preparation-pancreatic-acinar-cells-for-purpose-calcium-imaging-cell

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Tufts University Medical Center

The mechanisms by which reflux of bile acids into the pancreas induces pancreatitis are unknown. We reasoned that key events responsible for this phenomenon might be mediated by Gpbar1, a recently identified and widely expressed G-protein-coupled, cell surface bile acid receptor.

Biliary acute pancreatitis in mice is mediated by the G-protein-coupled cell surface bile acid receptor Gpbar1.

Les m&eacute;canismes par lequel reflux de la bile induit des acides dans le pancr&eacute;as pancr&eacute;atite sont inconnus. Nous avons pens&eacute; que les &eacute;v&eacute;nements cl&eacute;s responsables car ce ph&eacute;nom&egrave;ne pourrait &ecirc;tre m&eacute;di&eacute; par les Gpbar1, a r&eacute;cemment identifi&eacute; et largement exprim&eacute; de G-prot&eacute;ine-coupl&eacute;, cellule r&eacute;cepteurs de surface des acides biliaires.

La pancr&eacute;atite aigu&euml; biliaire chez la souris est m&eacute;di&eacute;e par les r&eacute;cepteurs de surface des acides biliaires cellule coupl&eacute;s aux prot&eacute;ines G Gpbar1.

I meccanismi da cui reflusso di bile acidi nel pancreas induce la pancreatite sono sconosciuti. Abbiamo motivato che gli eventi chiave responsabili per questo fenomeno potrebbe essere mediato da Gpbar1, un recentemente identificato e ampiamente espresso G-proteina-accoppiato, cella del recettore superficiale degli acidi biliari.

Pancreatite acuta biliare nei topi &egrave; mediata dal recettore superficiale degli acidi biliari G-protein-coupled cell Gpbar1.

Arthritis is one of the hallmarks of late-stage Lyme disease. Previous studies have shown that infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in degradation of proteoglycans and collagen in cartilage. B burgdorferi do not appear to produce any exported proteases capable of digesting proteoglycans and collagen, but instead, induce and activate host proteases, such as matrix metalloproteinases (MMPs), which results in cartilage degradation. The role of aggrecanases in Lyme arthritis has not yet been determined. We therefore sought to delineate the contribution of aggrecanases to joint destruction in Lyme arthritis.

Role of aggrecanase 1 in Lyme arthritis.

L&#39;arthrite est l&#39;une des principales caract&eacute;ristiques du stade avanc&eacute; de la maladie de Lyme. Des &eacute;tudes ant&eacute;rieures ont montr&eacute; que l&#39;infection par Borrelia burgdorferi, l&#39;agent causal de la maladie de Lyme, se traduit par d&eacute;gradation des prot&eacute;oglycanes et du collag&egrave;ne dans le cartilage. B burgdorferi ne semblent pas produire tout export&eacute;s prot&eacute;ases capables de dig&eacute;rer des prot&eacute;oglycanes et du collag&egrave;ne, mais au lieu de cela, induire et activer les prot&eacute;ases de l&#39;h&ocirc;te, tels que les m&eacute;talloprot&eacute;inases matricielles (MMP), qui se traduit par la d&eacute;gradation du cartilage. Le r&ocirc;le d&#39;aggrecanases dans l&#39;arthrite de Lyme n&#39;a pas encore &eacute;t&eacute; d&eacute;termin&eacute;. Nous avons donc cherch&eacute; &agrave; d&eacute;limiter la contribution d&#39;aggrecanases &agrave; la destruction des articulations dans l&#39;arthrite de Lyme.

R&ocirc;le d&#39;aggrecanase 1 l&#39;arthrite de Lyme.

L&#39;artrite &egrave; una delle caratteristiche della fase tardiva la malattia di Lyme. Studi precedenti hanno mostrato che l&#39;infezione da Borrelia burgdorferi, l&#39;agente eziologico della malattia di Lyme, risultati nella degradazione dei proteoglicani e collagene nella cartilagine. B burgdorferi non sembrano produrre qualsiasi esportate proteasi in grado di digerire proteoglicani e collagene, ma invece, indurre e attivare le proteasi host, ad esempio matrix metalloproteinasi (MMP), che si traduce nella degradazione della cartilagine. Il ruolo di aggrecanases nell&#39;artrite di Lyme non &egrave; stato ancora determinato. Abbiamo quindi cercato di delineare il contributo di aggrecanases a distruzione articolare nell&#39;artrite di Lyme.

Ruolo del aggrecanase 1 in artrite di Lyme.

Nous avons &eacute;mis l&#39;hypoth&egrave;se que la co-localisation des zymog&egrave;nes digestifs avec des hydrolases lysosomales, qui se produit durant les premiers stades de chaque mod&egrave;le de pancr&eacute;atite exp&eacute;rimentale, facilite l&#39;activation de ces zymog&egrave;nes en hydrolases lysosomales telles que la cathepsine B et que cette activation d&eacute;clenche une pancr&eacute;atite aigu&euml; par conduisant &agrave; des l&eacute;sions des cellules acineuses. Certains, cependant, ont fait valoir que le ph&eacute;nom&egrave;ne de colocalisation peut &ecirc;tre le r&eacute;sultat, plut&ocirc;t que la cause, de l&#39;activation de zymog&egrave;ne au cours de la pancr&eacute;atite. Pour r&eacute;soudre cette controverse et explorer les relations de cause &agrave; effet entre l&#39;activation de zymog&egrave;ne et autres &eacute;v&eacute;nements pr&eacute;coces de la pancr&eacute;atite, nous avons induit pancr&eacute;atite chez les souris par stimulation s&eacute;cr&eacute;tagogue supramaximal r&eacute;p&eacute;t&eacute;es avec c&eacute;rul&eacute;ine. Certains animaux sont pr&eacute;-trait&eacute;es avec l&#39;inhibiteur de la cathepsine B CA-074 moi pour inhiber la cathepsine B, &eacute;viter toute activation intra-pancr&eacute;atique des zymog&egrave;nes digestifs et r&eacute;duire la gravit&eacute; de la pancr&eacute;atite. Nous montrons que l&#39;inhibition de la cathepsine B par un pr&eacute;traitement avec CA-074 m&#39;emp&ecirc;che l&#39;activation de zymog&egrave;ne intra-pancr&eacute;atique et r&eacute;duit la fragilit&eacute; organites, mais il ne modifient la ph&eacute;nom&egrave;ne de colocalisation induite par le c&eacute;rul&eacute;ine ni sous-cellulaire redistribution d&#39;actine F ni emp&ecirc;cher l&#39;activation induite par la c&eacute;rul&eacute;ine d&#39;expression NF-kappaB, ERK1/2 et JNK ou sur-exprim&eacute;s de cytochemokines. Nous concluons 1) que le ph&eacute;nom&egrave;ne de la colocalisation, la redistribution de l&#39;actine F, activation des facteurs de transcription pro-inflammatoires et expression augment&eacute;e de cytochemokines ne sont pas les r&eacute;sultats de l&#39;activation de zymog&egrave;ne, et 2) que ces &eacute;v&eacute;nements pr&eacute;coces dans la pancr&eacute;atite ne d&eacute;pendent pas de l&#39;activit&eacute; cathepsine B. En revanche, zymog&egrave;ne activation et la fragilit&eacute; accrue des organites subcellulaire au cours de la pancr&eacute;atite induite par le c&eacute;rul&eacute;ine reposent sur l&#39;activit&eacute; de la cathepsine B.

Relations de cause &agrave; effet entre activation zymog&egrave;ne et autres &eacute;v&eacute;nements pr&eacute;coces dans la pancr&eacute;atite aigu&euml; induite par s&eacute;cr&eacute;tagogue.

We have hypothesized that the colocalization of digestive zymogens with lysosomal hydrolases, which occurs during the early stages of every experimental pancreatitis model, facilitates activation of those zymogens by lysosomal hydrolases such as cathepsin B and that this activation triggers acute pancreatitis by leading to acinar cell injury. Some, however, have argued that the colocalization phenomenon may be the result, rather than the cause, of zymogen activation during pancreatitis. To resolve this controversy and explore the causal relationships between zymogen activation and other early pancreatitis events, we induced pancreatitis in mice by repeated supramaximal secretagogue stimulation with caerulein. Some animals were pretreated with the cathepsin B inhibitor CA-074 me to inhibit cathepsin B, prevent intrapancreatic activation of digestive zymogens, and reduce the severity of pancreatitis. We show that inhibition of cathepsin B by pretreatment with CA-074 me prevents intrapancreatic zymogen activation and reduces organellar fragility, but it does not alter the caerulein-induced colocalization phenomenon or subcellular F-actin redistribution or prevent caerulein-induced activation of NF-kappaB, ERK1/2, and JNK or upregulated expression of cytochemokines. We conclude 1) that the colocalization phenomenon, F-actin redistribution, activation of proinflammatory transcription factors, and upregulated expression of cytochemokines are not the results of zymogen activation, and 2) that these early events in pancreatitis are not dependent on cathepsin B activity. In contrast, zymogen activation and increased subcellular organellar fragility during caerulein-induced pancreatitis are dependent on cathepsin B activity.

Cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis.

Noi abbiamo ipotizzato che la presenza di zimogeni digestivi con idrolasi lisosomiale, che si verifica durante le prime fasi di ogni modello sperimentale pancreatite, facilita l&#39;attivazione di quei zimogeni da idrolasi lisosomiale come catepsina B e che questa attivazione innesca una pancreatite acuta da leader a lesione delle cellule acinose. Alcuni, tuttavia, hanno sostenuto che il fenomeno di presenza pu&ograve; essere il risultato, piuttosto che la causa dell&#39;attivazione di zimogeno durante la pancreatite. Per risolvere questa controversia ed esplorare le relazioni causali tra attivazione zimogeno e altri primi eventi di pancreatite, abbiamo indotta pancreatite nei topi da stimolo secretagogo agonistica ripetute con caerulein. Alcuni animali sono stati pretrattati con inibitore catepsina B CA-074 me per inibire la catepsina B, prevenire intrapancreatico attivazione degli zimogeni digestivi e ridurre la gravit&agrave; della pancreatite. Ci mostrano che l&#39;inibizione della catepsina B dal pretrattamento con CA-074 mi impedisce l&#39;attivazione zimogeno intrapancreatico e riduce la fragilit&agrave; organellare, ma non alterare il fenomeno di presenza caerulein-indotta o subcellulari ridistribuzione di F-actina o prevenire caerulein indotta da attivazione di NF-kappaB, ERK1/2 e JNK o sovraregolati espressione di cytochemokines. Concludiamo 1) che il fenomeno di presenza, la ridistribuzione di F-actina, attivazione di fattori di trascrizione proinfiammatorie e sovraregolati espressione di cytochemokines non sono i risultati dell&#39;attivazione zimogeno, e 2) che questi primi eventi nella pancreatite non sono dipendenti da attivit&agrave; catepsina B. In contrasto, attivazione zimogeno e aumentata fragilit&agrave; organellare sottocellulari durante la pancreatite indotta da caerulein sono dipendenti da attivit&agrave; catepsina B.

Relazioni di causa-effetto tra l&#39;attivazione di zimogeno e altri primi eventi nella pancreatite acuta indotta da secretagogo.

Pancreatic and lung inflammation during acute pancreatitis is a poorly understood, but clinically important, phenomenon. The proto-oncogene Tpl2 (tumor progression locus-2) has recently been shown to have important immunomodulatory effects on some inflammatory processes, but its importance to pancreatitis has not been previously examined. Our studies were designed to (a) define the effects of Tpl2 on pancreatic and lung inflammation during pancreatitis and (b) identify mechanisms and cell types responsible for those effects. We examined pancreatitis-associated Tpl2 effects in wild type and Tpl2(-/-) mice subjected to either secretagogue-induced or bile salt-induced pancreatitis. To determine the myeloid or non-myeloid lineage of cells responsible for the Tpl2 effects, we used Tpl2(-/-) chimeric mice generated by lethal irradiation followed by bone marrow transplantation. Mechanisms responsible for the effects of Tpl2 ablation on caerulein-induced proinflammatory events were evaluated under in vivo and in vitro conditions using the techniques of electrophoretic mobility shift assay, immunoblot analysis, and quantitative reverse transcription-PCR. We found that Tpl2 ablation markedly reduced pancreatic and lung inflammation in these two dissimilar models of pancreatitis, but it did not alter pancreatic injury/necrosis in either model. The reduction in caerulein-induced pancreatic inflammation is dependent upon Tpl2 ablation in non-myeloid cells and is associated with both in vivo and in vitro inhibition of MEK, JNK, and AP-1 activation and the expression of MCP-1, MIP-2, and interleukin-6. Non-myeloid cell expression of Tpl2 regulates pancreatic inflammation during pancreatitis by mediating proinflammatory signals and the generation of neutrophil chemoattracting factors.

Tumor progression locus-2 is a critical regulator of pancreatic and lung inflammation during acute pancreatitis.

Inflammation pancr&eacute;atique et du poumon au cours de la pancr&eacute;atite aigu&euml; est un ph&eacute;nom&egrave;ne mal compris, mais cliniquement important. Le proto-oncog&egrave;ne Tpl2 (locus de progression tumorale-2) a &eacute;t&eacute; r&eacute;cemment montr&eacute; des effets immunomodulateurs important sur certains processus inflammatoires, mais son importance pour la pancr&eacute;atite n&#39;a pas &eacute;t&eacute; examin&eacute;e pr&eacute;c&eacute;demment. Nos &eacute;tudes visaient &agrave; (a) d&eacute;finir les effets de Tpl2 sur le pancr&eacute;as et l&#39;inflammation pulmonaire au cours de la pancr&eacute;atite et (b) identifier les m&eacute;canismes et les types de cellules responsables de ces effets. Nous avons examin&eacute; la pancr&eacute;atite associ&eacute;e &agrave; des effets de Tpl2 dans le type sauvage et souris de tpl2(-/-) soumises &agrave; une pancr&eacute;atite induite par s&eacute;cr&eacute;tagogue ou induite par les sels biliaires. Pour d&eacute;terminer la lign&eacute;e my&eacute;lo&iuml;de ou non my&eacute;lo&iuml;de des cellules responsables des effets Tpl2, nous avons utilis&eacute; Tpl2(-/-) souris chim&eacute;riques g&eacute;n&eacute;r&eacute;s par irradiation mortelle suivies de greffe de moelle osseuse. Les m&eacute;canismes responsables des effets de l&#39;ablation Tpl2 pro-inflammatoires induite par la c&eacute;rul&eacute;ine &eacute;v&eacute;nements ont &eacute;t&eacute; &eacute;valu&eacute;es dans des conditions in vivo et in vitro, en utilisant les techniques d&#39;analyse de d&eacute;calage de mobilit&eacute; &eacute;lectrophor&eacute;tique, immunobuvardage et reverse transcription-PCR quantitative. Nous avons trouv&eacute; que l&#39;ablation Tpl2 une r&eacute;duction marqu&eacute;e du pancr&eacute;as et inflammation pulmonaire dans ces deux mod&egrave;les dissemblables de pancr&eacute;atite, mais il n&#39;a pas alt&eacute;r&eacute; la blessure/n&eacute;crose pancr&eacute;atique dans chaque mod&egrave;le. La r&eacute;duction de l&#39;inflammation pancr&eacute;atique induite par le c&eacute;rul&eacute;ine d&eacute;pend de l&#39;ablation Tpl2 dans les cellules non my&eacute;lo&iuml;des et est associ&eacute;e &agrave; la fois in vivo et in vitro inhibition de l&#39;activation de MEK, JNK et AP-1 et l&#39;expression de MCP-1, MIP-2 et l&#39;interleukine-6. Expression de Tpl2 cellules non my&eacute;lo&iuml;de r&eacute;gule l&#39;inflammation pancr&eacute;atique au cours de la pancr&eacute;atite de m&eacute;diateurs pro-inflammatoires signaux et la production de facteurs de chimiotactisme neutrophile.

Tumeur progression locus-2 est un r&eacute;gulateur essentiel du pancr&eacute;as et inflammation pulmonaire au cours de la pancr&eacute;atite aigu&euml;.

Infiammazione del polmone e pancreatico durante la pancreatite acuta &egrave; un fenomeno scarsamente comprensibile, ma clinicamente importante. Il proto-oncogene Tpl2 (tumore progressione locus-2) ha recentemente dimostrato di avere effetti immunomodulatori importante su alcuni processi infiammatori, ma la sua importanza per la pancreatite non &egrave; stato esaminato in precedenza. I nostri studi sono stati progettati per (a) definire gli effetti di Tpl2 sul pancreas e infiammazione polmonare durante la pancreatite e (b) identificare meccanismi e tipi di cellule responsabili di tali effetti. Abbiamo esaminato la pancreatite associata Tpl2 effetti di tipo selvatico e Tpl2(-/-) topi sottoposti a secretagogue-indotta o indotta da sali biliari pancreatite. Per determinare il lineage mieloide o non-mieloidi di cellule responsabili degli effetti Tpl2, abbiamo usato Tpl2(-/-) mouse chimerici generati dall&#39;irradiazione letale seguite da trapianto di midollo osseo. Meccanismi responsabili per gli effetti dell&#39;ablazione Tpl2 su eventi proinfiammatorie indotta da caerulein sono stati valutati nelle condizioni in vivo e in vitro utilizzando le tecniche di analisi di spostamento di mobilit&agrave; elettroforetica, immunoblot analisi e trascrizione inversa-PCR quantitativa. Abbiamo trovato che l&#39;ablazione Tpl2 marcatamente ridotta del pancreas e infiammazione polmonare in questi due modelli dissimili di pancreatite, ma non alterava lesioni/necrosi pancreatica o modello. La riduzione dell&#39;infiammazione del pancreas caerulein-indotto &egrave; dipendente da ablazione Tpl2 in cellule non mieloidi ed &egrave; associata sia in vivo che in vitro inibizione dell&#39;attivazione di MEK, JNK e AP-1 e l&#39;espressione di MCP-1 e MIP-2, interleuchina-6. Espressione non-mieloide cellule di Tpl2 regola l&#39;infiammazione del pancreas durante la pancreatite da segnali proinfiammatori e la generazione dei neutrofili chemoattracting fattori di mediazione.

Locus di progressione tumorale-2 &egrave; un regolatore critico del pancreas e infiammazione polmonare durante la pancreatite acuta.

Most mechanistic studies of pancreatitis in mice employ the secretagogue-induced model. The currently reported studies were designed to develop an alternative, and possibly more clinically relevant, mouse model of pancreatitis.

A mouse model of acute biliary pancreatitis induced by retrograde pancreatic duct infusion of Na-taurocholate.

Des &eacute;tudes plus m&eacute;canistes de la pancr&eacute;atite chez les souris emploient le mod&egrave;le induite par s&eacute;cr&eacute;tagogue. Les &eacute;tudes actuellement signal&eacute;s visaient &agrave; &eacute;laborer un mod&egrave;le de souris alternatives et peut-&ecirc;tre plus cliniquement pertinents, de pancr&eacute;atite.

Un mod&egrave;le murin de la pancr&eacute;atite aigu&euml; biliaire induite par la perfusion de Na-taurocholate canal pancr&eacute;atique r&eacute;trograde.

Studi pi&ugrave; meccanicistici di pancreatite nei topi utilizzano il modello secretagogue-indotta. Gli studi attualmente segnalati sono stati progettati per sviluppare un modello alternativo e forse pi&ugrave; clinicamente rilevanti, mouse di pancreatite.

Un modello murino di pancreatite acuta biliare indotta da infusione retrograda dotto pancreatico di Na-taurocholate.

Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.

'Role reversal' for the receptor PAR1 in sepsis-induced vascular damage.

Une septic&eacute;mie est une maladie mortelle caract&eacute;ris&eacute;e par un d&eacute;rangement consid&eacute;rable des r&eacute;ponses pro-inflammatoires, anti-inflammatoire et coagulation. R&eacute;cepteur activ&eacute; par prot&eacute;ase 1 (PAR1), un r&eacute;gulateur important de la coagulation de sang et de la fonction de barri&egrave;re endoth&eacute;liale, a propos&eacute; de participer &agrave; des s&eacute;quelles l&eacute;tales d&#39;une septic&eacute;mie, mais on ne sait pas si l&#39;activation de PAR1 est b&eacute;n&eacute;fique ou nuisible. En utilisant une approche de peptide (pepducin) de p&eacute;n&eacute;tration cellulaire, nous fournir la preuve que PAR1 pass&eacute; d&#39;&ecirc;tre un r&eacute;cepteur vasculaire sans interruption &agrave; un r&eacute;cepteur de protection vasculaire au cours de la progression de l&#39;infection chez la souris. Contre toute attente, nous avons constat&eacute; que les effets protecteurs de PAR1 exigeait la transactivation de PAR2, voies de signalisation. Nos r&eacute;sultats sugg&egrave;rent th&eacute;rapeutique qui activent s&eacute;lectivement PAR1-PAR2 complexes peut &ecirc;tre b&eacute;n&eacute;fique dans le traitement du sepsis.

&laquo; Inversion des r&ocirc;les &raquo; pour les r&eacute;cepteurs PAR1 dans induite par l&#39;infection des blessures vasculaires.

Sepsi sono una malattia mortale caratterizzata da derangement considerevole delle risposte proinfiammatorie, anti-infiammatori e coagulazione. Recettori attivati da proteasi 1 (PAR1), un importante regolatore della barriera endoteliale funzione e sangue coagulazione, sono stato proposto di partecipare le sequele letale di sepsi, ma non &egrave; noto se l&#39;attivazione di PAR1 &egrave; benefico o nocivo. Utilizzando un approccio di peptide (pepducin) cellula-penetrante, forniamo prove che PAR1 passati da essere un recettore vascolare dirompente di un recettore vascolare-protettivo durante la progressione della sepsi nei topi. Inaspettatamente, abbiamo trovato che gli effetti protettivi di PAR1 necessaria transactivation di PAR2 vie di segnalazione. I nostri risultati suggeriscono terapie che attivare selettivamente PAR1 PAR2 complessi possono essere utile nel trattamento della sepsi.

&#39;Inversione di ruolo&#39; per il recettore PAR1 nella sepsi indotta da danni vascolari.

Protease-activated receptor-2 (PAR2) is a 7-transmembrane G-protein-coupled tethered ligand receptor that is expressed by pancreatic acinar and ductal cells. It can be physiologically activated by trypsin. Previously reported studies (Namkung, W., Han, W., Luo, X., Muallem, S., Cho, K. H., Kim, K. H., and Lee, M. G. (2004) Gastroenterology 126, 1844-1859; Sharma, A., Tao, X., Gopal, A., Ligon, B., Andrade-Gordon, P., Steer, M. L., and Perides, G. (2005) Am. J. Physiol. 288, G388-G395) have shown that PAR2 activation exerts a protective effect on the experimental model of pancreatitis induced by supramaximal secretagogue (caerulein) stimulation. We now show that PAR2 exerts a worsening effect on a different model of experimental pancreatitis, i.e. one induced by retrograde pancreatic ductal infusion of bile salts. In vitro studies using freshly prepared pancreatic acini show that genetic deletion of PAR2 reduces bile salt-induced pathological calcium transients, acinar cell injury, and activation of c-Jun N-terminal kinase, whereas genetic deletion of PAR2 has the opposite or no effect on these pancreatitis-related events when they are elicited, in vitro, by caerulein stimulation. Studies employing a combination of trypsin inhibition and activation of PAR2 with the activating peptide SLIGRL show that all these differences indeed depend on the activation of PAR2. These studies are the first to report that a single perturbation can have model-specific and opposite effects on pancreatitis, and they underscore the importance of performing mechanistic pancreatitis studies using two dissimilar models of the disease to detect idiosyncratic, model-specific events. We suggest PAR2 activation exerts a worsening effect on the severity of clinical pancreatitis and that interventions interfering with PAR2 activation may be of benefit in the treatment of patients with severe pancreatitis.

Protease-activated receptor-2 exerts contrasting model-specific effects on acute experimental pancreatitis.

Prot&eacute;ase-activated receptor-2 (PAR2) est un r&eacute;cepteur de 7 domaines transmembranaires coupl&eacute;s aux prot&eacute;ines G captif ligand qui est exprim&eacute; par les cellules acineuses et ductal pancr&eacute;atiques. Il peut &ecirc;tre physiologiquement activ&eacute; par la trypsine. &Eacute;tudes pr&eacute;c&eacute;demment signal&eacute;es (ndour, w., Han, w., Luo, x., Muallem, S., Cho, K. H., Kim, H. K. et Lee, M. G. (2004) gastro-ent&eacute;rologie 126, 1844-1859 ; Sharma, r., Tao, x., Guillaume, A., Leclerc, B., Andrade-Gordon, P., Steer, M. L. et Perides, g. (2005) AM j Physiol. 288, G388-G395) ont montr&eacute; que l&#39;activation de PAR2 exerce un effet protecteur sur le mod&egrave;le exp&eacute;rimental de pancr&eacute;atite induite par la stimulation de supramaximal s&eacute;cr&eacute;tagogue (c&eacute;rul&eacute;ine). Maintenant, nous montrons que PAR2 exerce un effet aggravant sur un mod&egrave;le diff&eacute;rent de la pancr&eacute;atite exp&eacute;rimentale, c&#39;est-&agrave;-dire un induite par la perfusion canalaire pancr&eacute;atique r&eacute;trograde des sels biliaires. &Eacute;tudes in vitro utilisant fra&icirc;chement pr&eacute;par&eacute;s acini pancr&eacute;atiques montrent que les d&eacute;l&eacute;tion g&eacute;n&eacute;tique de PAR2 r&eacute;duit transitoires de calcium pathologique induite sur les sels biliaires, l&eacute;sions des cellules acineuses et activation de la kinase c-Jun N-terminale, tandis que la d&eacute;l&eacute;tion g&eacute;n&eacute;tique de PAR2 a le contraire ou pas d&#39;effet sur ces &eacute;v&eacute;nements pancr&eacute;atite lorsqu&#39;ils sont stimul&eacute;s, in vitro, par stimulation de la c&eacute;rul&eacute;ine. &Eacute;tudes utilisant une combinaison de l&#39;inhibition de la trypsine et activation du PAR2 avec le spectacle SLIGRL peptide activant que toutes ces diff&eacute;rences d&eacute;pendent en effet de l&#39;activation du PAR2. Ces &eacute;tudes sont le premier &agrave; signaler qu&#39;une perturbation unique peut avoir sp&eacute;cifique au mod&egrave;le et inverse les effets sur la pancr&eacute;atite et ils soulignent l&#39;importance de la r&eacute;alisation d&#39;&eacute;tudes de pancr&eacute;atite m&eacute;caniste &agrave; l&#39;aide de deux mod&egrave;les diff&eacute;rents de la maladie afin de d&eacute;tecter les &eacute;v&eacute;nements idiosyncrasiques, sp&eacute;cifique au mod&egrave;le. Nous sugg&eacute;rons de PAR2 activation exerce un effet aggravant sur la gravit&eacute; de la pancr&eacute;atite clinique et que les interventions g&ecirc;nent-elles PAR2 activation peuvent &ecirc;tre b&eacute;n&eacute;fique dans le traitement des patients atteints de pancr&eacute;atite grave.

Prot&eacute;ase-activated receptor-2 exerce effets sp&eacute;cifiques sur la pancr&eacute;atite aigu&euml; exp&eacute;rimentale contrastants.

Recettori attivati da proteasi-2 (PAR2) &egrave; un recettore 7-transmembrana accoppiati a proteine G tethered ligando che &egrave; espresso dalle cellule acinose e duttale del pancreas. Fisiologicamente pu&ograve; essere attivato dalla tripsina. Precedentemente segnalati studi (Namkung, w., Han, w., Luo, x., Muallem, S., Cho, K. H., Kim, K. H. e Lee, gastroenterologia (2004) M. G. 126, 1844-1859; Sharma, A., Tao, x., Gopal, A., Ligon, B., Andrade-Gordon, P., manzo, M. L. e Perides, g. (2005) em. J. Pneumologia 288, G388-G395) hanno dimostrato che PAR2 attivazione esercita un effetto protettivo sul modello sperimentale di pancreatite indotta dalla stimolazione di agonistica secretagogo (caerulein). Mostriamo ora che PAR2 esercita un effetto di peggioramento su un modello diverso di pancreatite sperimentale, cio&egrave; uno indotta da retrogrado pancreatico duttale infusione di sali biliari. Studi in vitro usando appena preparati acini pancreatici mostrano che eliminazione genetica di PAR2 riduce transitori calcio patologica indotta da sali biliari, lesione delle cellule acinose e attivazione di c-Jun N-terminal kinase, considerando che la soppressione genetica di PAR2 ha opposto o nessun effetto su questi eventi correlati a pancreatite quando essi sono indotti, in vitro, da stimolazione caerulein. Studi che impiegano una combinazione di tripsina inibizione e attivazione di PAR2 con attivazione show SLIGRL del peptide che tutte queste differenze dipendono infatti l&#39;attivazione di PAR2. Questi studi sono la prima relazione che una singola perturbazione pu&ograve; avere specifiche modello opposto effetti sulla pancreatite e sottolineano l&#39;importanza di eseguire Studi meccanicistici pancreatite utilizzando due modelli dissimili della malattia per rilevare eventi idiosincratici, modello specifico. Consigliamo di attivazione PAR2 esercita un effetto di peggioramento della gravit&agrave; della pancreatite clinica e che gli interventi interferenti con PAR2 attivazione possono essere di beneficio nel trattamento di pazienti con pancreatite grave.

2-Recettori attivati da proteasi esercita effetti specifici del modello sulla pancreatite acuta sperimentale a contrasto.

Borrelia burgdorferi, the causative agent of Lyme arthritis, does not produce any exported proteases capable of degrading extracellular matrix despite the fact that it is able to disseminate from a skin insertion site to infect multiple organs. Prior studies have shown that B. burgdorferi induces the host protease, matrix metalloproteinase 9 (MMP-9), and suggested that the induction of MMP-9 may allow the organism to disseminate and produce local tissue destruction. We examined the role of MMP-9 in dissemination of B. burgdorferi and pathogenesis of Lyme arthritis. In a MMP-9(-/-) mouse model, MMP-9 was not required for the dissemination of the spirochete to distant sites. However, MMP-9(-/-) exhibited significantly decreased arthritis compared to wild-type mice. The decrease in arthritis was not due to an inability to control infection since the spirochete numbers in the joints were identical. Levels of inflammatory chemokines and cytokines were also similar in MMP-9(-/-) and wild-type mice. We examined whether decreased inflammation in MMP-9(-/-) mice may be the result of decreased production of neoattractants by MMP-9-dependent cleavage of collagen. MMP-9 cleavage of type I collagen results in increased monocyte chemoattraction. MMP-9 plays an important role in regulating inflammation in Lyme arthritis, potentially through the cleavage of type I collagen.

Matrix metalloproteinase 9 plays a key role in lyme arthritis but not in dissemination of Borrelia burgdorferi.

Borrelia burgdorferi, l&#39;agent causal de l&#39;arthrite de Lyme, ne produit pas de n&#39;importe quel export&eacute;s prot&eacute;ases capables de d&eacute;grader la matrice extracellulaire, malgr&eacute; le fait qu&#39;il est capable de diffuser sur un site d&#39;insertion peau d&#39;infecter plusieurs organes. Des &eacute;tudes ant&eacute;rieures ont montr&eacute; que b. burgdorferi induit la prot&eacute;ase de l&#39;h&ocirc;te, m&eacute;talloprot&eacute;inase matricielle 9 (MMP-9) et a sugg&eacute;r&eacute; que l&#39;induction de MMP-9 peut permettre &agrave; l&#39;organisme de diffuser et de produire la destruction des tissus locaux. Nous avons examin&eacute; le r&ocirc;le de MMP-9 dans la diffusion de b. burgdorferi et pathog&eacute;nie de l&#39;arthrite de Lyme. Dans un mod&egrave;le de souris MMP-9(-/-), MMP-9 n&#39;&eacute;tait pas n&eacute;cessaire pour la diffusion du spirochete aux sites &eacute;loign&eacute;s. Cependant, MMP-9(-/-) pr&eacute;sentait l&#39;arthrite une diminution importante par rapport aux souris de type sauvage. La diminution de l&#39;arthrite n&#39;&eacute;tait pas due &agrave; une incapacit&eacute; &agrave; contr&ocirc;ler l&#39;infection car les num&eacute;ros de spiroch&egrave;te dans les articulations &eacute;taient identiques. Niveaux de cytokines et de chimiokines inflammatoires &eacute;taient &eacute;galement similaires dans les MMP-9(-/-) et les souris de type sauvage. Nous avons examin&eacute; si une diminution inflammation chez la souris MMP-9(-/-) peut &ecirc;tre le r&eacute;sultat de la baisse de la production de neoattractants par clivage de MMP-9-d&eacute;pendante du collag&egrave;ne. Clivage de MMP-9 de type I r&eacute;sultats de collag&egrave;ne chez chimioattraction une augmentation des monocytes. MMP-9 joue un r&ocirc;le important dans la r&eacute;gulation de l&#39;inflammation dans l&#39;arthrite de Lyme, potentiellement par le clivage du collag&egrave;ne de type I.

M&eacute;talloprot&eacute;inase matricielle 9 joue un r&ocirc;le cl&eacute; dans l&#39;arthrite de lyme, mais non dans la diffusion de Borrelia burgdorferi.

Borrelia burgdorferi, l&#39;agente eziologico di artrite di Lyme, non produce alcun esportate proteasi in grado di degradare la matrice extracellulare, nonostante il fatto che &egrave; in grado di diffondere da un sito di inserimento della pelle per infettare diversi organi. Studi precedenti hanno dimostrato che b. burgdorferi induce la proteasi ospite, matrix metalloproteinase 9 (MMP-9) e ha suggerito che l&#39;induzione di MMP-9 pu&ograve; consentire l&#39;organismo diffondere e produrre distruzione tissutale locale. Abbiamo esaminato il ruolo di MMP-9 nella diffusione di b. burgdorferi e patogenesi dell&#39;artrite di Lyme. In un modello murino MMP-9(-/-), MMP-9 non era necessaria per la diffusione della spirocheta a siti distanti. Tuttavia, MMP-9(-/-) esposto artrite significativamente diminuito rispetto ai topi wild-type. La diminuzione di artrite non era a causa di un&#39;incapacit&agrave; di controllare l&#39;infezione dal momento che i numeri di spirochete nelle articolazioni erano identici. Livelli di citochine e di chemochine infiammatorie sono stati simili nei MMP-9(-/-) e nei topi wild-type. Abbiamo esaminato se diminuita infiammazione in topi MMP-9(-/-) pu&ograve; essere il risultato della diminuita produzione di neoattractants dal clivaggio di MMP-9-dipendente di collagene. MMP-9 clivaggio del tipo I risultati di collagene in aumento monociti chemoattraction. MMP-9 svolge un ruolo importante nel regolare l&#39;infiammazione nell&#39;artrite di Lyme, potenzialmente attraverso la fenditura del collagene di tipo I.

Matrix metalloproteinase 9 gioca un ruolo chiave nell&#39;artrite di lyme, ma non nella diffusione di Borrelia burgdorferi.

Mechanistic studies of acute pancreatitis require animal models because clinical material is generally not available during the early phases of the disease. Here we describe a protocol to induce biliary pancreatitis by retrogradely infusing bile acids into the pancreatic duct of anesthetized mice. The resulting model replicates events believed to be responsible for the onset of clinical biliary (i.e., gallstone) pancreatitis and creates highly reproducible pancreatitis with a severity that depends on the concentration of infused bile acid. Pancreatitis reaches its maximal level of severity within 24 h of induction, and it resolves over the subsequent week. This protocol enables the investigator to use genetically modified strains of mice, and it requires only relatively simple and easily learned techniques of small animal surgery. With practice and gentle technique, the surgery (from induction of anesthesia to completion of the infusion) can be completed within 25 min per animal.

Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct.

Des &eacute;tudes m&eacute;canistes de la pancr&eacute;atite aigu&euml; ont besoin animal mod&egrave;les parce que le mat&eacute;riel clinique n&#39;est g&eacute;n&eacute;ralement pas disponible durant les premi&egrave;res phases de la maladie. Nous d&eacute;crivons ici un protocole visant &agrave; induire la pancr&eacute;atite biliaire en infusant marqu&eacute;s des acides de bile dans le canal pancr&eacute;atique de souris anesth&eacute;si&eacute;s. Le mod&egrave;le obtenu r&eacute;plique les &eacute;v&eacute;nements cens&eacute;s &ecirc;tre responsables de l&#39;apparition des cliniques biliaire (p. ex., calculs biliaires) pancr&eacute;atite et cr&eacute;e la pancr&eacute;atite tr&egrave;s reproductible avec une s&eacute;v&eacute;rit&eacute; qui d&eacute;pend de la concentration d&#39;acide de bile infus&eacute;e. Pancr&eacute;atite atteint son niveau maximal de s&eacute;v&eacute;rit&eacute; dans les 24h de l&#39;induction, et elle est r&eacute;solue au cours de la semaine suivante. Ce protocole permet &agrave; l&#39;enqu&ecirc;teur d&#39;utiliser des souches de souris g&eacute;n&eacute;tiquement modifi&eacute;es, et n&eacute;cessite des techniques relativement simples et facilement savants seuls de petite chirurgie animale. Avec la pratique et la technique douce, la chirurgie (&agrave; partir de l&#39;induction de l&#39;anesth&eacute;sie jusqu&#39;&agrave; la fin de la perfusion) peut &ecirc;tre compl&eacute;t&eacute;e en 25 min par animal.

Pancr&eacute;atite biliaire aigu&euml; exp&eacute;rimentale induite par la perfusion r&eacute;trograde des acides biliaires dans le canal pancr&eacute;atique de la souris.

Studi meccanicistici di pancreatite acuta richiedono animale modelli perch&eacute; materiale clinico generalmente non &egrave; disponibile durante le prime fasi della malattia. Qui descriviamo un protocollo per indurre pancreatite biliare mediante infusione retrogradely acidi di bile nel dotto pancreatico di topi anestetizzati. Il modello risultante replica eventi ritenuti responsabile per l&#39;insorgenza di clinici biliare (cio&egrave;, colelitiasi) pancreatite e crea pancreatite altamente riproducibile con una gravit&agrave; che dipende dalla concentrazione dell&#39;acido di bile infuso. Pancreatite raggiunge il massimo livello di gravit&agrave; entro 24 h di induzione, e si risolve durante la settimana successiva. Questo protocollo consente l&#39;investigatore utilizzare ceppi geneticamente dei topi, e richiede sola relativamente semplice e facilmente appreso tecniche di chirurgia piccolo animali. Con la pratica e tecnica delicata, la chirurgia (dall&#39;induzione dell&#39;anestesia fino al completamento dell&#39;infusione) pu&ograve; essere completata entro 25 min per animale.

Sperimentale pancreatite acuta biliare indotta da infusione retrograda degli acidi di bile nel dotto pancreatico del mouse.

The roles of monocytes/macrophages and their mechanisms of action in the regulation of pancreatitis are poorly understood. To address these issues, we have employed genetically altered mouse strains that either express the human diphtheria toxin receptor (DTR) coupled to the CD11b promoter or have global deletion of TNF-Î±. Targeted, conditional depletion of monocytes/macrophages was achieved by administration of diphtheria toxin (DT) to CD11b-DTR mice. We show that in the absence of DT administration, pancreatitis is associated with an increase in pancreatic content of Ly-6C(hi) monocytes/macrophages but that this response is prevented by prior administration of DT to CD11b-DTR mice. DT administration also reduces pancreatic edema and acinar cell injury/necrosis in two dissimilar experimental models of acute pancreatitis (a secretagogue-induced model and a model elicited by retrograde pancreatic duct infusion of sodium taurocholate). In the secretagogue-elicited model, the DT-induced decrease in pancreatitis severity is reversed by adoptive transfer of purified Ly-6C(hi) monocytes harvested from non-DT-treated CD11b-DTR mice or by the transfer of purified Ly-6C(hi) monocytes harvested from TNF-Î±(+/+) donor mice, but it is not reversed by the transfer of Ly-6C(hi) monocytes harvested from TNF-Î±(-/-) donors. Our studies indicate that the Ly-6C(hi) monocyte subset regulates the severity of pancreatitis by promoting pancreatic edema and acinar cell injury/necrosis and that this phenomenon is dependent upon the expression of TNF-Î± by those cells. They suggest that therapies targeting Ly-6C(hi) monocytes and/or TNF-Î± expression by Ly-6C(hi) monocytes might prove beneficial in the prevention or treatment of acute pancreatitis.

TNF-alpha-dependent regulation of acute pancreatitis severity by Ly-6C(hi) monocytes in mice.

Les r&ocirc;les des monocytes/macrophages et leurs m&eacute;canismes d&#39;action dans la r&eacute;gulation de la pancr&eacute;atite sont mal compris. Pour r&eacute;soudre ces probl&egrave;mes, nous avons fait appel &agrave; des lign&eacute;es de souris g&eacute;n&eacute;tiquement modifi&eacute;es qui expriment le r&eacute;cepteur de la toxine dipht&eacute;rique humaine (DTR) coupl&eacute; au promoteur CD11b ou ont une suppression globale de TNF-&alpha;. Cibl&eacute;e, conditionnelle, appauvrissement des monocytes/macrophages a &eacute;t&eacute; atteint par l&#39;administration de la toxine dipht&eacute;rique (DT) &agrave; des souris CD11b-DTR. Nous montrent qu&#39;en l&#39;absence d&#39;administration DT, pancr&eacute;atite est associ&eacute;e &agrave; une augmentation dans le contenu du pancr&eacute;as des monocytes/macrophages de Ly-6C(hi) mais que cette r&eacute;ponse est emp&ecirc;ch&eacute;e par l&#39;administration pr&eacute;alable de DT aux souris CD11b-DTR. Administration DT r&eacute;duit &eacute;galement &oelig;d&egrave;me pancr&eacute;atique et blessures/n&eacute;crose des cellules acineuses dans deux diff&eacute;rents mod&egrave;les exp&eacute;rimentaux de la pancr&eacute;atite aigu&euml; (un mod&egrave;le induite par s&eacute;cr&eacute;tagogue et un mod&egrave;le induites par la perfusion du canal pancr&eacute;atique r&eacute;trograde de taurocholate de sodium). Dans le mod&egrave;le s&eacute;cr&eacute;tagogue-induites, la diminution induite par le DT de gravit&eacute; de la pancr&eacute;atite est renvers&eacute;e par un transfert adoptif de purifi&eacute;e Ly-6C(hi) monocytes r&eacute;colt&eacute;s chez des souris de CD11b-DTR DT non trait&eacute;es ou par le transfert des monocytes de Ly-6C(hi) purifi&eacute;s r&eacute;colt&eacute;es TNF-&alpha;(+/+) donneur souris, mais il n&#39;est pas invers&eacute;e par le transfert des monocytes Ly-6C(hi) r&eacute;colt&eacute;es TNF-&alpha;(-/-) donateurs. Nos &eacute;tudes indiquent que le sous-ensemble de monocyte Ly-6C(hi) r&eacute;gule la gravit&eacute; de la pancr&eacute;atite en promouvant l&#39;oed&egrave;me pancr&eacute;atique et blessures/la n&eacute;crose des cellules acineuses et que ce ph&eacute;nom&egrave;ne d&eacute;pend de l&#39;expression de TNF-&alpha; par ces cellules. Ils sugg&egrave;rent que les th&eacute;rapies ciblant des monocytes Ly-6C(hi) et/ou l&#39;expression de TNF-&alpha; par les monocytes Ly-6C(hi) pourraient se r&eacute;v&eacute;ler b&eacute;n&eacute;fiques dans la pr&eacute;vention ou le traitement de la pancr&eacute;atite aigu&euml;.

R&egrave;glement de TNF-alpha-d&eacute;pendant de la gravit&eacute; de la pancr&eacute;atite aigu&euml; par les monocytes Ly-6C(hi) chez la souris.

I ruoli dei monociti/macrofagi e loro meccanismi d&#39;azione nella regolazione della pancreatite sono capiti male. Per risolvere questi problemi, noi abbiamo impiegato ceppi di topo geneticamente modificato che esprimono il recettore di tossina di difterite umana (DTR) accoppiato al promotore CD11b o avere eliminazione globale di TNF-&alpha;. Mirate, condizionale deplezione dei monociti/macrofagi &egrave; stato ottenuto con la somministrazione di tossina difterica (DT) a topi CD11b-DTR. Ci mostrano che in assenza di amministrazione DT, pancreatite &egrave; associata con un aumento nel contenuto di pancreatico di Ly-6C(hi) monociti/macrofagi, ma che questa risposta &egrave; impedita dalla precedente amministrazione di DT a topi CD11b-DTR. Amministrazione DT riduce anche l&#39;edema del pancreas e delle cellule acinose lesioni/necrosi in due modelli sperimentali dissimili di pancreatite acuta (un secretagogo indotta da un modello e provocata dall&#39;infusione retrograda dotto pancreatico di taurocholate di sodio). Nel modello ha suscitato secretagogo, la diminuzione indotta da DT in gravit&agrave; pancreatite &egrave; invertita da trasferimento adottivo di purificato monociti Ly-6C(hi) raccolte da topi CD11b-DTR non-DT-trattati o dal trasferimento di purificato monociti Ly-6C(hi) raccolte da topi TNF-&alpha;(+/+) donatore, ma non &egrave; invertita dal trasferimento di monociti Ly-6C(hi) raccolte da donatori TNF-&alpha;(-/-). I nostri studi indicano che il sottoinsieme di monociti Ly-6C(hi) regola la gravit&agrave; della pancreatite promuovendo edema del pancreas e delle cellule acinose lesioni/necrosi e che questo fenomeno dipende dall&#39;espressione di TNF-&alpha; da quelle cellule. Essi suggeriscono che terapie targeting Ly-6C(hi) monociti e/o espressione di TNF-&alpha; da monociti Ly-6C(hi) potrebbero rivelarsi utile per la prevenzione o il trattamento di pancreatite acuta.

Regolamento di TNF-alfa-dipendente di gravit&agrave; pancreatite acuta da Ly-6C(hi) monociti nei topi.

Protease-activated receptor-2 (PAR2), a cell surface receptor for trypsin-like proteases, plays a key role in a number of acute and chronic inflammatory diseases of the joints, lungs, brain, gastrointestinal tract, and vascular systems. Despite considerable effort by the pharmaceutical industry, PAR2 has proven recalcitrant to targeting by small molecule inhibitors, which have been unable to effectively prevent the interaction of the protease-generated tethered ligand with the body of the receptor. Here, we report the development of first-in-class cell-penetrating lipopeptide "pepducin" antagonists of PAR2. The design of the third intracellular (i3) loop pepducins were based on a structural model of a PAR2 dimer and by mutating key pharmacophores in the receptor intracellular loops and analogous pepducins. Individual pharmacophores were identified, which controlled constitutive, agonist, and antagonist activities. This approach culminated in the identification of the P2pal-18S pepducin which completely suppressed trypsin and mast cell tryptase signaling through PAR2 in neutrophils and colon cancer cells. The PAR2 pepducin was highly efficacious in blocking PAR2-dependent inflammatory responses in mouse models. These effects were lost in PAR2-deficient and mast-cell-deficient mice, thereby validating the specificity of the pepducin in vivo. These data provide proof of concept that PAR2 pepducin antagonists may afford effective treatments of potentially debilitating inflammatory diseases and serve as a blueprint for developing highly potent and specific i3-loop-based pepducins for other G protein-coupled receptors (GPCRs).

Interdicting protease-activated receptor-2-driven inflammation with cell-penetrating pepducins.

Prot&eacute;ase-activated receptor-2 (PAR2), un r&eacute;cepteur de surface cellulaire pour les prot&eacute;ases de la trypsine, joue un r&ocirc;le cl&eacute; dans un certain nombre de maladies inflammatoires aigu&euml;s et chroniques des articulations, poumons, cerveau, tractus gastro-intestinal et syst&egrave;mes vasculaires. Malgr&eacute; des efforts consid&eacute;rables de l&#39;industrie pharmaceutique, PAR2 a prouv&eacute; r&eacute;calcitrants au ciblage de petites mol&eacute;cules inhibitrices, qui ont &eacute;t&eacute; incapables de pr&eacute;venir efficacement l&#39;interaction du ligand captif prot&eacute;ase g&eacute;n&eacute;r&eacute; avec le corps du receveur. Nous rapportons ici le d&eacute;veloppement de la premi&egrave;re cat&eacute;gorie p&eacute;n&eacute;tration cellulaire lipopeptide &laquo; pepducin &raquo; antagonistes de PAR2. La conception de la troisi&egrave;me pepducins de boucle intracellulaire (i3) reposaient sur un mod&egrave;le de structure d&#39;un dim&egrave;re de PAR2 et par mutation pharmacophores principaux dans la boucle intracellulaire du r&eacute;cepteur et pepducins analogues. Pharmacophores individuels ont &eacute;t&eacute; identifi&eacute;s, qui contr&ocirc;lait alors constitutive, agoniste et antagoniste des activit&eacute;s. Cette d&eacute;marche a abouti &agrave; l&#39;identification de le pepducin de P2pal de 18 ans qui a compl&egrave;tement supprim&eacute; la trypsine et la tryptase mastocytes signalisation par l&#39;interm&eacute;diaire de PAR2 dans les neutrophiles et les cellules de cancer du c&ocirc;lon. Le pepducin de PAR2 a &eacute;t&eacute; tr&egrave;s efficace dans le blocage des r&eacute;ponses inflammatoires PAR2-d&eacute;pendantes dans des mod&egrave;les murins. Ces effets ont &eacute;t&eacute; perdus dans des souris d&eacute;ficientes PAR2 et m&acirc;t-cellule-deficient, validant ainsi la sp&eacute;cificit&eacute; de le pepducin in vivo. Ces donn&eacute;es fournissent des preuves de concept que PAR2 pepducin antagonistes peuvent s&#39;offrir des traitements efficaces de potentiellement dommageable des maladies inflammatoires et servir de mod&egrave;le pour d&eacute;velopper pepducins i3-boucle-bas&eacute; tr&egrave;s puissant et sp&eacute;cifique pour les autres r&eacute;cepteurs de coupl&eacute;s aux prot&eacute;ines G (RCPG).

Interdire la prot&eacute;ase-activated receptor-2-conduit inflammation avec p&eacute;n&eacute;tration cellulaire pepducins.

Attivati da proteasi recettore-2 (PAR2), un recettore di superficie delle cellule per le proteasi tripsina-come, gioca un ruolo chiave in una serie di malattie infiammatorie acute e croniche delle articolazioni, polmoni, cervello, tratto gastrointestinale e sistemi vascolari. Nonostante i notevoli sforzi da parte dell&#39;industria farmaceutica, PAR2 ha dimostrato recalcitrante di targeting di piccole molecole inibitori, che sono stati in grado di prevenire efficacemente l&#39;interazione tra il ligando legato proteasi-generato con il corpo del recettore. Qui, segnaliamo lo sviluppo del primo-in-class cellula-penetrante lipopeptide &quot;pepducin&quot; antagonisti di PAR2. Il design del terzo pepducins loop intracellulare (i3) erano basati su un modello strutturale di un dimero PAR2 e mutando la chiave pharmacophores nel loop di recettore intracellulare e pepducins analoghe. Pharmacophores singoli sono stati identificati, che controllava costitutiva, agonista e antagonista attivit&agrave;. Questo approccio ha culminato nell&#39;identificazione della P2pal-18S pepducin che completamente soppressa tripsina e tryptase mast cell signaling attraverso PAR2 nei neutrofili e cellule tumorali del colon. Il pepducin PAR2 era altamente efficace nel bloccare le risposte infiammatorie PAR2-dipendente in modelli murini. Questi effetti sono stati persi nei topi PAR2-carenti e mast-cellule-carenti, quindi convalida la specificit&agrave; della pepducin in vivo. Questi dati forniscono la prova di concetto che PAR2 pepducin antagonisti possono permettersi trattamenti efficaci delle malattie infiammatorie potenzialmente invalidanti e servire come modello per lo sviluppo di pepducins i3-loop-based altamente potente e specifico per altri recettori accoppiati a proteine G (GPCR).

Rifugiarsi attivati da proteasi recettore-2-driven infiammazione con pepducins cellula-penetrante.

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 Î¼M and a peak-plateau signal at 500 Î¼M, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice.

La pancr&eacute;atite biliaire est l&#39;&eacute;tiologie la plus courante de pancr&eacute;atite aigu&euml;, mais son m&eacute;canisme physiopathologique reste incertaine. Ca(2+) signaux g&eacute;n&eacute;r&eacute;s dans les cellules acineuses du pancr&eacute;as amorcer la phase pr&eacute;coce de la pancr&eacute;atite, et les acides biliaires peuvent d&eacute;clencher la lib&eacute;ration de Ca(2+) intracellulaire anormale de cellules acineuses. Nous avons d&eacute;montr&eacute; pr&eacute;c&eacute;demment que Ca(2+) lib&eacute;r&eacute; via le canal de Ca(2+) intracellulaire, le r&eacute;cepteur &agrave; la ryanodine (RyR), contribue au signal Ca(2+) aberrant. Dans cette &eacute;tude, nous avons examin&eacute; si l&#39;inhibition RyR prot&egrave;ge contre les signaux Ca(2+) pathologique, l&eacute;sions des cellules acineuses et exposition acide biliaire de la pancr&eacute;atite. Le tauro-lithocholique acide de bile acide-3-sulfate (TLC) induit des oscillations intracellulaires de Ca(2+) &agrave; 50 &mu;M et un signal de cr&ecirc;te-plateau &agrave; 500 &mu;M et seulement la blessure de ce dernier induit des cellules acineuses, tel que d&eacute;termin&eacute; par la fuite de la lactate d&eacute;shydrog&eacute;nase (LDH). Un pr&eacute;traitement avec les inhibiteurs de RyR dantrol&egrave;ne ou ryanodine converti le signal de cr&ecirc;te-plateau &agrave; un mod&egrave;le pour la plupart oscillatoire (P 0,05 &lt;). Ils ont aussi r&eacute;duit fuite LDH cellules acineuses, blebbing basolat&eacute;rale et absorption de l&#39;iodure de propidium (P 0,05 &lt;). In vivo, une dose unique de dantrol&egrave;ne (5 mg/kg), 1 h avant, soit 2 h apr&egrave;s la perfusion de TLC intraductal, r&eacute;duit la gravit&eacute; de la pancr&eacute;atite jusqu&#39;au niveau du contr&ocirc;le (P 0,05 &lt;). Ces r&eacute;sultats sugg&egrave;rent que la gravit&eacute; de la pancr&eacute;atite biliaire peut &ecirc;tre am&eacute;lior&eacute;e par l&#39;utilisation clinique des inhibiteurs de RyR.

Les r&eacute;cepteurs de la ryanodine contribuent &agrave; signalisation calcique pathologique induite par l&#39;acide biliaire et pancr&eacute;atite chez les souris.

Pancreatite biliare &egrave; l&#39;eziologia pi&ugrave; comune di pancreatite acuta, ma il suo meccanismo fisiopatologico rimane poco chiaro. CA(2+) segnali generati all&#39;interno delle cellule acinose del pancreas avviare la prima fase della pancreatite, e gli acidi biliari pu&ograve; suscitare rilascio Ca(2+) intracellulare anomala delle cellule acinose. Abbiamo precedentemente dimostrato che Ca(2+) rilasciato tramite il canale Ca(2+) intracellulare, il recettore rianodinico (RyR), contribuisce al segnale Ca(2+) aberrante. In questo studio, abbiamo esaminato se RyR inibizione protegge contro patologica di segnali, lesione delle cellule acinose e pancreatite da acidi biliari esposizione Ca(2+). Gli acidi biliari tauro-lithocholic acido-3-solfato (TLCS) indotta da oscillazioni intracellulari di Ca(2+) a 50 &mu;M e un segnale di picco-altopiano a 500 &mu;M e solo l&#39;infortunio di quest&#39;ultima indotta delle cellule acinose, come determinato dalla perdita di Lattato deidrogenasi (LDH). Pretrattamento con gli inibitori di RyR dantrolene o rianodinico convertito il segnale di picco-altopiano in un modello prevalentemente oscillatorio (P &lt; 0,05). Hanno ridotto anche perdita di cellule acinose LDH, blebbing basolaterale e assorbimento di ioduro di propidium (P &lt; 0,05). In vivo, una singola dose di dantrolene (5 mg/kg), dato 1 h prima o 2 ore dopo infusione TLCS intraductal, ridotto la gravit&agrave; della pancreatite fino al livello del controllo (P &lt; 0,05). Questi risultati suggeriscono che la gravit&agrave; della pancreatite biliare pu&ograve; essere migliorata con l&#39;uso clinico di RyR inibitori.

Rianodinico recettori contribuiscono alla segnalazione di calcio patologica indotta da acidi biliari e pancreatite nei topi.

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca(2+). Thus, it would be clinically relevant to know the targets of this aberrant Ca(2+) signal. We hypothesized that the Ca(2+)-activated phosphatase calcineurin is such a Ca(2+) target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca(2+). Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-ÎºB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform AÎ² (CnAÎ²) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca(2+) generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.

Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin.

La pancr&eacute;atite biliaire est la principale cause de la pancr&eacute;atite aigu&euml; chez les enfants et les adultes. Un m&eacute;canisme propos&eacute; est le reflux de la bile dans le canal pancr&eacute;atique. Acides biliaires exposition cause un pr&eacute;judice de cellules acineuses du pancr&eacute;as par une hausse soutenue des Ca(2+) cytosolique. Ainsi, il serait cliniquement pertinent de savoir les cibles de ce signal aberrant de Ca(2+). Nous avons &eacute;mis l&#39;hypoth&egrave;se que la calcineurine phosphatase activ&eacute;e par Ca(2+) est telle une cible Ca(2+). Pour examiner l&#39;activation de la calcineurine, nous avons infect&eacute; des cellules acineuses primaires de souris avec un ad&eacute;novirus qui exprime le promoteur d&#39;un effecteur de la calcineurine en aval, facteur nucl&eacute;aire des lymphocytes T activ&eacute;s (NFAT). La bile acide taurolithocholique acide-3-sulfate (TLC) a &eacute;t&eacute; principalement utilis&eacute; pour examiner les r&eacute;ponses des acides biliaires. TLCS caus&eacute; activation calcineurine uniquement &agrave; des concentrations qui provoquent des l&eacute;sions des cellules acineuses. L&#39;activation de la calcineurine par TLC a &eacute;t&eacute; abolie par ch&eacute;lation Ca(2+) intracellulaire. Un pr&eacute;traitement avec bis(o-aminophenoxy)-1, 2 &eacute;thane-N, N, N&#39;, N&#39;-t&eacute;traac&eacute;tique (ester ac&eacute;toxym&eacute;thyl) (BAPTA-AM) ou les trois inhibiteurs de la calcineurine sp&eacute;cifiques FK506, cyclosporine A ou peptide inhibiteur de la calcineurine emp&ecirc;ch&eacute; l&eacute;sion induite par l&#39;acide biliaire des cellules acineuses mesur&eacute;e par absorption d&#39;iodure fuite et propidium lactate d&eacute;shydrog&eacute;nase. Les inhibiteurs de la calcineurine r&eacute;duit l&#39;activation intra-acinar du chymotrypsinog&egrave;ne dans les 30 minutes de l&#39;administration de TLC, et ils emp&ecirc;chait &eacute;galement l&#39;activation de NF-&kappa;B. In vivo, les souris qui ont re&ccedil;u le FK506 ou comportaient des lacunes dans la sous-unit&eacute; b&ecirc;ta-amylo&iuml;des (CnA&beta;) de la calcineurine isoforme avaient att&eacute;nu&eacute; la gravit&eacute; de la pancr&eacute;atite apr&egrave;s la perfusion de TLC ou acide taurocholique dans le canal pancr&eacute;atique. En r&eacute;sum&eacute;, nous d&eacute;montrons que des cellules acineuses calcineurine est activ&eacute;e en r&eacute;ponse &agrave; Ca(2+) g&eacute;n&eacute;r&eacute;s par l&#39;exposition de l&#39;acide biliaire, l&eacute;sions pancr&eacute;atiques induites par les acides biliaires d&eacute;pend de l&#39;activation de la calcineurine et inhibiteurs de la calcineurine peuvent pr&eacute;voir un traitement d&#39;appoint pour la pancr&eacute;atite biliaire.

Les acides biliaires induisent des l&eacute;sions des cellules acineuses du pancr&eacute;as et la pancr&eacute;atite en activant la calcineurine.

Pancreatite biliare &egrave; la principale causa di pancreatite acuta in adulti e bambini. Un meccanismo proposto &egrave; il reflusso di bile nel dotto pancreatico. Acido di bile esposizione provoca lesione delle cellule acinose del pancreas attraverso una crescita sostenuta in Ca(2+) citosolico. Quindi, sarebbe clinicamente rilevante conoscere gli obiettivi di questo aberrante segnale Ca(2+). Abbiamo ipotizzato che il Ca(2+) attivato fosfatasi calcineurina &egrave; una destinazione di Ca(2+). Per esaminare l&#39;attivazione della calcineurina, siamo infetti cellule acinose primarie da topi con un adenovirus esprimendo il promotore per un effector downstream calcineurina, fattore nucleare delle cellule T attivate (NFAT). L&#39;acido di bile taurolithocholic acido-3-solfato (TLCS) &egrave; stato utilizzato principalmente per esaminare le risposte degli acidi biliari. TLCS causato calcineurina attivazione solo a concentrazioni che causano lesioni delle cellule acinose. L&#39;attivazione della calcineurina da TLCS fu abolito da chelanti Ca(2+) intracellulare. Pretrattamento con 1,2-bis(o-aminophenoxy) etano-N, N, N&#39;, N&#39;-l&#39;acido etilendiamminotetraacetico (estere acetossimetil) (BAPTA-AM) o i tre inibitori della calcineurina specifico FK506, ciclosporina A o peptidi inibitori della calcineurina evitato lesioni biliari acido-indotta delle cellule acinose come misurato da assorbimento di ioduro di perdite e propidium Lattato deidrogenasi. Gli inibitori della calcineurina riduce l&#39;attivazione intra-acinar di chimotripsinogeno entro 30 min di amministrazione TLCS, e hanno impedito anche attivazione di NF-&kappa;B. In vivo, topi che ricevuto FK506 o erano carenti nella subunit&agrave; della calcineurina isoforma A&beta; (CnA&beta;) erano ridotta gravit&agrave; pancreatite dopo infusione di Acido taurocolico o TLCS nel dotto pancreatico. In sintesi, ci dimostrano che cellule acinose calcineurina &egrave; attivata in risposta a Ca(2+) generati dall&#39;esposizione degli acidi biliari, lesioni del pancreas indotta da acido di bile dipende dall&#39;attivazione della calcineurina e inibitori della calcineurina pu&ograve; fornire una terapia aggiuntiva per la pancreatite biliare.

Acidi biliari indurre lesioni delle cellule acinose del pancreas e pancreatite attivando calcineurina.

BACKGROUND: Ischemia-reperfusion injury worsens infarct size, a major determinant of morbidity and mortality after acute myocardial infarction (MI). We tested the hypothesis that reducing left ventricular (LV) wall stress with a percutaneous left atrial-to-femoral artery centrifugal (pLA-FA) bypass system, while delaying coronary reperfusion limits myocardial injury in a model of acute MI. METHODS AND RESULTS: MI was induced by balloon occlusion of the left anterior descending artery (LAD) in adult, male swine. In the MI group(n=4), 120 minutes of LAD occlusion was followed by 120 minutes of reperfusion without mechanical support. In the mechanically supported group (MI+Unload)(n=4), pLA-FA bypass was initiated after 120 minutes of ischemia and LAD occlusion prolonged for an additional 30 minutes, followed by 120 minutes of reperfusion with device support. All animals were sacrificed after reperfusion and infarct size quantified by triphenyltetrazolium chloride staining. Compared to baseline, mean LV wall stress and stroke work were not changed at any point in the MI group, but were decreased after reperfusion in the MI+Unload group (mean LV wall stress: 44658 vs 22963 dynes/cm(2); stroke work: 2823 vs 655 mmHg x mL, MI vs MI+Unload). Phosphorylation of reperfusion injury salvage kinase (RISK) pathway proteins from non-infarcted LV tissue was unchanged in the MI group, but increased in the MI+Unload group. Compared to MI alone, total infarct size was reduced in the MI+Unload group (49% vs 28%, MI vs MI+Unload). CONCLUSIONS: These data support that first unloading the LV despite delaying coronary reperfusion during an acute MI reduces myocardial injury.

Mechanically Unloading the Left Ventricle Before Coronary Reperfusion Reduces Left Ventricular Wall Stress and Myocardial Infarct Size.

Rappel des faits : Isch&eacute;mie-reperfusion s&#39;aggrave taille de l&#39;infarctus, un d&eacute;terminant majeur de la morbidit&eacute; et la mortalit&eacute; apr&egrave;s l&#39;infarctus aigu du myocarde (MI). Nous avons test&eacute; l&#39;hypoth&egrave;se que la r&eacute;duction ventriculaire tension pari&eacute;tale (VG) avec une gauche art&egrave;re auriculaire-&agrave;-f&eacute;morale percutan&eacute;e centrifuge (pLA-FA) a laiss&eacute; d&eacute;rivation syst&egrave;me, tandis que retarder la reperfusion coronaire limite des l&eacute;sions myocardiques dans un mod&egrave;le d&#39;aigu&euml; MI. m&eacute;thodes et r&eacute;sultats: MI a &eacute;t&eacute; induite par l&#39;occlusion de l&#39;art&egrave;re interventriculaire ant&eacute;rieure (LAD) chez les porcs adultes, m&acirc;les ballon. Dans le group(n=4) de MI, 120 minutes d&#39;occlusion de la DAG a &eacute;t&eacute; suivie de 120 minutes de reperfusion sans soutien m&eacute;canique. Dans le groupe m&eacute;caniquement (MI+Unload)(n=4), pLA-FA contournement a &eacute;t&eacute; lanc&eacute; apr&egrave;s 120 minutes d&#39;isch&eacute;mie et occlusion de la DAG prolong&eacute; pendant encore 30 minutes, suivie de 120 minutes de reperfusion avec le soutien de l&#39;appareil. Tous les animaux ont &eacute;t&eacute; sacrifi&eacute;s apr&egrave;s reperfusion et infarctus taille quantifi&eacute;e par coloration de chlorure de triph&eacute;nylt&eacute;trazolium. Par rapport au niveau de r&eacute;f&eacute;rence, LV mur contrainte moyenne et temps de travail n&#39;ont pas &eacute;t&eacute; modifi&eacute;es &agrave; tout moment dans le groupe de MI, mais ont diminu&eacute; apr&egrave;s la reperfusion du groupe MI + Unload (moyenne tension pari&eacute;tale VG: 44658 vs 22963 dynes/cm(2) ; travail de course : mmHg vs 655 2823 x mL, MI vs MI + Unload). La phosphorylation des prot&eacute;ines de voie des kinase (risque) de r&eacute;cup&eacute;ration de la blessure reperfusion du tissu non-infarci de LV est inchang&eacute;e dans le groupe de MI, mais augmente dans le groupe MI + Unload. Par rapport aux MI seul, taille de l&#39;infarctus totale a &eacute;t&eacute; r&eacute;duite dans le groupe MI + Unload (49 % contre 28 %, vs MI MI + Unload). CONCLUSIONS : Ces donn&eacute;es supportent que premier d&eacute;chargement le LV malgr&eacute; retardant la reperfusion coronaire lors d&#39;un infarctus aigu du myocarde r&eacute;duit les l&eacute;sions myocardiques.

D&eacute;chargement m&eacute;canique du ventricule gauche avant la Reperfusion coronaire r&eacute;duit le Stress de la paroi ventriculaire gauche et la taille de l&#39;infarctus du myocarde.

George Perides

Department of Surgery

Tufts University Medical Center

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

George Perides

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Surgery

Tufts University Medical Center

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

Department of Surgery