CCR2 knockout exacerbates cerulein-induced chronic pancreatitis with hyperglycemia via decreased GLP-1 receptor expression and insulin secretion.

Glucagon-like peptide-1 (GLP-1) promotes insulin release; however, the relationship between the GLP-1 signal and chronic pancreatitis is not well understood. Here we focus on chemokine (C-C motif) ligand 2 (CCL2) and its receptor (CCR2) axis, which regulates various immune cells, including macrophages, to clarify the mechanism of GLP-1-mediated insulin secretion in chronic pancreatitis in mice. One and multiple series of repetitive cerulein administrations were used to induce acute and chronic cerulein pancreatitis, respectively. Acute cerulein-administered CCR2-knockout (KO) mice showed suppressed infiltration of CD11b(+)Gr-1(low) macrophages and pancreatic inflammation and significantly upregulated insulin secretion compared with paired wild-type (WT) mice. However, chronic cerulein-administered CCR2-KO mice showed significantly increased infiltration of CD11b(+)/Gr-1(-) and CD11b(+)/Gr-1(high) cells, but not CD11b(+)/Gr-1(low) cells, in pancreas with severe inflammation and significantly decreased insulin secretion compared with their WT counterparts. Furthermore, although serum GLP-1 levels in chronic cerulein-administered WT and CCR2-KO mice were comparably upregulated after cerulein administrations, GLP-1 receptor levels in pancreases of chronic cerulein-administered CCR2-KO mice were significantly lower than in paired WT mice. Nevertheless, a significantly higher hyperglycemia level in chronic cerulein-administered CCR2-KO mice was markedly restored by treatment with a GLP-1 analog to a level comparable to the paired WT mice. Collectively, the CCR2/CCL2 axis-mediated CD11b(+)-cell migration to the pancreas is critically involved in chronic pancreatitis-mediated hyperglycemia through the modulation of GLP-1 receptor expression and insulin secretion.

Biliary findings assist in predicting enlargement of intraductal papillary mucinous neoplasms of the pancreas.

BACKGROUND & AIMS: There is controversy over the optimal management strategy for patients with branch-duct type intraductal papillary mucinous neoplasms of the pancreas (BD-IPMNs), precursors to pancreatic cancer. We aimed to identify factors associated with the presence of BD-IPMNs and changes in their diameter. METHODS: Two separate analyses were conducted in a cohort of patients who underwent magnetic resonance cholangiopancreatography (MRCP) in a single year (2006). MRCP findings and clinical outcomes of these patients were followed for a maximum of 6 years. We evaluated initial MRCP findings and demographics associated with the presence of BD-IPMNs at baseline and increase in BD-IPMN diameter over time. RESULTS: During the follow-up period, 154 patients developed BD-IPMN and 322 patients did not. Older age, diabetes mellitus, gallbladder adenomyomatosis, and absence of gallstones were associated with the presence of BD-IPMNs at baseline. Increases in diameter of BD-IPMNs were associated with 3 baseline factors: BD-IPMN diameter greater than 17 mm, gallbladder adenomyomatosis, and a common bile duct diameter less than 5.5 mm. Patients with BD-IPMNs could be stratified into 4 groups with varying risk for the enlargement of BD-IPMNs over time: those with 3 risk factors (hazard ratio [HR], 11.4; 95% confidence interval [CI], 3.4-37.8), 2 risk factors (HR, 4.7; 95% CI, 1.7-12.8), or 1 risk factor (HR, 3.1; 95% CI, 1.2-8.2) compared with those without risk factors. CONCLUSIONS: For patients with BD-IPMNs, careful follow-up evaluation is particularly important for those with BD-IPMN >17 mm in size, common bile duct diameter <5.5 mm, or gallbladder adenomyomatosis.

CCL2-induced migration and SOCS3-mediated activation of macrophages are involved in cerulein-induced pancreatitis in mice.

BACKGROUND & AIMS: Acute pancreatitis is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with recruitment of leukocytes. We investigated the pathologic roles of innate immune cells, especially macrophages, in cerulein- and L-arginine-induced acute pancreatitis in mice. METHODS: Acute pancreatitis was induced by sequential peritoneal administration of cerulein to mice. We determined serum concentrations of amylase and lipase, pancreatic pathology, and features of infiltrating mononuclear cells. We performed parabiosis surgery to assess the hemodynamics of pancreatic macrophages. RESULTS: Almost all types of immune cells, except for CD11b(high)CD11c(-) cells, were detected in the pancreas of healthy mice. However, activated CD11b(high)CD11c(-) cells, including Gr-1(low) macrophages and Gr-1(high) cells (granulocytes and myeloid-derived suppressor cells), were detected in damaged pancreas after cerulein administration. CCL2(-/-) mice given cerulein injections developed significantly less severe pancreatitis, with less infiltration of CD11b(high)CD11c(-)Gr-1(low) macrophages, but comparable infiltration of myeloid-derived suppressor cells, compared with cerulein-injected wild-type mice. Parabiosis and bone marrow analyses of these mice revealed that the CD11b(high)CD11c(-)Gr-1(low) macrophages had moved out of the bone marrow. Furthermore, mice with macrophage-specific deletion of suppressor of cytokine signaling 3 given injections of cerulein developed less severe pancreatitis and Gr-1(low) macrophage produced less tumor necrosis factor-α than wild-type mice given cerulein, although the absolute number of CD11b(high)CD11c(-)Gr-1(low) macrophages was comparable between strains. Induction of acute pancreatitis by L-arginine required induction of macrophage migration by CCL2, via the receptor CCR2. CONCLUSIONS: Cerulein induction of pancreatitis in mice involves migration of CD11b(high)CD11c(-)Gr-1(low) macrophage from the bone marrow (mediated by CCL2 via CCR2) and suppressor of cytokine signaling 3-dependent activation of macrophage. These findings might lead to new therapeutic strategies for acute pancreatitis.

Side population of pancreatic cancer cells predominates in TGF-beta-mediated epithelial to mesenchymal transition and invasion.

We report here side population (SP) cells, a cancer stem cell enriched fraction from pancreatic cancer cell line, have enormous superior potential of the epithelial to mesenchymal transition (EMT), invasion, and metastasis. In an isolated SP cell culture, the cells rapidly expressed and up-regulated E-cadherin, an epithelial phenotypic marker, and the cells formed tightly contacted cell cluster, which is a representative epithelial phenotypic appearance. When the SP cells were incubated in the presence of TGF-beta, SP cells changed their shape into mesenchymal-like appearance including spindle shaped assembly. This alteration was associated with significant reduction of E-cadherin expression level. TGF-beta induced EMT-associated gene alteration such as reduction of E-cadherin mRNA and induction of Snail mRNA and matrixmetalloproteinase (MMP)-2 mRNA. Finally, SP cells exerted notable matrigel invasion activity in response to TGF-beta treatment, whereas MP cells did not respond to TGF-beta-mediated invasion. In conclusion, these results suggest that SP cells from pancreatic cancer cell line possess superior potentials of phenotypic switch, i.e., EMT/MET, micro-invasion, and in vivo metastasis, as compared to MP cells. Because micro-invasion and metastasis are key mechanisms of cancer malignant potential, SP cells would be the attractive target for preventing cancer progression.

Induction of matrix metalloproteinase-1 gene transcription by tumour necrosis factor alpha via the p50/p50 homodimer of nuclear factor-kappa B in activated human hepatic stellate cells.

BACKGROUND/AIMS: Liver injury results in the activation of hepatic stellate cells (HSCs), which in turn produce matrix metalloproteinase (MMP) in response to pro-inflammatory cytokines for tissue remodelling. This study explored the transcriptional induction of the MMP-1 gene by tumour necrosis factor-alpha (TNF-alpha) in HSCs. METHODS: The LI90 human HSC line was used in the present study. Gelatin zymography, enzyme-linked immunosorbent assay, Northern blotting and gene promoter-reporter assays were used to analyse the induction of MMP-1 protein, mRNA expression and gene transcription respectively. Deletional or site-directed mutations were introduced into the promoter region and transiently transfected into LI90 cells to determine the cis-acting elements necessary for TNF-alpha inducibility. Gel shift mobility assays were used to determine the transcriptional factors involved in the TNF-alpha responsiveness. RESULTS: TNF-alpha upregulated MMP-1 protein and mRNA expression in a dose-dependent manner. A time-course experiment revealed a rapid induction of MMP-1 mRNA expression after TNF-alpha treatment. Mutation in a putative nuclear factor (NF)-kappaB-binding site at -2541 bp almost completely abolished the TNF-alpha response to MMP-1 gene-promoter activity, suggesting transcriptional regulation of MMP-1 expression by TNF-alpha via this site. Electrophoretic mobility shift assay and supershift assays indicated that this transcriptional regulation was regulated via the p50/p50 homodimer of NF-kappaB. CONCLUSIONS: MMP-1 gene expression might be induced by TNF-alpha via the p50/p50 homodimer of NF-kappaB in activated human HSCs.

[A case of tuberculous lymphadenitis found as an abdominal mass penetrating the duodenum].

A 42-year-old woman who complained of epigastralgia was referred to our hospital because of an abdominal mass found by ultrasonography. CT and MRI scans revealed that the abdominal mass, 4 cm in diameter, located on the left side of the right kidney. Gastroduodenoscopy detected a deep ulcerative lesion covered with a yellowish coat in the second portion of duodenum. A sonolucent area at the bottom of the ulcerative lesion seemed to expand to the abdominal mass on ultrasonic endoscopy. Tuberculosis was one possible differential diagnosis. Pathological examination including Ziehl-Neelsen staining using biopsy specimens taken from the bottom of the ulcerative lesion did not show tuberculosis infection. However, polymerase chain reaction analysis using the biopsy specimens revealed that gene expression of tuberculosis was positive. We determined that tuberculous lymphadenitis penetrated the duodenum forming an ulcerative lesion in the duodenum. The administration of anti-tubercular medicine for 6 months male the abdominal mass disappeared and the duodenal ulcerative lesion formed an ulcer scar. We report here a case of tuberculous lymphadenitis penetrating the duodenum which was successfully treated by conservative therapy without surgical treatment.

Ampullary Adenoma Treated by Endoscopic Double-Snare Retracting Papillectomy.

We report herein improved methods for the safe and successful completion of endoscopic papillectomy (EP). Between January 2008 and November 2011, 12 patients underwent double-snare retracting papillectomy for the treatment of lesions of the major duodenal papilla. The main outcomes were en bloc resection rates, pathological findings, and adverse events. All of the patients (mean age, 60.1 years; range, 38 to 80 years) were diagnosed with ampullary adenoma by endoscopic forceps biopsies prior to endoscopic snare papillectomy. En bloc resection by double-snare retracting papillectomy was successfully performed for all lesions (median size, 12.3 mm), comprising six tubular adenomas, one tubulovillous adenoma, three cases of epithelial atypia, one hamartomatous polyp, and one case of duodenitis with regenerative change. Significant hemorrhage and pancreatitis were observed in one case after EP. Adenoma recurrence occurred in three patients during follow-up (median, 28.5 months) at a mean interval of 2 months postoperatively (range, 1 to 3 months). No serious adverse events were observed. Double-snare retracting papillectomy is effective and feasible for treating lesions of the major duodenal papilla. Further treatment experience, including a single-arm phase II study, needs to be accumulated before conducting a randomized controlled study.

Transforming growth factor ß and Ras/MEK/ERK signaling regulate the expression level of a novel tumor suppressor Lefty.

OBJECTIVES: The objectives of the present study were (i) to identify a novel tumor suppressor gene whose expression level was regulated by transforming growth factor (TGF-ß) and (ii) to evaluate the effect of Ras/MEK/ERK signaling on TGF-ß-dependent Lefty up-regulation. METHODS: Human pancreatic cancer cell lines were used. The effect of Ras/MEK/ERK pathway on TGF-ß-mediated Lefty up-regulation was tested by adding K-ras small interfering RNA, MEK inhibitor U0126, or extracellular signal-regulated kinase (ERK) inhibitor LY294002. RESULTS: Transforming growth factor ß upregulated Lefty messenger RNA levels within 6 of the 7 cell lines. Lefty exerts an antagonistic effect against the tumor-promoting molecule, Nodal, as recombinant Lefty suppressed Nodal-mediated proliferation. Interestingly, inhibition of the Ras/MEK/ERK pathway dramatically enhanced TGF-mediated Lefty up-regulation, suggesting that Ras/MEK/ERK signaling suppresses TGF-ß-Lefty pathway. CONCLUSIONS: Our data suggest that Lefty is a novel TGF-ß target molecule that mediates growth inhibition of pancreatic cancer cells. In addition, activation of the Ras/MEK/ERK pathway serves as a mechanism by which pancreatic cancer escapes from growth inhibition by the TGF-ß-Lefty axis. The results imply a novel therapeutic strategy for pancreatic cancer, that is, combination treatment with Ras/MEK/ERK inhibitors and TGF-ß.

Hemobilia due to biliary intraepithelial neoplasia associated with Zollinger-Ellison syndrome.

A 58-year-old man was transferred to us from his local hospital because of failure to control his gastrointestinal bleeding by endoscopic hemostasis. Abdominal imaging suggested a hypervascular tumor of the pancreatic head (36 mm diameter), and laboratory testing showed an elevated serum gastrin level (17,800 pg/mL). Gastroduodenal endoscopy revealed multiple duodenal ulcers and active bleeding from the ampulla of Vater. The selective arterial secretagogue injection test suggested a gastrinoma in the pancreatic head, but no gastrinoma in the pancreatic tail. The patient was diagnosed with solitary pancreatic head gastrinoma complicated by hemosuccus pancreaticus, and pancreaticoduodenectomy was performed. Intraoperatively, the diagnosis was changed to primary peripancreatic lymph node gastrinoma without pancreatic involvement. The gastrointestinal bleeding stopped postoperatively and serum gastrin levels returned to normal. Histological examination of the surgical specimens revealed a small submucosal gastrinoma in the duodenum (7 mm diameter). The final diagnosis was microgastrinoma of the duodenum with peripancreatic lymph node metastasis. The cause of bleeding from the ampulla of Vater was initially obscure, but eventually a hemorrhagic erosion with moderate atypia was found in the common bile duct, indicating biliary intraepithelial neoplasia (BilIN). This is the first report of hemobilia due to BilIN with gastrinoma.

Prediction of metabolic syndrome using artificial neural network system based on clinical data including insulin resistance index and serum adiponectin.

OBJECTIVE: This study aimed to predict the 6-year incidence of metabolic syndrome (MetS) using an artificial neural network (ANN) system and multiple logistic regression (MLR) analysis based on clinical factors, including the insulin resistance index calculated by homeostasis model assessment (HOMA-IR). DESIGN: Subjects were recruited from participants in annual health check-ups in both 2000 and 2006. A total of 410 Japanese male teachers and other workers at Keio University, 30-59 years of age at baseline, participated in this retrospective cohort study. MEASUREMENTS: Clinical parameters were randomly divided into a training dataset and a validation dataset, and the ANN system and MLR analysis were applied to predict individual incidences. The leave some out cross validation method was used for validation. RESULTS: The sensitivity of the prediction was 0.27 for the MLR model and 0.93 for the ANN system, while specificities were 0.95 and 0.91, respectively. Sensitivity analysis employing the ANN system identified BMI, age, diastolic blood pressure, HDL-cholesterol, LDL-cholesterol and HOMA-IR as important predictors, suggesting these factors to be non-linearly related to the outcome. CONCLUSION: We successfully predicted the 6-year incidence of MetS using an ANN system based on clinical data, including HOMA-IR and serum adiponectin, in Japanese male subjects.

IgG4-negative autoimmune pancreatitis with sclerosing cholangitis and colitis: possible association with primary sclerosing cholangitis?

We report a case of autoimmune pancreatitis (AIP) with cholangiography and histopathology showing features characteristic of primary sclerosing cholangitis (PSC) and colitis. A 55-year-old previously-healthy man was diagnosed with anti-nuclear antibody (ANA)-positive AIP according to the finding of serum biochemistry, abdominal US (ultrasonography), CT (computed tomography) and ERCP (endoscopic retrograde cholangiopancreatography). However, bead-like strictures of intrahepatic bile ducts were also found and liver tissue showed onion skin-like periductal fibrosis but no anti-IgG4-positive cells. In addition, colon fiberscopy showed a pancolitis similar to ulcerative colitis indicating that, in this case, there may be an association with PSC. Here, we report a rare case of IgG4-negative AIP with sclerosing cholangitis and colitis with many clinical features that support an association with PSC.

Deficiency of inducible nitric oxide synthase exacerbates hepatic fibrosis in mice fed high-fat diet.

The role of inducible nitric oxide synthase (iNOS) in the progression of fibrosis during nonalcoholic steatohepatitis remains to be elucidated. This study examined the role of iNOS in the progression of fibrosis during steatohepatitis by comparing iNOS knockout (iNOS(-/-)) and wild-type (iNOS(+/+)) mice that were fed a high-fat diet. Severe fatty metamorphosis developed in the liver of iNOS(+/+) and iNOS(-/-) mice. Fibrotic changes were marked in iNOS(-/-) mice. Gelatin zymography showed that pro MMP-2 and pro MMP-9 protein expressions were more highly induced in iNOS(+/+) mice than in iNOS(-/-) mice. Active forms of MMP-2 and MMP-9 were clearly present only in the liver tissue of iNOS(+/+) mice. In situ zymography showed strong gelatinolytic activities in the liver tissue of iNOS(+/+) mice, but only spotty activity in iNOS(-/-)mice. iNOS may attenuate the progression of liver fibrosis in steatohepatitis, in part by inducing MMP-2 and MMP-9 expression and augmenting their activity.

Pro-inflammatory cytokine-induced matrix metalloproteinase-1 (MMP-1) secretion in human pancreatic periacinar myofibroblasts.

Matrix metalloproteinases (MMPs) are the proteases involved in the degradation of the extracellular matrix. MMP-1 is thought to be one of the key enzymes in fibrolysis, a process closely related to tissue remodeling. In the present study, we investigated MMP-1 secretion from human pancreatic periacinar myofibroblasts in response to pro-inflammatory cytokines IL-1beta and TNF-alpha. We also attempted to clarify the intracellular signaling pathways mediating the cytokine-induced MMP-1 secretion. MMP-1 secretion was measured by an enzyme-linked immunosorbent assay. MMP-1 molecules were analyzed by Western blotting. MMP-1 mRNA expression was evaluated by Northern blotting. IL-1l and TNF-alpha stimulated the MMP-1 secretion in a dose- and time-dependent manner. Ninety percent of MMP-1 was secreted as inactive form (pro-MMP-1). The effects of IL-1beta and TNF-alpha were significantly inhibited by PD98059 MEK/ERK inhibitor). In contrast, SB203580 (p38 MAPK inhibitor), GF109203X (PKC inhibitor), and PDTC (NF-kappaB inhibitor) did not alter the MMP-1 secretion induced by IL-1beta and TNF-alpha. These effects were also observed at them RNA level. In conclusion, in human pancreatic periacinar myofibroblasts, MMP-1 secretion was regulated by the pro-inflammatory cytokines via the MEK/ERK cascade. Thus, human pancreatic periacinar myofibroblasts may play an important role in the remodeling of damaged pancreatic tissue in chronic pancreatitis via MMP-1 secretion.