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Acid secretion by the parietal cell of the gastric mucosa depends on activation of the acid pump, the gastric H,K-ATPase. This enzyme is expressed in high quantities by the parietal cell. The site of acid secretion in the parietal cell is the secretory canaliculus. This specialized structure is considered to be an infolding of the apical plasma membrane. The active H,K-ATPase is present in the microvilli lining the secretory canaliculus. When inactive the pump is found in cytoplasmic tubules.

The key challenge in athlete's screening is the distinction between abnormal and normal which is hindered by the fact that the adaptation to sports activity in endurance athletes is different to that in power athletes. Especially cardiomyopathies provoke changes in ECG and echocardiography (echo) at an early stage when clinical symptoms are absent. ECG and echo data and their relationship to fitness peculiar to top handball players have never been described. We studied 291 male first league handball players (32 Olympians/47 national players) (25.3±4.4 years). Check up consisted of ECG, spiroergometry and echocardiography. None had T-wave inversions, 3.1% showed early repolarisation abnormalities in the precordial leads. Sokolow-Lyon voltage criterion for left ventricular hypertrophy was positive in 19.3%. Spiroergometry showed a maximum oxygen uptake (peakVO₂) of 50.3±7.7 ml/min/kg body weight. LVmass was increased in comparison to normal values. There was a correlation between peakVO₂ and LVindex (p

Cholecystokinin stimulates pancreatic zymogen secretion by binding with high affinity to a receptor on the pancreatic acinar cell. This receptor has been cloned and shown to be a CCK-A subtype. CCK also stimulates pepsinogen secretion from the gastric chief cell with high affinity. Using polymerase chain reaction with primers from the known sequence of the rat pancreatic CCK-A receptor cDNA, we prepared a 600 bp product from rat and rabbit stomach cDNA. From Southern analysis these represented a fragment of a gastric CCK-A receptor. PCR was then used to amplify a rabbit λZAP II gastric epithelial cDNA library with the same primers, and the product was identified by sequencing as representing a CCK-A receptor fragment. When this PCR product was used to screen the library, ten positive clones were identified in a screening of 4 · 105 plaques, and several of these were sequenced. All had essentially the same sequence contained within 2 of these clones consisted of 427 amino acids and was 92% homologous (87% identity) to the known rat pancreatic CCK-A sequence but only 43% homologous to the gastric CCK-B sequence. The cDNA was subcloned into a pcDNA1 expression vector and transiently expressed in the human embryonic kidney cell line, HK 293. The responses of intracellular Ca2+ in these transfected cells to CCK and gastrin were monitored using video imaging. On the average 40% of the cells responded to CCK-8 by a transient elevation of [Ca2+]i followed by a steady state plateau. CCK was a high and gastrin a low affinity ligand for this signal, corresponding to the actions of these ligands on pepsinogen secretion from chief cells and somatostatin release from D cells. Hence from sequence and second messenger responses, the clone represents the CCK-A receptor presumably responsible for pepsinogen secretion by gastric chief cells and somatostatin release from gastric D cells.

The hormone gastrin stimulates acid secretion by releasing histamine from gastric enterochromaffin-like (ECL) cells and induces ECL cell proliferation in vivo. This study uses a > 90% pure ECL cell preparation in culture to compare gastrin effects on histamine release, histidine decarboxylase (HDC) activity, and DNA synthesis. Gastrin and the cholecystokinin octapeptide (CCK-8, nonsulfated) induced histamine release from ECL cells (24-96 h of primary culture) within 5 min of incubation [concentration eliciting 50% of maximal response (EC50), 4 and 2 x 10(-11) M, respectively]. The CCK-B antagonist L-365,260 inhibited this effect [concentration inhibiting 50% of maximal response (IC50), 2 x 10(-8) M], whereas the CCK-A antagonist L-364,718 (10(-8) M) and the tyrosine kinase inhibitor genistein (10(-4) M) had no effect. Histamine release was associated with a biphasic elevation of intracellular Ca2+. Gastrin stimulated HDC activity two- to threefold after 60 min of incubation (EC50, 10(-10) M). Gastrin also increased DNA synthesis in ECL cells, with an EC50 of 1.7 x 10(-12) M as measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). Positive nuclear immunostaining increased two- to threefold in up to 20% of ECL cells after 48-96 h of incubation. This effect was inhibited by L-365,260 (IC50, 5 x 10(-9) M) and by genistein (10(-4) M) but was not altered by L-364,718 (10(-8) M). The antisecretory drugs omeprazole, lansoprazole, and pantoprazole did not affect BrdU incorporation in isolated ECL cells. In conclusion, acute and chronic gastrin effects on the ECL cell are mediated via CCK-B receptors but differ in apparent receptor affinity and signal transduction pathways.

Several antibodies against the gastric H+/K(+)-ATPase were analysed for the topological and sequence location of their epitopes. Topological mapping was done by comparing indirect immunofluorescent staining in intact and permeabilised rat parietal cells. Epitope definition was by Western analysis of intact and of trypsin or V8-proteinase-fragmented hog gastric ATPase combined with N-terminal sequencing of the fragments; by Western analysis of fragments of rabbit alpha subunit expressed in Escherichia coli; by analysis of rabbit alpha and beta subunits expressed in baculovirus-transfected SF 9 cells and by ELISA assay of synthetic octamers of one region of the hog alpha subunit. It was confirmed that the monoclonal antibody, mAb 95-111, recognised a cytoplasmic region between M4 and M5, close to the ATP-binding domain. The major epitope for monoclonal antibody mAb 12-18 was also in this region, but a second epitope was confirmed to be present in the M7/M8 region. The monoclonal antibody, mAb 146-14, was shown to recognise an extracytoplasmic epitope dependent on intact disulfide bonds, present in the rat and the rabbit, but absent in the hog beta subunit, due to non-conservative amino-acid substitutions. This antibody also recognised an epitope present in the alpha subunit of the H+/K(+)-ATPase at the M7 extracytoplasmic interface, perhaps indicating structural association of these two regions. The polyclonal antibody, pAb39, raised against the C-terminal portion of the enzyme, reacted only with the cytoplasmic surface of the H+/K(+)-ATPase, showing that the alpha subunit of the enzyme has an even number of membrane spanning segments.

The vascular system is lined by mitotically quiescent but metabolically active endothelial cells, which in addition to having a broad range of metabolic activities, provide a nonthrombogenic surface for blood flow. Beneath the endothelium, smooth muscle cells are found in the media of large vessels, and pericytes are found in close association with the endothelial cells of microvascular beds. The smooth muscle cells (pericytes) are thought to play major roles in maintaining vessel wall integrity, being responsible for the maintenance to the connective tissues of the vessel wall and in the control of vascular tone.8 Vascular cells (large and small vessel derived endothelial, pericyte, and smooth muscle cells) have been found to respond to injury in specific ways, depending upon the vascular bed and the cell type(s) injured. For example, following denudation injury evoked by angioplasty, endarterectomy or autologous or synthetic grafting, large vessel endothelial cells bordering the affected area will exhibit rapid sheet migration over the exposed extracellular matrix and proliferate in an attempt to reconstitute the normal continuous endothelial cell lining.15,20 The medial smooth muscle cells of large and medium-sized vessels respond to vessel injury by migrating into the intima, where they proliferate and synthesize matrix components, which results in the formation of a thickened intima which narrows the vessel lumen.34 In contrast, following soft tissue injury or in response to a variety of angiogenic factors, microvascular endothelial cells respond by freeing themselves from the constraints of their investing basement membranes. Following this, they migrate and proliferate in the surrounding three-dimensional interstitial stroma and ultimately form new microvessels.17

Cholangiocarcinomas arise from the epithelial cells of the bile ducts and are associated with poor prognosis. Despite new diagnostic approaches, the definite diagnosis of this malignancy continues to be challenging. Cholangiocarcinomas often grow longitudinally along the bile duct rather than in a radial direction. Thus, large tumor masses are frequently absent and imaging techniques, including ultrasound, CT, and MRI have only limited sensitivity. Tissue collection during endoscopic (ERCP) and/or percutaneous transhepatic (PTC) procedures are usually used to confirm a definitive diagnosis of cholangiocarcinoma. However, forceps biopsy and brush cytology provide positive results for malignancy in about only 50% of patients. Percutaneous and peroral cholangioscopy using fiber-optic techniques were therefore developed for direct visualization of the biliary tree, yielding additional information about endoscopic appearance and tumor extension, as well as a guided biopsy acquistion. Finally, endoscopic ultrasonography (EUS) complements endoscopic and percutaneous approaches and may provide a tissue diagnosis of tumors in the biliary region through fine-needle aspiration. In the future, new techniques allowing for early detection, including molecular markers, should be developed to improve the diagnostic sensitivity in this increasing tumor entity.

To analyze the in vitro activity of moxifloxacin and piperacillin/sulbactam against pathogens isolated from patients with acute cholangitis.In this prospective study a total of 65 patients with acute cholangitis due to biliary stone obstruction (n = 7), benign biliary stricture (n = 16), and malignant biliary stricture (n = 42) were investigated with regard to spectrum of bacterial infection and antibiotic resistance. Pathogens were isolated from bile cultures in all study patients. In 22 febrile patients, blood cultures were also obtained. In vitro activity of moxifloxacin and piperacillin/sulbactam was determined by agar diffusion.Thirty-one out of 65 patients had positive bile and/or blood cultures. In 31 patients, 63 isolates with 17 different species were identified. The predominant strains were Enterococcus species (26/63), E.coli (13/63) and Klebsiella species (8/63). A comparable in vitro activity of moxifloxacin and piperacillin/sulbactam was observed for E.coli and Klebsiella species. In contrast, Enterococcus species had higher resistances towards moxifloxacin. Overall bacteria showed antibiotic resistances in vitro of 34.9% for piperacillin/sulbactam and 36.5% for moxifloxacin.Enterococcus species, E.coli and Klebsiella species were the most common bacteria isolated from bile and/or blood from patients with acute cholangitis. Overall, a mixed infection with several species was observed, and bacteria showed a comparable in vitro activity for piperacillin/sulbactam and moxifloxacin.

AIM: To evaluate the sensitivity of brush cytology and forceps biopsy in a homogeneous patient group with hilar cholangiocarcinoma. METHODS: Brush cytology and forceps biopsy were routinely performed in patients with suspected malignant biliary strictures. Fifty-eight consecutive patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) including forceps biopsy and brush cytology in patients with hilar cholangiocarcinoma between 1995-2005. RESULTS: Positive results for malignancy were obtained in 24/58 patients (41.4%) by brush cytology and in 31/58 patients (53.4%) by forceps biopsy. The combination of both techniques brush cytology and forceps biopsy resulted only in a minor increase in diagnostic sensitivity to 60.3% (35/58 patients). In 20/58 patients (34.5%), diagnosis were obtained by both positive cytology and positive histology, in 11/58 (19%) by positive histology (negative cytology) and only 4/58 patients (6.9%) were confirmed by positive cytology (negative histology). CONCLUSION: Brush cytology and forceps biopsy have only limited sensitivity for the diagnosis of malignant hilar tumors. In our eyes, additional diagnostic techniques should be evaluated and should become routine in patients with negative cytological and histological findings.

Bombesin-like peptides as well as receptor-independent activators were tested for their effect on gastrin release from acutely dispersed rat gastric G-cells. The amphibian peptide bombesin as well as its mammalian analogues neuromedin B and neuromedin C stimulated gastrin release. Maximal responses were achieved with 10(-9) M bombesin (191.0 +/- 16.8% of basal release), 10(-8) M neuromedin C(205.9 +/- 17.6%) and 10(-7) M neuromedin B (162.2 +/- 10.4%), respectively. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and the synthetic diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) are receptor-independent activators of the protein kinase C. Both TPA (10(-6) M) and OAG (10(-5) M) stimulated gastrin release to 214.0 +/- 29.3% and 198.2 +/- 20.8% of basal, respectively. Calcium ionophore A23187 (10(-5) M) was the most effective stimulant tested (364.7 +/- 39.6%). Its effect was reversed by the calmodulin antagonist W 7 (10(-6)-10(-5) M). Finally, forskolin (10(-5) M), a direct activator of cAMP-formation, as well as the cAMP-analogue dbcAMP (10(-3) M) induced gastrin release. IN conclusion, neuromedin B is less potent and less effective than neuromedin C and bombesin in stimulating rat gastric G-cells. In addition, gastrin release is activated by calcium- and phospholipid-dependent as well as by cAMP-induced cellular signal transduction mechanisms.

A 53−year−old woman with a 17−year his− tory of multiple sclerosis, presented with abdominal pain and bowel distension. The computed tomography (CT) scan showed massive dilation of the colon (Fig− ure 1). The patient had a long history of chronic intestinal pseudo−obstruction, which so far had always resolved after co− lonoscopic decompression and placement of a colon tube. This time once again, after endoscopic evacuation of the trapped air, the patient s situation improved rapidly. In the course of the hospital stay it be− came clear that decompression was need− ed every third day. Therefore, we aimed for a permanent solution, and we chose an endoscopic approach.

Successfull Treatment of Globus Sensation By Argon-Plasma-Coagulation of Gastric Inlet Patches in the Cervical Esophagus A Pilot Trial Alexander Meining, Monther Bajbouj, Martin Preeg, Wolfgang Huber, Heinz Hoefler, Christian Prinz, Roland Schmid Background: It has been mentioned that globus sensation may be caused by gastroesophageal reflux disease. In addition, it has also been suggested, that the presence of a gastric inlet patch can by associated with such symptoms. So far, there are no reports, as to whether ablation of the cervical heterotopic mucosa leads to improvement of chronic globus sensation or other atypical reflux symptoms. Methods: Ten patients with a histologically proven gastric inlet patch and chronic globus sensation and/or sore throat despite therapy with protone-pump inhibitors underwent Argon-plasma-coagulation (APC) of the heterotopic mucosa. Before therapy, a thoroughly examination including barium swallow, laryngoscopy, manometry and 24h-two-channel-pH-metry was performed. A questionnaire was applied for assessment of typical and atypical reflux symptoms based on a visual analogue scale ranging from 0 to 10. Follow-up examinations were performed four and eight weeks after APC-therapy. Results: In all patients, ablation of the gastic inlet patch resulted in a remarkable reduction of symptoms scores for ‘‘globus’’ and/or ‘‘sore throat’’ (median scores for ‘‘globus’’: 2.7 prior and 0.0 eight weeks after therapy; p Z 0.015; median scores for ‘‘sore throat’’: 2.8 prior and 0.0 after therapy; p Z 0.018). Other symptoms assesed such as ‘‘heartburn’’, ‘‘chronic cough’’, ‘‘asthma’’, ‘‘hoarseness’’ or ‘‘burning tongue’’ did not improve significantly after therapy. Data obtained by other examinations in particular pH-metry were not associated with any improvement of symptoms. Conclusion: Our preliminary data suggest that upper endoscopy for screening of gastric inlet patches should be performed in every patient with globus or sore throat sensation. Because of its high effectiveness, APC-treatment might then be considered as the therapy of choice.

Four Membrane-Associated Antigens of Helicobacter pylori Suitable for Diagnosis of Both Infection and Eradication. Petra Voland, II Medical Dept, Munich Germany; David Weeks, VAGLAI-IS and Univ of CA, Los Angeles, Los Angeles, CA; Dino Vaira, Dept of Medicine, Orsola Hosp, Bologna Italy; Nina Neumayer, Dept of Medicine II, Munich Germany; George Sachs, VAGLAHS and Univ of CA, Los Angeles, Los Angeles, CA; Christian Pdnz, Oept of Medicine II, Teeh Univ, Munich Germany

Background. Human and rat fundic mucosa contain Enterochromaffinlike(ECL) cells. We have previously shown that these cells undergo apoptosis in the presence of TNF-a, a cytokine that is released during H. pylori infection in the gastric mucosa. Of further interest is basic FGF which is detected in ECL cell tumors. The aim of our current study was to elucidate the mechanisms of TNF-a induced apoptotis in ECL-cells and the role of bFGF during this reponse. MethodslResults. ECL-cells were isolated from rat fundic mucosa to a purity> 90% and cultured up to 24 hr. Western Blotting showed a strong signal from unphosphorylated lKBa at 0 min. After 5 min, the signal was decreased and after 10 min, unphosphorylated lKBa-was no longer detectable, indicating that IKBa was strongly phosphorylated, which in tum leads to the activation of NFKB. To determine NFKB-activation more directly, we performed electromobility-shift assays (EMSA) using cell extracts pretreated with vehicle for I hr, then treated with TNF-a (25 ng/mL) or vehicle for another hour. The EMSAs revealed a specific activation of the NFKB subunits p50 and p65. In order to investigate the functional role of NFKB in the mediation of pro-apoptotic TNF-a effects, we measured ECL-cell apoptosis (programed cell death) after preincubation (I hr) with the specific NFKB-inhibitors MGl32 (5 mM) and PSI (6 mM) and subsequent incubation with TNF-a (25 ng/mL, I hr). Cells incubated without inhibitors and/or TNF-a served as controls. Using the TUNEL technique, TNF-a was found to cause apoptosis in 16% ofECL cells compared to a basal rate of6%. MGI32 and PSI alone did not induce apoptosis (total of 5-8% of ECL cells), but were able to inhibit TNF-a-induced apoptosis completely. For MG132, these results were repeated using an ELISA-assay based upon the detection of nucleosomes which also showed complete inhibition ofTNF-a effects. The observations were verified using the EMSA technique mentioned above. To elucidate the role of basic FGF, we tested bFGF effects on TNF-a induced apoptosis. Using the ELISA, we found that bFGF at 10-100 ng/mL completely blocked TNF-a induced cell death. Conclusion. TNF-a induced apoptosis of ECL cells is mediated by phosphorylation of IKBa and subsequent activation of NFKB. bFGF inhibits TNF-a induced ECL cell death, which may also playa role for the persistence of ECL cells during chronic gastric inflammation (DFG Pr 41112-2).

Background. Human and rat fundic mucosa contain Enterochromaffinlike(ECL) cells. We have previously shown that these cells undergo apoptosis in the presence of TNF-a, a cytokine that is released during H. pylori infection in the gastric mucosa. Of further interest is basic FGF which is detected in ECL cell tumors. The aim of our current study was to elucidate the mechanisms of TNF-a induced apoptotis in ECL-cells and the role of bFGF during this reponse. MethodslResults. ECL-cells were isolated from rat fundic mucosa to a purity> 90% and cultured up to 24 hr. Western Blotting showed a strong signal from unphosphorylated lKBa at 0 min. After 5 min, the signal was decreased and after 10 min, unphosphorylated lKBa-was no longer detectable, indicating that IKBa was strongly phosphorylated, which in tum leads to the activation of NFKB. To determine NFKB-activation more directly, we performed electromobility-shift assays (EMSA) using cell extracts pretreated with vehicle for I hr, then treated with TNF-a (25 ng/mL) or vehicle for another hour. The EMSAs revealed a specific activation of the NFKB subunits p50 and p65. In order to investigate the functional role of NFKB in the mediation of pro-apoptotic TNF-a effects, we measured ECL-cell apoptosis (programed cell death) after preincubation (I hr) with the specific NFKB-inhibitors MGl32 (5 mM) and PSI (6 mM) and subsequent incubation with TNF-a (25 ng/mL, I hr). Cells incubated without inhibitors and/or TNF-a served as controls. Using the TUNEL technique, TNF-a was found to cause apoptosis in 16% ofECL cells compared to a basal rate of6%. MGI32 and PSI alone did not induce apoptosis (total of 5-8% of ECL cells), but were able to inhibit TNF-a-induced apoptosis completely. For MG132, these results were repeated using an ELISA-assay based upon the detection of nucleosomes which also showed complete inhibition ofTNF-a effects. The observations were verified using the EMSA technique mentioned above. To elucidate the role of basic FGF, we tested bFGF effects on TNF-a induced apoptosis. Using the ELISA, we found that bFGF at 10-100 ng/mL completely blocked TNF-a induced cell death. Conclusion. TNF-a induced apoptosis of ECL cells is mediated by phosphorylation of IKBa and subsequent activation of NFKB. bFGF inhibits TNF-a induced ECL cell death, which may also playa role for the persistence of ECL cells during chronic gastric inflammation (DFG Pr 41112-2).