Clinical studies on duct occlusion with prolamine

To prevent recurrence the pancreatic duct occlusion firstly was employed in the treatment of chronic pancreatitis. In combination with a Whipple's procedure a duct occlusion of the situ remaining pancreatic tail was performed in 141 cases since 1978. In the late follow-up only one patient developed a recurrent pancreatitis. In patients with pre-operatively normal glucose tolerance no post-operative diabetes was induced by this method. On the basis of these findings ductal occlusion also was carried out in a case of simultaneous pancreas-kidney-transplantation. Whereas the kidney did not function satisfactorily, the grafted pancreas with obstructed duct showed good endocrine function for over 9 weeks. The patient, who previously required injections of 60 IU insulin daily, needed no insulin during this period. The serum insulin level in several determinations was markedly above that of healthy normal people (65,3 microU/ml). Because of an infection which was uncontrollable under immunosuppression, the pancreas had to be removed again.     

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.     

Mesenchymal epimorphin is important for pancreatic duct morphogenesis

Epithelial-mesenchymal interactions are crucial for the proper development of many organs, including the pancreas. Within the pancreas, the ducts are thought to harbor stem/progenitor cells, and possibly to give rise to pancreatic ductal carcinoma. Little is known about the mechanism of formation of pancreatic ducts in the embryo. Pancreatic mesenchyme contains numerous soluble factors which help to sustain the growth and differentiation of exocrine and endocrine structures. Here, we report that one such morphoregulatory mesenchymal protein, epimorphin, plays an important role during pancreatic ductal proliferation and differentiation. We found that epimorphin is expressed in pancreatic mesenchyme during early stages of development, and at mesenchymal-epithelial interfaces surrounding the ducts at later stages. Strong upregulation of epimorphin expression was seen during in vitro pancreatic duct differentiation. Similarly, in vitro pancreatic duct formation was inhibited by a neutralizing antibody against epimorphin, whereas addition of recombinant epimorphin partially rescued duct formation. Together, our study demonstrates the role of epimorphin in pancreatic ductal morphogenesis.     

Pancreatic duct occlusion in the rat: report and assessment of a new technique

Temporary reduction of the exocrine pancreatic secretion may be desirable in various experimental models. In the rat this can be achieved by obstructing the connection between the pancreas and the duodenum. A new, simple technique of pancreatic duct occlusion using metal hemostatic clips is described. The reduction of secretion produced by the procedure was assessed by measuring duodenal protein, amylase, and trypsin during stimulation with cholecystokinin. Stimulated duodenal amylase activity 1 and 4 weeks following duct occlusion was reduced by approximately 80% compared with sham-operated controls, whereas proteolytic activity was reduced by 96 and 60%, respectively. The magnitude and duration of pancreatic insufficiency achieved by this technique is equivalent to that achieved with more complicated methods.     

Pancreatic duct occlusion with Ethibloc. An experimental study in juvenile pigs

In the transplantation of vascularized pancreatic grafts severe problems are related to the exocclusion is an improved alternative to duct ligation in producing atrophy of the exocrine pancreas while leaving the endocrine pancreas intact. Fractional growth rate in duct-ligated and duct-occluded animals was reduced to 1/3 - 1/4 of that of sham operated controls. Fasting blood glucose, fasting insulin, sum of blood glucose and glucose elimination rate during an intravenous glucose tolerance test remained normal in the duct-ligated and Ethibloc-occluded animals. There was a diminished insulin response to a maximal glucose load. In spite of this, the glucose tolerance remained virtually normal. The volume of the pancreas was reduced to 1/3 of its normal size after both experimental procedures. Histologically, the islets appeared to remain normal, while the exocrine portion of the gland was replaced by fibrous tissue. No traces of the active compound, Ethibloc, remained after 4 weeks. This study shows that pancreatic duct occlusion with Ethibloc results in impairment of endocrine function. Consequently, Ethibloc duct occlusion does not seem to be a superior alternative to other methods of producing exocrine atrophy in organs intended for transplantation.     

Effects of pancreatic duct ligation on pancreatic response to bombesin

To examine mechanisms that might be related to biliary pancreatitis, we examined the effects of pancreatic duct ligation (PDL) with pancreatic stimulation in vivo. PDL alone caused no increase in pancreatic levels of trypsinogen activation peptide (TAP), trypsin, or chymotrypsin and did not initiate pancreatitis. Although bombesin caused zymogen activation within the pancreas, the increases were slight and it did not cause pancreatitis. However, the combination of PDL with bombesin resulted in prominent increases in pancreatic TAP, trypsin, chymotrypsin, and the appearance of TAP in acinar cells and caused pancreatitis. Disruption of the apical actin network in the acinar cell was observed when PDL was combined with bombesin but not with PDL or bombesin alone. These studies suggest that when PDL is combined with pancreatic acinar cell stimulation, it can promote zymogen activation, the retention of active enzymes in acinar cells, and the development of acute pancreatitis.     

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging,Cell Injury Measurements,and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.     

Morphological changes in the rat exocrine pancreas after pancreatic duct ligation

In the present study, morphological changes of the exocrine pancreas in rats after pancreatic duct ligation were examined with light microscopy (hematoxylin-eosin, TUNEL, and PCNA staining) and scanning electron microscopy in order to elucidate the effects of increased pancreatic duct pressure. On the fifth day after pancreatic duct ligation, ductular proliferation, periductal fibrosis, and disappearance of acini were observed. TUNEL and PCNA staining demonstrated many apoptotic acinar cells and proliferating ductal cells immediately after ligation, which reached a maximal number on the 2nd or 3rd day. Tortuous or helical interlobular pancreatic ducts with inner surfaces containing many crater-like depressions and long cilia were found after ligation. These changes were almost identical to those observed in the pancreatic tissue of model chronic pancreatitis rats, WBN/Kob rats, and stroke-prone spontaneously hypertensive (SHRSP) rats. In summary, the morphological changes observed after pancreatic duct ligation were similar to those of chronic pancreatitis, therefore, the characteristic changes of pancreatic ducts observed in chronic pancreatitis may be caused by increased pancreatic duct pressure.     

THE NON-HUMAN PRIMATE ENDOCRINE PANCREAS: DEVELOPMENT,REGENERATION POTENTIAL AND METAPLASIA

An investigation into the development of the Vervet monkey endocrine pancreas revealed a sequence of occurrence of pancreatic peptides that differed from previous reports in mice, dog and human with PP and somatostatin occurring before glucagon and insulin. All four pancreatic peptides were identified, immunohistochemically, in only one of the pancreatic primordial buds, before fusion of the two buds to form the pancreas. This questions the hypothesis that the heterogeneous endocrine cell distribution seen in the adult pancreas is due to the contribution of only PP cells by the ventral bud and non-PP cells by the dorsal bud. Co-localization of glucagon and PP was observed extensively in the developing pancreas and the predominant expression of one over the other in an apparently organized non-random manner accounted for the glucagon- and PP-rich areas seen in the developing pancreas. A small number of cells immunoreactive to glucagon and PP were also observed in the adult. Reports of plasticity of differentiation of other pancreatic cells led us to investigate regeneration potential of the adult monkey pancreas. Partial obstruction of the Vervet monkey main pancreatic duct, by cellophane wrapping, resulted in duct cell proliferation and differentiation to form new endocrine tissue in a way that mimics normal organogenesis. Focal areas of hepatocytes were found in the regenerated pancreas of one monkey, illustrating further the latent developmental capabilities of adult pancreas cells. These findings could lead to interesting new therapies for pancreas and liver disease.     

Experimental studies on pancreatic duct occlusion with prolamine

The effect of the occlusion of the pancreatic duct system with prolamine (Ethibloc) has been studied in animal experiments with dogs and mini-pigs. The solution becomes solid in the duct system and becomes disintegrated again within 11 days. This time, however, is sufficient to keep a high-grade atrophy of the exocrine parenchyma. With this method one doesn't risk the provocation of an acute pancreatitis. The endocrine function of the atrophied glands is satisfactory, no animal became diabetic. The basal jugular vein insulin shows no difference to that of the control group, but nevertheless the mean whole pancreas hormone content is reduced for insulin and somatostatin, but not for glucagon.     

Neogenesis and proliferation of beta-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of beta-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a beta-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of beta-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of beta-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted beta-cell differentiation (mainly from duct cells) and proliferation of pre-existing beta-cells, resulting in an increase of the beta-cell mass that improved glucose tolerance in diabetic mice.     

The IIIb isoform of fibroblast growth factor receptor 2 is required for proper growth and branching of pancreatic ductal epithelium but not for differentiation of exocrine or endocrine cells

Fibroblast growth factors (Fgfs) and their receptors have been implicated in embryonic pancreas development. Recently it was shown that Fgf10, a major ligand for the IIIb isoform of fibroblast growth factor receptor 2 (Fgfr2b), has an important regulatory role in early pancreas development. The aim of our study was to define the role of Fgfr2b in pancreas development by analyzing the phenotype of Fgfr2b (-/-) mice. Pancreases of Fgfr2b (-/-) embryos were noticeably smaller than the wild type littermates during embryogenesis, and pancreatic ductal branching as well as duct cell proliferation was significantly reduced. However, both exocrine and endocrine pancreatic differentiation occurred relatively normally. Exogenous addition of Fgfr2b ligands (Fgf7 and Fgf10) stimulated duct cell proliferation and inhibited endocrine cell differentiation in the ex vivo embryonic organ cultures of wild type pancreas. Our results thus suggest that Fgfr2b-mediated signaling plays a major role in pancreatic ductal proliferation and branching morphogenesis, but has little effect on endocrine and exocrine differentiation.     

Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3

Regulatory proteins have been identified in embryonic development of the endocrine pancreas. It is unknown whether these factors can also play a role in the formation of pancreatic endocrine cells from postnatal nonendocrine cells. The present study demonstrates that adult human pancreatic duct cells can be converted into insulin-expressing cells after ectopic, adenovirus-mediated expression of the class B basic helix-loop-helix factor neurogenin 3 (ngn3), which is a critical factor in embryogenesis of the mouse endocrine pancreas. Infection with adenovirus ngn3 (Adngn3) induced gene and/or protein expression of NeuroD/beta2, Pax4, Nkx2.2, Pax6, and Nkx6.1, all known to be essential for beta-cell differentiation in mouse embryos. Expression of ngn3 in adult human duct cells induced Notch ligands Dll1 and Dll4 and neuroendocrine- and beta-cell-specific markers: it increased the percentage of synaptophysin- and insulin-positive cells 15-fold in ngn3-infected versus control cells. Infection with NeuroD/beta2 (a downstream target of ngn3) induced similar effects. These data indicate that the Delta-Notch pathway, which controls embryonic development of the mouse endocrine pancreas, can also operate in adult human duct cells driving them to a neuroendocrine phenotype with the formation of insulin-expressing cells.     

Regulation of pancreatic duct cell differentiation by phosphatidylinositol-3 kinase

We have previously demonstrated that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling is essential for pancreatic regeneration after partial pancreatectomy in mice. In the present study, we examined a role of PI3K/Akt signaling for pancreatic duct cell differentiation into insulin-producing cells. Epithelial-like cells were isolated from mouse pancreas and confirmed to be positive for a duct cell marker cytokeratin-20 (CK-20) but negative for insulin. Incubation of these cells with epidermal growth factor, exhibited a gradual increase in Akt phosphorylation and expression of pancreatic duodenal homeobox-1 (PDX-1), a regulator of β-cell differentiation. Three weeks later, these CK-20-positive cells were noted to express insulin as determined by immunofluorescent double-staining. Akt phosphorylation, PDX-1 expression, and insulin production were effectively reduced by blocking the PI3K/Akt pathway using siRNA to the p85α regulatory subunit of PI3K. Our results demonstrate that PI3K/Akt activation has a critical role for pancreatic duct cell differentiation into insulin-producing cells.     

Cloning and immunolocalization of a rat pancreatic Na(+) bicarbonate cotransporter.

In the rat, pancreatic HCO(-)(3) secretion is believed to be mediated by duct cells with an apical Cl(-)/HCO(-)(3) exchanger acting in parallel with a cAMP-activated Cl(-) channel and protons being extruded through a basolateral Na(+)/H(+) exchanger. However, this may not be the only mechanism for HCO(-)(3) secretion by the rat pancreas. Recently, several members of electrogenic Na(+)/HCO(-)(3) cotransporters (NBC) have been cloned. Here we report the cloning of a NBC from rat pancreas (rpNBC). This rpNBC is 99% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas, prostate, and a minor clone in kidney]. The longer NBC isoforms are identical to the rat and human kidney-specific forms (kNBC; 1035 aa; 116 kDa) at the approximately 980 C-terminal aa's and are unique (with different lengths) at the initial N-terminus. Using polyclonal antibodies to the common N- and C-termini of rat kidney NBC, a approximately 130-kDa protein band was labeled by immunoblotting of rat pancreas homogenate and was enriched in the plasma membrane fraction. Immunofluorescence and immunoperoxidase light microscopy of rat pancreatic tissue with both antibodies revealed basolateral labeling of acinar cells. Labeling of both apical and basolateral membranes was found in centroacinar cells, intra- and extralobular duct, and main duct cells. The specificity of the antibody labeling was confirmed by antibody preabsorption experiments with the fusion protein used for immunization. The data suggest that rpNBC likely plays a more important role in the transport of HCO(-)(3) by rat pancreatic acinar and duct cells than previously believed.     

Early changes in pancreatic acinar cell calcium signaling after pancreatic duct obstruction

Intracellular Ca(2+)-changes not only participate in important signaling pathways but have also been implicated in a number of disease states including acute pancreatitis. To investigate the underlying mechanisms in an experimental model mimicking human gallstone-induced pancreatitis, we ligated the pancreatic duct of Sprague-Dawley rats and NMRI mice for up to 6 h and studied intrapancreatic changes including the dynamics of [Ca(2+)](i) in isolated acini. In contrast to bile duct ligation, pancreatic duct obstruction induced intra-pancreatic trypsinogen activation, leukocytosis, hyperamylasemia, and pancreatic edema and increased lung myeloperoxidase activity. Although resting [Ca(2+)](i) in isolated acini rose by 45% to 205 +/- 7 nmol, the acetylcholine- and cholecystokinin (CCK)-stimulated calcium peaks as well as the amylase secretion declined, but neither the [Ca(2+)](i)-signaling pattern nor the amylase output in response to the Ca(2+)-ATPase inhibitor thapsigargin nor the secretin-stimulated amylase release were impaired by pancreatic duct ligation. On the single cell level pancreatic duct ligation reduced the percentage of cells in which submaximal secretagogue stimulation was followed by a physiological response (i.e. Ca(2+) oscillations) and increased the percentage of cells with a pathological response (i.e. peak plateau or absent Ca(2+) signal). Moreover, it reduced the frequency and amplitude of Ca(2+) oscillation as well as the capacitative Ca(2+) influx in response to secretagogue stimulation. Serum pancreatic enzyme elevation as well as trypsinogen activation was significantly reduced by pretreatment of animals with the calcium chelator BAPTA-AM. These experiments suggest that pancreatic duct obstruction rapidly changes the physiological response of the exocrine pancreas to a Ca(2+)-signaling pattern that has been associated with premature digestive enzyme activation and the onset of pancreatitis, both of which can be prevented by administration of an intracellular calcium chelator.     

Combined use of brushing cytology and endoscopic retrograde pancreatography for the early detection of pancreatic cancer

Seventy-two cases of pancreatic cancer were examined by brushing cytology combined with endoscopic retrograde pancreatography. The results of this combined method were better than those reported for the exfoliative cytologic study of pancreatic fluid. The method detected a minute cancer of the main pancreatic duct that was not detected with any other method. The cells obtained by this technique had very well-preserved cytoplasm and nuclear chromatin, which facilitated making a correct diagnosis. Though this method can be applied only to the main pancreatic duct, it is effective for the diagnosis of ductal cell carcinoma, especially those located at the head of the pancreas, which is the most common site for pancreatic cancer. It is a safe procedure, with no complications seen in this series. The differentiation of carcinoma cells from the benign atypical cells of chronic pancreatitis is illustrated and emphasized.     

Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening

The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells.