Abrahim I. Orabi

A Comparison of Presentation and Management Trends in Acute Pancreatitis Between Infants/Toddlers and Older Children

Objectives: Acute pancreatitis is a necroinflammatory disease that leads to 210,000 hospitalizations in the United States annually. Recent reports suggest that there may be important differences in clinical features between infants/toddlers and older children. Thus, in this study we make a direct comparison between the pediatric age groups in presentation and management trends of acute pancreatitis. Patients and Methods: We examined all children (ages 0 to 20 years) admitted to Yale-New Haven Childrens Hospital with pancreatitis between 1994 and 2007. Results: Two hundred seventy-one cases met inclusion criteria for acute pancreatitis. Infants and toddlers manifested fewer signs and symptoms of abdominal pain, epigastric tenderness, and nausea compared with older children (43% vs 93%; 57% vs 90%; and 29% vs 76%, respectively; P < 0.05 for all comparisons). They were more likely to be diagnosed by serum lipase than by amylase and to undergo radiographic evaluation (P < 0.05). They had a longer hospital stay (19.5 vs 4 days; P < 0.05) and were less likely to be directly transitioned to oral nutrition (14% vs 71%; P < 0.05). Conclusions: Infants and toddlers with acute pancreatitis present with fewer classical symptoms and are managed differently from older children. We believe these data will be helpful in evaluating and understanding treatment practices in this age group.

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

Background: Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca2+. Results: Pharmacologic and genetic inhibition of the Ca2+-activated phosphatase calcineurin dramatically reduces acinar cell injury and in vivo pancreatitis resulting from bile acid exposure. Conclusion: Acinar cell calcineurin mediates acinar cell injury and pancreatitis resulting from bile acid exposure. Significance: Calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis. 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 Ca2+. Thus, it would be clinically relevant to know the targets of this aberrant Ca2+ signal. We hypothesized that the Ca2+-activated phosphatase calcineurin is such a Ca2+ 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 Ca2+. 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-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aβ (CnAβ) 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 Ca2+ 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.

Novel Characterization of Drug-associated Pancreatitis in Children

Background and Objectives: Medications are a major cause of acute pancreatitis; however, little is known about their influence in children. Our primary aims were to identify common comorbidities and concomitant pancreatitis etiologies in children with drug-associated pancreatitis. Our secondary aims were to identify the most commonly associated drugs in the different age groups, evaluate management practices, and compare drug-associated cases with non–drug-associated cases. Patients and Methods: In the present study, we examined children (ages 0–20 years) admitted to Yale-New Haven Childrens Hospital with pancreatitis between 1994 and 2007. Results: Of a total of 271 pediatric cases, drugs were associated with pancreatitis in 25.6% (55). The 3 most common comorbidities in children with drug-associated pancreatitis were seizure disorders, acute lymphocytic leukemia, and Crohn disease. One third of drug-associated cases had an additional pancreatitis etiology. The most commonly associated drugs were valproic acid and corticosteroids. Compared with non–drug-associated cases, children with drug-associated cases were more likely to undergo CT scanning (54.5% vs 28.4%; P < 0.001), stay in the hospital longer (10 vs 4 days; P < 0.001), and transition to parenteral nutrition from a nil per os status (37.5% vs 21.2%; P < 0.05). There was a higher frequency of valproic acid–associated cases in children younger than 11 years (29.4% vs 9.5% in the 11- to 20-year-old age group). Conclusions: Our study underscores the importance of considering drugs as a cause and a contributor to pancreatitis in children, particularly valproic acid in young children.

The Association of Primary Hyperparathyroidism With Pancreatitis

The association between primary hyperparathyroidism (PHPT) and acute or chronic pancreatitis is controversial. For this reason, we conducted a review of the literature over the past 30 years to explore the relationship between these 2 disorders. Ten retrospective studies each with >50 patients diagnosed with PHPT were identified. With the notable exception of 2 studies, the rate of pancreatitis among patients with PHPT was higher than that reported in general among hospitalized patients without PHPT. A higher serum calcium level may contribute to pancreatitis in these cases, along with additional genetic or environmental insults. Hypercalcemia may predispose the pancreatic acinar cell to abnormal, sustained calcium levels, lead to premature pancreatic protease activation, and pancreatitis. Although there was only short-term follow-up, most reports cited that definitive treatment of PHPT by parathyroidectomy led to the resolution of pancreatitis attacks. The published cohorts of patients with PHPT and pancreatitis are subject to bias, because serum calcium screening was not universally performed among all control nonpancreatitis patients to evaluate for PHPT. However, the pooled clinical and experimental data suggest an association between PHPT and pancreatitis and implicate hypercalcemia. For clinicians, it is important to recognize pancreatitis in patients with PHPT and, conversely, to consider PHPT by checking serum calcium levels in patients, who present with an unexplained pancreatitis.

Ethanol Enhances Carbachol-induced Protease Activation and Accelerates Ca2+ Waves in Isolated Rat Pancreatic Acini

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70–90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca2+ within the pancreatic acinar cell. An important conductor of this Ca2+ is the basolaterally localized, intracellular Ca2+ channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca2+ signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca2+. Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 μm). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca2+ wave from 9 to 18 μm/s (p < 0.0005; n = 18–22 cells/group); an increase in Ca2+ wave speed was also observed with a change from physiologic concentrations of carbachol (1 μm) to a supraphysiologic concentration (1 mm) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n = 10–16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca2+ waves.

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

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 μM and a peak-plateau signal at 500 μM, 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.

Dantrolene mitigates caerulein-induced pancreatitis in vivo in mice.

Acute pancreatitis is a painful, inflammatory disorder for which adequate treatments are lacking. An early, critical step in its development is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. This Ca(2+) release is modulated by the intracellular Ca(2+) channel the ryanodine receptor (RYR). We have previously shown that RYR inhibition reduces pathological intra-acinar protease activation, an early marker of pancreatitis. In this study, we examined whether pretreatment with the RYR inhibitor dantrolene attenuates the severity of caerulein-induced pancreatitis in mice. Immunofluorescent labeling for RYR from mouse pancreatic sections showed localization to the basolateral region of the acinar cell. After 1 h of caerulein hyperstimulation in vivo, dantrolene 1) reduced pancreatic trypsin activity by 59% (P < 0.05) and 2) mitigated early ultrastructural derangements within the acinar cell. Eight hours after pancreatitis induction, dantrolene reduced pancreatic trypsin activity and serum amylase by 61 and 32%, respectively (P < 0.05). At this later time point, overall histological severity of pancreatitis was reduced by 63% with dantrolene pretreatment (P < 0.05). TUNEL-positive cells were reduced by 58% (P < 0.05). These data suggest that the RYR plays an important role in mediating early acinar cell events during in vivo pancreatitis and contributes to disease severity. Blockade of Ca(2+) signals and particularly RYR-Ca(2+) may be useful as prophylactic treatment for this disease in high-risk settings for pancreatitis.

Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin

BACKGROUND & AIMS Radiocontrast agents are required for radiographic procedures, but these agents can injure tissues by unknown mechanisms. We investigated whether exposure of pancreatic tissues to radiocontrast agents during endoscopic retrograde cholangiopancreatography (ERCP) causes pancreatic inflammation, and studied the effects of these agents on human cell lines and in mice. METHODS We exposed mouse and human acinar cells to the radiocontrast agent iohexol (Omnipaque; GE Healthcare, Princeton, NJ) and measured intracellular release of Ca(2+), calcineurin activation (using a luciferase reporter), activation of nuclear factor-κB (NF-κB, using a luciferase reporter), and cell necrosis (via propidium iodide uptake). We infused the radiocontrast agent into the pancreatic ducts of wild-type mice (C57BL/6) to create a mouse model of post-ERCP pancreatitis; some mice were given intraperitoneal injections of the calcineurin inhibitor FK506 before and after infusion of the radiocontrast agent. CnAβ(-/-) mice also were used. This experiment also was performed in mice given infusions of adeno-associated virus 6-NF-κB-luciferase, to assess activation of this transcription factor in vivo. RESULTS Incubation of mouse and human acinar cells, but not HEK293 or COS7 cells, with iohexol led to a peak and then plateau in Ca(2+) signaling, along with activation of the transcription factors NF-κB and nuclear factor of activated T cells. Suppressing Ca(2+) signaling or calcineurin with BAPTA, cyclosporine A, or FK506 prevented activation of NF-κB and acinar cell injury. Calcineurin Aβ-deficient mice were protected against induction of pancreatic inflammation by iohexol. The calcineurin inhibitor FK506 prevented contrast-induced activation of NF-κB in pancreata of mice, this was observed by live imaging of mice given infusions of adeno-associated virus 6-NF-κB-luciferase. CONCLUSIONS Radiocontrast agents cause pancreatic inflammation in mice, via activation of NF-κB, Ca(2+) signaling, and calcineurin. Calcineurin inhibitors might be developed to prevent post-ERCP pancreatitis in patients.

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

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 β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ-/-) 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 CnAβ-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAβ-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.

Pancreatic Acinar Cell Nuclear Factor κB Activation Because of Bile Acid Exposure Is Dependent on Calcineurin

Background: Bile acids cause activation of NF-κB and lead to injury in pancreatic acinar cells, but the mechanism is unknown. Results: Pharmacologic and genetic inhibition of calcineurin reduces bile acid-induced NF-κB activation and PKC-δ translocation. Conclusion: Calcineurin facilitates bile-induced NF-κB activation. The mechanism is at the level of PKC activation. Significance: We have identified a novel mechanism by which bile acids facilitate NF-κB activation in pancreatic acinar cells. 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-κB. In this study, we examined the role of the downstream Ca2+ target calcineurin on NF-κB translocation. Freshly isolated mouse acinar cells were infected for 24 h with an adenovirus expressing an NF-κB luciferase reporter. The bile acid taurolithocholic acid-3-sulfate caused NF-κB activation at concentrations (500 μm) that were associated with cell injury. We show that the NF-κB inhibitor Bay 11-7082 (1 μm) blocked translocation and injury. Pretreatment with the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, the calcineurin inhibitors FK506 and cyclosporine A, or use of acinar cells from calcineurin Aβ-deficient mice each led to reduced NF-κB activation with taurolithocholic acid-3-sulfate. Importantly, these manipulations did not affect LPS-induced NF-κB activation. A critical upstream regulator of NF-κB 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-δ isoform. In summary, bile-induced NF-κB 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.