Rat pancreatitis-associated protein is expressed in relation to severity of experimental pancreatitis.

To study the relation between expression of the pancreatitis-associated protein (PAP) and severity of pancreatitis in rats, different degrees of experimental pancreatitis were induced by a 1-, 3-, or 5-h cerulein infusion (5 micrograms kg-1 h-1). This treatment decreased pancreatic volume secretion to below 10%. Immediately after infusion, the secretion rate increased to approximately 50% of control. Within 1 day, volume and bicarbonate secretion rates were not different from controls. At this point, protein secretion amounted to 30% of control, but only in animals receiving the 3- or 5-h dose. The values increased to 40-60% within 3 days. In all groups, the isoenzyme pattern was not influenced by the cerulein treatment. One day after induction of pancreatitis, the PAP was found in pancreatic juice in concentrations related to the dose of cerulein given. By immunohistochemical techniques, the protein was localized over acinar cells, but was not detectable in interstitial tissue, islets, or in the healthy exocrine pancreas. Pathomorphologic alterations in the pancreas were quantified by a scoring system. One and 2 days after the treatment, a more severe pancreatitis and more elevated levels of PAP were found in animals treated with the higher dose of cerulein. It is concluded that PAP is expressed in the pancreas in relation to the severity of cerulein-induced pancreatitis.

Nucleolar organizer regions and glycoprotein-hormone alpha-chain reaction as markers of malignancy in endocrine tumours of the pancreas.

The value of silver staining of nucleolar organizer regions (AgNORs) and human chorionic gonadotropin alpha-chain reaction (HCG-alpha) as markers of malignancy was investigated in 60 primary pancreatic endocrine tumours, 37 of which had metastasized at the time of surgery, and in one of which metastases developed 4 years after surgery. Assessment of AgNORs by digital image analysis revealed few but large AgNORs (mean number 2.5 +/- 1.1; mean area 0.32 +/- 0.1 microns 2) in the 22 benign tumours and many but small AgNORs (mean number 5.1 +/- 1.9, P < 0.05; mean area 0.18 +/- 0.09 microns 2, P < 0.01) in the malignant tumours. Quantification of the number of AgNORs per tumour cell nucleolus (AgNOR distribution score) showed that 96% (26/27) of tumours exhibiting at least 5% of cells with more than six AgNORs per nucleolus showed metastases either at the time of diagnosis or up to 4 years after surgery. HCG-alpha immunoreactive cells were present in 25/38 (66%) malignant tumours and in 4/22 (18%) benign tumours. Combined evaluation of AgNOR distribution and HCG-alpha scores showed a high positive predictive value of 96% in cases with a raised AgNOR distribution score irrespective of the HCG-alpha status. A good negative predictive value (81%) was, however, only obtained if both parameters, AgNOR distribution and HCG-alpha scores, were negative. Thus, investigation of AgNORs and HCG-alpha is helpful in predicting malignancy in a high percentage of pancreatic endocrine tumours.

Glucagon-like peptide-1 cells in the gastrointestinal tract and pancreas of rat, pig and man.

A highly specific monoclonal antibody directed against the C-terminal part of glucagon-like peptide-1 (GLP-1) was raised to immunohistochemically evaluate the distribution of GLP-1 containing cells in the entire gastrointestinal tract including pancreas of rat, pig and man. In the pancreas GLP-1-immunoreactive cells were found variously shaped and predominantly located in the periphery of the islets. Ultrastructurally, GLP-1 was co-localized with glucagon in the alpha-granula of A-cells and was mainly restricted to the electrondense core. In the intestine open type cells reaching the lumen via a slender apical process were stained with the GLP-1 antibody. They occurred in all parts of the crypts but predominantly in the basal portion. The density of GLP-1 immunoreactive cells varied between species in a characteristic order: rat greater than pig greater than man. In pig and human gut a large number of cells occurred in the distal jejunum and ileum. A continuous increase of cell densities was found from the proximal to the distal colon resulting in highest numbers in the rectum. In rats the highest cell density occurred in the ileum. Again, a continuous increase of GLP-1-positive cell numbers was evident from the proximal to the distal portion of small and large bowel. GLP-1 was partly co-localized with PYY. The GLP-1 positive cells appeared electronmicroscopically as L-cells with the typical large granula. This morphological data indicates that GLP-1-releasing cells in the small intestine are appropriately positioned in the distal part to sense and respond to the presence of nutrients that have escaped the absorptive surface of the upper small intestine.

Recurrent acute pancreatitis and intraluminal duodenal diverticulum.

An intraluminal duodenal diverticulum (IDD) and a partial pancreas divisum were diagnosed in a 22-year-old man who exhibited recurrent attacks of acute pancreatitis. Resection of the diverticulum resulted in a complete disappearance of symptoms. The possible etiological relationship between IDD and recurrent acute pancreatitis is discussed.

Hormone-induced pancreatitis.

Intravenous infusion of the synthetic cholecystokinin analogue cerulein at a dose of 0.25 micrograms/kg/h causes maximal stimulation of pancreatic exocrine secretion. The infusion of supramaximal doses of cerulein (5 and 10 micrograms/kg/h) induces a significant increase in pancreatic enzymes in blood, and interstitial edema and inflammatory cell infiltration. This model of hormone-induced pancreatitis works in rats, mice, dogs and hamsters. Besides intravenous infusion, repeated intraperitoneal injections can also be used for induction of pancreatitis. In the early phase of cerulein-induced pancreatitis, large autophagic vacuoles result from fusion of zymogen granules within the acinar cell. This is accompanied by an increase in lysosomal enzyme activity and activation of trypsinogen which finally leads to cellular necrosis. All animals survive the induction of pancreatitis. The pancreas completely regenerates within 6 days after induction of pancreatitis. This model of experimental pancreatitis favors the analysis of intracellular events in the early phase of pancreatitis.

Lung injury in acute experimental pancreatitis in rats. I. Morphological studies.

The pathogenesis of pancreatitis-related pulmonary injury was studied at the light- and electronmicroscopic level. Experimental pancreatitis was induced in rats by infusion of supramaximal doses of cerulein for 12 h. Investigations were carried out 3, 6, and 12 h after the start of infusion and 12, 48, and 72 h after the end of pancreatitis induction. Initial manifestations of pancreatitis-associated lung injury revealed a pronounced clustering of polymorphonuclear leukocytes in pulmonary microvessels, followed by severe damage of alveolar endothelial cells. Consecutively, the increase in vascular permeability of the lung resulted in interstitial edema formation. Structural changes were maximal after 12 h and reversed completely after 84 h. In conclusion, the structural appearance of pulmonary injury in cerulein-induced pancreatitis was similar to that reported in early stages of the adult respiratory distress syndrome (ARDS). It is suggested that polymorphonuclear granulocytes play a crucial role in the pathogenesis of pancreatitis-related lung injury.

Lung injury in acute experimental pancreatitis in rats. II. Functional studies.

In this study we report the functional changes in isolated perfused lungs from rats with cerulein-induced experimental pancreatitis. Rat lungs isolated immediately after the cerulein infusion demonstrated decreased pressor responses to angiotensin II (A II) and acute hypoxia (FIO2: 0.0). The lung wet- to dry-weight ratio was increased, as was the lung-leak index, consistent with high-permeability edema formation in the lung. Neither saline-solution infusion for 12 h nor perfusion with cerulein of rat lungs isolated from untreated animals caused lung injury or functional alterations. The changes in pulmonary vascular reactivity were normalized 48-72 h after induction of pancreatitis. In conclusion, we describe an animal model of pancreatitis and reversible, ARDS-like lung injury.

Evidence of intracellular activation of serine proteases in acute cerulein-induced pancreatitis in rats.

It is believed that activation of zymogen proteases occurs in the early development of acute pancreatitis. This hypothesis was proved on subcellular fractions of rat pancreas after induction of pancreatitis by infusion of high doses of cerulein for 2 h. Secretory enzyme activities were measured spectrophotometrically in subcellular fractions obtained by differential ultracentrifugation. Additionally, trypsin and chymotrypsin activities were detected by enzyme blots after isoelectric focusing. Finally immunoblotting (Western-blot analysis) for amylase, lipase, trypsin/ogen, and chymotrypsin/ogen was carried out on fractions separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In cerulein pancreatitis, subcellular fractions of secretory granules and vacuoles showed significant amounts of free trypsin and chymotrypsin activities compared with controls. The presence of free activities of serine proteases was paralleled by the appearance of numerous low molecular weight peptides detected by 2-dimensional electrophoresis and SDS-PAGE, which in part represented proteolytically cleaved secretory proteins. It is concluded that the intracellular activation of serine proteases that occurs in cerulein pancreatitis could contribute to further acinar cell destruction.

Effects of positive end-expiratory pressure (PEEP) ventilation on the exocrine pancreas in minipigs.

The effect of positive end-expiratory pressure (PEEP) ventilation on the pancreas was studied in 22 anesthetized minipigs. Five pigs were treated with intermittent positive pressure ventilation (IPPV) for 21 h (Group I) and six pigs were treated with a PEEP of 15 cm H2O for 21 h (Group II). We next explored the influence of PEEP ventilation while stimulating the pancreas with Ceruletide, a synthetic cholecystokinin (CCK) analog, at 2.3 micrograms/kg per h.i.v. for 3 h. Ventilation with IPPV for 4 h (n = 5, group III) was compared with PEEP of 15 cm H2O for 4 h (n = 6, group IV). Changes in serum-lipase were observed in all groups. The average lipase level rose from 12.6 U/l to 67 U/l group I and from 15.1 U/l to 129.2 U/l in group II (P = 0.025 for group I vs group II). In group IV (PEEP and Ceruletide), the mean lipase level rose about six times more than in group III (IPPV and Ceruletide). The difference was significant (P less than 0.00005). By three-factor ANOVA analysis, effects due to the drug (P less than 0.000006) and to PEEP (P less than 0.038) as well as a threefold interaction could be demonstrated, i.e., using Ceruletide a greater PEEP effect on lipase than without the drug (P less than 0.023) took place. There were no significant histological changes of the pancreas in groups I and III. In group II (21 h PEEP), vacuolization of acinar cells was evident; on the ultrastructural level, indication was given that these vacuoles derived both from the Golgi apparatus and from fusion of individual zymogen granules. In group IV (PEEP and Ceruletide), the focal appearance of fatty-tissue necrosis and acinar cells as well as hemorrhage of the gland was observed. We conclude that PEEP ventilation impairs the function of the pancreas and produces even more deleterious effects when the gland is stimulated. These effects should be considered when PEEP is used in clinical practice in the treatment of acute pancreatitis.

Mechanism of acute pancreatitis. Cellular and subcellular events.

A membrane-bound system through which secretory and lysosomal proteins travel in a vectorial fashion is essential for the preserved integrity of pancreatic acinar cells. This system is composed of an ordered array of compartments, such as the rough endoplasmic reticulum, the Golgi complex, lysosomes, and secretory granules. As a principle, in acute pancreatitis the final steps of this transport seem to be disturbed. Caerulein-induced pancreatitis is a valuable experimental model for studying altered intracellular transport, and compartmentation of lysosomal and digestive enzymes. The formation of enlarged secretory vacuoles containing lysosomal and digestive enzymes is paralleled by the activation of lysosomes and degradation of cellular organelles in autophagosomes. On the level of secretory and autophagic vacuoles, activation of serine proteases occurs, which in addition to increasing lysosomal enzyme activities can represent the initial stage for acinar cell destruction and the development of pancreatitis.

[Ultrastructural diagnosis of neuroendocrine tumors using fine needle biopsy]. / Ultrastrukturelle Diagnostik neuroendokriner Tumoren an Feinnadelpunktaten.

Ultrasonic-guided fine-needle-biopsies of primary pancreatic tumours or liver metastasis were performed in 13 patients with neuroendocrine tumours of the gastrointestinal tract (5 carcinoids, 3 gastrinoma, 1 PPoma, 1 calcitoninoma, 1 insulinoma, and 2 non-functional tumours). Specimens obtained were examined on the light- and electronmicroscopic level. In all cases ultrastructural examination sufficiently revealed the correct diagnosis, due to the presence of cytoplasmic neuroendocrine granules within the tumour cells. Additionally performed immunocytochemical investigations at the ultrastructural level enabled the discrimination of gastrinomas, insulinomas, and PPomas. In contrast, light-microscopic examination was less sensitive for tumour classification. It is concluded that ultrastructural investigation of fine needle biopsies represents a valuable method to sufficiently discriminate neuroendocrine neoplasms.

[Paralytic ileus as an initial manifestation of malignant VIPoma of the pancreas--case report with review of the literature]. / Paralytischer Ileus als Erstmanifestation eines malignen VIPoms des Pankreas--Fallbeschreibung mit Literaturübersicht.

A 57-year old patient with a paralytic ileus of unknown origin was admitted to the intensive care unit. Because of the laboratory findings with therapy resistant hypokalemia, hypercalcemia and metabolic acidosis a VIPoma was suspected. Therapy with somatostatin resulted in correction of laboratory abnormalities and in normalization of gastrointestinal motility. Plasma concentrations of VIP and PP were elevated, ultrasonography revealed a pancreatic tumor. Postsurgical examination of the removal tumor tissue confirmed the diagnosis of a malignant VIPoma. Clinical symptoms, laboratory findings with and without somatostatin-therapy and immunhistochemical properties are described.

Localization of lysosomal and digestive enzymes in cytoplasmic vacuoles in caerulein-pancreatitis.

Intracellular localization and enzymatic activities of lysosomal enzymes (cathepsin B, N-acetyl-beta-glucosaminidase, and beta-glucuronidase) were studied in control rats and after induction of caerulein pancreatitis. In control rats high enzymatic activities were found in the postnuclear 1000 g fraction (purified zymogen granules). The corresponding subcellular fraction in pancreatitis animals additionally contained larger secretory vacuoles and autophagosomes and revealed a marked increase in lysosomal enzyme activities. Immunolabelling studies at the ultrastructural level for trypsinogen and cathepsin B demonstrated a colocalization of lysosomal and digestive enzymes in zymogen granules in healthy controls. After induction of pancreatitis immunolabelling still demonstrated a colocalisation of cathepsin B and trypsinogen in secretory granules and newly formed Golgi-derived secretory vacuoles. Concomitantly appearing autophagosomes were, however, only labelled for cathepsin B. It is concluded that segregation of lysosomal and digestive enzymes is incomplete in normal acinar cells resulting in a colocalization in zymogen granules. In pancreatitis colocalization in secretory granules is maintained, whereas only lysosomal enzymes were sufficiently transferred into autophagic vacuoles. No indication for impaired mechanisms of molecular sorting of lysosomal and digestive enzymes in caerulein-induced pancreatitis was found.

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas.

Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.

Caerulein-induced acute pancreatitis in rats: changes in glycoprotein-composition of subcellular membrane systems in acinar cells.

Caerulein-induced acute pancreatitis is characterized by the occurrence of two membrane-bound vacuolar systems in acinar cells. Beside digestive enzymes containing secretory vacuoles, lysosomal autophagic structures can be identified at the ultrastructural level. In the present study glycoconjugate patterns of the surrounding membranes were characterized by ultrastructural lectin-binding experiments using five colloidal-gold labeled lectins with distinct sugar specificities. Furthermore, the profile of membrane glycoproteins of isolated vacuolar fractions was studied by SDS-PAGE and lectin-blotting. In pancreatitis, membranes of secretory vacuoles showed a significant lower degree of lectin-binding compared to normal zymogen granules. In contrast, newly appearing autophagic vacuoles in pancreatitis revealed a strong membrane labelling for most lectins used. The pattern of membrane glycoproteins of secretory and autophagic vacuoles as determined by SDS-PAGE and lectin-blotting differed from those of normal zymogen granules resembling the protein profile of smooth microsomes. Since this pattern requires a previous passage through Golgi stacks, it is assumed that the two types of vacuoles derive from Golgi elements. For the pathogenesis of caerulein pancreatitis these vacuolar post-Golgi structures seem to play an important role.

Cerulein-induced acute pancreatitis in rats: evidence for reduced calcium affinity of secretory granule membranes.

Membranes of secretory granules in pancreatic acinar cells seem to be interrelated in the regulation of intragranule Ca2+ concentrations. Since low intragranule Ca2+ levels are involved in zymogen stabilization versus autoactivation of proteases, a disturbance of the Ca2(+)-regulating system in secretory granules could be invoked to account for uncontrolled proenzyme activation. This is proposed as the initial mechanism in the pathogenesis of acute pancreatitis. Using pancreatic subcellular fractions obtained from control rats and after induction of acute cerulein pancreatitis we found a markedly reduced Ca2+ affinity of membranes from the secretory granule fraction in pancreatitis. The strong Ca2+ binding of control zymogen granule membranes primarily seemed to be a function of non-proteinacous membrane components, e.g. phosphatidylinositols. It is suggested, that part of the inner surface of membranes from secretory granules acts as a calcium-buffering system that works in synergy with other protective mechanisms to stabilize the zymogen granule population. In cerulein pancreatitis there seemed to be an imbalance of this system.

Histochemical and ultrastructural characteristics of tubular complexes in human acute pancreatitis.

The morphologic characteristics of ductlike tubular complexes were studied in human acute pancreatitis. Pancreatic specimens were obtained from 10 patients who were operated on for acute pancreatitis. Immunocytochemistry for pancreatic enzymes, keratin, actin, and carcinoembryonic antigen were combined with lectin-binding studies and ultrastructural investigations. Irrespective of clinical onset and duration of pancreatitis, tubular complexes situated in the vicinity of fat necrosis were observed in all patients. Intermediate forms of ductlike structures were characterized by widening of acinar lumina, decreased height of acinar cells, and large autophagic vacuoles. These structures bound all of the lectins employed and retained their immunoreactivity to secretory proteins. Typical tubular complexes were composed of low cuboidal or flattened cells surrounding a large acinar lumen. They revealed a loss for pancreatic enzymes, a reduced lectin-binding for L-fucose and N-acetylgalactosamine, and an increase for cytoskeletal proteins (keratin, actin). It is concluded that tubular complexes in human acute pancreatitis represent degenerating acinar cells which lost their secretory and membrane characteristics.

Immunocytochemical and morphometric analysis of acinar zymogen granules in human acute pancreatitis.

In the present study fine structural changes of acinar zymogen granules were investigated in human acute pancreatitis. Pancreatic tissue was obtained at surgery from 6 patients, prepared for ultrastructural analysis, and stained immunocytochemically for trypsinogen. Stereological parameters of zymogen granules were evaluated. The density of the immunocytochemical labelling for trypsinogen was estimated over zymogen granules, the rough endoplasmic reticulum, Golgi apparatus and the acinar lumina. In acute pancreatitis the number of zymogen granules was diminished and their size reduced. The density of the labelling for trypsinogen was unchanged over zymogen granules but showed a significant reduction over the rough endoplasmic reticulum, Golgi apparatus, and the acinar lumina. In general the integrity of zymogen granules was well preserved. Focally degenerative changes of zymogen granules and large autophagosomes were observed. From the immunogold labelling a disturbance of enzyme synthesis and secretion was suggested. Evidence is given that a disruption of the zymogen granule membranes and a fusion with lysosomal bodies might contribute to the pathogenesis of human acute pancreatitis.

[Age-related changes in glycosaminoglycans in the cell nuclei of the rat liver--biochemical and histochemical pilot studies]. / Alternsabhängige Veränderungen der Glycosaminoglycane in Zellkernen der Rattenleber--biochemische und histochemische Pilotstudien.

Cell nuclei of the livers of 12 male Wistar rats aged 24 months, and 16 male Wistar rats aged 4 months were isolated. 0.4 microCi [3H]-glucosamine/kg body weight were administered intraperitoneally. The livers of four animals were used for each biochemical analysis. The tissue was degraded by proteolysis, the glycosaminoglycans (GAGs) were isolated and the GAG types were determined by enzyme digestion, nitrite degradation and radiometry. The amount of GAGs was photometrically determined in single samples of pooled material of each of the two age groups. In paraffin sections (from other male Wistar rats of a different age), the influence of enzymes on specific GAG staining was observed. Most of the radioactivity was found in heparan sulfate (HS) and a lower content in chondroitin sulfates (CSs). In HS the incorporation increased with increasing age. The amount of HS showed no age-related differences. The results indicate an age-related activation of the HS metabolism. The GAG pattern and the age-related changes of the GAG types in total tissue (earlier results) are different from those in the nuclei. By histochemical methods, we observed a small but distinct effect of heparitinase in the cell nuclei.