Autocrine loop between TGF-beta1 and IL-1beta through Smad3- and ERK-dependent pathways in rat pancreatic stellate cells.

Pancreatic stellate cells (PSCs) are activated during pancreatitis and promote pancreatic fibrosis by producing and secreting ECMs such as collagen and fibronectin. IL-1beta has been assumed to participate in pancreatic fibrosis by activating PSCs. Activated PSCs secrete various cytokines that regulate PSC function. In this study, we have examined IL-1beta secretion from culture-activated PSCs as well as its regulatory mechanism. RT-PCR and ELISA have demonstrated that PSCs express IL-1beta mRNA and secrete IL-1beta peptide. Inhibition of TGF-beta(1) activity secreted from PSCs by TGF-beta(1)-neutralizing antibody attenuated IL-1beta secretion from PSCs. Exogenous TGF-beta(1) increased IL-1beta expression and secretion by PSCs in a dose-dependent manner. Adenovirus-mediated expression of dominant-negative (dn)Smad2/3 expression reduced both basal and TGF-beta(1)-stimulated IL-1beta expression and secretion by PSCs. Coexpression of Smad3 with dnSmad2/3 restored IL-1beta expression and secretion by PSCs, which were attenuated by dnSmad2/3 expression. In contrast, coexpression of Smad2 with dnSmad2/3 did not alter them. Furthermore, inhibition of IL-1beta activity secreted from PSCs by IL-1beta-neutralizing antibody attenuated TGF-beta(1) secretion from PSCs. Exogenous IL-1beta enhanced TGF-beta(1) expression and secretion by PSCs. IL-1beta activated ERK, and PD-98059, a MEK1 inhibitor, blocked IL-1beta enhancement of TGF-beta(1) expression and secretion by PSCs. We propose that an autocrine loop exists between TGF-beta(1) and IL-1beta in activated PSCs through Smad3- and ERK-dependent pathways.

[Ultrasonography with liquid type CalorieMate for gallbladder motility].

We usually use yolks to assess gallbladder motility by ultrasonography. In this study, we evaluated liquid type CalorieMate as a simple oral stimulus instead of yolks. The volunteers (n = 27) underwent ultrasonography before, 30 min after, and 60 min after taking liquid type CalorieMate. Gallbladder volume and the ejection fraction were measured by ellipsoid method. The mean fasting gallbladder volume, 30-min ejection fraction, and 60-min one were 13.5 ml, 53%, and 62%, respectively. These results were similar to the previous reports by yolks. If the fasting volume is lower than 4ml, they should take re-examination after longer fast to reduce the influence of the dinner the day before the exam. In conclusion, liquid type CalorieMate is useful stimulus to assess gallbladder motility.

Gastrointestinal angiodysplasia in a patient with type 2 von Willebrand's disease and analysis of exon 28 of the von Willebrand factor gene.

Although the association between gastrointestinal angiodysplasia and von Willebrand's disease has been suggested, molecular mechanisms involved in the formation of angiodysplasia in patients with von Willebrand's disease remained undetermined. We examined exon 28 of the von Willebrand factor gene in a patient with both von Willebrand's disease and recurrent bleeding from angiodysplasia in the duodenum as well as his father's, and found a point mutation, C 3916-->T (amino acid substitution; Arg 543-->Trp), in the A1 domain of the von Willebrand factor gene. This mutation was identical with a previously reported mutation in a patient with von Willebrand's disease complicated with gastrointestinal angiodysplasia.

Angiotensin II stimulates DNA synthesis of rat pancreatic stellate cells by activating ERK through EGF receptor transactivation.

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [3H]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation-ERK activation pathway.

Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions.

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-beta(1) (TGF-beta(1)) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-beta(1) mRNA expression level or the amount of autocrine TGF-beta(1) peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-beta(1) increased TGFbeta(1) mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-beta(1), and consequently attenuated TGF-beta(1) enhancement of its own mRNA expression in PSCs. We propose that TGF-beta(1) differentially regulates PSC activation, proliferation, and TGF-beta(1) mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.

Design of long-acting formulation of protein drugs with a double-layer structure and its application to rhG-CSF.

In order to design a sustained-release formulation of protein drugs characterized by excellent long-acting properties without an initial burst, a new double-layer minipellet (DL-MP) in which the lateral side of a matrix-type sustained-release formulation 'minipellet' using collagen as a carrier was coated with collagen was designed, and its performance was evaluated. In a DL-MP using bovine serum albumin (BSA) as a model drug, the initial burst observed with a single-layer minipellet (SL-MP) was effectively inhibited in an in vitro release test, and the addition of additives such as chondroitin sulfate (CS) permitted control of release rate. This formulation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was then prepared, and its characteristics were determined in normal rats. It was found that blood rhG-CSF concentration was maintained for about 1 week after administration of a DL-MP with additional CS, with persistent increase in white cell count. The results of this study indicated that DL-MP was useful as a long-acting formulation of rhG-CSF characterized by excellent long acting properties without an initial burst.

Biliary guidewire facilitates bile duct biopsy and endoscopic drainage.

BACKGROUND: The introduction of a guidewire through bile duct strictures may facilitate transpapillary bile duct biopsy and subsequent biliary drainage. METHODS: Endoscopic bile duct biopsy was attempted in 61 patients with bile duct strictures. After the introduction of a guidewire into the bile duct, biopsy forceps were inserted via the papilla. Both devices were inserted through the working channel (3.2 mm in diameter) of a conventional duodenoscope. After the procedure, an endoscopic naso-biliary drainage catheter was advanced along the guidewire. The success rate of inserting the biopsy forceps, the sensitivity of the biopsy, and the success rate of endoscopic biliary drainage after the biopsy were analyzed prospectively. RESULTS: The final diagnosis was malignant strictures in 50 patients and benign strictures in 11. The success rate of inserting biopsy forceps without performing endoscopic papillary balloon dilation was 85%. The sensitivity of the biopsy for primary bile duct cancer (83%) was significantly higher (P < 0.05) than that of pancreatic cancer (47%). All patients had successful endoscopic biliary drainage after the procedure. CONCLUSION: A previously placed guidewire facilitates insertion of biopsy forceps and endoscopic biliary drainage. The histological diagnosis of cancer is more likely with bile duct cancer than with pancreatic cancer.