この作品は、CatalystのCo-感染症や合併症の助成を通じて、健康研究のカナダの協会によってサポートされていました。ヒト赤血球は、センターHospitalierエトワールラヴァル大学血液バンクから提供されました。

<ol>
	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Malaria Report. , (2005).</li><li>Kublin, J. G., Patnaik, P., Jere, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Plasmodium+falciparum+malaria+on+concentration+of+HIV-1-RNA+in+the+blood+of+adults+in+rural+Malawi:+a+prospective+cohort+study.">Effect of Plasmodium falciparum malaria on concentration of HIV-1-RNA in the blood of adults in rural Malawi: a prospective cohort study.</a> Lancet. 365, 233-240 (2005).</li><li>Cuadros, D. F., Branscum, A. J., Crowley, P. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+by+malaria+antigens+renders+mononuclear+cells+susceptible+to+HIV+infection+and+re-activates+replication+of+endogenous+HIV+in+cells+from+HIV-infected+adults.">Activation by malaria antigens renders mononuclear cells susceptible to HIV infection and re-activates replication of endogenous HIV in cells from HIV-infected adults.</a> Parasite Immunol. 26, 213-217 (2004).</li><li>Diou, J., Tardif, M. R., Barat, C., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+derived+from+hemozoin-loaded+monocytes+display+a+partial+maturation+phenotype+that+promotes+HIV-1+trans-infection+of+CD4++T+cells+and+virus+replication.">Dendritic cells derived from hemozoin-loaded monocytes display a partial maturation phenotype that promotes HIV-1 trans-infection of CD4+ T cells and virus replication.</a> J. Immunol. 184, 2899-2907 (2010).</li><li>Diou, J., Tardif, M. R., Barat, C., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+hemozoin+modulates+susceptibility+of+immature+monocyte-derived+dendritic+cells+to+HIV-1+infection+by+inducing+a+mature-like+phenotype.">Malaria hemozoin modulates susceptibility of immature monocyte-derived dendritic cells to HIV-1 infection by inducing a mature-like phenotype.</a> Cell Microbiol. 12, 615-625 (2010).</li><li>Diou, J., Gauthier, S., Tardif, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+the+malaria+pigment+hemozoin+renders+human+macrophages+less+permissive+to+HIV-1+infection.">Ingestion of the malaria pigment hemozoin renders human macrophages less permissive to HIV-1 infection.</a> Virology. 395, 56-66 (2009).</li><li>Davies, J. Q., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+human+macrophages.">Isolation and culture of human macrophages.</a> Methods Mol. Biol. 290, 105-116 (2005).</li><li>Fortin, J. F., Cantin, R., Lamontagne, G., Tremblay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+ICAM-1+glycoproteins+incorporated+on+human+immunodeficiency+virus+type+1+are+biologically+active+and+enhance+viral+infectivity.">Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity.</a> J. Virol. 71, 3588-3596 (1997).</li><li>Bounou, S., Leclerc, J. E., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+host+ICAM-1+in+laboratory+and+clinical+strains+of+human+immunodeficiency+virus+type+1+increases+virus+infectivity+and+CD4(+)-T-cell+depletion+in+human+lymphoid+tissue,+a+major+site+of+replication+in+vivo.">Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo.</a> J. Virol. 76, 1004-1014 (2002).</li><li>Wei, X., Decker, J. M., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+resistant+human+immunodeficiency+virus+type+1+in+patients+receiving+fusion+inhibitor+(T-20)+monotherapy.">Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.</a> Antimicrob. Agents Chemother. 46, 1896-1905 (2002).</li><li>Trager, W., Jensen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+malaria+parasites+in+continuous+culture.">Human malaria parasites in continuous culture.</a> Science. 193, 673-675 (1976).</li><li>Turrini, F., Ginsburg, H., Bussolino, F., Pescarmona, G. P., Serra, M. V., Arese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+Plasmodium+falciparum-infected+human+red+blood+cells+by+human+monocytes:+involvement+of+immune+and+nonimmune+determinants+and+dependence+on+parasite+developmental+stage.">Phagocytosis of Plasmodium falciparum-infected human red blood cells by human monocytes: involvement of immune and nonimmune determinants and dependence on parasite developmental stage.</a> Blood. 80, 801-808 (1992).</li><li>Akkina, R. K., Walton, R. M., Chen, M. L., Li, Q. X., Planelles, V., Chen, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+CD34++cells+with+a+human+immunodeficiency+virus+type+1-based+retroviral+vector+pseudotyped+with+vesicular+stomatitis+virus+envelope+glycoprotein+G.">High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.</a> J. Virol. 70, 2581-2585 (1996).</li><li>Zhao, C., Papadopoulou, B., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+infantum+enhances+human+immunodeficiency+virus+type-1+replication+in+primary+human+macrophages+through+a+complex+cytokine+network.">Leishmania infantum enhances human immunodeficiency virus type-1 replication in primary human macrophages through a complex cytokine network.</a> Clinical Immunol. 113, 81-88 (2004).</li></ol>

利害の衝突が宣言されません。

我々は、ウイルス遺伝子の発現または単球由来マクロファージにおける子孫ウイルスの生産と複製のどちらかを分析することにより、HIV-1ウイルスのサイクル、すなわち上のマラリア原虫の影響を分析するためにここで2つの異なるアプローチを説明しました。同様のアプローチは、他のHIV-1寄生虫の共感染16のために使用されている。しかし、これらの新しいデータはマラリア、HIV-1の共感染の調査では一歩前進です。 実際 、Diou ら 8は、HIV-1複製に、hemozoinの効果ではなく、ライブの寄生虫を研究した。我々の結果と一致して、彼らはhemozoinはウイルスMDMによる生産ではなく、MDMSを阻害すること自体が十分であったことを観察した。 記載された実験レイアウトを使用して、我々は観察されたP.熱帯熱マラリア原虫はマクロファージにおけるHIV-1複製サイクルを明確に有害な効果を発揮する：ウイルス産生の有意な阻害は、マクロファージの寄生虫に事前に暴露さで観察される（Figur電子2A）およびウイルス転写における寄生虫の具体的な影響は、図2Bに示されています。しかし、我々は、ウイルスの侵入または融合（decapsidation）上、またはそのようなタンパク質合成またはウイルス粒子のアセンブリおよび出芽などのポスト統合メカニズムに寄生虫の任意の追加の影響を除外することはできません。さらに、寄生虫、ウイルスと同時にまたは次のMDM感染症のいずれかで追加された場合、HIV-1複製に異なる影響が得られるであろうことが可能である。 私たちは 、in vitro共感染モデルでのHIV-1 / Pの詳細な調査を実行する強力なツールを提供しています宿主細胞内での熱帯熱マラリア原虫の相互作用。例えば、宿主細胞ゲノムへのウイルスretrotranscriptionの具体的な手順とウイルスゲノムの統合を標的とし、定量化できるような定量的リアルタイムPCRのような他の技術とこの実験的なレイアウトの組み合わせは、非常に可能であり、さらにinsiが得られるはず共同の感染症に関与するメカニズムにghts。また、ウイルスのサイクル（ウイルス融合、decapsidation、エントリの定量）の初期段階を評価するための特異的なアッセイは、さらにウイルス複製に及ぼす影響を分析するためにこの基本的なプロトコルに適用することができます。トリチウム標識ヌクレオチドは、おそらくHIV-1 p24抗原のELISAを置き換えることができますを使用して実際にも、標準的なHIV-1逆転写定量アッセイ：これらの変更は、このプロトコルは、HIV-1定量と検出に関する方法を柔軟に表示されます。 MDMに加えて、我々のシステムがこのような単球や樹状細胞などのHIV-1マラリアの相互作用に関連する他の細胞型への適応になることを期待しています。 MDMは、接着細胞であるという事実は、任意のマクロファージを除去することなく、で撮影されていない、またはMDMとの接触にあることをIRBCの外に洗濯しやすいようにできるように、その操作を容易にします。このような利点は、それ自体を複雑に、懸濁液中で培養される樹状細胞や単球には適用されませんでしたそのような細胞とuRBCと自由IRBCのparation、我々は、現在この問題に取り組んでいる。私たちのシステムはまた、そのような共培養実験におけるCD4陽性T細胞のような他の免疫細胞におけるHIV-1ウイルスのサイクルでマラリアにさらされた単球由来細胞の効果など、より複雑な相互作用を研究するのに便利かもしれません。最後に、我々のプロトコルは、Pの影響を見て実験に適している可能性がHIV-1感染者のためのPBMCにおけるHIV-1再活性化で熱帯熱マラリア原虫の iRBCs。 倫理文ヒトPBMCは、センターHospitalierエトワールラヴァル大学研究センターの倫理委員会のガイドラインに従って、健康なドナーの血液から得られた。書面による同意は、すべての血液ドナーから得られた。

<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> RPMI 1640 </td><td>マルチセル</td><td> 350から000-CL </td><td></td></tr><tr><td> PBS-エンドトキシンフリー</td><td>シグマ</td><td> D8662 </td><td></td></tr><tr><td> FBS </td><td> Wisent </td><td> 080から150 </td><td> 30分間56℃での熱不活化</td></tr><tr><td> Accutase </td><td> eBiosciences </td><td> 00-4555-56 </td><td></td></tr><tr><td> Albumax II </td><td>ギブコ</td><td> 11021-037 </td><td> RPMI 1640に溶解し、フィルター滅菌</td></tr><tr><td>リンパ球分離媒体</td><td>マルチセル</td><td> 305から010-CL </td><td></td></tr><tr><td>白色ルミノメータープレート</td><td>プロメガ</td><td> Z3291 </td><td></td></tr><tr><td> CSのMac分離columms </td><td>ミルテニーバイオテク</td><td> 130-041-305 </td><td></td></tr><tr><td> VarioMacsセパレータ</td><td>ミルテニーバイオテク</td><td> 130-090-282 </td><td></td></tr><tr><td>ペニシリン/ストレプトマイシン溶液</td><td> Wisent </td><td> 450から201-EL </td><td></td></tr><tr><td> D-ソルビトール</td><td> Sigma-Aldrich社</td><td> S1876 </td><td></td></tr><tr><td>セルスクレーパー（25センチ） </td><td>ザルスタット</td><td> 83.1830 </td><td></td></tr><tr><td> 24ウェル細胞培養プレート</td><td> BDファルコン</td><td> 353047 </td><td></td></tr><tr><td> 150×20 mmの培養皿</td><td>ザルスタット</td><td> 83.1803.003 </td><td></td></tr><tr><td> M-CSF </td><td> Genscript </td><td> Z02001 </td><td></td></tr><tr><td> HEPES </td><td> Sigma-Aldrich社</td><td> H4034 </td><td></td></tr><tr><td>ヒト血清AB + </td><td>バレーメディカル</td><td> HP1022 </td><td></td></tr><tr><td> HBSS </td><td> Wisent </td><td> 311から505-CL </td><td></td></tr><tr><td>ゲンタマイシン</td><td> Sigma-Aldrich社</td><td> G1397 </td><td></td></tr><tr><td>ヒポキサン​​チン</td><td> Sigma-Aldrich社</td><td> H9636 </td><td></td></tr><tr><td> NaHCO 3を </td><td> Sigma-Aldrich社</td><td> S5761 </td><td></td></tr><tr><td>ルシフェラーゼアッセイシステム</td><td>プロメガ</td><td> E1500 </td><td></td></tr><tr><td>ヒト赤血球</td><td></td><td></td><td>得は、哲血液バンクから精製された。 </td></tr></tbody></table>

an in vitro co-infection model to study plasmodium falciparum-hiv-1 interactions in human primary monocyte-derived immune cells

熱帯熱マラリア原虫 、マラリアの最悪の形態の病原体、およびヒト免疫不全ウイルス1型（HIV-1）は、毎年1〜4万人が死亡の合計責任がある、世界的に最も重要な健康問題の一つです。特に開発途上地域、サハラ以南のアフリカでの大規模な重複のために、マラリア、HIV-1との共感染が一般的ですが、2つの疾患の間の相互作用は十分に理解されています。疫学的な報告書は、マラリア感染は一過性HIV-1複製を高めると共感染した個体2,3のHIV-1ウイルス量を増加させることが示唆されている。このウイルス血症は、抗マラリア薬による治療後数週間にわたって高いままなので、この現象は疾患の進行および伝送に影響を及ぼす可能性があります。 これらの観​​察の背後にある細胞の免疫学的メカニズムは、ほとんど研究されていない。 in vitro試験investig のいくつかのHIV-1にマラリアの影響をatingは、水溶性のマラリア抗原への曝露が免疫細胞にHIV-1感染と再活性化を高めることができることを実証した。しかし、これらの研究は、Pの全細胞抽出物を使用熱帯熱マラリア原虫シゾント段階の寄生虫および末梢血単核細胞（PBMC）、マラリア成分（s）が観測された効果とどのようなターゲット·ホスト細胞は4,5であったために責任があった解読が困難となっています。最近の仕事はマラリア色素の未熟単球由来樹状細胞の暴露を実証してきましたhemozoinは、CD4 + T細胞の6,7にHIV-1を転送する能力を増加したが、それはマクロファージ8のHIV-1感染を減少させている。この複雑なプロセス、マラリア原虫と異なった、関連するヒト初代細胞集団におけるHIV-1の間の相互作用の体系的な分析に光を当てるために決定的に必要とされる。 Hの影響を調査するためのいくつかのテクニック微生物の貪食におけるIV-1とHIV-1複製にそのような病原体の効果が記載されている。ここではPの影響を調査する手法を提案ヒトの第一次単球由来マクロファージにおけるHIV-1の複製に熱帯熱マラリア原虫に感染した赤血球。感染したマクロファージによって放出ウイルス量への影響は測定することによって決定される一方、HIV-1転写/翻訳のイベントの寄生虫暴露の影響は、ルシフェラーゼレポーター遺伝子は、env遺伝子に置き換えられていた単一サイクルシュードウイルスを使用して監視されてい細胞上清中のELISAによるp24のHIV-1キャプシドタンパク質。

Watch this Scientific Journal Video about An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells at JoVE.com

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

我々は開発した<em&gt; in vitroで</emの影響を研究する&gt;マラリア、HIV-1の共感染モデル<em&gt;熱帯熱マラリア原虫（Plasmodium falciparum）</em&gt;ヒトの第一次単球由来マクロファージにおけるHIV-1複製サイクルで。この汎用性の高いシステムを簡単​​にHIV-1感染に感受性で他の主要な細胞型に適合させることができます。

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 ...

an-vitro-co-infection-model-to-study-plasmodium-falciparum-hiv-1

Research

JoVE Journal

Immunology and Infection

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

13.6K ビュー.  CHUL (CHUQ), Quebec City, Quebec, Canada. 我々は開発した in vitroでマラリア、HIV-1の共感染モデル熱帯熱マラリア原虫（Plasmodium falciparum）ヒトの第一次単球由来マクロファージにおけるHIV-1複製サイクルで。この汎用性の高いシステムを簡単​​にHIV-1感染に感受性で他の主要な細胞型に適合させることができます。

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

免疫・感染学

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

プライマリーヒトT細胞のpiggyBacトランスポゾンシステムの変更

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

不定冠詞<em&gt;インビトロ</emヒトマクロファージの分化と偏光の不均一性を研究するために&gt;モデル

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from 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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

アン<em&gt;インビトロ</em&gt; HIV重感染のコンテキストでのマラリアに対する免疫応答を測定するためのモデル

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

全血からのヒト単球由来樹状細胞の生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

病原体、共生生物、およびホストの腸上皮間の相互作用を研究する人間 Colonoid 単分子膜

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

自己免疫性糖尿病モデルにおける機能試験用抗原特異的一次マウス細胞傷害性T細胞の高効率生成

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.