Relationship of C282Y HFE Mutations, Hepatic Iron Deposition and Histologic Features in Patients With Nonalcoholic Fatty Liver Disease

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feature of cholestasis is increased serum bile acids (BAs), which normally have detergent effects in the intestine to aid in the break down of dietary fats. Our aims were to 1) assess the integrity of the BBB in a rodent model of biliary obstruction 2) evaluate the effects of BAs in BBB permeability In Vitro, and 3) determine the effects of biliary obstruction and BAs on the tight junctions of the BBB In Vivo and In Vitro. Methods: Rats underwent bile duct ligation (BDL) for 7 days. BBB integrity was assessed by trypan blue exclusion and albumin IHC and total serum BAs were assessed by enzymatic assays. The permeability of a confluent monolayer of rat brain microvessel endothelial cells (RBMEC) to fluorescein dextrin and the transepithelial electrical resistance (TEER) was assessed after treatment with 50% serum from control and BDL rats and after treatment with various BAs (10-100 μM). RBMEC cell viability was assessed by MTS assays after BA treatment (10-100 μM). The expression of Claudin 5 an integral tight junction protein was assessed in rat brain microvessels isolated from rats after BDL surgery and in a confluent RBMEC monolayer treated with 50% serum from control and BDL rats and after treatment with various BAs (10-100 μM). Results: Consistent with previous results, serum BAs increased after BDL which was paralleled by increased BBB permeability. There was a decrease in TEER and increased dextrin permeability in RBMEC monolayers after treatment with serum from BDL rats compared to control serum and also after treatment with cholic acid (CA), deoxycholate (DCA) and chenodeoxycholate (CDCA), but not after ursodeoxycholate (UDCA). The effects of BDL serum and of the BAs on permeability could not be attributed to decreases in RBMEC cell viability. However, there was a decrease in Claudin 5 expression and location in the tight junctions in brain microvessels isolated from BDL animals and in the RBMEC monolayers treated with BDL serum or CA, DCA and CDCA. Conclusion: Our data suggests that the increase in circulating serum BAs may be contributing to the increased permeability of the BBB seen during obstructive cholestasis. This is possibly via a mechanism involving decreased Claudin 5 expression which compromises the integrity of the tight junctions between the brain microvessel endothelial cells.

AASLD Abstracts

Histidine Decarboxylase Supports the Tumor Microenvironment in Cholangiocarcinogenesis by Increased Cholangiocyte VEGF and Mast Cell Mediator Expression Yuyan Han, Taylor Francis, Fanyin Meng, Dustin Staloch, Heather Francis Cholangiocarcinoma (CCA) is a deadly biliary cancer. Histamine (HA) and the enzyme responsible for HA synthesis, histidine decarboxylase (HDC) regulate CCA growth. Mast cells (MC) support the tumor microenvironment by increasing VEGF expression and inducing MC mediator (tryptase and chymase) release. This is regulated by HDC. We have shown that (i) HDC is overexpressed in CCA and (ii) chronic HA treatment increases CCA growth via increased VEGF expression. We propose the novel hypothesis that CCA express MC mediators and that HDC inhibition alter the tumor microenvironment thus decreasing tumor growth. To evaluate the presence of mast cell mediators, we measured tryptase and chymase expression in (i) normal and CCA cells by immunoblotting and immunofluorescence; and (ii) human tumor CCA biopsy arrays by immunohistochemistry. In Vitro studies: Mz-ChA1 cells were stably transfected with an HDC shRNA (Mz-HDC) or empty vector (Mz-neg). Cells were evaluated for (i) HDC expression (by qPCR and immunoblots); (ii) proliferation by PCNA immunoblots; and (iii) VEGF-A/C, VEGFR-2/3 and chymase and tryptase by qPCR. In In Vivo studies: 5 x 106 of our genetically modified (Mz-neg and Mz-HDC) cells were suspended in extracellular matrix gel (250 μl) and injected s.c. in the hind flanks of nu/nu mice. After development, tumor volumes were recorded for 42 days. Tumors were extracted and prepared for immunoblots and qPCR. Paraffin embedded tumor tissues were used for immunofluorescent imaging of chymase and tryptase, co-stained with the cholangiocyte marker, CK-19. CCA cell lines express increased levels of tryptase and chymase compared to normal and immunoreactivity for the MC proteases was increased in human CCA samples compared to nonmalignant. In Vitro, shRNA transfection reduced HDC gene (~50%) and protein (100%) expression compared to Mz-neg. Loss of HDC induced a decrease in (i) growth; (ii) gene expression of VEGF-A/C and VEGFR-2/3; and (iii) gene and protein expression of chymase and tryptase compared to Mz-neg. In Vivo, tumor growth in MzHDC was significantly less aggressive compared to Mz-neg implanted tumors. In tumors from Mz-HDC we found a decrease in (i) proliferation; (ii) VEGF-A/C and VEGFR-2/3 gene expression; and (iii) chymase and tryptase gene and protein expression compared to Mzneg. In tumor sections we found a co-localization of CK-19 and chymase and tryptase expression that was decreased in Mz-HDC compared to Mz-neg. CCA express MC proteases. Inhibition of HDC decreases the proliferative capacity of CCA tumor growth both In Vitro and In Vivo by altering components of the tumor microenvironment. This is coupled with HDC-dependent decreased angiogenic potential and reduced MC proteases. This data supports the potential role of an autocrine pathway in CCA that is able to alter tumor microenvironment mediators and decrease tumor growth.

Su1661 Carbon Monoxide Interacts With Nitric Oxide to Induce Choleresis by Increasing Biliary Secretions of Glutathione Disulfide and Bicarbonate Chiung-Yu Chen, Chi-Ya Kao, Shu-Chu Shiesh Background: The effects of carbon monoxide (CO) on bile formation are inconsistent among previous studies. Nitric oxide (NO) stimulated glutathione disulfide (GSSG) excretion and bile flow in liver-perfused rat models. As CO and NO gas molecules interact with each other in many aspects, we aimed to investigate the In Vivo effect of CO on bile excretion and whether its effect is through the interactions of NO. Methods: Male Sprague-Dawley rats, weighing 250-275 g, were used. Anesthesia was induced by intra-peritoneal injection of zoletil 50 and maintained with supplemental intra-peritoneal bolus injections of ketamine for 4 hours. Dichloromethane (DCM) was administered via gastric feeding to yield CO. LNAME was injected intra-peritoneally to inhibit the activity of nitric oxide synthase (NOS). The rats were divided into DCM in corn oil treated- (n = 7), DCM plus L-NAME treated(n = 6), and corn oil treated-group (n = 8). The levels of carboxyhemoglobin (COHb) in arterial blood were monitored throughout the experiment. Measurements of serum levels of nitrite and nitrate (NOx) were performed by capillary electrophoresis, and biliary glutathione and GSSG by liquid chromatography-tandem mass spectrometry. The hepatic expressions of inducible NOS and Mrp2 were examined by western blot. The bile output as well as biliary levels of bile salts, bilirubin, and bicarbonate were measured. The fold changes of these analytes from the basal levels were compared between groups by using MannWhitney U test. Results: Levels of COHb began to rise at 2-h and up to 10% at 4-h after DCM supplement. The increased COHb was not influenced by the L-NAME treatment. The DCM group had a significantly higher hepatic expression of iNOS and serum level of NOx, compared to control group (224.0±17.1 μmol/l vs. 165.6±19.8 μmol/l, p<0.05). Bile flow increased starting at 2-h after DCM supplement (cumulated fold change at 4-h in DCM vs. control: 4.43±0.2 vs. 3.29±0.13, p<0.005). L-NAME treatment restored the bile output to its basal level. The increase of bile output was associated with an increase of biliary bicarbonate (cumulated fold change at 4-h in DCM vs. control: 3.88±0.17 vs. 3.18±0.12, p<0.01) and GSSG (cumulated fold change at 4-h in DCM vs. control: 1.08±0.30 vs. 0.41±0.06, p<0.01). The hepatic Mrp2 expression was up-regulated by DCM supplement. The treatment of L-NAME abolished the CO-induced increment of glutathione and bicarbonate excretion. Conclusion: Our study showed that CO stimulates bile excretion by increasing GSSG and bicarbonate excretion. CO also stimulated NO release by inducing hepatic inducible NOS. L-NAME treatment abolished the choleretic effect of CO. As NO can enhance bile formation by increasing GSSG secretion of hepatocytes and bicarbonate secretion of cholangiocytes, the effect of CO on biliary excretion seems mainly from the action of NO.

Su1659 Differential Gene Expression of Sphingosine Kinases and Sphingosine-1 Phosphate Receptors in Cultured Neu-Transformed Versus SpontaneouslyTransformed Rat Cholangiocytes and Corresponding Cholangiocarcinomas Xiqiao Zhou, Ruihua Shi, Xuan Wang, Deanna J. Campbell, Elaine Studer, Phillip Hylemon, Alphonse E. Sirica, Huiping Zhou Sphingosine kinases (SPHKs) have been linked to the development and progression of various types of human cancers. Recent studies also reported that sphingosine-1 phosphate (S1P) receptor-mediated signaling pathways are involved in tumor progression. However, the roles of sphingosine kinases and S1P receptors in cholangiocarcinoma progression remain unknown. We have established a rat cholangiocarcinoma model based on bile duct inoculation of spontaneously-transformed low grade malignant rat BDE1 cholangiocytes (BDEspC) and high grade malignant erbB-1/neu-transformed BDE1 cholangiocytes (BDEneuC). We also established tumor derived cholangiocarcinoma cell lines (BDEspT and BDEneuT). This study characterized the expression of SPHK1, SPHK2, and S1P receptors in cholangiocarcinoma cell lines and examined the association between expression of SPHKs and S1P receptors and progression of cholangiocarcinoma. Methods: BDEspC, BDEspT, BDEneuC and BDEneuT cells were used in this study. The expression of SPHK1, SPHK2, S1P1, S1P2, S1P3, S1P4, and S1P5 was determined by RT-PCR using gene specific primers. The relative mRNA levels of SPHK1, SPHK2, S1P1, S1P2, and S1P5 were determined by real time RT-PCR. The activation of ERK and AKT signaling pathways was determined by Western blot analysis. Results: Both SPHK1 and SPHK2 were expressed in the cholangiocytes cell lines. The major S1P receptors expressed in cholangiocytes were S1P1, S1P2, and S1P5. Real-time RT-PCR indicated that, compared to those in BDEspC and BDEspT, SPHK1 mRNA was markedly upregulated by 5.6-and 5.7-fold and SPHK2 mRNA was increased by 1.8and 2.3-fold in BDEneuC and BDEneuT, respectively. In addition, S1P1 was significantly upregulated 4.7- and 11-fold, while S1P2 and S1P5 were increased about 2- to 3-fold in BDEneuC and BDEneuT. The upregulation of SPHKs and S1P receptors was correlated to the increase of ERK and AKT activation in BDEneuC and BDEneuT. Conclusions: These results indicate that the expression levels of SPHKs and S1P receptors, especially SPHK1 and S1P1, are associated with cholangiocarcinoma progression. SPHKs and S1P receptors might represent novel and valuable predictors of disease progression and adjuvant therapy for cholangiocarcinoma patients as well as potential targets for new drug development.

Su1852 Relationship of C282Y HFE Mutations, Hepatic Iron Deposition and Histologic Features in Patients With Nonalcoholic Fatty Liver Disease James E. Nelson, Matthew M. Yeh, Kris V. Kowdley Background: The relationship between HFE gene mutations, body iron stores and NASH pathophysiology remains controversial despite numerous studies. Most previous reports had several limitations including small sample size, lack of uniform criteria for the diagnosis of NASH and lack of a standardized liver histological scoring system for NASH and iron deposition. Aim: The goal of this study was to investigate the relationship between HFE mutations and the presence and pattern of hepatic iron deposition and histologic features of NASH in the large, well characterized NASH CRN cohort. Methods: SNP genotyping for the two common HFE mutations C282Y (rs1800562) and H63D (rs1799945) was performed in 787 adult (≥18 yrs) NASH CRN subjects (enrolled in the Database and PIVENS studies) with biopsy proven NAFLD and hepatic iron staining results. Real time genotyping assays were performed using 10 ng of genomic DNA and fluorescently labeled MGB-Eclipse probes (Nanogen Bioscience). Clinical, histologic, laboratory and anthropometric data were compared between different genotypes using Chi2, Fisher's exact, Wilcoxon rank sum and Kruskal-Wallis tests. Multivariate logistic regression was used to determine the independent association of each HFE genotype and histological variables. Results: The prevalence of HFE

Su1660 Increased Serum Bile Acids After Extraheptic Biliary Obstruction Causes Leakiness to the Blood Brain Barrier via the Disruption of Tight Junctions Matthew Quinn, Gabriel A. Frampton, Hae Yong Pae, Darijana Horvat, Li Huang, Sharon DeMorrow The brain is protected by a barrier of blood vessels and glia (blood brain barrier; BBB) that tightly controls the passage of molecules into the brain. Neurological changes have been observed during obstructive cholestasis, including leakiness of the BBB, and a suppressed HPA axis, however the mechanism by which these changes occur is unknown. A common

AASLD Abstracts

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mutations was as follows: C282Y/wt, n=89, 13.0%; H63D/wt, n=174, 22.3%; H63D/H63D, n=17, 2.2%; C282Y/H63D, n=13, 1.7%. The majority of patients with HFE mutations were Caucasian (90%) and 40% of all Caucasians had at least one HFE mutation. Patients with C282Y/wt or C282Y/H63D genotypes were more likely to have hepatocellular (HC) iron deposition (34% and 54%, respectively vs 21%, p≤0.007) and a higher grade of HC iron (mean grade 0.51 and 1.15, respectively vs 0.27, p≤0.016) compared to wt/wt genotypes. H63D/wt or H63D/H63D genotypes were not associated with either the presence or grade of HC iron. Subjects with C282Y mutations had lower serum ALT (p=0.035) and AST (p= 0.013) levels than wt/wt patients and were less likely to have a definitive diagnosis of NASH after adjustment for age, gender, BMI and presence of diabetes (OR 0.63, 95% CI 0.410.95, p=0.029). No differences in serum ferritin or iron or the presence of nonparenchymal iron deposition or advanced fibrosis was observed for HFE genotypes compared to wt/wt, except C282Y/H63D patients had a higher TS (p=0.003). Conclusions: Presence of C282Y but not H63D HFE mutations is associated with HC iron deposition in patients with NAFLD. HFE mutations are not associated with nonparenchymal iron deposition or advanced histologic features of NASH. However, C282Y mutations were independently associated with a lower risk of a definitive NASH diagnosis and also lower serum aminotransferase levels, suggesting that mild HC iron deposition may not contribute to NASH pathogenesis in US patients.

activity of heme oxygenase) in HMOX1 between WD and control groups were found (OR 0.53; 95% CI 0.16-1.175, p=0.30). Conclusions: Data from our study suggest that an increased oxidative stress contributes more importantly to the neurological manifestation of WD. However, (GT)n variations in HMOX1 does not seem to play any major role in pathogenesis of WD. Supported by grant IGA MZCR NT 11247 Su1855

Wilson disease (WD) is an autosomal recessive inherited disorder of hepatic copper metabolism. The clinical presentation of WD is highly variable. While some patients become symptomatic in childhood or early adulthood, others present much later, as late as in their 70-ies. Factors that influence clinical presentation are largely unknown. Methods: The time of onset of clinical symptom in relation to gender was analyzed in a large cohort of patients in an ongoing multinational study with clinical and genetic data of 1210 patients (1048 were index patients, 162 were siblings of index cases; mostly from Germany, Austria, Hungary, Poland, the Czech Republic and Slovakia, the Balkans and Benelux countries). Diagnosis of WD was based on the criteria of the International WD study group (Ferenci et al; Liver International 2003). For this study only index cases were evaluated. Disease presentation was defined based on the first initial symptoms as hepatic or neurologic. Results: Overall, as expected, overall gender distribution was equal (519 female/528 male patients), but there was a difference in the gender distribution according presentation. 325 of the 519 females (62.6%, 95% CI: 58.3-66.8%) but only 295/529 males (55.8%; 51.4-60.0; p< 0.05) presented with liver disease. This difference was mostly due to a higher frequency of fulminant WD in females (n=42, 8.1%; 5.9-10.9%) than in males (n=12, 2.3%; 1.2-4.0; p<0.05). Independent of gender more paediatric and young adult patients (≤ 20a) presented with liver disease (again being more common in females [221/294, 75.2%] than in males [208/320, 65.0%; p<0.05]) than with neurologic disease,. In patients presenting in adulthood (> 20a), neurologic disease was more common. Liver disease tended to be more frequent in females (95/ 213, 44.6% vs. 76/197, 38.8%; NS). The distribution of WD gene mutations was not different among male or female patients. Conclusion: Gender has a significant effect on the clinical presentation of WD. Females more commonly present with liver disease (including fulminant WD) than males; this effect is particularly true in children and young adults. The impact of sex hormones on WD should be further explored.

Su1853 Molecular Characterization of Copper Induced Carcinogenesis in Atp7b-/Mouse Liver Dominik Huster, Wiebke Schirrmeister Wilson disease (WD) is a severe genetic disorder associated with accumulation of copper in the liver and brain caused by ATP7B gene mutations. An excellent model for hepatic WD is the Atp7b-/-mouse. These mice show all pathologic manifestations described in WD patients most important chronic hepatitis. Further, Atp7b-/-mice develop liver tumors from about 40 weeks of age. Nevertheless, the copper induced molecular changes which lead to tumor development are poorly understood. In an attempt to characterize these tumors, livers from 60 weeks old Atp7b-/- and wild-type mice were extracted, micro-dissected into tumor and surrounding tissue, and gene expression was studied using microarrays (Affymetrix®). The microarray analysis was confirmed by RT-PCR and immunohistochemistry. First we compared gene expression profiles of liver tumors (Atp7b-/-mice) with liver tissue of wild-type mice. A total of 81 differentially expressed genes associated with tumorigenesis were identified. A pathway dramatically affected by copper accumulation and chronic inflammation was the canonical Wnt signaling pathway which plays an important role in tumor development. Significant upregulation of various genes of this pathway (e.g. Wnt5a (3.1fold up), SOX4 (3.3fold up) and SMAD4 (2.5fold up)) leads to a similar upregulation of Wnt target genes such as Cyclin D2 (2.3fold up) and D1 (1.7fold up), c-jun (4.4fold up) and MMP-7 (12fold up), each of which enhances proliferation and migration of cells. The anti-apoptotic regulators SPP-1 (7.2fold up), Bcl-2 (2.8fold up) and Bcl-6 (3.1fold up) were up-regulated significantly, leading to inhibition of apoptosis. Similar observations were made when liver tumors were compared with surrounding tissue of Atp7b-/-mice. Furthermore, we sought to determine, whether these tumors are to be classified as cholangiocarcinoma (CC) or hepatocellular carcinoma (HCC). From histological evaluation, tumor cells showed features of hepatocytes, but tumor architecture resembled cholangiocarcinoma. Therefore we investigated several tumor markers to characterize these tumors on the molecular level. Important markers such as C/EBP (2.4fold down), HNF-4 (2.2fold down), Glypican 1 (2.1fold up), CEACAM1 (3.3fold down) and CK-19 (7.9fold up) showed significant expression changes in tumors compared to wild-type livers. In conclusion we found that copper accumulation and chronic inflammation in Atp7b-/-mice result in development of tumors with features of both entities i.e. CC and HCC. Our findings underline the potential of this mouse model to study carcinogenesis associated with copper toxicity and chronic inflammation. This model may be important for development of innovative tumor prevention and therapy strategies.

Su1856 Effect of Ambiguous Hemochromatosis Gene Test Results on Physician Utilization: A Population Based Study on 20,306 Participants Mark Speechley, David Alter, Helen Guo, Helen Harrison, Paul Adams Background: Genetic testing is becoming more commonly used including direct to consumer testing. The impact of genetic testing and notification of genetic test results on physician utilization after the test has not been clearly established. Methods: A primary care- based cohort of 20,306 participants (HEIRS Study, Ontario site) were tested for the C282Y and H63D mutation of the HFE gene and for abnormal serum ferritin levels. For this substudy, participants were linked to Ontario single-payer physician billing data. The primary outcome variable was total number of physician claims per patient. Physician claims for the 12 months after notification of test results were compared across 5 genotype/phenotype groups. C282Y homozygotes were not considered in this analysis since they had further investigations as part of the HEIRS study. Non C282Y homozygotes were notified of their genetic test results by mail. Number of claims was modeled using multiple Poisson regression. Co-variates included age, gender, baseline log serum ferritin and number of physician claims during the 12 months before notification of results. First order interactions were included. The reference group had no HFE mutations (wild type) and a normal serum ferritin. Results:(Table 1) Conclusions: Participants who were mailed letters indicating an ambiguous hemochromatosis gene test and a normal serum ferritin had statistically significantly higher average physician utilization of 3.5 %. At the population level, this is concerning because there is no reason to expect medical follow-up to have positive effects on health status in these otherwise healthy people. Moreover, this group is consuming scarce resources that might be better devoted to confirming elevated iron values and providing treatment, and to providing genetic counseling for those with known risk-conferring genotypes. The health effects, if any, of Increased utilization in heterozygotes or those with mild ferritin elevations are unknown but are unlikely to be large at the population level. Ambiguous genetic test results may directly increase health care costs through increased physician utilization and should be considered when assessing the total cost implications of widespread genetic testing.

Su1854 Total Antioxidative Capacity in Serum Correlates With the Phenotypic Manifestation of Wilson Disease Radan Bruha, Libor Vitek, Zdenek Marecek, Lenka Pospisilova, Sona Nevsimalova, Pavel Martasek, Jaromir Petrtyl, Petr Urbanek, Alena Jiraskova, Peter Ferenci Background & Aims: Wilson disease (WD) is an inherited disorder of copper metabolism with variable symptoms. In addition to a genetic predisposition, other factors are likely to contribute to its clinical manifestation. The susceptibility to oxidative stress could be one of these factors as WD is associated with increased oxidative stress due to high copper load. Heme oxygenase-1 (encoded by HMOX1), involved in the heme degradation pathway, belongs to the most important oxidative stress defense systems. The objective of the study was to assess whether oxidative stress and inflammatory markers, as well as microsatellite variations in the promoter regions of HMOX1 gene are associated with phenotypic manifestation of WD. Methods: In 56 patients with WD (29 men and; 26 with hepatic form, 22 with neurologic form and 8 in asymptomatic phase of WD; mean age 38.5±12 years), total serum peroxyl radical scavenging activity (PRSA) measured as a relative proportion of chain breaking antioxidant consumption present in serum to that of Trolox and inflammatory parameters (hs-CRP, IL-1 β, IL-2, IL-6, IL-10 and TNF-α) were analyzed and related to the clinical manifestation, severity of neurologic symptoms, mutations to the ATP7B gene and (GT)n dinucleotide variations in promoter region for HMOX1 gene. The control group for TAC and inflammatory parameters consisted of 50 age- and sex-matched healthy individuals; the control group for evaluation of HMOX1 gene variation consisted of 986 healthy age- and sex-matched controls. Results: WD patients had a significantly lower PRSA (p<0.00001), lower IL-10 levels (p=0.039) and higher IL-1β (p=0.019) and IL-6 (p=0.005) levels compared to the control subjects. TNF-α, hs-CRP and IL-2 did not differ from controls. Patients with the neurological form of WD had a significantly lower PRSA than those with the hepatic form (p<0.001) and severity of neurological symptoms was associated with lower PRSA (p= 0.04). No relationship between inflammatory parameters and clinical symptoms was found. No significant differences in frequencies of L-allele (S/L, M/L, L/L, associated with lower

Su1857 Health Related Quality of Life in Children With Autoimmune Liver Disease Reema Gulati, Kadakkal R. Radhakrishnan, Vera Hupertz, Robert Wyllie, Naim Alkhouri, Sarah Worley, Ariel E. Feldstein Health related quality of life (HRQOL), is a pivotal outcome indicator of health care interventions in children. No studies to date have evaluated HRQOL in children with autoimmune liver disease (AILD). Aim: To determine the HRQOL in children with AILD- Autoimmune

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AASLD Abstracts

AASLD Abstracts

Gender Influences the Clinical Presentation of Wilson Disease (WD) Peter Ferenci, Karl Heinz Weiss, Anna Czlonkowska, Roderick Houwen, Ferenc Szalay, Radan Bruha, Rudolf Stauber, Wolfgang Stremmel