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Supplementary Table 1 from Serotonin Metabolism Is Dysregulated in Cholangiocarcinoma, which Has Implications for Tumor Growth

Supplementary Figure 5 from Serotonin Metabolism Is Dysregulated in Cholangiocarcinoma, which Has Implications for Tumor Growth

Supplementary Figure 1 from Serotonin Metabolism Is Dysregulated in Cholangiocarcinoma, which Has Implications for Tumor Growth

Supplementary Figure 2 from Serotonin Metabolism Is Dysregulated in Cholangiocarcinoma, which Has Implications for Tumor Growth

Supplementary Figure Legends 1-5 from Serotonin Metabolism Is Dysregulated in Cholangiocarcinoma, which Has Implications for Tumor Growth

Supplementary Figure 3 from Serotonin Metabolism Is Dysregulated in Cholangiocarcinoma, which Has Implications for Tumor Growth

Supplementary Figure 4 from Serotonin Metabolism Is Dysregulated in Cholangiocarcinoma, which Has Implications for Tumor Growth

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: One of substudies of the prospective trials aimed to evaluate the usefulness of serial [18F] 2-fluoro-2-deoxy-D-glucose-positron emission tomography ([18F] FDG-PET) for predicting pathological complete response (pCR) in stage II/III breast cancer with preoperative chemotherapy (PST). Methods: Serial PET was undertaken in 57 breast cancer patients enrolled in three different neoadjuvant trials: 35 patients from a phase II study with paclitaxel/gemcitabine/trastuzumab with ClinicalTrial.gov NCT 00532857, 9 patients from a phase Ib study with paclitaxel/gemcitabine/lapatinib with ClinicalTrial.gov NCT 01133912, and 13 patients from a phase Ib with paclitaxel/gemcitabine/sunitinib with ClinicalTrial.gov NCT0 1070706. All patients received 6 cycles of PST followed by surgery and radiotherapy. We assessed the peak standardized uptake value (SUVp) in the primary tumor at the baseline and after the 2nd cycle (37 patients) or after completion (20 patients) of 6 cycles of PST, and calculated the reduction rate (RR) of the SUVp. Pathological response was classified into pCR and non-pCR. To compare the mean of SUVp and RR of SUVp between different response groups, two-way tables and chi-square tests were used Results: Fifteen (40.6%) of 37 patients who took repeat PET after the 2nd PST and 15 (75%) of 20 patients after completion of PST achieved a pCR with overall pCR rate of 52.6% in the primary tumor. In patients with repeat PET after the 2nd PST, post-treatment SUVp and RR of the SUVp in primary tumors were significantly different by the pathological response (post-treatment SUVp, 1.54 ± 0.63 in pCR vs 2.54 ± 1.06 in non-pCR, P=0.002; RR of the SUVp, 79.2% ± 11.9% in pCR vs 68.9% ± 15.4% in non-pCR, P=0.03). However, in patients with repeat PET after completion of PST, there were no statistical differences of these values (post-treatment SUVp, 1.09 ± 0.63 in pCR vs 1.29 ± 0.36 in non-pCR, P=0.42; RR of the SUVp, 83.7% ± 14.0% in pCR vs 67.5% ± 21.1% in non-pCR, P=0.17) Conclusions: This study demonstrated that repeat PET after the 2nd cycle of PST, not after completion of PST could predict pCR in stage II/III breast cancer with preoperative chemotherapy. Acknowledgement NCC Grant #0910320. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-01.

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.

We tested the hypothesis that well-differentiated gallbladder epithelial cells (GBECs) are capable of engrafting and surviving in murine liver and acquire phenotypic characteristics of hepatocytes. GBECs isolated from transgenic mice that constitutively express green fluorescent protein (GFP) were either cultured before transplantation or transplanted immediately following isolation. Recipient mice with severe-combined immunodeficiency underwent retrorsine treatment and either partial hepatectomy before transplantation or carbon tetrachloride treatment following transplantation. From 1 to 4 months following transplantation, the livers of recipient mice contained discrete colonies of GFP+ cells. Most GFP+ cells surrounded vesicles, were epithelial cell-like in morphology, and expressed the biliary epithelial markers cytokeratin 19 and carbonic anhydrase IV. Subpopulations of GFP+ cells resembled hepatocytes morphologically and expressed the hepatocyte-specific markers connexin-32 and hepatic nuclear factor-4α, but not cytokeratin 19 or carbonic anhydrase IV. At 4 months, cells in GFP + colonies were not actively proliferating as determined by proliferating cell nuclear antigen expression. Thus, GBECs are capable of engrafting and surviving in damaged mouse livers, and some can differentiate into cells with hepatocyte-like features. These findings suggest that environmental cues in the recipient liver are sufficient to allow a subpopulation of donor GBECs to differentiate into hepatocyte-like cells in the absence of exogenous transcriptional reprogramming. GBECs might be used as donor cells in a cell transplantation approach for the treatment of liver disease. Copyright © American Society for Investigative Pathology., link_to_subscribed_fulltext

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

We often encounter the situation that discrete count data have a large portion of zeros. In this case, it is not appropriate to analyze the data based on standard regression models such as the poisson or negative binomial regression models. In this article, we consider Bayesian analysis for two commonly used models. They are zero-inflated poisson and negative binomial regression models. We use the Bayes factor as a model selection tool and computation is proceeded via Markov chain Monte Carlo methods. Crash count data are analyzed to support theoretical results.

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.

The cellular and molecular mechanisms of fibrogenesis in the extrahepatic biliary epithelium are not known. Transforming growth factor-beta1 (TGF-beta1) is a cytokine implicated in signaling pathways that mediate collagen formation. An observation that paclitaxel (PT), applied topically into the rat common bile duct, inhibited stricture formation led us to hypothesize that PT's effects might be due to interruption of TGF-beta1 signaling between biliary epithelial cells and subepithelial myofibroblasts.We tested this hypothesis using an in vitro cell-culture model in which murine gallbladder epithelial cells (GBEC) are cultured separately or cocultured with human gallbladder myofibroblasts (GBMF).Exposure to Escherichia coli lipopolysaccharide (LPS) enhanced TGF-beta1 mRNA expression and stimulated TGF-beta1 protein secretion into both apical and basolateral compartments in GBEC. This effect was more prominent with basolateral secretion and was also more pronounced in the coculture system. In GBMF, collagen I mRNA expression and protein secretion were stimulated by treatment with LPS or TGF-beta1. GBMF also expressed TGF-beta1 mRNA, whose levels were enhanced by exposure to either LPS or exogenous TGF-beta1. PT inhibited LPS-induced TGF-beta1 mRNA expression and protein secretion in GBEC in both culture systems. Tumor necrosis factor-alpha mRNA expression and protein secretion were not affected by PT in GBEC, demonstrating that the effects were specific for TGF-beta1. PT also inhibited LPS- and TGF-beta1-induced collagen I mRNA expression and protein secretion in GBMF.These findings support a model in which GBEC communicate with subepithelial GBMF via TGF-beta1, leading to collagen deposition and fibrosis, and in which GBMF possess autocrine mechanisms involving TGF-beta1 that could regulate collagen production. PT inhibits these fibrogenic pathways.

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.

This work examines three related, but previously unexplored, aspects of membrane biophysics and colloid science in the context of atherosclerosis. First, we show that sphingomyelinase (SMase)-induced aggregation of low density lipoproteins (LDLs), coupled with LDL exposure to cholesterol esterase (CEase), results in nucleation of cholesterol crystals, long considered the hallmark of atherosclerosis. In particular, this study reveals that the order of enzyme addition does not effect the propensity of LDL to nucleate cholesterol crystals, raising the possibility that nucleation can proceed from either the intra- or extracellular space. Second, we demonstrate that ceramide-rich aggregates of LDL release cholesterol to neighboring vesicles far more rapidly, and to a greater extent, than does native LDL. A likely explanation for this observation is displacement of cholesterol from SM-Chol rafts by "raft-loving" ceramide. Third, we demonstrate that a time-independent Förster resonance energy transfer (FRET) assay, based on dehydroergosterol and dansylated lecithin and used previously to study cholesterol nanodomains, can be used to measure raft sizes (on the order of 10 nm) in model membrane systems. Taken together, these observations point to the possibility of an extracellular nucleation mechanism and underscore the important role that biological colloids play in human disease.

Gallstones are strongly associated with higher parity in women. This study prospectively assessed the incidence, natural history, and risk factors for biliary sludge and stones during pregnancy and the postpartum in 3,254 women at an army medical center. Women with a prior cholecystectomy or with stones at their first study ultrasound were excluded. Gallbladder ultrasound and subject questionnaires were obtained in each trimester and at 4 to 6 weeks postpartum. Serum glucose, lipids, insulin, leptin, estradiol, and progesterone were measured at 26 to 28 weeks' gestation. A nested case-control study was done to examine the effects of serum leptin and insulin on incident gallbladder disease. At least two study ultrasounds were available for 3,254 women. Sludge or stones had been found on at least one study ultrasound in 5.1% by the second trimester, 7.9% by the third trimester, and 10.2% by 4 to 6 weeks postpartum. Regression of sludge and stones was common, such that overall 4.2% had new sludge or stones on the postpartum ultrasound. Twenty-eight women (0.8%) underwent cholecystectomy within the first year postpartum. Prepregnancy body mass index was a strong predictor of incident gallbladder disease (P < .001). Serum leptin was independently associated with gallbladder disease (odds ratio per 1 ng/dL increase, 1.05; 95% CI, 1.01, 1.11), even after adjusting for body mass index. In conclusion, incident gallbladder sludge and stones are common in pregnancy and the postpartum, and cholecystectomy is frequently done within the first year postpartum. Prepregnancy obesity and serum leptin are strong risk factors for pregnancy-associated gallbladder disease.

Because hypersecretion of gallbladder (GB) mucus occurs in gallstone formation and because binding of Ca(2+) to biliary lipids only accounts for 50% of the total Ca(2+) in GB bile, we investigated the binding of Ca(2+) to human biliary mucin. Biliary mucin was purified from GB bile and binding to Ca(2+) studied. Scatchard plot analysis suggested two binding sites. Removal of sialic acid by neuraminidase resulted in 10% reduction of Ca(2+) binding, whereas, sulfatase treatment reduced Ca(2+) binding by 30%. Using a hypotonic NaCl solution, Ca(2+) binding to mucin increased curvilinearly with mucin concentration. However, binding decreased with increasing ionic strength of the NaCl solution. We conclude that binding of Ca(2+) to mucin is effected mainly through sulfate. Binding to Ca(2+) can be displaced by Na(+). Ca(2+) binding to mucins is enhanced in the setting of low Na(+) concentrations. This phenomenon has pathophysiologic implications for the formation of thick mucus in cystic fibrosis epithelia.

In this work, we use two vertically-coupled square two-dimensional lattices to simulate membrane bilayers containing a uniform size distribution of cholesterol immiscible domains of a predetermined size distribution. We substitute cholesterols and phospholipids with their fluorescent analogs and calculate the efficiency of energy transfer as a function of acceptor concentration for four membrane configurations. The simulated efficiency of energy transfer as a function of acceptor concentration data is then fit with an analytical FRET model to estimate the domain size, in the same manner in which experimental FRET data is analyzed. The fitted model parameters (domain size and donor partition coefficient) are compared to the simulation inputs to test the applicability of the FRET model to estimating the size of laterally phase separated cholesterol domains. We show that the FRET model yields good size estimates for domains that range between 1 and 25 nm. We also find that the assumed fluorophore configuration in the FRET model leads to a constant under-prediction of these values. Finally, we demonstrate that when two parameters are open to the fit, the FRET model adequately predicts the donor partition coefficient in addition to the domain size.

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.

Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5-20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.

Biliary sludge develops commonly in critically ill patients and may be associated with biliary colic, acute pancreatitis or acute cholecystitis. Sludge often resolves upon resolution of the underlying pathogenetic factor. It is generally diagnosed on sonography. Treatment of sludge itself is unnecessary unless further complications develop. Acute acalculous cholecystitis also develops frequently in critically ill patients. It may be difficult to diagnose in these patients, manifesting only as unexplained fever, leukocytosis or sepsis. Sonography and hepatobiliary scintigraphy are the most useful diagnostic tests. Management decisions should take into account the underlying co-morbid conditions. For many patients, percutaneous cholecystostomy may be the best management option. Cholecystostomy may also provide definitive drainage as patients recover and underlying critical illness resolves.

We present evidence that laterally phase separated cholesterol domains constitute a new, equilibrium phase in biological membranes. The domains are characterized in multi-lamellar vesicles (MLV) made of cholesterol and dimyristoylphosphatidylcholine (DMPC) but are also shown to exist in biologically relevant, egg lecithin systems containing a mixture of phospholipids. This work utilizes the fluorescent membrane probes 1-acyl-2-[12-[(5-dimethylamino-1-naphthalenesufonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DANSYL), and ergosta-5,7,9(11),22-tetraen-3β-ol (ERGO), which have been shown to be minimally invasive mimics of native membrane lipids. The highlight of the work is a heating-induced alleviation of a DANSYL blue shift at relatively high (but undersaturated) cholesterol loadings, which is reversible through at least three heating and cooling cycles. Comparison of the DANSYL spectral shifts with published DMPC–cholesterol phase diagrams shows unequivocally that the spectral results cannot be explained in terms of previously understood phase behavior. Rather, a lateral phase separation occurs within the vesicle bilayer, giving rise to cholesterol micro-domains. The cholesterol domains appear to coexist with, and should not be confused with, the well-known liquid-order phase that arises because of the cholesterol condensation effect. Additional studies involving ERGO–DANSYL energy transfer show a sequestration of probes within the bilayer, confirming the DANSYL spectral data, and a model that includes domains provides the best description of measured energy transfer efficiencies. Best fits of the energy transfer data, using a mathematical model developed to account for the presence of domains, indicates the domain size to be in the range 10–20 nm.

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.

Background. The cell type responsible for the desmoplastic reaction surrounding human pancreatic carcinoma is unknown. Hepatic stellate cells, which activate to a myofibroblast-like form, are responsible for collagen deposition in cirrhosis and around hepatocellular carcinomas. Recently, pancreatic stellate cells have been described and implicated in the fibrosis of chronic pancreatitis. We sought to determine whether these cells are responsible for the scirrhous reaction surrounding pancreatic adenocarcinomas. Methods. Archival formalin-fixed, paraffin-embedded pancreatic tissues from 10 patients undergoing pancreaticoduodenectomy for ductal adenocarcinoma and from 2 patients with pancreatic islet cell tumors were examined immunohistochemically for alpha-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SMMHC), procollagen I, collagen IV, and endothelial cell markers, von Willebrand factor and cluster of differentiation 31. Results. In non-neoplastic areas, staining for α-SMA and SMMHC was confined to interlobular septal regions. In contrast, the desmoplastic reaction surrounding all 10 pancreatic adenocarcinoma specimens displayed intense interstitial staining for α-SMA, SMMHC, and collagen IV but no staining for von Willebrand factor and cluster of differentiation 31. Procollagen I staining localized intracellularly to fibroblast-shaped cells within this α-SMA/SMMHC-positive scirrhous region. Islet cell tumors demonstrated an increase in α-SMA staining, although this was not as marked as in ductal adenocarcinomas. Conclusions. A massive increase in myofibroblast activity, compatible with the activation of stellate cells, is associated with the deposition of collagen types I and IV in the desmoplastic reaction around pancreatic adenocarcinomas. (Surgery 2002;131:129-34.)

Background & Aims: Although cholesterol is the most abundant sterol in animal tissues, oxidized products of cholesterol (oxysterols) also occur in mammalian organs and blood and are cytotoxic, atherogenic, and carcinogenic. However, the presence of oxysterols in bile or gallstones has never been reported. Methods: Fresh human bile and gallstones were collected. Sterol content and structure were analyzed using gas chromatography/mass spectrometry (GC/MS). Bacterial DNA was extracted from human gallstones. Results: GC/MS identified cholesta-4,6-diene-3-one and cholest-4-ene-3-one, with several as yet unidentified oxysterols in bile and stone samples. Several plant and fungal sterols were also present in gallstones. When 102 human gallstones were analyzed for oxysterols, they were markedly higher (as percent of total sterols) in pigment gallstones, where bacterial DNA is most abundant. Conclusions: These observations suggest biliary oxysterols are associated with the presence of bacteria and may play a role in the pathogenesis of gallstones and biliary tract cancers.

Intestinal epithelial cells secrete a protective luminal mucus barrier inhibiting viral gene transfer. Quiescent, polarized monolayers of primary epithelial cells from dog gallbladder and human colon are efficiently transduced through the apical mucus side by lentivirus vectors, suggesting their application to intestinal gene therapy.

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.

We demonstrate Förster resonance energy transfer from dehydroergosterol to dansylated lecithin in lecithin-cholesterol vesicles and characterize the vesicles in the presence of the pro-nucleating enzyme, phospholipase C (PLC). Exposure to phospholipase C causes a temporary decrease in the dehydroergosterol to dansyl fluorescence ratio followed by an increase to and above the initial value. The temporary decrease in the fluorescence ratio results from an increase in the dansylated lecithin intensity that coincides with a dansyl blue shift. The extent of the blue shift correlates with the level of diacylglycerol generated in situ by PLC, suggesting an increased association between dansylated lecithin and cholesterol as membrane fluidity increases and membrane polarity decreases. The subsequent increase in the fluorescence ratio results from both an increase in the dehydroergsterol intensity and a concomitant decrease in the dansylated lecithin intensity of equal magnitude. This signifies a reduction in energy transfer from dehydroergosterol to dansylated lecithin and indicates an increased separation between the two fluorophores. The increase in the fluorescence ratio persists beyond the time scales for vesicle aggregation and fusion, as measured by turbidity, and precedes the onset of macroscopic cholesterol crystals observed with an optical microscope. Thus, the increased separation between dehydroergosterol and dansylated lecithin is consistent with a mechanism of cholesterol nucleation from the vesicles. Moreover, the onset and rate of increase in the fluorescence ratio correlate with the cholesterol:lecithin mole ratio of the vesicles. Fluorescence energy transfer from dehydroergosterol to dansylated lecithin therefore shows potential as a methodology for measuring cholesterol nucleation in model bile.—Wrenn, S. P., E. W. Kaler, and S. P. Lee. A fluorescence energy transfer study of lecithin-cholesterol vesicles in the presence of phospholipase C. J. Lipid Res. 1999. 40: 1483–1494.

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.

The nucleation of cholesterol crystals is an obligatory precursor to cholesterol gallstone formation. Nucleation, in turn, is believed to be preceded by aggregation and fusion of cholesterol-rich vesicles. We have investigated the effects of two putative pro-nucleating proteins, a concanavalin A-binding protein fraction and a calcium-binding protein, on the stability of sonicated small unilamellar cholesterol–lecithin vesicles. Vesicle aggregation is followed by monitoring absorbance, and upon addition of the concanavalin A-binding protein fraction the absorbance of a vesicle dispersion increases continuously with time. Vesicle fusion is probed by a fluorescence contents-mixing assay. Vesicles apparently fuse slowly after the addition of the concanavalin A-binding protein, although inner filter effects confound the quantitative measurement of fusion rates. The rates of change of absorbance and fluorescence increase with the concentration of the protein, and the second-order dimerization rate constant increases with both the protein concentration and the cholesterol content of the vesicles. On the other hand, the calcium-binding protein has no effect on the stability of the vesicle dispersion. This protein may therefore affect cholesterol crystal formation not by promoting the nucleation process, but by enhancing crystal growth and packaging. Our results demonstrate that biliary proteins can destabilize lipid vesicles and that different proteins play different roles in the mechanism of cholesterol gallstone formation.

Normal dog gallbladder epithelial cells in long-term culture were used as a model to study the morphologic, genetic, and secretory processes associated with the progression to cancer formation. Dog gallbladder epithelial cells cultured on collagen-coated plates grew into polarized monolayers, could be passaged repeatedly, and showed the typical morphological profile of well-differentiated columnar epithelial cells. After cells were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 10(-5) mol/L for 48 hours, the treated cells grew on plastic and could be cloned. Flow cytometry revealed emergence of an aneuploid cell subpopulation. In organotypic culture, treated cells showed a pseudostratified appearance, with cellular and nuclear pleomorphism. Large and hyperchromatic nuclei were present as well as increased mitotic rate. The proteins of MNNG-treated dog gallbladder epithelial cells showed increased phosphorylation of tyrosine residues. Treated cells showed a decrease in mucin secretion in response to prostaglandin E2, manifesting an altered pattern of mucin secretion. Transforming growth factor-beta failed to inhibit cell proliferation in the MNNG-treated cells compared with the prominent inhibition in normal cells. Together, the data reflected changes representing preliminary steps on the pathway to develop cancer cells. Our results indicate that carcinogenic chemicals can cause measurable chromosomal and cellular modifications to normal biliary epithelial cells in vitro. This model may be useful in understanding the sequential steps in carcinogenesis and affords an opportunity to study chromosomal damage, cytokinetics, changes in molecular genetic markers, and expression, as well as cell biological function during cellular transformation.

The liver secretes cholesterol and lecithin in the form of mixed vesicles during the formation of bile. When exposed to bile salts, these metastable vesicles undergo various structural rearrangements. We have examined the effects of three different bile salts, taurocholate (TC), tauroursodeoxycholate (TUDC), and taurodeoxycholate (TDC), on the stability of sonicated lecithin vesicles containing various amounts of cholesterol. Vesicle growth was probed by turbidity measurements, quasi-elastic light scattering, and a resonance energy transfer lipid-mixing assay. Leakage of internal contents was monitored by encapsulation of fluorescence probes in vesicles. At low bile salt-to-lecithin ratios (TC/L or TUDC/L1), pure lecithin vesicles do not grow, but exhibit slow intervesicular mixing of lipids as well as gradual leakage. At high BS/L (TC/L or TUDC/L5), pure lecithin vesicles are solubilized into mixed micelles with a concomitant decrease in the overall particle size. In this regime, extensive leakage and lipid mixing occur instantaneously after exposure to bile salt. At intermediate BS/L (1TC/L or TUDC/L5), vesicles grow with time, and the rates of both leakage and lipid mixing are rapid. The data suggest that vesicles grow by the transfer of lecithin and cholesterol via diffusion in the aqueous medium. The addition of cholesterol to lecithin vesicles reduces leakage dramatically and increases the amount of BS required for complete solubilization of vesicles. The more hydrophobic TDC induces vesicle growth at a lower BS/L than does TC or TUDC. These results demonstrate the physiologic forms of lipid microstructures during bile formation and explain how the hydrophilic-hydrophobic balance of BS mixtures may profoundly affect the early stages of CH gallstone formation.

Mice with targeted disruption of the cftr gene show pathophysiologic changes in the gallbladder, which correlate with hepatobiliary disease seen in cystic fibrosis patients. As gallbladder epithelium secretes mucin, and as this epithelium consists of a relatively homogenous cell type, study of CFTR function in these cells would be beneficial to delineate the complex cellular functions of this protein. The size and anatomic location of the murine gallbladder makes such studies difficult in vivo. Therefore, the need exists for in vitro models of gallbladder epithelium. We describe a method to isolate and culture murine gallbladder epithelium from wild-type and CF mice. Cells were grown in a monolayer on porous inserts over a feeder layer of fibroblasts. These nontransformed cells can be successively passaged and maintain a well-differentiated epithelial cell phenotype as shown by morphologic criteria, characterized by polarized columnar epithelial cells with prominent microvilli and intercellular junctions. Organotypic cultures showed columnar cells simulating in vivo morphology. This culture system should be valuable in delineating cellular processes relating to CFTR in gallbladder epithelium.