Quantitative analysis of carcinoembryonic antigen messenger RNA in peripheral venous blood and portal blood of patients with pancreatic ductal adenocarcinoma.

One major therapeutic failure of pancreatic adenocarcinoma treatment is metastasis to the liver. To screen patients with high risk for such hematogenous dissemination, we previously developed a very sensitive system to detect carcinoembryonic antigen (CEA) in blood. For a more practical application, we improved this system by making it quantitative and capable of analyzing both preoperative peripheral blood and intraoperative portal blood for the presence of CEA mRNA. CEA mRNA was not detected in the peripheral venous blood of any of the three patients examined, but it was identified in the portal blood without fail. In addition, the quantities of CEA mRNA identified in the portal blood before and after pancreatectomy were different. This study suggests that analysis of the portal blood seems to be important for the precise evaluation of hematogenous dissemination and of the pathophysiology of pancreatic ductal adenocarcinoma.

Auto-induction and growth stimulatory effect of betacellulin in human pancreatic cancer cells.

Betacellulin (BTC) was identified in mouse pancreatic beta cell tumors as a member of the epidermal growth factor (EGF) family, and was found to bind and activate the EGF receptor. BTC is also expressed in some human malignancies and may have an important role in tumor growth progression. We examined whether BTC and EGF have a growth stimulatory effect on human pancreatic cancer cell lines both in vitro and in vivo. We also investigated the BTC expression and autonomous induction of BTC in pancreatic cancer cells. in vitro, both BTC and EGF had almost the same proliferative effect on Panc-1, MIA PaCa-2 and AsPC-1. in vivo, in a Panc-1 inoculated athymic mice model, BTC-treated tumors grew approximately five times larger than in control. Immunocytochemistry showed that BTC expression occurred in three pancreatic cancer cell lines, with MIA PaCa-2 showing the strongest intensity. Semi-quantitative RT-PCR of MIA Paca-2 showed that mRNA levels of BTC gradually increased after treatment with 1 nM BTC. Immunocytochemistry also demonstrated that the intensity of BTC-like immunoreactivity was increased when treated with 1 nM BTC but was reduced after treatment with 100 nM of AG1478, an EGF receptor tyrosine kinase inhibitor. BTC has thus a significant growth stimulatory effect on pancreatic cancer cells and might function as an autocrine and paracrine growth factor. BTC expression in pancreatic cancer cells is, at least in part, controlled by an auto-induction mechanism.

Effect of a new immunosuppressive agent, FTY720, on survival of islet allografts.

A newly developed immunosuppressant, FTY720, has a unique mechanism that is quite different from those of conventional immunosuppressants, and is presumed to be mediated through decreases in the number of peripheral lymphocytes, especially helper T cells. This study was performed to ascertain whether this innovative drug could prolong islet allograft survival. The donors were inbred Lewis rats and the recipients were ACI rats rendered hyperglycemic with intravenous streptozotocin. In the study group, FTY720 dissolved in distilled water was orally administered at a dose of 5 mg/kg to the recipient ACI rats 1 day before and on the day of grafting. In the control group, only distilled water was orally administered to the recipient ACI rats on the day before and the day of grafting. Two thousand islets were transplanted into the portal vein of the recipient rats in the study and control groups immediately after isolation. The graft survival time in the study group was significantly longer than that in the control group, indicating that FTY720 retains a potent effect on the prolongation of islet allograft survival. FTY720 could become a useful immunosuppressant for future clinical islet allotransplantation.

Improved large-scale isolation of breeder porcine islets: possibility of harvesting from nonheart-beating donor.

To establish a large-scale isolation procedure for adult porcine islets usable as a donor source for xenotransplantation and as a model of human islet isolation, we improved several characteristics of the conventional isolation procedure. At a slaughterhouse we first selected a breeder pig over 1.5 years old (and over 200 kg in weight) with warm ischemic time (WIT) of 15 +/- 2 minutes as nonheart-beating donors. Then, we made a special enzymic mixture that consisted of collagenase S-1 (260 U/mg, NittaZelatin, Japan), collagenase P (1.86 U/ml Lyo Boehringer-Mannheim, USA), DNase (Sigma, St. Louis, Mo), Disparse (NittaZelatin, Japan), and protease inhibitor (Sigma). Third, this mixture was injected very gently into the pancreatic duct at the time of pancreatic harvesting. To prevent overdigestion of the pancreas, the mixture was first cooled to less than 10 degrees C. Fourth, during the warm digestion of pancreas, the pancreas with the enzymic mixture was quietly put in a water bath at 37 degrees C without mechanical shaking. Fifth, we purified the islets with a COBE 2991 cell processor by the Dextran 70 gradient method, because Dextran 70 is very cheap and has the same purification effect as the Ficoll gradient. The results of 10 consecutive breeder porcine islet isolations are reported. The total yield of isolations of islets over 50 microm in the longest diameter after staining with Dithizone (DTZ) was 85,900 +/- 19,954 islets, 291,667 +/- 240,452 IEQ (2,900 +/- 2,324 IEQ/g). The purity of the isolated islets was very high: 90.2 +/- 3.8%. Glucose stimulation during in vitro incubation induced significant insulin release from isolated breeder porcine islets. In two of the diabetic rats receiving encapsulated islets grafts using a mesh-reinforced polyvinyl alcohol hydrogel bag (MRPB), a prominent reduction in serum glucose levels (less than 200 mg/dL) persisted for 13 and 19 days, respectively, after intraperitoneal xenotransplantation islets without immunosuppression. In conclusion, we succeeded in a more efficient and less-expensive isolation of a large amount of adult porcine islets from a nonheart-beating donor.

Effects of cyclosporine and tacrolimus (FK 506) on acute pancreatitis in mice.

OBJECTIVE: To use mice to examine the effects of cyclosporine and tacrolimus (FK 506) on two forms of acute pancreatitis often seen after clinical organ transplantation. METHODS AND DESIGN: In the first experiment, male CD-1 mice received cyclosporine (10 mg/kg), tacrolimus (0.32 mg/kg), or saline solution (control) subcutaneously once a day for 10 days. On the 11th day, acute edematous pancreatitis was induced by ceruletide (cerulein). In the second experiment, female ICR mice were fed with a choline-deficient, ethionine-supplemented (CDE) diet for 72 hours to induce necrotizing pancreatitis. After 30 hours on the CDE diet, the mice received cyclosporine (10 mg/kg), tacrolimus (0.32 mg/kg), or saline solution (control) subcutaneously twice daily for 3 days. RESULTS: The pancreatic dry-to-wet weight ratios after ceruletide injections significantly decreased in mice treated with cyclosporine but did not with tacrolimus. Cyclosporine also significantly increased serum amylase levels, but tacrolimus did not. Cyclosporine or tacrolimus alone did not produce pancreatitis. In the CDE diet groups there was a significant difference in survival among the cyclosporine-treated, the tacrolimus-treated, and the control groups. CONCLUSIONS: Cyclosporine or tacrolimus given alone does not induce acute pancreatitis. In contrast, cyclosporine can adversely affect the course of acute edematous pancreatitis, and both immunosuppressants may worsen the survival of mice with acute hemorrhagic necrotizing pancreatitis. This study also demonstrated that the deteriorating effect of tacrolimus is less potent than that of cyclosporine.

Effects of synthetic kassinin on splanchnic circulation and exocrine pancreas in dogs.

Effects of intravenously administered synthetic kassinin on splanchnic circulation and exocrine pancreatic secretion were examined in six anesthetized dogs. Kassinin caused dose-related increases in the blood flow in superior mesenteric artery and portal vein, and produced an initial increase followed by a decrease in pancreatic blood flow, but did not affect the exocrine pancreatic secretion. This study demonstrates that kassinin affects splanchnic blood flow in dogs, and suggests that kassinin or a kassinin-like substance functions as a neuropeptide controlling the splanchnic circulation in mammalian species.

Effect of synthetic neuromedin-N, a novel neurotensin-like peptide, on exocrine pancreatic secretion and splanchnic blood flow in dogs.

Neuromedin-N, a novel neurotensin-like peptide, has recently been identified from porcine spinal cord by using a bioassay for a stimulatory effect on guinea pig ileum. The amino acid sequence of the peptide has been determined to be Lys-Ile-Pro-Tyr-Ile-Leu, which is quite homologous to the COOH-terminal sequence of neurotensin. In this study, the effect of neuromedin-N on pancreas and splanchnic blood flow was investigated in eight dogs. Intravenous injections of graded doses of synthetic neuromedin-N caused a dose-dependent decrease of systemic arterial pressure and a dose-dependent increase in both portal and superior mesenteric arterial blood flow, which were measured with transit time ultrasonic flow meter. Volume and protein output of the pancreatic juice were also increased significantly by Neuromedin-N. Pancreatic capillary blood flow measured with laser Doppler flowmetry was increased in a dose-related manner. The present study first demonstrated that neuromedin-N retains a potent stimulatory effect on the pancreas and splanchnic circulation, indicating that this peptide is one of the biological active forms of neurotensin-like peptide in mammals. This study also leads to the suggestion that this peptide possesses physiological significance as a novel neuropeptide.

Inhibitory action of islet amyloid polypeptide and calcitonin gene-related peptide on release of insulin from the isolated perfused rat pancreas.

Islet (or insulinoma) amyloid polypeptide (IAPP) is a 37-residue peptide recently purified from amyloid deposits in the pancreas of patients with type 2 diabetes and from amyloid deposits of a human insulinoma. IAPP immunoreactivity has been identified in islet B cells of diabetic and nondiabetic humans. IAPP is structurally similar to calcitonin gene-related peptide (CGRP). The purpose of this study was to examine the effects of IAPP and CGRP on glucose- and carbachol-stimulated release of insulin and pancreatic polypeptide (PP) from the isolated perfused rat pancreas. IAPP and CGRP, at 10(-7) M, failed to inhibit glucose-stimulated (16.7 mM) release of insulin. At the same concentration, however, IAPP significantly (p less than 0.05) inhibited carbachol-stimulated (10(-7) M) release of insulin by 30%, and CGRP significantly inhibited carbachol-stimulated release of insulin by 33% when compared with the control group. IAPP also significantly decreased carbachol-stimulated release release of PP. IAPP and CGRP, at 10(-8) M, did not inhibit carbachol-stimulated release of insulin and PP. These results suggest that IAPP and CGRP may have roles in the regulation of secretion of insulin. IAPP may inhibit secretion of insulin, at least in part, by blocking cholinergic mechanisms.

Microcirculatory response of the pancreas to feeding, sham feeding, and truncal vagotomy in conscious dogs.

In this study, changes in the microcirculatory dynamics of the pancreas in response to normal feeding, sham feeding, and truncal vagotomy were investigated to elucidate the involvement of a neural mechanism in the physiological modulation of pancreatic blood flow in the conscious state. Continuous measurement of changes in the microcirculation of the pancreas was performed in conscious dogs by the thermoelectric method. A meat meal was given to six normal dogs, seven dogs constructed with external esophagostomy (sham feeding), and four dogs with truncal vagotomy. In response to normal feeding, pancreatic blood flow attained the peak increase of 65.2 +/- 6.2%, showing a significant and biphasic response until approximately 120 min. After sham feeding, pancreatic blood flow was significantly increased with peak values of 89.0 +/- 19.0%, but thereafter showed a rapid decrease, returning to the basal level already at 7.2 +/- 1.1 min. Although truncal vagotomy significantly and greatly reduced the peak increase of pancreatic blood flow to 28.2 +/- 5.1%, blood flow showed still a significant and sustained elevation above basal. This study provides evidence for the involvement of the neural mechanism in the physiological modulation of the microcirculation of the pancreas in the conscious state. The results strongly suggest that the cephalic phase of the increase in pancreatic blood flow is vagally mediated.

Chronic effect of oral cholestyramine, a bile salt sequestrant, and exogenous cholecystokinin on insulin release in rats.

Oral cholestyramine, a bile salt sequestrant, stimulates pancreatic exocrine secretion and growth chiefly by increasing cholecystokinin (CCK) release. In this report, we examine pancreatic insulin content and insulin release from the isolated perfused pancreas in rats given oral cholestyramine (4%, wt/wt) or subcutaneous CCK-8 (1 micrograms/kg every 8 h) for 2 weeks. Cholestyramine significantly increased pancreatic weight by 32%. CCK administration significantly increased pancreatic weight by 15%. Total pancreatic content of protein and DNA were also increased significantly by cholestyramine and pancreatic protein content was increased significantly by CCK administration. Total pancreatic insulin content was not affected by cholestyramine or CCK. Both cholestyramine and CCK significantly increased the first phase of glucose (8.4 mM)-stimulated release of insulin [mean insulin output (ng/min): control, 2.0 +/- 0.1; cholestyramine, 2.7 +/- 0.2; CCK, 2.6 +/- 0.2]. Cholestyramine also significantly enhanced the second phase of glucose-stimulated release of insulin. Insulin release stimulated by CCK-8 (10(-10) M) was not affected by oral cholestyramine or CCK treatment. These findings indicate that oral cholestyramine and exogenous CCK have a stimulatory effect on beta cell function. Since pancreatic insulin content was not affected by cholestyramine and CCK treatment, cholestyramine and CCK may increase the sensitivity of beta cells to glucose. The absence of a stimulatory effect of cholestyramine and CCK administration on insulin release in response to CCK-8 may be related to a down-regulation of CCK receptors on beta cells.

Synthetic neuromedin C stimulates exocrine pancreatic secretion in dogs and rats.

We have examined the effect of neuromedin C on exocrine pancreatic secretion both in vivo and in vitro, and compared its bioactivity with those of related peptides. In anesthetized dogs, neuromedin C caused a dose-dependent initial reduction of pancreatic blood flow and an increase in secretin-stimulated exocrine pancreatic secretion, and had almost the same potency as gastrin-releasing peptide (GRP) in decreasing pancreatic blood flow. A potent stimulatory effect on exocrine pancreatic secretion was found in conscious dogs accompanied by a significant elevation in the circulating cholecystokinin (CCK) levels. In isolated rat pancreatic acini, amylase was released dose-dependently in response to neuromedin C. This study demonstrates that neuromedin C (a smaller molecular form of GRP) possesses potent bioactivity on exocrine pancreas and suggests that two factors may be involved in the mechanism by which this peptide effects exocrine secretion, namely; direct stimulation on acinar cells and stimulation of CCK release.

Effect of synthetic human cholecystokinin-33 on pancreatic blood flow in dogs.

We have examined the effect of synthetic human cholecystokinin (CCK-33 and CCK-8) on pancreatic blood flow and protein output in anesthetized dogs. Human CCK-33 and CCK-8 increased pancreatic blood flow and protein output in a dose-related manner. There were no significant differences in increasing pancreatic blood flow between human CCK-33 and CCK-8, and increases in blood flow were closely related to the increase of the pancreatic enzyme secretion. L-364,718 (20 nmol/kg) caused a potent inhibition of CCK-stimulated pancreatic blood flow as well as protein output. The degree of inhibition by L-364,718 was dependent on the amount of CCK infused. This study demonstrates that increasing effect on pancreatic blood flow may be one of the biological actions of CCK mediated via CCK receptor. The CCK-33, one of longer molecular forms of CCK, is an important biological stimulator of pancreatic blood flow as well as of exocrine pancreatic secretion.

Experimental hybrid islet transplantation: application of polyvinyl alcohol membrane for entrapment of islets.

In this study, we first examined in vitro a polyvinyl alcohol membrane to be used to contain hybrid islet cells, and second we tested a bioartificial pancreas with entrapment of pancreatic islets in polyvinyl alcohol membrane in rats with experimentally induced diabetes. The permeability of the polyvinyl alcohol membrane to different substances was studied in a two-cell chamber system. Glucose, insulin, and nutrients passed through the membrane easily, whereas the passage of immunoglobulin G was completely prevented, indicating that this membrane could be effective in protecting the bioartificial pancreas from immunorejection. Approximately 2,000 islets collected from three Sprague-Dawley rats were enclosed in a mesh-reinforced polyvinyl alcohol tube and transplanted into the peritoneal cavity of six Wistar rats with streptozotocin-induced diabetes. Their nonfasting serum glucose levels were significantly decreased for at least 12 days. Six diabetic rats receiving intraperitoneal transplantation of free islets without the tube showed a slight but significant decrease in nonfasting serum glucose levels for only 3 days. One diabetic rat with transplantation of the bioartificial pancreas had a significant and sustained decrease in nonfasting glucose levels from pretransplanted levels of 440-500 mg/dl to a mean value of 162 +/- 13 mg/dl for over 3 months without immunosuppression. The bioartificial pancreas was then removed, and glucose levels gradually increased to over 500 mg/dl. The results of the present study suggest that a bioartificial pancreas with entrapment of islets in a polyvinyl alcohol membrane could be a promising therapeutic approach to diabetes mellitus.

Effect of synthetic neuromedin U-8 and U-25, novel peptides identified in porcine spinal cord, on splanchnic circulation in dogs.

Two novel peptides which exert a potent stimulant effect on rat uterus smooth muscle have recently been identified in porcine spinal cord. These peptides designated neuromedin U-8 and U-25 have been reported to exert a hypertensive effect in rats. But further biological activities are not known. In the present study, the effect of these peptides on blood flow in portal vein, superior mesenteric artery and pancreatic tissue and on blood pressure were examined in dogs, utilizing recently developed ultrasonic transit time volume flow meter and laser Doppler flow meter. Neuromedin Us potently reduced blood flow in superior mesenteric artery. The minimum reductions could be observed even at very small doses of neuromedin U-25 (32 fmol/kg) and U-8 (90 fmol/kg), while the maximal reductions of 48.4 and 51.0% were attained at the doses of 320 pmol/kg (U-25) and 900 pmol/kg (U-8), respectively. These peptides also reduced portal vein blood flow, and the maximal reductions of 42.1 and 37.2% were attained at the doses of 32 pmol/kg (U-25) and 90 pmol/kg (U-8), respectively. On the other hand, blood flow in pancreatic tissue increased slightly with the maximal increases of 13.8% at 3.2 pmol/kg (U-25) and 11.8% at 9 pmol/kg (U-8), respectively. The maximal increases of blood pressure were 5.2% at 320 pmol/kg (U-25) and 4.3% at 90 pmol/kg (U-8). Furthermore, neither neuromedin U-25 nor U-8 influenced the axillary artery blood flow, suggesting their selective effect on splanchnic blood flow. Because of the potent and probably selective activity on splanchnic circulation, neuromedin U-25 and U-8 may well be recognized as physiologically significant novel neuropeptides or hormones.

Effect of human epidermal growth factor (hEGF) on splanchnic circulation in dogs.

The effect of intravenous administration of human epidermal growth factor on the splanchnic blood flows was examined in anesthetized dogs, using an ultrasonic transit-time volume flow meter. Human epidermal growth factor (0.1, 0.5 and 1 microgram/kg) significantly increased blood flows in the portal vein (36.9 +/- 7.4% at 1 microgram/kg) and the superior mesenteric artery (49.0 +/- 16.8% at 1 microgram/kg). Systemic blood pressure monitored simultaneously was significantly decreased (8.4 +/- 1.2% at 1 microgram/kg). This study is the first to demonstrate that intravenous administration of epidermal growth factor increases the portal venous blood flow.

Effect of synthetic physalaemin on splanchnic circulation in dogs.

Physalaemin has been reported as one of the most potent vasodilator and hypotensive peptides (1-4). In spite of these studies, however, the effect of the peptide on splanchnic circulation is not known precisely. In the present study, the effect of synthetic physalaemin on superior mesenteric arterial blood flow, portal venous blood flow and pancreatic capillary blood flow was investigated in dogs. Dose dependent increases of superior mesenteric arterial blood flow and portal venous blood flow were induced in response to physalaemin (0.1-10.0 ng/kg). Superior mesenteric arterial blood flow and portal venous blood flow attained maximal increases of 77 +/- 8.9% and 70 +/- 8.6%, respectively, at a dose of 5 ng/kg. Physalaemin caused a dose-related decrease in systemic arterial blood pressure. Pancreatic capillary blood flow did not show significant change with the administration of physalaemin. These data suggest that physalaemin may play some physiological roles in the regulation of splanchnic circulation.

Effects of endothelin on microcirculation of the pancreas.

Endothelin, a newly described endothelial-derived peptide, has potent vasoconstrictive properties and has been speculated to play a physiological role in the regulation of blood flow in some organs. The present study was designed to evaluate the effects of endothelin-1, endothelin-2 and endothelin-3 on the pancreatic microcirculation. Pancreatic tissue blood flow was measured by a laser Doppler flow meter in anesthetized dogs and endothelin-1, endothelin-2 or endothelin-3 was injected intravenously in graduated doses. Endothelins induced dose-dependent decreases in pancreatic tissue blood flow. Endothelin-1, endothelin-2 and endothelin-3 at a dose of 100 pmol/kg reduced pancreatic blood flow by 45.4%, 19.6% and 51.9%, respectively, whereas systemic arterial blood pressure was not significantly affected. When endothelin-3 was administered at a dose of 1000 pmol/kg, pancreatic blood flow was decreased by 73.5% with a concomitant increase of systemic arterial blood pressure by 17.6%. Endothelins potently decreased pancreatic tissue blood flow, suggesting a possible role of these agents in regulating the pancreatic microcirculation.

Results of pancreatectomy with radiation therapy for pancreatic cancer.

BACKGROUND/AIMS: Surgical resection provides the only chance for long-term survival for adenocarcinoma of the pancreas. However, a major problem of surgery appears to be post-resection recurrence. The purpose of the current study was to evaluate the efficacy of intraoperative radiotherapy (IORT) and/or external beam radiotherapy (EBRT) in combination with resective surgery. METHODOLOGY: 86 patients received radiotherapy combined with resection surgery and 64 patients received surgery alone. Of 86 patients, 37 received postoperative EBRT, 14 received IORT, and 31 received both. The primary outcome variable analyzed was survival. RESULTS: The survival curve in patients who received radiotherapy combined with resective surgery was significantly better than that in patients who only received surgery (MST: 12.8 vs. 7.9 months). The number of patients in both groups was equally populated on the basis of potentially prognostic factors such as gender and age of patients, type of resection, vascular resection, location of the tumor, and pTNM classification. Among the subgroups of the patients, adjuvant radiotherapy showed significant survival benefit in the patient group with pT3 tumor, pN positive and R0 surgery. CONCLUSIONS: Adjuvant radiotherapy including TORT with resection surgery for adenocarcinoma of the pancreas is accomplished with acceptable risk and provides significant survival benefit.

Insulin release from a bioartificial pancreas using a mesh reinforced polyvinyl alcohol hydrogel tube. An in vitro study.

Islet transplantation with a bioartificial pancreas is a potential alternative to whole pancreas transplantation. The authors constructed a bioartificial pancreas using mesh reinforced polyvinyl alcohol hydrogel tubes (MRPT), in an attempt to clarify the in vitro responsiveness to glucose of islets seeded in the MRPT. When the MRPT were perfused in a small chamber with buffer containing 3.3 mmol or 16.7 mmol glucose, insulin release from the MRPT began to increase at 9 +/- 3 min, reaching a plateau at approximately 40 min after the glucose concentration in the perfusate increased from 3.3 to 16.7 mmol. When MRPT seeded with islets were subjected to static incubation in buffer containing 3.3 mmol or 16.7 mmol glucose, insulin release from the MRPT remained elevated for 3 hr of high glucose stimulation, the amount of secreted insulin depending upon the number of islets seeded. Although pre incubation of semipermeable membranes in culture medium containing fetal bovine serum prior to seeding with islets has recently been reported to improve insulin release, the authors found that such pre treatment of the MRPT did not have a beneficial effect. Their in vitro findings in this study suggest that the bioartificial pancreas using MRPT could be a promising therapeutic approach to human diabetes mellitus.

[Study on splanchnic circulation: measurement of the liver blood flow].

In anesthetized patients during abdominal surgery, hepatic artery and portal vein flows were measured simultaneously utilizing an ultrasonic transit-time volume flowmeter. The total hepatic blood flow was 994.6 +/- 52.4 ml/min. The hepatic artery flow and the portal vein flow were 260.0 +/- 23.8 ml/min and 730.8 +/- 41.3 ml/min, respectively. The ratio of hepatic artery flow to portal vein flow was 0.37 +/- 0.04. A significant increase in hepatic artery flow (p less than 0.01) followed portal vein occlusion, whereas no significant change was observed in portal vein flow after hepatic artery occlusion. Common hepatic artery occlusion resulted in a significant decrease in hepatic artery flow (p less than 0.05), but no significant change was observed in portal vein flow. The present study firstly demonstrated that ultrasonic transit-time volume flowmeter is a device to quantitatively assess hepatic artery and portal vein flows with good reproducibility and stability in human subjects. This easy and simple technique seemed to have wide clinical application to abdominal surgery and would have a promising in studying splanchnic blood flows in various situations such as in cases of hepatectomy and portal hypertension.