an-il-8-transiently-transgenized-mouse-model-for-vivo-long-term

An IL-8 Transiently Transgenized Mouse Model for the <em>In Vivo</em> Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Chiesi Farmaceutici S.p.A.

Edotecarin (J-107088, formerly ED-749) is a potent indolocarbazole topoisomerase-I inhibitor that has the potential to treat solid tumors. The current studies evaluated the potency and antitumor activity of edotecarin, as a single agent and in combination with capecitabine or docetaxel.

Antitumor activity of edotecarin in breast carcinoma models.

In vivo imaging of apoptosis in a preclinical setting in anticancer drug development could provide remarkable advantages in terms of translational medicine. So far, several imaging technologies with different probes have been used to achieve this goal. Here we describe a bioluminescence imaging approach that uses a new formulation of Z-DEVD-aminoluciferin, a caspase 3/7 substrate, to monitor in vivo apoptosis in tumor cells engineered to express luciferase. Upon apoptosis induction, Z-DEVD-aminoluciferin is cleaved by caspase 3/7 releasing aminoluciferin that is now free to react with luciferase generating measurable light. Thus, the activation of caspase 3/7 can be measured by quantifying the bioluminescent signal. Using this approach, we have been able to monitor caspase-3 activation and subsequent apoptosis induction after camptothecin and temozolomide treatment on xenograft mouse models of colon cancer and glioblastoma, respectively. Treated mice showed more than 2-fold induction of Z-DEVD-aminoluciferin luminescent signal when compared to the untreated group. Combining D: -luciferin that measures the total tumor burden, with Z-DEVD-aminoluciferin that assesses apoptosis induction via caspase activation, we confirmed that it is possible to follow non-invasively tumor growth inhibition and induction of apoptosis after treatment in the same animal over time. Moreover, here we have proved that following early apoptosis induction by caspase 3 activation is a good biomarker that accurately predicts tumor growth inhibition by anti-cancer drugs in engineered colon cancer and glioblastoma cell lines and in their respective mouse xenograft models.

In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation.

NF-&kappa; B &#x7684;&#x6FC0;&#x6D3B;&#x662F;&#x5728;&#x708E;&#x75C7;&#x53CD;&#x5E94;&#x4E2D;&#x7684;&#x5173;&#x952E;&#x4FE1;&#x53F7;&#x4E8B;&#x4EF6;&#x548C;&#x4E00;&#x4E9B;&#x75C5;&#x7406;&#x80BA;&#x75BE;&#x75C5;&#x6709;&#x7275;&#x8FDE;&#x3002;&#x8F6C;&#xFF0C;&#x4F7F;&#x80BA;&#x7EC4;&#x7EC7; NF-&kappa; B &#x6D3B;&#x6027;&#x7684;&#x8BC4;&#x4F30;&#xFF0C;&#x6211;&#x4EEC;&#x67D3;&#x8367;&#x5149;&#x7D20;&#x9176;&#x57FA;&#x4E8E; NF-&kappa; B &#x8BB0;&#x8005;&#x5230;&#x5C0F;&#x9F20;&#x7684;&#x80BA;&#x90E8;&#x6216; Raw264.7 &#x7EC6;&#x80DE;&#x7684;&#x57F9;&#x517B;&#x3002;&#x8F6C;&#x67D3;&#x7684;&#x9F20;&#x80BA;&#x7EC4;&#x7EC7;&#x4E2D;&#x663E;&#x793A;&#x7279;&#x5B9A;&#x7684;&#x8367;&#x5149;&#x7D20;&#x9176;&#x8868;&#x8FBE;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x8FD9;&#x4E9B;&#x5C0F;&#x9F20;&#x6A21;&#x578B;&#xFF0C;&#x7814;&#x7A76;&#x4E86;&#x52A8;&#x529B;&#x5B66;&#x7684; NF-&kappa; B &#x6FC0;&#x6D3B;&#x8102;&#x591A;&#x7CD6; (LPS) &#x63A5;&#x89E6;&#x4E4B;&#x540E;&#x3002;Raw264.7 &#x7EC6;&#x80DE;&#x8868;&#x8FBE;&#x8367;&#x5149;&#x7D20;&#x9176;&#x540E;&#x66B4;&#x9732;&#x4E8E; LPS &#x548C; NF-&kappa; B &#x80BA;&#x540E; LPS &#x6311;&#x6218;&#x3001; &#x5EFA;&#x7ACB;&#x751F;&#x7269;&#x53D1;&#x5149;&#x6210;&#x50CF;&#x6280;&#x672F;&#x63D0;&#x4F9B;&#x4E86;&#x7528;&#x4E8E;&#x8DDF;&#x8E2A; NF-&kappa; B &#x7684;&#x6FC0;&#x6D3B;&#x901A;&#x8DEF;&#x8DB3;&#x591F;&#x7075;&#x654F;&#x5EA6;&#x8BB0;&#x8005;&#x5C0F;&#x9F20;&#x8868;&#x8FBE;&#x8367;&#x5149;&#x7D20;&#x9176;&#x5242;&#x91CF;&#x4F9D;&#x8D56;&#x6027;&#x589E;&#x52A0;&#x3002;&#x5F71;&#x54CD; NF-&kappa; B &#x901A;&#x8DEF;&#x7684;&#x5E72;&#x9884;&#x63AA;&#x65BD;&#x662F;&#x6709;&#x5E0C;&#x671B;&#x7684;&#x4E34;&#x5E8A;&#x6CBB;&#x7597;&#x5B66;&#xFF0C;&#x56E0;&#x6B64;&#x6211;&#x4EEC;&#x8FDB;&#x4E00;&#x6B65;&#x5BA1;&#x67E5; MLN120B &#x548C;&#x7CD6;&#x539F;&#x5408;&#x6210;&#x9176;&#x6FC0;&#x9176; 3 beta TDZD 8 &#x5BF9;&#x6291;&#x5236; NF-&kappa; B &#x7684;&#x6FC0;&#x6D3B; IKK-2 &#x6291;&#x5236;&#x4F5C;&#x7528;&#x7684;&#x5F71;&#x54CD;&#x3002;&#x9884;&#x5904;&#x7406;&#x4E0E; MLN120B &#x6216; TDZD-8 &#x51CF;&#x6BD2; NF-&kappa; B &#x6D3B;&#x5316;&#x7684;&#x80BA;&#x7EC4;&#x7EC7;&#xFF0C;&#x4F34;&#x968F;&#x6709;&#x6291;&#x5236;&#x708E;&#x75C7;&#x8D8B;&#x5316;&#x56E0;&#x5B50; MIP 1&szlig; &#x548C;&#x6297;&#x708E;&#x75C7;&#x7EC6;&#x80DE;&#x56E0;&#x5B50; IL-10 &#x611F;&#x5E94;&#x3002;&#x603B;&#x4E4B;&#xFF0C;&#x6211;&#x4EEC;&#x5EFA;&#x7ACB;&#x4E86;&#x975E;&#x4FB5;&#x5165;&#x6027;&#x548C;&#x7EB5;&#x5411;&#x8BC4;&#x4F30; NF-&kappa; B &#x7684;&#x6FC0;&#x6D3B;&#x548C;&#x8C03;&#x8282;&#x6025;&#x6027;&#x80BA;&#x635F;&#x4F24;&#x5F71;&#x50CF;&#x5B66;&#x57FA;&#x7840;&#x7684;&#x65B9;&#x6CD5;&#x3002;&#x8FD9;&#x79CD;&#x505A;&#x6CD5;&#x5C06;&#x8FDB;&#x4E00;&#x6B65;&#x52A0;&#x5F3A;&#x5404;&#x79CD;&#x708E;&#x75C7;&#x6761;&#x4EF6;&#x4E0B;&#x7684; NF-&kappa; B &#x89C4;&#x5F8B;&#x7814;&#x7A76;&#x3002;

&#x5F71;&#x50CF;&#x5B66;&#x80BA;&#x9ECF;&#x6DB2;&#x548C; MLN120B &#x548C; TDZD-8 &#x7684;&#x7597;&#x6548;&#x3002;

NF-ÎºB activation is a critical signaling event in the inflammatory response and has been implicated in a number of pathological lung diseases. To enable the assessment of NF-ÎºB activity in the lungs, we transfected a luciferase based NF-ÎºB reporter into the lungs of mice or into Raw264.7 cells in culture. The transfected mice showed specific luciferase expression in the pulmonary tissues. Using these mouse models, we studied the kinetics of NF-ÎºB activation following exposure to lipopolysaccharide (LPS). The Raw264.7 cells expressed a dose-dependent increase in luciferase following exposure to LPS and the NF-ÎºB reporter mice expressed luciferase in the lungs following LPS challenge, establishing that bioluminescence imaging provides adequate sensitivity for tracking the NF-ÎºB activation pathway. Interventions affecting the NF-ÎºB pathway are promising clinical therapeutics, thus we further examined the effect of IKK-2 inhibition by MLN120B and glycogen synthase kinase 3 beta inhibition by TDZD-8 on NF-ÎºB activation. Pre-treatment with either MLN120B or TDZD-8 attenuated NF-ÎºB activation in the pulmonary tissues, which was accompanied with suppression of pro-inflammatory chemokine MIP-1ÃŸ and induction of anti-inflammatory cytokine IL-10. In summary, we have established an imaging based approach for non-invasive and longitudinal assessment of NF-ÎºB activation and regulation during acute lung injury. This approach will potentiate further studies on NF-ÎºB regulation under various inflammatory conditions.

Imaging pulmonary NF-kappaB activation and therapeutic effects of MLN120B and TDZD-8.

L&#x26;#39;activation de NF-kB est un &#xE9;v&#xE9;nement critique de signalisation dans la r&#xE9;ponse inflammatoire et a &#xE9;t&#xE9; impliqu&#xE9; dans un certain nombre de maladies pulmonaires pathologiques. Pour permettre l&#x26;#39;&#xE9;valuation de l&#x26;#39;activit&#xE9; de NF-kB dans les poumons, nous avons transfect&#xE9; une lucif&#xE9;rase en fonction de NF-kB rapporteur dans les poumons de souris ou en RAW264.7 cellules en culture. Les souris transfect&#xE9;es a montr&#xE9; une expression sp&#xE9;cifique de la lucif&#xE9;rase dans les tissus pulmonaires. En utilisant ces mod&#xE8;les de souris, nous avons &#xE9;tudi&#xE9; la cin&#xE9;tique de l&#x26;#39;activation de NF-kB apr&#xE8;s une exposition au lipopolysaccharide (LPS). Les cellules RAW264.7 exprim&#xE9; une augmentation dose-d&#xE9;pendante apr&#xE8;s l&#x26;#39;exposition au LPS lucif&#xE9;rase et les souris reporter NF-kB exprim&#xE9;es lucif&#xE9;rase dans les poumons &#xE0; la suite d&#xE9;fi LPS, &#xE9;tablissant que l&#x26;#39;imagerie par bioluminescence fournit une sensibilit&#xE9; suffisante pour assurer le suivi de la voie d&#x26;#39;activation de NF-kB. Les interventions qui affectent la voie NF-kB sont prometteurs th&#xE9;rapeutiques cliniques, ainsi nous avons encore examin&#xE9; l&#x26;#39;effet de IKK-2 et l&#x26;#39;inhibition par MLN120B glycog&#xE8;ne synthase kinase 3 b&#xEA;ta par l&#x26;#39;inhibition TDZD-8 sur l&#x26;#39;activation de NF-kB. Pr&#xE9;-traitement avec soit MLN120B ou TDZD-8 att&#xE9;nu&#xE9; activation de NF-kB dans les tissus pulmonaires, qui a &#xE9;t&#xE9; accompagn&#xE9;e de la suppression de la chimiokine pro-inflammatoire MIP-1&#xDF; et l&#x26;#39;induction de cytokines anti-inflammatoires IL-10. En r&#xE9;sum&#xE9;, nous avons &#xE9;tabli une approche bas&#xE9;e sur l&#x26;#39;imagerie pour l&#x26;#39;&#xE9;valuation non invasive et longitudinale de NF-kB activation et la r&#xE9;gulation au cours des l&#xE9;sions pulmonaires aigu&#xEB;s. Cette approche permettra de potentialiser de nouvelles &#xE9;tudes sur la r&#xE9;glementation NF-kB dans diverses conditions inflammatoires.

D&#x26;#39;imagerie pulmonaire NF-kappaB activation et les effets th&#xE9;rapeutiques de MLN120B et TDZD-8

NF-&kappa;B Aktivierung ist ein wichtiges Signal Ereignis in die entz&uuml;ndliche Reaktion und hat verwickelt wurde, in einer Reihe von pathologischen Lungenkrankheiten. Um die Beurteilung der Aktivit&auml;t von NF-&kappa;B in der Lunge zu aktivieren, die wir transfected Luciferase basierte NF-&kappa;B Reporter in die Lungen von M&auml;usen oder in Raw264.7 Zellen in Kultur. Die transfizierte M&auml;use zeigten spezifische Luciferase Ausdruck im pulmonale Gewebe. Verwenden diese Mausmodelle, studierte wir die Kinetik der NF-&kappa;B Aktivierung nach Exposition gegen&uuml;ber Lipopolysaccharid (LPS). Die Raw264.7 Zellen ausgedr&uuml;ckt einen dosisabh&auml;ngiger Anstieg der Luciferase nach Exposition gegen&uuml;ber LPS und die NF-&kappa;B Reporter M&auml;use ausgedr&uuml;ckt Luciferase in der Lunge nach LPS-Herausforderung, dass Biolumineszenz-Imaging bietet ausreichende Empfindlichkeit zum Nachverfolgen des NF-&kappa;B Aktivierung Weges. Ma&szlig;nahmen, die Auswirkungen auf die NF-&kappa;B-Signalweg sind vielversprechende klinische Therapeutik, damit wir weiter untersuchten den Effekt einer IKK-2-Hemmung von MLN120B und Glykogen-Synthase Kinase 3 Beta Hemmung von TDZD-8 auf NF-&kappa;B Aktivierung. Vorbehandlung mit entweder MLN120B oder TDZD-8 abgeschw&auml;cht NF-&kappa;B Aktivierung des Lungen-Gewebes, die mit der Unterdr&uuml;ckung von Pro-inflammatorische Chemokine MIP-1&szlig; und Induktion von entz&uuml;ndungshemmenden Zytokin IL-10 begleitet wurde. Zusammenfassend l&auml;sst sich sagen haben wir einen bildgebenden Ansatz f&uuml;r non-invasive und l&auml;ngs Bewertung von NF-&kappa;B Aktivierung und Regulierung w&auml;hrend acute Respiratory Distress Syndrome etabliert. Dieser Ansatz wird weitere Studien &uuml;ber NF-&kappa;B-Verordnung unter verschiedenen entz&uuml;ndlichen Erkrankungen potenzieren.

Imaging, pulmonale NF-KappaB Aktivierung und therapeutischen Wirkungen des MLN120B und TDZD-8.

Attivazione di NF-&kappa;B &egrave; un evento critico segnalazione nella risposta infiammatoria ed &egrave; stato implicato in un certo numero di malattie polmonari patologici. Per consentire la valutazione delle attivit&agrave; di NF-&kappa;B nei polmoni, noi transfected un reporter di NF-&kappa;B luciferase basato nei polmoni di topi o in Raw264.7 cellule in coltura. I mouse transfected ha mostrato espressione luciferase specifici nei tessuti polmonari. Utilizzando questi modelli murini, abbiamo studiato la cinetica di attivazione di NF-&kappa;B dopo esposizione al lipopolisaccaride (LPS). Le cellule Raw264.7 espresso un aumento dose-dipendente della luciferasi dopo esposizione a LPS e il luciferase NF-&kappa;B reporter topi espressi nei polmoni in seguito a LPS sfida, stabilendo che imaging bioluminescenza fornisce un&#39;adeguata sensibilit&agrave; per il monitoraggio del percorso di attivazione di NF-&kappa;B. Gli interventi che interessano il pathway NF-&kappa;B sono promettenti terapie cliniche, cos&igrave; abbiamo ulteriormente esaminato l&#39;effetto di inibizione IKK-2 da MLN120B e glicogeno sintasi chinasi 3 beta inibizione di TDZD-8 sull&#39;attivazione di NF-&kappa;B. Il pretrattamento con MLN120B o TDZD-8 attenuato attivazione di NF-&kappa;B nei tessuti polmonari, che era accompagnato con soppressione del pro-infiammatorie delle chemochine MIP-1&szlig; e induzione di citochine anti-infiammatorie IL-10. In sintesi, abbiamo stabilito un approccio basato su imaging per la valutazione non invasiva e longitudinale del regolamento durante il danno polmonare acuto e attivazione di NF-&kappa;B. Questo approccio sar&agrave; potenziare ulteriormente gli studi sul regolamento NF-&kappa;B in varie condizioni infiammatorie.

Imaging polmonare attivazione di NF-kappaB e gli effetti terapeutici di MLN120B e di TDZD-8.

NF-&#x3BA;B&#x306E;&#x6D3B;&#x6027;&#x5316;&#x200B;&#x200B;&#x306F;&#x708E;&#x75C7;&#x53CD;&#x200B;&#x200B;&#x5FDC;&#x306E;&#x91CD;&#x8981;&#x306A;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x3067;&#x3042;&#x308A;&#x3001;&#x75C5;&#x7684;&#x306A;&#x80BA;&#x75BE;&#x60A3;&#x306E;&#x6570;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x80BA;&#x306E;NF-&#x3BA;B&#x6D3B;&#x6027;&#x306E;&#x8A55;&#x4FA1;&#x3092;&#x6709;&#x52B9;&#x306B;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x6211;&#x3005;&#x306F;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x80BA;&#x306B;&#x307E;&#x305F;&#x306F;&#x57F9;&#x990A;&#x4E2D;&#x306E;RAW264.7&#x7D30;&#x80DE;&#x3078;&#x306E;&#x30EB;&#x30B7;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;NF-&#x3BA;B&#x30EC;&#x30DD;&#x30FC;&#x30BF;&#x30FC;&#x3092;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30AF;&#x30C8;&#x3057;&#x305F;&#x3002;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30AF;&#x30C8;&#x3057;&#x305F;&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x3001;&#x80BA;&#x7D44;&#x7E54;&#x5185;&#x306E;&#x7279;&#x5B9A;&#x306E;&#x30EB;&#x30B7;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;&#x767A;&#x73FE;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x30E2;&#x30C7;&#x30EB;&#x3092;&#x7528;&#x3044;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;&#x30EA;&#x30DD;&#x30DD;&#x30EA;&#x30B5;&#x30C3;&#x30AB;&#x30E9;&#x30A4;&#x30C9;&#xFF08;LPS&#xFF09;&#x3078;&#x306E;&#x66DD;&#x9732;&#x5F8C;&#x306B;NF-&#x3BA;B&#x6D3B;&#x6027;&#x5316;&#x306E;&#x52D5;&#x614B;&#x3092;&#x8ABF;&#x3079;&#x305F;&#x3002; RAW264.7&#x7D30;&#x80DE;&#x306F;&#x3001;NF-&#x3BA;B&#x6D3B;&#x6027;&#x5316;&#x7D4C;&#x8DEF;&#x3092;&#x8FFD;&#x8DE1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5341;&#x5206;&#x306A;&#x611F;&#x5EA6;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x751F;&#x7269;&#x767A;&#x5149;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x3092;&#x78BA;&#x7ACB;&#x3057;&#x3001;LPS&#x30C1;&#x30E3;&#x30EC;&#x30F3;&#x30B8;&#x5F8C;&#x306B;&#x80BA;&#x306B;&#x30EB;&#x30B7;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;&#x767A;&#x73FE;&#x3057;&#x3001;LPS&#x304A;&#x3088;&#x3073;NF-&#x3BA;B&#x30EC;&#x30DD;&#x30FC;&#x30BF;&#x30FC;&#x30DE;&#x30A6;&#x30B9;&#x306B;&#x30EB;&#x30B7;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;&#x306E;&#x66B4;&#x9732;&#x306E;&#x7528;&#x91CF;&#x4F9D;&#x5B58;&#x7684;&#x5897;&#x52A0;&#x3092;&#x8868;&#x660E;&#x3057;&#x305F;&#x3002; NF-&#x3BA;B&#x7D4C;&#x8DEF;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x306E;&#x4ECB;&#x5165;&#x306F;&#x3001;&#x81E8;&#x5E8A;&#x6CBB;&#x7642;&#x3092;&#x7D04;&#x675F;&#x3057;&#x3066;&#x3001;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;&#x3055;&#x3089;&#x306B;NF-&#x3BA;B&#x306E;&#x6D3B;&#x6027;&#x5316;&#x200B;&#x200B;&#x306B;TDZD-8&#x3067;MLN120B&#x3068;&#x30B0;&#x30EA;&#x30B3;&#x30FC;&#x30B2;&#x30F3;&#x30B7;&#x30F3;&#x30BF;&#x30FC;&#x30BC;&#x30AD;&#x30CA;&#x30FC;&#x30BC;3&#x3B2;&#x963B;&#x5BB3;&#x306B;&#x3088;&#x308B;IKK-2&#x963B;&#x5BB3;&#x306E;&#x52B9;&#x679C;&#x3092;&#x691C;&#x8A0E;&#x3057;&#x305F;&#x3002; MLN120B&#x307E;&#x305F;&#x306F;&#x708E;&#x75C7;&#x8A98;&#x767A;&#x6027;&#x30B1;&#x30E2;&#x30AB;&#x30A4;&#x30F3;MIP-1&#x3B2;&#x304A;&#x3088;&#x3073;&#x6297;&#x708E;&#x75C7;&#x6027;&#x30B5;&#x30A4;&#x30C8;&#x30AB;&#x30A4;&#x30F3;IL-10&#x306E;&#x8A98;&#x5C0E;&#x306E;&#x6291;&#x5236;&#x3092;&#x4F34;&#x3063;&#x305F;&#x80BA;&#x7D44;&#x7E54;&#x4E2D;&#x306E;TDZD-8&#x6E1B;&#x8870;NF-&#x3BA;B&#x306E;&#x6D3B;&#x6027;&#x5316;&#x200B;&#x200B;&#x3001;&#x306E;&#x3044;&#x305A;&#x308C;&#x304B;&#x3067;&#x524D;&#x51E6;&#x7406;&#x3002;&#x8981;&#x7D04;&#x3059;&#x308B;&#x3068;&#x3001;&#x6211;&#x3005;&#x306F;NF-&#x3BA;B&#x306E;&#x6D3B;&#x6027;&#x5316;&#x200B;&#x200B;&#x3068;&#x6025;&#x6027;&#x80BA;&#x50B7;&#x5BB3;&#x6642;&#x306E;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x975E;&#x4FB5;&#x8972;&#x7684;&#x3068;&#x7E26;&#x8A55;&#x4FA1;&#x306E;&#x305F;&#x3081;&#x306E;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3092;&#x78BA;&#x7ACB;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x3001;&#x7A2E;&#x3005;&#x306E;&#x708E;&#x75C7;&#x6027;&#x6761;&#x4EF6;&#x306E;&#x4E0B;&#x3067;NF-&#x3BA;B&#x306E;&#x8ABF;&#x7BC0;&#x306B;&#x95A2;&#x3059;&#x308B;&#x3055;&#x3089;&#x306A;&#x308B;&#x7814;&#x7A76;&#x3092;&#x5897;&#x5F37;&#x3057;&#x307E;&#x3059;&#x3002;

MLN120B&#x3068;TDZD-8&#x306E;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x80BA;&#x306E;NF-&#x3BA;B&#x306E;&#x6D3B;&#x6027;&#x5316;&#x200B;&#x200B;&#x3068;&#x6CBB;&#x7642;&#x52B9;&#x679C;

NF &kappa;B &#xD65C;&#xC131;&#xD654; &#xC5FC;&#xC99D; &#xBC18;&#xC751;&#xC5D0;&#xC11C; &#xC911;&#xC694; &#xD55C; &#xC2E0;&#xD638; &#xC774;&#xBCA4;&#xD2B8; &#xC774;&#xBA70; &#xBCD1; &#xC801;&#xC778; &#xD3D0; &#xC9C8;&#xD658; &#xC218;&#xC5D0; &#xC5F0;&#xB8E8; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xD3D0;&#xC5D0;&#xC11C; NF &kappa;B &#xD65C;&#xB3D9;&#xC758; &#xD3C9;&#xAC00;, &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB3C4;&#xB85D; &#xC6B0;&#xB9AC; &#xBB38;&#xD654;&#xC5D0;&#xC11C; Raw264.7 &#xC138;&#xD3EC; &#xB610;&#xB294; &#xC950;&#xC758; &#xD3D0;&#xC5D0; &#xAE30;&#xBC18; &#xB8E8;&#xC2DC;&#xD398;&#xB77C;&#xC544;&#xC81C; NF &kappa;B &#xAE30;&#xC790; &#xD398;. Transfected &#xC950; &#xD3D0; &#xC870;&#xC9C1;&#xC5D0; &#xD2B9;&#xC815; &#xB8E8;&#xC2DC;&#xD398;&#xB77C;&#xC544;&#xC81C; &#xC2DD;&#xC744; &#xBCF4;&#xC5EC;&#xC8FC;&#xC5C8;&#xB2E4;. &#xC774; &#xB9C8;&#xC6B0;&#xC2A4; &#xBAA8;&#xB378;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xC6B0;&#xB9AC; lipopolysaccharide (LPS)&#xC5D0; &#xB178;&#xCD9C;&#xC5D0; &#xB530;&#xB77C; NF &kappa;B &#xD65C;&#xC131;&#xD654; &#xC18D;&#xB3C4; &#xB860;&#xC744; &#xACF5;&#xBD80; &#xD588;&#xB2E4;. Raw264.7 &#xC138;&#xD3EC; &#xD45C;&#xD604; &#xB8E8;&#xC2DC;&#xD398;&#xB77C;&#xC544;&#xC81C; LPS, &#xD3D0; LPS &#xB3C4;&#xC804;, &#xC0DD;&#xBB3C; &#xBC1C;&#xAD11; &#xC601;&#xC0C1; &#xC81C;&#xACF5; NF &kappa;B &#xD65C;&#xC131;&#xD654; &#xACBD;&#xB85C; &#xCD94;&#xC801;&#xC5D0; &#xB300; &#xD55C; &#xC801;&#xC808; &#xD55C; &#xAC10;&#xB3C4; &#xC124;&#xC815;&#xC5D0;&#xC11C; NF &kappa;B &#xAE30;&#xC790; &#xC950; &#xD45C;&#xD604; &#xB8E8;&#xC2DC;&#xD398;&#xB77C;&#xC544;&#xC81C;&#xC758; &#xB178;&#xCD9C;&#xC5D0; &#xB530;&#xB77C; &#xBCF5;&#xC6A9;&#xB7C9; &#xC758;&#xC874; &#xC99D;&#xAC00;. NF &kappa;B &#xD1B5;&#xB85C; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xAC1C;&#xC785; MLN120B &#xBC0F; glycogen synthase &#xB2C8; 3 &#xBCA0;&#xD0C0; &#xC800;&#xD574; TDZD 8 NF &kappa;B &#xD65C;&#xC131;&#xD654;&#xC5D0; &#xC758;&#xD574; IKK-2 &#xC5B5;&#xC81C;&#xC758; &#xD6A8;&#xACFC; &#xAC80;&#xC0AC; &#xC784;&#xC0C1; &#xCE58;&#xB8CC; &#xD559;, &#xB530;&#xB77C;&#xC11C; &#xC6B0;&#xB9AC;&#xB294; &#xCD94;&#xAC00; &#xC57D;&#xC18D;. MLN120B &#xB610;&#xB294; TDZD-8 &#xC804;&#xCC98;&#xB9AC; &#xAC10;&#xC1E0; &#xD3D0; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; NF-&kappa;B &#xC815;&#xD488; &#xC778;&#xC99D; &#xD504;&#xB85C;-&#xC5FC;&#xC99D; &#xC131; chemokine MIP 1&szlig;&#xC758; &#xC5B5;&#xC81C; &#xBC0F; &#xD56D; &#xC5FC;&#xC99D; &#xC131; cytokine IL-10&#xC758; &#xC720;&#xB3C4; &#xB3D9;&#xBC18; &#xD588;&#xB2E4;. &#xC694;&#xC57D; &#xD558;&#xC790;&#xBA74;, &#xC6B0;&#xB9AC; NF &kappa;B &#xD65C;&#xC131;&#xD654; &#xBC0F; &#xAE09;&#xC131; &#xD3D0; &#xBD80;&#xC0C1; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xADDC;&#xC81C;&#xC758; &#xBB34;&#xB09C; &#xD558; &#xACE0; &#xACBD;&#xB3C4; &#xD3C9;&#xAC00; &#xB300; &#xD55C; &#xC774;&#xBBF8;&#xC9D5; &#xAE30;&#xBC18;&#xB41C; &#xC811;&#xADFC;&#xC744; &#xC124;&#xB9BD; &#xD588;&#xB2E4;. &#xC774; &#xC774;&#xB807;&#xAC8C; &#xB2E4;&#xC591; &#xD55C; &#xC5FC;&#xC99D; &#xC131; &#xC870;&#xAC74;&#xC5D0;&#xC11C; NF-&kappa;B &#xADDC;&#xC815;&#xC5D0; &#xB300; &#xD55C; &#xC5F0;&#xAD6C; &#xB354; &#xC57D;&#xD6A8; &#xAC83;&#xC785;&#xB2C8;&#xB2E4;.

&#xC774;&#xBBF8;&#xC9D5; &#xD3D0; NF kappaB &#xD65C;&#xC131;&#xD654; &#xBC0F; MLN120B &#xBC0F; TDZD-8&#xC758; &#xCE58;&#xB8CC; &#xD6A8;&#xACFC;.

NF-kB activaci&#xF3;n es un acontecimiento cr&#xED;tico de se&#xF1;alizaci&#xF3;n en la respuesta inflamatoria y ha sido implicada en un n&#xFA;mero de enfermedades pulmonares patol&#xF3;gicos. Para posibilitar la evaluaci&#xF3;n de la actividad de NF-kB en los pulmones, transfectadas una luciferasa base NF-kB reportero en los pulmones de los ratones o en RAW264.7 c&#xE9;lulas en cultivo. Los ratones transfectadas mostraron expresi&#xF3;n de luciferasa espec&#xED;fica en los tejidos pulmonares. El uso de estos modelos de ratones, se estudi&#xF3; la cin&#xE9;tica de activaci&#xF3;n de NF-kB tras la exposici&#xF3;n a lipopolisac&#xE1;rido (LPS). Las c&#xE9;lulas RAW264.7 expresa un aumento dosis-dependiente en la exposici&#xF3;n luciferasa siguiente a LPS y los ratones reportero del NF-kB expresadas luciferasa en los pulmones despu&#xE9;s de LPS, se establece que las im&#xE1;genes de bioluminiscencia proporciona una sensibilidad adecuada para el seguimiento de la v&#xED;a de activaci&#xF3;n de NF-kB. Las intervenciones que afectan a la ruta de NF-kB se promete la terap&#xE9;utica cl&#xED;nica, por lo que continu&#xF3; examinando el efecto de IKK-2 y la inhibici&#xF3;n por MLN120B gluc&#xF3;geno sintasa quinasa 3 inhibici&#xF3;n de la beta por TDZD-8 sobre la activaci&#xF3;n de NF-kB. El pre-tratamiento, ya sea con o MLN120B TDZD-8 atenuada activaci&#xF3;n de NF-kB en los tejidos pulmonares, que fue acompa&#xF1;ada de la supresi&#xF3;n de la pro-inflamatorias quimiocinas MIP-1&#xDF; y la inducci&#xF3;n de citoquinas antiinflamatorias IL-10. En resumen, hemos establecido un enfoque basado en im&#xE1;genes para la evaluaci&#xF3;n no invasiva y longitudinal de la activaci&#xF3;n de NF-kB y la regulaci&#xF3;n durante la lesi&#xF3;n pulmonar aguda. Este enfoque se potencian nuevos estudios sobre regulaci&#xF3;n de NF-kB en diversas condiciones inflamatorias.

Imagen pulmonar activaci&#xF3;n de NF-kappaB y los efectos terap&#xE9;uticos de MLN120B y TDZD 8-

One of the most remarkable properties of interleukin 8 (CXCL8/IL-8), a chemokine with known additional functions also in angiogenesis and tissue remodeling, is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by several folds in response to proinflammatory cytokines, bacterial or viral products, and cellular stress. Although mouse cells do not bear a clear homologous IL-8 gene, the murine transcriptional apparatus may well be capable of activating or repressing a heterologous IL-8 gene promoter driving a reporter gene. In order to induce a transient transgenic expression, mice were systemically injected with a bovine IL-8 promoter-luciferase construct. Subsequently mice were monitored for luciferase expression in the lung by in vivo bioluminescent image analysis over an extended period of time (up to 60 days). We demonstrate that the bovine IL-8 promoter-luciferase construct is transiently and robustly activated 3-5 hours after LPS and TNF-α instillation into the lung, peaking at 35 days after construct delivery. Bovine IL-8 promoter-luciferase activation correlates with white blood cell and neutrophil infiltration into the lung. This study demonstrates that a small experimental rodent model can be utilized for non-invasively monitoring, through a reporter gene system, the activation of an IL-8 promoter region derived from a larger size animal (bovine). This proof of principle study has the potential to be utilized also for studying primate IL-8 promoter regions.

In vivo imaging of transiently transgenized mice with a bovine interleukin 8 (CXCL8) promoter/luciferase reporter construct.

Polymorphonuclear cells diapedesis has an important contribution to the induced Mannhemia haemolytica (M. haemolytica) infection lung inflammation and IL-8 is the primary polymorphonuclear chemoattractant. Using a bovine IL-8/luciferase transiently transgenized mouse model, the orchestration among M. haemolytica, IL-8 promoter activation and neutrophilia was followed in real time by in vivo image analysis.

Enlightened Mannhemia haemolytica lung inflammation in bovinized mice.

Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver.

In vivo image analysis of BoHV-4-based vector in mice.

Raising of alpacas as exotic livestock for wool and meat production and as companion animals is growing in importance in the United States, Europe and Australia. Furthermore the alpaca, as well as the rest of the camelids, possesses the peculiarity of producing single-chain antibodies from which nanobodies can be generated. Nanobodies, due to their structural simplicity and reduced size, are very versatile in terms of manipulation and bio-therapeutic exploitation. In fact the biotech companies involved in nanobody production and application continue to grow in number and size. Hence, the development of reagents and tools to assist in the further growth of this new scientific and entrepreneurial reality is becoming a necessity. These are needed mainly to address alpaca disease diagnosis and prophylaxis, and to develop alpaca immunization strategies for nanobody generation. For instance an immortalized alpaca cell line would be extremely valuable. In the present work the first stabilized alpaca cell line from alpaca skin stromal cells (ASSCs) was generated and characterized. This cell line was shown to be suitable for replication of viruses bovine herpesvirus-1, bovine viral diarrhea virus and caprine herpesvirus-1 and the endocellular parasite Neospora caninum. Moreover ASSCs were easy to transfect and transduce by several methods. These two latter characteristics are extremely useful when recombinant antigens need to be produced in a host homologous system. This work could be considered as a starting point for the expansion of the biotechnologies linked to alpaca farming and industry.

Generation and characterization of the first immortalized alpaca cell line suitable for diagnostic and immunization studies.

We studied in vivo the potential involvement of nuclear factor-κB (NF-κB) pathway in the molecular mechanism of the anti-inflammatory and immunomodulatory activity of azithromycin in the lung. Mice transiently transfected with the luciferase gene under the control of a NF-κB responsive element were used to assess in vivo NF-κB activation by bioluminescence imaging. Bioluminescence as well as inflammatory cells and concentrations of proinflammatory cytokines in bronchoalveolar lavage fluids, were monitored in an acute model of pulmonary inflammation resulting from intratracheal instillation of lipopolysaccharide. Lipopolysaccharide (LPS) instillation induced a marked increase in lung bioluminescence in mice transiently transfected with the luciferase gene under the control of an NF-κB responsive element, with significant luciferase expression in resident cells such as endothelial and epithelial cells, as assessed by duoplex immunofluorescence staining. Activation of NF-κB and inflammatory cell lung infiltration linearly correlated when different doses of bortezomib were used to inhibit NF-κB activation. Pretreatment with azithromycin significantly decreased lung bioluminescence and airways cell infiltration induced by LPS, also reducing proinflammatory cytokines concentrations in bronchoalveolar lavages and inhibiting NF-κB nuclear translocation. The results obtained using a novel approach to monitor NF-κB activation, provided, for the first time, in vivo evidence that azithromycin treatment results in pulmonary anti-inflammatory activity associated with the inhibition of NF-κB activation in the lung.

Azithromycin inhibits nuclear factor-κB activation during lung inflammation: an in vivo imaging study.

CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide] is a novel phosphodiesterase 4 (PDE4) inhibitor designed for use in pulmonary diseases by inhaled administration. Intratracheal administration of CHF6001 to ovalbumin-sensitized Brown-Norway rats suppressed the antigen-induced decline of lung functions (ED50 = 0.1 µmol/kg) and antigen-induced eosinophilia (ED50 = 0.03 µmol/kg) when administered (0.09 μmol/kg) up to 24 hours before antigen challenge, in agreement with CHF6001-sustained lung concentrations up to 72 hours after intratracheal treatment (mean residence time 26 hours). Intranasal, once daily administration of CHF6001 inhibited neutrophil infiltration observed after 11 days of tobacco smoke exposure in mice, both upon prophylactic (0.15-0.45 µmol/kg per day) or interventional (0.045-0.45 µmol/kg per day) treatment. CHF6001 was ineffective in reversing ketamine/xylazine-induced anesthesia (a surrogate of emesis in rat) up to 5 µmol/kg administered intratracheally, a dose 50- to 150-fold higher than anti-inflammatory ED50 observed in rats. When given topically to ferrets, no emesis and nausea were evident up to 10 to 20 µmol/kg, respectively, whereas the PDE4 inhibitor GSK-256066 (6-[3-(dimethylcarbamoyl)phenyl]sulfonyl-4-(3-methoxyanilino)-8-methylquinoline-3-carboxamide) induced nausea at 1 µmol/kg intratracheally. A 14-day inhalation toxicology study in rats showed a no-observed-adverse-effect level dose of 4.4 µmol/kg per day for CHF6001, lower than the 0.015 μmol/kg per day for GSK-256066. CHF6001 was found effective and extremely well tolerated upon topical administration in relevant animal models, and may represent a step forward in PDE4 inhibition for the treatment of asthma and chronic obstructive respiratory disease.

CHF6001 II: a novel phosphodiesterase 4 inhibitor, suitable for topical pulmonary administration--in vivo preclinical pharmacology profile defines a potent anti-inflammatory compound with a wide therapeutic window.

Chronic inflammation of the airways is a central component in lung diseases and is frequently associated with bacterial infections. Monitoring the pro-inflammatory capability of bacterial virulence factors in vivo is challenging and usually requires invasive methods.

In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.

Asthma is a multifactorial disease for which a variety of mouse models have been developed. A major drawback of these models is represented by the transient nature of the airway pathology peaking 24-72 h after challenge and resolving in 1-2 weeks. We characterized the temporal evolution of pulmonary inflammation and tissue remodeling in a recently described mouse model of chronic asthma (8 week treatment with 3 allergens: Dust mite, Ragweed, and Aspergillus; DRA).

Monitoring inflammation and airway remodeling by fluorescence molecular tomography in a chronic asthma model.

Assessment of asthmatic inflammation using hybrid fluorescence molecular tomography-x-ray computed tomography.

Experimentally, lung inflammation in laboratory animals is usually detected by the presence of inflammatory markers, such as immune cells and cytokines, in the bronchoalveolar lavage fluid (BALF) of sacrificed animals. This method, although extensively used, is time, money and animal life consuming, especially when applied to genetically modified animals. Thus a new and more convenient approach, based on in vivo imaging analysis, has been set up to evaluate the inflammatory response in the lung of CFTR-deficient (CF) mice, a murine model of cystic fibrosis.

In vivo monitoring of lung inflammation in CFTR-deficient mice.

Idiopathic pulmonary fibrosis is a very common interstitial lung disease derived from chronic inflammatory insults, characterized by massive scar tissue deposition that causes the progressive loss of lung function and subsequent death for respiratory failure. Bleomycin is used as the standard agent to induce experimental pulmonary fibrosis in animal models for the study of its pathogenesis. However, to visualize the establishment of lung fibrosis after treatment, the animal sacrifice is necessary. Thus, the aim of this study was to avoid this limitation by using an innovative approach based on a double bleomycin treatment protocol, along with the in vivo images analysis of bleomycin-treated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on double bleomycin-treated mice to investigate, in real time, the correlation between bleomycin treatment, inflammation, tissue remodeling and fibrosis. Bioluminescence emitted by the lungs of bleomycin-treated mice, corroborated by fluorescent molecular tomography, successfully allowed real time monitoring of fibrosis establishment. The reporter gene technology experienced in this work could represent an advanced functional approach for real time non-invasive assessment of disease evolution during therapy, in a reliable and translational living animal model.

Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice.

Article	Year
Antitumor activity of edotecarin in breast carcinoma models. Cancer chemotherapy and pharmacology\| PubMed ID: 17089166	2007
In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation. Apoptosis : an international journal on programmed cell death\| PubMed ID: 21082356	2011
Imaging pulmonary NF-kappaB activation and therapeutic effects of MLN120B and TDZD-8. PloS one\| PubMed ID: 21966423	2011
In vivo imaging of transiently transgenized mice with a bovine interleukin 8 (CXCL8) promoter/luciferase reporter construct. PloS one\| PubMed ID: 22761878	2012
Enlightened Mannhemia haemolytica lung inflammation in bovinized mice. Veterinary research\| PubMed ID: 24460618	2014
In vivo image analysis of BoHV-4-based vector in mice. PloS one\| PubMed ID: 24752229	2014
Generation and characterization of the first immortalized alpaca cell line suitable for diagnostic and immunization studies. PloS one\| PubMed ID: 25140515	2014
Azithromycin inhibits nuclear factor-κB activation during lung inflammation: an in vivo imaging study. Pharmacology research & perspectives\| PubMed ID: 25505605	2014
CHF6001 II: a novel phosphodiesterase 4 inhibitor, suitable for topical pulmonary administration--in vivo preclinical pharmacology profile defines a potent anti-inflammatory compound with a ... The Journal of pharmacology and experimental therapeutics\| PubMed ID: 25576073	2015
In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin. Journal of translational medicine\| PubMed ID: 26239109	2015
Monitoring inflammation and airway remodeling by fluorescence molecular tomography in a chronic asthma model. Journal of translational medicine\| PubMed ID: 26496719	2015
Assessment of asthmatic inflammation using hybrid fluorescence molecular tomography-x-ray computed tomography. Journal of biomedical optics\| PubMed ID: 26803669	2016
In vivo monitoring of lung inflammation in CFTR-deficient mice. Journal of translational medicine\| PubMed ID: 27468800	2016
Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice. Frontiers in immunology\| PubMed ID: 28298912	2017

Fabio Stellari

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JoVE Journal Publications

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