Abstract: IgE-mediated allergies, such as allergic rhinoconjunctivitis and asthma, have become highly prevalent, today affecting up to 35% of the population in industrialized countries. Allergen immunotherapy (also called hyposensitization therapy, desensitization or allergen-specific immunotherapy), the administration of gradually increasing amounts of an allergen, either subcutaneously or via the sublingual or oral route is effective. However, only few allergy patients (<5%) choose immunotherapy, as treatment duration is over years and because allergen administrations are associated with local and in some cases even systemic allergic side effects due to allergen accidentally reaching the circulation. Therefore, ideally the allergen should be administered to a site that contains high numbers of potent antigen-presenting cells in order to enhance efficacy and shorten treatment duration, and ideally that site should also be nonvascularized in order to prevent both systemic distribution of the allergen and systemic allergic side effects. The epidermis, a nonvascularized multilayer epithelium that contains high numbers of potent antigen-presenting Langerhans cells, could therefore be an interesting administration route.
Abstract: In order to understand motivations and barriers to vaccination, and to identify people's intentions to get vaccinated for season 2007-8, influenza vaccination coverage was assessed in the United Kingdom (UK) from 2001 to 2007. Between 2001 and 2007 representative household surveys were performed by telephone interview with 12,143 individuals aged 16 or older. The overall influenza vaccination coverage rate dropped non-significantly from 25.9% in 2005-6 to 25.0% in 2006-7 (p=0.510). In the elderly (>/=65 years) the rate decreased from 78.1% to 65.3% (p=0.001), and the odds ratio of being vaccinated compared to those not belonging to any of the risk groups targeted by vaccination decreased from 36.6 to 19.9. Healthcare workers and chronically ill persons had odds ratios of 2.0 and 15.5, respectively. The most important reason for getting vaccinated was a recommendation by the family doctor or nurse, and this was also perceived as the major encouraging factor for vaccination. No recommendation from the family doctor was the main reason for not getting vaccinated. A total of 38.4% of the respondents intended to get immunised against influenza in 2007-8. From 2001 to 2006 a slightly increasing trend (p for trend across seasons <0.0001) in vaccination coverage was observed in the UK, but in 2006-7 the rates returned to the level of 2004-5. Less media attention to the threat of avian influenza after 2005 may have contributed to the recent decrease of vaccination rates.
Abstract: Influenza is a considerable health problem all over the world. Vaccination is the most important measure for preventing influenza and reducing morbidity and mortality. The aims of this study were to assess influenza vaccination coverage from 2001 to 2007 in Germany, to understand motivations and barriers to vaccination, and to identify vaccination intentions for season 2007/08.
Abstract: The 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.
Abstract: PURPOSE: To prospectively determine the safety, feasibility and efficacy of a new atherectomy device. Methods. Seventy patients with 93 symptomatic femoro-popliteal lesions underwent percutaneous atherectomy with Redha-cut, a metallic, flexible catheter. The device works on pull-back with two umbrella-like blades and enables cutting, retaining and removing of material. Quantitative analysis of the angiograms was performed. RESULTS: Obtaining of atherectomized samples was successful in all cases. The mean atherectomy time was 5 +/- 2 minutes. The stenoses were reduced from 74 +/- 2.3% before to 26 +/- 2% after atherectomy (p < 0.001). Subsequent PTA, which was performed to optimize the results, further decreased the degree of stenosis to 17% (p > 0.5). No perforations, acute closure or early re-thrombosis occurred. Cumulative patency rates according to the findings of non-invasiveive examinations were 83% at 6 months and 66% at 12 months. Conclusions. The Redha-cut device is a simple, safe and efficacious tool for biopsy sampling and atherectomy. Further studies randomizing patients either to balloon or atherectomy are planned to compare this technique with others in regard to long-term patency.
Abstract: In 11 female and 13 male patients 29 symptomatic femoropopliteal occlusions were treated with a novel intraluminal endarterectomy device (REDHA-CUT). The pre-existing stenoses (71 +/- 9%) could be reduced to 29 +/- 18% (means +/- s.d.). The arteriectomy procedure with this device took on average 5 +/- 3 min. Neither perforations nor dissections nor distal embolisms or any other complications were observed. In comparison with other percutaneous transluminal angioplasty procedures the clinical use of the novel REDHA-CUT device is safe, simple and fast and allows for the retrieval of plaque biopsies.
Abstract: 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.
Abstract: Contrast echocardiography with sonicated radiographic contrast agents has been used for the qualitative and quantitative determination of myocardial blood flow. One major problem has been the size of the microbubbles since only bubbles smaller than 8 microns are expected to pass the capillary bed and larger bubbles may obstruct the capillaries and, thus, alter myocardial blood flow. These techniques have been used for several years, but their reliability has not yet been assessed accurately. Five different methods for the production of sonicated radiographic contrast agents (methods 1-3 from the literature, and 4 and 5 from our laboratory; M1-5) were evaluated for their use in quantitative contrast echocardiography. The sonication of non-ionic X-ray contrast media was performed with a standard titanium probe (20 kHz) for methods 1-4, with variation in the sonication time and the number of sonication jets used for each method. In M5, we used bubbles that were produced by the insufflation of oxygen in the X-ray contrast agent; large (> 8 microns) bubbles were destroyed by sonication at 380 kHz (resonance method). Mean bubble size was determined by computerized videomicroscopy. The effect of bubble size on the backscatter of the ultrasonic signal was calculated for each method. Mean bubble size (+/- 1 SD) ranged between 11.5 +/- 4 microns and 16.1 +/- 14 microns for M1-M5. The best values, i.e., the smallest bubbles, were found with M4 (prepressurized contrast medium). Assuming capillary passage for bubbles smaller than 8 microns, only 14%-48% of the bubbles were smaller than 8 microns (M1-M5). The best results with regard to bubble size (< or = 8 microns) were observed with M5 (48% < or = 8 microns). In regard to the influence of bubble size on the backscatter of the ultrasonic signal, 56%-98.5% of the signal was produced by bubbles larger than 15 microns (M1-5) but the best results were obtained with M4. It is concluded that capillary-passage of sonicated microbubbles (< or = 8 microns) can be expected in only 14%-48% of the bubbles for the five different sonication techniques. More than 50% of all microbubbles produced by these techniques are larger than the expected 8 microns. These large bubbles are responsible for the backscatter of the ultrasonic signal in the vast majority of cases. Thus, the sonication of radiographic contrast agents appears to be inappropriate for the production of uniformly small microbubbles and, thus, this method is not suitable for quantitative measurements of coronary blood flow.
Abstract: Studies in humans in vivo have demonstrated that substances found in grapefruit juice may increase the bioavailability of dihydropyridine derivatives as a result of the inhibition of liver enzyme activities by flavonoids found in grapefruit. Since the metabolism of dihydropyridine drugs is mediated by cytochrome P-450 (CYP) 3A4, it has been hypothesized that flavonoids may also influence the metabolism of other drugs, such as midazolam and quinidine, which are biotransformed by the same CYP isoform. Three flavonoids, kaempferol, naringenin and quercetin, are found in grapefruit juice but not in orange juice. The effect of these substances on the metabolism of midazolam and quinidine has been investigated in human liver microsomes. In the concentration range 10-160 microM the inhibitory potential of flavonoids was the same for both of the tested drugs; it decreased in the order quercetin >> kaempferol > naringenin. The data suggest that the flavonoids found in grapefruit juice may influence the kinetics of midazolam and quinidine in man.
Abstract: 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-microns 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 alpha-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 alpha-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.
Abstract: 1. Theophylline metabolism was studied using seven human cytochrome P-450 isoforms (CYPs), namely CYP1A1, 1A2, 2A6, 2B6, 2D6, 2E1 and 3A4, and microsomal epoxide hydroxylase (EH), expressed in human B-lymphoblastoid cell lines. 2. At a high theophylline concentration of 10 mM four CYPs (1A1, 1A2, 2D6, 2E1) catalyzed the metabolism of theophylline. 3. Theophylline had the highest affinity (apparent Km range 0.2-1.0 mM) for the CYP1A subfamily and the kinetics of metabolic formation mediated by CYP1A2 indicated substrate-inhibition (Ki range 9-16 mM). 4. CYP1A2 catalyzed the demethylation of theophylline as well as its hydroxylation, and was associated with the highest intrinsic clearance (1995 l h-1 per mol CYP) to 1,3-dimethyluric acid (DMU). Therefore, this isoform can be considered to be the most important enzyme involved in theophylline metabolism in vitro. 5. CYP2E1 was responsible for a relatively high intrinsic clearance by 8-hydroxylation (289 l h-1 per mol CYP). The apparent Km value of this reaction was about 15 mM, suggesting that CYP2E1 may be the low-affinity high-capacity isoform involved in theophylline metabolism. 6. The affinity of theophylline for CYP1A1 was comparable with that of its homologue 1A2. When induced, the participation of CYP1A1 in theophylline metabolism may be important. 7. CYP2D6 played only a minor role and CYP3A4 was not active in the in vitro metabolism of theophylline. 8. Our findings confirm the major role of CYP1A2 in theophylline metabolism and explain why in vivo the elimination kinetics of theophylline are non-linear and in vitro theophylline metabolism by human liver microsomes does not obey monophasic kinetics. 9. The data suggest also that not only tobacco smoking but also chronic alcohol intake may influence theophylline elimination in man as ethanol induces CYP2E1.
Abstract: 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.
Abstract: The efficacy of long-term chemotherapy in nonresectable alveolar echinococcosis is debated, particularly because of the difficulty in defining therapeutic success. In this study the effect of chemotherapy on the parasitic mass was evaluated in a series of 37 patients. The patients had larval lesions documented by serial computed tomography studies at least 1.5 yr after chemotherapy (mean = 6.4 yr, range = 1.5 to 10.7 yr). The therapeutic regimen consisted of mebendazole (n = 34) or albendazole (n = 3) as previously described. The maximal areas of the parasitic lesions were assessed morphometrically by means of digital image analysis, utilizing the point-integration method, before and after chemotherapy. Marked regression of larval tissue occurred in 18 patients (group A; 48.6%), stationary lesions were noted in 13 patients (group C; 35.1%) and progression was found in 6 patients (group B; 16.2%). The three groups did not differ significantly with regard to age, plasma drug levels, duration of chemotherapy or initial size and composition of lesions (e.g., cystic cavities, degree of calcification). Despite morphologically successful chemotherapy in moist patients (e.g., 84%; groups A and C), late cholestatic complications after 1.5 to 11 yr of chemotherapy occurred in 10 patients (group A, n = 7; group C, n = 3; 4 of them died) and esophageal variceal bleeding occurred in 3 patients (relieved by sclerotherapy). These late complications were probably mainly due to posttherapy fibrosis of hilar structures. In conclusion, our data support the efficacy of chemotherapy. However, chemotherapy is not curative, and severe late complications were observed in patients with hilar (fibrotic) involvement.
Abstract: Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the proteins, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.
Abstract: Purified pig pancreatic zymogen granules were subjected to free flow electrophoresis (FFE) in an acetate buffer system (acetic acid/NaOH, pH 5.5) to detect the presence or absence of more than one population or zymogen granules. Pig pancreatic zymogen granules were purified by differential and density gradient centrifugation and subjected to FFE. Fractions were analyzed for protein, alpha-amylase (EC 3.2.1.1) and 5'-nucleotidase (EC 3.1.3.5) as marker enzymes for zymogen granule content and membranes, respectively. Only one distinct peak, with coincident alpha-amylase and 5'-nucleotidase activity, and most protein was detected, which reflects the presence of a single population of intact zymogen granules. This was confirmed by electron microscopy. When the granules were incubated with different lectins before FFE, the one distinct peak representing intact zymogen granules was shifted towards the cathode in the case of concanavalin A (Con A) and Ricinus communis agglutinin 120 (RCA 120). No splitting of the peak occurred. Our results do not support the hypothesis of a coexistence of more than one distinct population of zymogen granules.
Abstract: The association of hypercalcemia and acute pancreatitis had been experimentally reproduced in cats by local infusions of the divalent cation calcium whereas the monovalent cation potassium did not induce any pancreatic pathology. The purpose of the present study was therefore to investigate the role of further divalent cations in order to determine the relevance of ion valency for pancreatitis induction. Anesthetized male SIV-rats received divalent cations, of which a role in the pancreas had already been reported in the literature, through retrograde infusions into the splenic artery at a dose of 0.6 mmol/kgh for 3 hours and at a flow of 0.5-1.0 ml/h. The pancreas was then removed for morphologic studies. In the animals treated with calcium and manganese, pancreas showed a hemorrhagic necrosis of the acinar lobuli with leucocytic infiltrates. The barium treated animals spontaneously died after 49 +/- 15 minutes and revealed acute pancreatitis in the perfused, but not in the residual pancreas. Zinc at the initial dose induced an immediate heparin-refractory blood-clotting with subsequent ischemic necrosis whereas a lower dosis (0.002 mmol/kgh) led to an acute pancreatitis as seen after calcium. The magnesium treated animals and the controls did not reveal any pathology. We conclude that some divalent cations may induce an acute pancreatitis, but that the induction is not dependent on the cation valency.
Abstract: The adequacy of retrograde delivery of cardioplegic solution to the right ventricle ist controversial. To evaluate this issue, we excised the plegic heart in 11 bovine experiments and infused an India ink solution (10 ml of India ink in 300 ml NaCl 0.9%) into the coronary sinus (n = 7) at a pressure of 60 cm H2O and into the aortic root (n = 4) at a pressure of 120 cm H2O. After fixation, the ventricles were cut in 11 transversal slices. The portion of coloured (= perfused) ventricular myocardium was calculated with computer-aided morphometric analysis. With antegrade infusion, 95 +/- 5% (mean +/- standard deviation) of the left ventricular volume (left ventricular free wall plus interventricular septum) was stained, with retrograde infusion 94 +/- 3%. Perfusion of the right ventricle was significantly lower with retrograde infusion (antegrade infusion 93 +/- 8%, retrograde 45 +/- 13%, p < 0.001), especially in the basal segments (basal vs. apical: 16 +/- 26% vs. 82 +/- 5%, p < 0.001). The adequate delivery of retrograde infusion to the left ventricle and septum allows good left ventricular myocardial protection with retrograde cardioplegia. Because the retrograde delivery to the right ventricle is markedly inadequate and nonuniform, the quality of right ventricular protection with retrograde cardioplegia has to be questioned.
Abstract: In continuation of our preceding studies, we disclose long-term experiments to test more extensively the protective power of the novel 4H Glove. 2 compounds encountered in modern electron microscopy technique, 1-hexadecene (1-HD) and 2-hydroxyethyl acrylate (2-HEA), which elicit allergic and/or toxic reactions in laboratory workers, were tested for their penetration through the 4H Glove material 1-HD has not to our knowledge been tested on any glove material. 2-HEA was known to have a breakthrough time exceeding 240 min; in contrast, it penetrates the common latex or vinyl gloves within minutes. When exposing the outside of a 4H Glove to 2-HEA for 200 h at 21 degrees C and then attaching the reverse unexposed side to a sensitized volunteer's forearm, 30 min of contact elicited a barely visible reddening of the skin at the contact site which disappeared within 24 h; 90 min of contact caused a somewhat stronger reddening limited to the contact area, which disappeared within 2 days. By the same criteria, 1-HD did not penetrate through the 4H Glove in 200 h. In an additional experiment, breakthrough time of 1-HD on latex gloves was less than 30 min.
Abstract: Several case reports exist which demonstrate cholesterol crystals to be the cause of acute pancreatitis in humans. The crystals have been found intravascularly in the pancreas and at least in one case the origin of the crystals was known. We have undertaken to experimentally reproduce this pathogenetic mechanism in the rat. 30 rats were subjected to the following procedures: under anaesthesia the splenic artery was distally ligated and proximally cannulated; 16 control animals were injected via the cannula with saline (8 rats), particle free cholesterol saturated physiologic saline (3 rats) or nothing (5 rats). 14 rats were injected with ca. 400 microliters of a dilute suspension of cholesterol crystals of 5-40 microns diameter in cholesterol saturated physiologic saline. The abdomen was closed and after 24 h all animals were killed. Acute pancreatitis was diagnosed in all animals that received cholesterol microcrystals and in none of the controls. The diagnosis was based on macroscopic and histological findings. Acute pancreatitis was of focal, disseminated, necrotic type with oedema and moderate haemorrhage and fat necrosis. Only those parts of the pancreas were affected which were supplied by branches of the distal splenic artery used for retrograde injections of cholesterol crystals. This model supports the notion that microembolic or microthrombotic events play a pivotal role in the pathogenesis of spontaneous acute pancreatitis.
Abstract: 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, Cl, 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.
Abstract: 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.
Abstract: We investigated intestinal permeability in healthy adult volunteers, and in patients with Crohn's disease, ulcerative colitis, idiopathic sprue and idiopathic hyperamylasemia by oral administration of 10 g low molecular weight polyethylene glycol (PEG-400) and quantitation of its renal excretion over the subsequent 6 hours by high performance liquid chromatography (gel permeation HPLC). The mean amount of PEG-400 excreted during the first 6 hours by the 12 patients with Crohn's disease (3.1 +/- 0.3 g, mean +/- SE) and the 8 patients with ulcerative colitis (2.6 +/- 0.3 g) was not significantly different from the amount excreted by the 24 healthy volunteers (2.9 +/- 0.1 g). The 3 patients with idiopathic sprue excreted significantly less (1.4 +/- 0.3 g, p less than 0.05) and the 3 patients with idiopathic hyperamylasemia significantly more (4.0 +/- 0.4 g, p less than 0.05) than the healthy controls. In conclusion, no alteration of intestinal permeability could be demonstrated in patients with Crohn's disease or ulcerative colitis. However, intestinal permeability was decreased in patients with idiopathic sprue and increased in those with idiopathic hyperamylasemia.
Abstract: 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 microns polystyrene microspheres retrogradely into the distal splenic artery of rats, thus incompletely blocking blood perfusion in 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 less than 0.0005, highly significant (chi 2 analysis) and shows that acute experimental pancreatitis can indeed be induced by partially blocking the arterial blood supply within the organ.
Abstract: (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. The aim of our report is to review briefly the toxicological properties and occupational hazards of the chemicals involved, summarize our own experiences with resins and protective devices in this respect, give guidelines for safe embedding and pass on these data to all interested researchers in order that workers are not discouraged from using (meth)acrylate embedding media, but know the risks and how to minimize them.
Abstract: This report is about occupational contact dermatitis found in 3 out of 6 workers of a chemistry laboratory using Lowicryl embedding media, which contain (meth)acrylate monomer mixtures of known composition. The notation (meth)acrylates is used to refer to both acrylates and methacrylates. (Meth)acrylate monomers will polymerize in the absence of oxygen when induced by metal ions, peroxides, heat or ultraviolet light. The monomers are of low viscosity and remain in the liquid state at temperatures far below 0 degree C. The volatile compounds, some of which exhibit a most pungent odour, have a tendency to penetrate all tissue and to permeate into the finest fissures, a property which makes them suitable as sealants, glues, embedding material, etc. This and their toxicity may represent a danger to the health of individuals who need to work with them, especially if no precautions are taken. We show with patch testing that one patient reacted strongly to the compound 2-hydroxyethyl acrylate at the dilutions tested (0.5 and 1% v/v), but not at all to 10 other (meth)acrylates. In the same test, 3 volunteer controls were negative to 2-hydroxyethyl acrylate. We demonstrate that at maximum working concentration, 2-hydroxyethyl acrylate penetrates both latex and vinyl gloves and elicits irritant/allergic reactions on the patient and irritant reactions on a control. Finally, we discuss the necessary protective measures.
Abstract: Pancreatic calcifications are virtually pathognomonic of chronic pancreatitis and develop in up to 90% of patients with alcoholic chronic pancreatitis in series with long-term results. We investigated the natural course of pancreatic calcification in a prospective longitudinal study over the past 23 yr. All patients were studied at regular intervals with particular regard to etiology, clinical findings, surgery, pancreatic function, and pancreatic calcification visible by x-ray (e.g., film series in three projections centered on the pancreas). We evaluated the findings of 107 patients with x-ray documentation of pancreatic calcification in at least three film series over a period of 4 yr or longer. Eighty-four patients had alcoholic chronic pancreatitis (group A) and 23 patients had nonalcoholic chronic pancreatitis (group B). Four hundred seventy-two film series of group A and one hundred forty-two film series of group B were reviewed independently by two expert teams. Both series were graded according to a score system in terms of intensity and distribution of pancreatic calcification (correlation of grading r = 0.91). The duration of calcification averaged 10 yr in group A and 12.6 yr in group B. Similar dynamic changes of pancreatic calcification were noted in groups A and B. Chronologically, three phases of evolution could be distinguished. After an initial increase (phase 1), greater than 50% of cases reached a plateau of stationary calcification (phase 2). Approximately one-third of cases showed a marked decrease of calcification in late phases of chronic pancreatitis (phase 3). Dissolution of pancreatic stones was related primarily to duration of chronic pancreatitis (duration of calcification and marked pancreatic dysfunction), and occurred frequently (but not exclusively) in patients after ductal drainage procedures. These results indicate that spontaneous dissolution of pancreatic stones is a rather common biologic phenomenon. The factors responsible for dissolution of stones remain to be elucidated.
Abstract: Since 1976 60 patients with inoperable alveolar echinococcosis caused by Echinococcus multilocularis were treated with high doses of mebendazole and examined at regular intervals prospectively according to our protocol regarding clinical course, liver function, morphology, immunologically and plasma mebendazole levels. The average duration of disease was 8(1-19) years, the average duration of chemotherapy was 4.25 (0.75-9) years. The long term results showed a correlation of the clinical course with the mean plasma mebendazole levels and the duration of chemotherapy, respectively. Death (n = 5) or transient progression of the disease process (n = 14) was observed primarily in patients with low plasma mebendazole levels in the early course and within the first two years of chemotherapy. Only 9 patients showed a decrease of the parasite mass. Immundiagnosis (total serum IgE and serum antibodies against Echinococcus antigen) gave some information with regard to therapy results, but only in the long-term course. The cumulative survival of the patients under study was 96% at 5 years and 84% at 10 years, respectively which is markedly higher compared to historical control series with a letality of greater than 90% within 10 years.
Abstract: Controversies in the literature regarding definition, diagnosis, and therapy of chronic pancreatitis may be related in part to differences in the natural history of alcoholic and idiopathic (nonalcoholic) chronic pancreatitis. In order to evaluate this problem the long-term course of 205 patients with alcoholic (85.4% with calcifications) (group A) and 82 patients with idiopathic (nonalcoholic) chronic pancreatitis (76.8% with calcifications) (group B) has been analyzed prospectively since 1963. The patients were studied at regular intervals with particular regard to pain, pancreatic exocrine, and endocrine function and calcifications. The observation time was 2 years or longer in 230 patients with a median observation time of 6.7 years from diagnosis in group A and 10.6 years in group B. In group B over 50% of the cases had primary painless chronic pancreatitis. Progressive deterioration of exocrine and endocrine function was observed in both groups. However, in group A the rate of progression of exocrine dysfunction after diagnosis was more rapid and the incidence of diabetes in relation to marked exocrine insufficiency was much higher than in group B. Steatorrhea preceded diabetes in 56% (group A) and 80% (group B), respectively. Onset of pancreatic calcifications was closely associated with pancreatic exocrine insufficiency in group A in contrast to group B. In addition lasting pain relief occurred spontaneously in about 30% of patients in group B despite a normal exocrine function for 6 years or longer which is in disaccord with the results in alcoholic chronic pancreatitis. In conclusion group A and B have many features in common, in particular the high incidence of pancreatic calcifications and the progressive pancreatic dysfunction. However, the long-term profile of both groups differs in some important aspects, particularly in the clinical pattern and in the rate of progression of pancreatic dysfunction and morphology. These differences should be appreciated in the discussion of problems regarding definition, diagnosis, and surgical therapy of chronic pancreatitis.
Abstract: Quantitative measurement of vessel tortuosity and its variation on fundus photographs is a sensitive means of obtaining information about the course of an asphyctic event in newborns, virtually independent of bias produced by the photographic process. We subdivide a tortuous vessel into single arcs and measure the chord length and the arrow height (Pfeilhöhe) of every arc on a projected image of the film. From these figures, a fairly accurate value for the relative length increase of the arc, as compared with the chord, can be derived by a simple approximation formula [Eq. (5)]. It is shown that neglect of the third dimension, not visible on an ordinary photograph, entails only a small error. Trained observers achieve results reproducible to about 1% in relative length variation.
Abstract: A micromethod for the isolation of brush border membrane fragments from single peroral duodenal biopsies, and their subsequent analysis by polyacrylamide gel electrophoresis is described. The quantity of biopsy material used varied between 5 and 15 mg wet weight, leaving enough mucosa for histological examination. By cutting the gels longitudinally into two halves it was possible to identify several maltases, sucrase, isomaltase and lactase and to correlate these enzymatic activities with distinct co-migrating protein peaks. For alkaline phosphatase and enterokinase this correlation was not possible. This method is suitable for the study on single biopsies of the molecular alterations occurring in the various congenital enzyme deficiencies of the human small intestine.
Abstract: The incorporation of [14C]glucosamine into brush border glycoproteins by human small intestinal mucosa in organ culture has been investigated. The experiments were based on the observations that (1) isolated brush border membrane fragments from cultured explants showed an unchanged pattern of protein bands and brush border enzyme activities on sodium dodecyl sulfate/polyacrylamide gels after electrophoresis and (2) the rate of overall [14C]glucosamine incorporation measured in the tissue homogenate remained constant up to 48 h. After 24 h of culture, the radioactivity peaks on gels due to incorporation of [14C]glucosamine were found exclusively in the high molecular weight region and corresponded to protein bands identified as maltase-glucoamylase, lactase, sucrase-isomaltase, enterokinase and alkaline phosphatase. Enzymatic activity could not be assigned to the three remaining labelled bands. Most of these glycoproteins were already labelled after 5 h. Newly glycosylated brush border enzymes remained predominantly associated with the brush border membrane of intact cells with little release into the medium up to 24 h.
Abstract: In a child with hereditary sucrase-isomaltase deficiency immunoreactive enzyme was present in the intact duodenal mucosa. Polyacrylamide gel electrophoresis carried out with membrane fragments of an intestinal biopsy showed an abnormal protein band without enzyme activity. The mucosa had a relatively high residual isomaltase activity which was recovered from the gel in a position suggesting higher than normal molecular weight. The results indicated that in this patient the primary structural defect was in the sucrase moiety which was enzymatically inactive. The isomaltase subunits may have aggregated into a large molecular weight complex because of unavailability of their partners. The observation also provided evidence for separate biosynthesis of the two moieties of the sucrase-isomaltase complex.
Abstract: Brush-border membrane proteins of the small-bowel mucosa were separated on polyacrylamide gels from intestinal biopsy specimens obtained from four children with congenital lactose malabsorption and from two adults with specific hypolactasia. In three patients with the congenital type of lactase deficiency the protein band corresponding to brush-border lactase was reduced in intensity, but was never completely absent. No difference in gel patterns was detected when this pattern in congenital deficiency was compared to that obtained from the two patients with adult-type selective hypolactasia. In one patient with congenital lactose malabsorption the protein band corresponding to lactase activity was not detectable. The findings suggest that the mechanisms leading to low lactase activity in the congenital and adult forms of lactose intolerance are similar.
