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Ornithine decarboxylase (ODC) plays a critical role in cell proliferation and is overexpressed in a variety of cancers. Furthermore, γ-aminobutyric acid (GABA) content and glutamate decarboxylase (GAD) activity are increased in neoplastic tissues in colon and breast cancer. However, few studies have examined these molecules in gallbladder cancer specimens. We observed the expression levels of ODC and GAD65 in benign and malignant lesions of the gallbladder and investigated their clinicopathological significance for the first time. The expression levels of ODC and GAD65 in specimens from gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenomatous polyps (n=15) and chronic cholecystitis (n=35) were detected using immunohistochemical methods. Kaplan-Meier survival and Cox regression analyses were carried out to explore the clinical and pathological correlations. The levels of positive staining of ODC and GAD65 were significantly higher in gallbladder adenocarcinoma than in peritumoral tissues, adenomatous polyps and chronic cholecystitis. The Kaplan‑Meier survival analysis and Cox regression analysis showed that the expression of ODC and GAD65 correlated significantly with the one-year survival rate and the mean survival time of the patients postoperatively. We conclude that the overexpression of ODC and GAD65 are significant in the carcinogenesis and progression of gallbladder adenocarcinoma. They may be important biological markers for the evaluation of biological behaviors and the prognosis of gallbladder adenocarcinoma.

A 62-year-old woman presented with a markedly increased serum ALP level of skeletal origin during a regular follow-up of chronic hepatitis C. Serum calcium, phosphorus, and intact-PTH levels were normal and bone turnover markers were increased. Her generalized bone density was diffusely increased. These findings were consistent with hepatitis C-associated osteosclerosis (HCAO). She underwent cholecystectomy, as gallbladder cancer was suspected; however, histopathological findings demonstrated xanthogranulomatous cholecystitis. After cholecystectomy, serum ALP level and bone turnover markers were gradually decreased. This may indicate the existence of a novel osteogenic factor in the gallbladder in HCAO.

Carcinoma of the gallbladder is the third most common cancer of the gastrointestinal tract. Recent studies have shown increased arginase activity in various malignancies. The main aim of this study was to evaluate whether arginase activity increases in carcinoma of the gallbladder. The arginase activity was evaluated in serum and gallbladder tissue in 22 patients with histologically proven carcinoma of the gallbladder and 20 patients with cholecystitis using spectrophotometry and western blot assay. The Student's t-test, analysis of variance, and Student-Newman-Keuls test were used for comparison of data and for statistical significance. The mean tissue arginase and serum arginase activity (118.64+/-17.45 and 15.91+/-1.91, respectively) in cases of carcinoma of the gallbladder were significantly higher in comparison with cholecystitis (86.37+/-4.45 and 12.73+/-0.72, respectively). Subgroup analysis showed stage III gallbladder carcinoma had the maximum tissue arginase activity (142.00+/-21.68 U/g of tissue) followed by stage II (124.15+/-19.88) and stage I (108.46+/-6.73). This significant rise in mean tissue arginase and serum arginase activity in patients with gallbladder cancer probably supports an association between arginase activity and the malignancy.

The correlation between matrix metalloproteinase (MMP)-2, MMP-9, and MMP-14 expression on the prognostic parameters of gallbladder carcinoma (GBC) and their role in carcinogenesis were evaluated. Carcinomas of the gallbladder (n=20) and chronic cholecystitis (n=10) were studied for the expression of MMP-2, MMP-9, and MMP-14 by immunohistochemistry. In all of the cases, metaplastic and dysplastic epithelial alterations, and (in GBC histologic type, grade of differentiation, level of infiltration, perineural and angiolymphatic invasion, liver invasion, and lymph node involvement were noted. MMP-2, MMP-9, MMP-14 were expressed in tumor epithelium in 9 (45%), 20 (100%), and 20 (100%) of the cases, respectively. MMP stromal expression including muscle layer, vascular endothelium, fibroblasts, and lymphoid cells were detected in all cases. MMP-2 was not expressed in normal, metaplastic, and dysplastic epithelia. In contrast, MMP-9 and MMP-14 immunoreactivities were present in antral-type metaplastic areas as moderate (grade 2) and strong in dysplastic epithelia (grade 3). Only in mucinous-type GBC was the expression of the MMPs lower than in the other types. No significant correlation was detected with the grade of differentiation, level of infiltration, perineural and angiolymphatic invasion, liver invasion, or lymph node involvement. These data suggest that MMP-9 and MMP-14 overexpression may have an important role in tumorigenesis. MMP-2, MMP-9, and MMP-14 were expressed in GBC epithelium but also the expression in the stromal component may be essential for the malignant potential of GBC.

Background and Objectives: It was proposed that occult pancreaticobiliary reflux (OPBR) was associated with precancerous mucosal changes in the gallbladder, hence the importance of this disorder. There are no published reports investigating the incidence of OPBR in patients operated on for the entire spectrum of benign gallbladder diseases and gallbladder cancer. Our aim was to determine the incidence of OPBR and measure the levels of active pancreatic enzymes (amylase and lipase) in gallbladder bile of patients undergoing cholecystectomy for benign and malignant gallbladder diseases., Methods: One hundred eight patients with normal pancreaticobiliary junction evidenced by operative cholangiography were included in the study., Results: According to gallbladder bile amylase and lipase levels, 84.2% and 89% patients respectively had OPBR. OPBR was present in all gallbladder cancer patients; in these patients the biliary levels of amylase and lipase were significantly higher than the levels found in patients with benign gallbladder pathology (P < 0.0001)., Conclusions: OPBR could lead to inflammatory changes of the biliary epithelium and progress towards the development of precancerous mucosal changes and gallbladder cancer. The reason why such high levels of pancreatic enzymes are regurgitated into the biliary tree of patients with gallbladder cancer should be clarified., (Copyright 2007 Wiley-Liss, Inc.)

This study examines the hypothesis that cholecystitis down-regulates Guinea pig gallbladder (GPGB) smooth muscle cholecystokinin (CCK)-stimulated prostaglandin (PG) release. Guinea pig gallbladder from Control and 48 h bile duct ligated (BDL) animals were placed in cell culture and grown to confluence. The cultures underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacyclin Synthase (PS), or were incubated with CCK at 10(-8)M or 10(-6)M with and without indomethacin for 1h and analyzed for release of 6-keto-PGF1alpha, PGE2 and TxB2 by EIA. BDL increased Guinea pig gallbladder cell culture basal PGE2 and PGI2 release which was in part due to increased COX-2 content. CCK incubation down-regulated BDL Guinea pig gallbladder cell culture release of 6-keto-PGF1alpha and PGE2 and down-regulated COX-2 content but did not alter the Control group. The decrease in CCK-mediated BDL cell Guinea pig gallbladder release may be an endogenous mechanism to limit physiologic derangements induced by increased endogenous gallbladder PG synthesis during early acute cholecystitis.

Background: Two acyl-coenzyme A:cholesterol acyltransferase (ACAT) genes, ACAT1 and ACAT2, have been identified that encode 2 proteins responsible for intracellular cholesterol esterification., Methods and Results: In this study, immunohistology was used to establish their cellular localization in human liver biopsies. ACAT2 protein expression was confined to hepatocytes, whereas ACAT1 protein was found in Kupffer cells only. Studies with a highly specific ACAT2 inhibitor, pyripyropene A, in microsomal activity assays demonstrated that ACAT2 activity was highly variable among individual human liver samples, whereas ACAT1 activity was more similar in all specimens. ACAT2 provided the major cholesterol-esterifying activity in 3 of 4 human liver samples examined., Conclusions: The data suggest that in diseases in which dysregulation of cholesterol metabolism occurs, such as hypercholesterolemia and atherosclerosis, ACAT2 should be considered a target for prevention and treatment.

Our goal has been to investigate the expression and correlated significance of inducible nitric oxide synthase (iNOS) and P53, Bax in benign and malignant gallbladder diseases. We detected the expression of iNOS, P53 and Bax in the gallbladder wall by SP immunohistochemistry in 16 cases of chronic cholecystitis, 11 cases of chronic cholecystitis with adenomyoma and 24 cases of gallbladder adenocarcinoma. The percentage of positively marked tumor cells was counted under microscope and the intensity of immunoreactivity was graded. SPSS10.0 statistical software was applied for statistical analysis. In this study, we found that: (1) Both benign and malignant diseased gallbladder wall expressed iNOS and Bax. Compared to benign diseased gallbladders, their expression in adenocarcinoma was decreased (p < 0.05), P53 was expressed strongly only in nuclei of adenocarcinoma cells of some cases. (2) In benign and malignant diseased gallbladders, iNOS expression was related positively to Bax (p < 0.01), the expression of P53 and Bax had a negative relationship (p < 0.01). The results suggested that both chronic cholecystitis and chronic cholecystitis with adenomyoma carry the risk of becoming malignant, especially the latter. NO is an important mediated molecule in cancer, there are intimate relationships between gallbladder cancer and apoptosis.

The COX expressions were evaluated separately in the epithelium and in the stroma of gallbladder cancer, chronic cholecystitis, xanthogranulomatous cholecystitis (XGC) and the normal gallbladder. In normal gallbladder COX-2 expression rate was significantly higher in the epithelium than in the stroma. The COX-2 expression rate in the epithelium of non-cancerous adjacent epithelium to cancerous lesion was significantly lower than those not only of cancer, but also chronic cholecystitis, XGC and normal gallbladder. In stroma, the COX-2 expression rate in cancer, chronic cholecystitis and XGC were significantly higher than that of the normal gallbladder. The rate in non-cancerous adjacent stroma to cancer is significantly lower than that of cancer and XGC. However, the difference of rate between of normal and of chronic cholecystitis was not significant. The COX-2 expression rates were significantly higher in both the epithelium and the stroma in the well and moderately differentiated cancer group than in the poorly and undifferentiated cancer group. Our results suggest that COX-2 expression in the gallbladder may be regulated by various factors and not directly related to carcinogenesis. The significance of its repression in the non-cancerous adjacent tissue to cancer lesion should be re-evaluated.

Telomerase is detected by the telomeric repeat amplification protocol (TRAP) assay in more than 85% of primary cancers. In the present study, we determined telomerase activity using exfoliated bile cells obtained from biliary tract neoplasia specimens. The aim of this study was to provide additional information regarding minimally invasive approaches to the detection of biliary tract cancer in combination with routine cytologic examination. We analyzed for telomerase activity bile juice from patients with gallbladder carcinoma, cholangiocarcinoma, cholecystitis and cholangitis. Semiquantitative determination of telomerase activity was performed using both a fluorescence-based TRAP assay on cell extracts and at the cellular level by an in situ TRAP assay. The fluorescence-based TRAP assay detected bile telomerase activity in samples from 4 of 10 patients with biliary tract cancer. In contrast, the in situ TRAP assay detected telomerase positive cells in samples from 6 of 10 patients with biliary tract cancer. However, only one of these samples showed class V cytology. A combination of semiquantitative analysis and an in situ TRAP assay to detect telomerase positive cells may improve the diagnosis of biliary tract cancers with the combination of routine cytologic examination.

Activity was studied of blood serum plasmic enzymes L-serine and L-threonine dehydrogenazes (SDG and ThDG) in 92 liquidators of aftermath of the Chernobyl atomic power plant breakdown, presenting with chronic non-calculous cholecystitis during the stage of moderately severe exacerbation with no clinical and laboratory and sonographic signs of affection of the liver. A quarter of the examinees demonstrated an increased activity of the enzymes under study, which fact is regarded by the authors as a preclinical sign of reactive hepatitis. Recommendations are given as to the outpatient registration and prophylactic management and therapy of those persons having taken part in the elimination of the effects of the Chernobyl accident, presenting with biliary pathologies.

Background & Aims: Nitric oxide is synthesized from L-arginine and is metabolized to nitrate and nitrite. This study evaluates the effects of a pharmacological blockade of NO synthesis on fluid transport by the inflamed gallbladder mucosa., Methods: Experiments were performed in cats with cholecystitis and in control animals. NO synthase activity was measured in gallbladder tissue; the enzyme was characterized by immunoblotting techniques and localized by immunofluorescence. Fluid transport and release of nitrate and nitrite by the gallbladder mucosa and bile and bile salt secretion from the liver were registered simultaneously in vivo., Results: Fluid secretion in inflamed gallbladders was reversed to a net absorption in response to the NO synthase blockers N omega-nitro-L-arginine and aminoguanidine, and formation of nitrate was reduced. The effects were reversed by L-arginine. Increased levels of inducible NO synthase in inflamed gallbladders were shown by immunoblotting, by immunofluorescence (mainly in macrophages), and by Ca(2+)-independent [3H]citrulline formation from [3H]arginine. The NO synthase blockers had no effect on gallbladder fluid transport in normal gallbladders., Conclusions: Increased levels of inducible NO synthase activity are shown in inflamed gallbladders, and a pharmacological blockade of this enzyme blocks fluid secretion and decreases nitrate release from the mucosa.

Acute enzymatic cholecystitis is a rare form of an acute enzymatic-inflammatory process of the gallbladder. In 782 patients with ultrasound signs of cholecystitis, acute enzymatic cholecystitis has been found in 10 cases (1.2%). The authors describe the most common ultrasound signs of acute enzymatic cholecystitis and its morphologic interpretation. The data given in the article help early diagnosis of acute enzymatic cholecystitis and to start the treatment in time.

In patients with purulent inflammation foci of various location, reduction in the number of natural killers (NK) and in the activity of alpha-glycerophosphatedehydrogenase (alpha-GPDH) and adenosinetriphosphatase (ATP-ase) of lymphocytes was revealed. Maximum reduction in indices of NK, alpha-GPDH and ATP-ase was noted in patients with diffuse peritonitis, phlegmonous-gangrenous cholecystitis, destructive appendicitis. After treatment, the mentioned indices increased, but by the time of discharge of the patients from the hospital they were lower than the normal ones.

A study to develop a sensitive method for measuring beta-lactamase activity in bile was conducted. Since separation of substrate from biological components is required to increase the assay sensitivity and to achieve an accurate assay of beta-lactamase activity, high performance liquid chromatography (HPLC) was used for separation and analysis of the substrates (cephaloridine for cephalosporinase, benzylpenicillin for penicillinase and cefuloxime for cefuloximase). In addition, conditions for increased assay sensitivity were also studied and optimal substrate concentrations and reaction times were determined. beta-Lactamase activity of 0.05 munit/ml in bile was detected using the HPLC assay method which is a significant improvement when compared to the direct spectrophotometric method which has a detection limit of approximately 10 munit/ml.

Dynamics of glutathione-related enzymes activity was studied in erythrocytes of 22 dogs with destructive form of cholecystitis. As clinical symptoms of intoxication developed the enzymatic activity was decreased. In the animals with purulent-inflammatory complications distinct decrease was detected in activity of glutathione reductase (by 54.3%), glutathione-S-transferase--by 46.94% and glutathione peroxidase--by 42.1% (P less than 0.05). These data suggest that specific methods should be chosen for correction of impairments in the enzymatic activity in order to improve the treatment course efficiency as well as for prophylaxis of complications. The procedure developed for cholecystitis treatment, which involved channelled transport of antibiotics by means of autologous erythrocyte ghosts, proved to be more effective as compared with routine methods as shown by evaluation of the animals clinical state as well as by dynamics of hepato-specific enzymes activity and the glutathione-related enzymes activity. This procedure may be used in clinical practice; the laboratory tests described may serve for evaluation of the treatment course efficiency.

In the experiment on 30 mongrel dogs, acute enzymatic cholecystitis (AEC) was modelled by means of intraduodenal connection with a fluoroplastic catheter of the common bile duct and pancreatic duct. The animals died at day 3-5 from transudative biliary peritonitis. A correlation between the severity of the clinical course, pronouncement of enzymologic shifts (increase in activity of amylase and phospholipase A2, content of biliary malonic dialdehyde) and morphological changes in the gallbladder and common bile duct in the form of necrosis and round-cell infiltration of the mucosa was revealed. Only decompression of the bile duct within the first 6 h from the moment of AEC development contributes to regression of clinical and enzymologic disorders and recovery of the animals.

Four patients with acquired immunodeficiency syndrome developed severe abdominal pain and fever due to acute acalculous cholecystitis. In all patients, preoperative laboratory data showed elevation of alkaline phosphatase and gamma-glutamyltransferase levels. Endoscopic or intraoperative cholangiography showed signs of intrahepatic and extrahepatic cholangitis. Cholecystectomy was performed and prompt relief of symptoms was achieved in all patients; no postoperative complication was observed. One patient did not develop any recurrence during an 18-month period of follow-up; two patients died 2 and 3 months after the operation. One patient developed recurrent abdominal pain and cholestasis 4 months after the operation, with dilatation of the common bile duct and papillary stenosis due to progression of cholangitis. These observations suggest that cholangitis is frequently associated with cholecystitis in patients with the acquired immunodeficiency syndrome. Its pathogenesis is not known.

In analysis of the results of clinical, laparoscopic and intraoperative investigations in 70 patients with acute obstructive, and 68--with acute enzymatic cholecystitis, it was established that for differential diagnosis of acute enzymatic cholecystitis, of most importance were the intractable pain in the right hypochondrium, early appearance of peritoneal symptoms, hyaloid edema of the gallbladder wall and hepatoduodenal ligament, exudate in the subhepatic space with a dash of bile, and detection of the phospholipase activity and high content of malonic dialdehyde in the cystic bile.

Esterases active at neutral and acid pH have been demonstrated in human gallbladder epithelium. Reaction to a wide variety of inhibitors and activators suggests that they probably represent the same enzyme with two active sites while a close correlation with acid phosphatase, a known lysosomal enzyme, suggests a lysosomal localization. Esterase activity in the gallbladder mucosa in significantly decreased in chronic cholecystitis. Polyacrylamide gel electrophoresis suggests that cholesterol stones may be associated with a specific type of isoenzyme pattern. Esterase positive cells are present in the inflammatory infiltrate in the lamina propria of the gallbladder in most cases of chronic cholecystitis. Naphthol AS-D chloracetate esterase staining indicates that mast cells form a large part of the infiltrate. Bile acids modulate the enzyme activity in vitro. Although the exact substrate specificity is not known, several possibilities are discussed, implicating esterases in the pathogenesis of chronic cholecystitis.

The positional specificity of the phospholipase A in human gallbladder epithelium was studied by using biosynthetically radioabeled diacylphosphoglycerides as substrates. Diacylphosphoglyceride in 14C-palmitic acid-labeled, autoclaved E.coli was hydrolyzed under the formation of monoacylphosphoglyceride and fatty acid that were both radiolabled. In contrast, diacylphosphoglyceride in 14C-oleate-labeled bacteria was hydrolyzed so as to give radiolabel in the fatty acid only. Since 14C-palmitate occupies predominantly the 1-acyl position and 14C oleate the 2-acyl position of the major E. coli diacylphosphoglycerides, these findings suggest that: 1) the phospholipase attacks and 2-position of diacylphosphoglycerides, and 2) a complete deacylation of diacylphosphoglycerides in the gallbladder wall is brought about by the combined action of phospholipase A2 and lysophospholipase, the latter being able to hydrolyze the 1-acyllysophosphoglyceride. It appears, therefore, that the biochemical preequisites for a local formation and degreadation of lysolecithin in the gallbladder itself are met by the positional specificity of theenzymes present. This finding further substantiates the hypothesis that lysolecithin is an adjustable mediator of aseptic cholecystitis.

Serum cholinesterase activity (ChE) was studied in 84 patients with chronic liver diseases (cirrhosis, chronic hepatitis, chronic cholecystocholangiohepatitis, hepatolenticular degeneration). Most pronounced alterations were found in cirrhosis. Significant difference is not established between cardiac and "noncardiac" cirrhosis but is well established between decompensated and compensated cirrhosis. As a rule ChE is normal in chronic hepatitis and cholecystocholangio-hepatitis. The values in agressive and persisting hepatitis do not differ significantly. Essential correlation of ChE with serum albumins is established. The diagnostic ChE value is confronted with that of the routine laboratory indices. Critical values are established (1500 ME, 1100 ME resp) that may be helpful in the differentiation of cirrhosis from chronic hepatitis, compensated from decompensated cirrhosis resp.

An investigation of the lysozyme activity of the hepatic tissue, B-bile and C-bile has shown that in mechanical jaundice the lysozyme activity of the hepatic tissue is increased. A reliable decrease of the C-bile lysozyme activity is noted in patients with purulent cholangitis. In spite of adequate methods of external drainage of bile ducts in purulent cholangitis the bacterial activity of bile due to lysozyme is recovered slowly. The results obtained allow to make the degree of inflammation of bile ducts objective and to optimize the duration of antibacterial therapy.

Aseptic inflammation in the gallbladder wall was studied in vitro using rabbit gallbladders. Wall permeability changes, measured as changes in the transmural passage of radioactively labelled hippuran and biligraphin as well as enzyme (acid phosphatase) release from wall to lumen were registered as parameters permitting quantitative assessment of an inflammatory process. Lysolecithin, a surface-active substance of possible significance in the pathogenesis of acute cholecystitis was chosen as inflammation-inducing agent. When 6-20 mg/ml lysolecithin was instilled into the gallbladders, dose-dependent increases in permeability and enzyme release occurred. No effects as compared with controls were seen when 2 mg/ml was instilled. Lysolecithin-treated gallbladders showed morphological signs of inflammation as opposed to untreated. The data reported are consistent with earlier observations that lysolecithin may cause inflammatory lesions to the gallbladder wall. The experimental model used may prove useful in examining the effects of aggressive and protective agents upon the gallbladder mucous membrane.

Histochemically, alkaline phosphatase was localised in epithelial brush-border and capillary endothelium of the lamina propria in 41 gallbladders studied. Three distinct patterns of the brush-border enzyme distribution were observed, namely, complete delineation, patchy localisation and a brush-border totally devoid of enzyme activity. Where no histochemical activity was found, biliary chenodeoxycholate was 50 per cent. higher than in cases of patchy distribution and twice as high as in cases with complete staining of the brush-border. Conversely, biliary cholate levels were significantly higher in completely stained cases. Biliary cholesterol was higher in the cases with no histochemical activity and this was reflected in a significantly higher lithogenic index. Significant quantities of cholesterol esters were found in bile from the focal activity group only. Total alkaline phosphatase activity assayed in gallbladders from 112 patients with cholecystitis and/or lithiasis showed that activity was significantly lower in patients with pigment stones than patients with cholecystitis and/or cholesterol stones. This activity was not related to biliary lipid composition in the 77 bile samples analysed. This evidence indicates that biliary lipid composition is associated with the histochemical distribution of alkaline phosphatase in gallbladder mucosa.

It is often difficult to differentiate acute pancreatitis (A.P.) from some other acute abdominal diseases, when there is an elevated serum amylase. In contrast, the renal clearance of amylase, expressed as a percentage of creatinine clearance, can separate patients with A.P. from patients with acute colecistitis, common duct stone without pancreatitis, hyperamylasemia after biliary surgery, acute peptic ulcer and acute salivary diseases.

The authors have studied the activity of enzymes of pentosophosphate pathway in patients with different diseases of bile outflow tracts. An increased activity of the enzymes under consideration was found after an operation for acute phlegmonous, acute gangrenous and chronic cholecystitis, chronic cholecystopancreatitis, mechanic jaundice. It is the authors' opinion that the changes revealed are not specific, and they can not be considered a diagnostic test in bile passages diseases, however, these tests may serve as the control of the efficacy of therapy and functional state of the liver.

Determination of B-glucuronidase activity was carried out in 203 subjects including 60 controls. A modified technic for assaying B-glucuronidase activity was used that employs glass column chromatography and Sephadex. Although the lowest mean value was in the group of patients with cancer of the pancreas, there was wide range of activity of B-glucuronidase in all groups and subgroups and considerable overlap in values between them. It was concluded, therefore, that B-glucuronidase activity assay of the duodenal contents was of no value as a diagnostic test for cancer of the pancreas or for distinguishing between the several diseases affecting the pancreas.