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Background & Aims: Obesity and type 2 diabetes increase hepatocellular carcinoma (HCC) incidence in humans and accelerate diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. We investigated whether exercise reduces HCC development in obese/diabetic Alms1 mutant (foz/foz) mice and studied protective mechanisms., Methods: We measured HCC development in DEN-injected male foz/foz and wild-type (WT) littermates housed with or without an exercise wheel from week 4 until 12 or 24 weeks, and in foz/foz mice pair-fed to WT littermates. We also studied HCC development in DEN-injected Jnk1 -/- .foz/foz mice generated by cross breeding, as well as their genetic controls. Dysplastic hepatocytes were identified by glutathione-S-transferase pi form (GST-pi) immunohistochemistry, liver nodules were counted, and HCC was analysed by histopathology., Results: Exercising foz/foz mice maintained similar weight as WT mice up to 10 weeks, but then gained weight and were obese by 24 weeks; a similar body weight profile was obtained by pair-feeding foz/foz mice to WT. At 12 weeks, livers of exercising foz/foz mice exhibited fewer GST-pi positive hepatocytes than sedentary counterparts; by 24 weeks, fewer exercising foz/foz mice developed HCC (15% vs. 64%, p <0.05). Conversely, pair-feeding foz/foz mice failed to reduce HCC incidence. In these insulin-resistant foz/foz mice, exercise failed to activate hepatic AMPK or Akt/mTORC1. Instead, it improved insulin sensitivity, ameliorated steatosis and liver injury, activated p53 to increase p27 expression, and prevented JNK activation. This was associated with suppression of hepatocellular proliferation. DEN-injected Jnk1 -/- .foz/foz mice failed to develop liver tumours or HCC at 24 weeks., Conclusions: Direct effects of exercise dampen proliferation of dysplastic hepatocytes to reduce 3-month dysplastic foci and 6-month incidence of DEN-induced HCC in obese, insulin-resistant mice. The effects of exercise that potentially slow hepatocarcinogenesis include p53-mediated induction of p27 and prevention of JNK activation., Lay Summary: Fatty liver disease commonly occurs alongside obesity and diabetes, contributing to rapidly increasing rates of liver cancer throughout the world. Herein, we show that exercise reduces the incidence and progression of hepatocellular carcinoma in mouse models. The effect of exercise on cancer risk was shown to be independent of changes in weight. Exercise could be a protective mechanism against liver cancer in at-risk individuals., Competing Interests: Conflict of interests The authors have no conflict of interest to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. All rights reserved.)

It is unclear what drives the development of fibrosing nonalcoholic steatohepatitis (NASH). We aimed to determine whether cholesterol crystallization within hepatocyte lipid droplets (LDs) distinguishes patients with fibrosing NASH from patients with isolated hepatic steatosis and to study pathways leading to cholesterol accumulation in hepatocyte LDs . Patients with fibrosing NASH (n = 16) were compared to patients with isolated steatosis (n = 14). Almost all patients with fibrosing NASH had free cholesterol staining by filipin (16/16) and cholesterol crystals (15/16) in hepatocyte LDs, mostly in association with the LD membrane, compared to only 3/14 with cholesterol crystals and 3/14 with faint filipin staining in patients with isolated steatosis ( P  < 0.05). We were unable to identify significant differences in the expression of genes in liver tissue related to cholesterol homeostasis or LD proteins between patients with fibrosing NASH and isolated steatosis. Human hepatoma cell line (HepG2) cells were supplemented with low-density lipoprotein (LDL)-cholesterol and oleic acid to develop large LDs, similar to those observed in patients with NASH. Fluorescent markers were used to track the uptake and intracellular trafficking of LDL-cholesterol. LDL-cholesterol was taken up by HepG2 cells and transported through the endosomal-lysosomal compartment directly to LDs, suggesting direct contact sites between late endosomes and LDs. Exposure of HepG2 cells to LDL-cholesterol resulted in a high concentration of cholesterol and cholesterol crystallization in LDs. Conclusion: Excess cholesterol is stored in the liver primarily within hepatocyte LDs where it can crystallize. Our findings are best explained by direct transport of cholesterol from late endosomes/lysosomes to LDs in hepatocytes. We found a strong association between the presence of LD cholesterol crystals and the development of fibrosing NASH in humans, suggesting a causal relationship.

The underlining mechanisms of dietary cholesterol and nonalcoholic steatohepatitis (NASH) in contributing to hepatocellular carcinoma (HCC) remain undefined. Here we demonstrated that high-fat-non-cholesterol-fed mice developed simple steatosis, whilst high-fat-high-cholesterol-fed mice developed NASH. Moreover, dietary cholesterol induced larger and more numerous NASH-HCCs than non-cholesterol-induced steatosis-HCCs in diethylnitrosamine-treated mice. NASH-HCCs displayed significantly more aberrant gene expression-enriched signaling pathways and more non-synonymous somatic mutations than steatosis-HCCs (335 ± 84/sample vs 43 ± 13/sample). Integrated genetic and expressional alterations in NASH-HCCs affected distinct genes pertinent to five pathways: calcium, insulin, cell adhesion, axon guidance and metabolism. Some of the novel aberrant gene expression, mutations and core oncogenic pathways identified in cholesterol-associated NASH-HCCs in mice were confirmed in human NASH-HCCs, which included metabolism-related genes (ALDH18A1, CAD, CHKA, POLD4, PSPH and SQLE) and recurrently mutated genes (RYR1, MTOR, SDK1, CACNA1H and RYR2). These findings add insights into the link of cholesterol to NASH and NASH-HCC and provide potential therapeutic targets.

Lipotoxicity associated with insulin resistance is central to nonalcoholic steatohepatitis (NASH) pathogenesis. To date, only weight loss fully reverses NASH pathology, but mixed peroxisome proliferator-activated receptor-alpha/delta (PPAR-α/δ) agonists show some efficacy. Seladelpar (MBX-8025), a selective PPAR-δ agonist, improves atherogenic dyslipidemia. We therefore used this agent to test whether selective PPAR-δ activation can reverse hepatic lipotoxicity and NASH in an obese, dyslipidemic, and diabetic mouse model. From weaning, female Alms1 mutant ( foz/foz ) mice and wild-type littermates were fed an atherogenic diet for 16 weeks; groups (n = 8-12) were then randomized to receive MBX-8025 (10 mg/kg) or vehicle (1% methylcellulose) by gavage for 8 weeks. Despite minimally altering body weight, MBX-8025 normalized hyperglycemia, hyperinsulinemia, and glucose disposal in foz/foz mice. Serum alanine aminotransferase ranged 300-600 U/L in vehicle-treated foz/foz mice; MBX-8025 reduced alanine aminotransferase by 50%. In addition, MBX-8025 normalized serum lipids and hepatic levels of free cholesterol and other lipotoxic lipids that were increased in vehicle-treated foz/foz versus wild-type mice. This abolished hepatocyte ballooning and apoptosis, substantially reduced steatosis and liver inflammation, and improved liver fibrosis. In vehicle-treated foz/foz mice, the mean nonalcoholic fatty liver disease activity score was 6.9, indicating NASH; MBX-8025 reversed NASH in all foz/foz mice (nonalcoholic fatty liver disease activity score 3.13). Conclusion : Seladelpar improves insulin sensitivity and reverses dyslipidemia and hepatic storage of lipotoxic lipids to improve NASH pathology in atherogenic diet-fed obese diabetic mice. Selective PPAR-δ agonists act independently of weight reduction, but counter lipotoxicity related to insulin resistance, thereby providing a novel therapy for NASH. ( Hepatology Communications 2017;1:663-674).

Background and aims : TLR9 deletion protects against steatohepatitis due to choline-amino acid depletion and high-fat diet. We measured TLR9 in human non-alcoholic steatohepatitis (NASH) livers, and tested whether TLR9 mediates inflammatory recruitment in three murine models of non-alcoholic fatty liver disease (NAFLD). Methods : We assayed TLR mRNA in liver biopsies from bariatric surgery patients. Wild-type ( Wt ), appetite-dysregulated Alms1 mutant (foz/foz), Tlr9 -/- , and Tlr9 -/- foz/foz C57BL6/J mice and bone marrow (BM) chimeras were fed 0.2% cholesterol, high-fat, high sucrose (atherogenic[Ath]) diet or chow, and NAFLD activity score (NAS)/NASH pathology, macrophage/neutrophil infiltration, cytokines/chemokines, and cell death markers measured in livers. Results : Hepatic TLR9 and TLR4 mRNA were increased in human NASH but not simple steatosis, and in Ath-fed foz/foz mice with metabolic syndrome-related NASH. Ath-fed Tlr9 -/- mice showed simple steatosis and less Th1 cytokines than Wt. Tlr9 -/- foz/foz mice were obese and diabetic, but necroinflammatory changes were less severe than Tlr9 +/+ .foz/foz mice. TLR9-expressing myeloid cells were critical for Th1 cytokine production in BM chimeras. BM macrophages from Tlr9 -/- mice showed M2 polarization, were resistant to M1 activation by necrotic hepatocytes/other pro-inflammatory triggers, and provoked less neutrophil chemotaxis than Wt Livers from Ath-fed Tlr9 -/- mice appeared to exhibit more markers of necroptosis [receptor interacting protein kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] than Wt , and ∼25% showed portal foci of mononuclear cells unrelated to NASH pathology., Conclusion: Our novel clinical data and studies in overnutrition models, including those with diabetes and metabolic syndrome, clarify TLR9 as a pro-inflammatory trigger in NASH. This response is mediated via M1-macrophages and neutrophil chemotaxis., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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.

Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥ 3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation.

The oxysterols cholestan-3β,5α,6β-triol (Triol) and 3-keto-cholest-4-ene (3K4) are increased in Opisthorchis viverrini-associated hamster cholangiocarcinoma and induce DNA damage and apoptosis via a mitochondria-dependent mechanism in MMNK-1 human cholangiocytes. Based on these observations, we hypothesized that chronic exposure of cholangiocytes to these pathogenic oxysterols may allow a growth advantage to a subset of these cells through selection for resistance to apoptosis, thereby contributing to cholangiocarcinogenesis. To test this hypothesis, we cultured MMNK-1 cells long-term in the presence of Triol. Alteration in survival and apoptotic factors of Triol-exposed cells were examined. Cells cultured long-term in the presence of Triol were resistant to H2O2-induced apoptosis, and demonstrated an increase in the phosphorylation of p38-α, CREB, ERK1/2 and c-Jun. Elevations in the ratio of Bcl-2/Bax and in the protein levels of anti-apoptotic factors including cIAP2, clusterin, and survivin were detected. These results show that long-term exposure of MNNK-1 cells to low doses of Triol selects for kinase-signaling molecules which regulate resistance to apoptosis and thereby enhance cell survival. Clonal expansion of such apoptosis-resistant cells may contribute to the genesis of cholangiocarcinoma., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background & Aims: We have recently showed that hyperinsulinemia promotes hepatic free cholesterol (FC) accumulation in obese, insulin-resistant Alms1 mutant (foz/foz) mice with NASH. Here we tested whether cholesterol-lowering drugs reduce stress-activated c-Jun N-terminal kinase (JNK) activation, hepatocyte injury/apoptosis, inflammation, and fibrosis in this metabolic syndrome NASH model., Methods: Female foz/foz and WT mice were fed HF (0.2% cholesterol) 16 weeks, before adding ezetimibe (5 mg/kg), atorvastatin (20 mg/kg), or both to diet, another 8 weeks. Hepatic lipidomic analysis, ALT, liver histology, Sirius Red morphometry, hepatic mRNA and protein expression and immunohistochemistry (IHC) for apoptosis (M30), macrophages (F4/80), and polymorphs (myeloperoxidase) were determined., Results: In mice with NASH, ezetimibe/atorvastatin combination normalized hepatic FC but did not alter saturated free fatty acids (FFA) and had minimal effects on other lipids; ezetimibe and atorvastatin had similar but less profound effects. Pharmacological lowering of FC abolished JNK activation, improved serum ALT, apoptosis, liver inflammation/NAFLD activity score, designation as "NASH", macrophage chemotactic protein-1 expression, reduced macrophage and polymorph populations, and liver fibrosis., Conclusions: Cholesterol lowering with ezetimibe/atorvastatin combination reverses hepatic FC but not saturated FFA accumulation. This dampens JNK activation, ALT release, hepatocyte apoptosis, and inflammatory recruitment, with reversal of steatohepatitis pathology and liver fibrosis. Ezetimibe/statin combination is a potent, mechanism-based treatment that could reverse NASH and liver fibrosis., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Alms1 mutant (foz/foz) mice develop hyperphagic obesity, diabetes, metabolic syndrome, and fatty liver (steatosis). High-fat (HF) feeding converts pathology from bland steatosis to nonalcoholic steatohepatitis (NASH) with fibrosis, which leads to cirrhosis in humans., Objective: We sought to establish how dietary composition contributes to NASH pathogenesis., Design and Methods: foz/foz mice were fed HF diet or chow 24 weeks, or switched HF to chow after 12 weeks. Serum ALT, NAFLD activity score (NAS), fibrosis severity, neutrophil, macrophage and apoptosis immunohistochemistry, uncoupling protein (UCP)2, ATP, NF-κB activation/expression of chemokines/adhesion molecules/fibrogenic pathways were determined., Result: HF intake upregulated liver fatty acid and cholesterol transporter, CD36. Dietary switch expanded adipose tissue and decreased hepatomegaly by lowering triglyceride, cholesterol ester, free cholesterol and diacylglyceride content of liver. There was no change in lipogenesis or fatty acid oxidation pathways; instead, CD36 was suppressed. These diet-induced changes in hepatic lipids improved NAS, reduced neutrophil infiltration, normalized UCP2 and increased ATP; this facilitated apoptosis with a change in macrophage phenotype favoring M2 cells. Dietary switch also abrogated NF-κB activation and chemokine/adhesion molecule expression, and arrested fibrosis by dampening stellate cell activation., Conclusion: Reversion to a physiological dietary composition after HF feeding in foz/foz mice alters body weight distribution but not obesity. This attenuates NASH severity and fibrotic progression by suppressing NF-κB activation and reducing neutrophil and macrophage activation. However, adipose inflammation persists and is associated with continuing apoptosis in the residual fatty liver disease. Taken together, these findings indicate that other measures, such as weight reduction, may be required to fully reverse obesity-related NASH., (Copyright © 2013 The Obesity Society.)

We sought to determine whether hepatic cholesterol crystals are present in patients or mice with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NASH), and whether their presence or distribution correlates with the presence of NASH as compared with simple steatosis. We identified, by filipin staining, free cholesterol within hepatocyte lipid droplets in patients with NASH and in C57BL/6J mice that developed NASH following a high-fat high-cholesterol diet. Under polarized light these lipid droplets exhibited strong birefringence suggesting that some of the cholesterol was present in the form of crystals. Activated Kupffer cells aggregated around dead hepatocytes that included strongly birefringent cholesterol crystals, forming "crown-like structures" similar to those recently described in inflamed visceral adipose tissue. These Kupffer cells appeared to process the lipid of dead hepatocytes turning it into activated lipid-laden "foam cells" with numerous small cholesterol-containing droplets. In contrast, hepatocyte lipid droplets in patients and mice with simple steatosis did not exhibit cholesterol crystals and their Kupffer cells did not form crown-like structures or transform into foam cells. Our results suggest that cholesterol crystallization within hepatocyte lipid droplets and aggregation and activation of Kupffer cells in crown-like structures around such droplets represent an important, novel mechanism for progression of simple steatosis to NASH.

Unlabelled: The majority of patients with nonalcoholic fatty liver disease (NAFLD) have "simple steatosis," which is defined by hepatic steatosis in the absence of substantial inflammation or fibrosis and is considered to be benign. However, 10%-30% of patients with NAFLD progress to fibrosing nonalcoholic steatohepatitis (NASH), which is characterized by varying degrees of hepatic inflammation and fibrosis, in addition to hepatic steatosis, and can lead to cirrhosis. The cause(s) of progression to fibrosing steatohepatitis are unclear. We aimed to test the relative contributions of dietary fat and dietary cholesterol and their interaction on the development of NASH. We assigned C57BL/6J mice to four diets for 30 weeks: control (4% fat and 0% cholesterol); high cholesterol (HC; 4% fat and 1% cholesterol); high fat (HF; 15% fat and 0% cholesterol); and high fat, high cholesterol (HFHC; 15% fat and 1% cholesterol). The HF and HC diets led to increased hepatic fat deposition with little inflammation and no fibrosis (i.e., simple hepatic steatosis). However, the HFHC diet led to significantly more profound hepatic steatosis, substantial inflammation, and perisinusoidal fibrosis (i.e., steatohepatitis), associated with adipose tissue inflammation and a reduction in plasma adiponectin levels. In addition, the HFHC diet led to other features of human NASH, including hypercholesterolemia and obesity. Hepatic and metabolic effects induced by dietary fat and cholesterol together were more than twice as great as the sum of the separate effects of each dietary component alone, demonstrating significant positive interaction., Conclusion: Dietary fat and dietary cholesterol interact synergistically to induce the metabolic and hepatic features of NASH, whereas neither factor alone is sufficient to cause NASH in mice., (Copyright © 2012 American Association for the Study of Liver Diseases.)

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,N(6)-etheno-2'-deoxyadenosine, 3,N(4)-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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Bile acids are implicated as aetiological factors in many types of gastrointestinal tract cancer including cholangiocarcinoma (CCA). Alterations in bile acid concentrations may affect the pathogenesis of these different types of cancer. Our aim was to determine the bile acid profile in gallbladder bile from patients who underwent liver resection. Thirty-seven patients with cholangiocarcinoma, 5 with hepatocellular carcinoma, and 7 with benign biliary diseases were studied. High pressure liquid chromatography was used to analyze conjugated and unconjugated bile acids. CCA patients with low (≤ 2 mg/dl) and high (>2 mg/dl) levels of total serum bilirubin had significantly higher total bile acid and conjugated bile acid concentrations than the benign biliary disease group. Markedly elevated levels of cholic and chenodeoxycholic acid were found in CCA cases with high levels of total serum bilirubin. Concentrations of total bile acids and primary bile acid were correlated with serum cholesterol, bilirubin and ALP in CCA. Notably, correlation of the carcinoembryonic antigen, a tumor marker, was found with level of total bile acids and chenodeoxycholic acid. These findings suggest a different pattern of bile acid concentration in cancer patients compared to patients with benign biliary diseases. Thus, accumulation of certain bile acids may be involved in carcinogenesis.

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., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.

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.

We determined whether extrahepatic biliary epithelial cells can differentiate into cells with phenotypic features of hepatocytes. Gallbladders were removed from transgenic mice expressing hepatocyte-specific beta-galactosidase (beta-Gal) and cultured under standard conditions and under experimental conditions designed to induce differentiation into a hepatocyte-like phenotype. Gallbladder epithelial cells (GBEC) cultured under standard conditions exhibited no beta-Gal activity. beta-Gal expression was prominent in 50% of cells cultured under experimental conditions. Similar morphological changes were observed in GBEC from green fluorescent protein transgenic mice cultured under experimental conditions. These cells showed higher levels of mRNA for genes expressed in hepatocytes, but not in GBEC, including aldolase B, albumin, hepatocyte nuclear factor-4alpha, aldehyde dehydrogenase 1, and glutamine synthetase, and they synthesized bile acids. Additional functional evidence of a hepatocyte-like phenotype included LDL uptake and enhanced benzodiazepine metabolism. Connexin-32 expression was evident in murine hepatocytes and in cells cultured under experimental conditions, but not in cells cultured under standard conditions. Notch 1, 2, and 3 and Notch ligand Jagged 1 mRNAs were downregulated in these cells compared with cells cultured under standard conditions. CD34, alpha-fetoprotein, and Sca-1 mRNA were not expressed in cells cultured under standard conditions, suggesting that the hepatocyte-like cells did not arise from hematopoietic stem cells or oval cells. These results point to future avenues for investigation into the potential use of GBEC in the treatment of liver disease.

Oxysterols are naturally occurring intermediates in the conversion of cholesterol to bile acids, the major route for elimination of cholesterol. Additionally, they are important signaling agents, particularly in control of cholesterol synthesis; however, some species also are cytotoxic and carcinogenic. Oxysterols in plasma, contained in oxidized low-density lipoprotein, are strongly correlated with atherosclerosis. Oxysterols are found in infected human bile and the oxysterol content in gallstones correlates with bacterial DNA in the stones. Here we demonstrate that human leukocytes, activated by the presence of bacterial lipopolysaccharide, are able to oxidize cholesterol to a variety of oxysterols, including species known to be carcinogenic.

Background: We hypothesized that ileal stem cell clusters transplanted into a segment of jejunum can be used to treat bile acid malabsorption., Study Design: In adult Lewis rats, a 15-cm segment of jejunum was isolated with its blood circulation left intact and partially stripped of enterocytes using luminal high-velocity perfusions with 3mmol/L ethylenediamine tetra-acetic acid solutions. Continuity was restored by anastomosing the proximal and distal gut. Ileal stem cell clusters were harvested from neonatal Lewis rats and transplanted into the stripped segments to generate a "neoileum." After 4weeks, recipients underwent resection of the native ileum, and the isolated neoileum was anastomosed in its place. After an additional 4weeks, a 48-hour stool collection was performed. The engrafted segment was harvested for taurocholate uptake studies, ileal bile acid transporter (IBAT) protein by immunohistomorphometry, and IBAT mRNA quantitation by reverse transcription polymerease chain reaction. Data were analyzed by ANOVA/t-test. Rats undergoing ileectomy, jejunectomy, or sham operations served as controls., Results: Total bile acid loss in the stool was markedly lower in rats with a neoileum compared with rats with an ileectomy (p < 0.001). Total taurocholate uptake was notably increased in the neoileum compared with the jejunum (p < 0.001). IBAT protein signal intensity was considerably higher in the neoileum compared with jejunum (p < 0.001). IBAT mRNA amounts in the neoileal group were comparable with those in normal rat ileum and were considerably higher (p = 0.003) than in the jejunum., Conclusions: Ileal stem cell clusters were used to establish a new zone of bile acid uptake and IBAT expression in a jejunal segment. This neoileum eliminated loss of bile acids in the stool after ileectomy. This is the first time that transplantation of intestinal stem cell clusters has been shown to correct a clinical malabsorption syndrome.

Objectives: 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., Methods: 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., Results: 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, p < 0.05, and 22.0 +/- 25.0 x 10(-3)% of cholesterol vs 1.6 +/- 1.2 x 10(-3)% of cholesterol, p < 0.05, 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.

Objectives: 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., Methods: 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., Results: 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%., Conclusions: 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.

We report problems encountered during preparation of tritium-labeled unconjugated bilirubin ((3)H-UCB) from precursor (3)H-5-aminolevulinic acid ((3)H-ALA) in 2 dogs with external biliary drainage installed into the animals under general anesthesia. Under prolonged sedation, 12.9 or 14.0 mCi of (3)H-ALA was administered intravenously in two divided doses, and bile was collected for 9 hours. In one animal, taurocholate (TC) infusion was needed to maintain bile flow. (3)H-UCB was isolated from the bile and recrystallized with the improved method of Webster et al (Webster CC, Tiribelli C, Ostrow JD. J Lab Clin Med 2001;137:370-3). Based on radioactivity and pigment content, hourly bile collections were pooled to optimize specific activities. Surprisingly, in the first dog, only 2.9% of injected radioactivity was recovered in bile and only 14.1% in urine, and the specific activities of the crystalline (3)H-UCB from the two pools were only 39.5 and 30.0 x 10(3) dpm/microg. High-performance liquid chromatography analysis revealed that only 4% of ALA degraded during 5 minutes in injection solution at pH 6.8. The low incorporation of (3)H-ALA and low specific activity of (3)H-UCB was apparently caused mainly by prior degradation and exchange of labile tritium of the (3)H-ALA and probably by enhanced endogenous ALA synthesis caused by the anesthetic/sedative agents. Revised procedures in the second dog improved the incorporation of (3)H-ALA to 11.9% excreted in bile and the specific activity of the crystalline (3)H-UCB to 122.0 and 50.8 x 10(3) dpm/microg, while urinary excretion of tritium increased to 28.5%. These experiences emphasize possible pitfalls in preparing (3)H-UCB by biosynthetic labeling from (3)H-ALA administered to dogs.

Background and 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.

Objective: Numerous investigators have proposed a role for bacteria in biliary lithogenesis. We hypothesized that bacterial DNA is present in gallstones, and that categorical differences exist between gallstone type and the frequency of bacterial sequences., Methods: Polymerase chain reaction (PCR) was used to amplify bacterial 16S rRNA and uidA (encoding Escherichia coli [E. coli] beta-glucuronidase) genes in different types of gallstones. PCR products were sequenced., Results: Bacterial 16S rRNA and uidA DNA sequences in E. coli were detected in all brown pigment, common bile duct, and mixed cholesterol gallstones (n = 14). In contrast, only one (14%) of seven pure cholesterol gallstones yielded a PCR product. Most (88%) mixed cholesterol gallstones yielded PCR amplification products from their central, as well as their outer, portions. Sequenced products possessed 88-98% identity to 16S rRNA genes of E. coli and Pseudomonas species., Conclusions: Bacterial DNA sequences are usually present in mixed cholesterol (to 95% cholesterol content), brown pigment, and common bile duct, but rarely in pure cholesterol gallstones. The presence of bacterial beta-glucuronidase is also suggested. The role of bacteria and their products in the formation of mixed cholesterol gallstones, which comprise the majority of cholesterol gallstones, warrants further study.

There is experimental and epidemiological evidence that antioxidant vitamins can inhibit carcinogenesis. Since immortalization by Human Papilloma Virus (HPV) is one possible early step towards carcinogenesis in oral epithelia, we studied the differential effect of vitamins A, C and E on HPV-immortalized oral epithelial cells (IHGK) as compared to the normal counterpart. The dose response was determined by morphology, cell cycle by flow cytometry, and growth curve by cell number. The optimum dose in terms of inhibitory effect vs. toxicity was determined for each vitamin by morphology. Optimum doses were: vitamin A--1.4 x 10(-5) M, vitamin C--10(-3) M, and vitamin E--10(-6) M for both HPV-immortalized and normal cells. Growth curve showed reduction of proliferation by all three vitamins, with vitamins A and E more effective than C for both cell types. Flow cytometry showed that vitamins A and E reduced the percentage of cells at G2 phase of cell cycle and indicated arrest in the S phase. This effect was greatest in the immortalized cells with a 50% and 35% decrease of G2 for vitamins A and E respectively, whereas the normal counterpart showed a 48% decrease for A and a 12% increase for E. By organotypic culture, the morphology was not markedly different between the vitamin-treated and the control cells, except for a slight increase in the keratinization of normal cells with vitamin A. Also noted was a reduction in number of cell layers from five layers or more for controls to only one or two for vitamin E. In conclusion, we have demonstrated that the antioxidant vitamins inhibit proliferation, and show a preferential effect on IHGK cells.

We used confluent cultures of dog gallbladder epithelial cells, stimulated by conditioned medium from a culture of human neonatal foreskin fibroblasts, to establish the presence of inducible nitric oxide synthase (NOS, EC 1.14.13.39). Assay was by conversion of radiolabeled arginine to citrulline. By 4 days after addition of the conditioned medium, a relatively high level of activity was observed. However, further study showed that the enzyme did not require addition of the usual cofactors for maximal activity (NADPH, FAD, FMN and tetrahydrobiopterin) and was stable in the absence of anti-proteolytic agents. Our suspicion that this enzyme might not be NOS but arginine deiminase (EC 3.5.3.6) was confirmed by enzyme purification and by the liberation of ammonia during enzyme reaction. This enzyme, which is absent from primates and virtually confined to single-cell organisms, suggested the presence of Mycoplasma, a common contaminant of cell cultures, and it was subsequently confirmed that the fibroblast culture was a source of Mycoplasma. With the widespread interest in nitric oxide and NOS, and common use of the convenient [3H]arginine assay, there is a considerable danger of the two enzymes being confused. At the very least, it is necessary to check for activity in the absence of added cofactors.

Biliary lipid composition and bile flow are altered after orthotopic liver transplantation. Cyclosporine may have additional effects on biliary lipid composition and secretion. We studied the effects of liver transplantation, allograft function, and cyclosporine on biliary lipids in humans. Changes in lipid composition and secretion were correlated with serum cyclosporine levels, clinical events, and allograft function. Bile samples were withdrawn via a T-tube at interval time points in 17 patients during the first 3 months posttransplantation. Total and individual bile acid, cholesterol, and phospholipid were determined using high-performance liquid chromatography. Biliary lipid profiles were then correlated with clinical events, serum cyclosporine levels, and other clinical laboratory values. Biliary lipid concentrations decreased in 3 patients during periods of graft dysfunction (acute cellular rejection, drug-induced hepatitis, and inferior vena caval thrombosis) and increased with resolution of the graft injury. Serum cyclosporine levels were positively correlated with total bile acid, cholesterol, and phospholipid concentrations in bile. There was no relationship between the composition of secreted bile acids and serum cyclosporine levels. Bile acid, cholesterol, and phospholipid secretion were not uncoupled in the presence of cyclosporine. We concluded that (1) a decrease in biliary lipid concentrations may be an indicator of worsened graft function in some allografts; (2) biliary lipid concentrations are correlated with increasing cyclosporine levels; and (3) bile acid composition is unchanged, and uncoupling of secretion of other biliary lipids is not observed in the presence of cyclosporine., (Copyright 1998 W.B. Saunders Company.)

Recent advancements in liver transplantation have resulted in extended survival both for grafts and recipients. Such improvement, together with the shortage of donor organs has prompted expansion of the donor pool to include less than ideal donors, especially in life-threatening situations. The use of older liver donors has been associated with lower long-term survival. However, potential morbidity such as gallstone formation has not been explored. We analyzed bile composition in a child who developed cholesterol gallstones in the proximal bile duct two years after undergoing emergency liver transplantation with a liver from a 78-year-old donor. Oral administration of ursodeoxycholic acid (ursodiol) shifted the cholesterol composition of the bile from a supersaturated, potentially crystallized state to a liquid (micellar) state. Unlike cyclosporin A, FK506 showed an increase in the proportion of chenodeoxycholic acid and a decrease in the proportion of cholic acid, and thus may exhibit minimal or no hepatotoxic effect. Thus, in donor livers with factors known to be associated with cholesterol gallstone formation (such as age, sex, or obesity), one may consider analyzing the bile composition at the time of procurement. Depending on cholesterol and bile acid composition the use of FK506 with or without addition of ursodeoxycholic acid may be warranted.

Background and Aim: The recent introduction of new measurement technology (using ion specific electrodes) makes intraoperative evaluation of blood ionized magnesium (Mg2+, or iMg)--the bioactive fraction of circulation magnesium--possible. The goals of this study were: (1) to examine the longitudinal pattern(s) of change in blood iMg during cardiopulmonary bypass (CPB); and (2) to determine the relationship of iMg to Ca2+ (iCa), K, pH, Na, and hematocrit (Hct) during CPB., Methods: Blood was collected serially before, during, and after CPB on 30 patients undergoing elective coronary artery bypass graft procedures and the iMg was measured with an AVL Scientific Corp., model 988-4 instrument., Results: Overall, 73% of iMg results were abnormally low, 50% during CPB. Some cases had both hypo- and hyperionized magnesemic episodes. There were low iCa during CPB in 97% of cases. Using Spearman's rank order correlations and p < 0.05, iMg and K were directly correlated before, during, and after bypass, suggesting their parallel movement between tissue and blood. iMg and iCa were directly correlated before, and inversely correlated after, CPB, but unassociated during bypass. iMg and Na were inversely correlated after bypass in all cases. iMg was inversely correlated to pH and positively correlated to Hct during CPB only, and only in patients with concurrent association of iMg and iCa., Conclusions: Blood iMg depletion occurs frequently in CPB patients. iMg changes are not readily predictable. The association of intraoperative iMg depletion with postsurgical atrial fibrillation--reported to have a hypomagnesemic connection- should be investigated.

Recent evidence suggests that loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene plays a role in colorectal tumorigenesis and other cancers. However, little is known as to whether the APC gene contributes to the pathogenesis of oral squamous cell carcinoma. To assess involvement of both the APC gene and the human papillomavirus (HPV) in the development of oral pre-malignant and malignant lesions, we analysed DNA from 14 paired oral normal and pre-malignant or malignant paraffin-embedded biopsy specimens, and DNA from cultured normal and HPV 16-immortalised oral epithelial cells for the presence of LOH of APC and for HPV infection, using PCR based techniques. LOH of APC occurred in 80% of cases of oral epithelial dysplasia, 67% of carcinoma in situ, 50% of invasive squamous cell carcinoma cases, and in the HPV 16-immortalised oral epithelial cells. HPV was detected in half of the biopsy specimens, with HPV 16 as the dominant type. More than half of the carcinoma cases were found to contain both LOH of APC and HPV infection. These results suggest that LOH of APC is an early event during oral tumorigenesis. Our findings also suggest a strong correlation between HPV infection, particularly HPV 16, and LOH of the APC gene in oral squamous cell carcinomas.

Cyclosporine, an immunosuppressive agent widely used in organ transplantation, has several undesirable side effects, including gingival hyperplasia, which occurs in up to 70% of patients. Another complication associated with use of cyclosporine and other immunosuppressants is an increased incidence of malignancies. Long-term use of cyclosporine also is associated with a spectrum of hyperproliferative disorders ranging from reactive lymphoid hyperplasia to aggressive malignant lymphomas. While cyclosporine-related lymphoproliferative disorders have been widely reported, they have not been described in the oral cavity as the first manifestation of this disease. We report on two cardiac transplantation patients with a history of cyclosporine use who presented initially with oral symptoms of lymphoproliferative disorder. Both had erythematous to cyanotic and hyperplastic gingiva. On gingivectomy, the fixed tissue was soft, glistening, and tan colored, in contrast to the usual firm, white, cyclosporine-associated, benign gingival fibrous hyperplasia. Histologically, a dense, diffuse infiltrate of lymphoplasmacytoid cells with vesicular nuclei, prominent nucleoli, a moderate amount of cytoplasm, and high mitotic activity was observed. Immunocytochemical studies confirmed that the cells were monoclonal for lambda light chains in one patient and kappa light chains in the other. The cells from one patient were positive for CD45, while both patients were negative for CD20 and all nonhematopoietic antigens tested. Both tissues were strongly positive for Epstein-Barr virus. Morphology and immunocytochemistry findings are consistent with a posttransplant lymphoproliferative disorder. These are the first two reported cases of cyclosporine-associated posttransplant lymphoproliferative disorders presenting as gingival hyperplasia.

The marine alga Chlorella minutissima contains DGTS (diacylglyceryl-N,N,N-trimethylhomoserine) as a major component (up to 44% of total lipids). This lipid is absent from other members of the Chlorococcales, except for C. fusca, which contains DGTS as 1.3% of total lipids. Contrary to expectation, the DGTS is accompanied by PC (phosphatidylcholine) as the major phospholipid. DGTS is normally highly saturated in the C-1 position of glycerol, but in C. minutissima, both C-1 and C-2 are acylated with EPA (eicosapentaenoic acid, 20:5) in the major molecular species (over 90% of total). The DGTS level shows a marked rhythmic fluctuation with time which is inversely correlated with the level of MGDG (monogalactosyldiacylglycerol), the other major lipid. Improved NMR data and the first electrospray MS data on this lipid are presented.

We have modified methods of growing human gallbladder epithelial cells in monolayer and organotypic culture. These cells were grown in the presence of fetal bovine serum and with coculture of feeder layers of human gallbladder fibroblasts. Human gallbladders were obtained from cholecystectomy specimens, and the cells were dissociated with trypsin/EDTA. Cells, which were grown with feeder layer on collagen-coated plates in the presence of 10% FBS, grew rapidly and formed islands of cuboidal cells with morphology typical of epithelial cells in culture. They could be passaged up to four times. The cells were also successfully grown by organotypic technique producing a monolayer of tall, columnar, palisade, epithelial cells. These cells, both in monolayer and in organotypic culture, were positive to antibodies for simple epithelial keratin and negative to antibody for vimentin or any of the mesenchymal antibodies. These cells respond to agonists (prostaglandin E2, isoproterenol) by the intracellular generation of cAMP. Secreted mucin on the apical surface stained strongly with periodic acid-Schiff. Organotypic culture of human gallbladder epithelium may serve as a cell preparation for the study of pathobiology of columnar epithelial cells.

The column method of Christner and Fetter (Steroids 24: 327, 1974) has been modified to give a simple, rapid assay for unconjugated estriol in serum. Estriol is isolated from serum by being retained on a Sephadex column, while estriol conjugates and serum proteins are eluted. The sample and labeled estriol compete for antibody on the column. Antibody is eluted, removing proportional amounts of sample and labeled estriol. Our modifications include using stable serum-based standards, shortening the incubation to 10 min, complete removal of conjugates, and regeneration of the columns so that they can be used repeatedly. The assay, which can be completed in 2 h, has a mid-range interassay CV of 8.2%.

Time studies of crepenynic acid synthesis inCrepis rubra show that this acid is not present in the seed for several days after, flowering commences but builds up rapidly between the 14th and 28th days to become the major fatty acid of the seed oil.Radioactive tracer studies clearly demonstrate that the acetylenic bond is introduced into the carbon chain of a preformed long-chain fatty acid rather than built in during formation of the carbon chain. The nearest precursor found is oleic acid. There is no conversion to crepenynic acid by seed preparations ofcis,cis-linoleic acid,cis,trans (trans,cis)-linoleic acid, orcis-12,13-epoxy-oleic acid. Possible biosynthetic pathways to explain these results are suggested.The crepenynic acid is chiefly, but not entirely, in triglycerides in the seed oil, and it has been shown to be esterified in the 2- and 3- positions of the triglyceride.

1. The bacterium Acetobacter xylinum produces extracellular cellulose microfibrils that form a pellicle in the medium enmeshing the bacterial cells. These microfibrils may show some localized alignment, which can be seen as birefringence when the culture is viewed between crossed Polaroid sheets. 2. An increase in birefringence can be induced by the addition of small amounts of certain classes of lipids, particularly sterols, to the cultures. 3. A crude lipid extract from Acetobacter cells induced greatly increased birefringence when added to fresh cultures of this organism. 4. When the bacterial lipids were fractionated, most of the activity was recovered in a complex, polar lipid. The lipid is secreted into the medium during growth and is unstable. The non-saponifiable portion of this lipid is shown to be a 1:1 mixture of a saturated and a monounsaturated C(35) tetrahydroxy terpene with a hopane ring system in the accompanying paper by Förster et al. (1973). The saturated molecule is referred to as tetrahydroxybacteriohopane. 5. Tetrahydroxybacteriohopane is itself capable of inducing birefringence in cultures as is 22-hydroxyhopane, which was also isolated from the non-saponifiable fraction of the total lipids. 6. The mechanism of induction of birefringence (orientation of microfibrils) is not known. This is unlikely to be a specific effect, since all the above compounds are active (intact lipid, tetrahydroxybacteriohopane, 22-hydroxyhopane), as are other classes of lipid. It is suggested, however, that a common mechanism may be involved and that similar compounds may be concerned with control of microfibril alignment in the cells of higher plants.

A novel C(35) terpene and its monounsaturated analogue were isolated from cultures of Acetobacter xylinum, together with traces of their C(36) homologues. These substances were found to be hopane derivatives substituted by a five-carbon chain bearing four vicinal hydroxyl groups. For the parent hydrocarbon the term bacteriohopane is proposed. The elucidation of the structures utilized high-resolution mass spectrometry of the terpenes, degradation to C(32) hydrocarbons and detailed mass-spectrometric comparison of these with C(32) hydrocarbons synthesized from known pentacyclic triterpenes. High-resolution mass-spectral data of the terpenes are presented. N.m.r. data are in agreement with the proposed structures, which are further supported by the isolation from the same organism of 22-hydroxyhopane and derivative hopene(s).