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 ...

Children's Hospital of Pittsburgh of UPMC

Previous studies characterized B cell-dependent and B cell-independent models of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. To further characterize the B cell response generated in these two models, the serum antibody response and the B cell surface immunoglobulin (Ig) repertoire were analyzed following immunization of wild-type C57BL/6 mice with either recombinant myelin oligodendrocyte glycoprotein (MOG; B cell-dependent EAE) or the encephalitogenic MOG(35-55) peptide (B cell-independent EAE). Plasma ELISA revealed responses to unique linear epitopes of MOG following immunization with recombinant MOG that were absent in MOG(35-55)-immunized animals. B cell repertoire analysis by RT-PCR identified a unique response restricted to 7183 Ig heavy chain variable gene family in mice immunized with recombinant MOG that was not observed in MOG(35-55)-immunized mice. These insights could aid in the identification of the relevant B cell populations important to the pathogenesis of B cell-dependent EAE and in the mechanisms by which these B cell populations contribute to disease.

Unique B cell responses in B cell-dependent and B cell-independent EAE.

Des &eacute;tudes ant&eacute;rieures caract&eacute;risent d&eacute;pendant des lymphocytes B et les mod&egrave;les B cellule ind&eacute;pendante de l&#39;enc&eacute;phalomy&eacute;lite allergique exp&eacute;rimentale (EAE) chez les souris C57BL/6. Afin de caract&eacute;riser la r&eacute;ponse des lymphocytes B g&eacute;n&eacute;r&eacute;e dans ces deux mod&egrave;les, la r&eacute;ponse en anticorps s&eacute;riques et du r&eacute;pertoire de surface immunoglobulines (Ig) de cellules B ont &eacute;t&eacute; analys&eacute;s suivant l&#39;immunisation de souris C57BL/6 sauvage avec une glycoprot&eacute;ine d&#39;oligodendrocyte recombinant de la my&eacute;line (MOG ; B cell-dependent EAE) ou l&#39;encephalitogenic MOG(35-55) peptide (ind&eacute;pendante de la cellule B EAE). Plasma ELISA a r&eacute;v&eacute;l&eacute; responses to uniques &eacute;pitopes lin&eacute;aires de MOG apr&egrave;s vaccination par MOG recombinant qui &eacute;taient absentes chez les animaux vaccin&eacute;s MOG(35-55). Lymphocytes b r&eacute;pertoire analyse par RT-PCR a permis une r&eacute;ponse unique limit&eacute;e &agrave; 7183 famille de g&egrave;ne variable de cha&icirc;ne lourde Ig chez les souris immunis&eacute;es avec MOG recombinant qui n&#39;a pas &eacute;t&eacute; observ&eacute;e chez les souris vaccin&eacute;es MOG(35-55). Ces id&eacute;es pourraient aider &agrave; l&#39;identification des populations pertinentes des lymphocytes B importantes dans la pathogen&egrave;se de l&#39;EAE d&eacute;pendant des lymphocytes B et dans les m&eacute;canismes par lesquels ces populations de lymphocytes B contribuent &agrave; la maladie.

Lymphocytes B unique en d&eacute;pendant des lymphocytes B et lymphocytes B ind&eacute;pendant EAE.

Studi precedenti caratterizzato dalle cellule di B e modelli delle cellule di B-indipendente di encefalomielite autoimmune sperimentale (EAE) nei topi C57BL/6. Per caratterizzare ulteriormente la risposta delle cellule B generata in questi due modelli, la risposta anticorpale sierica e il repertorio di superficie immunoglobuline (Ig) delle cellule di B sono stati analizzati seguendo l&#39;immunizzazione dei topi C57BL/6 wild-type con o glicoproteina del oligodendrocyte di mielina ricombinante (MOG; B Cell-dependent EAE) o il encephalitogenic MOG(35-55) peptide (indipendente dalla cellula B EAE). Plasma ELISA rivelato responses to unici epitopi lineari di MOG dopo immunizzazione con MOG ricombinante che erano assenti negli animali immunizzati MOG(35-55). Analisi repertorio delle cellule b mediante RT-PCR identificato una risposta unica limitata a 7183 famiglia del gene variabile nei topi immunizzati con MOG ricombinante che non &egrave; stata osservata nei topi immunizzati MOG(35-55) catena pesante della Ig. Queste intuizioni potrebbero aiutare nell&#39;identificazione delle popolazioni pertinenti delle cellule B importante per la patogenesi di EAE B cellula-dipendente e nei meccanismi con cui queste popolazioni di cellule B contribuiscono alla malattia.

Risposte uniche delle cellule di B in cellule B-dipendente e indipendente dalla cellula B EAE.

The approved immunomodulatory agents for the treatment of multiple sclerosis (MS) are only partially effective. It is thought that the combination of immunomodulatory and neuroprotective strategies is necessary to prevent or reverse disease progression. Irradiation with far red/near infrared light, termed photobiomodulation, is a therapeutic approach for inflammatory and neurodegenerative diseases. Data suggests that near-infrared light functions through neuroprotective and anti-inflammatory mechanisms. We sought to investigate the clinical effect of photobiomodulation in the Experimental Autoimmune Encephalomyelitis (EAE) model of multiple sclerosis.

Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by photobiomodulation induced by 670 nm light.

Les agents immunomodulateurs approuv&eacute;s pour le traitement de la scl&eacute;rose en plaques (MS) sont seulement partiellement efficaces. On pense que la combinaison de strat&eacute;gies neuroprotectrices et immunomodulatrices est n&eacute;cessaire pour pr&eacute;venir ou inverser la progression de la maladie. Irradiation avec rouge loin/pr&egrave;s de la lumi&egrave;re infrarouge, appel&eacute;e photobiomodulation, est une approche th&eacute;rapeutique pour inflammatoires et les maladies neurod&eacute;g&eacute;n&eacute;ratives. Les donn&eacute;es sugg&egrave;rent que la lumi&egrave;re infrarouge proche fonctionne par le biais de m&eacute;canismes neuroprotecteurs et anti-inflammatoire. Nous avons cherch&eacute; &agrave; &eacute;tudier l&#39;effet clinique de photobiomodulation dans le mod&egrave;le de l&#39;enc&eacute;phalomy&eacute;lite auto-immune exp&eacute;rimentale (EAE) de scl&eacute;rose en plaques.

Am&eacute;lioration de l&#39;enc&eacute;phalomy&eacute;lite allergique exp&eacute;rimentale chez la souris C57BL/6 par photobiomodulation induite par la lumi&egrave;re 670 nm.

Gli agenti immunomodulatori approvato per il trattamento della sclerosi multipla (SM) sono solo parzialmente efficaci. Si &egrave; pensato che la combinazione di strategie immunomodulatori e neuroprotective &egrave; necessaria per prevenire o invertire la progressione della malattia. Irradiazione con rosso lontano/vicino a luce infrarossa, chiamato fotobiomodulazione, &egrave; un approccio terapeutico per infiammatori e malattie neurodegenerative. I dati suggeriscono che il vicino infrarosso luce funzioni attraverso meccanismi neuroprotective ed antinfiammatorio. Abbiamo cercato di indagare l&#39;effetto clinico di fotobiomodulazione nel modello di encefalomielite Autoimmune sperimentale (EAE) di sclerosi multipla.

Miglioramento di encefalomielite autoimmune sperimentale nei topi C57BL/6 di fotobiomodulazione indotta dalla luce di 670 nm.

Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca(2+) is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca(2+) signaling is the Ca(2+)-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the Î²-isoform of the catalytic A subunit (CnAÎ²) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAÎ²-deficient mice (CnAÎ²-/-) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAÎ²-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAÎ²-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.

Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury.

La pancr&eacute;atite aigu&euml; est un fardeau majeur pour la sant&eacute;, pour lesquels il n&#39;y a actuellement aucune des th&eacute;rapies cibl&eacute;es. Activation pr&eacute;matur&eacute;e des proenzymes digestives ou zymog&egrave;nes, dans les cellules acineuses du pancr&eacute;as est un &eacute;v&eacute;nement pr&eacute;coce et critique dans cette maladie. Une grande amplitude, hausse soutenue des cellules acineuses Ca(2+) est requise pour l&#39;activation de zymog&egrave;ne. Nous avons pr&eacute;c&eacute;demment montr&eacute; dans un mod&egrave;le de pancr&eacute;atite induite par la chol&eacute;cystokinine qu&#39;une cible potentielle de cette signalisation aberrante de Ca(2+) est la calcineurine phosphatase activ&eacute;e par Ca(2+) (Cn). Toutefois, dans cette &eacute;tude, nous avons examin&eacute; le r&ocirc;le du Cn sur l&#39;activation de zymog&egrave;ne tant de blessures, dans la condition clinique pertinente de stimulation neurog&egrave;ne (en donnant le carbachol analogiques de l&#39;ac&eacute;tylcholine) en utilisant trois diff&eacute;rents inhibiteurs de la Cn ou cellules acineuses Cn d&eacute;ficientes. Dans les cellules acineuses souris fra&icirc;chement isol&eacute;es, un pr&eacute;traitement avec FK506, peptide inhibiteur de la calcineurine (CiP) ou cyclosporine (CsA) a bloqu&eacute; activation zymog&egrave;ne intra-acinar (n = 3 ; P &lt; 0,05). Les inhibiteurs de la Cn r&eacute;duit aussi la fuite de la lactate d&eacute;shydrog&eacute;nase (LDH) par 79 %, 62 % et 63 %, respectivement (n = 3 ; P &lt; 0,05). Des diverses isoformes Cn, l&#39;isoforme &beta; de la catalytique une sous-unit&eacute; (CnA&beta;) a &eacute;t&eacute; fortement exprim&eacute;e dans les cellules acineuses de souris. Pour cette raison, nous avons obtenu des cellules acineuses de souris d&eacute;ficientes en CnA&beta; (CnA&beta;-/-) et a observ&eacute; une r&eacute;duction de 84 % et 50 % dans l&#39;activation de la trypsine et la chymotrypsine, respectivement, comparativement aux t&eacute;moins de type sauvage (n = 3 ; P &lt; 0,05). Communiqu&eacute; de la LDH dans les cellules d&eacute;ficientes en CnA&beta; a &eacute;t&eacute; r&eacute;duite de 50 % (n = 2 ; P &lt; 0,05). Les cellules d&eacute;ficientes en CnA&beta; sont &eacute;galement prot&eacute;g&eacute;s contre l&#39;activation de zymog&egrave;ne et la l&eacute;sion cellulaire induite par la c&eacute;rul&eacute;ine analogue de la chol&eacute;cystokinine. Ce qui est important, s&eacute;cr&eacute;tion d&#39;amylase n&#39;est g&eacute;n&eacute;ralement pas affect&eacute;e par les inhibiteurs de la Cn ou Cn carence. Ces donn&eacute;es fournissent des preuves g&eacute;n&eacute;tiques et pharmacologiques qui implique Cn des blessures intra-acinar zymog&egrave;ne activation et cellulaires au cours de la pancr&eacute;atite.

Inhibition pharmacologique et g&eacute;n&eacute;tique de la calcineurine prot&egrave;ge contre l&#39;activation induite par le carbachol de zymog&egrave;ne pathologiques et des l&eacute;sions des cellules acineuses.

Pancreatite acuta &egrave; un onere maggiore salute per cui non ci sono attualmente terapie mirate. Attivazione precoce di proenzymes digestivi, o zimogeni, all&#39;interno delle cellule acinose del pancreas &egrave; un evento precoce e fondamentale in questa malattia. Una grande ampiezza, sostenuto aumento delle cellule acinose Ca(2+) &egrave; richiesto per l&#39;attivazione di zimogeno. Vi abbiamo mostrato in precedenza in un modello di pancreatite indotta colecistochinina che un potenziale bersaglio di questa segnalazione aberrante di Ca(2+) &egrave; il Ca(2+) attivato fosfatasi calcineurina (Cn). Tuttavia, in questo studio, abbiamo esaminato il ruolo di Cn su attivazione zimogeno e del pregiudizio, nella condizione clinicamente rilevante di stimolazione neurogena (fornendo il carbachol analogico acetilcolina) utilizzando tre diversi inibitori Cn o cellule acinose Cn-carenti. Nelle cellule acinose del mouse appena isolato, pretrattamento con FK506, peptidi inibitori della calcineurina (CiP) o ciclosporina (CsA) bloccato attivazione intra-acinar zimogeno (n = 3; P &lt; 0,05). Anche gli inibitori Cn ridotta dispersione della Lattato deidrogenasi (LDH) 79%, 62% e 63%, rispettivamente (n = 3; P &lt; 0,05). Delle diverse isoforme di Cn, l&#39;isoforma &beta; del catalitico A subunit&agrave; (CnA&beta;) era fortemente espresso nelle cellule acinose del mouse. Per questo motivo, abbiamo ottenuto le cellule acinose dal mouse CnA&beta;-carenti (CnA&beta;-/-) e osservate un 84% e 50% riduzione nell&#39;attivazione della tripsina e chimotripsina, rispettivamente, rispetto al wild-type controlli (n = 3; P &lt; 0,05). Rilascio LDH nelle cellule CnA&beta;-carente &egrave; stato ridotto del 50% (n = 2; P &lt; 0,05). Le cellule CnA&beta;-carenti erano protetti anche contro zimogeno attivazione e lesione cellulare indotta dalla colecistochinina analogico caerulein. Soprattutto, la secrezione di amilasi generalmente non fu influenzata dal Cn inibitori o deficit di Cn. Questi dati forniscono prove farmacologiche sia genetica che implica Cn lesioni delle cellule e l&#39;attivazione di zimogeno intra-acinar durante la pancreatite.

Inibizione farmacologica e genetica della calcineurina protegge contro attivazione indotta carbachol patologica zimogeno e lesione delle cellule acinose.

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.

Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca(2+). These aberrant Ca(2+) elevations are triggered by release of Ca(2+) from apical Ca(2+) pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca(2+) release channel (IP3R2(-/-)). Using live acinar cell Ca(2+) imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca(2+) signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2(-/-) and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca(2+) signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes.

IP3 receptor type 2 deficiency is associated with a secretory defect in the pancreatic acinar cell and an accumulation of zymogen granules.

La pancr&eacute;atite aigu&euml; est une maladie mortelle douloureuse du pancr&eacute;as dont l&#39;&eacute;tiologie est souvent multifactorielle. Il est d&#39;une grande importance pour comprendre l&#39;interaction entre les facteurs qui pr&eacute;disposent les patients afin de d&eacute;velopper la maladie. Un de ces facteurs est une &eacute;l&eacute;vation excessive des cellules acineuses du pancr&eacute;as Ca(2+). Ces &eacute;l&eacute;vations Ca(2+) aberrantes sont d&eacute;clench&eacute;es par la lib&eacute;ration de Ca(2+) partir d&#39;un m&eacute;lange de Ca(2+) apical qui sont bloqu&eacute;es par les types de r&eacute;cepteurs (IP3R) inositol 1,4,5-trisphosphate 2 et 3. Dans cette &eacute;tude, nous avons examin&eacute; le r&ocirc;le de IP3R de type 2 (IP3R2) &agrave; l&#39;aide de souris d&eacute;ficientes en ce canal de diffusion de le Ca(2+) (IP3R2(-/-)). Imagerie Ca(2+) des cellules acineuses vivantes, nous avons constat&eacute; que la perte de IP3R2 r&eacute;duit l&#39;amplitude du signal Ca(2+) apical et a retard&eacute; son initiation. Cela a &eacute;t&eacute; associ&eacute;e &agrave; une r&eacute;duction de la lib&eacute;ration d&#39;amylase stimul&eacute;e par le carbachol et une accumulation de zymog&egrave;ne (ZGs). Plus pr&eacute;cis&eacute;ment, il y avait une augmentation du nombre de ZGs 2 fois (P &lt; 0,05) et une expansion de l&#39;aire de piscine ZG au sein de la cellule. Il y a aussi une augmentation de 1.6 et 2.6 fois en amylase cellulaire et le trypsinog&egrave;ne, respectivement. Cependant, les souris n&#39;avaient pas de preuve de dommage du pancr&eacute;as au d&eacute;part, autre qu&#39;un niveau d&#39;amylase s&eacute;rique. De plus, r&eacute;sultats de pancr&eacute;atite en utilisant un mod&egrave;le d&#39;hyperstimulation c&eacute;rul&eacute;ine doux &eacute;taient similaires entre les IP3R2(-/-) et les souris de type sauvage. En r&eacute;sum&eacute;, IP3R2 module signaux Ca(2+) cellules acineuses apicale et la s&eacute;cr&eacute;tion des enzymes pancr&eacute;atiques. Cellules acineuses IP3R d&eacute;ficientes accumulent ZGs, mais les souris ne succombent pas aux dommages pancr&eacute;atiques ou pire des r&eacute;sultats de pancr&eacute;atite.

D&eacute;ficit de type 2 r&eacute;cepteurs IP3 est associ&eacute; &agrave; un d&eacute;faut de s&eacute;cr&eacute;tion dans les cellules acineuses du pancr&eacute;as et une accumulation de zymog&egrave;ne.

Pancreatite acuta &egrave; un disturbo doloroso e life-threatening del pancreas cui eziologia &egrave; spesso multifattoriale. &Egrave; di grande importanza per comprendere l&#39;interazione tra i fattori che predispongono i pazienti a sviluppare la malattia. Un tale fattore &egrave; un aumento eccessivo delle cellule acinose del pancreas Ca(2+). Queste elevazioni Ca(2+) aberranti sono attivate dal rilascio di Ca(2+) dalle piscine Ca(2+) apicale che sono dipendenti dai tipi di recettori (IP3R) inositolo 1.4.5-trifosfato 2 e 3. In questo studio, abbiamo esaminato il ruolo di IP3R tipo 2 (IP3R2) usando i mouse carenti in questo canale di rilascio del Ca(2+) (IP3R2(-/-)). Utilizzando imaging Ca(2+) diretta delle cellule acinose abbiamo scoperto che la perdita di IP3R2 ridotto l&#39;ampiezza del segnale Ca(2+) apicale e ha causato un ritardo nella sua iniziazione. Questa &egrave; stata associata con una riduzione del rilascio di amilasi carbachol-stimolato e un accumulo di granuli di zimogeno (ZGs). In particolare, c&#39;&egrave; stato un aumento di 2 volte il numero di ZGs (P &lt; 0,05) e un&#39;espansione della zona piscina ZG all&#39;interno della cellula. C&#39;era anche un 1.6 - 2.6 volte aumento amilasi cellulare e tripsinogeno, rispettivamente. Tuttavia, i topi non hanno evidenza di lesioni del pancreas al basale, diverso da un livello di amilasi sierica elevata. Ulteriormente, esiti pancreatite utilizzando un modello di iperstimolazione lieve caerulein erano simili tra IP3R2(-/-) e topi wild type. In sintesi, IP3R2 modula segnali Ca(2+) apicale delle cellule acinose e la secrezione di enzimi pancreatici. IP3R-deficienti cellule acinose accumulano ZGs, ma i topi non soccombere ai danni del pancreas o peggio esiti pancreatite.

Deficit di tipo 2 recettore IP3 &egrave; associato un difetto secretivo nel cellule acinose del pancreas e un accumulo di granuli di zimogeno.

Biliary pancreatitis is the most common etiology of acute pancreatitis, accounting for 30-60% of cases. A dominant theory for the development of biliary pancreatitis is the reflux of bile into the pancreatic duct and subsequent exposure to pancreatic acinar cells. Bile acids are known to induce aberrant Ca2+ signals in acinar cells as well as nuclear translocation of NF-ÎºB. In this study, we examined the role of the downstream Ca2+ target calcineurin on NF-ÎºB translocation. Freshly isolated mouse acinar cells were infected for 24 hr with an adenovirus expressing an NF-ÎºB luciferase reporter. The bile acid taurolithocholic acid-3-sulfate (TLCS) caused NF-ÎºB activation at concentrations (500 uM) that were associated with cell injury. We show that the NF-ÎºB inhibitor Bay 11-7082 (1 uM) blocked translocation and injury. Pretreatment with the Ca2+ chelator BAPTA, the calcineurin inhibitors, FK506 and cyclosporine A, or use of acinar cells from calcineurin AÎ²-deficient mice each led to reduced NF-ÎºB activation with TLCS. Importantly, these maniuplations did not affect LPS-induced NF-ÎºB activation. A critical upstream regulator of NF-ÎºB activation is protein kinase C, which translocates to the membranes of various organelles in the active state. We demonstrate that pharmacologic and genetic inhibition of calcineurin blocks translocation of the PKC-Î´ isoform. In summary, bile-induced NF-ÎºB activation and acinar cell injury are mediated by calcineurin, and a mechanism for this important early inflammatory response appears to be upstream at the level of PKC translocation.

Pancreatic acinar cell NF-ÎºB activation due to bile acid exposure is dependent on calcineurin.

La pancr&eacute;atite biliaire est l&#39;&eacute;tiologie la plus courante de pancr&eacute;atite aigu&euml;, avec 30-60 % des cas. Une th&eacute;orie dominante pour le d&eacute;veloppement de la pancr&eacute;atite biliaire est le reflux de la bile dans le canal pancr&eacute;atique et l&#39;exposition subs&eacute;quente &agrave; cellules acineuses du pancr&eacute;as. Les acides biliaires sont connus pour induire des signaux de Ca2 + aberrants dans les cellules acineuses ainsi que la translocation nucl&eacute;aire du NF-&kappa;B. Dans cette &eacute;tude, nous avons examin&eacute; le r&ocirc;le de la cible calcineurine de Ca2 + en aval sur la translocation de NF-&kappa;B. Les cellules acineuses souris fra&icirc;chement isol&eacute;es ont &eacute;t&eacute; infect&eacute;s pendant 24 heures avec un ad&eacute;novirus qui exprime un rapporteur lucif&eacute;rase de NF-&kappa;B. La bile acide taurolithocholique acide-3-sulfate (TLC) caus&eacute; d&#39;activation de NF-&kappa;B aux concentrations (500 uM) qui &eacute;taient associ&eacute;s &agrave; des l&eacute;sions des cellules. Nous montrons que l&#39;inhibiteur de NF-&kappa;B Bay 11-7082 (1 uM) bloqu&eacute; translocation et blessures. Un pr&eacute;traitement avec le ch&eacute;lateur du Ca2 + BAPTA, les inhibiteurs de la calcineurine, FK506 et cyclosporine A ou utilisation des cellules acineuses de la calcineurine souris A&beta;-d&eacute;ficientes chaque conduit &agrave; l&#39;activation de NF-&kappa;B r&eacute;duite avec TLC. Ce qui est important, ces maniuplations n&#39;affecte pas d&#39;activation de NF-&kappa;B induite par LPS. Un critique r&eacute;gulateur en amont de l&#39;activation de NF-&kappa;B est la prot&eacute;ine kinase C, lequel translocation aux membranes des organites diff&eacute;rents dans l&#39;&eacute;tat actif. Nous d&eacute;montrons que l&#39;inhibition pharmacologique et g&eacute;n&eacute;tique de la calcineurine translocation de blocs de l&#39;isoforme de la PKC-&delta;. En r&eacute;sum&eacute;, bile induite par l&#39;activation de NF-&kappa;B et cellules acineuses blessures sont v&eacute;hicul&eacute;s par la calcineurine, et un m&eacute;canisme pour cette r&eacute;ponse inflammatoire d&eacute;but important semble &ecirc;tre en amont au niveau de la translocation de la PKC.

Les cellules acineuses du pancr&eacute;as activation de NF-&kappa;B dus &agrave; l&#39;exposition des acides biliaires sont tributaire de la calcineurine.

Kamaldeen A. Muili

Rangos Research Center,
Pediatric Gastroenterology,
Hepatology,
and Nutrition,
Rangos Research Center, Pediatric Gastroenterology, Hepatology, and Nutrition

Children's Hospital of Pittsburgh of UPMC

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

Kamaldeen A. Muili

Rangos Research Center,Pediatric Gastroenterology,Hepatology,and Nutrition,Rangos Research Center, Pediatric Gastroenterology, Hepatology, and Nutrition

Children's Hospital of Pittsburgh of UPMC

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

Rangos Research Center,
Pediatric Gastroenterology,
Hepatology,
and Nutrition,
Rangos Research Center, Pediatric Gastroenterology, Hepatology, and Nutrition