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Proprotein convertase subtilisin/kexin type 9 (Pcsk9) binds to hepatic low-density lipoprotein receptor (LDLR) and induces its internalization and degradation. Pcsk9 inhibition increases LDLR expression by hepatocytes, which causes increased uptake of circulating LDL, thereby reducing plasma LDL-cholesterol. However, by increasing the uptake of LDL by the liver, Pcsk9 inhibition increases the exposure of the liver to cholesterol, which may result in higher risk of steatohepatitis and ever carcinogenesis. We compared Pcsk9-/- knockout (KO) mice and appropriate wild-type (WT) controls of the same strain assigned to a high-fat (15%, wt/wt) diet for 9 months supplemented with 0.25%, 0.5%, or 0.75% dietary cholesterol. Pcsk9 KO mice on a high-fat, high-cholesterol diet exhibited higher levels of hepatic free cholesterol loading and hepatic cholesterol crystallization than their WT counterparts. Pcsk9 KO mice developed crown-like structures of macrophages surrounding cholesterol crystal-containing lipid droplets and hepatocytes, exhibited higher levels of apoptosis, and developed significantly more hepatic inflammation and fibrosis consistent with fibrosing steatohepatitis, including 5-fold and 11-fold more fibrosis at 0.5% and 0.75% dietary cholesterol, respectively. When injected with diethylnitrosamine, a hepatic carcinogen, early-in-life Pcsk9 KO mice were more likely to develop liver cancer than WT mice. Conclusion: Pcsk9 KO mice on high-cholesterol diets developed increased hepatic free cholesterol and cholesterol crystals and fibrosing steatohepatitis with a higher predisposition to liver cancer compared with WT mice. Future studies should evaluate whether patients on long-term treatment with anti-PSCK9 monoclonal antibodies are at increased risk of hepatic steatosis, steatohepatitis or liver cancer, while accounting for concurrent use of statins.

We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ≥0.5%, whereas mice on lower-cholesterol diets developed only simple steatosis. Hepatic cholesterol crystals and crown-like structures also developed at a dietary cholesterol concentration ≥0.5%. Crown-like structures consisted of activated Kupffer cells (KCs) staining positive for NLRP3 and activated caspase 1, which surrounded and processed cholesterol crystal-containing remnant LDs of dead hepatocytes. The KCs processed LDs at the center of crown-like structures in the extracellular space by lysosomal enzymes, ultimately transforming into lipid-laden foam cells. When HepG2 cells were exposed to LDL cholesterol, they developed cholesterol crystals in LD membranes, which caused activation of THP1 cells (macrophages) grown in coculture; upregulation of TNF-alpha, NLRP3, and interleukin 1beta (IL1β) mRNA; and secretion of IL-1beta. In conclusion, cholesterol crystals form on the LD membrane of hepatocytes and cause activation and cholesterol loading of KCs that surround and process these LDs by lysosomal enzymes.

Background : We investigated the relationship between resumption or persistence of menstruation after chemotherapy and disease free survival (DFS) in premenopausal patients with early breast cancer. Patients and methods : A total of 843 patients diagnosed with a stage I to III breast cancer between March 2001 and December 2006 were included in this study. All patients received cytotoxic chemotherapy after surgery; 411 (48.8%) with anthracycline based, 416 (49.3%) with anthracycline and taxane containing, and 16 (1.9%) with other regimens. We reviewed the medical records with a long term follow-up. Results : The median age of patients was 41 years (range, 21–54 years) and the median follow-up duration was 6.2 years (range, 0.7−10.4 years). Of all, 632 (75%) patients were hormone receptor (HR) positive who received tamoxifen therapy upon completion of chemotherapy. The chemotherapy induced amenorrhea (CIA) rate was 78.1% (n=658) and 52.4% (n= 442) experienced the resumption of period during the long term follow-up. One hundred two (12.1%) patients had persistent menstruation without CIA. The disease free survival (DFS) was significantly affected by the younger age (≤ 35 years) (HR=1.58, [95% CI, 1.10−2.70], P=0.014), advanced stage (stage 3) (HR=4.45, [95% CI, 3.17−6.25], P Conclusions : A considerable proportion of premenopausal patients treated by chemotherapy experienced resuming period after CIA. The resumption or persistence of menstruation was a poor prognostic factor for disease free survival in premenopausal patients with early breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-24.

Purpose Invasive lobular carcinoma (ILC) is known to be the second most frequent histologic subtype, occupying 10% of invasive breast cancer in the Western countries. The present study was designed to assess the clinical characteristics and outcomes of ILC compared to general invasive ductal carcinoma (IDC) and the luminal A subtype (LA-IDC). Methods The study population included d 2916 patients with invasive breast cancer consecutively diagnosed at the National Cancer Center, Korea between 2001 and 2008. The clinicopathological characteristics and clinical outcomes were retrospectively reviewed. Results There were 83 pts (2.8%) diagnosed with ILC and 1,088 pts (37.3%) with LA-IDC. Mean age was 48.2 years of all patients, 48.3 years of ILC group and 47.9 years of LA-IDC group. The ILC patients presented with a larger tumor size (≥T2, 59.8% vs. 38.8%, P=0.001), lower histologic grade (HG 1 or 2, 90.4% vs 64.4%, P Conclusions ILC is a very rare histologic subtype of breast cancer in Korea compared to the Western countries and has distinctive clinicopathological characteristics similar to those of LA-IDC. Acknowledgement: supported by NCC grant #0910320 Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-09-14.

Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor, and at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore, it is imperative that alternative treatment options are explored. We present novel data indicating that the metabolism of serotonin is dysregulated in cholangiocarcinoma cell lines, compared with normal cholangiocytes, and tissue and bile from cholangiocarcinoma patients. Specifically, there was an increased expression of tryptophan hydroxylase 1 and a suppression of monoamine oxidase A expression (enzymes responsible for the synthesis and degradation of serotonin, respectively) in cholangiocarcinoma. This resulted in an increased secretion of serotonin from cholangiocarcinoma and increased serotonin in the bile from cholangiocarcinoma patients. Increased local serotonin release may have implications on cholangiocarcinoma cell growth. Serotonin administration increased cholangiocarcinoma cell growth in vitro, whereas inhibition of serotonin synthesis decreases tumor cell growth both in vitro and in vivo. The data presented here represent the first evidence that serotonin metabolism is dysregulated in cholangiocarcinoma and that modulation of serotonin synthesis may represent an alternative target for the development of therapeutic strategies. © 2008 American Association for Cancer Research., link_to_subscribed_fulltext

Background/Aims We determined the effects of dietary lipid composition on steatohepatitis development with particular attention to the nature of lipid molecules that accumulate in the liver and pathways of hepatic triglyceride synthesis. Methods Mice were fed methionine and choline deficient (MCD) diets supplemented with 20% fat as lard (saturated) or olive oil (monounsaturated), for 3 weeks. Results Irrespective of dietary lipid composition, MCD-fed mice developed steatosis, ballooning degeneration and lobular inflammation. MCD-feeding increased hepatic free fatty acid (FFA) levels 2–3-fold, as well as total triglyceride levels. Hepatic FFA composition was characterized by increased ratio of monounsaturated: saturated FFA. There were reduced nuclear levels of the lipogenic transcription factor sterol regulatory element binding protein-1 in MCD-fed mice, but no consistent reduction in fatty acid synthesis genes (acetyl-CoA carboxylase and fatty acid synthase). Consistent with pathways of hepatic triglyceride synthesis, expression of diacylglycerol acyltransferase-1 and -2 was increased, as were delta-5- and delta-6- fatty acid desaturase mRNA levels. Conclusions In this nutritional model of steatohepatitis, accumulation of FFA occurs despite substantial suppression of lipogenesis and induction of triglyceride synthesis genes. Accumulation of FFA supports a lipotoxicity mechanism for liver injury in this form of fatty liver disease.

OBJECTIVES: The presence of elevated serum aminotransferase activity is a sign of possible underlying liver disease. We aimed to describe the prevalence and associations of elevated serum aminotransferase activity in a recent, nationally representative U.S. survey. METHODS: We described the prevalence and predictors of elevated alanine aminotransferase (ALT >43 IU/L) or elevated aspartate aminotransferase (AST >40 IU/L) activity among 6,823 participants of the National Health and Nutrition Examination Survey (NHANES) conducted between 1999 and 2002. We compared our findings to the results already published based on the NHANES conducted between 1988 and 1994. RESULTS: In NHANES 1999-2002, the prevalences of elevated ALT, AST, or either ALT or AST were 8.9%, 4.9%, and 9.8%, respectively, in the entire population and 7.3%, 3.6%, and 8.1%, respectively, after excluding participants who tested positive for hepatitis C virus (HCV) antibody or reported excessive alcohol consumption. Strong predictors of elevated ALT activity included increasing waist circumference and body mass index, alcohol consumption, male sex, Mexican American ethnicity, decreasing age, and presence of HCV antibody. In NHANES 1988-1994, which employed a different assay methodology, the prevalences of elevated aminotransferases were approximately half of the prevalences we describe in NHANES 1999-2002, but the predictors of elevated ALT activity were similar. CONCLUSIONS: The current prevalence of elevated ALT activity in the United States (8.9%) is more than double that of previously available estimates. This prevalence is very high (7.3%) even among persons without viral hepatitis C or excessive alcohol consumption and is strongly associated with risk factors for nonalcoholic fatty liver disease.

Cyclooxygenase-2 (COX-2), which is expressed by cholangiocytes in biliary tract disorders, has recently been implicated in biliary tract carcinogenesis. The mechanisms responsible for this COX-2 expression remain unclear. In human diseases, bile contains oxygenated derivatives of cholesterol (oxysterols) which possess diverse biological properties. Therefore, we determined if oxysterols modulate COX-2 expression. The effect of an oxysterol (22(R)-hydroxycholesterol, 22-HC) on COX-2 expression in KMBC cells, a human cholangiocarcinoma cell line, was examined. 22-HC enhanced COX-2 protein expression. This oxysterol activated p42/44 and p38 MAPK, but not JNK 1/2. A p42/44 MAPK inhibitor did not block COX-2 induction, while p38 MAPK inhibitor effectively attenuated COX-2 induction. Although COX-2 mRNA levels were increased by 22-HC, this increase was not transcriptionally regulated, as 22-OH did not increase activity in a COX-2 promoter gene assay. In contrast, COX-2 mRNA stability was augmented by 22-HC treatment, and this effect was reversed by a p38 MAPK inhibitor. In conclusion, the results demonstrate that the oxysterol 22-HC stabilizes COX-2 mRNA via a p38 MAPK-dependent mechanism. This enhanced COX-2 protein expression by oxysterols may participate in the genesis and progression of cholangiocarcinoma.

Introduction: Calcium-activated chloride conductance has been identified in normal pancreatic duct cells. Recent in vitro evidence suggests that angiotensin II (AngII) stimulates pancreatic secretion in both cystic fibrosis (CFPAC) and transformed pancreatic cells. Aims: To investigate calcium-mediated stimulatory effects of AngII in both nontransformed dog pancreatic duct epithelial (DPDE) and CFPAC cells. Methods: Western blots were performed in both cells seeking AngII receptors. In additional studies, DPDE and CFPAC cells were grown on vitrogen-coated glass cover slips and loaded with Indo-1-AM dye. Cells were placed in a confocal microscope's perfusion chamber and perfused with 100 μM AngII or ATP (control). Cells were excited with UV light, and intracellular calcium ([Ca +2 ] i ) was read using fluorescence emission at 405 and 530 nm. Finally, single channels in the DPDE cells were examined using cell-attached patch clamps. Current amplitude histograms provided estimates of the conductance and open probability of channels. Results: Western blots demonstrated presence of both AT 1 and AT 2 AngII receptors in DPDE and CFPAC cells; the density of AT 1 receptors appeared lower than that of AT 2 receptors. Basal intracellular calcium concentrations did not differ between DPDE (109 ± 11 nM) and CFPAC (103 ± 8 nM) cells. AngII significantly increased measured intracellular calcium concentrations in both DPDE (909 ± 98 nM) and CFPAC (879 ± 207 nM) cells, as did ATP (DPDE = 1722 ± 228 nM; CFPAC = 1522 ± 245 nM). In the patch clamp studies, a variety of different channels were observed; they appeared to be an 11pS nonselective cation (NSC) channel, a 4.6pS Na + channel, a 3pS anion channel, and an 8pS chloride channel. The latter channel had characteristics similar to cystic fibrosis transmembrane conductance regulator (CFTR). Apical or basolateral application of AngII activated both the llpS NSC and the 3pS channels. Conclusion: In nontransformed DPDE and CFPAC cells, specific AngII receptors mediate increases in [Ca +2 ] i . The latter effect of AngII may elicit activation of calcium-mediated chloride channels, suggesting a role for AngII as an alternative mediator of pancreatic ductal secretion.

In order to investigate oxysterol-mediated effects on the biliary system, we studied the effects of cholestan-3beta,5alpha,6beta-triol (TriolC) and 7-ketocholesterol (7KC) on gallbladder epithelial cells. We compared their cell proliferation effects in cultured dog gallbladder epithelial cells (DGBE) to their effects in cultured human pulmonary artery endothelial cells (HPAE). Oxysterols inhibited cell proliferation in a dose-dependent fashion. Oxysterols inhibited cell growth to 50% of control at a higher dose for DGBE cells than for HPAE cells. TriolC was more cytotoxic than 7KC. We also investigated the effect of oxysterols on bile salt-induced mucin secretion by DGBE cells. TriolC suppressed mucin secretion by DGBE cells, whereas 7KC did not. These findings support the hypothesis that biliary oxysterols affect gallbladder mucosal function.

The E4 allele of apolipoprotein E (apoE4) has previously been associated with symptomatic gallstone disease. The aim of this study was to determine if apoE4 is associated with the development of gallbladder sludge and/or stones during pregnancy. We conducted a nested case-control study based on an ongoing cohort study of gallbladder disease in pregnancy. Women in this study receive gallbladder ultrasounds in each trimester of pregnancy. Cases (n = 52) were defined as women with incident gallbladder sludge or stones diagnosed at the third trimester ultrasound. Controls (n = 104) were defined as women without gallbladder sludge or stones on any of 3 study ultrasounds. ApoE genotyping was performed from stored white blood cell pellets. Data were analyzed by stratified analysis and multivariate logistic regression. Cases and controls were similar in baseline characteristics. Forty-two women had sludge, 6 had gallstones, and 4 had both sludge and stones. After adjusting for risk factors such as age, parity, and body mass index, the odds ratio (OR) for the association between heterozygosity or homozygosity for the apoE4 allele and incident gallbladder sludge or stones was 0.91 (95% confidence interval [CI], 0.41-2.02). Further adjustment for family medical history and serum lipid levels did not substantially change these results (OR, 0.73; 95% CI, 0.29-1.82). In conclusion, apoE4 appears to have little or no overall association with the development of new gallbladder sludge or stones in pregnancy. However, an effect could not be ruled out in certain subgroups, such as blacks or women who are homozygous for apoE4.

Bile supersaturation is necessary for cholesterol gallstones to form. Not all people with supersaturated bile form gallstones, however, and additional factors must be present. The role of pronucleating substances has been extensively studied. Of these, proteins, especially mucin, are best understood. Mucin is secreted by the gallbladder epithelium and may act as a nidus for crystal nucleation. Other proteins that may act as pronucleators include alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, phospholipase C, and a small calcium binding protein. The role of antinucleating factors is less well understood. Certain drugs, including octreotide and ceftriaxone, may also predispose to stone formation. Another local factor is gallbladder stasis, a well-known risk factor for pigment stone formation. More recent research has focused on the role of bacterial infection, which has long been believed to be a factor in pigment gallstone formation. Newer data also support a role for infection in cholesterol gallstone pathogenesis. Additionally, genetic factors that may predispose a patient to cholesterol gallstones have been identified in mice and in humans.

Extracellular triphosphate nucleotides, such as ATP, may regulate various cellular functions through specific cell surface receptors. We examine in this report the different secretory effects of ATP and analogs on nontransformed dog pancreatic duct epithelial cells (PDEC). We observed that 1) ATP, UTP, adenosine 5′- O-(3-thiotriphosphate), and, to a lesser extent, β,γ-methylene-ATP, but not adenosine, stimulated125I−efflux from PDEC, suggesting a primary role for P2Y2receptors, 2) ATP-stimulated125I−efflux was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, and DIDS, suggesting mediation through Ca2+-activated Cl−channels, 3) ATP stimulated an86Rb+efflux sensitive to BaCl2and charybdotoxin, thus likely occurring through Ca2+-activated K+channels, 4) serosal or luminal addition of UTP activated apical Cl−conductance and basolateral K+conductance when nystatin-permeabilized PDEC were studied in an Ussing chamber, suggesting the expression of P2Y2receptors on both sides of the cell, 5) ATP stimulated mucin secretion, and 6) ATP increases intracellular Ca2+concentration ([Ca2+]i). In conclusion, ATP and UTP interact with P2Y2receptors on nontransformed PDEC to increase [Ca2+]i, stimulate mucin secretion, and activate ion conductances; these findings have implications for pancreatic exocrine function in both health and disease, such as cystic fibrosis.

Objective To evaluate the effect of a physical activity intervention upon the incidence of gallbladder sludge or stones during pregnancy. Study Design Pregnant women without gallstones were randomized to an intervention to increase moderate to vigorous physical activity or control. Intervention group women received motivational materials and small-group instruction to increase physical activity. Gallbladder ultrasound and blood draws were obtained at entry, 18 weeks' gestation, and 36 weeks' gestation. Results In all, 591 were randomized to the intervention and 605 women to control groups. Women in the intervention group reported modestly higher levels of physical activity compared with control women, and fewer women in the intervention group reported no physical activity during pregnancy. The incidence of gallbladder sludge or stones was similar in intervention and control groups at 18 weeks (4.8% versus 5.4%; relative risk 0.89; 95% confidence interval 0.53, 1.47) and 36 weeks (4.3% versus 3.3%; relative risk 1.31; 95% confidence interval 0.70, 2.54). Fasting glucose, lipid, insulin, leptin, and adiponectin levels were similar in the two groups, as was insulin sensitivity and the incidence of gestational diabetes. Conclusion An intervention to increase moderate to vigorous physical activity did not decrease the incidence of gallbladder sludge or stones during pregnancy and did not result in improvement in maternal metabolic measures.

The effect of aspirin on the rate of secretion and amino acid composition of human ileal mucin was studied, using subjects with ileal conduits as a model system in which mucin secreted from the ileal conduit tissue is flushed out in the urine and can be measured and analysed. Aspirin (600 mg per day, administered orally) increased the daily mucin output by 37-104% in subjects by days 3 or 4, but thereafter the mucin output declined to below the baseline level by day 10. Mucin samples, purified from the ileal conduit urine during the control period and during aspirin administration, were compared. There were no discernible changes in the degree of polymerisation or the density, but during aspirin administration the amino acid composition was significantly changed, and in particular threonine and proline were enriched. One possible explanation, consistent with the compositional analyses, is that the N- and C-terminal regions of the mucin subunits have been cleaved off and lost during aspirin administration. The observed changes in mucin secretion may have implications for the mechanism of the toxic effects of aspirin on the small intestine by altering the barrier properties of the mucus layer.

Gallstones, particularly cholesterol gallstones, are common in Western populations and may cause symptoms such as biliary colic or complications such as acute cholecystitis or gallstone pancreatitis. Recent studies have allowed for a better understanding of the risk of symptoms or complications in patients with gallstones. In addition, newer data suggest an association of gallstones with overall mortality, cardiovascular disease, gastrointestinal cancers, and non-alcoholic fatty liver disease. Knowledge of appropriate indications and timing of cholecystectomy, particularly for mild biliary pancreatitis, has gradually accumulated. Lastly, there are exciting possibilities for novel agents to treat or prevent cholesterol stone disease. This review covers new advances in our understanding of the natural history, clinical associations, and management of gallstone disease.

A variety of diseases are included under the umbrella term ‘cholangitis’, including hepatobiliary diseases with an autoimmune pathogenesis (such as primary sclerosing cholangitis, primary biliary cholangitis, and IgG4-associated sclerosing cholangitis) and disease processes associated with intraductal stones and infectious etiologies (such as ascending bacterial cholangitis, recurrent pyogenic cholangitis, and liver fluke-associated cholangitis). Recent advances in the pathophysiologic bases of these disorders, particularly with respect to the autoimmune variety, are allowing improved diagnosis and prognostication as well as providing the opportunity to refine and re-imagine treatment modalities. The aim of this review is to highlight selected advances in cholangitis research that point to novel insights into the pathophysiology, diagnosis, and treatment of this diverse array of disorders.

Mucin is the main secretory product of gallbladder epithelial cells. Increased gallbladder mucus secretion has been implicated in gallstone formation in humans. The mechanisms underlying control of mucin synthesis and secretion by the gallbladder are not known. This study aimed to elucidate the efficacy of a panel of secretagogues to stimulate mucin secretion and to determine the intracellular second messengers involved. Studies were carried out on normal well-differentiated epithelial cells from dog gallbladder grown in monolayer culture. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) as measured by radioimmunoassay increased in response to prostaglandin (PG) E2, PGE1, vasoactive intestinal peptide, epinephrine, and isoproterenol. The greatest effect, a 37-fold increase in cAMP level, was noted with PGE2 at 1.0 microM concentration. In contrast, three breakdown products of phosphatidylinositol (inositol triphosphate, inositol bisphosphate and inositol monophosphate) were not detected with any of the secretagogues tested. Assay of mucin secretion using tritiated N-acetyl-D-glucosamine, a mucin precursor, showed that the same secretagogues noted to increase intracellular cAMP led to an increase in mucin secretion. No correlation was noted, however, between the magnitude of the intracellular cAMP rise and the amount of mucin secreted. A membrane-permeable form of cAMP, dibutyryl cAMP, mimicked PGE2-induced mucin secretion. The results unequivocally show that secretagogue-stimulated mucin secretion in these normal gallbladder epithelial cells can proceed via a cAMP signal transduction pathway.

Oxysterols are cholesterol oxidation products that are generated by enzymatic reactions through cytochrome P450 family enzymes or by non-enzymatic reactions involving reactive oxygen and nitrogen species. Oxysterols have been identified in bile in the setting of chronic inflammation, suggesting that biliary epithelial cells are chronically exposed to these compounds in certain clinical settings. We hypothesized that biliary oxysterols resulting from liver fluke infection participate in cholangiocarcinogenesis. Using gas chromatography/mass spectrometry, we identified oxysterols in livers from hamsters infected with Opisthorchis viverrini that develop cholangiocarcinoma. Five oxysterols were found: 7-keto-cholesta-3,5-diene (7KD), 3-keto-cholest-4-ene (3K4), 3-keto-cholest-7-ene (3K7), 3-keto-cholesta-4,6-diene (3KD), and cholestan-3β,5α,6β-triol (Triol). Triol and 3K4 were found at significantly higher levels in the livers of hamsters with O. viverrini-induced cholangiocarcinoma. We therefore investigated the effects of Triol and 3K4 on induction of cholangiocarcinogenesis using an in vitro human cholangiocyte culture model. Triol- and 3K4-treated cells underwent apoptosis. Western blot analysis showed significantly increased levels of Bax and decreased levels of Bcl-2 in these cells. Increased cytochrome c release from mitochondria was found following treatment with Triol and 3K4. Triol and 3K4 also induced formation of the DNA adducts 1,N6-etheno-2′-deoxyadenosine, 3,N4-etheno-2′-deoxycytidine and 8-oxo-7,8-dihydro-2′-deoxyguanosine in cholangiocytes. The data suggest that Triol and 3K4 cause DNA damage via oxidative stress. Chronic liver fluke infection increases production of the oxysterols Triol and 3K4 in the setting of chronic inflammation in the biliary system. These oxysterols induce apoptosis and DNA damage in cholangiocytes. Insufficient and impaired DNA repair of such mutated cells may enhance clonal expansion and further drive the change in cellular phenotype from normal to malignant.

Background & Aims Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to the pathogenesis of NASH. Methods Alms1 mutant ( foz/foz ) and wild-type NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice. Results Hepatic cholesterol accumulation was attributed to up-regulation of low-density lipoprotein receptor via activation of sterol regulatory element binding protein 2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and low-density lipoprotein receptor and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol levels exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis. Conclusions In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH.

In about 20 to 40 percent of cases of acute pancreatitis, no cause can be found, and these are labeled idiopathic. In this study, we sought to determine the frequency with which patients with acute idiopathic pancreatitis have biliary sludge, a suspension of cholesterol monohydrate crystals or calcium bilirubinate granules that is found predominantly in the gallbladder.Between 1980 and 1988, we prospectively studied 86 patients who had acute pancreatitis. In patients with no known cause of pancreatitis and no ultrasonographic evidence of gallstones or dilatation of the biliary ducts, we determined how often biliary sludge was present and its subsequent fate by repeated microscopical examinations of bile samples and abdominal ultrasonography. The outcome of patients treated by cholecystectomy or papillotomy was compared with that of untreated patients.The pancreatitis was considered idiopathic in 31 of the 86 patients (36 percent), of whom 23 had microscopical evidence of biliary sludge. Biliary sludge was detected by ultrasonography in only 11 of the 23 patients (48 percent). The sludge detected by ultrasonography was composed of calcium bilirubinate granules in 10 and cholesterol monohydrate crystals in 1 (P = 0.003). Calcium bilirubinate granules were found more frequently in men (nine men vs. four women, P less than 0.001). Of the 21 patients in whom biliary sludge was the only finding (2 patients also had dilasted bile ducts when restudied), the 6 treated by cholecystectomy and the 4 treated by papillotomy had fewer recurrences of acute pancreatitis during follow-up (up to seven years) than the 11 untreated patients (P = 0.011). The presence of biliary sludge appeared to increase the likelihood of recurrent attacks of pancreatitis (P = 0.020).Biliary sludge is an underestimated cause of acute idiopathic pancreatitis.

Background and Aim: Little is known about the role of platelet-derived growth factor (PDGF) in biliary fibrosis in the setting of bacterial colonization of the biliary tree. We therefore sought to investigate whether exposure to bacterial lipopolysaccharide (LPS) alters PDGF isoform and receptor expression in cultured rat common bile duct fibroblasts (CBDF) and normal rat cholangiocytes (NRC). Methods: Collagen content in cells and media was assessed by colorimetric assay and gel electrophoresis. mRNA levels of PDGF-A and -B, and PDGF-Receptors (PDGF-R) α and β were measured by relative quantitative real-time PCR. Protein levels of PDGF-AA, AB and BB were measured by ELISA, and PDGF-Rα and PDGF-Rβ by Western blot. Results: In CBDF, LPS increased total soluble collagen synthesis and secretion. PDGF-Rα and β mRNA and protein were also increased by LPS treatment in CBDF. Lipopolysaccharide treatment elicited an increase in PDGF-A and -B mRNA levels in CBDF. In NRC, levels of PDGF-A mRNA increased in a dose-dependent fashion following LPS treatment, whereas PDGF-B mRNA showed no response. PDGF-AA secretion was higher by CBDF than by NRC. PDGF-BB levels were also higher in CBDF than in NRC. While PDGF-BB levels did not respond to LPS treatment in CBDF, there was a dose-dependent response of this isoform to LPS in NRC. Intracellular and secreted PDGF-AB increased with LPS treatment in NRC. Conclusions: These results support a model in which chronic bacterial colonization of the biliary tree induces fibrosis through PDGF-dependent mechanisms.

Little is known about the impact of dietary factors on the progression of liver disease. Our aim was to determine whether dietary intake was associated with the risk of cirrhosis-related or liver cancer–related death or hospitalization in the U.S. population. Participants included 9221 persons aged 25-74 years without evidence of cirrhosis at entry into the study or during the first 5 years of follow-up, who were subsequently followed for a mean of 13.3 years as part of the first National Health and Nutrition Examination Survey. Dietary intake was ascertained at baseline using a 24-hour dietary recall questionnaire. During follow-up, 123 of 9221 participants had a diagnosis of cirrhosis (n = 118) or liver cancer (n = 5) in hospitalization records or death certificates, including 36 who were diagnosed only on the basis of death certificates. Participants who reported a diet high in protein were at a higher risk of hospitalization or death due to cirrhosis or liver cancer (P = 0.001), whereas those who reported a diet high in carbohydrates were at a lower risk (P = 0.003), after adjusting for potential confounders (daily consumption of protein, carbohydrate, fat, tea or coffee, and alcohol, gender, race, age, educational attainment, U.S. geographical region, diabetes, body mass index, and subscapular-to-triceps skinfold ratio). Although total fat consumption was not significantly associated with the risk of cirrhosis or liver cancer, cholesterol consumption was associated with higher risk (P = 0.007), whereas serum cholesterol level was not associated with risk of cirrhosis or liver cancer. Conclusion: Diet may be an important and potentially modifiable determinant of liver disease. (HEPATOLOGY 2009.)

Amyloid-β (Aβ) is cleared from the brain by both proteolytic digestion and transport across the blood-brain-barrier into the peripheral circulatory system. To investigate the role peripheral Aβ levels play in regulating Aβ brain clearance, we measured the clearance of [125I]-Aβ-{1-40 injected into the brains of liver-ligated rats that allowed peripheral Aβ levels to be maintained at elevated levels for approximately one hour with/without a single peripheral bolus of unlabeled Aβ-{1-40. We found that elevating peripheral Aβlevels significantly decreased [125I]-Aβ-{1-40 brain clearance, thus supporting the hypothesis that peripheral Aβ levels regulate Aβ clearance from the central nervous system. © 2009 - IOS Press and the authors. All rights reserved., link_to_subscribed_fulltext

Although many putative sterol transporters influencing cholesterol absorption and physical–chemical factors affecting dietary cholesterol absorption have been extensively investigated, it is still unclear how biliary cholesterol contributes to the regulation of intestinal cholesterol absorption. We studied whether the gallbladder can modulate the microaggregates of cholesterol carriers, which may in turn influence the intestinal absorption of biliary cholesterol. Supersaturated, crystallized, or micellar model biles were delivered via a duodenal catheter to conscious, freely moving C57L mice daily for 2 days. Intestinal uptake and absorption of biliary cholesterol and its fecal excretion, as well as expression levels of intestinal sterol transporters, were analyzed. Cholesterol uptake and absorption by the enterocyte were dramatically reduced in mice treated with crystallized biles compared with supersaturated biles. This correlated with the higher cumulative radioactivity of cholesterol recovered in the feces at 24 hours. Such findings were absent with the added reference compound sitostanol. After removing cholesterol crystals from crystallized biles, micellar biles showed essentially identical effects on intestinal absorption but with lower fecal cholesterol excretion compared with the original samples containing crystals. Expression levels of the jejunal Abcg5 (ATP-binding cassette transporter G5) and Abcg8, but not Npc1l1 (Niemann-Pick C1 like 1), were significantly increased by supersaturated biles compared with crystallized biles. Conclusion: Different physical forms of biliary cholesterol dramatically determine intestinal uptake and absorption of cholesterol. Solid plate-like cholesterol monohydrate crystals in bile are probably not absorbed and are totally excreted in feces from the body. The gallbladder may have a role in regulating cholesterol homeostasis by modulating the physical forms of biliary cholesterol. (HEPATOLOGY 2008.)

Background & Aims: Insulin resistance is associated with prevalent gallstones, but its effect on initial gallstone formation is not well-understood. Methods: We conducted a nested case-control study to examine whether insulin resistance is a risk factor for initial gallbladder sludge and stone formation during pregnancy. Cases were 205 women with new gallbladder sludge and stones during pregnancy and the early postpartum. Controls were 443 randomly selected women without sludge or stones during pregnancy. Gallbladder ultrasounds were obtained during each trimester and at 4-6 weeks post partum. Fasting serum glucose, lipids, and insulin were measured at 26-28 weeks gestation. Insulin resistance was measured by the homeostasis model. Logistic regression was used to identify independent risk factors for gallstone formation. Results: Insulin resistance was significantly greater in cases than in controls on univariate analysis (P < .001). Pre-pregnancy body mass index was strongly associated with gallstone formation on univariate analysis (P < .001), but this association was diminished after adjusting for insulin resistance (P = .01). On multivariate analysis, insulin resistance was significantly associated with gallstone formation (P = .004), even after adjustment for pre-pregnancy body mass index and other confounding factors including high-density lipoprotein cholesterol and physical activity. This association was strongest in women with pre-pregnancy body mass index, link_to_subscribed_fulltext

Mucus of cystic fibrosis patients exhibits altered biochemical composition and biophysical behavior, but the causal relationships between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the abnormal mucus seen in various organ systems remain unclear. We used cultured gallbladder epithelial cells (GBEC) from wild-type and Cftr(−/−) mice to investigate mucin gene and protein expression, kinetics of postexocytotic mucous granule content expansion, and biochemical and ionic compositions of secreted mucins. Muc1, Muc3, Muc4, Muc5ac, and Muc5b mRNA levels were significantly lower in Cftr(−/−) GBEC compared with wild-type cells, whereas Muc2 mRNA levels were higher in Cftr(−/−) cells. Quantitative immunoblotting demonstrated a trend toward lower MUC1, MUC2, MUC3, MUC5AC, and MUC5B mucin levels in Cftr(−/−) cells compared with cells from wild-type mice. In contrast, the levels of secreted MUC1, MUC3, MUC5B, and MUC6 mucins were significantly higher from Cftr(−/−) cells; a trend toward higher levels of secreted MUC2 and MUC5AC was also noted from Cftr(−/−) cells. Cftr(−/−) cells demonstrated slower postexocytotic mucous granule content expansion. Calcium concentration was significantly elevated in the mucous gel secreted by Cftr(−/−) cells compared with wild-type cells. Secreted mucins from Cftr(−/−) cells contained higher sulfate concentrations. Thus absence of CFTR is associated with pleiotropic effects on mucins in murine GBEC.

In the United States, elevated serum alanine aminotransferase (ALT) activity in the absence of viral hepatitis or excessive alcohol consumption is most commonly attributed to nonalcoholic fatty liver disease (NAFLD). NAFLD is related to predictors of coronary heart disease (CHD) such as insulin resistance and central obesity. We examined the association between elevated serum ALT activity and the 10-year risk of CHD as estimated using the Framingham risk score (FRS). We performed a cross-sectional analysis comparing participants in the Third National Health and Nutrition Examination Survey with normal and elevated ALT activity (>43 IU/L), examining the mean levels of FRS. Among participants without viral hepatitis or excessive alcohol consumption, those with elevated ALT activity (n = 267) had a higher FRS than those with normal ALT activity (n = 7,259), both among men (mean difference in FRS 0.25, 95% CI 0.07-0.4; hazard ratio for CHD 1.28, 95% CI 1.07-1.5) and women (mean difference in FRS 0.76, 95% CI 0.4-1.1; hazard ratio for CHD 2.14, 95% CI 1.5-3.0). The ALT threshold for increased risk of CHD was higher in men (>43 IU/L) than in women (>30 IU/L). Elevated ALT activity was not associated with higher FRS among nonobese participants with viral hepatitis or excessive alcohol consumption. In conclusion, individuals with elevated serum ALT activity in the absence of viral hepatitis or excessive alcohol consumption, most of whom have NAFLD, have an increased calculated risk of CHD. This association is more prominent in women. (HEPATOLOGY 2006;43:1145–1151.)

The gallbladder is traditionally regarded as an absorptive organ. There is increasing evidence that the gallbladder mucosa can have a secretory function. We studied a patient with primary sclerosing cholangitis whose gallbladder was excluded from his extrahepatic bile ducts by stricture formation. He was admitted into hospital because of cholecystitis and cholangitis and required separate drainage tubes into his gallbladder and common hepatic duct. This unique combination of drains afforded the opportunity to examine hepatic bile and gallbladder secretion. We analyzed samples for fluid volume, protein, electrolyte concentrations and biliary lipids. The simultaneous, yet separate, drainage from the gallbladder and the liver had a striking difference. The former was colorless to opalescent; the latter always golden brown. Hepatic bile flow was continuous but gallbladder drainage was variable in volume, intermittent, and occurred only after a meal. The gallbladder fluid had no bilirubin, bile salts, cholesterol, or phospholipids and had the ionic profile of an extracellular fluid. It was alkaline and contained abundant bicarbonate. We have shown that the gallbladder can secrete. In addition, these observations may also have important implications in the pathogenesis and prevention of gallbladder sludge and stones.

We studied 22 patients with symptomatic microlithiasis to determine whether a contributory role of the gallbladder in the early stage of cholesterol gallstone formation exists. We compared the merits of different methods (ultrasonography and microscopy) and sources (hepatic or gallbladder) of bile samples for diagnosing microlithiasis.Paired hepatic and gallbladder bile samples were studied with polarizing microscopy. Nucleation time, bile salts, phospholipid, cholesterol, cholesterol saturation index (CSI), bilirubin, total protein, albumin and mucin concentration were measured. All patients had abdominal ultrasound examination.With polarizing microscopy as the standard, ultrasonography was positive in 13 patients (59%) and negative in 9 (41%). All gallbladder bile samples were positive for microlithiasis by microscopy. Only one hepatic bile sample was positive (P.0001). There was a disproportional enrichment of total protein, albumin, and mucin (P.05) in the gallbladder bile and a conversion of bilirubin diglucuronide to monoglucuronide (P.01). Gallbladder samples had lower CSI but a faster nucleation time (P.001), which correlates inversely with CSI, total protein, and mucin concentration.Biochemical composition and physical chemical behavior of hepatic bile are modified during residence in the gallbladder, contributing to sludge formation. Gallbladder bile has a lower calculated CSI, higher deconjugation of bilirubin, protein and mucin concentration and crystals were present. Hepatic bile samples are inappropriate for microscopic detection of microlithiasis.

Background & Aims: Nonalcoholic fatty liver disease has been defined by the presence of hepatic steatosis in the absence of other chronic liver diseases. We sought to determine whether obesity, insulin resistance, and the metabolic syndrome, which are the main risk factors for nonalcoholic fatty liver disease, are associated with similar elevations in serum alanine aminotransferase activity in persons with and those without other causes of chronic liver disease. Methods: Adult participants of the third National Health and Nutrition Examination Survey were divided into those with causes of chronic liver disease (n = 1037), defined as viral hepatitis, excessive alcohol consumption, or increased transferrin-iron saturation, and those without (n = 8004). Results: Among persons with other causes of chronic liver disease, obesity (adjusted odds ratio, 4.9; 95% confidence interval, 2.5–9.4), insulin resistance (adjusted odds ratio, 6.8; 95% confidence interval, 3.0–15.5, comparing the highest and the lowest quartile), and the metabolic syndrome (adjusted odds ratio, 3.3; 95% confidence interval, 1.4–8.0) were all strongly associated with increased alanine aminotransferase activity (>43 IU/L). Among persons without other causes of chronic liver disease, statistically similar associations were identified. Conclusions: Obesity, insulin resistance, and the metabolic syndrome are strong predictors of increased alanine aminotransferase activity in the US population, both in persons with and in persons without other causes of chronic liver disease. We hypothesize that metabolic fatty liver disease related to these conditions is the cause of the increased alanine aminotransferase activity and may be underrecognized in persons with other causes of chronic liver disease.

Oxysterols are ubiquitous in the body and are potential cytotoxic agents in addition to being metabolic regulators. Although bile contains high concentrations of cholesterol, oxysterol concentrations in bile and the effect of infection on oxysterol levels have not been measured, nor has their origin been studied. The purpose of this study was to determine if infection of the biliary tract was associated with increased concentrations of oxysterols in the bile and, if so, which oxysterols showed a significant change.Hepatic bile was obtained from eight patients with biliary tract disease by means of a naso-biliary catheter. Oxysterols were extracted and purified by solid-phase extraction, derivatized and measured by gas chromatography-mass spectrometry.The following were quantified in hepatic bile: 7-alpha-hydroxycholesterol, 7-beta-hydroxycholesterol, cholestan-3-beta,5-alpha,6-beta-triol, 25-hydroxycholesterol, 26-hydroxycholesterol, 7-ketocholesterol, and 7-alpha-hydroxy-4-cholesten-3-one. Total oxysterols in hepatic bile ranged from 0.133 mumol/L to 7.748 mumol/L (1.47 +/- 2.55 mumol/L). Levels of 7-alpha-hydroxycholesterol and 7-beta-hydroxycholesterol were increased in infected bile (14.2 +/- 15.1 x 10(-3)% of cholesterol vs 1.9 +/- 0.5 x 10(-3)% of cholesterol, p0.05, and 22.0 +/- 25.0 x 10(-3)% of cholesterol vs 1.6 +/- 1.2 x 10(-3)% of cholesterol, p0.05, respectively). Serum C-reactive protein levels correlated positively with biliary levels of 7-alpha-hydroxycholesterol (R = 0.948), 7-beta-hydroxycholesterol (R = 0.976), cholestan-3-beta,5-alpha,6-beta-triol (R = 0.823), 7-alpha-hydroxy-4-cholesten-3-one (R = 0.846,) and 7-ketocholesterol (R = 0.973). Different oxysterols were found in gallstones, chiefly 3-keto-cholest-4-ene (624 +/- 316 parts per million [ppm] of dry weight), 3-keto-cholesta-4,6-diene (240 +/- 329 ppm) and 7-keto-cholesterol (77 +/- 81 ppm). Incubation of human leukocytes with model bile in the presence of bacterial lipopolysaccharide resulted in changes in sterol composition, including increases in oxysterols. We have identified and quantified oxysterols from uninfected and infected human hepatic bile and from gallstones and gallbladder bile. Biliary infection may be involved in the biogenesis of oxysterols in bile through the production of reactive oxygen species from activated leukocytes.

Biliary casts develop in up to 18% of liver transplant recipients. Casts are associated with morbidity, graft failure, need for retransplantation, and mortality. Proposed etiological mechanisms include acute cellular rejection, ischemia, infection, and biliary obstruction. We aimed to identify clinical features associated with biliary cast formation, review treatments, and analyze the biochemical composition of casts at a single, large, liver transplant center.Patient records were reviewed retrospectively to identify patients who developed casts. Data were collected with attention to ischemia, rejection, obstruction, infection, immunosuppression, postoperative biliary drain use, and cast-directed management, and were compared with data from controls. Cast specimens, retrieved at cholangiography, were analyzed with chromatography techniques.Ischemic factors were noted in 70% (7/10) of cast patients versus 15% (6/40) of controls (OR = 13.2; 95% CI = 2.7-66.0; p = 0.001). Biliary strictures were present in 50% of cast patients versus 10% of controls (OR = 9.0; 95% CI = 1.8-45.2; p = 0.01). Differences in cold ischemia time, acute cellular rejection, cyclosporin use, infection, and postoperative biliary drain use were not significant. Casts were successfully treated by endoscopic and percutaneous methods in 60% of patients. One patient died of cast-related complications (mortality 10%). Four casts were in satisfactory condition for biochemical analysis. Bilirubin was the main component ( approximately 10-50%). Bile acid synthesis products and cholesterol comprised smaller percentages, and protein comprised only 5-10%.Biliary casts are more likely to develop in the setting of hepatic ischemia and biliary strictures. Endoscopic and percutaneous cast extraction might achieve favorable results and should be attempted before surgical therapy.

Gallstones are extremely common in Western societies. Approximately l5% of Americans have gallstones, and approximately 650,000 to 700,000 cholecystectomies are performed every year.1. More than 98% of all biliary tract disorders are in some way related to gallstones. Symptoms and complications related to gallstones are among the most costly digestive disorders, at an estimated annual cost of almost $6.5 billion, exceeding the combined total for chronic liver disease and cirrhosis ($1.6 billion), chronic hepatitis C ($0.8 billion) and diseases of the pancreas ($2.2 billion).2 Stones are most commonly found in the gallbladder, but gallstones can pass through the cystic duct to become intrahepatic or extrahepatic bile duct stones. Ten to fifteen percent of people with gallbladder stones will harbor concomitant common bile duct stones. In some situations, bile duct stones can develop as primary intrahepatic or extrahepatic stones without involving the gallbladder. Primary bile duct concretions are much more common in patients of Asian descent compared with those of European descent. In Western societies, gallstones are composed primarily of cholesterol. EPIDEMIOLOGY OF CHOLESTEROL GALLSTONES The epidemiology of cholesterol gallstones has been examined in multiple studies. Risk factors for gallstones include behavioral factors such as nutrition, obesity, weight loss, and physical activity as well as biologic factors such as increasing age, female sex, race, and serum lipid levels.

Proliferative cholangitis (PC) leads to biliary stricture, which is the main cause of hepatolithiasis, recurrent cholangitis, and biliary cirrhosis. The aim of this study was to determine whether local delivery of paclitaxel, which inhibits cell proliferation by overstabilization of microtubules, prevents PC in a rat model.PC was induced by introducing a fine nylon thread into the bile duct in a rat. Paclitaxel (100 microl of 10, 100, and 1000 micromol/l) or solvent vehicle was administered into the bile duct for 15 min. One week after treatment, histopathologic examination and 5-bromodeoxyuridine (BrdU) labeling of the bile duct were performed.In comparison with the control, the mean thickness of the bile duct was reduced by 29% in the 1000 micromol/l paclitaxel-treated group (2.61 +/- 0.31 microm vs 3.67 +/- 0.25 micro m, P0.05). The luminal area increased ( P0.0001) and the grade of epithelial-glandular proliferation was decreased ( P0.01) as the dose of paclitaxel increased. Ductal fibrosis and inflammatory cell infiltration were similar in both groups. The BrdU labeling index was significantly lower in the paclitaxel-treated group ( P0.05).Local delivery of paclitaxel suppressed PC in a rat model by the inhibition of epithelial-glandular proliferation and may offer an effective therapeutic option for biliary stricture.

Gall-bladder epithelial cells (GBEC) are exposed to high concentrations of cholesterol in bile. Whereas cholesterol absorption by GBEC is established, the fate of this absorbed cholesterol is not known. The aim of this study was to determine whether ABCA1 (ATP-binding cassette transporter A1) mediates cholesterol efflux in GBEC. Polarized canine GBEC were cultured on porous membrane filters allowing separate access to apical (AP) and basolateral (BL) compartments. After AP loading of cells with model bile and [14C]cholesterol, cholesterol efflux was measured. Cholesterol loading together with 8-bromo-cAMP treatment, which increased ABCA1 expression, led to a significant increase in cholesterol efflux with apolipoprotein A-I (apoA-I) as the acceptor. Cholesterol efflux was observed predominantly into the BL compartment. Similar results were found for phospholipid efflux. Confocal immunofluorescence microscopy showed a predominantly BL ABCA1 localization. Interestingly, apoA-I added to either the AP or the BL compartments elicited BL lipid efflux with cAMP treatment. No paracellular or transcellular passage of 125I-apoA-I occurred. Ligands for the nuclear hormone receptors liver X receptor α (LXRα) and retinoid X receptor (RXR) elicited AP and BL cholesterol efflux, suggesting the involvement of both ABCA1- and non-ABCA1-mediated pathways. In summary, BL cholesterol/phospholipid efflux consistent with an ABCA1-mediated mechanism occurs in GBEC. This efflux pathway is stimulated by cAMP and by LXRα/RXR ligands, and in the case of the cAMP pathway appears to involve a role for biliary apoA-I.

These studies investigated the growth characteristics and functional properties of isolated canine pancreatic ductal epithelial cells. Cells were isolated from the accessory pancreatic duct and cultured by using three conditions: on vitrogen-coated petri dishes with fibroblast conditioned medium (nonpolarized); in vitrogen-coated Transwells above a fibroblast feeder layer (polarized); or as organotypic rafts above a fibroblast-embedded collagen layer (polarized). Growth characteristics, transepithelial resistances, and carbonic anhydrase and cyclic adenosine monophosphate (AMP) responses were evaluated. Under polarized conditions, the cells grew as monolayers with columnar epithelial characteristics. The monolayers developed high transepithelial resistance and became impervious to the passage of horseradish peroxidase. Epithelial growth factor (EGF) (2 ng/ml) stimulated ductal cell growth and accelerated the formation of a high-resistance monolayer. Forskolin (10 microM) rapidly decreased transepithelial resistance. Carbonic anhydrase activity, which was lower in nonpolarized compared with polarized conditions, was stimulated by carbachol (175 microM). Secretin, however, did not stimulate carbonic anhydrase activity in these cells. Although secretin stimulated adenylyl cyclase activity in early-passage cells, this response was lost in later-passage cells. Both vasoactive intestinal polypeptide (VIP; 1 microM) and forskolin (10 microM) consistently increased adenylyl cyclase activity. Isolated canine pancreatic ductal epithelial cells proliferate in vitro, develop high-resistance epithelial monolayers, and respond to stimuli that activate adenylyl cyclase. These cells should provide a useful model for regulatory studies of ductal cell functions.

Biliary infection is associated with mucin hypersecretion by the biliary epithelium. Mucins have been identified as potent pronucleators of cholesterol in bile. The aim of the present study was to determine whether lipopolysaccharides (LPS) from different bacteria are capable of stimulating mucin secretion by cultured dog gallbladder epithelial (DGBE) cells, and to investigate the mechanism by which LPS stimulate mucin secretion. Mucin secretion by confluent monolayers of DGBE cells was quantified by measuring the secretion of [3H]-N-acetyl-D-glucosamine-labeled glycoproteins. Cell viability was evaluated by measuring the leakage of the enzyme, lactate dehydrogenase (LDH), into the culture medium. LPS, derived from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (200 microg/mL), all caused an increase in mucin secretion by the DGBE cells, without causing concomitant cell lysis. LPS from E. coli was found to be the most potent stimulator of mucin secretion, and increased mucin secretion by the DGBE cells to 252% +/- 14% of control. LPS from E. coli had no effect on intracellular cyclic adenosine monophosphate (cAMP) levels in the DGBE cells. Addition of the nitric oxide (NO)-releasing compound, NOR-4 (0.125-1 mmol/L), to the cells did not result in increased mucin secretion, and the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) (4 or 10 mmol/L), did not inhibit the LPS-stimulated mucin secretion. Exogenous tumor necrosis factor alpha (TNF-alpha) (1-10 ng/mL) did cause a minor increase in mucin secretion by the DGBE cells, but the effect of LPS from E. coli on mucin secretion could not be inhibited by preincubation with a TNF-alpha antibody (10 microg/mL). We conclude that LPS stimulates mucin secretion by the gallbladder epithelium. Whether this stimulation is mediated by TNF-alpha remains to be determined.

Cl- secretion by pancreatic duct epithelial cells (PDEC) regulates cellular HCO3- secretion, an important component of the exocrine pancreas. In cystic fibrosis, for example, impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel results in decreased pancreatic secretion and secondary pancreatic insufficiency. Studies of ion transport by PDEC have been hindered by the lack of a practical in vitro model. We have successfully cultured nontransformed dog PDEC on Vitrogen-coated permeable membranes overlying a feeder layer of myofibroblasts and report the characterization of Cl- channels in these cells. Cl- conductance, assessed through efflux of 125I from PDEC, was stimulated by agents acting via adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+. The Cl- conductances activated by cAMP and Ca2+ were distinct, since they were differentially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, to a lesser extent, by 5-nitro-2-(3-phenylpropylamino)benzoic acid and diphenylamine-2 carboxylate. Patch-clamp studies confirmed the presence of Cl- channels activated by cAMP and Ca2+, with differential inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of CFTR Cl- channels in PDEC was confirmed by immunoblotting. These cultured PDEC are an optimal model for studies of pancreatic duct secretion.

To study the morphologic and genetic events associated with the carcinogenic process in the pancreas, we have isolated and cultured a cell line of dog pancreatic duct epithelial cells and treated these cells with a carcinogen. The pancreatic duct epithelial cells were plated onto Vitrogen-coated Transwell inserts suspended above a feeder layer of human gallbladder myofibroblasts. The epithelial cells grew steadily into polarized monolayers, could be passaged repeatedly, and demonstrated the typical morphologic, immunohistochemical, and flow cytometric profile of normal well-differentiated columnar pancreatic epithelial cells. After being treated with 10 -5 M N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 48 h, the treated cells grew on plastic surfaces. When grown in organotypic culture, the MNNG-treated cells were cuboidal with a multilayered, pseudostratified architecture. Flow cytometry demonstrated aneuploidy and a high percentage of the cells in S phase after reaching confluency, in sharp contrast to untreated cells. Cytogenetic analysis of the MNNG-treated cells revealed frequent chromosomal trisomy and tetrasomy. The secretion of mucin was also different in the MNNG-treated cells versus the untreated cells. The cultured pancreatic epithelial cells may be useful as an assay system to study the genotoxicity of known and potential carcinogens.

We have analyzed the chemical composition of primary intrahepatic stones from 72 Korean patients. Two types of concretions have been identified: brown pigment (calcium bilirubinate) stones and black-colored mixed stones. Brown pigment stones were found in 68% of all cases and the remainder (32%) consisted of mixed stones. Intrahepatic mixed stones had mean cholesterol and bilirubin contents of 46.6% and 25.9%, respectively, whereas calcium bilirubinate stones had mean cholesterol and bilirubin contents of 14.1% and 43.6%, respectively. Intrahepatic mixed stones had a smooth black-colored surface and on cross section, exhibited a distinct outer shell surrounding an inner yellow, cholesterol-rich body. The finding of intrahepatic mixed stones with high cholesterol content suggests that primary hepatolithiasis may result from at least two different conditions or disorders and points to different approaches to their treatment.

Hypersecretion of gallbladder mucin has been proposed as a pathogenic factor in gallstone formation. We investigated whether mucin secretion is modulated by biliary constituents using normal, well-differentiated dog gallbladder epithelial cells. Model biles or bile salts were applied to monolayers of epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-D-glucosamine-labeled glycoproteins. Model biles with different cholesterol saturation indices increased mucin secretion by the cells to an average 251% after 5 hours of incubation (P < 0.01). Mucin secretion remained elevated during a 24-hour period, suggesting a sustained effect on mucin secretion. There was no relation between the cholesterol or phospholipid concentration and the extent of stimulation of mucin secretion. Taurocholate caused a dose-dependent increase in mucin secretion, suggesting that bile salt was the bile component responsible for the stimulatory effect. At a concentration of 0.5 mmol/L, only the more hydrophobic bile salts taurochenodeoxycholate and taurodeoxycholate, but not the hydrophylic bile salts taurocholate and tauroursodeoxycholate, stimulated mucin secretion (P < 0.01). Bile salts play an important role in the regulation of mucin secretion. A shift in the bile salt composition of bile towards the more hydrophobic bile salts may cause mucin hypersecretion, thereby initiating cholesterol gallstone formation