Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Otto von Guericke University Magdeburg

University of Magdeburg

Bone marrow-derived progenitor cells have been implicated to contribute to neointima formation, but the time course and extent of their accumulation and differentiation into vascular cells and, most importantly, the long-term contribution of bone marrow-derived progenitor cells to the vascular lesion remain undefined.

Time-course analysis on the differentiation of bone marrow-derived progenitor cells into smooth muscle cells during neointima formation.

The endothelial nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA) is a well-established risk factor for oxidative stress, vascular dysfunction, and congestive heart failure. The aim of the present study was to determine the impact of rapid atrial pacing (RAP) on ADMA levels and eNOS expression.

The impact of rapid atrial pacing on ADMA and endothelial NOS.

L&#39;endothelial nitric oxide synthase (eNOS) inhibiteur dim&eacute;thylarginine asym&eacute;trique (ADMA) est un facteur de risque bien &eacute;tabli pour le stress oxydatif, dysfonction vasculaire et une insuffisance cardiaque congestive. Le but de cette &eacute;tude &eacute;tait de d&eacute;terminer l&#39;impact de la rapide une stimulation auriculaire (RAP) sur l&#39;expression de niveaux et eNOS ADMA.

L&#39;impact de la stimulation auriculaire rapide sur ADMA et NOS endoth&eacute;liales.

L&#39;endoteliale di ossido nitrico sintasi (eNOS) inibitore dimethylarginine asimmetrico (ADMA) &egrave; un fattore di rischio ben consolidato per lo stress ossidativo, disfunzione vascolare e insufficienza cardiaca congestizia. Lo scopo del presente studio era di determinare l&#39;impatto della rapida atriale ritmo (RAP) sull&#39;espressione di livelli ed eNOS ADMA.

L&#39;effetto di stimolazione atriale rapida su ADMA e NOS endoteliale.

&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x53C2;&#x4E0E;&#x5404;&#x79CD;&#x5404;&#x6837;&#x7684;&#x751F;&#x7406;&#x548C;&#x75C5;&#x7406;&#x8FC7;&#x7A0B;&#xFF0C;&#x5176;&#x4E2D;&#x8BB8;&#x591A;&#x5728;&#x5C0F;&#x9F20;&#x6A21;&#x578B;&#x7684;&#x7814;&#x7A76;&#x3002;&#x5F53;&#x524D;&#x534F;&#x8BAE;&#x6765;&#x9694;&#x79BB;&#x5C0F;&#x9F20;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x5F88;&#x5C11;&#xFF0C;&#x5BFC;&#x81F4;&#x4EE4;&#x4EBA;&#x4E0D;&#x6EE1;&#x610F;&#x7EC6;&#x80DE;&#x4EA7;&#x7387;&#x548C;&#x7EAF;&#x5EA6;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x5C06;&#x4ECB;&#x7ECD;&#x4E00;&#x79CD;&#x65B0;&#x7684;&#x65B9;&#x6CD5;&#xFF0C;&#x4EE5;&#x6709;&#x6548;&#x5730;&#x4F7F;&#x5927;&#x6279;&#x6210;&#x719F;&#x708E;&#x75C7;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x533A;&#x522B;&#x4E8E;&#x5F02;&#x6784;&#x9AA8;&#x9AD3;&#x7EC6;&#x80DE;&#x60AC;&#x6D6E;&#x6DB2;&#x3002;&#x9AA8;&#x9AD3;&#x7EC6;&#x80DE;&#x60AC;&#x6D6E;&#x6DB2;&#x88AB;&#x9694;&#x79BB;&#x7684; Balb/c &#x4ECE;&#x5395;&#x80A1;&#x9AA8;&#x548C;&#x80EB;&#x9AA8;&#x548C; C57Bl/6 &#x5C0F;&#x9F20;&#xFF0C;&#x8F85;&#x4E4B;&#x4EE5;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x96C6;&#x843D;&#x523A;&#x6FC0;&#x56E0;&#x5B50; (M-CSF)&#xFF0C;&#x548C;&#x88AB;&#x8D85;&#x4F4E;&#x9644;&#x4EF6;&#x8868;&#x9762;&#x6291;&#x5236;&#x9075;&#x5B88;&#x4ECB;&#x5BFC;&#x7684;&#x6210;&#x719F;&#x7EC6;&#x80DE;&#x3002;&#x5355;&#x5143;&#x683C;&#x5728;&#x6307;&#x5B9A;&#x7684;&#x65F6;&#x95F4;&#x70B9;&#x6536;&#x83B7;&#x4E86;&#x3001; &#x672F;&#x7684;&#x5206;&#x5316;&#x8FC7;&#x7A0B;&#x4E2D;&#xFF0C;&#x65F6;&#x95F4;&#x7EBF;&#x5206;&#x6790;&#x548C;&#x968F;&#x540E;&#x88AB;&#x5E7F;&#x6CDB; phenotyped &#x6765;&#x9A8C;&#x8BC1;&#x5176; monocytotic &#x5C5E;&#x6027;&#x3002;&#x4E3A;&#x4E86;&#x786E;&#x8BA4;&#x4E0B;&#x6E38;&#x7684;&#x517C;&#x5BB9;&#x6027;&#xFF0C;&#x6211;&#x4EEC;&#x6D4B;&#x8BD5;&#x4E86;&#x5178;&#x578B;&#x7684;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x7684;&#x884C;&#x4E3A;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x534F;&#x8BAE;&#x4EA7;&#x751F;&#x4E86; 24 &plusmn; 6 &times; 10(6) &#x5206;&#x5316;&#x7EC6;&#x80DE;&#x6BCF;&#x6350;&#x52A9;&#x9F20;&#x6807;&#xFF0C;&#x6BD4;&#x4EE5;&#x524D;&#x4F7F;&#x7528;&#x83B7;&#x5F97;&#x7684;&#x6536;&#x76CA;&#x7387;&#x6240;&#x8FF0;&#x5916;&#x5468;&#x8840;&#x5206;&#x79BB;&#x65B9;&#x6CD5;&#x9AD8; 10 &#x500D;&#x3002;&#x5206;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x5305;&#x62EC;&#x5927;&#x7EA6; 47%&plusmn; 12%&#x5355;&#xFF0C;&#x5176;&#x4F59;&#x5747;&#x4E3A;&#x6210;&#x719F;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x3002;&#x6211;&#x4EEC;&#x53EF;&#x4EE5;&#x4E0A;&#x5347;&#x5230; 86%&plusmn; 6%&#x7684;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x7EAF;&#x5EA6;&#x6D88;&#x8017;&#x9057;&#x6C11;&#x5DE8;&#x566C;&#x7EC6;&#x80DE;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x9AA8;&#x9AD3;&#x5355;&#x6838;&#x7EC6;&#x80DE; (BMDMs) &#x662F;&#x6709;&#x5438;&#x5F15;&#x529B;&#x7684;&#x5DE5;&#x5177;&#xFF0C;&#x4F8B;&#x5982;&#xFF0C;&#x7814;&#x7A76;&#x5148;&#x5929;&#x548C;&#x81EA;&#x9002;&#x5E94;&#x7684;&#x514D;&#x75AB;&#x7CFB;&#x7EDF;&#x3001; &#x52A8;&#x8109;&#x7CA5;&#x6837;&#x786C;&#x5316;&#x548C;&#x7EC6;&#x80DE;&#x8FC1;&#x79FB;&#x671F;&#x95F4;&#x611F;&#x67D3;&#x3002;&#x6B64;&#x5916;&#xFF0C;BMDM &#x79FB;&#x690D;&#x53EF;&#x7528;&#x4E8E;&#x6D4B;&#x8BD5;&#x5C0F;&#x8BF4;&#xFF0C;&#x6CBB;&#x7597;&#x4F53;&#x5185;&#x65B9;&#x6CD5;&#x5728;&#x5C0F;&#x9F20;&#x75BE;&#x75C5;&#x6A21;&#x578B;&#x3002;

&#x4E00;&#x4EE3;&#x7684;&#x6210;&#x719F;&#x5C0F;&#x9F20;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x4ECE;&#x5F02;&#x6784;&#x9AA8;&#x9AD3;&#x548C;&#x5B83;&#x4EEC;&#x7684;&#x5C5E;&#x6027;&#x7684;&#x8BF4;&#x660E;&#x3002;

Monocytes are involved in a wide range of physiological and pathological processes, many of which are studied in mouse models. Current protocols to isolate murine monocytes are few and result in unsatisfactory cell yield and purity. Here, we describe a novel approach to efficiently differentiate large numbers of mature inflammatory monocytes from heterogeneous bone marrow cell suspensions. Bone marrow cell suspensions were isolated by flushing femurs and tibias from Balb/c and C57Bl/6 mice, supplemented with macrophage colony-stimulating factor (M-CSF), and were cultured on ultra-low attachment surfaces to inhibit adherence-mediated maturation. Cells were harvested at indicated time points, underwent time-line analysis of the differentiation processes, and were subsequently extensively phenotyped to verify their monocytotic properties. In order to confirm downstream compatibility, we tested for typical monocyte behavior. Our protocol yielded 24 Â± 6 Ã— 10(6) differentiated cells per donor mouse, 10-fold higher than yields obtained using previously described peripheral blood isolation methods. Differentiated cells consisted of approximately 47% Â± 12% monocytes, the rest being mature macrophages. We increased monocyte purity to 86% Â± 6% by depleting adherent macrophages. Our findings indicate that bone marrow-derived monocytes (BMDMs) are an attractive tool to study, for example, the innate and adaptive immune system, atherosclerosis, and cellular migration during infection. Moreover, BMDM transplantation could be used to test novel, therapeutic in vivo approaches in mice disease models.

Generation of mature murine monocytes from heterogeneous bone marrow and description of their properties.

Monocytes sont impliqu&eacute;s dans un large &eacute;ventail de processus physiologiques et pathologiques, dont beaucoup sont &eacute;tudi&eacute;s dans des mod&egrave;les murins. Protocoles courants pour isoler les monocytes murins sont peu nombreux et entra&icirc;nent rendement insatisfaisant cellulaire et la puret&eacute;. Nous d&eacute;crivons ici une nouvelle approche pour diff&eacute;rencier efficacement un grand nombre de monocytes inflammatoires matures des suspensions de cellules h&eacute;t&eacute;rog&egrave;nes de la moelle osseuse. Suspensions de cellules de moelle osseuse ont &eacute;t&eacute; isol&eacute;es en rin&ccedil;ant le f&eacute;mur et le tibia de souris Balb/c et les souris C57Bl/6, additionn&eacute;s de macrophage colony-stimulating factor (M-CSF) et ont &eacute;t&eacute; cultiv&eacute;s sur des surfaces tr&egrave;s faible attachement &agrave; inhiber la maturation induite par l&#39;adh&eacute;sion. Les cellules ont &eacute;t&eacute; r&eacute;colt&eacute;es aux moments indiqu&eacute;s, a subi une analyse chronologique des processus de diff&eacute;renciation et ont ensuite &eacute;t&eacute; largement ph&eacute;notyp&eacute;es pour v&eacute;rifier leurs propri&eacute;t&eacute;s monocytotic. Afin de confirmer la compatibilit&eacute; en aval, nous avons test&eacute; pour un comportement typique de monocytes. Notre protocole donn&eacute; 24 &plusmn; 6 &times; 10 (6) diff&eacute;renci&eacute;s de cellules par souris donneur 10 fois sup&eacute;rieure aux rendements obtenus &agrave; l&#39;aide pr&eacute;c&eacute;demment d&eacute;crit les m&eacute;thodes d&#39;isolation de sang p&eacute;riph&eacute;rique. Les cellules diff&eacute;renci&eacute;es se composait d&#39;environ 47 % &plusmn; 12 % monocytes, le reste &eacute;tant des macrophages matures. Nous avons augment&eacute; la puret&eacute; des monocytes &agrave; 86 &plusmn; 6 % en r&eacute;duisant les macrophages adh&eacute;rentes. Nos r&eacute;sultats indiquent que les monocytes de moelle osseuse (BMDMs) sont un outil int&eacute;ressant pour &eacute;tudier, par exemple, le syst&egrave;me immunitaire inn&eacute; et adaptatif, l&#39;ath&eacute;roscl&eacute;rose et la migration cellulaire au cours de l&#39;infection. En outre, transplantation BMDM pourrait servir &agrave; tester le roman, des approches th&eacute;rapeutiques in vivo dans des mod&egrave;les de maladies des souris.

G&eacute;n&eacute;ration de matures monocytes murins de moelle h&eacute;t&eacute;rog&egrave;ne et la description de leurs propri&eacute;t&eacute;s.

Monozyten sind in einer Vielzahl von physiologischen und pathologischen Prozessen, beteiligt, von die viele in Maus-Modellen studiert werden. Aktuelle Protokolle murine Monozyten zu isolieren sind wenige und f&uuml;hren zu unbefriedigenden Zelle Ausbeute und Reinheit. Hier beschreiben wir einen neuartigen Ansatz, um effizient gro&szlig;e Anzahl von Reifen entz&uuml;ndliche Monozyten von heterogenen Knochenmark Zellsuspensionen zu unterscheiden. Knochenmark-Zellsuspensionen wurden durch Sp&uuml;lung Oberschenkel- und Wadenbein von Balb/c isoliert und C57Bl/6 M&auml;usen, erg&auml;nzt durch Makrophagen-Kolonie-stimulierende Faktor (M-CSF) und wurden kultivierten auf extrem niedrigen Anlage Oberfl&auml;chen festhalten-vermittelte Reifung zu hemmen. Zellen zu angegebenen Zeitpunkten geerntet wurden, erfuhr Zeitleiste Analyse die Differenzierungsprozesse und waren anschlie&szlig;end ausgiebig phenotyped ihre monocytotic Eigenschaften zu &uuml;berpr&uuml;fen. Um nachgeschaltete Kompatibilit&auml;t zu best&auml;tigen, haben wir f&uuml;r typische Monozyte Verhalten getestet. Unser Protokoll ergab 24 &plusmn; 6 &times; 10 6 differenzierte Zellen pro Spender Maus, 10-fache h&ouml;her als Ertr&auml;ge, die mit zuvor ermittelt peripherem Blut Isolierung Methoden beschrieben. Differenzierte Zellen bestand aus etwa 47 % &plusmn; 12 % Monozyten, der Rest wird Reifen Makrophagen. Wir haben um abbauende adh&auml;rente Makrophagen Monozyte Reinheit bis 86 % &plusmn; 6 % gesteigert. Unsere Ergebnisse zeigen, dass Knochenmark abgeleitete Monozyten (BMDMs) einem attraktiven Werkzeug, z. B. der angeborenen und erworbenen Immunsystems, Atherosklerose und zellul&auml;re Migration w&auml;hrend der Infektion zu studieren. Dar&uuml;ber hinaus konnte BMDM Transplantation zum Roman, Therapieans&auml;tze in-vivo in M&auml;usen Krankheitsmodelle testen verwendet werden.

Generation von Reifen murinen Monozyten von heterogenen Knochenmark und Beschreibung ihrer Eigenschaften.

Monociti sono coinvolti in una vasta gamma di processi fisiologici e patologici, molte delle quali vengono studiate in modelli murini. Protocolli per isolare i monociti murini sono pochi e risultano insoddisfacente cella resa e purezza. Qui, descriviamo un nuovo approccio per differenziare in modo efficiente un gran numero di monociti maturi infiammatorie da sospensioni di cellule di midollo osseo eterogenei. Sospensioni di cellule del midollo osseo sono stati isolati da vampate di calore femori e tibie da Balb/c e topi C57Bl/6, completati con macrofagi colony-stimulating factor (M-CSF) e sono stati coltivati su superfici ultra-accessorio per inibire la maturazione di aderenza-mediata. Le cellule sono state raccolte a punti di tempo indicato, sub&igrave; la linea del tempo analisi dei processi di differenziazione e successivamente sono stati ampiamente phenotyped per verificare le propriet&agrave; monocytotic. Al fine di confermare la compatibilit&agrave; a valle, abbiamo testato per comportamento tipico monociti. Il nostro protocollo ha reso 24 &plusmn; 6 &times; 10 differenziato cellule per mouse donatore, 10 volte superiore a rendimenti ottenuti utilizzando precedentemente descritti metodi di isolamento del sangue periferico. Le cellule differenziate consistevano di circa 47% &plusmn; 12% monociti, il resto essendo macrofagi maturi. Abbiamo aumentato la purezza di monociti a 86% &plusmn; 6% da esaurimento macrofagi aderenti. I nostri risultati indicano che i monociti derivate dal midollo osseo (BMDMs) sono un interessante strumento per studiare, per esempio, il sistema immunitario innato e adattivo, aterosclerosi e migrazione cellulare durante l&#39;infezione. Inoltre, il trapianto BMDM potrebbe essere utilizzato per verificare il romanzo, approcci terapeutici in vivo in modelli di topi malattia.

Generazione di monociti maturi murini da eterogeneo del midollo osseo e la descrizione delle loro propriet&agrave;.

&#x5358;&#x7403;&#x306F;&#x3001;&#x591A;&#x304F;&#x306F;&#x30DE;&#x30A6;&#x30B9; &#x30E2;&#x30C7;&#x30EB;&#x3067;&#x8ABF;&#x3079;&#x305F;&#x751F;&#x7406;&#x7684;&#x304A;&#x3088;&#x3073;&#x75C5;&#x7406;&#x5B66;&#x7684;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x5E83;&#x3044;&#x7BC4;&#x56F2;&#x3067;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30DE;&#x30A6;&#x30B9;&#x5358;&#x7403;&#x3092;&#x5206;&#x96E2;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x73FE;&#x5728;&#x306E;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x306F;&#x5C11;&#x306A;&#x304F;&#x3001;&#x4E0D;&#x6E80;&#x8DB3;&#x306A;&#x30BB;&#x30EB;&#x306E;&#x53CE;&#x91CF;&#x3068;&#x7D14;&#x5EA6;&#x306E;&#x7D50;&#x679C;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x52B9;&#x7387;&#x7684;&#x306B;&#x591A;&#x6570;&#x306E;&#x6210;&#x719F;&#x3057;&#x305F;&#x708E;&#x75C7;&#x6027;&#x5358;&#x7403;&#x7570;&#x7A2E;&#x9AA8;&#x9AC4;&#x7D30;&#x80DE;&#x61F8;&#x6FC1;&#x6DB2;&#x304B;&#x3089;&#x533A;&#x5225;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x65AC;&#x65B0;&#x306A;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;Balb/c &#x304B;&#x3089;&#x30D5;&#x30E9;&#x30C3;&#x30B7;&#x30E5; femur &#x306E;&#x8907;&#x6570;&#x5F62;&#x3068;&#x30DE;&#x30A6;&#x30B9;&#x30EC;&#x30B7;&#x30D4;&#x30A8;&#x30F3;&#x30C8;&#x9AA8;&#x9AC4;&#x7D30;&#x80DE;&#x61F8;&#x6FC1;&#x6DB2;&#x304C;&#x9694;&#x96E2;&#x3055;&#x308C;&#x305F;&#x3068; c57bl/6 &#x30DE;&#x30A6;&#x30B9;&#x3001;&#x5927;&#x98DF;&#x7D30;&#x80DE;&#x306E;&#x30B3;&#x30ED;&#x30CB;&#x30FC;&#x523A;&#x6FC0;&#x3068;&#x88DC;&#x8DB3;&#x56E0;&#x5B50; (M-CSF) &#x3068;&#x8D85;&#x4F4E;&#x6DFB;&#x4ED8;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x306E;&#x8868;&#x9762;&#x306B;&#x4ED8;&#x7740;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x6210;&#x719F;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;&#x57F9;&#x990A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D30;&#x80DE;&#x306F;&#x3001;&#x793A;&#x3055;&#x308C;&#x305F;&#x6642;&#x9593;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x3067;&#x53CE;&#x7A6B;&#x3055;&#x308C;&#x305F;&#x5206;&#x5316;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x30BF;&#x30A4;&#x30E0; &#x30E9;&#x30A4;&#x30F3;&#x5206;&#x6790;&#x3092;&#x65BD;&#x884C;&#x3057;&#x305F;&#x3057;&#x3001;&#x305D;&#x306E;&#x5F8C;&#x306E; monocytotic &#x30D7;&#x30ED;&#x30D1;&#x30C6;&#x30A3;&#x3092;&#x78BA;&#x8A8D;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x5E83;&#x304F; phenotyped &#x3060;&#x3063;&#x305F;&#x3002;&#x4E0B;&#x4F4D;&#x4E92;&#x63DB;&#x6027;&#x3092;&#x78BA;&#x8A8D;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x306F;&#x3001;&#x5178;&#x578B;&#x7684;&#x306A;&#x5358;&#x7403;&#x306E;&#x52D5;&#x4F5C;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x79C1;&#x9054;&#x306E;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB; &#x30C9;&#x30CA;&#x30FC; &#x30DE;&#x30A6;&#x30B9;&#x672B;&#x68A2;&#x8840;&#x306E;&#x5206;&#x96E2;&#x65B9;&#x6CD5;&#x4EE5;&#x524D;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x5F97;&#x3089;&#x308C;&#x308B;&#x5229;&#x56DE;&#x308A;&#x3092;&#x8AAC;&#x660E;&#x3088;&#x308A;&#x3082; 10 &#x500D;&#x9AD8;&#x3044;&#x3042;&#x305F;&#x308A; 24 &plusmn; 6 &times; 10(6) &#x5206;&#x5316;&#x7D30;&#x80DE;&#x304C;&#x5F97;&#x3089;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x5206;&#x5316;&#x7D30;&#x80DE;&#x5358;&#x7D04; 47 &#xFF05; &plusmn; 12% &#x7403;&#x306E;&#x6210;&#x719F;&#x3057;&#x305F;&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x3055;&#x308C;&#x3066;&#x6B8B;&#x308A;&#x306E;&#x90E8;&#x5206;&#x304B;&#x3089;&#x6210;&#x3063;&#x3066;&#x3044;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F;&#x5358;&#x7403;&#x7D14;&#x5EA6; 86% &plusmn; 6% &#x306B;&#x4ED8;&#x7740;&#x6027;&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8;&#x3092;&#x67AF;&#x6E07;&#x3055;&#x305B;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x5897;&#x52A0;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x9AA8;&#x9AC4;&#x7531;&#x6765;&#x306E;&#x5358;&#x7403; (BMDMs)&#x3001;&#x305F;&#x3068;&#x3048;&#x3070;&#x3001;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x3068;&#x9069;&#x5FDC;&#x514D;&#x75AB;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3001;&#x52D5;&#x8108;&#x786C;&#x5316;&#x3001;&#x304A;&#x3088;&#x3073;&#x611F;&#x67D3;&#x6642;&#x306E;&#x7D30;&#x80DE;&#x306E;&#x79FB;&#x884C;&#x3092;&#x691C;&#x8A0E;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x9B45;&#x529B;&#x7684;&#x306A;&#x30C4;&#x30FC;&#x30EB;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x307E;&#x305F;&#x3001;BMDM &#x306E;&#x79FB;&#x690D;&#x3001;&#x6CBB;&#x7642;&#x306E;&#x65B0;&#x305F;&#x306A; in vivo &#x30DE;&#x30A6;&#x30B9;&#x75C5;&#x6C17;&#x30E2;&#x30C7;&#x30EB;&#x3067;&#x30C6;&#x30B9;&#x30C8;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x6210;&#x719F;&#x30DE;&#x30A6;&#x30B9;&#x5358;&#x7403;&#x304B;&#x3089;&#x7570;&#x7A2E;&#x9AA8;&#x9AC4;&#x3068;&#x305D;&#x306E;&#x30D7;&#x30ED;&#x30D1;&#x30C6;&#x30A3;&#x306E;&#x8AAC;&#x660E;&#x306E;&#x751F;&#x6210;&#x3002;

Monocytes &#xB2E4;&#xC591; &#xD55C; &#xB9C8;&#xC6B0;&#xC2A4; &#xBAA8;&#xB378;&#xC5D0;&#xC11C; &#xACF5;&#xBD80; &#xD558;&#xB294; &#xB9CE;&#xC740; &#xC0DD;&#xB9AC; &#xC801; &#xBC0F; &#xBCD1; &#xC801;&#xC778; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; &#xD3EC;&#xD568; &#xB41C;&#xB2E4;. &#xD604;&#xC7AC; murine monocytes&#xB97C; &#xBD84;&#xB9AC; &#xD558;&#xB294; &#xBA87; &#xAC00;&#xC9C0; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;&#xACFC; &#xBD88;&#xB9CC;&#xC871; &#xC140; &#xC218;&#xC728; &#xBC0F; &#xC21C;&#xB3C4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xD6A8;&#xC728;&#xC801;&#xC73C;&#xB85C; &#xC720;&#xD615;&#xC774; &#xB2E4;&#xB978; &#xACE8; &#xC218; &#xC138;&#xD3EC; &#xC815;&#xC9C0;&#xC5D0;&#xC11C; &#xC131;&#xC219;&#xD55C; &#xC790;&#xADF9;&#xC801;&#xC778; monocytes&#xC758; &#xB9CE;&#xC740; &#xC218;&#xB97C; &#xCC28;&#xBCC4;&#xD654; &#xD558;&#xB294; &#xB370; &#xC0C8;&#xB85C;&#xC6B4; &#xC811;&#xADFC; &#xBC29;&#xC2DD;&#xC744; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;. &#xACE8; &#xC218; &#xC138;&#xD3EC; &#xC815;&#xC9C0; femurs &#xBC0F; tibias Balb/c&#xC5D0;&#xC11C; &#xD64D; &#xC870; &#xD558; &#xC5EC; &#xACE0;&#xB9BD; &#xB418;&#xC5C8;&#xB2E4; C57Bl/6 &#xB9C8;&#xC6B0;&#xC2A4; &#xB300; &#xC2DD; &#xC138;&#xD3EC; &#xCF5C;&#xB85C; &#xB2C8; &#xC790;&#xADF9; &#xBCF4;&#xC644; &#xC694;&#xC18C; (CSF M), &#xADF8;&#xB9AC;&#xACE0; &#xC131;&#xC219; &#xC900;&#xC218; &#xC911;&#xC7AC;&#xB97C; &#xC5B5;&#xC81C; &#xD558;&#xAE30; &#xC704;&#xD574; &#xCD08; &#xC800; &#xCCA8;&#xBD80; &#xD45C;&#xBA74;&#xC5D0; &#xACBD;&#xC791; &#xD588;&#xB2E4;. &#xC140; &#xD45C;&#xC2DC; &#xC2DC;&#xAC04; &#xC9C0;&#xC810;&#xC5D0;&#xC11C;&#xAC00; &#xAC77;&#xC774; &#xD588;&#xB2E4; &#xCC28;&#xBCC4;&#xD654; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xC758; &#xD0C0;&#xC784; &#xB77C;&#xC778; &#xBD84;&#xC11D;&#xC744; &#xBC1B;&#xC558;&#xB2E4; &#xADF8;&#xB9AC;&#xACE0; &#xC774;&#xD6C4;&#xC5D0; &#xAD11;&#xBC94;&#xC704; &#xD558; &#xAC8C; phenotyped&#xC758; monocytotic &#xC18D;&#xC131;&#xC744; &#xD655;&#xC778; &#xD588;&#xB2E4;. &#xB2E4;&#xC6B4;&#xC2A4;&#xD2B8;&#xB9BC; &#xD638;&#xD658;&#xC131; &#xD655;&#xC778;&#xC744; &#xC704;&#xD574; &#xC77C;&#xBC18;&#xC801;&#xC778; monocyte &#xB3D9;&#xC791;&#xC5D0; &#xB300; &#xD55C; &#xD14C;&#xC2A4;&#xD2B8;. &#xC6B0;&#xB9AC;&#xC758; &#xD504;&#xB85C;&#xD1A0;&#xCF5C; &#xAE30;&#xC99D;&#xC790; &#xB9C8;&#xC6B0;&#xC2A4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC774;&#xC804;&#xC5D0; &#xAC00;&#xC838;&#xC628; &#xC218;&#xC775;&#xB960; &#xC8FC;&#xBCC0; &#xD608;&#xC561; &#xACA9;&#xB9AC; &#xBC29;&#xBC95; &#xBCF4;&#xB2E4; 10 &#xBC30; &#xB192;&#xC740; &#xB2F9; 24 &plusmn; 6 &times; &#xC2DC;&#xBBFC;&#xAD8C;&#xC774; &#xBD84;&#xD654; &#xC138;&#xD3EC;&#xB97C; &#xC5BB;&#xC9C0; &#xBABB;&#xD588;&#xB2E4;. &#xCC28;&#xBCC4;&#xD654; &#xB41C; &#xC140; &#xC131;&#xC219;&#xD55C; &#xB300; &#xC2DD; &#xC138;&#xD3EC;&#xAC00; &#xB418; &#xACE0; &#xB098;&#xBA38;&#xC9C0; &#xC57D; 47% &plusmn; 12 %monocytes &#xAD6C;&#xC131; &#xB418;&#xC5B4; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xAC00; &#xBD80;&#xCC29; &#xD55C; &#xB300; &#xC2DD; &#xC138;&#xD3EC;&#xB97C; &#xC5C6;&#xC560;&#xACE0; monocyte &#xC21C;&#xB3C4; 86% &plusmn; 6%&#xB85C; &#xC99D;&#xAC00;. &#xC6B0;&#xB9AC;&#xC758; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xACE8; &#xC218; &#xC720;&#xB798; monocytes (BMDMs)&#xB294; &#xC5F0;&#xAD6C;, &#xC608;&#xB97C; &#xB4E4;&#xC5B4;, &#xD0C0;&#xACE0; &#xB09C;&#xACFC; &#xC801;&#xC751; &#xBA74;&#xC5ED; &#xC2DC;&#xC2A4;&#xD15C;, &#xB3D9;&#xB9E5; &#xACBD;&#xD654; &#xC99D; &#xBC0F; &#xAC10;&#xC5FC; &#xC2DC; &#xC138;&#xD3EC; &#xB9C8;&#xC774;&#xADF8;&#xB808;&#xC774;&#xC158; &#xD558;&#xB294; &#xB9E4;&#xB825;&#xC801;&#xC778; &#xB3C4;&#xAD6C;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, BMDM &#xC774;&#xC2DD; &#xC18C;&#xC124;, &#xC950; &#xC9C8;&#xBCD1; &#xBAA8;&#xB378;&#xC5D0;&#xC11C; &#xCE58;&#xB8CC; &#xC0DD;&#xCCB4; &#xC870;&#xAC74; &#xC811;&#xADFC; &#xD14C;&#xC2A4;&#xD2B8;&#xB97C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xB2E4;&#xB978; &#xC720;&#xD615;&#xC758; &#xACE8; &#xC218; &#xBC0F; &#xADF8;&#xB4E4;&#xC758; &#xC18D;&#xC131;&#xC758; &#xC124;&#xBA85;&#xC5D0;&#xC11C; &#xC131;&#xC219;&#xD55C; murine monocytes&#xC758; &#xC138;&#xB300;.

Los monocitos participan en una amplia gama de procesos fisiol&oacute;gicos y patol&oacute;gicos, muchos de los cuales se estudian en modelos de rat&oacute;n. Los protocolos actuales para aislar los monocitos murinos son pocos y resultan en rendimiento insatisfactorio de la c&eacute;lula y pureza. Aqu&iacute;, describimos un nuevo enfoque para distinguir eficientemente grandes cantidades de monocitos inflamatorios maduros de suspensiones celulares de m&eacute;dula heterog&eacute;neos. Suspensiones de c&eacute;lulas de m&eacute;dula &oacute;sea se aislaron por lavado de f&eacute;mur y tibia de Balb/c y ratones C57Bl/6, complementados con estimulante de colonias de macr&oacute;fagos (M-CSF) del factor y fueron cultivadas en superficies de ultrabajo accesorio para inhibir la adherencia mediada por maduraci&oacute;n. Las c&eacute;lulas fueron cosechadas en los momentos indicados, sometieron a an&aacute;lisis de la l&iacute;nea de tiempo de los procesos de diferenciaci&oacute;n y posteriormente fueron ampliamente fenotipadas para comprobar sus propiedades monocytotic. Con el fin de confirmar la compatibilidad descendente, probamos para el comportamiento t&iacute;pico del monocito. Nuestro protocolo rindi&oacute; 24 &plusmn; 6 &times; 10 (6) distinguidas las c&eacute;lulas por el rat&oacute;n de la donante, diez veces superior a los rendimientos obtenidos anteriormente describen m&eacute;todos de aislamiento de sangre perif&eacute;rica. Las c&eacute;lulas diferenciadas consisti&oacute; en aproximadamente 47% &plusmn; 12% monocitos, el resto eran maduros macr&oacute;fagos. Incrementamos pureza de monocitos al 86% &plusmn; 6% por agotamiento de macr&oacute;fagos adherentes. Nuestros resultados indican que los monocitos de m&eacute;dula &oacute;sea (BMDMs) son una atractiva herramienta para estudiar, por ejemplo, el sistema inmune innato y adaptativo, aterosclerosis y migraci&oacute;n celular durante la infecci&oacute;n. Por otra parte, trasplante de BMDM podr&iacute;a utilizarse para probar la novela, enfoques terap&eacute;uticos in vivo en modelos de ratones de la enfermedad.

Generaci&oacute;n de monocitos maduros murinos de m&eacute;dula heterog&eacute;nea y descripci&oacute;n de sus propiedades.

Therapeutic augmentation of collateral artery growth (arteriogenesis) is of tremendous clinical interest. Since monocytes home to areas of arteriogenesis and create a local arteriogeneic milieu by secreting a wide range of growth factors, we followed the idea of utilizing these cells for augmentation of collateral growth. For that purpose, we adoptively transferred both syngeneic (same strain) and allogeneic (different strain) bone marrow derived monocytes (BMDMs) into balb/c mice 24 h after femoral artery ligation. Restoration of hind-limb perfusion was determined by Laser Doppler Perfusion Imaging and histological workup. While syngeneic cell transplantation did not augment arteriogenesis in comparison to non-transplanted animals (PI = 0.56 ± 0.06 vs. 0.48 ± 0.09, respectively, ns), allogeneic monocytes massively promoted the collateralization (PI = 0.85 ± 0.14, p < 0.001). Homed monocytes were visualized near growing collateral vessels by staining the cells with the lipophil fluorochrome DiI prior to transplantation. To analyze whether the effect of allogeneic BMDM transplantations is due to local inflammation triggered by a host-versus-graft reaction, transplant recipients were pre-treated with the immunosuppressive drug cyclosporine A, which completely prevented the effect of allogeineic monocyte transplantation (PI = 0.45 ± 0.06, p < 0.001). Here, we have demonstrated murine allogeneic monocytes to be an attractive way to trigger local inflammatory responses near growing collateral vessels and stimulate their adaption, overcoming the endogenous restriction of collateral vessel growth.

Transplantation of bone marrow derived monocytes: a novel approach for augmentation of arteriogenesis in a murine model of femoral artery ligation.

The pathogenesis of collateral growth (arteriogenesis) has been linked to both the innate and adaptive immune systems. While therapeutic approaches for the augmentation of arteriogenesis have focused on innate immunity, exploiting both innate and adaptive immune responses has not been examined. We hypothesized that tetanus toxoid (tt) immunization of mice followed by transplantation of monocytes (Mo) exposed ex vivo to tt augments arteriogenesis after ligation of the hind limb.

Tetanus toxoid-pulsed monocyte vaccination for augmentation of collateral vessel growth.

Senescence or biological aging impacts a vast variety of molecular and cellular processes. To date, it is unknown whether CD4(+) Th cells display an age-dependent bias for development into specific subpopulations. In this study, we show the appearance of a distinct CD4(+) T cell subset expressing IL-4 at an early stage of development in infant adenoids and cord blood that is lost during aging. We identified by flow cytometric, fluorescent microscopic, immunoblot, and mass spectrometric analysis a population of CD4(+) T cells that expressed an unglycosylated isoform of IL-4. This T cell subpopulation was found in neonatal but not in adult CD4(+) T cells. Furthermore, we show that the mRNA of the Th2 master transcription factor GATA3 is preferentially expressed in neonatal CD4(+) T cells. The Th2 phenotype of the IL-4(+)CD4(+) T cells could be reinforced in the presence of TGF-β. Although the IL-4(+)CD4(+) T cells most likely originate from CD31(+)CD4(+) T recent thymic emigrants, CD31 was downregulated prior to secretion of IL-4. Notably, the secretion of IL-4 requires a so far unidentified trigger in neonatal T cells. This emphasizes that cytokine expression and secretion are differentially regulated processes. Our data support the hypothesis of an endogenously poised cytokine profile in neonates and suggest a link between cytokine production and the developmental stage of an organism. The determination of the IL-4 isoform-expressing cells in humans might allow the identification of Th2 precursor cells, which could provide novel intervention strategies directed against Th2-driven immunopathologies such as allergies.

CD4+ T cells from human neonates and infants are poised spontaneously to run a nonclassical IL-4 program.

The NF-κB signaling pathway is central for the innate immune response and its deregulation is found in multiple disorders such as autoimmune, chronic inflammatory and metabolic diseases. IKKγ/NEMO is essential for NF-κB activation and NEMO dysfunction in humans has been linked to so-called progeria syndromes, which are characterized by advanced ageing due to age-dependent inflammatory diseases. It has been suggested that glycogen synthase kinase-3β (GSK-3β) participates in NF-κB regulation but the exact mechanism remained incompletely understood. In this study, we identified NEMO as a GSK-3β substrate that is phosphorylated at serine 8, 17, 31 and 43 located within its N-terminal domain. The kinase forms a complex with wild-type NEMO while point mutations of NEMO at the specific serines abrogated GSK-3β binding and subsequent phosphorylation of NEMO resulting in its destabilization. However, K63-linked polyubiquitination was augmented in mutated NEMO explaining an increased binding to IKKα and IKKβ. Even IκBα was found degraded. Still, TNFα-stimulated NF-κB activation was impaired pointing towards an un-controlled signalling process. Our data suggest that GSK-3β is critically important for ordered NF-κB signalling through modulation of NEMO phosphorylation.

GSK-3β controls NF-kappaB activity via IKKγ/NEMO.

Deregulated proliferation is key to tumor progression. Although unrestricted proliferation of solid tumor cells correlates with the cold-shock protein Y-box (YB)-binding protein-1 accumulation in the nuclei, little is known about its expression and function in hematopoietic malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL). Here we show that YB-1 protein is highly enriched in the nuclei of activated T cells and malignant human T-ALL cell lines but not in resting T cells. YB-1 S(102) mutations that either mimic (S102D) or prevent phosphorylation (S102N) led to accumulation of YB-1 in the nucleus of T cells or strictly excluded it, respectively. Inactivation of ribosomal S6 kinase (RSK) was sufficient to abrogate T-cell and T-ALL cell proliferation, suggesting that RSK mediates cell-cycle progression, possibly dependent on YB-1-phosphorylation. Indeed, phosphomimetic YB-1(S102D) enhanced proliferation implying that S(102) phosphorylation is a prerequisite for malignant T-cell proliferation. At initial diagnosis of T-ALL, YB-1 localization was significantly altered in the nuclei of tumor blasts derived from bone marrow or peripheral blood. Our data show deregulated YB-1 in the nucleus as a yet unreported characteristic of T-ALL blasts and may refine strategies to restrict progression of hematopoietic tumors.

Soenke Weinert

Department for Cardiology,
Angiology and Pneumology,
Department for Cardiology, Angiology and Pneumology

Otto von Guericke University Magdeburg

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

Soenke Weinert

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department for Cardiology,Angiology and Pneumology,Department for Cardiology, Angiology and Pneumology

Otto von Guericke University Magdeburg

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

Department for Cardiology,
Angiology and Pneumology,
Department for Cardiology, Angiology and Pneumology