サンプルの上清中のヘモグロビンの割合（ステップ7）、総ヘモグロビン（ステップ6）8、11、12から溶解した細胞の量を定量化する。 </li></ol> Ⅱ。 MTSのカードを使用してメジャーとマイナー抗原のカモフラージュ<ol><li>場所は50エッペンドルフチュー​​ブに変更されたRBC（10％ヘマトクリット）HPGμlの、1分間1,000×gで遠心分離し、上清を除去します。 </li><li>細胞ペレットにMTS希釈液の110μlを添加し、内と外、ピペッティングにより細胞懸濁液を均質化する。 </li><li>各ミニゲルカラムに細胞懸濁液11μlを添加する。添加の間にゲルを触れないようにしてください。 </li><li>遠心分離機のカードホルダーにMTSのカードを見つけて、6分間156×gで遠心分離します。 </li><li>表面抗原の保護は製造に基づいて、ミニゲルカラムにおけるRBCの位置から決定されるrを説明。 </li></ol> Ⅲ。水性二相分配測定<ol><li>デキストラン500kDaの13による（5％デキストラン500K、4％PEG 8K、150mMのNaCl、10mMのリン酸）から成る/ PEG 8 kDaの2相分離システムを準備します。 </li><li>二相系（下におけるデキストラン）を取得するために室温で10分間、433×gで遠心分離します。 </li><li>慎重に二つの相を分離します。 </li><li>標識されたチューブ（凹状の底付き）に上位のPEG相のアリコート1.0mlを、フリックで優しく20 20パーセントヘマトクリットHPG改変RBCの添加および混合セルを追加します。 </li><li>下相0.5mlに細胞が含まれており、フリックで優しくシステムを混在させる上相の0.5 mlを加える。 </li><li>分離して、システム内のセルの位置を決定するシステムのための〜2分間ベンチにチューブを置きます。 RBCのローカリゼーションは、（上部のPEG相で、低いデキストラン相または両方で）修飾の程度への関数であり、分子HPGの重量。 </li></ol> Ⅳ。浸透圧抵抗の測定<ol><li> 0〜0.9（W / V）％の範囲の濃度のNaCl溶液を準備します。 </li><li> 1.5mlのエッペンドルフチュー​​ブにNaCl溶液の代わりに0.4ミリリットル。 </li><li> NaCl溶液中に赤血球（20％ヘマトクリット値）の20μlを添加する。 </li><li>反転し、30分間37℃の水浴中にそれらを置くことによって注意深くミックス細胞。 </li><li>逆さにして慎重に細胞を一時停止します。 RBC懸濁液50μlを取り、キュベットに入れ、1ミリリットルDrabkinの試薬に追加します。 inとoutペッティングによりDrabkinの試薬で細胞を混ぜる。ベックマン·コールターのDU 730 UV / VISリーダーを用いて540nmでヘモグロビンcyanoderivati​​veの吸光度を測定します。 </li><li>上清中のヘモグロビンの量を定量化するために、1分間、1,000×gで細胞を遠心分離します。キュベット内のDrabkinの試薬1mlに上清を200μl加え。 inとoutペッティングによりDrabkinの試薬で細胞を混ぜる。 hemoglの吸光度を測定するベックマン·コールターのDU 730 UV / VISリーダーを用いて540nmにおけるobin cyanoderivati​​ve。 </li><li>上清中のヘモグロビン量の比（ステップ6）からの細胞懸濁液（ステップ5）中のヘモグロビンの総量に対して異なるNaCl溶液中に溶解した細胞の量を計算します。 </li></ol> Vのフローサイトメトリー測定 - アカゲザル-D（RHD）抗原の保護<ol><li> 5コントロールのμlまたは25μlの全体積の0.5％BSA添加PBSバッファへの三連でHPG修正された赤血球（20％ヘマトクリット）を追加します。 </li><li>商Biodiagnostics（PA、USA）から購入したFITCモノクローナル抗アカゲザルD（RHD）の25μlを添加する。 </li><li>水浴中で37℃で30分間混合物をインキュベートする。 </li><li> 1.5ミリリットルのPBS緩衝液と混合して2回洗浄する。上清を除去するために、1分間1,000×gで遠心分離します。 </li><li> 1mlの生理食塩水に赤血球をサスペンドし、4mlのフローBDのフローサイトメトリーチューブに懸濁液を移す。 </li><li> FACSCanto II floのを使用して分析するワットメーター、RBCの制御ゲートに5000のイベントを取得し、分析のために全体のイベントを使用します。 </li></ol> Ⅵ。フローサイトメトリーの測定 - CD47の発現<ol><li> 44μlの全体積の0.5％BSA添加PBSバッファへ三重で1.54制御μlまたはHPG修正赤血球（20％ヘマトクリット）を追加します。 </li><li> BD Biosciences社（ニュージャージー州、米国）から購入したマウス抗ヒトCD47のラベルが付いたフィコエリトリン（PE）の6μlを添加する。 </li><li>水浴中で37℃で30分間混合物をインキュベートする。 </li><li> 2回1.5ミリリットルPBS緩衝液で混合物を洗浄します。上清を除去するために、1分間1,000×gで遠心分離します。 </li><li> 1mlの生理食塩水に赤血球を一時停止し、細胞凝集を最小限に抑えるために26 G 5月8日針を通過。 </li><li> 4mlのフローBDのフローサイトメトリーチューブに懸濁液を移す。 </li><li> RBCの制御ゲートに10,000のイベントを取得し、FACSCanto IIフローサイトメーターを用いて分析します。 </li></ol>

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ユニバーサルドナーの赤血球は輸血療法のための増強血液の可用性と安全性に大きな可能性を秘めている。 RBCはまた、彼らの長い循環と内在する生体適合性14、15に起因する有望な薬物送達ビヒクルと見なされます。本書で提示実験は、修正された赤血球をHPGのin vitroでの特性評価を行った。 in vitroでのプロパティと HPG修正赤血球の in vivoでの循環におけ...

<table border="1"><tbody><tr><td>グリシドール</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td>トリメチロールプロパン</td><td>フルカ</td><td></td><td> （ON、カナダ） </td></tr><tr><td>カリウムメチ</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td>無水ピリジン</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td> 4 - ジメチルアミノピリジン</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td>無水コハク酸</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td>アセトン</td><td>フィッシャー·サイエンティフィック</td><td></td><td> （ON、カナダ） </td></tr><tr><td>無水ジメチルホルムアミド</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td> N-ヒドロキシ</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td> N、N&#39;-ジイソプロピルカルボジイミド</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td> MTSのカード</td><td></td><td></td><td>マイクロタイピングシステム（MTS）カード（フロリダ州、アメリカ） </td></tr><tr><td>デキストラン500kDaの</td><td>ファルマシア·ファインケミカル</td><td></td><td> （スウェーデン） </td></tr><tr><td> PEG 8 kDaの</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td> FITCモノクローナル抗アカゲザルD（RHD） </td><td>商Biodiagnostics </td><td></td><td> （米国ペンシルバニア州） </td></tr><tr><td> PEモノクローナル抗CD47 </td><td> BD Biosciences社</td><td></td><td> （米国ニュージャージー州） </td></tr><tr><td> Drabkinの試薬</td><td>シグマアルドリッチ</td><td></td><td> （ON、カナダ） </td></tr><tr><td></td><td></td><td></td><td> テーブル。赤血球膜とその分析にポリマーをHPGのグラフト化のために使用される化学薬品および試薬。 </td></tr></tbody></table>

antigens protected functional red blood cells by the membrane grafting of compact hyperbranched polyglycerols

赤血球（RBC）輸血は、サラセミアメジャーおよび鎌状赤血球貧血の1から3のように、急性および慢性の医学的な問題の数の治療のために不可欠です。 RBC表面上の抗原の多数の存在（〜308既知の抗原4）のために、慢性的な輸血療法の患者は、輸血赤血球4,5のマイナー抗原のミス·マッチに起因する同種抗体を開発しています。ポリエチレングリコール（PEG）およびハイパーブランチポリ（HPG）などの親水性ポリマーのグラフト化することは、酸素、ブドウ糖、イオン3のような小さな分子の通過に影響を与えることなく、表面抗原と抗体の相互作用を防止するRBC膜上の除外層を形成する。現時点では方法だとRBC表面上の抗原の数が多い（タンパク質と炭水化物ベース）の存在とdによって提示された困難な課題の一部にユニバーサル赤血球ドナー細胞の生成のため利用できませんこのような方法のevelopmentが大幅に輸血の安全性を向上させ、飛躍的に赤血球の可用性および使用を改善します。本報告では、HPGとその評価のグラフト膜によって保護された官能赤血球抗原を開発するために使用されている実験が掲載されています。 HPGsは非常にコンパクトなポリマー6,7、生体適合性であり、脂質膜8,9とマスク赤血球表面抗原10,11を囲む細胞糖衣内に配置されると予想されます。

アカゲザルD抗原およびCD47 RBC表面タンパク質の迷彩は、蛍光標識されたモノクローナル抗体を用いたフローサイトメトリーによって定量化した、代表的な結果を図1に示されている。 HPG-グラフト赤血球（灰色）の場合では、信号の強度は表面タンパク質のマスキングを示す細胞表面への抗体への結合の減少を示す制御赤血球（赤＆緑）に比べて（ピークが左にシフト）減少。 ...

Watch this Scientific Journal Video about Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols at JoVE.com

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

多分岐ポリグリセロールと赤血球（RBC）（HPG）の細胞膜修飾が提示される。修正された赤血球は、水性二相のパーティショニング、浸透圧抵抗と補体媒介細胞溶解を特徴としていた。表面タンパク質と抗原の迷彩は、フローサイトメトリーとマイクロタイピングシステム（MTS）の血液表現型カードを使用して評価した。

抗原は、コンパクトな多分岐ポリグリセロールのグラフト膜によって機能赤血球を保護

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

antigens-protected-functional-red-blood-cells-membrane-grafting

Research

JoVE Journal

Immunology and Infection

18.0K ビュー.  University of British Columbia. 多分岐ポリグリセロールと赤血球（RBC）（HPG）の細胞膜修飾が提示される。修正された赤血球は、水性二相のパーティショニング、浸透圧抵抗と補体媒介細胞溶解を特徴としていた。表面タンパク質と抗原の迷彩は、フローサイトメトリーとマイクロタイピングシステム（MTS）の血液表現型カードを使用して評価した。

ビデオ: Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

補体の枯渇により特定の細胞集団の枯渇

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

免疫・感染学

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

蛍光活性化細胞選別（FACS）によって特定の細胞集団の精製

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

拡大、精製、及びヒト末梢血NK細胞の機能的評価

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

赤血球上の抗体誘導性補体活性化の定量的検出のための方法

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

における骨髄細胞ダイナミクスのリアルタイムイメージング<em&gt;のApc<sup&gt;最小/ +</sup</em&gt;腸腫瘍ディスク共焦点顕微鏡スピニングすることにより

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

過ヨウ素酸シッフ染色で末梢血単核細胞中のグリコーゲンの検出

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

がん抗原の機構理事T細胞受容体の認識を決定するためにX線結晶学、生物物理学、および機能アッセイを使用して、

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.