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Background & Aims: Tumor necrosis factor (TNF)-alpha contributes to the development of acute pancreatitis. Because TNF-alpha is involved in the control of apoptosis, we studied its interaction with the pancreatic apoptotic pathway., Methods: Pancreatic acinar AR4-2J cells were used. Apoptosis was monitored by morphologic and biochemical criteria., Results: TNF-alpha induced apoptosis in AR4-2J cells. Induction was strongly enhanced in cells treated with actinomycin D, suggesting that TNF-alpha activated concomitantly an antiapoptotic mechanism through newly synthesized proteins. This mechanism involved activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein (MAP) kinases because their inhibition worsened TNF-alpha-induced apoptosis. The antiapoptotic pancreatitis-associated protein (PAP) I is a candidate for mediating TNF-alpha activity. Its expression is induced by TNF-alpha, and cells overexpressing PAP I show significantly less apoptosis on exposure to TNF-alpha. We examined whether TNF-alpha induction of PAP I expression was mediated by NF-kappaB or MAP kinases by using specific inhibitors of both pathways. Inhibition of NF-kappaB had no effect. However, inhibitors of MEK1 eliminated PAP I induction., Conclusions: TNF-alpha induces concomitantly proapoptotic and antiapoptotic mechanisms in pancreatic AR4-2J cells. Antiapoptotic mechanisms are mediated by NF-kappaB and MAP kinases, and PAP I is one of the effectors of apoptosis inhibition.

Background & Aims: Homeobox genes are involved in establishing and maintaining differentiated patterns in adult tissues. Cdx1 might carry out that function in the intestinal epithelium because its expression is specific to that tissue and increases during development., Methods: Cdx1 expression was induced in IEC-6 intestinal epithelial cells by stable transfection, and subsequent changes in cell growth, resistance to apoptosis, migration, and differentiation were monitored., Results: Compared with control, IEC-6/Cdx1 cells proliferated more rapidly, were more resistant to apoptosis, and migrated 3-4 times faster, as shown by an in vitro wound assay. IEC-6/Cdx1 cells in culture formed multilayers. Morphology of the top layer was similar to that of columnar epithelium, with cells showing typical features of differentiated enterocytes, including complex junctions and well-developed microvilli with glycocalix. Expression of 2 markers of enterocyte differentiation, aminopeptidase N and villin, was induced in IEC-6/Cdx1 cells. Aminopeptidase N was targeted to the basolateral membrane, and villin was localized to the cytoplasm. Actin filaments, which were mostly present in transcytoplasmic stress fibers in control cells, were redistributed to the cortex in Cdx1-transfected cells., Conclusions: Cdx1 expression in IEC-6 cells induces phenotypic changes characteristic of differentiating enterocytes, suggesting an important role for Cdx1 in the transition from stem cells to proliferating/transit cells.

Background/aims: The pancreatitis-associated protein (PAP) is undetectable in normal pancreatic secretion and overexpressed in the acute phase of pancreatitis. We investigated whether serum PAP could be an indicator of the course of acute pancreatitis., Methods: Serum PAP was retrospectively monitored in 98 patients with acute pancreatitis during their stay in the hospital. Patients were classified according to the severity of their disease as group I (< or = 1 complication), group II (> or = 2 complications), or group III (lethal pancreatitis)., Results: At admission, 34% of patients, all from group I, had normal PAP values (< 10 micrograms/L). None of them developed complications. They had a significantly shorter stay in the hospital than patients with elevated PAP (6.2 days vs. 14.9 days). In all patients, serum PAP increased after admission to a maximum, which correlated significantly to the severity of the disease. Average peak values were 22.2 micrograms/L and 240.0 micrograms/L in group I patients with normal or high PAP at admission, 963.0 micrograms/L and 1436.0 micrograms/L in groups II and III. Serum PAP decreased steadily during recovery., Conclusions: Monitoring serum PAP in patients with acute pancreatitis would provide (1) at admission, selection of most patients who will not develop complications; (2) a dynamic assessment of severity; and (3) anticipation of the patient's recovery.

Pancreatic juice is naturally supersaturated in calcium and bicarbonate ions. A mechanism controlling CaCO3 crystal formation and growth is therefore necessary to prevent duct clogging. The present study shows that lithostathine, a glycoprotein present in human pancreatic juice at a concentration in the range of 10 mumol/L, could be involved in such a control. Lithostathine in concentrations greater than 1.5 mumol/L significantly delayed crystal nucleation and inhibited growth of preformed CaCO3 crystals from supersaturated solutions. Adsorption of lithostathine on crystals was shown by immunodetection. Albumin also adsorbed on CaCO3 crystals, but neither albumin nor other pancreatic secretory proteins inhibited crystal nucleation or growth. Lithostathine adsorbed to sites specifically inhibiting crystal growth with a dissociation constant (Kd) = 0.9 x 10(-6) mol/L. The glycosylated amino-terminal undecapeptide generated by limited trypsin hydrolysis inhibited CaCO3 crystal growth with a Kd = 3.0 x 10(-6) mol/L, similar to that of lithostathine. On the contrary, the carboxy-terminal polypeptide was inactive. A synthetic undecapeptide identical to the N-terminal end but not glycosylated was equally active. The activity disappeared upon digestion of the undecapeptide with V8 protease. The N-terminal undecapeptide of lithostathine is therefore essential to the inhibitory activity of the protein on CaCO3 crystal growth.

After pancreas transplantation, signs of acute pancreatitis are found in the grafted tissue. Pancreatic juice secreted from this organ was analyzed by gel electrophoresis. Initially, the pattern of secretory proteins was similar to that of the juice collected from normal individuals, but high levels of albumin were present. Within 2 days after reperfusion of the grafted pancreas, proteins of molecular weights 17,000-20,000 increased remarkably. Separation by two-dimensional gel electrophoresis showed that this was largely due to the appearance of new a protein, present neither in the juice collected immediately after reperfusion nor in normal pancreatic juice. After transfer onto nitrocellulose, this additional protein was detected by antibodies directed against the recently described rat "pancreatitis-associated protein." Maximal amounts of approximately 7.5% of total secretory protein were found 5 days after transplantation. The concentration of the protein decreased in the further course but was still detectable after 45 days. The isoelectric point (7.1) and the molecular weight (17,500) were similar to those of the rat protein. It is concluded that after inflammation induced by pancreatic transplantation, the human pancreas secretes high amounts of a protein not present in normal juice. Because of its similarities to the rat pancreatitis-associated protein it is designated human pancreatitis-associated protein.

A new protein was purified from the pancreatic juice of rats with acute pancreatitis. That protein, not detectable in control animals, was called "pancreatitis-associated protein." It was first observed 6 hours after induction of experimental pancreatitis with taurocholate or cerulein, reached maximal levels of 45 micrograms/mg protein in zymogen granules and 1.8 micrograms/mg protein in pancreatic tissue during the acute phase (48 hours), and disappeared during recovery (day 5). It was never detected in spleen, liver, kidney, heart, or lung. The detection limit of the assay system was 12 ng/mg protein, so that pancreatitis-associated protein levels increased at least 100-fold in pancreatic tissue during the acute phase. The molecular weight (12,000) and isoelectric point (8.2) were determined by two-dimensional gel electrophoresis. Subcellular fractionation and immunoelectron microscopy showed that the protein was synthesized on the rough endoplasmic reticulum and stored in zymogen granules before being secreted, similar to other pancreatic secretory proteins. Immunoblotting and two-dimensional gel electrophoresis revealed that the same protein was synthesized upon induction of pancreatitis by cerulein infusion, by retrograde injection of bile acids, or pancreatitis induced by pancreatic surgery. The pancreatitis-associated protein is therefore an acute-phase protein that differs from other proteins of that family because of its exocrine nature.

We have previously shown that the pancreatic stone protein (PSP) is an inhibitor of calcium carbonate crystal growth and may participate in the stabilization of the normally supersaturated pancreatic juice. Our aim in this study was to determine if PSP is a normal secretory product of the human pancreas by determining if the normal human pancreas contains a messenger RNA coding for PSP. Human pancreatic messenger RNAs were used to direct protein synthesis in a cell-free translation system. Immunoprecipitation of translation products with a monospecific antibody directed against the PSP yielded a product migrating as a single homogeneous band on sodium dodecyl sulfate-polyacrylamide gels. This product has a molecular weight of 16,000, the value expected for pre-PSP. Products selected by immunoprecipitation with antitrypsin-1 antibodies also migrated as a single band, with a molecular weight of 27,000. It is concluded that a messenger RNA coding for pre-PSP, distinct from the messenger RNA coding for pretrypsinogen, is present in human pancreas. These results support the hypothesis that PSP is a molecular entity, and not a degradation product of trypsinogen 1 or another pancreatic protein.

Duodenal juice was collected from 50 healthy patients in the basal state, and after intravenous injection of 1 clinical unit of secretin kg-1 + 3 Crick-Harper-Raper units of cholecystokinin-pancreozymin (CCK-PZ) kg-1. Amylase to chymotrypsin and amylase to lipase ratios were significantly decreased by stimulation. Pure pancreatic juice was collected from 7 healthy subjects by endoscopic retrograde catheterization of the papilla. Secretin (1 clinical unit kg-1 hr-1) was infused intravenously during 30 min, and a single intravenous injection of CCK-PZ (3 Crick-Harper-Raper units kg-1) was done at the 15th min of the secretin infusion. Chymotrypsinogen and lipase secretions did not parallel that of amylase during CCK-PZ stimulation. As in the first set of experiments, amylase secretion was proportionally less stimulated by CCK-PZ injection than were chymotrypsinogen and lipase secretions. However, in approximately 20% of subjects the secretion of the enzymes was parallel in both duodenal and pancreatic juice.