Andreas U. Freiburghaus

Injection of Microspheres into Pancreatic Arteries Causes Acute Hemorrhagic Pancreatitis in the Rat: A New Animal Model

Alterations in the vascular bed of the pancreas or disturbances of the blood coagulation system are mostly considered to be sequelae of acute pancreatitis, but it seems that impairment of the pancreatic blood supply can per se lead to acute hemorrhagic pancreatitis. To test this hypothesis with a new animal model, we injected 20 μm polystyrene microspheres retrogradely into the distal splenic artery of rats, thus incompletely blocking blood perfusionin the splenic portion of the pancreas. Eight of eight rats (100%) subjected to microsphere injection developed acute hemorrhagic pancreatitis by 27 h after surgery, when they were killed, but none of the six sham-operated control animals (0%) showed macroscopic signs of pancreatitis. Blood amylase levels at death were 3,087 ± 650 I.U./L (mean ± SEM) and the histologic severity score for pancreatitis was 10.8 ± 1.0 (mean ± SEM), whereas in the six control rats amylase levels were 1,375 ± 158 I.U./L and the histology score was only 1.7 ± 1.0. The result is, with p < 0.0005, highly significant (x2 analysis) and shows that acute experimental pancreatitis can indeed be induced by partially blocking the arterial blood supply within the organ.

A universal method for two-dimensional polyacrylamide gel electrophoresis of membrane proteins using isoelectric focusing on immobilized pH gradients in the first dimension.

Two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension, initially applied for the separation of soluble and total cellular proteins, has been extended to the analysis of membrane proteins. We show that the usual procedures lead to artifacts and irreproducible results due to aggregation and precipitation of proteins and protein-phospholipid complexes during isoelectric focusing (first dimension) and sodium dodecyl sulfate (SDS) gel electrophoresis (second dimension). Optimized solubilization procedures for hydrophobic membrane proteins are presented and the use of dilute samples is shown to be essential to overcome the major problems in isoelectric focusing. Increased volumes of samples dissolved in rehydration buffer are applied by direct rehydration of dry immobilized pH gradient (IPG) gels. Isoelectric focusing in 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) without urea gives good results as does 2% Nonidet-P40 with 8 M urea. Heat denaturation should be avoided. An optimized equilibration procedure for IPG gel strips in SDS sample buffer prior to separation in the second dimension was developed that minimizes loss of proteins and results in high-resolution two-dimensional electropherographic maps with a minimum of streaking. The gel strips are partially dehydrated at 40 degrees C and shortly reswollen in situ on the SDS slab gel in SDS-sample buffer containing agarose.

Occupational risks of (meth)acrylate compounds in embedding media for electron microscopy

(Meth)acrylates are the main constituents of embedding media widely used in electron microscopy research for low‐temperature embedding of biological tissue. (Meth)acrylates toxicology is still incompletely understood and therefore an estimation of health hazards involved in handling must be inaccurate. (Meth)acrylate monomers are known to be harmful to skin and other tissues and may sensitize workers. Since low‐temperature electron microscopy techniques have gained popularity in research laboratories, it is important to establish safety rules for handling the (meth)acrylate‐containing solutions.

Influenza vaccination coverage rates in Austria in 2006/07 – a representative cross-sectional telephone survey

ZusammenfassungZiel der Studie war die Erfassung der Influenza-Durchimpfungsrate in Österreich während der Saison 2006/07 und die Erhebung von Motiven für und gegen die Impfung. 2000 Telefon-Interviews mit Personen über 15 Jahren wurden durchgeführt. Die Durchimpfungsrate in der Gesamtpopulation lag bei 17,8 %. Bei den über 65-jährigen waren 32,1 % geimpft, die adjustierten Quotenverhältnisse der Geimpften in dieser Gruppe, verglichen mit denen, die nicht zu einer Hochrisikogruppe gehören, lag bei 3,8. Bei der Gruppe der chronisch Kranken und dem Personal des Gesundheitswesens ergaben sich adjustierte Quotenverhältnisse von 2,6 bzw. 1,5, während geimpfte chronisch Kranke ältere Personen ein adjustiertes Quotenverhältnis von 7,0 aufwiesen. Das Ansteckungsrisiko zu vermindern war der häufigste Grund, sich impfen zu lassen (35,2 %), eine Empfehlung des Hausarztes erwies sich als der wichtigste motivierende Faktor für die Impfung (46,4 %). Der Hauptgrund, sich nicht impfen zu lassen, war Desinteresse (>50 %). Die Durchimpfungsrate in Österreich während der Saison 2006/07 war niedrig und weit hinter den Empfehlungen der WHO.SummaryThe objective of the study was to evaluate influenza vaccination coverage in Austria in the season of 2006/07 and to understand motivations and barriers. Two-thousand telephone interviews with individuals over 15 years of age were conducted. The overall influenza vaccination coverage rate was 17.8%. In the elderly (>65 years) the rate was 32.1%, and the adjusted odds ratio of being vaccinated, compared to those not belonging to a high-risk group, was 3.8. Chronically ill persons and health care workers had adjusted odds ratios of 2.6 and 1.5, respectively, while chronically ill elderly persons had an odds ratio of vaccination of 7.0. Minimizing the risk of contracting influenza was the most frequent reason for getting vaccinated (35.2%), and a recommendation by the family doctor was perceived as the major encouraging factor for vaccination (46.4%). The main reason for not getting vaccinated was indifference (>50%). Vaccination coverage in Austria in 2006/07 was low and far behind WHO objectives.

Does acute pancreatitis progress to chronic pancreatitis : a microvascular pancreatitis model in the rat

Ischemia as a causative factor for acute pancreatitis has been discussed for decades but has only recently gained wider acceptance. Chronic pancreatitis, however, has rarely been attributed to ischemic injury. While experimental evidence is available for the ischemic pathogenesis of acute pancreatitis, no studies have been reported about pancreatic ischemia as a single cause of chronic pancreatitis. Also, the progression from acute to chronic pancreatitis has been a very controversial issue. To address both questions we have injected polystyrene microspheres of 20-pm diameter into the pancreatic branches of the splenic artery of 36 rats. Thirteen more rats were sham operated and injected with saline. The animals were killed at 1,2, 3, and 9 weeks after operation and macroscopically and histologically examined, and serum a-amylase and weight gain were determined. For the pancreas the following parameters were assessed using a score from 0 (no change) to 4 (severe change): atrophy, hemorrhage, edema, fat necrosis, acinar necrosis, polymorphonuclear infiltration, mononuclear infiltration, interstitial fibrosis, and ductal changes. While no difference between control and experiment was observed for serum a-amylase, weight gain, edema, and hemorrhage, persistent differences were evident for the parameters characteristic of chronic pancreatitis, most significantly for interstitial fibrosis, ductal changes, mononuclear infiltration, acinar necrosis, and atrophy. No spontaneous deaths occurred. The severity of the lesions remained stationary after the first week. Our work shows for the first time that pancreatic ischemia by microvascular hypoperfusion can cause histopathologic changes characteristic of chronic pancreatitis and that these changes follow acute necrotizing pancreatitis.

Element distribution in organelles of pancreatic acinar cells of rat, mouse, and pig investigated by energy-dispersive X-ray microanalysis.

The exocrine pancreas was long thought to be composed of identical subunits, the acinar cells that store the inactive forms of the digestive enzymes in zymogen granules (ZGs). These were generally seen as a homogeneous population of vesicles. This homogeneity was recently questioned: Digestive demands are answered by the release of specific enzymes and immunocytochemical labeling showed distinctive nonidentical populations of ZGs. We have aimed at finding concomitant differences in element contents. We analyzed by energy-dispersive x-ray microanalysis (EDX) the subcellular distribution of elements in acinar cells of resting and stimulated rat, resting mouse, and resting pig pancreas and compared the results with values from the literature. We found large variances in the concentrations of Na, Mg, P, S, Cl, K, and Ca in cytoplasm rich in endoplasmic reticulum (C/E), whereas the concentrations of P, C1, K, and Ca in mitochondria and ZGs had surprisingly small variations. Na and Mg were detected in measurable amounts only in C/E and mitochondria and Ca was detectable only in ZGs. We could not find any other elements. We have not found clearly distinguishable populations of ZGs. We critically discuss our findings in comparison with the literature. Many discrepancies can be explained by the different preparation procedures. We show that it is questionable to present absolute values of concentration in biological specimens on the basis of EDX. The technique should, in our opinion, be used only for the study of relative concentrations.

Identification of a glycosylphosphatidylinositol-anchored glycoprotein with nucleoside phosphatase activity on the membrane of pig pancreatic zymogen granules.

The molecular events between the second messenger-mediated triggering of regulated exocytosis and the subsequent fusion of the secretory granules with the apical plasma membrane are unclear. The glycoprotein GP-2, the most abundant of the very few proteins of the pancreatic zymogen granule membrane has been cloned and sequenced in dog and rat, but no (enzymatic) function has so far been ascribed to it. Nucleoside phosphatase activities associated with the pig zymogen granule membrane were recently assumed to be related to GP-2. To identify the protein(s) carrying these activities we have used a novel combination of native and denaturing one- and two-dimensional polyacrylamide gel electrophoresis in the detergents CHAPS, Triton X-100 or SDS. Histochemical examination on the gels and incubation with lectins and phosphatidylinositol phospholipase-C have allowed characterization of the protein with the nucleoside di- and tri-phosphatase activities. SDS-PAGE of the single protein spot with nucleoside phosphatase activity excised from Triton X-100 2-dimensional gels showed the presence of 92 kDa and 67 kDa glycoproteins. The isolated protein had an isoelectric point of 5.2, formed high molecular weight complexes, was shown to be glycosylphosphatidylinositol-anchored and contained complex carbohydrate structures. It hydrolyses di- and tri-phosphate nucleotides in dependence of the glycosylphosphatidylinositol anchor and is sensitive to non-mitochondrial diphosphohydrolase inhibitors. In summary, this paper identifies GP-2 as a nucleoside phosphatase within the zymogen granule membrane, suggesting it may be involved in energy-requiring processes on the cytosolic side of the granules.

Nucleoside phosphatase activities on pig pancreas zymogen granule membranes analyzed by nondenaturing polyacrylamide gel electrophoresis

The membrane of the pancreatic zymogen granule plays an important part in the sequence of storage, transport and exocytosis of digestive enzymes. While much is known on stimulus-secretion coupling, very little is understood about how the storage organelles move in the cytoplasm to the luminal plasma membrane and why and how they fuse with it to release the contents. It is assumed that nucleoside phosphatases are involved in these energy consuming processes. Pancreatic zymogen granule membranes contain one major glycoprotein, GP-2, and a few minor proteins all with unknown functions. In order to identify functions we have purified zymogen granule membranes from pig pancreas, solubilized the proteins under non-denaturing conditions with the detergent CHAPS and characterized the extracted proteins by polyacrylamide gel electrophoresis, histochemistry and lectins. Three major protein bands, often fused in one broad band, revealed enzymatic activity for adenosine-, cytidine-, inositol- and guanidine- di- and triphosphates by the precipitation of liberated phosphate by Pb(NO3)2. This activity was sensitive to known ATP diphosphohydrolase inhibitors. The band with activity arises from a 92 kDa glycoprotein. A different narrow band showed monophosphatase activity for AMP, GMP, IMP and CMP. Some of the activities were inhibited by different lectins, indicating glycosyl groups near the active site. Electron microscopical cytochemistry confirmed a nucleoside phosphatase activity on granule membranes. Our results show for the first time that the nucleoside phosphatase activity of the zymogen granule membranes is carried by a 92 kDa glycoprotein, probably the known self-associating form of GP-2. The hydrolysis of tri- and diphosphate nucleotides could provide the energy required by exocytosis.