Calcium concentration and artificial precipitates in human pancreatic juice.

We studied the role of the increase in the calcium concentration in pure pancreatic juice of alcoholic noncalcified chronic pancreatitis. Pure pancreatic juice was obtained endoscopically. The pancreatic juice from patients with chronic pancreatitis was adjusted to pH 7.5; then the calcium concentration was adjusted to 0.4, 2.9, 5.4, or 10.4 mmol/L. Artificial precipitates were produced by incubation of the samples at 37 degrees C for 6 hours. Proteins in the artificial precipitates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein patterns were compared with the patterns of natural protein plugs from patients with chronic pancreatitis. The amount of the precipitate increased as the added calcium increased. The protein patterns of SDS-PAGE of the artificial precipitates were similar to those of protein plugs. Albumin, a-amylase, lipase, trypsinogen, and chymotrypsinogen were identified by immunoblotting both in the precipitate and in the protein plug. The increased calcium concentrations in pancreatic juice induced the formation of precipitates whose protein composition was similar to that of protein plugs. An increased calcium concentration in human pancreatic juice may play an important role in the pathogenesis of protein plugs.

Enhanced interstitial collagenase (matrix metalloproteinase-13) production of Kupffer cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis.

Hepatic fibrosis results from an imbalance between fibrogenesis and fibrolysis in the liver. It remains uninvestigated whether Kupffer cells produce matrix metalloproteinase-13 (MMP-13), which mainly hydrolyzes extracellular matrix (ECM). We sought to determine the role of Kupffer cells in fibrogenesis/fibrolysis. In vivo, we used the rat model of pig serum-induced liver fibrosis. A subset was treated with gadolinium chloride (GdCl(3)), which specifically acts on Kupffer cells. Administration of GdCl(3) remarkably decreased the hydroxyproline content of the liver and increased the expression of MMP-13 mRNA in the liver without a difference in procollagen type I and tissue inhibitors of metalloproteinase-1 (TIMP-1) mRNA expression on Northern blot analysis with the elimination of ED2-positive cells. In vitro, addition of GdCl(3) to isolated Kupffer cells showed increased type I collagen-degrading activity in a dose-dependent manner as well as MMP-13 mRNA expression on Northern blot analysis. It is concluded that Kupffer cells are a major source of MMP-13 and modulation of Kupffer cells by GdCl(3) prevents liver fibrosis with increased expression of MMP-13 mRNA and protein, whereas procollagen type I and TIMP-1 mRNA, which encode two major effectors of fibrogenesis, were unchanged. This is the first report showing that Kupffer cells produce interstitial collagenase (MMP-13) resulting in the reduction of ECM. This discovery may provide new insights into therapy for hepatic fibrosis.

Detection of K-ras and p53 gene mutations in pancreatic juice for the diagnosis of intraductal papillary mucinous tumors.

The aim of this study was to investigate mutations of the K-ras oncogene and the p53 tumor suppressor gene in pancreatic juice and to evaluate our method for the diagnosis of intraductal papillary mucinous tumors (IPMT). Pancreatic juice was collected endoscopically from 12 patients with IPMT who underwent surgical resection (eight carcinomas and four adenomas) and eight cases without evident pancreatic diseases. DNA was extracted and both genes were examined by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. In addition, surgically resected specimens were analyzed for both genes by the same methods, and p53 overexpression was investigated immunohistochemically. K-ras point mutations were detected in pancreatic juice from all 12 patients (100%) and p53 mutations were detected in five of 12 (42%). They were detected not only in carcinoma but also in adenoma and there was no difference between the mutations detected in pancreatic juice and surgical specimens. No mutations were found in any cases without pancreatic diseases. These findings suggest that alterations of K-ras and p53 gene are common events in the development of IPMT and that genetic analysis of them in pancreatic juice can be a useful tool for the clinical diagnosis of IPMT before surgery.

Expression of vascular endothelial growth factor correlates with tumor progression in gallbladder cancer.

Angiogenesis must occur for malignant tumors to proliferate and vascular endothelial growth factor (VEGF) is now believed to be central to this process. Immunohistochemical staining for VEGF was performed on surgical resection specimens from 50 patients with gallbladder cancer. VEGF-positive rate was 38%. Comparison of clinicopathologic parameters between the groups with and without VEGF expression showed significant differences in tumor size, lymphatic invasion and disease stage. Survival rate was worse in the patients whose tumors demonstrated VEGF expression. It is suggested that VEGF is correlated with tumor progression and may be used as a prognostic indicator.

Two-dimensional zymography for analysis of proteolytic enzymes in human pure pancreatic juice.

Proteolytic enzymes in human pure pancreatic juice (PPJ), which was collected by cannulating the main pancreatic duct using endoscopy, were investigated by two-dimensional zymography (2-DZ). 2-DZ was carried out by combining isoelectric focusing (IEF) in the first dimension with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, using gels containing casein or gelatin as a substrate for the proteolytic enzymes. After electrophoresis, the gels were incubated in Triton X-100 followed by incubation at 37 degrees C in Tris buffer (pH 8.5) containing CaCl2. By staining the gels with Coomassie Brilliant Blue (CBB R-250), proteolytic enzymes were detected as clear spots and zones against a blue background. Proteinase inhibitors, such as a cysteine proteinase inhibitor (E-64), a metalloproteinase inhibitor (EDTA), and a serine proteinase inhibitor (Pefabloc SC), were added to PPJ in order to determine the types of proteinases. In patients with pancreatic cancer, spots of molecular weight (Mr) 70,000 and isoelectric points (pI) 5.3-5.5 were clearly detected on the gels containing casein and gelatin, while these spots were not detected in the PPJ from healthy subjects. The proteolytic activities of these spots were strongly inhibited by EDTA and Pefabloc SC but not E-64. These results suggest that the spots of Mr 70,000 and pI 5.3-5.5 in PPJ of pancreatic cancer might be matrix metalloproteinase 2, which is a candidate for tumor-associated proteinase. 2-DZ proved to be a tool for analysis of proteolytic enzymes in PPJ and for the clinical diagnosis of pancreatic cancer.

p53 mutations in two patients with intraductal papillary adenoma of the pancreas.

There has been no report on p53 gene mutation in benign human pancreatic intraductal tumors. We examined pancreatic juice and tissue specimens from two patients with intraductal papillary adenoma of the pancreas by polymerase chain reaction-single-strand conformation polymorphism analysis and direct sequencing and found point mutations of p53 gene resulting in amino acid substitutions in exons 6 and 8. Thus, p53 gene mutation may be an early event in the neoplastic process of some pancreatic intraductal tumors and may play an important role in tumorigenesis.