Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

University Health Network

The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.

Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and prevents autoimmunity.

&#x75C5;&#x7406;&#x8840;&#x7BA1;&#x751F;&#x6210;&#x6709;&#x52A9;&#x4E8E;&#x4E0E;&#x70DF;&#x8349;&#x6709;&#x5173;&#x7684;&#x75BE;&#x75C5;&#xFF0C;&#x5982;&#x6076;&#x6027;&#x80BF;&#x7624;&#x3001; &#x52A8;&#x8109;&#x7CA5;&#x6837;&#x786C;&#x5316;&#x548C;&#x5E74;&#x9F84;&#x76F8;&#x5173;&#x6027;&#x9EC4;&#x6591;&#x53D8;&#x6027;&#x3002;&#x5C3C;&#x53E4;&#x4E01;&#x4F5C;&#x7528;&#x4E8E;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x70DF;&#x78B1;&#x578B;&#x4E59;&#x9170;&#x80C6;&#x78B1;&#x53D7;&#x4F53; (nAChRs)&#xFF0C;&#x6FC0;&#x6D3B;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x5E76;&#x589E;&#x52A0;&#x75C5;&#x7406;&#x8840;&#x7BA1;&#x751F;&#x6210;&#x3002;&#x5728;&#x5F53;&#x524D;&#x7684;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x7814;&#x7A76;&#x4E86; nAChR &#x4E9A;&#x57FA;&#x53C2;&#x4E0E;&#x8FD9;&#x4E9B;&#x884C;&#x52A8;&#x3002;&#x6211;&#x4EEC;&#x68C0;&#x6D4B;&#x5230; alpha(2)&#x3001; alpha(4)&#x3001; &#x4F3D;&#x739B;&#x53CA;&#x5728;&#x56DB;&#x79CD;&#x4E0D;&#x540C;&#x7C7B;&#x578B;&#x7684; ECs &#x4E09;&#x89D2;&#x6D32;&#x9664;&#x5916;&#x7684;&#x6240;&#x6709;&#x54FA;&#x4E73;&#x52A8;&#x7269; nAChR &#x4E9A;&#x57FA; mRNA&#x3002;&#x4F7F;&#x7528; siRNA &#x7684;&#x65B9;&#x6CD5;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0; alpha(7) nAChR &#x626E;&#x6F14;&#x4E3B;&#x5BFC;&#x89D2;&#x8272;&#x4E2D;&#x70DF;&#x78B1;&#x8BF1;&#x53D1;&#x7EC6;&#x80DE;&#x5185;&#x4FE1;&#x53F7;&#x4F20;&#x9012; (&#x7531;&#x7EC6;&#x80DE;&#x5185;&#x9499;&#x79BB;&#x5B50;&#x548C;&#x6CA1;&#x6709;&#x6210;&#x50CF;&#x548C;&#x86CB;&#x767D;&#x7684;&#x8868;&#x8FBE;&#x548C;&#x78F7;&#x9178;&#x5316;&#x7684;&#x7814;&#x7A76;&#x8BC4;&#x4F30;&#xFF09;&#xFF0C;&#x4EE5;&#x53CA;&#x5C3C;&#x53E4;&#x4E01;&#x6FC0;&#x6D3B; EC &#x51FD;&#x6570; &#xFF08;&#x6269;&#x6563;&#x3001; &#x751F;&#x5B58;&#x3001; &#x8FC1;&#x79FB;&#x548C;&#x7BA1;&#x5F62;&#x6210;&#xFF09;&#x3002;Alpha(9) &#x548C; alpha(7) nAChRs &#x6709;&#x53CD;&#x5BF9;&#x5BF9;&#x5C3C;&#x53E4;&#x4E01;&#x8BF1;&#x5BFC;&#x7EC6;&#x80DE;&#x589E;&#x6B96;&#x548C;&#x5B58;&#x6D3B;&#x7684;&#x5F71;&#x54CD;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x8868;&#x660E; alpha(7) nAChR &#x4ECB;&#x5BFC;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x5BF9;&#x5C3C;&#x53E4;&#x4E01;&#x7684;&#x5F71;&#x54CD;&#x7684;&#x5173;&#x952E;&#x4F5C;&#x7528;&#x3002;&#x5176;&#x4ED6;&#x4E9A;&#x57FA;&#x53D1;&#x6325;&#x8C03;&#x8282;&#x4F5C;&#x7528;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x53EF;&#x80FD;&#x6CBB;&#x7597;&#x5F71;&#x54CD;&#x75BE;&#x75C5;&#x75C5;&#x7406;&#x8840;&#x7BA1;&#x5F62;&#x6210;&#x7684;&#x7279;&#x70B9;&#x3002;

&#x80C6;&#x78B1;&#x80FD;&#x8C03;&#x5236;&#x7684;&#x8840;&#x7BA1;&#x751F;&#x6210;&#xFF1A; 7 &#x70DF;&#x78B1;&#x578B;&#x4E59;&#x9170;&#x80C6;&#x78B1;&#x53D7;&#x4F53;&#x7684;&#x4F5C;&#x7528;&#x3002;

Pathological angiogenesis contributes to tobacco-related diseases such as malignancy, atherosclerosis and age-related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except alpha(2), alpha(4), gamma, and delta in four different types of ECs. Using siRNA methodology, we found that the alpha(7) nAChR plays a dominant role in nicotine-induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine-activated EC functions (proliferation, survival, migration, and tube formation). The alpha(9) and alpha(7) nAChRs have opposing effects on nicotine-induced cell proliferation and survival. Our studies reveal a critical role for the alpha(7) nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis.

Cholinergic modulation of angiogenesis: role of the 7 nicotinic acetylcholine receptor.

L&#x26;#39;angiogen&#xE8;se pathologique contribue &#xE0; maladies li&#xE9;es au tabac comme une tumeur maligne, l&#x26;#39;ath&#xE9;roscl&#xE9;rose et la d&#xE9;g&#xE9;n&#xE9;rescence maculaire li&#xE9;e &#xE0; l&#x26;#39;&#xE2;ge. La nicotine agit sur endoth&#xE9;liales r&#xE9;cepteurs nicotiniques d&#x26;#39;ac&#xE9;tylcholine (nAChR) pour activer les cellules endoth&#xE9;liales et d&#x26;#39;augmenter angiogen&#xE8;se pathologique. Dans l&#x26;#39;&#xE9;tude actuelle, nous avons &#xE9;tudi&#xE9; les sous-unit&#xE9;s nAChR impliqu&#xE9;s dans ces actions. Nous avons d&#xE9;tect&#xE9; l&#x26;#39;ARNm pour tous les sous-unit&#xE9;s nAChR mammif&#xE8;res sauf alpha (2), l&#x26;#39;alpha (4), gamma et delta dans quatre diff&#xE9;rents types de contraceptifs d&#x26;#39;urgence. En utilisant une m&#xE9;thodologie siARN, nous avons constat&#xE9; que l&#x26;#39;alpha (7) nAChR joue un r&#xF4;le dominant dans la signalisation cellulaire induite par la nicotine (&#xE9;valu&#xE9;e par le calcium intracellulaire et de NO imagerie, et des &#xE9;tudes d&#x26;#39;expression de prot&#xE9;ines et de la phosphorylation), ainsi que la nicotine activ&#xE9;s fonctions communautaires ( prolif&#xE9;ration, la survie, la migration et la formation du tube). L&#x26;#39;alpha (9) et alpha (7) nAChR ont des effets oppos&#xE9;s sur la prolif&#xE9;ration cellulaire induite par la nicotine et la survie. Nos &#xE9;tudes r&#xE9;v&#xE8;lent un r&#xF4;le crucial pour l&#x26;#39;alpha (7) nAChR dans la m&#xE9;diation des effets de la nicotine sur l&#x26;#39;endoth&#xE9;lium. Autres sous-unit&#xE9;s jouent un r&#xF4;le modulateur. Ces r&#xE9;sultats peuvent avoir des implications th&#xE9;rapeutiques pour des maladies caract&#xE9;ris&#xE9;es par une angiogen&#xE8;se pathologique.

Cholinergique modulation de l&#x26;#39;angiogen&#xE8;se: R&#xF4;le de r&#xE9;cepteur de l&#x26;#39;ac&#xE9;tylcholine nicotinique 7

Pathologische Angiogenesis tr&auml;gt zur Tabak-Erkrankungen wie B&ouml;sartigkeit, Atherosklerose und altersbedingte Makula-Degeneration. Nikotin wirkt auf endothelial nikotinische Acetylcholin-Rezeptoren (nAChRs), Endothelzellen zu aktivieren und pathologischen Angiogenese zu erweitern. In der aktuellen Studie untersucht wir nAChR Untereinheiten dieser Aktionen beteiligt. Wir erkannten mRNA f&uuml;r alle S&auml;ugetier-nAChR Untereinheiten au&szlig;er alpha(2), alpha(4), Gamma und Delta in vier verschiedene Typen von ECs. Mit SiRNA-Methodik, fanden wir, dass die alpha(7) nAChR eine beherrschende Rolle bei Nikotin-induced Zelle Signalisierung (gepr&uuml;ft durch intrazellul&auml;re Kalzium und keine Bildgebung und Studium der Proteinexpression und Phosphorylierung) sowie Nikotin aktivierte EG-Funktionen (Verbreitung, &uuml;berleben, Migration und Gef&auml;&szlig;-Anordnung) spielt. Die alpha(9) und alpha(7) nAChRs haben gegens&auml;tzliche Wirkungen auf Nikotin-induzierte Zellproliferation und &uuml;berleben. Unsere Studien zeigen eine entscheidende Rolle f&uuml;r die alpha(7)-nAChR, bei der Vermittlung von der Wirkungen von Nikotin auf das Endothel. Andere Untereinheiten Rolle eine modulierende. Diese Erkenntnisse k&ouml;nnen Krankheiten mit pathologischen Angiogenese therapeutische auswirken.

Cholinerge Modulation der Angiogenese: Rolle der 7 nikotinische Acetylcholin-Rezeptor.

L&#39;angiogenesi patologica contribuisce a malattie legate al tabacco come tumori maligni, l&#39;aterosclerosi e la degenerazione maculare legata all&#39;et&agrave;. Nicotine agisce sui recettori endoteliali nicotinici acetilcolina (nAChRs) per attivare le cellule endoteliali e di aumentare l&#39;angiogenesi patologica. In questo studio, abbiamo studiato le subunit&agrave; nAChR coinvolte in queste azioni. Abbiamo rilevato mRNA per tutti i mammiferi nAChR subunit&agrave; tranne alpha(2), alpha(4), gamma e delta in quattro diversi tipi di ECs. Utilizzando la metodologia di siRNA, abbiamo trovato che i nAChR alpha(7) gioca un ruolo dominante nella segnalazione cellulare indotta da nicotina (valutata da calcio intracellulare e non imaging e gli studi di espressione della proteina e fosforilazione), oltre a funzioni di CE attivati da nicotina (proliferazione, sopravvivenza, migrazione e formazione del tubo). Il nAChRs alpha(9) e alpha(7) hanno opposti effetti sulla proliferazione cellulare indotta da nicotina e sopravvivenza. I nostri studi rivelano un ruolo critico per i nAChR alpha(7) nel mediare gli effetti della nicotina sull&#39;endotelio. Altre subunit&agrave; gioca un ruolo modulatore. Questi risultati possono avere implicazioni terapeutiche per malattie caratterizzate da angiogenesi patologica.

Modulazione colinergica dell&#39;angiogenesi: ruolo del recettore 7 nicotinici acetilcolina.

&#x75C5;&#x7684;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x306F;&#x3001;&#x60AA;&#x6027;&#x816B;&#x760D;&#x3001;&#x52D5;&#x8108;&#x786C;&#x5316;&#x3084;&#x52A0;&#x9F62;&#x9EC4;&#x6591;&#x5909;&#x6027;&#x75C7;&#x306A;&#x3069;&#x306E;&#x305F;&#x3070;&#x3053;&#x95A2;&#x9023;&#x75BE;&#x60A3;&#x306B;&#x8CA2;&#x732E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x306E;&#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x6027;&#x30A2;&#x30BB;&#x30C1;&#x30EB;&#x30B3;&#x30EA;&#x30F3;&#x53D7;&#x5BB9;&#x4F53;&#xFF08;nAChRs&#xFF09;&#x306E;&#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x4F5C;&#x7528;&#x306F;&#x3001;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x3092;&#x6D3B;&#x6027;&#x5316;&#x3057;&#x3001;&#x75C5;&#x7684;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x3092;&#x5897;&#x5F37;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3002;&#x73FE;&#x5728;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30A2;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x306E;nAChR&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x3092;&#x8ABF;&#x3079;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;EC&#x306E;4&#x3064;&#x306E;&#x7570;&#x306A;&#x308B;&#x30BF;&#x30A4;&#x30D7;&#x306E;&#x30A2;&#x30EB;&#x30D5;&#x30A1;&#xFF08;2&#xFF09;&#x3001;&#x30A2;&#x30EB;&#x30D5;&#x30A1;&#xFF08;4&#xFF09;&#x3001;&#x30AC;&#x30F3;&#x30DE;&#x3001;&#x30C7;&#x30EB;&#x30BF;&#x3092;&#x9664;&#x304F;&#x3059;&#x3079;&#x3066;&#x306E;&#x54FA;&#x4E73;&#x985E;&#x306E;nAChR&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;mRNA&#x3092;&#x691C;&#x51FA;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002; siRNA&#x306E;&#x624B;&#x6CD5;&#x3092;&#x7528;&#x3044;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;&#x3B1;&#xFF08;7&#xFF09;nAChR&#x306E;&#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x8A98;&#x767A;&#x6027;&#x306E;&#x7D30;&#x80DE;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#xFF08;&#x7D30;&#x80DE;&#x5185;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x53CA;&#x3073;NO&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x3001;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x767A;&#x73FE;&#x3068;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306E;&#x7814;&#x7A76;&#x306B;&#x3088;&#x308A;&#x8A55;&#x4FA1;&#xFF09;&#x3068;&#x540C;&#x69D8;&#x306B;&#x3001;&#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x6D3B;&#x6027;&#x5316;EC&#x95A2;&#x6570;&#x3067;&#x652F;&#x914D;&#x7684;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x304C;&#x308F;&#x304B;&#x3063;&#x305F;&#xFF08;&#x5897;&#x6B96;&#x3001;&#x751F;&#x5B58;&#x3001;&#x904A;&#x8D70;&#x3001;&#x30C1;&#x30E5;&#x30FC;&#x30D6;&#x5F62;&#x6210;&#xFF09;&#x3002; &#x3B1;&#xFF08;9&#xFF09;&#x3068;&#x30A2;&#x30EB;&#x30D5;&#x30A1;&#xFF08;7&#xFF09;nAChRs&#x306F;&#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x8A98;&#x767A;&#x6027;&#x306E;&#x7D30;&#x80DE;&#x5897;&#x6B96;&#x304A;&#x3088;&#x3073;&#x751F;&#x5B58;&#x306B;&#x53CA;&#x307C;&#x3059;&#x5F71;&#x97FF;&#x306B;&#x53CD;&#x5BFE;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005;&#x306E;&#x7814;&#x7A76;&#x306F;&#x3001;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x306B;&#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x306E;&#x52B9;&#x679C;&#x3092;&#x5A92;&#x4ECB;&#x3059;&#x308B;&#x30A2;&#x30EB;&#x30D5;&#x30A1;&#xFF08;7&#xFF09;nAChR&#x306E;&#x305F;&#x3081;&#x306E;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x305F;&#x3002;&#x4ED6;&#x306E;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306F;&#x3001;&#x8ABF;&#x7BC0;&#x306E;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x77E5;&#x898B;&#x306F;&#x3001;&#x75C5;&#x7684;&#x306A;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x306B;&#x3088;&#x3063;&#x3066;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x308B;&#x75BE;&#x60A3;&#x306E;&#x6CBB;&#x7642;&#x306B;&#x5F71;&#x97FF;&#x304C;&#x3042;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;

&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x306E;&#x30B3;&#x30EA;&#x30F3;&#x4F5C;&#x52D5;&#x6027;&#x8ABF;&#x7BC0;&#xFF1A;7&#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x6027;&#x30A2;&#x30BB;&#x30C1;&#x30EB;&#x30B3;&#x30EA;&#x30F3;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x5F79;&#x5272;

&#xBCD1; &#xC801;&#xC778; &#xC2E0;&#xC0DD; &#xC545;&#xC131; &#xC885;&#xC591;, &#xB3D9;&#xB9E5; &#xACBD;&#xD654; &#xC99D;, &#xC5F0;&#xB839; &#xAD00;&#xB828; &#xD669; &#xBC18; &#xBCC0;&#xC131; &#xB4F1; &#xB2F4;&#xBC30; &#xAD00;&#xB828; &#xC9C8;&#xBCD1;&#xC5D0; &#xAE30;&#xC5EC; &#xD558; &#xACE0; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB2C8;&#xCF54;&#xD2F4; &#xB0B4; &#xD53C; nicotinic &#xC544; &#xC138; &#xD2F8; &#xCF5C;&#xB9B0; &#xC218;&#xC6A9; &#xCCB4; (nAChRs) &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xB97C; &#xD65C;&#xC131;&#xD654; &#xD558; &#xACE0; &#xBCD1; &#xC801;&#xC778; &#xC2E0;&#xC0DD; &#xBCF4;&#xAC15;&#xC5D0; &#xC5ED;&#xD560;&#xC744; &#xD569;&#xB2C8;&#xB2E4;. &#xD604;&#xC7AC; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; nAChR subunits &#xC774;&#xB7EC;&#xD55C; &#xC791;&#xC5C5;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xACF5;&#xBD80;. &#xC6B0;&#xB9AC;&#xB294; alpha(2), alpha(4), &#xAC10;&#xB9C8; &#xBC0F; &#xB378;&#xD0C0; Ecs&#xC758; 4 &#xC885;&#xB958;&#xC5D0;&#xC11C;&#xB97C; &#xC81C;&#xC678; &#xD558; &#xACE0; &#xBAA8;&#xB4E0; &#xD3EC;&#xC720;&#xB958; nAChR subunits&#xC5D0; &#xB300; &#xD55C; Mrna&#xB97C; &#xAC10;&#xC9C0; &#xD569;&#xB2C8;&#xB2E4;. SiRNA &#xBC29;&#xBC95;&#xB860;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xC6B0;&#xB9AC;&#xB294; alpha(7) nAChR EC &#xAE30;&#xB2A5; &#xB2C8;&#xCF54;&#xD2F4; &#xD65C;&#xC131;&#xD654; (&#xC99D;&#xC2DD;, &#xC0DD;&#xC874;, &#xB9C8;&#xC774;&#xADF8;&#xB808;&#xC774;&#xC158; &#xBC0F; &#xD29C;&#xBE0C; &#xD615;&#xC131;) &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xB2C8;&#xCF54;&#xD2F4; &#xC720;&#xB3C4; &#xC138;&#xD3EC; &#xC2E0;&#xD638; (&#xC138;&#xD3EC;&#xB0B4; &#xCE7C;&#xC298; &#xC544;&#xBB34; &#xC774;&#xBBF8;&#xC9D5;, &#xACE0; &#xB2E8;&#xBC31;&#xC9C8; &#xC2DD; &#xBC0F; &#xC778; &#xC0B0;&#xD654;&#xC5D0; &#xC758;&#xD574; &#xD3C9;&#xAC00;)&#xC5D0;&#xC11C; &#xC9C0;&#xBC30;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4; &#xBC1C;&#xACAC;. Alpha(9) &#xBC0F; alpha(7) nAChRs &#xB2C8;&#xCF54;&#xD2F4; &#xC720;&#xB3C4; &#xB41C; &#xC138;&#xD3EC; &#xC99D;&#xC2DD;&#xACFC; &#xC0DD;&#xC874;&#xC5D0; &#xB300; &#xD55C; &#xD6A8;&#xACFC; &#xBC18;&#xB300; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xC5F0;&#xAD6C;&#xB294; alpha(7) nAChR endothelium&#xC5D0; &#xB2C8;&#xCF54;&#xD2F4;&#xC758; &#xD6A8;&#xACFC; &#xC911;&#xC7AC;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xACF5;&#xAC1C;. &#xB2E4;&#xB978; &#xD558;&#xC704; &#xB2E8;&#xC704; modulatory &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xC9C8;&#xBCD1; &#xBCD1; &#xC801;&#xC778; &#xC2E0;&#xC0DD;&#xC774; &#xD2B9;&#xC9D5;&#xC5D0; &#xB300; &#xD55C; &#xCE58;&#xB8CC; &#xC801; &#xC758;&#xBBF8; &#xC788;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC2E0;&#xC0DD;&#xC758; cholinergic &#xBCC0;&#xC870;: 7 nicotinic &#xC544; &#xC138; &#xD2F8; &#xCF5C;&#xB9B0; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC5ED;&#xD560;.

Angiog&#xE9;nesis patol&#xF3;gica contribuye a las enfermedades relacionadas con el tabaco, como c&#xE1;ncer, la aterosclerosis y la relacionada con la edad degeneraci&#xF3;n macular. La nicotina act&#xFA;a sobre el endotelio receptores nicot&#xED;nicos de acetilcolina (nAChR) para activar las c&#xE9;lulas endoteliales y aumentar la angiog&#xE9;nesis patol&#xF3;gica. En el presente estudio, se estudiaron las subunidades de nAChR que participan en estas acciones. Hemos detectado mRNA de todas las subunidades de nAChR mam&#xED;feros excepto alfa (2), alfa (4), gamma y delta en cuatro tipos diferentes de EC. Utilizando la metodolog&#xED;a del siRNA, encontramos que el alfa (7) nAChR juega un papel dominante en la se&#xF1;alizaci&#xF3;n celular inducida por la nicotina (evaluado por el calcio intracelular y la imagen NO, y los estudios de expresi&#xF3;n de la prote&#xED;na y la fosforilaci&#xF3;n), as&#xED; como la nicotina activadas las funciones de la CE ( proliferaci&#xF3;n, la supervivencia, la migraci&#xF3;n y la formaci&#xF3;n del tubo). La alfa (9) y nAChRs alfa (7) tienen efectos opuestos sobre la proliferaci&#xF3;n celular inducida por la nicotina y la supervivencia. Nuestros estudios revelan un papel fundamental para el alfa (7) nAChR en la mediaci&#xF3;n de los efectos de la nicotina sobre el endotelio. Subunidades Otros juegan un papel modulador. Estos hallazgos pueden tener implicaciones terap&#xE9;uticas para las enfermedades que se caracterizan por la angiog&#xE9;nesis patol&#xF3;gica.

Colin&#xE9;rgica de modulaci&#xF3;n de la angiog&#xE9;nesis: Papel de los receptores de acetilcolina nicot&#xED;nico 7

Broadly neutralizing antibodies (bNt Abs) against HIV-1 are rarely produced during natural infection, and efforts to induce such Abs by vaccination have been unsuccessful. Thus, elucidating the nature and cellular origins of bNt Abs is a high priority for vaccine research. As the bNt monoclonal Abs (MAbs) 2F5, 4E10 and 2G12 have been reported to bind select autoantigens, we investigated whether these MAbs display a broader range of autoreactivity and how their autoreactivity compares with that of pathogenic autoAbs.

Reactivity profiles of broadly neutralizing anti-HIV-1 antibodies are distinct from those of pathogenic autoantibodies.

Antibody-mediated rejection (AMR) is gaining increasing recognition as a major complication after heart transplantation, posing a significant risk for allograft failure, cardiac allograft vasculopathy, and poor survival. AMR results from activation of the humoral immune arm and the production of donor-specific antibodies (DSA) that bind to the cardiac allograft causing myocardial injury predominantly through complement activation. The diagnosis of AMR has evolved from a clinical diagnosis involving allograft dysfunction and the presence of DSA to a primarily pathologic diagnosis based on histopathology and immunopathology. Treatment for AMR is multifaceted, targeting inhibition of the humoral immune system at different levels with emerging agents including proteasome and complement inhibitors showing particular promise. While there have been significant advances in our current understanding of the pathogenesis, diagnosis, and treatment of AMR, further research is required to determine optimal diagnostic tools, therapeutic agents, and timing of treatment.

Antibody-mediated rejection: an evolving entity in heart transplantation.

Women develop certain autoimmune diseases more often than men. It has been hypothesized that this may relate to the development of more robust T-helper (Th)1 responses in women. To test whether women exhibit a Th1 bias, we isolated naïve cluster of differentiation (CD)4(+) T cells from peripheral blood of healthy women and men and measured the proliferation and cytokine production by these cells in response to submaximal amounts of anti-CD3 and anti-CD28. We observed that CD4(+) T cells from women produced higher levels of IFNγ as well as tended to proliferate more than male CD4(+) T cells. Intriguingly, male CD4(+) T cells instead had a predilection toward IL-17A production. This sex dichotomy in Th cytokine production was found to be even more striking in the Swiss/Jackson Laboratory (SJL) mouse. Studies in mice and humans indicated that the sexual dimorphism in Th1 and Th17 cytokine production was dependent on the androgen status and the T-cell expression of peroxisome proliferator activated receptor (PPAR)α and PPARγ. Androgens increased PPARα and decreased PPARγ expression by human CD4(+) T cells. PPARα siRNA-mediated knockdown had the effect of increasing IFNγ by male CD4(+) T cells, while transfection of CD4(+) T cells with PPARγ siRNAs increased IL-17A production uniquely by female T cells. Together, our observations indicate that human T cells exhibit a sex difference in the production of IFNγ and IL-17A that may be driven by expressions of PPARα and PPARγ.

Peroxisome proliferator-activated receptor (PPAR)α and -γ regulate IFNγ and IL-17A production by human T cells in a sex-specific way.

β-adrenergic receptors (β-ARs) are model G-protein coupled receptors that mediate signal transduction in the sympathetic nervous system. Despite the widespread clinical use of agents that target β-ARs, the signaling pathways that operate downstream of β-AR stimulation have not yet been completely elucidated. Here, we utilized a lysate microarray approach to obtain a broad-scale perspective of phosphoprotein signaling downstream of β-AR. We monitored the time course of phosphorylation states of 54 proteins after β-AR activation mouse embryonic fibroblast (MEF) cells. In response to stimulation with the non-selective β-AR agonist isoproterenol, we observed previously described phosphorylation events such as ERK1/2(T202/Y204) and CREB(S133), but also novel phosphorylation events such as Cdc2(Y15) and Pyk2(Y402). All of these events were mediated through cAMP and PKA as they were reproduced by stimulation with the adenylyl cyclase activator forskolin and were blocked by treatment with H89, a PKA inhibitor. In addition, we also observed a number of novel isoproterenol-induced protein dephosphorylation events in target substrates of the PI3K/AKT pathway: GSK3β(S9), 4E-BP1(S65), and p70s6k(T389). These dephosphorylations were dependent on cAMP, but were independent of PKA and correlated with reduced PI3K/AKT activity. Isoproterenol stimulation also led to a cAMP-dependent dephosphorylation of PP1α(T320), a modification known to correlate with enhanced activity of this phosphatase. Dephosphorylation of PP1α coincided with the secondary decline in phosphorylation of some PKA-phosphorylated substrates, suggesting that PP1α may act in a feedback loop to return these phosphorylations to baseline. In summary, lysate microarrays are a powerful tool to profile phosphoprotein signaling and have provided a broad-scale perspective of how β-AR signaling can regulate key pathways involved in cell growth and metabolism.

Broad-scale phosphoprotein profiling of beta adrenergic receptor (β-AR) signaling reveals novel phosphorylation and dephosphorylation events.

Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long-term immunosuppression. To investigate mechanisms of rapamycin-induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC-mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4(+) and CD8(+) immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin-induced, donor-specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen-like protein 2 (FGL2). Depletion of Treg cells with anti-CD25 (PC61) and treatment with anti-FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co-expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual-staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg-associated genes (foxp3, lag3, tgf-β and fgl2) had increased expression and pro-inflammatory genes (ifn-γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin-induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.

The regulatory T cell effector molecule fibrinogen-like protein 2 is necessary for the development of rapamycin-induced tolerance to fully MHC-mismatched murine cardiac allografts.

Although the newer continuous-flow left ventricular assist devices (CF-LVADs) provide clinical advantages over the pulsatile pumps, the effects of low pulsatility on inflammation are incompletely understood. The objective of our study was to examine the levels of inflammatory mediators in CF-LVAD recipients compared with both healthy control subjects and heart failure patients who were candidates for CF-LVAD support. Plasma levels of chemokines, cytokines, and inflammatory markers were measured in 18 CF-LVAD recipients and compared with those of 14 healthy control subjects and 14 heart failure patients who were candidates for CF-LVADs. The levels of granulocyte macrophage-colony stimulating factor, macrophage inflammatory proteins-1β, and macrophage-derived chemokine were significantly higher in the CF-LVAD group compared with both the heart failure and the healthy control groups, whereas no significant differences were observed between the healthy control subjects and the heart failure groups. Compared with the healthy controls, C-reactive protein, interferon gamma-induced protein-10, monocyte chemotactic protein-1, and interleukin-8 levels were significantly higher in both the CF-LVAD and heart failure groups, but no significant differences were observed between the CF-LVAD recipients and the heart failure patients. Inflammatory markers were elevated in CF-LVAD recipients compared with healthy control subjects and the heart failure patients. Further studies should investigate the clinical implications of elevated levels of inflammation in CF-LVAD recipients.

Markers of inflammation in recipients of continuous-flow left ventricular assist devices.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are critical to the maintenance of immune tolerance. Treg are known to utilize a number of molecular pathways to control immune responses and maintain immune homeostasis. Fibrinogen-like protein 2 (FGL2) has been identified by a number of investigators as an important immunosuppressive effector of Treg, which exerts its immunoregulatory activity by binding to inhibitory FcγRIIB receptors expressed on antigen-presenting cells including dendritic cells, endothelial cells, and B cells. More recently, it has been suggested that FGL2 accounts for the immunosuppressive activity of a highly suppressive subset of Treg that express T cell immunoreceptor with Ig and ITIM domains (TIGIT). Here we discuss the important role of Treg and FGL2 in preventing alloimmune and autoimmune disease. The FGL2-FcγRIIB pathway is also known to be utilized by viruses and tumor cells to evade immune surveillance. Moving forward, therapies based on modulation of the FGL2-FcγRIIB pathway hold promise for the treatment of a wide variety of conditions ranging from autoimmunity to cancer.

Role of Regulatory T Cells (Treg) and the Treg Effector Molecule Fibrinogen-like Protein 2 in Alloimmunity and Autoimmunity.

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR.

Andrzej Chruscinski

Multi-Organ Transplant Program

University Health Network

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Andrzej Chruscinski

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University Health Network

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Multi-Organ Transplant Program