Rolipram attenuates bile duct ligation-induced liver injury in rats: a potential pathogenic role of PDE4.

Anti-inflammatory and antifibrotic effects of the broad spectrum phosphodiesterase (PDE) inhibitor pentoxifylline have suggested an important role for cyclic nucleotides in the pathogenesis of hepatic fibrosis; however, studies examining the role of specific PDEs are lacking. Endotoxemia and Toll-like receptor 4 (TLR4)-mediated inflammatory and profibrotic signaling play a major role in the development of hepatic fibrosis. Because cAMP-specific PDE4 critically regulates lipopolysaccharide (LPS)-TLR4-induced inflammatory cytokine expression, its pathogenic role in bile duct ligation-induced hepatic injury and fibrogenesis in Sprague-Dawley rats was examined. Initiation of cholestatic liver injury and fibrosis was accompanied by a significant induction of PDE4A, B, and D expression and activity. Treatment with the PDE4-specific inhibitor rolipram significantly decreased liver PDE4 activity, hepatic inflammatory and profibrotic cytokine expression, injury, and fibrosis. At the cellular level, in relevance to endotoxemia and inflammatory cytokine production, PDE4B was observed to play a major regulatory role in the LPS-inducible tumor necrosis factor (TNF) production by isolated Kupffer cells. Moreover, PDE4 expression was also involved in the in vitro activation and transdifferentiation of isolated hepatic stellate cells (HSCs). Particularly, PDE4A, B, and D upregulation preceded induction of the HSC activation marker α-smooth muscle actin (α-SMA). In vitro treatment of HSCs with rolipram effectively attenuated α-SMA, collagen expression, and accompanying morphologic changes. Overall, these data strongly suggest that upregulation of PDE4 expression during cholestatic liver injury plays a potential pathogenic role in the development of inflammation, injury, and fibrosis.

Immunohistochemical analysis of cyclooxygenase-2 and vascular endothelial growth factor in pancreaticobiliary maljunction.

Recent studies have elucidated that cyclooxygenase (COX)-2 is strongly related to cancer progression or development by means of its anti-apoptotic effect, enhancement of angiogenesis or decrease of cell-to-cell adhesive activity. However, there is no report on the relationship between COX-2 expression and angiogenesis in pancreaticobiliary maljunction (PBM). We examined the correlation between the overexpression of COX-2 and vascular endothelial growth factor (VEGF) in 65 lesions from 30 patients with PBM immunohistochemically. The positive expression of COX-2 was found in 20% of regenerative epithelium, 11.1% of hyperplasia without atypia, 86.4% of hyperplasia with mild atypia, 75% of dysplasia, and 75% of cancerous lesions. VEGF was highly expressed in 80% of regenerative epithelium, 27.8% of hyperplasia without atypia, 86.4% of hyperplasia with mild atypia, 66.7% of dysplasia, and 75% of cancerous lesions. The positive rate of both COX-2 and VEFG expression was significantly higher in hyperplasia with atypia, dysplasia and cancerous lesions than that in hyperplasia without atypia. In addition, there was a statistically significant correlation between COX-2 and VEGF overexpression among all lesions. In 6 of 8 patients of various histological types, both COX-2 and VEGF were stained in almost exactly the same locations. In addition, there were no significant differences between the degree of inflammatory cell infiltration in the surrounding stroma and the expression of COX-2 and VEGF, respectively. These results demonstrated a strong relationship between COX-2 and VEGF overexpression in PBM. Therefore, chemoprevention via the suppression of angiogenesis by means of COX-2 inhibitor may be effective in PBM.

Detection of telomerase reverse transcriptase mRNA in biopsy specimens and bile for diagnosis of biliary tract cancers.

Human telomerase reverse transcriptase (hTERT), which codes for the catalytic subunit of telomerase, is essential for telomerase activity. Recent studies revealed that levels of hTERT mRNA as well as telomerase activity are high in neoplasm. The purpose of this study was to correlate the expression of hTERT mRNA with telomerase activity in biopsy specimens and bile from biliary tract cancers and to evaluate the potential diagnostic value of hTERT mRNA analysis for biliary malignancy. We analyzed hTERT mRNA and telomerase activity in biopsy specimens and exfoliated bile cells from patients with cholangiocarcinoma, gallbladder carcinoma and bile duct stones. hTERT was detected by either nested reverse transcriptase-polymerase chain reaction (PCR) or real-time PCR. Telomerase activity was examined by a fluorescence-based telomeric repeat amplification protocol assay. Six of 10 malignant biopsy specimens had detectable hTERT and 7 of 10 had telomerase activity. All cases with hTERT expression had telomerase activity. In bile, 7 of 10 malignant patients had detectable hTERT and 3 of 10 had telomerase activity. Importantly, there were no false positive results in tissue specimens or bile examined in 6 non-cancerous cases. In conclusion, the detection of hTERT mRNA in biopsy specimens and bile cells, in combination with routine histologic and cytologic examination may improve the diagnosis of biliary tract cancers.

A model of bile duct hyperplasia in the rat induced by diethylnitrosamine and selective cytotoxicity.

Adult Wistar rats were subjected to a chemical carcinogenesis regimen involving initiation with diethylnitrosamine (DEN) and cytotoxic selection of initiated cells following partial hepatectomy. The livers of treated rats exhibited sequential changes of vacuolar degeneration and hepatocellular nodular hyperplasia up to 5 mth after completion of the experimental regimen. The hyperplastic nodules regressed slowly at that time. Cystic bile duct hyperplasia emerged with high frequency between 5 and 15 mth after completion of the regimen. The nitrosamine-initiated nodular and biliary hyperplasias could not be unequivocally accepted as preneoplastic lesions since frankly neoplastic transformation under these conditions was a relatively rare occurrence.

Changes in serum succinyltrialanine p-nitroanilide hydrolysing activity in hepatobiliary diseases.

Serum succinyltrialanine p-nitroanilide hydrolysing activity was elevated in patients with hepatobiliary diseases. The highest activities were seen in acute cholangitis and intrahepatic cholestasis. The change in succinyltrialanine p-nitroanilide hydrolysing activity was closely associated with those in gamma-glutamyltranspeptidase and alkaline phosphatase activities. In some cases, however, the former was more sensitive than the latter.

[Hepatobiliary alternations in leptospirosis convalescents]. / Porazheniia gepatobiliarnoi sistemy u bol'nykh, perenesshikh leptospiroz.

AIM: To study hepatic function in leptospirosis convalescents. MATERIALS AND METHODS: Clinical laboratory and device examinations were performed in 121 leptospirosis convalescents. RESULTS: The majority of the examinees retained biochemical signs of cholestatic, mesenchymal-inflammatory and moderate cytolytic syndromes secondary to hepatic impairment. Ultrasound and radionuclide investigations detected biliary disorders and secondary chronic hepatitis in many of leptospirosis convalescents. CONCLUSION: It is shown that leptospirosis rehabilitation must be targeted. Reasons of development of chronic hepatic and biliary lesions in population of the north Caucus and Kuban in the absence of viral hepatitis markers are suggested.

[Antioxidant liver enzymes in chronic liver disease]. / Antioksidantnye énzimy pecheni pri ee khronicheskom porazhenii.

Biopsy of the liver of 73 patients with chronic affection of the hepatobiliary system was conducted to study the enzymatic system of inactivation of the active forms of hepatic oxygen according to the stage of the chronic process. Reduced activity of superoxide dismutase and catalase and disruption of their relationship in chronic active hepatitis were revealed. Significantly diminished rate of inactivation of superoxide radicals was encountered in fibrosis and primary biliary cirrhosis. The informative importance of the catalase/NADP H-peroxidase index in appraising chronic affection of the hepatic tissue is shown. It is concluded that the interrelation of the antioxidative enzymes as a system of antioxidant protection of hepatocytes is impaired in chronic diseases of the liver.

Time profiles of the expression of metalloproteinases, tissue inhibitors of metalloproteases, cytokines and collagens in hamsters infected with Opisthorchis viverrini with special reference to peribiliary fibrosis and liver injury.

The liver fluke Opisthorchis viverrini is endemic in southeastern Asia, and causes cholangiocarcinoma and liver fibrosis. We investigated the time profile of the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in relation to peribiliary fibrosis in O. viverrini-infected hamsters. Hepatic mRNA expression of MMPs, TIMPs, cytokines and collagens I and III was assessed by quantitative reverse transcription-PCR. Zymography and immunohistochemistry were also used to examine MMPs-2 and -9 expression. After infection, an increase of peribiliary fibrosis was time-dependent. Opisthorhis viverrini-induced gene expression in hamster liver, with increased mRNA expression levels of IL-1beta, TNF-alpha, TGF-beta, and collagens I and III, was observed at 21 days p.i. Expression of MMPs-2, -13 and -14 and TIMPs-1 and -3 genes, was significantly higher at 1 month, and maximal levels of most MMPs (MMPs-2, -9, -13 and -14) were observed at 2 months p.i. The cytoplasmic levels of MMP-2 and MMP-9 were similar to mRNA expression. Immunohistochemistry revealed that MMP-9 was expressed mainly in the cytoplasm of inflammatory cells at the invasive front of the fibrous area. In contrast, the highest levels of mRNA expression of TIMPs-2 and -3, and TGF-beta were observed 10 months p.i. Concentration of TIMP-2 protein in the plasma correlated with its transcriptional level (r=0.320, P=0.040). Peribiliary fibrosis correlated positively with liver hydroxyproline content (r=0.846, P<0.001), plasma hydroxyproline concentration (r=0.770, P<0.001), plasma TIMP-2 level (r=0.335, P=0.046), and mRNA expression levels of MMP-7 (r=0.511, P=0.006), TIMP-1 (r=0.320, P=0.040), TIMP-2 (r=0.428, P=0.026), and TIMP-3 (r=0.553, P=0.003). This study suggests that expression of MMPs is associated with an inflammatory reaction in the early phase and TIMPs expression at the late phase may contribute to both fibrosis and liver injury. MMPs and TIMPs may serve as diagnostic markers for the severity of O. viverrini-induced liver injury.

Occult pancreaticobiliary reflux in gallbladder cancer and benign gallbladder diseases.

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.

Variations in human liver fucosyltransferase activities in hepatobiliary diseases.

The hyperfucosylation of a number of glycoconjugates observed in liver diseases involves the action of several specific fucosyltransferases (F.T.) notably responsible for synthesizing histo-blood group antigens. We determined the activities of alpha 3, alpha 2 and alpha 3/4 F.T. in 35 liver biopsy samples from patients with fatty liver, alcoholic or post-hepatic liver cirrhosis, primary or secondary biliary cirrhosis, acute hepatitis or a normal liver. F.T. activities were measured by transfer of GDP [14C] fucose to asialotransferrin for alpha 3 F.T., to phenyl beta-D-galactoside for alpha 2 F.T. and to 2' fucosyllactose for alpha 3/4 F.T. The diseased liver extracts showed an early increase in non-Le gene-associated alpha 3 F.T. activity (p = 0.001), which was related to the number of steatosic hepatocytes and the degree of intralobular inflammatory infiltration. Overexpression of this alpha 3 F.T. provides an explanation for the strong expression of 3-fucosyl lactosamine structures described in several hepatobiliary diseases. alpha 2 F.T. levels were significantly elevated in the two groups of liver cirrhosis and acute hepatitis (p = 0.05), but not enough to consider alpha 2 F.T. as a sensitive feature of mesenchymal cell injury. All Lewis-positive biopsies displaying biliary alterations showed increased Le gene-encoded alpha 3/4 F.T. activity (p = 0.001), which was related to the intensity of neoductular proliferation. Elevated levels of alpha 3/4 F.T. may be a very early sign of biliary regeneration.

Serum alkaline phosphatase isoenzymes in hepatobiliary disorders resolved by use of immobilized pH gradients.

This new method for fractionating alkaline phosphatase isoforms in hepatobiliary disorders is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. The high-Mr alkaline phosphatase forms typical of hepatobiliary disease (greater than 1 mega-dalton), which cannot migrate into the Immobiline gel, are disaggregated in zwitterionic detergents (the most effective being sulfobetaine 3-12)--20 g/L in the sample, 5 g/L in the gel--suggesting that they are still complexed with membrane fragments or that they tend to aggregate spontaneously in solution. These isoforms focus in the pI 5-6 range (while alkaline phosphatases in normal serum focus in the pI 4-5 interval) in immobilized pH gradients, but behave as strongly acidic components by agarose isoelectric focusing in the presence of carrier ampholytes, suggesting that they are strongly complexed with the latter. On treatment with neuraminidase, the low-pI isoforms in normal serum focus in the pI 5-6 range typical of the hepatobiliary isoforms, suggesting that the latter are poorly glycosylated. By a second-dimension run, in a porosity gradient, followed by activity staining, all alkaline phosphatase forms that have entered the Immobiline gel in the first dimension (normal forms and high-Mr species) exhibit the same Mr (ca. 140,000 Da), suggesting that no new chains are synthesized in hepatobiliary disorders.

Macro creatine kinases: results of isoenzyme electrophoresis and differentiation of the immunoglobulin-bound type by radioassay.

In 2.9% of sera from 1253 unselected patients we detected two different types of macromolecular creatine kinases (CK; EC 2.7.3.2). One macro type was represented by immunoglobulin-linked CK: in sera containing macro CK-BB isoenzyme, 125I-labeled CK-BB was bound with high affinity to the immunoglobulin fraction. Furthermore, during electrophoresis, macro CK-BB mostly migrated between CK-MB and CK-MM, and was fixed to Protein A from Staphylococcus aureus. We therefore propose radioelectrophoresis as a specific, highly sensitive, and simple method for detecting this type of macro CK. This form occurs predominantly in elderly women, is not correlated to any specific disease, and persists in blood over a long period of time. In contrast, a second type (macro-CK type 2) never bound radiolabeled CK isoenzymes, and was not adsorbed to Protein A. Electrophoretic migration of this macro-CK type 2 was generally cathodic to CK-MM. We observed this type in severely ill patients, frequently those suffering from malignant tumors. Clinical observations and biochemical data suggest that macro-CK type 2 is of mitochondrial origin.

Long-term soft-tissue effects of biliary extracorporeal shock waves: an animal study.

Although delayed effects of renal extracorporeal shock-wave lithotripsy (ESWL) have been reported, the long-term soft-tissue effects after biliary ESWL have not been investigated. We report soft-tissue effects seen up to 1 year after biliary extracorporeal shock waves were administered to 18 Yucatan pigs. The gallbladder received 4000 electromagnetic shock waves from a Siemens Lithostar overhead module. Blood samples were drawn from each pig for hematologic, coagulation, and biochemical profiles immediately before and then at prescribed time intervals after administration of shock waves. Autopsy and histopathologic examination of the gallbladder and surrounding organs were performed. Kidneys and adrenal glands also were examined in five pigs followed up for 1 year. There were no gross or microscopic abnormalities in 11 animals, including all five animals in the 1-year group in which kidneys and adrenal glands also were normal. One animal (3-week group) had two 2-mm foci of parenchymal necrosis in the right lobe of the liver, probably related to ischemia after shock-wave therapy. Transient rise in liver and pancreatic enzyme levels was seen in most animals after administration of shock waves. The levels returned to normal within 2 months in all but one animal. We conclude that biliary ESWL with the Lithostar Plus does not produce long-term histologic evidence of organ damage in Yucatan pigs.

Intestinal metaplasia and altered enzyme expression in propylnitrosamine-induced Syrian hamster cholangiocellular and gallbladder lesions.

Intrahepatic bile duct and gallbladder preneoplastic and neoplastic lesions induced in Syrian golden hamsters by propylnitrosamine treatment were investigated for the presence of polysaccharides and assayed immunohistochemically for expression of the enzymes glucose-6-phosphate dehydrogenase (G6PD) and glutathione-S-transferase (GST) molecular forms. On the basis of an increase in G6PD and the GST-placental form, a sequence of altered cell populations ranging from simple ductular proliferation through dysplasia and cholangiofibroma to cholangiocellular carcinoma could be established, the latter three lesions being characterized by marked increase in polysaccharide production. While similar goblet cell (intestinal) metaplasia and increased polysaccharide storage were also evident in carcinogen-induced gallbladder lesions G6PD and GST-P expression was decreased when compared with control epithelium.

beta-Glucuronidase in common duct bile, methodological aspects, variation of pH optima and relation to gallstones.

beta-Glucuronidase of human or bacterial origin may deconjugate bilirubin diglucuronide, causing pigment gallstones. Intrinsic interference by biliary compounds must be minimized for accurate assay of beta-glucuronidase. We report a modified ion-pair extraction of interfering substances by tetrahexylammonium chloride (THAC) in ethyl acetate in the presence of albumin, and a microtitre plate assay for biliary beta-glucuronidase activity in bile with the substrate p-nitrophenol-glucuronide. Adding albumin improved the recovery of beta-glucuronidase activity to 99.8% (CV 1.9%), and 92.2% of the bilirubin in bile samples was extracted in one step. Competitive inhibition was overcome by increasing the substrate concentration. In endoscopically obtained common duct bile from 44 patients, five different beta-glucuronidase activity peaks were identified, at pH 3.9, 4.8, 5.3, 5.8 and 7.2. The pH profiles were classified into one bacterial pattern and five patterns for presumed human beta-glucuronidase. Of the latter patterns, four displayed dual activity peaks. In a second sample, obtained at follow up in four patients, their original pH profile was maintained. In conclusion, using the modified purification and assay system, we found functionally diverse subcategories of human beta-glucuronidase with respect to activity at variable pH. Our results indicate that several pH optima have to be taken into consideration in order to clarify the role of human biliary beta-glucuronidase in the pathogenesis of pigment gallstones. Bacterial beta-glucuronidase activity was associated with duodenal diverticula (p < 0.05) and common duct stones (p < 0.05).

Serum gamma glutamyl transferase as a specific indicator of bile duct lesions in the rat liver.

Serum gamma-glutamyl transferase (GGT), a marker of hepatic injury used extensively in humans, has been used rarely in rats because its specificity has not been previously defined. Studies were designed for investigation of the specificity of serum GGT activity with the use of cell type specific hepatotoxicants in Fischer 344 rats. Single necrogenic doses of CCl4, allyl alcohol (AA), and alpha-naphthylisothiocyanate (ANIT) were used to produce cell specific injury in centrilobular hepatocytes, periportal hepatocytes, and bile duct cells, respectively. Administration of CCl4 markedly increased serum activities of alanine aminotransferase (ALT), alkaline phosphatase (AP), and serum bile acid concentrations within 24 hours but had no effect on serum GGT activity. ANIT treatment increased serum GGT and AP activities and bile acid concentration 24 hours following administration. Allyl alcohol administration increased serum ALT activity but had no effect on GGT activity. Administration of ANIT in the diet at 0.01%, 0.022%, 0.047%, and 0.1% for 2, 4, and 6 weeks produced dose- and time-dependent increases in serum GGT activity which strongly correlated with quantitative increases in hepatic bile duct volume, which was determined morphometrically. These observations support the use of serum GGT activity in the rat as diagnostic of bile duct cell necrosis when increases are detected shortly after the insult and as an indicator of possible bile duct hyperplasia.

[Changes of the liver and bile ducts in patients with acute intestinal diseases]. / Izmeneniia pecheni i zhelchnykh putei u bol'nykh s ostrymi kishechnymi zabolevaniiami.

Patients with acute intestinal infections, in particular, salmonellosis showed in 1/3 during the acute period an insignificant increase of bilirubin and alaninaminotransferase. During early reconvalescence the majority of patients revealed functional changes of the biliary tract, mainly, in the form of hypotensive-hypokinetic type of dyskinesia.