我々はHBEC - 5iは細胞のためにサンフランシスコCandal、CDC技術移転オフィス、ジョージア州アトランタに感謝。この作品は、ウェルカムトラスト（ACおよびJARへの基本的な生物医学科学のシニアフェローシップ、助成金番号084226から4年間の博士課程の学生の身分）によって賄われていた。

一般的な推奨事項人間の脳の微小血管内皮細胞（HBEC - 5i）の文化は、以前は次の6に記載されている、8。P.熱帯熱マラリア原虫は、9と同様に培養した。 HBEC - 5iおよびP.両方熱帯熱マラリア原虫の培養は常に無菌状態に維持する必要があります。すべての試薬は37℃であらかじめ温めたされるべき我々は定期的にPCRによるマイコプラズマ汚染10（Minevera Biolabs社、製造元の指示に従います）をチェックすることをお勧めします。プロトコールを図1に要約される。 1。内皮細胞のルーチン培養<ol><li>必要な試薬を準備します。 </li></ol><table border="1"><tbody><tr><td> 準備する培地 </td><td> 試薬 </td><td> 数量 </td></tr><tr><td> &quot;不完全なDMEM&quot; </td><td> DMEM - F12ハム</td><td> 500ミリリットル</td></tr><tr><td> ＆ニッポン放送p; </td><td> L -グルタミン200mmの</td><td> 5ミリリットル</td></tr><tr><td></td><td>ペニシリン/ストレプトマイシン100X </td><td> 5ミリリットル</td></tr><tr><td></td><td> NaOHを1M </td><td> 1.3ミリリットル（7.4にpHを調整） </td></tr><tr><td> &quot;完全DMEM&quot; </td><td> &quot;不完全なDMEM&quot; </td><td> 450ミリリットル</td></tr><tr><td></td><td>胎児ウシ血清熱不活化</td><td> 50ミリリットル</td></tr><tr><td></td><td>内皮細胞成長サプリメント</td><td> 5ミリリットル</td></tr></tbody></table> <ol start="2"><li>文化、5％CO 2で37℃インキュベーターで10ミリリットルDMEM完全培地で通気孔25センチメートル2フラスコでHBEC - 5I。 </li><li>継代数の細胞がコンフルエントになる。吸引して、古い培地を除去し、二回DMEM不完全培地または組織培養グレードのPBSを（のCa 2 +およびMg 2 +フリー）予め温めておいた37℃使用して洗う</li><li>予め温めておいた試しに1mlの追加フラスコの全体をカバーし、37〜2分間インキュベートするpsin - EDTA（0.025％トリプシン、0.5 EDTA）、スワール℃に</li><li>細胞の少なくとも90％が分離されていることを倒立顕微鏡下で確認してください。必要に応じて、軽く接着細胞を取り除くために、フラスコの底をノック。 15ミリリットルコニカルチューブにトリプシンおよび転送細胞をブロックするDMEM完全培地10mlのを追加。室温（RT）で300 gで4分間遠心。 </li><li>上清を捨て、DMEM完全培地10mlのでペレットを再懸濁します。徹底的に細胞を再懸濁するために上下ソリューションをピペット。 </li><li>新しい培養フラスコに細胞懸濁液の1または2 mlを加え、文化を維持するために新鮮な培地の8ミリリットルを追加します。倒立顕微鏡下で毎日文化の成長を評価し、それが黄色に変わる前に、2,3日ごとに1培地を変更してください。 </li></ol> 2。選択のための内皮細胞の準備<ol><li>前のSelectIOの日の2日前まで<html> nは、一つ（またはそれ以上）60ミリメートルペトリ皿に滅菌PBSで希釈したフィブロネクチン（濃度2μg/ cm 2）を加える。 37℃で5〜20分間の一品をインキュベート° Cは、その後の月のために4℃で保存し、一度、再使用可能なフィブロネクチンソリューションを、削除してください。 </li><li>継代数の細胞は、セクション1.3で説明。 1.5〜。と仮定すると100パーセントコンフルエント細胞が分離し、10ミリリットルDMEM完全培地（1.6節。、コンフルエントが低い場合に培地の同等のボリュームで再懸濁し、コンフルエンシーが80％だった場合、例えば 8ミリリットルで再懸濁）に再懸濁し、懸濁の1.5ミリリットルに追加フィブロネクチンコートしたペトリ皿とDMEM完全培地の別の1.5ミリリットルの細胞。 </li><li>インキュベーターでシードペトリ皿を置きます。注：理想的には、細胞集密度は二日後に選択の時点で約90％になります。 </li><li> 省略可能 。 HBEC - 5iはアクティブにするには、前の選択に50μg/ml24時間の最終濃度でTNF（腫瘍壊死因子）サイトカインを加える。 </li></ol>e_title&quot;&gt; 3。 熱帯熱マラリア原虫のルーチン培養<ol><li>必要な試薬を準備します（ここで下の表を参照）。白血球除去フィルター（一般的なマラリア原虫の培養のための出版物11&quot;マラリア研究の方法&quot;を参照）を通過させることによって全血（グループO +）を分離することによって、ヒト赤血球（RBC）を準備します。 5分間400gで遠心分離と10mlのRPMI不完全でそれらを再懸濁することにより回RBCを洗ってください。 4で洗浄赤血球を保つ° Cの不完全培地で50％のヘマトクリット値で。 </li></ol><table border="1"><tbody><tr><td> 準備する培地 </td><td> 試薬 </td><td> 数量 </td></tr><tr><td> &quot;不完全なRPMI&quot; </td><td> RPMI 1640（炭酸水素） </td><td> 500ミリリットル</td></tr><tr><td></td><td> HEPES 1M </td><td> 12.5ミリリットル</td></tr><tr><td></td><td>グルコース20パーセント</td><td> 5ミリリットル</td></tr><tr><td></TD&gt; <td> L -グルタミン200mmの</td><td> 5ミリリットル</td></tr><tr><td></td><td>ゲンタマイシン50mg/ml </td><td> 250μlの</td></tr><tr><td></td><td> NaOHを1M </td><td> 0.7ミリリットル（7.2にpHを調整） </td></tr><tr><td> &quot;RPMIを完了&quot; </td><td> &quot;不完全なRPMI&quot; </td><td> 450ミリリットル</td></tr><tr><td></td><td>ヒト（非免疫）プール血清</td><td> 50ミリリットル</td></tr></tbody></table> <ol start="2"><li>文化P. 37 2％のヘマトクリット値とインキュベートでRPMI完全培地℃、3％CO 2、1％O 2、および96％N 2。と熱帯熱マラリア感染赤血球寄生虫（図2）の開発の段階を評価するためのギムザ塗抹標本11は毎日行います。 </li><li>定期的に（およそ週一回）ソルビトール処理12で文化を同期させる。前日に選択アッセイ、5％の寄生虫以上のリングステージの培養のための目的（理想的に少なくとも10％）。 </li></ol> 4。 P.の選択内皮細胞へのcytoadhesionのための熱帯熱マラリア原虫 <ol><li> HBEC - 5iは文化が（理想的には90％）50から100パーセントでコンフルエントになるはずながら、アッセイの日には、寄生虫の文化は、色素栄養型の段階（ 図2）（理想的に10％の寄生虫）になっているはず。寄生虫文化の30μlの血中血球容積はHBEC - 5iはペトリ皿ごとに必要とされる。 </li><li>寄生虫の文化の1.5ミリリットル（5分間500g）を遠心分離によって二回寄生虫を洗ってください。上清を捨て、焼きたての、暖め、DMEM不完全培地10mlので懸濁します。洗浄をもう一度繰り返す。 1％BSAを持つ不完全なDMEM中1.5ミリリットルと30μlの血中血球容積を再懸濁します。 </li><li>二回培地を吸引し、不完全なDMEMの3ミリリットルを追加するHBEC - 5iはコーティングされたペトリ皿を洗ってください。 </li><li> 37 HBEC - 5iは皿とインキュベート寄生虫の解決° 75分のためのCを追加。インキュベーション中に（30および60分後）二回寄生虫を再懸濁します優しく四方の料理だけでなく、時計回りと反時計回りに揺動して。 </li><li>インキュベーション後、暖かいDMEM不完全培地、および穏やかなロッキングの3mlを追加するには、プラスチック製パスツールピペットを用いて、培地を吸引し皿を5回洗浄する。多くの皿が使用されている場合は、そのような37℃の水で満たされた大きなフラスコ℃と、暖かい表面にそれらを保つ</li><li>倒立顕微鏡下で料理を確認してください。多くの非感染赤血球がまだ表示されている場合、上記のようなより多くの洗浄を行います。汚染の危険を避けるために、非常に慎重にペトリ皿を扱う。 </li><li>皿から培地を除去し、新鮮なuRBCの40μlの血中血球容積で暖かいRPMI完全培地の3ミリリットルを追加します。 </li><li>それは、3分間、気密インキュベーションチャンバー内にガスを料理を置き、37℃で一晩インキュベーターにチャンバーを配置。 </li><li>翌日、セクション4.4で説明したと同様の方法で、RPMI不完全培地で洗浄することによって寄生虫を収穫。しかし、より積極的に。ケーpはすべての培地は、15ミリリットルコニカルチューブに（再懸濁されたRBCを含む）を使用。すべてRBCが皿から削除されていることを倒立顕微鏡下で確認してください。 </li><li>ペレット寄生虫は、5ミリリットルRPMI完全培地に再懸濁し、上清を、破棄し、通常の培養用フラスコ中で混合物を配置。 </li></ol> 5。代表的な結果選択されていない寄生虫はHBEC - 5I（図3A）に結合する低レベルを示しています。このように、選択の第1ラウンド後に、非常に少数の寄生虫を収穫され、それが選択の第二ラウンドのための十分な寄生虫に到達するための培養の月間ほどかかる場合があります。選択の各ラウンドの後、より多くの寄生虫は、内皮細胞に結合し、選択はより短い時間間隔で繰り返すことができます。選択の4-5ラウンド後に、高結合寄生虫の個体数が得られる（図3）。 私たちの手で、HBEC - 5IまたはTNF活性HBEC - 5i]のHB3 - HBECの結合は、SIMであったILARレベル（データは示さず）。 ここで記述されたプロトコルは、4 P.でテストされています熱帯熱マラリア原虫株 ：HB3、IT/FCR3、3D7とDD2（表1）。唯一の後者は、さらに選択の5ラウンドの後、HBEC - 5Iにバインドできない証明した。 HB3の寄生虫はまた、ヒト皮膚と肺微小血管内皮細胞（HDMECとHPMEC）への細胞接着のために選択した。ここに記載された方法を使用して、選択の4ラウンド、後に、高結合人口はHDMECとHPMEC（図4）の両方が得られた。 <img alt="図1" src="/files/ftp_upload/3122/3122fig1.jpg" /> 図1。選考プロセスの概要。 <img alt="図2" src="/files/ftp_upload/3122/3122fig2.jpg" /> P.の図2。発展段階熱帯熱マラリア原虫は赤血球に感染する 。ギムザ塗抹標本では1000X倍率で顕微鏡下で可視化した。 <img alt="図3" src="/files/ftp_upload/3122/3122fig3.jpg" /> 図3。HBEC - 5i]の結合寄生虫の典型的な例 。 （A）青の層は、ギムザでグルタルアルデヒドとステンドグラスでHBEC - 5iは固定です1000X倍率で顕微鏡下で可視化した。 uRBCとバインドされていないiRBCが洗い流された後、撮影された。左側のパネルでは、単一のPを示しています。 HBEC - 5Iにバインドされている熱帯熱マラリア原虫 HB3（未選択）寄生虫。右側のパネルではHB3 - HBEC寄生虫は5ラウンドのために選択されていた。 （B）データは2つの独立した実験、重複で実行される各の平均を表しています。内皮細胞当たりの結合寄生虫の数をカウントした。 <img alt="表1" src="/files/ftp_upload/3122/3122table1.jpg" /> 表1。成功 HB3もTNF活性化HBに選択されていることをHBEC - 5I（注） への結合のために選択された熱帯熱マラリア原虫株の概要EC - 5iは。選択の5ラウンドの後、DD2株が選択されていない- DD2に比べHBEC - 5Iへの結合の増加を示さなかった。 <img alt="図4" src="/files/ftp_upload/3122/3122fig4.jpg" /> 図4 Pの結合選択の4ラウンドの前と後の熱帯熱マラリア HB3皮膚に寄生虫（HDMEC）と肺（HPMEC）内皮細胞、。HDMECとHPMECのための培養液がわずかに異なりますが（供給者の指示を参照）、選択のために使用されるプロトコルがあるとして同一であったHBEC - 5iは。 400Xの倍率で撮影した画像。 <table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> DMEM - F12ハム</td><td>シグマ</td><td> D6421 </td><td> DMEM完全培地用</td></tr><tr><td> L -グルタミン200mmの</td><td>GIBCO </td><td> 25030 </td><td> DMEMおよびRPMI完全培地用</td></tr><tr><td>ペニシリン/ストレプトマイシン100X（10000単位/ mlおよび10mg/ml） </td><td> ScienCell </td><td> 0503 </td><td> DMEM完全培地用</td></tr><tr><td>胎児ウシ血清熱不活化</td><td> ScienCell </td><td> 0025 </td><td> DMEM完全培地用</td></tr><tr><td>トリプシン- EDTA（0.025％トリプシン、0.5 EDTA） </td><td> ScienCell </td><td> 0103 </td><td></td></tr><tr><td>内皮細胞成長サプリメント</td><td> ScienCell </td><td> 1052 </td><td> DMEM完全培地用</td></tr><tr><td>組織培養は、60 mm X 15ミリメートルのペトリ皿を扱わ</td><td> BD </td><td> 353002 </td><td></td></tr><tr><td>ヒトフィブロネクチン</td><td>ミリポア</td><td> FC010 </td><td>濃度2μg/ cm 2で使用</td></tr><tr><td> TNF </td><td> R＆Dシステム</td><td> 210 - TA </td><td>オプションは、50μg/ mlで使用</td></tr><tr><td> RPMI 1640 </td><td>ロンザ</td><td> BE12 - 167F </td><td> RPMI完全培地用</td></tr><tr><td>ゲンタマイシン50mg/ml </td><td>ロンザ</td><td> 17 - 518Z </td><td> RPMI完全培地用</td></tr><tr><td> HEPES 1M </td><td>ロンザ</td><td> BE17 - 737E </td><td> RPMI完全培地用</td></tr><tr><td> HDMEC </td><td> ScienCell </td><td> 2000 </td><td>一次細胞株</td></tr><tr><td> HPMEC </td><td> ScienCell </td><td> 3000 </td><td>一次細胞株</td></tr><tr><td> HBEC - 5I </td><td></td><td></td><td>フランシスコCandal（fcandal@cdc.gov）から入手</td></tr></tbody></table> 表2。マテリアル

<ol>
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Microbiol. , (2009).</li><li>Nakamura, K., Hasler, T., Morehead, K., Howard, R. J., Aikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum-infected+erythrocyte+receptor(s)+for+CD36+and+thrombospondin+are+restricted+to+knobs+on+the+erythrocyte+surface.">Plasmodium falciparum-infected erythrocyte receptor(s) for CD36 and thrombospondin are restricted to knobs on the erythrocyte surface.</a> J. Histochem. Cytochem. 40, 1419-1422 (1992).</li><li>Wassmer, S. C., Cianciolo, G. J., Combes, V., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LMP-420,+a+new+therapeutic+approach+for+cerebral+malaria.">LMP-420, a new therapeutic approach for cerebral malaria.</a> Med. Sci. (Paris). 22, 343-345 (2006).</li><li>Wassmer, S. C., Combes, V., Candal, F. J., Juhan-Vague, I., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+potentiate+brain+endothelial+alterations+induced+by+Plasmodium+falciparum.">Platelets potentiate brain endothelial alterations induced by Plasmodium falciparum.</a> Infect. Immun. 74, 645-653 (2006).</li></ol>

我々は、開示することは何もない。

脳マラリアの特徴は、P.の隔離です。脳内で熱帯熱マラリア iRBC微小血管系2、3。しかしながら、P.の in vitro 培養における HBEC - 5iは、人間の脳微小血管内皮細胞のモデルにのみ不十分cytoadhere 熱帯熱マラリア 。ここでは、表現型より&quot;のような生体内 &quot;、HBEC - 5Iにバインドするための人口を豊かにする簡単なアッセイを開発した。 P. 4のうち三熱帯熱マラリア原虫株は正常にこのメソッドを使用して選択した。さらに、HB3もこのプロトコルは、様々な寄生虫や内皮細胞のタイプに使用できることを示す、HDMECとHPMECに選ばれました。 別の仮説は、DD2歪みと結合性の欠如を説明することがあります。この寄生虫のラインがknoblessであるという事実が、最も可能性が高い、これは深く細胞接着13、14を妨げる。 我々は選択のプロと一緒に培養する選択されていない寄生虫をお勧めします比較のために制御を提供するcess。これは、寄生虫リガンドの候補を発見するのを期待して、結合および非結合寄生虫のトランスクリプトームを比較し、例えば、できるようになります。 TNFは脳マラリアの患者では高いレベルにあるサイトカインであるとHBEC - 5I（Claessens ら 、準備中、8、15、16）の多くの表面タンパク質の発現を誘導する表示されています。このケースでは、バインドされたiRBCの量は、活性化HBEC - 5Iに比べて通常のHBEC - 5Iで同様であった。 この&quot;隔離モデルは&quot;またiRBCと内皮細胞間の分子相互作用だけでなく、推定上の抗細胞接着の薬の効果を研究するために使用することができます。このケースでは、我々はより小さいような&quot;8ウェルチャンバースライド&quot;（BD 354628）のような井戸、または&quot;CultureWell&quot;（シグマC7735 - 20EA）のHBEC - 5iはメッキをお勧めします。

selection of plasmodium falciparum parasites for cytoadhesion to human brain endothelial cells

ほとんどの人間のマラリアの死亡は血液段階熱帯熱マラリア原虫の寄生虫によって引き起こされます。脳マラリア、疾患のほとんどの生命を脅かす合併症は、脳2-4の微小血管系における色 ​​素栄養型の段階での熱帯熱マラリア原虫感染赤血球（iRBC）の蓄積によって特徴付けられる。この微小血管閉塞（隔離は）アシドーシス、低酸素症と有害な炎症性サイトカイン（5レビュー）につながる。隔離は、人間の体2、3のほとんどの微小血管の組織に含まれています。 iRBCが血管壁に付着するメカニズムはまだよくわかっていない。 不死化ヒト脳微小血管内皮細胞株（HBEC - 5iが）血液脳関門6のin vitroモデルとして使用されています。しかし、 熱帯熱マラリア原虫 iRBCは密sequestratとは異なり、in vitroで HBEC - 5Iにわずかしかアタッチ脳マラリアのケースで発生するイオン。そこで、（豊かに）様々なPを選択するためにパンアッセイを開発HBEC - 5Iへの接着のための熱帯熱マラリアは系統、 生体内で発生するものがのより多くの担当者に高い結合寄生虫の個体数を取得するため。 色素栄養型の段階で寄生虫の文化のサンプル（iRBCと感染していない赤血球の混合物）をペトリ皿上に成長させたHBEC - 5Iの層で洗浄し、インキュベートする。インキュベーション後、皿を優しくuRBCとバインドされていないiRBCから無料で洗浄される。新鮮なuRBCは一晩HBEC - 5iおよびインキュベーションに接続されているいくつかのiRBCに追加されます。バーストシゾント段階の寄生虫として、メロゾイトreinvade RBC及びこれらの環状期の寄生虫は、次の日に収穫されます。十分な材料が得られるまでの寄生虫は、培養する（通常は2〜4週間）と選択の新ラウンドを行うことができます。 P.に応じて熱帯熱マラリア原虫株の、選択の4〜7ラウンドは、人口を取得するために必要とされるほとんどの寄生虫はHBEC - 5Iに結合する場所。結合表現型は、徐々にこうしてHBEC - 5Iで通常の選択は表現型を維持するために必要とされ、変異表面抗原の遺伝子発現のスイッチを示し、数週間後に失われます。 要約すると、我々は、P.をレンダリング選択アッセイを開発熱帯熱マラリアは寄生虫より&quot;脳マラリアの接着剤&quot;の表現型を。我々は、4 P.のうち3つの選択することができたHBEC - 5Iで熱帯熱マラリア原虫株 。このアッセイはまた、正常ヒト皮膚と肺内皮細胞への結合のための寄生虫を選択するために使用されています。重要なのは、このメソッドは、脳の内皮に結合するための候補寄生虫リガンドを同定するために、組織特異的な寄生虫の集団を選択するために使用することができます。さらに、このアッセイは、推定反隔離薬7の画面に使用することができます。

Watch this Scientific Journal Video about Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells at JoVE.com

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

不定冠詞<em&gt; in vitroで</em脳マラリアの隔離のための&gt;モデルが記載されている<sup&gt; 1</sup&gt;。<em&gt;熱帯熱マラリア原虫</em&gt;感染した赤血球は、不死化ヒト脳微小血管内皮細胞への結合のために選択されています。選択された寄生虫は、異なる表現型を示す。選択プロセスは、様々な使用して適用することができます。<em&gt; P.熱帯熱マラリア原虫</em&gt;株と血管内皮細胞株。

の選択熱帯熱マラリア原虫</em人間の脳内皮細胞へのCytoadhesion用>寄生虫

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

selection-plasmodium-falciparum-parasites-for-cytoadhesion-to-human

Research

JoVE Journal

Immunology and Infection

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

18.9K ビュー.  University of Edinburgh. 不定冠詞 in vitroでモデルが記載されている 1。熱帯熱マラリア原虫感染した赤血球は、不死化ヒト脳微小血管内皮細胞への結合のために選択されています。選択された寄生虫は、異なる表現型を示す。選択プロセスは、様々な使用して適用することができます。 P.熱帯熱マラリア原虫

ビデオ: Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

不定冠詞<em&gt;のin vitro</em研究する&gt;共同感染モデル<em&gt;熱帯熱マラリア原虫（Plasmodium falciparum）</emヒトの第一次単球由来の免疫細胞に&gt;-HIV-1の相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

免疫・感染学

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

の血小板媒介クランピングのためのシンプルなプロトコル<em&gt;熱帯熱マラリア原虫</emリソース悪い設定で、&gt;感染した赤血球

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脳から隔離された白血球の単離と解析<em&gt;マラリアベルゲイ</em&gt; ANKA感染マウス

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

の分離<em&gt;熱帯熱マラリア原虫（Plasmodium falciparum）</em磁気手段によって&gt;後期感染赤血球

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

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 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

高収率の精製<em&gt;熱帯熱マラリア原虫</emオプソニン抗体アッセイにおける使用のための&gt;メロゾイト

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

小さなRNAおよびリボソームの占有の調節の役割を調査する方法<em&gt;熱帯熱マラリア原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

一定の流れの原発性ヒト内皮細胞の肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

髄膜炎菌 人工多能性幹細胞由来脳内皮細胞の感染

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

の選択熱帯熱マラリア原虫寄生虫