Oxidative Stress Activates Membrane Ion Channels in Human Biliary Epithelial Cancer Cells (Mz-Cha-1).

Oxidative stress is known to contribute to cell damage. In several cell types, one of the earliest effects of oxidative stress is a rapid and substantial decrease in cell volume, which in turn regulates a broad range of cell functions, including development of apoptosis. Since volume regulation is closely coupled to membrane ion permeability, activation of ion channels may play an important role in oxidative stress-related cell injury. Oxidative stress plays a major role in a variety of liver diseases and bile duct epithelia cells (BDE) represent an important site of injury. We, therefore, investigated the functional interactions of oxidative stress, cell volume and ion permeability in a BDE model. Whole-cell patch clamp studies were performed in the human Mz-Cha 1 cell line. Oxidative stress was produced by addition of H2O2 to the bath solution. Changes of intracellular Ca(2+) concentration and of crosssectional area (for calculating cell volume) were monitored by laser scanning microscopy. Exposure of Mz-Cha 1 cells to H2O2 resulted in cell shrinkage and increase of the intracellular Ca(2+) concentration. Patch-clamp studies revealed that exposure to H2O2 also resulted in the activation of ion currents with a threshold of 10(-6) M H2O2. Ion substitution studies and blocker experiments identified the currents as representing an increase in membrane K(+) and Cl-permeability. Interestingly both ion channel activation and cell shrinkage had a close relationship to the applied H2O2 concentration and were significantly inhibited by intracellular Ca(2+) chelation. These data imply that in a BDE model, oxidative stress leads to cell shrinkage through activation of Ca(2+)-dependent K(+) and Cl(-) currents. Since cell shrinkage has been associated with increased cell damage, the opening of these ion channels might contribute to the high susceptibility of biliary epithelial cancer cells to oxidative stress.

Sequential chemotherapies for advanced gastric cancer: a retrospective analysis of 111 patients.

BACKGROUND: The role of second-line chemotherapy in advanced gastric cancer is not yet fully established. PATIENTS AND METHODS: We analysed 111 patients with advanced gastric cancer treated at the University Hospital Heidelberg (51) and the private oncology practice Bottrop/Dorsten (60) between 2001 and 2011, comparing the outcome of patients with first-line chemotherapy and those who received second-line chemotherapy. RESULTS: Thirty-six patients were treated with one chemotherapy regimen, 75 patients received at least two different chemotherapies. Patients who received one chemotherapy regimen were older (median age 69 years) and had a shorter overall survival (6 months) than patients receiving sequential chemotherapies [median age 61 years, p = 0.009, overall survival 14 months (2-42), p = 0.001]. Under second-line chemotherapy, partial response was observed in 25 patients (33%) and stable disease for ≥3 months in 26 patients (35%). Patients treated before 2005 had a slightly better overall survival than patients treated in or after 2005. Survival was not influenced by the treatment centre (primary or tertiary), but was influenced by former surgery. CONCLUSION: The prognosis of advanced gastric cancer is still poor. Selected patients may benefit from individualized salvage chemotherapy after failure of first-line chemotherapy.

Extracellular ATP induces cytoplasmic and nuclear Ca2+ transients via P2Y2 receptor in human biliary epithelial cancer cells (Mz-Cha-1).

Extracellular nucleotides such as adenosine triphosphate (ATP) play a role in biliary epithelial cell function. Since nucleotide receptors are potential targets for various diseases related to epithelial cell dysfunction and cancer, the purpose of this study was to investigate the expression and to functionally characterize the nucleotide receptor subtypes in biliary epithelial cancer cells (Mz-Cha-1). Extracellular ATP dose-dependently resulted in an intracellular Ca(2+) increase (mean effective concentration (EC(50)) 40 µM). Uridine triphosphate (UTP) produced a similar Ca(2+) response and cross-desensitation was observed. The rank order of tested agonists was ATP=UTP>> adenosine>ADP=AMP>α,ß-methylene-ATP. This confirms the functional expression of purinoceptor P2Y2 and P2Y4 in biliary epithelial cancer cell membranes. mRNAs for P2Y1, P2Y2, P2Y4 and P2Y6 purinergic receptor subtypes were found, whereas western blot analysis suggested only the expression of P2Y2 receptors. Confocal imaging and nuclear staining was used to compartmentalize ATP-induced cytosolic and nuclear Ca(2+)-transients, indicating a role for secretory ATP in regulating nuclear function, by increasing nuclear Ca(2+) concentrations. These data define the expression profile of P2Y receptors on human biliary epithelial cancer cells and indicate P2Y2 receptors as being potential targets in new treatment strategies for biliary cancer.

Value of lipopolysaccharide binding protein, interleukin-6 and C-reactive protein as biomarkers of severity in acute diverticulitis: a prospective study.

BACKGROUND: New biomarkers have the capability to predict severity and outcome of infectious diseases. Lipopolysaccharide binding protein (LBP) and Interleukin 6 (IL-6) were determined as new markers in patients with acute diverticulitis and were compared with standard markers such as C-reactive protein (CRP) and white blood cell count (WBC). METHODS: CRP, IL-6, WBC and LBP were measured in 38 patients at hospital admission and every second day and after colonoscopy. Multi-slice CT scans, ultrasound and early colonoscopy were performed to confirm diagnosis and to detect complications (perforations, stenosis). RESULTS: CRP, IL-6 and LBP levels one correlated highly with each other and were equally influenced by antibiotic therapy. WBC changes were unremarkable. Severity of the disease (sealed- or non-perforation) was not reflected by the biomarkers. In non-perforated patients, colonoscopy was performed on day 6 (median) after admission with a success rate of 93%. Sealed-perforated patients were examined on median day 11 with a success rate of 60% (p > 0.001). Failure in all cases was due to sigmoidal stenosis requiring surgery. In a receiver-operating characteristic curve analysis (ROC), LBP on day one performed best in predicting colonic steno-sis with an area under the curve of 0.88 (95% CI 0.73 - 0.03 p < 0.02). CONCLUSIONS: CRP, IL-6, and LBP can be used to monitor diverticulitis. Initial LBP values in patients with acute diverticulitis may also be usefully in detecting candidates for surgical intervention.

Adipocyte-specific inactivation of Acyl-CoA synthetase fatty acid transport protein 4 (Fatp4) in mice causes adipose hypertrophy and alterations in metabolism of complex lipids under high fat diet.

Fatp4 exhibits acyl-CoA synthetase activity and is thereby able to catalyze the activation of fatty acids for further metabolism. However, its actual function in most tissues remains unresolved, and its role in cellular fatty acid uptake is still controversial. To characterize Fatp4 functions in adipocytes in vivo, we generated a mouse line with adipocyte-specific inactivation of the Fatp4 gene (Fatp4(A-/-)). Under standard conditions mutant mice showed no phenotypical aberrance. Uptake of radiolabeled palmitic and lignoceric acid into adipose tissue of Fatp4(A-/-) mice was unchanged. When exposed to a diet enriched in long chain fatty acids, Fatp4(A-/-) mice gained more body weight compared with control mice, although they were not consuming more food. Pronounced obesity was accompanied by a thicker layer of subcutaneous fat and greater adipocyte circumference, although expression of genes involved in de novo lipogenesis was not changed. However, the increase in total fat mass was contrasted by a significant decrease in various phospholipids, sphingomyelin, and cholesteryl esters in adipocytes. Livers of Fatp4-deficient animals under a high fat diet exhibited a higher degree of fatty degeneration. Nonetheless, no evidence for changes in insulin sensitivity and adipose inflammation was found. In summary, the results of this study confirm that Fatp4 is not crucial for fatty acid uptake into adipocytes. Instead, under the condition of a diet enriched in long chain fatty acids, adipocyte-specific Fatp4 deficiency results in adipose hypertrophy and profound alterations in the metabolism of complex lipids.

Oxidative stress reduces Na+/H+ exchange (NHE) activity in a biliary epithelial cancer cell line (Mz-Cha-1).

In cholangiocarcinogenesis, chronic inflammation and oxidative stress play a key role. The Na(+)/H(+) exchanger (NHE) forms a potential link between control of intra- and pericellular pH and tumor development. Therefore, the effects of oxidant stress were determined by the use of tert-butyl hydroperoxide (t-BOOH) on Na(+)/H(+) exchange in a biliary epithelial cancer cell line (Mz-Cha-1). The cells were exposed to the hydroperoxide and the rate of recovery from acidosis was determined by the use of the pH-sensitive fluorochrome 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). t-BOOH reduced Na(+)/H(+) exchange activity in a dose-dependent manner. At 4 mM t-BOOH, Na(+)/H(+) exchange activity was virtually absent. This was accompanied by an increase in cytotoxicity (MTT assay). Glutathione repletion and intracellular Ca(++) chelation partially restored the Na(+)/H(+) exchange activity. Hydroperoxide seemed neither to alter the intracellular signal transduction pathways (cAMP and Ca(++) oscillations) nor the membrane distribution of the exchanger (immunostaining). Decrease in Na(+)/H(+) exchange activity in this model of oxidant stress may represent an early perturbation of membrane function, and the functional integrity of Na(+)/H(+) exchange could therefore be dependent on the glutathione redox system.

Gamma-hydroxybutyric acid versus clomethiazole for the treatment of alcohol withdrawal syndrome in a medical intensive care unit: an open, single-center randomized study.

BACKGROUND: Clomethiazole (CLO) has been shown to be effective in treating alcohol withdrawal syndrome (AWS). Gamma-Hydroxybutyric acid (GHB) has also been introduced in the treatment of alcoholic patients and is effective in surgical intensive care unit (ICU) patients in preventing and treating AWS. There are no comparative studies between CLO and GHB in a medical ICU setting. METHODS: Twenty-six alcoholic patients with severe AWS and concomitant medical diseases were randomally enrolled in the study. CLO was given orally to 12 patients in a dosage of 250 mg every 4 hours as a liquid; GHB (initially 30 mg/kg body weight (BW) followed by 15 mg/kg BW) was administered intravenously to 14 patients. Four major AWS symptoms (tremor, sweating, nausea, restlessness) were scored, and the administration of additional medication was registered. RESULTS: GHB was more effective in treating AWS symptoms. In the GHB group, AWS score dropped from 6.6 +/- 2.6 to 1.8 +/- 2.1 (p <.01), while in the CLO group, the score dropped from 6 +/- 2.5 to 4.1 +/- 2.4 (n. s.). Differences between groups were significant (p =.021, two-way ANOVA). The treatment did not alter outcome or the duration of ICU stay. No serious side effects were detected. CONCLUSION: GHB effectively controls AWS symptoms in medical ICU patients. The rapid initial treatment response of GHB in contrast to CLO has no influence on duration of patient withdrawal.

Risk factors for colonic diverticular bleeding: a Westernized community based hospital study.

AIM: To evaluate the risk factors-other than nonsteroidal anti-inflammatory drugs-for colonic diverticular bleeding in a westernized population. METHODS: One hundred and forty patients, treated for symptomatic diverticular disease in a community based hospital, were included. Thirty (21%) had signs of diverticular bleeding. Age, gender, and the results of colonoscopy were collected and compared to a group of patients with nonbleeding symptomatic diverticulosis. Records were reviewed for comorbidities, such as obesity, alcohol consumption, smoking habits and metabolic diseases. Special emphasis was put on arterial hypertension, cardiovascular events, diabetes mellitus, hyperuricemia and hypercholesterinemia. RESULTS: There was no difference between patients with diverticular hemorrhage and those with nonbleeding symptomatic diverticulosis regarding gender ratio (male/female 9/21 vs 47/63) and diverticular localisation. Bleeding patients differed in respect to age (73.4+/-9.9 vs 67. 8+/-13.0, P<0.013). Significant differences were found between both groups regarding the presence of hyperuricemia and use of steroids and nonsteroidal anti-inflammatory drugs. Patients with three concomitant metabolic diseases were also identified as being at risk of bleeding. A forward stepwise logistic regression analysis revealed steroids, hyperuricemia and the use of calcium-channel blockers as independent risk factors of bleeding. CONCLUSION: Beside nonsteroidal anti-inflammatory steroid drug use, antihypertensive medication and concomitant arteriosclerotic diseases are risk factors for colonic diverticular hemorrhage. Our results support the hypothesis of an altered arteriosclerotic vessel as the source of bleeding.

Alcohol binging causes peliosis hepatis during azathioprine therapy in Crohn's disease.

Patients with inflammatory bowel disease have normal life expectancy and, due to modern immunosuppressive therapies, also a normal quality of life. Since mostly young people are affected, their social behaviour suits this environment. Alcohol binging is an increasingly disturbing factor among young people. We describe a patient with Crohn's disease, treated with azathioprine, who developed peliosis hepatis after three epsiodes of alcohol binging. Liver toxicity was not observed previously during the course of the treatment. Azathioprine-induced peliosis hepatis is thought to be idiosyncratic in humans. From animal studies, however, it is clear that hepatic depletion of glutathione leads to azathioprine toxicity to the sinusoidal endothelial cells. Damage of these cells causes peliosis hepatis. Since alcohol binging leads to hepatic glutathione depletion, we conclude that in our patient the episodes of binging have reduced liver gluathione content and therefore this has increased azathioprine toxicity causing peliosis hepatis. The problem of alcohol binging has not yet been addressed in IBD patients undertaking immunosuppressive therapy. This should be reviewed in future considerations regarding patients advice.

Serum levels of soluble Fas, nitric oxide and cytokines in acute decompensated cirrhotic patients.

AIM: To evaluate plasma levels of nitrite/nitrate (NOx), soluble Fas (sFas) antigen, tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) in patients with compensated and acute decompensated cirrhosis and to evaluate mediators causing acute decompensation in liver cirrhosis. METHODS: This prospective study was conducted in the medical intensive care unit of an academic tertiary center. Fifty-five patients with acute decompensation (gastrointestinal hemorrhage, encephalopathy, hydropic decompensation) and twenty-five patients with compensated liver cirrhosis were included. Blood samples were taken for analyses of sFas, Nox, IL-6, TNF-alpha. Liver enzymes and kidney functions were also tested. RESULTS: In patients with acute decompensation, plasma sFas levels were higher than in non-decompensated patients (15305 +/- 4646 vs 12458 +/- 4322 pg/mL, P < 0.05). This was also true for the subgroup of patients with alcoholic liver cirrhosis (P < 0.05). The other mediators were not different and none of the parameters predicted survival, except for ALT (alanine-aminotransferase). In patients with portal-hypertension-induced acute hemorrhage, NOx levels were significantly lower than in patients with other forms of decompensation (70.8 +/- 48.3 vs 112.9 +/- 74.9 pg/mL, P < 0.05). When NOx levels were normalized to creatinine levels, the difference disappeared. IL-6, TNF-alpha and sFas were not different between bleeders and non-bleeders. In decompensated patients sFas, IL-6 and NOx levels correlated positively with creatinine levels, while IL-6 levels were dependent on Child class. CONCLUSION: In acute decompensated cirrhotic patients sFas is increased, suggesting a role of apoptosis in this process and patients with acute bleeding have lower NOx levels. However, in this acute complex clinical situation, kidney function seems to have a predominant influence on mediator levels.

Ulcerative jejunoileitis and enteropathy-associated T-cell lymphoma.

Ulcerative jejunoileitis and enteropathy-associated T-cell lymphoma are rare conditions described in patients with refractory coeliac disease. Ulcerations affect the small bowel and are unrelated to drugs, ischaemia, infections or other known causes. We describe a female patient with an unclassified enteropathy who experienced several episodes of jejunoileal ulcerations. Several resections of the small bowel segments were necessary. The repetitive ulcerations were either from cytotoxic T cells, the patient developed a T-cell lymphoma, and malignant cells could be detected at the bottom of the ulcers, or from acid-producing cells in areas of gastric metaplasia. Two mechanisms might thus be responsible for the occurrence of repetitive ulceration, and require different treatment strategies. The patient is currently being treated with proton pump inhibitors, oral steroids and parenteral nutrition.