Papillary hyperplasia of the gallbladder in pancreaticobiliary maljunction represents a senescence-related lesion induced by lysolecithin.

Cellular senescence, an irreversible growth arrest, is considered to play as safeguard against malignant progression, though such a mechanism is speculative in human carcinogenesis. In gallbladder carcinoma, cholecystolithiasis and pancreaticobiliary maljunction (PBM) are major risk factors. Here, by using 113 surgically resected gallbladders and cultures of human gallbladder epithelial cells (HGECs) and gallbladder carcinoma cell line (TGBC2TKB), we examined carcinogenesis with respect to cellular senescence. Among 15 cases of PBM in which carcinoma was found in 4 cases, nonneoplastic gallbladder mucosa showed diffuse papillary hyperplasia (PHP). PHP was not found in gallbladders with cholecystolithiasis. Interestingly, PHP exhibited senescent features such as expression of p16(INK4A) and low cell proliferative activity. In contrast, EZH2, a polycomb group protein, was overexpressed in intraepithelial neoplasm and carcinoma in gallbladders with cholecystolithiasis. In PBM, EZH2 was expressed only in carcinoma foci but not in PHP. Cultured HGECs treated with lysolecithin, the level of which is elevated in gallbladder bile of PBM, showed increased expression of p16(INK4A) and senescence-associated beta-galactosidase. Conversely, enforced overexpression of EZH2 in senescent HGECs reduced p16(INK4A) expression. A knockdown of EZH2 in cultured TGBC2TKB cells increased p16(INK4a) expression. In conclusion, PHP in PBM may act as a barrier to malignant transformation for decades. EZH2 may be responsible for the escape from cellular senescence followed by malignant transformation in the gallbladder of PBM.

Limy bile syndrome presenting as acute acalculous cholecystitis.

A 35-year-old woman presented to the surgical clinic complaining of right hypochondrial pain for 4 days. Abdominal examination revealed tenderness on deep palpation in the right hypochonrdium, with no palpable organs or masses. The patient had repeated attacks of the same pain that mandated repeated admissions to the emergency hospital and treated conservatively. The white blood cell count was 13 000 cells/µL. Ultrasound examination of the abdomen showed thick-walled gall bladder, thick bile, with no visible stones and acalculous cholecystitis was the diagnosis. Decision done for laparoscopic cholecystectomy. After removal of the gall bladder and opening the bladder, a thick milky contents was found to fill the gall bladder with no stones. The diagnosis of limy bile syndrome then done. Histopathological examination of the gall bladder showed features of chronic cholecystitis with no malignancy. The patient discharged on the third postoperative day with no complications.

Regulation of fibrogenesis during the early phase of common bile duct obstruction.

BACKGROUND: Both nitric oxide (NO) and prostaglandins have been proposed as inhibitor substances involved in collagen deposition in the hepatic parenchyma. The possible reciprocal connections between NO and eicosanoids in the development of liver fibrosis were investigated during the initial phase of common bile duct obstructions. METHODS: A total of 30 male albino guinea pigs were randomly and equally assigned to three groups. Group 1 underwent sham laparotomy. Group 2 and group 3 were subjected to permanent common bile duct ligature for 24 and 72 h, respectively. Changes in the liver prostaglandin E(2) (PGE(2)), leukotriene C(4), malondialdehyde contents and plasma nitrite plus nitrate concentrations were measured. To evaluate the extent of hepatic fibrosis, histological assessment of liver was confirmed with the equivalent hydroxyproline contents of liver. RESULTS: Twenty-four hours after ligature, the amount of malondialdehyde and PGE(2) and plasma nitrite plus nitrate concentrations increased significantly, whereas liver hydroxyproline contents did not change. However, 72 h after ligature (Group 3), lipid peroxidation and collagen deposition were significantly higher than that of the group 2 animals. The PGE(2) : leukotriene C(4) ratio peaked at 24 h and later decreased, whereas PGE(2) : NO ratio remained unchanged in both group 2 and group 3 animals. CONCLUSIONS: The initiation of collagen synthesis occurred in portal tract as early as within the first 72 h of bile duct obstruction. The optimum function of reactive oxygen species on the stellate cell activation might be determined by the interaction between NO and PGE(2).

Expression and prognostic significance of 14-3-3sigma and ERM family protein expression in periampullary neoplasms.

Aberrant gene expression in pancreatic ductal adenocarcinomas contributes to the dismal outcome of patients who develop this disease. The 5' region of 14-3-3sigma (stratifin) is hypomethylated in pancreatic adenocarcinomas and is associated with gene overexpression. In multiple experimental systems, ezrin (ERM, Radixin, Moesin) has been identified as being important in the metastatic behavior of pancreatic and other cancers. We investigated the prognostic significance of aberrant expression of 14-3-3sigma and the ERM proteins (Ezrin, radixin, Moesin) in a series of invasive periampullary adenocarcinomas including 300 infiltrating pancreatic adenocarcinomas, 54 ampullary adenocarcinomas, and 33 noninvasive intraductal papillary mucinous neoplasms from patients who underwent pancreaticoduodenal resection at The Johns Hopkins Hospital, Baltimore, MD, between 1991 and 2003. Two-hundred fourty-four (82%) primary infiltrating adenocarcinomas of the pancreas demonstrated positive expression of the 14-3-3sigma, 45 (15%) showed weak immunolabelling, and 9 (3%) were negative. 201 (68%) showed positive immunolabeling of the ERM proteins, 75 (25%) demonstrated weak expression and 20 (7%) no expression. A similar proportion of ampullary cancers showed 14-3-3sigma and ERM protein expression. Expression of 14-3-3sigma and ERM protein was more likely in poorly differentiated cancers (p = 0.00005), and their expression was associated with poor survival in univariate analysis (p = 0.09). By multivariate analysis, patients whose cancers expressed 14-3-3sigma, but not ERM tended to have a poorer prognosis (Hazard ratio, 1.4; 0.9-2.2, p = 0.14). Aberrant expression of 14-3-3sigma may contribute to the outcome of patients with pancreatic ductal adenocarcinoma.

The novel formulation design of self-emulsifying drug delivery systems (SEDDS) type O/W microemulsion II: stable gastrointestinal absorption of a poorly water soluble new compound, ER-1258 in bile-fistula rats.

The stabilization effect of the novel self-emulsifying drug delivery systems (SEDDS) type O/W microemulsion on the gastrointestinal absorption of a poorly water soluble new compound, ER-1258 was examined by bile-fistula model rats. In the components of this formulation, medium chain fatty acid triglyceride (MCT), diglyceryl monooleate (DGMO-C), polyoxyethylene hydrogenated castor oil 40 (HCO-40) and ethanol were used as an oil, a lipophilic surfactant, a hydrophilic surfactant and a solubilizer at the mixture ratio of 25/5/45/25 w/w%, respectively. The ratios of AUC in the non-treated rats to that in the bile-fistula rats were 5.1, 12.1 and 3.0 for the suspension, the oily solution and the SEDDS type O/W microemulsion, respectively. The risk from which the difference between individuals of the compound absorption amounts resulting from the flow of the bile secretion serves as the maximum was high in order of oily solution>suspension>SEDDS type O/W microemulsion. Therefore, it was verified that the SEDDS type O/W microemulsion was able to reduce this risk, compared with the other formulations. When short chain fatty acid triglyceride (Triacetin) was used as an oil, the similar effect was demonstrated in the formulation composed of sorbitan sesquioleate (SO-15) as a lipophilic surfactant and polyoxyethylene hydrogenated castor oil 60 (HCO-60) or polyoxyethylene 20 sorbitan monooleate (TO-10M) as a hydrophilic surfactant.

Evidence for accelerated generation of hydroxyl radicals in experimental obstructive jaundice of rats.

We present evidence herein of the accelerated generation of hydroxyl radical (.OH) in the plasma and the liver tissue of common bile duct ligated (CBDL) rats, a model for experimental obstructive jaundice. .OH production in the plasma was monitored in vivo by the identification of dihydroxybenzoates in plasma [2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (2,5-DHB)] using high performance liquid chromatography (HPLC). The average concentrations of 2,3-DHB and 2,5-DHB produced in the plasma of the controls were 33+/-3 microM and 232+/-34 microM (n = 15), respectively, whereas their respective concentrations increased to 149+/-28 microM and 604+/-88 microM in the CBDL rats (n = 19). Furthermore, we also observed a time-dependent decreasing trend of 2,3-DHB and 2,5-DHB production after surgical removal of the ligation of the experimental animals. In addition, the generation of .OH in the liver tissue was studied by using dimethyl sulfoxide (DMSO) as a molecular probe and measuring the amount of methanesulfinic acid (MSA), the product of the trapping reaction. The net production of MSA in the liver tissue of the control rats was 1.22+/-0.05 O.D. unit/g protein (n = 5), whereas its respective concentration of MSA in the liver tissue of CBDL rats increased to 2.05+/-0.15 O.D. unit/g protein (n = 5). In addition, we showed that CBDL rats receiving a pretreatment of mannitol, an .OH scavenger, resulted in the decreased production of MSA. Electron micrographic study indicated that the most prominent change observed in CBDL rats was the alteration of mitochondria, which were swollen with distorted cristae. Meanwhile, the bile canaliculi were moderately more dilated than that of the controls, and an increased neutrophil peripheral blood count was found in CBDL rats when compared to the controls. Taken together, our data suggest that accelerated generation of .OH in the CBDL rats is obvious and may play a key role in the pathogenesis of liver damage associated with obstructive jaundice.

Sirolimus/cyclosporine/tacrolimus interactions on bile flow and biliary excretion of immunosuppressants in a subchronic bile fistula rat model.

The new immunosuppressive agent sirolimus generally is combined in transplant patients with cyclosporine and tacrolimus which both exhibit cholestatic effects. Nothing is known about possible cholestatic effects of these combinations which might be important for biliary excretion of endogenous compounds as well as of immunosuppressants. Rats were daily treated with sirolimus (1 mg kg(-1) p.o.), cyclosporine (10 mg kg(-1) i.p.), tacrolimus (1 mg kg(-1) i.p.), or a combination of sirolimus with cyclosporine or tacrolimus. After 14 days a bile fistula was installed to investigate the effects of the immunosuppressants and their combinations on bile flow and on biliary excretion of bile salts, cholesterol, and immunosuppressants. Cyclosporine as well as tacrolimus reduced bile flow (-22%; -18%), biliary excretion of bile salts (-15%;-36%) and cholesterol (-15%; -47%). Sirolimus decreased bile flow by 10%, but had no effect on cholesterol or bile salt excretion. Combination of sirolimus/cyclosporine decreased bile flow and biliary bile salt excretion to the same extent as cyclosporine alone, but led to a 2 fold increase of biliary cholesterol excretion. Combination of sirolimus/tacrolimus reduced bile flow only by 7.5% and did not change biliary bile salt and cholesterol excretion. Sirolimus enhanced blood concentrations of cyclosporine (+40%) and tacrolimus (+57%). Sirolimus blood concentration was increased by cyclosporine (+400%), but was not affected by tacrolimus. We conclude that a combination of sirolimus/tacrolimus could be the better alternative to the cotreatment of sirolimus/cyclosporine in cholestatic patients and in those facing difficulties in reaching therapeutic ranges of sirolimus blood concentration.

The effect of lethal endotoxaemia on the liver glycogen metabolism in guineapigs with common bile duct obstruction.

The effect of hepatocellular trauma due to bile reflux and endotoxaemia on liver glycogen metabolism was studied in guineapigs with common bile duct obstruction. Considerable drops in basal glycogen levels of hepatocytes from bile duct ligated (BDL) animals were recorded in comparison with the sham operated (SHAM) ones. However, the regurgitation of bile did not affect the basal blood glucose concentrations of BDL guineapigs. The circulating glucose was consumed, mainly reflecting the level of energy requirement of the peripheral tissues in the endotoxaemic SHAM pair-fed animals and the BDL group. The hepatic glycogen stores failed to prevent the SHAM group from becoming hypoglycaemic at the end of the eighth hour after endotoxin administration. Enhancement in glucose consumption and diminished liver glycogen indicated the necessity of glucose intake in the early phase of extrahepatic bile duct obstruction. It was concluded that both endogenous and exogenous glucose have limited value in improving energy metabolism in lethal endotoxaemia following bile duct obstruction.

Copper kinetics in infantile hepatobiliary disease.

Copper metabolism was investigated in 33 infants with hepatobiliary disease. In 25 paients with biliary atresia, 37-135 days old, hepatic copper ranged from 5 to 133 microgram Cu/g wet weight (mean:43.3 microgram). In over two-thirds of the liver samples copper content was elevated above normal. There was no correlation between hepatic copper concentration and patients' age or degree of liver fibrosis. The mean hepatic copper content was also elevated in six infants with other hepatobiliary diseases (mean:41.6 microgram). Serial assays of copper excretion in bile were made in 25 infants having Kasai procedures for biliary atresia. The average daily excretion of biliary copper in 12 patients with successful operations was 3.3 to 33.7 microgram (mean:15.1 microgram), whereas in 13 patients without postoperative bile drainage, the daily values were 0.3-6.4 microgram (mean:2.7 microgram) (p < 0.0001). In five patients with active bile excretion who had repeated liver biopsies there was a steady decrease in hepatic copper concentration. The results indicate that derangement of copper homeostasis occurs frequently in infants with hepatobiliary disease and that in patients with biliary atresia successful reestablishment of bile flow effects a return toward normal copper metabolism probably because of enhanced biliary excretion.

Hepatic mitochondrial changes in experimental obstructive jaundice complicated by biliary infection.

The effects of biliary infection on the structural and functional changes in the liver in obstructive jaundice was studied using rats as experimental animals. Biliary infection with obstructive jaundice was induced in the animals by injecting Escherichia coli into the common bile duct through a nylon tube cannulated into the duct. This was followed by the clamping of the tube. For the controls, the tube was clamped in the absence of Escherichia coli. After 3, 7, 14 and 21 days, the animals were sacrificed, and some serum enzyme activities, histological changes in the liver, and the coupling efficiency of mitochondria isolated from the liver were investigated. The phosphorylating ability of hepatic mitochondria was more seriously affected when the obstruction was complicated by cholangitis. We suggest that, when associated with obstructive jaundice, biliary infection should be carefully treated prior to hepatectomy.

Pharmacokinetics of lidocaine and its major metabolite- monoethylglycinexylidide (MEGX) in rabbits with experimental common bile duct obstruction.

The aim of this study was to evaluate, using an experimental model, the effect of obstructive cholestasis on the pharmacokinetics of lidocaine and the formation rate of its major metabolite- monoethylglycinexylidide (MEGX)-in rabbits. The investigation was carried out on 20 rabbits, randomly divided into two groups: a control one sham-operated and an experimental one-animals with biliary duct ligation. The measurements, i.e. laboratory and pharmacodynamic tests, as well as pharmacokinetic assays were performed prior to the operation as well as 10-12 days after the bile duct ligation. At the end stage of the study, livers were examined macro- and microscopically and biochemical analysis of the liver microsomes were performed. Lidocaine was given intravenously, as a bolus of 6 mg/kg. Blood for pharmacokinetic assay was sampled within 6 h following the drug administration, and MEGX concentration was evaluated 15 min after lidocaine had been administered. The immunofluorescence polarization method was employed for determination of lidocaine and MEGX concentrations. The one-compartment open model was used for calculations.

[Biogenic amines in duodenal contents in various forms of biliary tract dyskinesia]. / Biogennye aminy v duodenal'nom soderzhimom pri raznykh formakh diskinezii zhelchevyvodiashchikh putei.

The concentration and secretion of adrenaline and noradrenaline with different portions of the bile seem to significantly reflect the function of the vegetative innervation apparatus of the biliary tract. A correlation was established between the pattern of motor and tonic disorders and the pattern of catecholamine secretion during multi-stage duodenal exploration. The data obtained make it possible to outline approaches to adequate therapy.

[Morphofunctional characteristics of endocrine gastric cells in chronic biliary pancreatitis]. / Morfofunktsional'nye osobennosti éndokrinnykh kletok zheludka pri khronicheskom biliarnom pankreatite.

Clinicoendoscopic morphological and morphofunctional studies in 50 patients with chronic cholecystitis and 50 patients with chronic biliary pancreatitis have demonstrated that morphometric analysis of the antral stomach endocrine cells secreting melatonin, neurotensin and somatostatin can be used in addition to standard laboratory and device methods for differentiation chronic biliary pancreatitis with biliary pathology.

Participation of bone marrow cells in biliary fibrosis after bile duct ligation.

BACKGROUND AND AIM: Bone marrow derived cells are involved in the process of hepatic fibrosis secondary to chronic injury. However, it is not yet known how quickly this event occurs in acute fibrosis models. The purpose of this study was to determine the role of bone marrow cells in rapid fibrosis following bile duct ligation in mice using green fluorescent protein (GFP) expressing bone marrow cells. METHOD: After whole body irradiation, 1 x 10(6) donor whole bone marrow cells from green fluorescent protein(+/-) mice were transplanted into C57BL/6 recipients via the tail vein. Four weeks after bone marrow transplantation, chimeric mice were subjected to common bile duct ligation, and livers of these animals were histologically examined after bile duct ligation using anti-fibroblast specific protein (FSP)-1 antibody and anti-alpha-smooth muscle actin (alpha-SMA) antibody. RESULTS: Periductal fibrosis consisting of fibroblast specific protein-positive cells was demonstrated histologically as early as day 7. Most of the fibrotic cells were green fluorescent protein-negative, however, a significant number of cells were green fluorescent protein-positive and some were also anti-FSP or alpha-SMA-positive. CONCLUSION: Differentiation of bone marrow derived cells into activated fibroblast and myofibroblast-like phenotypes occurs in the very early course of periductal fibrosis following bile duct ligation, suggesting a new strategy for prevention of biliary fibrosis by inhibiting migration of bone marrow cells to liver.