The structural integrity of the islet in vitro: the effect of incubation temperature.

The purpose of this study was to examine the effect of incubation temperature on the structural integrity of the islet during culture. Islets were isolated from the pancreas of the Syrian golden hamster and cultured in a collagen gel for < or =12 days at 24 degrees C or 37 degrees C. At 24 degrees C, cells in the islet periphery died, leading to a complete disintegration of the mantle region in 37.4+/-5.6% of the islets. In comparison, at 37 degrees C, few islets exhibited mantle disintegration (p<0.001). Insulin immunoreactivity was distributed nonhomogeneously in islets at 24 degrees C, and the intensity of the staining, by using a semiquantitative scale (0-3), was +1. Islets cultured at 37 degrees C had a normal homogeneous distribution of insulin immunoreactivity with a score of +3. As the pancreas is a complex gland composed of different cell types, and cell-cell interactions are known to be important in the maintenance of cell survival, additional experiments were repeated to include the coculture of islets with duct epithelial cells. The proportion of islets that developed mantle disintegration was now reduced to 2.5+/-0.3% (p<0.001), comparable to that seen at 37 degrees C. Similar results were obtained for islets cultured in the presence of duct-conditioned medium (DCM). Together with the preservation of the islet mantle, islets cultured in the presence of duct epithelial cells or DCM had a normal homogeneous distribution of insulin immunoreactivity, with a staining intensity of +3. We conclude that incubation temperature has a profound effect on the structural integrity of islets, and that the detrimental effects of low-temperature culture can be mitigated by coculture of islets with secretory products derived from pancreatic ductal cells. These data provide evidence for a trophic relation between pancreatic islets and ductal epithelium.

Early changes of gene expression during cerulein supramaximal stimulation.

Administration of supramaximal doses of cerulein results in acute interstitial pancreatitis. To understand the pathogenesis of this disease, it would be of great importance to elucidate the changes during the early phase of the process. We report changes of gene expression in the pancreas during the first 6 h of cerulein supramaximal stimulation. The expression of genes, including the secretory enzyme amylase, the lysosomal enzyme cathepsin B, as well as the housekeeping genes beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPD), was investigated in this study. The most prominent alteration in gene expression is beta-actin messenger RNA (mRNA), which increased continuously after cerulein infusion. Immunostaining for beta-actin was observed along the membrane of large cytoplasmic vacuoles in pancreatic acinar cells. The level of amylase mRNA decreased during the first 30 min of cerulein infusion, recovered to the control level at 1 h and increased twofold at 2 h. An obvious increase in cathepsin B mRNA was observed after 3 h of cerulein infusion and reached sixfold of the control at 6 h. A significant increase of GAPD mRNA level was observed at 6 h of cerulein stimulation. In conclusion, this study provides direct evidence that the changes in gene expression, such as cathepsin B and amylase, after supramaximal cerulein stimulation, are regulated at the transcriptional level. It also suggests that beta-actin is involved in the formation of cytoplasmic vacuoles during supramaximal cerulein administration. Finally, this study indicates that beta-actin and GAPD may not be appropriate as RNA-loading controls for Northern blot analysis of pancreatic tissue.

Pancreatic schwannoma: report of two cases and review of the literature.

Solitary intrapancreatic schwannoma is a rare tumor. We present two patients with this tumor and review 13 previously reported cases from the English-language literature. While the final diagnosis was made based on pathological examination of the tumors, both computed tomography scan and magnetic resonance imaging helped establish the benign nature of the lesion, narrow the differential diagnosis, and define the anatomical locations of the small tumors. Both tumors were treated by enucleation from the surrounding pancreatic parenchyma, and both patients, after 2 years of follow-up, are alive and well. It is concluded that multimodality radiologic investigations are useful in the workup of unusual pancreatic masses. In addition, based on the known biologic behavior of schwannomas occurring elsewhere in the body, simple enucleation, rather than more radical resection, is likely to be adequate therapy for these tumors.

Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters.

Induction of islet neogenesis by cellophane wrapping (CW) reverses streptozotocin-induced (STZ) diabetes. Administration of Ilotropin, a protein extract isolated from CW pancreata, causes recapitulation of normal islet ontogeny and reverses STZ diabetes, reducing mortality by 50%. We investigated the hypothesis that a novel gene encoding a constituent of Ilotropin was expressed in the hamster pancreas undergoing islet neogenesis. Islet neogenesis associated protein (INGAP) is a product of a novel gene expressed in regenerating hamster pancreas. Northern blot analysis showed a strong single transcript of 850 bp at 1 and 2 d after CW that disappeared by the 6th day and was absent from untreated control pancreata. INGAP gene is expressed in acinar cells, but not in islets. Western blot analysis demonstrated the presence of INGAP in Ilotropin but not in extracts from control pancreata. A synthetic pentadecapeptide, corresponding to a region unique to INGAP, stimulated a 2.4-fold increase in [3H]thymidine incorporation into hamster duct epithelium in primary culture and a rat pancreatic duct cell line but had no effect on a hamster insulinoma tumor cell line. A portion of human INGAP gene was cloned and appears to be highly homologous to the hamster gene. This data suggests that the INGAP gene is a novel pancreatic gene expressed during islet neogenesis whose protein product is a constituent of Ilotropin and is capable of initiating duct cell proliferation, a prerequisite for islet neogenesis.

Phenotypic modulation of hamster acinar cells by culture in collagen matrix.

The aim of this study was to assess the effect of different culture conditions on the survival and morphological phenotype of cultured acinar cells. Acinar fragments isolated from hamster pancreas were embedded in rat-tail collagen. Four groups were established: Medium 1-5% NuSerum + basic medium (basic medium = DMEM/F12 supplemented with dexamethasone, 3-isobutyl-2-methylxanthine, and antibiotics); Medium 2-10% NuSerum + basic medium. Medium 3-Medium 2 supplemented with epidermal growth factor and cholera toxin; and Medium 4:-Medium 3 supplemented with soybean trypsin inhibitor. Freshly isolated acinar cells were retrieved morphologically intact. In Medium 1, more than 80% of cells retained a normal histological appearance at 34 days in culture. Immunostaining for amylase was observed at the apical pole of the cells. The remaining cells showed variable degrees of degeneration. In Medium 2, approximately 50% of acinar cells appeared normal at 34 days in culture, while the remainder were severely degenerated. A few cystic structures were also observed. Positive immunostaining for amylase was limited to the cells with a normal histological appearance. The cells grown in Media 3 and 4 had similar courses of morphological changes. After 8 days in culture, most acinar fragments disappeared and were replaced by cystic structures, lined by a single layer of cuboidal cells. Some amylase-positive immunoreactive cells were integral components of the cystic wall. Cellular amylase activity was a function of the different culture media, a more rapid decrease in amylase activity being observed in Media 3 and 4. Uptake of [3H]thymidine did not show any significant differences between the media. It was also found that the ductlike cells cultured in Medium 4 had a limited capacity to redifferentiate into acinar cells. This study shows that the acinar cell phenotype can be maintained in vitro for more than 1 month. This study also suggests that ductal-like epithelial structures arise from transformation of acinar cells.

Transdifferentiation of human islets to pancreatic ductal cells in collagen matrix culture.

Transdifferentiation is a change from one differentiated phenotype to another, involving morphological and functional phenotypic markers. Stability of the cellular phenotype is probably related to the extracellular milieu, as well as cytoplasmic and nuclear components that interact to control gene expression, and the conversion of cell phenotype is likely to be accomplished by selective enhancement of gene expression, which controls the terminal developmental commitment of cells. In this paper, we show the induction of cultured human islets cells to alter their usual phenotypic expression and attain morphological and functional characteristics of duct cells. Islets were isolated by collagenase digestion of pancreata that were removed from cadaveric organ donors. The islets were purified on a two-step density gradient of bovine serum albumin and were then placed into a three-dimensional rat-tail collagen gel matrix supplemented with NuSerum epithelial growth factor and cholera toxin. During the initial 96 h of culture, the islets underwent a cystic transformation that was associated with (1) the maintenance of immunoreactivity for neuron-specific enolase, an endocrine cell marker, but a progressive loss of insulin gene expression, (2) a loss of immunoreactivity for insulin protein, and (3) the appearance of CK-19, a marker for ductal cells. After the transformation was complete, the cells had the ultrastructural appearance of primitive duct-like cells. Cyst enlargement after the initial 96 h was associated, at least in part, with cell replication, as reflected in the 1500% increase in the incorporation of tritiated thymidine. These experiments are consistent with the transdifferentiation of an islet cell to a ductal cell. The exact mechanisms involved still need to be fully elucidated.

Induction of islet cell differentiation and new islet formation in the hamster--further support for a ductular origin.

Partial obstruction of the adult hamster pancreas leads to islet cell differentiation and new islet formation. From morphologic and morphometric observations, we have tentatively identified the source of the new islet tissue to be from cells in the ducts. In this study, in vivo labeling with a single pulse of tritiated thymidine after partial duct obstruction was used to ascertain whether newly formed islet cells were in fact derived from cells in the ductal epithelium. Supportive evidence for this formulation was also sought using immunocytochemistry for islet hormones and in situ hybridization for glucagon and insulin mRNA to probe areas of proliferating duct cells. Endocrine cell differentiation was observed as a migration of cells out from small ducts beginning at about 10 days after obstruction. Duct and islet cell labeling indices (LI;%) in control animals remained at a low level (0.25 +/- 0.01 and 0.26 +/- 0.03, respectively) throughout the experiment. In contrast, at 2 weeks after partial obstruction, the duct and islet cell LI were 4.2 +/- 0.7 and 0.80 +/- 0.1 (p < 0.05 vs. control). After 2 weeks, there was a rapid and significant 86% decline in the duct cell LI to a low of 0.6 +/- 0.2 at 8 weeks, which was accompanied by a comparable, but reciprocal, 113% increase in the islet cell LI to a high of 1.7 +/- 0.8 (p < 0.05). In situ hybridization demonstrated glucagon and insulin mRNA-positive cells within intralobular ducts as early as 6 and 8 days, respectively, after obstruction. Glucagon and insulin peptides appeared in these cells at approximately 8 and 10 days, respectively, as cells migrated out from the duct wall. This study provides additional evidence that further supports our concept that pancreatic endocrine cell differentiation in this model reiterates the normal ontogeny of beta cell differentiation from cells in the ductular epithelium.

Alterations in pancreatic microcirculation and expression of endothelin-1 in a model of chronic pancreatitis.

Chronic pancreatitis (CP) is characterized by the presence of an inflammatory infiltrate with progressive destruction of acinar cells and fibrosis. The finding that endothelin-1 (ET-1), an endothelium-derived peptide having vasoconstrictive and mitogenic properties, reduces pancreatic blood flow (PBF) in normal rats suggested that the peptide may be associated with the reduced PBF seen in animal models of CP and with the morphological abnormalities of the disease. This study investigates changes in blood flow to the pancreas and other abdominal organs in a rat model of CP and compares ET-1 production in the pancreata of these rats and normal controls. CP was induced in male Wistar rats by the injection of oleic acid into the common bile/pancreatic duct. The radiolabeled microsphere technique was employed to measure blood flow to the pancreas, duodenum, liver, spleen, and kidneys. Immunohistochemistry was used to investigate the cellular production of ET-1. After 3 weeks, significant decreases were noted in body weight, pancreatic weight, and pancreatic DNA, amylase, and protein content in the animals with CP. PBF was reduced by 64% and duodenal blood flow by 80% relative to those in control animals. Hepatic and splenic blood flows were increased by 91 and 88%, respectively, compared to those in controls. A 50% decrease in renal blood flows were increased by 91 and 88%, respectively, compared to those in controls. A 50% decrease in renal blood flow was also seen in the experimental group after 3 weeks. Pancreata from animals with CP stained diffusely for ET-1 in the cytoplasm of vascular endothelial, acinar, and ductal cells. In the control pancreata, focal staining for ET-1 was observed only in acinar cells. This difference was significant in endothelial and ductal cells. There was weak staining of islet cells in both groups. The results suggest that elevation in local production of ET-1 may be associated with the morphological and hemodynamic changes of CP.

Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16.

Pancreatic cancer is one of the most lethal cancers in humans. The majority of these cancers arise from the pancreatic duct epithelium. Research into the pathogenesis of pancreatic carcinoma has largely relied on animal models. In vitro models of pancreatic carcinogenesis using propagable cultured epithelial cells derived from the pancreatic ducts of rats and hamsters have been described. A human model, however, has been nonexistent due to the unavailability of propagable cultured duct epithelial cells derived from normal human pancreas. We report here a reproducible method for the long-term culture of pancreatic duct epithelial cells derived from normal and benign adult human pancreata by infection with a retrovirus containing the E6 and E7 genes of the human papilloma virus 16. One of these cell lines has become immortal and has propagated continuously for more than 20 passages. They remain anchorage dependent in their growth and nontumorigenic in nude mice. These cell lines and the methodology described here to establish them may provide new avenues for in vitro studies of the roles played by duct epithelium in human pancreatic diseases and cancers.

Islet-cell regeneration in the diabetic hamster pancreas with restoration of normoglycaemia can be induced by a local growth factor(s).

Partial pancreatic duct obstruction in the hamster leads to the induction of endocrine-cell differentiation and new islet formation. We prepared cytosolic extracts from the partially obstructed pancreas and identified one, which when administered i.p., produced significant increases in the incorporation of tritiated thymidine by ductular and islet cells, as well as a corresponding increase in islet mass. In this study, we evaluate the ability of this extract to reverse streptozotocin diabetes mellitus. Hamsters were treated i.p. twice daily for 7 weeks with either 0.9% NaCl (saline) (n = 10) or a cytosol extract (n = 10) prepared previously from partially obstructed hamster pancreata. All animals in the cytosol group survived vs only 60% of the saline group (p = 0.02). Random blood glucose levels were greater than 22.2 mmol/l in 90% of the saline group vs 40% in the cytosol group (p < 0.05). Pancreatic tissue from the surviving saline animals and from persistently hyperglycaemic cytosol-treated animals, showed intra-cytoplasmic vacuolation of islet cells, a characteristic lesion of sustained hyperglycaemic states. Vacuolation was not observed in normoglycaemic extract treated animals. Islets in hyperglycaemic animals demonstrated a profound decrease or absence of immunoreactive insulin, compared to an abundance of immunoreactive beta cells in cytosol-treated animals that reverted to normoglycaemia. In this group, single cells or nests of cells stained for insulin on glucagon cells were identified in ductal epithelium in association with cells budding from the duct. Morphometric analysis of pancreata in reverted cytosol-treated animals showed a new population of small islets compared with saline controls and an increased islet mass. In summary, streptozotocin diabetes can be reversed by new islet formation induced by local pancreatic growth factors, the exact nature of which remains to be determined.

Paracrine/autocrine regulation of pancreatic islet cell proliferation and differentiation in the hamster: studies using parabiosis.

Partial obstruction of the pancreatic duct in hamsters leads to new islet formation and reversal of streptozotocin diabetes. The purpose of this study was to delineate the mechanism by which endocrine cell proliferation and differentiation is mediated in this model. Six pairs of parabiotic hamsters were established and partial duct obstruction was inducted in 1 parabiont from each pair. At 6 weeks, the pancreatic weight (mg/100g bw); DNA (microgram/100g bw) and protein content (mg/100g bw) showed 28% (167 +/- 21 vs. 130 +/- 17), 32% (1,052 +/- 206 vs. 795 +/- 159), and 20% (25.4 +/- 6.6 vs. 21.2 +/- 1.9) increases (p < 0.05), respectively, over the non-wrapped parabionts. Morphometric analysis demonstrated the presence of new islets in the wrapped pancreata with a 100% increase in the number of islets/mm2 compared with non-wrapped controls (0.90 +/- 0.5 vs. 1.8 +/- 0.7, p < 0.01). A cytosol extract was prepared from duct-obstructed pancreases, and 4 microliters/g bw injected i.p. twice daily for 2 d produced significant increases in pancreatic weight and DNA content of 12% and 40%, respectively. Cytosol extract from non-wrapped pancreata had no effects compared with saline. When wrapped cytosol extract was injected for 21 d, the labeling index of ductular and islet cells (% cell nuclei labeled with 3H-TdR) was increased 10- and 6-fold respectively over controls (2.42 +/- 0.28 vs. 0.23 +/- 0.01 and 1.17 +/- 0.01 vs. 0.25 +/- 0.04, respectively, p < 0.01). The trophic effects observed in this model of islet cell proliferation and differentiation did not appear to be mediated by a humoral mechanism because the changes induced by partial obstruction were not observed in the non-operated parabiont. Control of pancreatic endocrine cell growth in this model appears to involve paracrine and/or autocrine regulatory mechanisms.

Prevention of diabetes in the spontaneously diabetic BB rat by the glutamine antimetabolite acivicin.

The autoimmune syndrome of the BB rat is associated with a marked increase in glutamine (Gln) metabolism in immune system cells of both diabetes-prone (BBdp) and diabetic (BBd) rats. To test whether inhibition of Gln metabolism prevents diabetes, 17 BBdp received acivicin (1 mg/kg) and 17 received saline subcutaneously every 2 days from age 48 days until diabetes onset or age 186 days. Twenty-seven non-diabetes-prone (BBn) rats served as controls. Acivicin caused some growth effects and a macrocytic anemia, but no other clinical or biochemical side effects. Only one acivicin-treated BBdp became diabetic (age 158 days), compared with saline-treated rats, of which 10 became diabetic and 2 became glucose intolerant (p < 0.001). Insulitis was moderate to severe in 88% of the saline-treated BBdp rats, but minimal in most acivicin-treated BBdp rats. Liver glutamine and glutamate tended to be higher in acivicin- than saline-treated BBdp rats. Acivicin caused no change in the proportions of T or B lymphocytes, NK cells, or macrophage phenotypes in spleen or blood; all BBdp rats were typically lymphopenic. Mitogenic responses of splenocytes in vitro were not affected. The results are consistent with the hypothesis that acivicin, by interfering with Gln metabolism, "targets" activated cells of the immune system and thereby attenuates the process and prevents overt diabetes, without major disturbance of Gln levels or generalized immunosuppression. This prevention is not due to a nutritional-growth retardation effect, as diabetes was prevented in females that showed no such effect.

Expression of endothelin-1 in pancreatic tissue of patients with chronic pancreatitis.

Chronic pancreatitis is characterized by the presence of an inflammatory infiltrate, progressive destruction of acinar cells, and fibrosis. The finding that endothelin-1, an endothelium-derived peptide with vasoconstrictive and mitogenic properties, reduces pancreatic blood flow in normal rats suggested that the peptide may be associated with the reduced pancreatic flow seen in animal models of chronic pancreatitis and in the morphological abnormalities of the disease. The aim of this study was to investigate sites of endothelin-1 expression in the pancreas of normal subjects and patients with chronic pancreatitis. The techniques of immunohistochemistry, in situ hybridization, and Northern blotting were used. Endothelin-1-like immunoreactivity was localized predominantly to islet cells both in normal subjects and in patients with chronic pancreatitis. Semi-quantitative analyses of immunostaining showed that endothelin-1-like immunoreactivity in islet cells of patients with chronic pancreatitis was greater than in normal subjects. Co-localization studies with glucagon, insulin, somatostatin, and pancreatic polypeptide showed that endothelin-1-like immunoreactivity co-exists with glucagon and insulin. There was no apparent co-existence of endothelin-1-like immunoreactivity with somatostatin or pancreatic polypeptide. Endothelin-1 mRNA was expressed in sites similar to those of the immunostaining, as well as in vascular endothelial cells. Northern blot analysis showed an increase in the expression of endothelin-1 mRNA in the patient population. There was a significant correlation between intensity of endothelin-1 immunostaining and severity of fibrosis in the patients with chronic pancreatitis. These findings suggest that an elevation in local expression of endothelin-1 may be associated with the morphological and haemodynamic changes of chronic pancreatitis.

Hepatocyte growth factor and Met receptor expression in human pancreatic carcinogenesis.

We have used immunohistochemistry to evaluate the clinicopathological significance of hepatocyte growth factor (HGF) and Met/HGF receptor expression in ductal lesions of 46 human pancreata. Normal duct epithelium shows no significant immunoreactivity for either HGF or Met. Strong immunostaining for HGF was respectively demonstrated in hyperplastic and severely dysplastic epithelia in 35.5 and 40% of cases with such duct lesions, whereas 37% of ductal adenocarcinoma showed diffuse HGF immunostaining. Positive Met immunostaining was demonstrated in 58, 80, and 78%, respectively, of specimens demonstrating the above duct lesions. Patients with resectable ductal adenocarcinoma demonstrating diffuse Met immunostaining have a significantly longer survival than those with tumors showing negative/focal staining (19.7 versus 8.1 months at P = 0.026). In contrast, HGF immunoreactivity did not significantly correlate with the survival of the patients. The results suggest that HGF and Met expression may play significant bifunctional roles during human pancreatic ductal carcinogenesis and progression. Whereas an upregulation of Met receptor expression and HGF-Met interaction may have an important pathogenetic role during the early stages of neoplastic promotion, a lack or subsequent loss of Met expression in invasive adenocarcinoma appears to result in a more aggressive clinical behavior.

Hepatocytic differentiation of cultured rat pancreatic ductal epithelial cells after in vivo implantation.

We have investigated the differentiation potential of propagable cultured rat pancreatic duct epithelial cells after in vivo implantation in isogeneic Fischer-344 rats. Cells genetically labeled with Escherichia coli beta-galactosidase (lacZ) reporter gene were embedded in a mixture of collagen and Matrigel (basement membrane matrix) and implanted either subcutaneously or intraperitoneally. Tissues from the two locations were harvested 4 to 8 weeks later. The great majority of the lacZ-labeled epithelial cells colonizing both sites phenotypically resembled hepatocytes, although they demonstrated different degrees of hepatocytic differentiation. Less than 5% of lacZ-labeled cells formed ductular structures. The hepatocyte-like cells from the subcutaneous implantation site expressed mixed phenotypes of both hepatocyte and ductal cell, including the expression of alpha-fetoprotein, tyrosine amino-transferase, gamma-glutamyl transpeptidase, carbonic anhydrase II, and cytokeratin 19. In contrast, the hepatocyte-like cells colonizing the mesentery showed the phenotype of mature hepatocytes, including an abundant glycogen storage and a lack of alpha-fetoprotein and carbonic anhydrase II expressions. Neither acinar cell nor endocrine differentiation was seen. These findings demonstrate that pancreatic ductal cells can be the progenitor cell for transdifferentiated hepatocytes.