Association of residual gastric acid secretion with persistent symptoms in gastroesophageal reflux disease patients receiving standard-dose proton pump inhibitor therapy.

BACKGROUND: Although a third of gastroesophageal reflux disease (GERD) patients are refractory to proton pump inhibitor (PPI) therapy, the underlying mechanism of the refractoriness remains unclear. We compared the level of gastric acid suppression during PPI treatment between responders and non-responders by directly measuring gastric acid secretion in GERD patients taking PPIs. METHODS: Seventy-five consecutive patients receiving standard-dose PPI therapy for GERD were prospectively recruited, irrespective of persistent GERD symptoms. They were asked about their GERD symptoms using a validated questionnaire, and simultaneously underwent both a routine endoscopic examination and a gastric acid secretory testing using an endoscopic gastrin test. Associations between residual gastric acid secretion during PPI treatment and persistent GERD symptoms were analyzed by a logistic regression analysis. RESULTS: Overall, 26 of 75 (34.7%) patients were judged to be positive for persistent GERD symptoms. The patients with and without persistent symptoms showed similar gastric acid secretion levels (1.3 [1.3] mEq/10 min vs. 1.4 [2.0] mEq/10 min). Sufficient gastric acid suppression, defined as < 0.6, was not significantly associated with persistent GERD symptoms (odds ratio 1.1, 95% confidence interval 0.40-3.5). CONCLUSIONS: This study provided solid evidence to support that the gastric acid suppression level during PPI treatment does not differ between patients with and without persistent GERD symptoms. The insignificant role of residual gastric acid in the persistent GERD symptoms suggests that the use of medications other than those that enhance gastric acid inhibitory effects would be an essential approach for the management of PPI-refractory GERD.

Glial fibrillary acidic protein promoter targets pancreatic stellate cells.

BACKGROUND: Pancreatic fibrosis is one of the clinical manifestations of chronic pancreatitis and pancreatic cancer. Pancreatic stellate cells (PSCs) have been recognised as principal effector cells in the development of pancreatic fibrosis. The ability to specifically address PSCs might offer a potential for developing a targeted therapy for pancreatic fibrosis. AIM: Characterisation of the 2.2kb hGFAP (human glial fibrillary acidic protein) promoter for its usefulness to express reporter genes specifically in PSCs in vitro and in vivo. METHODS: 2.2kb hGFAP-LacZ reporter expressions were examined in four immortalised PSC lines and two non-PSCs, meanwhile, GFAP-LacZ transgenic mice were used to detect LacZ reporter in pancreas tissue. Several kinase inhibitors, vitamin A and its metabolites were applied to study the regulation of 2.2kb hGFAP promoter in PSCs. RESULTS: Our results showed that the 2.2kb hGFAP promoter is capable of regulating the expression of reporter genes exclusively in immortalised and primary PSCs, as well as in PSCs of transgenic GFAP-LacZ mice. When a PSC cell line transfected with the LacZ reporter (SAM-K/LacZ/C1) was treated with different anti-fibrotic agents and kinase inhibitors, the transgenic beta-galactosidase activity was found to be regulated by multiple signalling pathways known to be involved in the PSC activation. CONCLUSIONS: This study provides the proof of concept for using the 2.2kb hGFAP promoter to specifically manipulate PSCs for the development of targeted gene and/or drug therapy in pancreatic fibrosis, and for the screening of anti-fibrotic agents.

Fibrinogen induces cytokine and collagen production in pancreatic stellate cells.

OBJECTIVE: Fibroblasts in the area of fibrosis in chronic pancreatitis and of the desmoplastic reaction associated with pancreatic cancer are now recognised as activated pancreatic stellate cells (PSCs). Recent studies have shown strong expression of fibrinogen, the central protein in the haemostasis pathway, in the stromal tissues of pancreatic cancer and chronic pancreatitis, suggesting that PSCs are embedded in and exposed to abundant fibrinogen in these pathological settings. The effects of fibrinogen on cell functions in PSCs were examined here. METHODS: PSCs were isolated from human pancreas tissues of patients undergoing operations for pancreatic cancer, and from rat pancreatic tissues. The effects of fibrinogen on key cell functions and activation of signalling pathways in PSCs were examined. RESULTS: Fibrinogen induced the production of interleukin 6 (IL6), interleukin 8 (IL8), monocyte chemoattractant protein-1, vascular endothelial growth factor, angiopoietin-1 and type I collagen, but not proliferation or intercellular adhesion molecule-1 expression. Fibrinogen increased alpha-smooth muscle actin expression and induced the activation of nuclear factor-kappaB (NF-kappaB), Akt and three classes of mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK)). Fibrinogen-induced IL6 and IL8 production was inhibited by antibodies against alpha(v)beta(3) and alpha(5)beta(1) integrins, suggesting that these integrins worked as counter receptors for fibrinogen in PSCs. In addition, fibrinogen-induced production of these cytokines was abolished by an inhibitor of NF-kappaB, and partially inhibited by inhibitors of ERK and p38 MAPK. CONCLUSION: Fibrinogen directly stimulated profibrogenic and proinflammatory functions in PSCs.

A loss-of-function p.G191R variant in the anionic trypsinogen (PRSS2) gene in Japanese patients with pancreatic disorders.

OBJECTIVE: There is a concept that pancreatitis results from an imbalance of proteases and their inhibitors within the pancreatic parenchyma. It has been recently shown that a loss-of-function variant, c.571G>A (p.G191R), in the anionic trypsinogen (PRSS2) gene protects against chronic pancreatitis in European populations. Here we examined the association of the p.G191R variant with pancreatic disorders in Japan. METHODS: Genomic DNA was prepared from 378 healthy controls and 604 patients with pancreatic disorders (241 patients with chronic pancreatitis, 174 with acute pancreatitis, and 189 with pancreatic neoplasm). Mutational analysis of the PRSS2 gene was performed by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. RESULTS: The heterozygous p.G191R variant was found in three of 241 (1.2%) patients with chronic pancreatitis, in seven of 174 (4.0%) patients with acute pancreatitis, and in 12 of 189 (6.3%) patients with pancreatic neoplasm. The p.G191R variant was found in 25 (two were homozygous and 23 were heterozygous) of 378 (6.6%) healthy controls. The p.G191R frequency in patients with chronic pancreatitis was lower than that in healthy controls (p = 0.001; odds ratio (OR) 0.178; 95% confidence interval (CI) = 0.057 to 0.561). The p.G191R frequency was lower in patients with alcoholic (0.9%; p = 0.015; OR, 0.132; 95% CI, 0.022 to 0.779) and idiopathic (1.0%; p = 0.025; OR, 0.144; 95% CI, 0.025 to 0.851) chronic pancreatitis than that in healthy controls. There were no statistical differences in the p.G191R frequency between healthy controls and patients with acute pancreatitis or with pancreatic neoplasm. Patients with alcoholic acute pancreatitis (n = 59) had no variant carrier, and the p.G191R frequency was lower than that in healthy controls (p = 0.035). CONCLUSION: The p.G191R variant protected against alcoholic and idiopathic chronic pancreatitis as well as alcoholic acute pancreatitis in Japan.

Tumor necrosis factor-related apoptosis-inducing ligand and its receptor expression and the pathway of apoptosis in human pancreatic cancer.

METHODOLOGY: The authors performed the reverse transcription-polymerase chain reaction (RT-PCR) in 17 cases of pancreatic ductal cell carcinoma (PDC) and five cases of normal pancreatic tissues to determine the expression of tumor necrosis factor -related apoptosis-inducing factor (TRAIL) and its five receptors in PDC. RESULTS: The expression of TRAIL and its receptors other than osteoprotegerin was found frequently in both PDC and normal tissues. whereas the expression of osteoprotegerin was detected only in PDC. The authors detected cancer cell death by TRAIL, ranging from 37% to 77% in all the PDC cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Hochest staining revealed that cell death was caused by apoptosis. Caspase-8 and caspase-3 and poly (ADP-ribose) polymerase cleavage was activated within 2 hours after stimulation with 200 ng/mL TRAIL. CONCLUSION: These findings suggest a relation between osteoprotegerin expression and the biologic aggressiveness of PDC and the involvement of caspase-8 and caspase-3 activation in the TRAIL-mediated apoptosis pathway in PDC.

Lysophosphatidylcholine activates transcription factor NF-kappaB and AP-1 in AR42J cells.

Phospholipase A2 (PLA2) has been suggested in the pathogenesis of acute pancreatitis, in part through the PLA2-generated phospholipid by-products, most notably lysophosphatidylcholine (lyso-PC). The effects of lyso-PC on pancreatic acinar cells other than necrosis are poorly characterized. Recent studies have suggested a role of the activation of transcription factors such as nuclear factor kappa B (NF-kappaB) for the pathogenesis of acute pancreatitis. Here we examined the effects of lyso-PC on the activation of transcriptional factors in rat pancreatic AR42J cells. Lyso-PC induced apoptosis at concentrations > or = 10 microM. At 10 and 25 microM, lyso-PC increased the NF-kappaB- and activator protein-1 (AP-1)-specific DNA binding activity as determined by electrophoretic mobility shift assay. Lyso-PC also increased the transcriptional activity of NF-kappaB and AP-1 as assessed by luciferase assay. Lyso-PC increased the mRNA level of pancreatitis-associated protein-I and c-jun. Lyso-PC activated three classes of mitogen activated protein kinases: extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase/stress-activated protein kinase and p38 kinases. Activation of transcription factors by lyso-PC was not altered by a specific platelet activating factor receptor antagonist, TCV-309, suggesting that the activation was independent of the platelet activating factor receptor. These molecular events may suggest a novel role of lyso-PC for the modulation of acinar cell function.

Rebamipide inhibits ceramide-induced interleukin-8 production in Kato III human gastric cancer cells.

Helicobacter pylori adheres to gastric epithelial cells and stimulates interleukin-8 production. Ceramide, a lipid second messenger, has become known as an important mediator of some actions of several cytokines. We have recently reported that H. pylori-dependent ceramide production may activate nuclear factor-kappaB and mediate interleukin-8 expression in human gastric cancer cell lines. In this study, we evaluated the effect of rebamipide, an antigastritis and antiulcer agent, on H. pylori-dependent ceramide production and subsequent interleukin-8 expression in Kato III cells. Rebamipide inhibited ceramide-induced interleukin-8 expression in a dose-dependent manner. Rebamipide decreased the ceramide-induced increase of the interleukin-8 mRNA level as assessed by Northern blotting. Rebamipide suppressed interleukin-8 gene transcription and nuclear factor-kappaB-dependent transcriptional activity as assessed by luciferase assay. Rebamipide inhibited the ceramide-induced degradation of IkappaB-alpha (a major cytoplasmic inhibitor of nuclear factor-kappaB), further supporting that rebamipide inhibits the activation of nuclear factor-kappaB. Rebamipide also inhibited the ceramide-dependent activation of mitogen-activated protein kinases. Furthermore, rebamipide significantly attenuated the H. pylori-dependent increase in the intracellular ceramide level. These results suggest a novel mechanism by which rebamipide may protect against the mucosal inflammation associated with H. pylori infection.

Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors.

BACKGROUND: Survivin is a new member of the inhibitor of apoptosis family of antiapoptotic proteins. This protein was expressed selectively in all the most common human carcinomas but not in normal adult tissues. To our knowledge, the relation between survivin expression and apoptosis or tumorigenesis has not yet been studied in pancreatic neoplasms. METHODS: The authors investigated the expression of survivin in 4 pancreatic carcinoma cell lines and 56 human pancreatic tissues (5 cases of normal, 12 cases of chronic pancreatitis [CP], 26 cases of pancreatic duct cell adenocarcinoma [PDC], 16 lesions of 13 intraductal papillary-mucinous tumor [IPMT] by immunohistochemistry, immunoblotting, and reverse transcription-polymerase chain reaction (RT-PCR) to examine the association of its expression with tumor apoptosis and/or tumorigenesis. RESULTS: Survivin expression was found in the tumor cells but not in the nonneoplastic pancreatic tissues (normal and CP tissues). Survivin expression was observed in 20 of 26 cases of PDC (76.9%) and in 9 of 16 IPMT lesions that ranged from adenoma to invasive (56.3%) by immunohistochemistry. Survivin was more frequently expressed in malignant tumors than in benign tumors (P = 0.0089). In PDC, high levels of survivin expression were associated significantly with a reduction in the apoptotic index of the tumor cells (0.445% +/- 0.150% vs. 0.961% +/- 0.378%; P < 0.0001). CONCLUSIONS: These data suggest that the expression of survivin may be upregulated during an early step of tumorigenesis and during the development of cancer by reducing the cancer cell apoptosis.

Analysis of the human pancreatic secretory trypsin inhibitor (PSTI) gene mutations in Japanese patients with chronic pancreatitis.

Chronic pancreatitis (CP) is a continuing or relapsing inflammatory disease of the pancreas. Several studies have demonstrated that mutations in the cationic trypsinogen (PRSS1) gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene are causative of the pathogenesis in a subset of hereditary and/or idiopathic CP cases. Recently, the N34S alteration of the pancreatic secretory trypsin inhibitor (PSTI) gene has been suggested to be closely associated with the pathogenesis of hereditary and/or idiopathic CP. Herein we analyzed genetic alterations of the PSTI gene in 32 unrelated Japanese CP patients who developed juvenile-onset CP or had a family history of CP; 5 patients were found to harbor alterations in this gene. In 3 of these 5 patients, heterozygous N34S alterations were found; this frequency is significantly lower than that in Caucasian patients reported previously. Moreover, a novel homozygous G-to-A transition in the promoter region of PSTI at 215bp upstream from the translation initiation site (-215G>A) was observed in 2 patients. We further surveyed the -215G>A alteration in 117 normal individuals; none of these individuals harbored this alteration. Our results suggested that the -215G>A alteration, as well as the N34S alteration, is a predisposing factor for CP.

Ascites of rat experimental model of severe acute pancreatitis induces lung injury.

The molecular mechanisms that lead from acute pancreatitis (AP) to multiple organ failure remain to be clarified. We previously reported that ascitic fluids from a rat model of severe acute pancreatitis (pancreatitis-associated ascitic fluids, PAAF) transcriptionally activated endothelial cells and leukocytes in vitro. To clarify the role of ascitic fluids on the development of multiple organ failure in AP, we examined the effects of PAAF on the prognosis and immunohistologic findings in cerulein pancreatitis, an experimental model of mild pancreatitis in vivo. Intraperitoneal injection of PAAF decreased the survival rates in a dose-dependent manner. Histologically, destruction of vessels, alveolar septal thickening, interstitial hypertrophy, and infiltration of inflammatory cells were prominent in the lung of PAAF-injected rats. Transcription factor, nuclear factor KB (NF-kappaB) was activated and the mRNA levels of tumor necrosis factor-alpha and interleukin-1beta were increased in the lung of the PAAF-injected rats. The permeability index assessed by Evans blue assay and the lung myeloperoxidase activity levels were significantly higher in the PAAF-injected rats than in controls. Inhibition of NF-kappaB ameliorated the histologic findings and improved the survival rates. Our results suggest that PAAF play a role in the pathogenesis of lung injury in severe AP, at least in part through the activation of NF-kappaB.

Lysophosphatidylcholine induces apoptosis in AR42J cells.

Phospholipase A2 (PLA2) has been suggested to play an important role in the pathogenesis of acute pancreatitis, in part through the PLA2-generated phospholipid by-products, most notably lysophosphatidylcholine (lyso-PC). The effects of lyso-PC on pancreatic acinar cells, other than the induction of necrosis, are poorly characterized. Here we examined the effects of lyso-PC on the induction of apoptosis in rat pancreatic AR42J cells. Lyso-PC induced apoptosis in a dose-dependent manner at 10 and 25 microM, but induced cell lysis at > or = 50 microM. Lyso-PC-induced (at 25 microM) apoptosis was not blocked by a protein kinase C inhibitor (staurosporine) or by inhibitors of caspases (acetyl-DEVD-aldehyde and benzoyloxycarbonyl-VAD-fluoromethylketone). Lyso-PC at 10 and 25 microM induced the expression of clusterin mRNA and wild-type p53. Apoptosis induction by lyso-PC (at 25 microM) was not inhibited by a specific antagonist of platelet-activating factor (PAF) receptor, suggesting that the action was independent of th

A case of hemosuccus pancreaticus associated with hereditary pancreatitis.

We report a 25-year-old male with hemosuccus pancreaticus associated with hereditary pancreatitis. He was originally diagnosed as having familial chronic pancreatitis at the age of 12, because his brother was also diagnosed as having pancreatitis. No history of pancreatitis was found in their parents. The patient was admitted because of a growing pancreatic pseudocyst. While he had undergone conservative treatment for the pseudocyst, computed tomography incidentally revealed a pancreatic pseudoaneurysm. Endoscopic examination revealed spontaneous bleeding from the major papilla. Interventional embolization was successfully performed. An R122H mutation in the cationic trypsinogen gene was identified in this patient, his brother, and his mother, indicating that they have hereditary pancreatitis. To our knowledge, this is the first report of hemosuccus pancreaticus associated with hereditary pancreatitis. Mutational screening is useful for the diagnosis of hereditary pancreatitis, especially in patients whose diagnosis is inconclusive based on the traditional clinical criteria.

Activation of adenosine A1-receptor pathway induces edema formation in the pancreas of rats.

BACKGROUND & AIMS: Adenosine has been shown to modulate various pathophysiologic conditions through receptor-mediated mechanisms. However, the role of adenosine in the pathogenesis of acute pancreatitis has not been described. We examined the effect of adenosine-receptor stimulation or inhibition on the pathologic changes of the pancreas. METHODS: Rats received intraperitoneal injections of selective agonists of A1, A2a, and A3 adenosine receptors: 2-chloro-N(6)-cyclopentyladenosine (CCPA), CGS-21680 (CGS), or 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-D-ribofuranuronamide (IB-MECA), respectively. Serum amylase activity and pathologic changes of the pancreas were evaluated. The effects of a specific A1-receptor antagonist (FK-838) on the pathologic findings of cerulein- and taurocholate-induced pancreatitis were also examined. RESULTS: Administration of a selective A1 agonist induced hyperamylasemia and morphologic changes in the pancreas characterized by interstitial edema and leukocyte infiltration; neither A2a nor A3 agonist produced such changes. Treatment with an A1-receptor antagonist significantly attenuated the outcome induced by A1 agonist stimulation. In addition, the A1-receptor antagonist significantly ameliorated pancreatic edema in both pancreatitis models, although it did not improve the acinar cell damage of the pancreas or the increase of serum amylase. CONCLUSIONS: Activation of the adenosine A1-receptor pathway may have an important role in the pathogenesis of acute pancreatitis.

Nitric oxide decreases endothelial activation by rat experimental severe pancreatitis-associated ascitic fluids.

To clarify the roles of nitric oxide (NO) in acute pancreatitis (AP), we examined the effects of NO on the endothelial activation induced by ascitic fluids from rats with experimental severe AP. Necrotizing hemorrhagic pancreatitis was induced in male Wistar rats with sodium taurocholate. Six hours later, peritoneal exudates were collected, centrifuged, and human umbilical vein endothelial cells were treated with the supernatants. Then (a) the mRNA level of endothelial-type NO synthase (ecNOS) was examined by reverse transcription-polymerase chain reaction; (b) effects of an NO donor, sodium nitroprusside (SNP) and an inhibitor of NOS, N(omega)-nitro-L-arginine (L-NNA) on the ascitic fluids-induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and interleukin-8 were assessed by enzyme-linked immunoassay; (c) nuclear translocation of nuclear factor-kappa B (NF-kappaB) was examined by electrophoretic mobility shift assay; and (d) effects of SNP and L-NNA on the adhesion of U937 cells to endothelial monolayer were assessed. The ecNOS mRNA level was decreased by the ascitic fluids; ascitic fluids-induced expression of adhesion molecules and interleukin-8 as well as the nuclear translocation of NF-kappaB were attenuated by SNP, whereas L-NNA augmented them; and the effects on the endothelial activation were paralleled by the altered adhesion of U937 cells to endothelium. The ability of NO to limit endothelial activation and inhibit leukocyte adhesion might contribute to its antiinflammatory properties in AP.

Ascites of severe acute pancreatitis in rats transcriptionally up-regulates expression of interleukin-6 and -8 in vascular endothelium and mononuclear leukocytes.

The molecular mechanisms that link acute pancreatitis and multiple organ failure remain unknown. We examined the effect of ascitic fluids prepared from rats with experimental necrotizing pancreatitis on the expression of interleukin (IL) -6 and IL-8 in human umbilical vein endothelial cells (HUVEC) and human monocytic THP-1 cells. Incubation of HUVEC or THP-1 cells with the ascitic fluids resulted in a concentration-dependent up-regulation of the cytokine expression with comparable mRNA induction. Electrophoretic mobility shift assay revealed that the ascitic fluids increased the nuclear factor-kappaB (NF-kappaB) and NF-IL6 binding activities. Intraperitoneal injection of ascitic fluids into healthy rats induced the activation of NF-kappaB in the infiltrating leukocytes in the lung. Our results suggested that ascitic fluids may play a role in the pathophysiology of severe acute pancreatitis through the activation of transcription factors and consequent cytokine productions in distant organs.

Fas ligand is frequently expressed in human pancreatic duct cell carcinoma.

Fas ligand (Fas-L) is a key molecule in normal immune development, homeostasis, modulation, and function. Recent reports suggested that tumor cells can evade immune attack by killing lymphocytes through expressing Fas-L on the tumor cell surface. The expression of Fas-L has been demonstrated in pancreatic cancer cell lines and in tissues. The messenger RNA (mRNA) and protein expression of Fas-L was investigated in four pancreatic cancer cell lines and 19 human pancreatic duct cell carcinomas (PDCs) by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. In addition, coculture assay of Fas-L and Fas-expressing PANC-1 cells and Fas-sensitive Jurkat cells was performed. Fas-L mRNA expression was observed in all four cell lines as well as in 10 PDC tissues, and the protein expression was frequently detected in the PDC tissues (16 of 19 cases). The coculture experiments showed that PANC-1 cells induced apoptosis of Jurkat cells, whereas the PANC-1 cells themselves and Jurkat cells cultured without the presence of PANC-1 showed few apoptotic changes. These findings suggest that Fas-L may play an important role in the ability of PDCs to escape from immune surveillance through the induction of apoptosis in tumor-attacking lymphocytes. The frequent expression of Fas-L in PDCs may partly explain the notorious biologic behavior of PDCs.

Ascitic fluid of experimental severe acute pancreatitis modulates the function of peritoneal macrophages.

Although the pathophysiology of acute pancreatitis appears to be greatly influenced by the production of ascites, little is known about the mechanism. To investigate the effects of pancreatitis-associated ascitic fluid (PAAF) on macrophage function, we examined the effects of PAAF obtained from a rat model of severe acute pancreatitis on the ability of peritoneal macrophages to produce tumor necrosis factor-alpha (TNF-alpha). In addition, we compared the responses of PAAF-treated and PAAF-untreated macrophages to lipopolysaccharide (LPS) by evaluating their TNF-alpha production and nuclear factor-kappaB (NFkappaB) activation. Incubation of peritoneal macrophages with the PAAF led to the rapid and prolonged activation of NF-kappaB and to TNF-alpha production. Pyrrolidine dithiocarbamate, a potent inhibitor of NF-kappaB activation, attenuated the macrophage TNF-alpha production by PAAF. Macrophages produced TNF-alpha in response to LPS, but the cytokine production was significantly reduced when macrophages were pretreated with PAAF. The suppression of TNF-alpha production by PAAF pretreatment accompanied the impairment of NF-kappaB activation in response to LPS. These results indicate that the PAAF of severe acute pancreatitis may play important roles in the pathologic course of this disease through its effects on macrophage function.