310 Impaired Bone Morphogenic Protein Signalling Underlies Hepcidin Deficiency in HFE Hereditary Haemochromatosis

providing a plausible explanation for the protective effect of O3FA during inflammatory liver diseases.

309 Prevalence of Hypertension in a Hereditary Haemachromatosis Population and the Influence of Treatment on Their Cardiovascular Risk Cliona M. Waterhouse, Barbara M. Ryan, Niall Breslin, Colm A. O'Morain

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Introduction: Previous reports have shown a modest association of ferritin and transferrin saturation with peripheral arterial disease (PAD). No studies, however, have examined the prevalence of hypertension in genetically confirmed hereditary haemachromatosis (HH) patients. Aims & Background: The aim of this study is to determine the prevalence of hypertension in our HH population, and to determine the effect of treatment of their underlying disorder on their blood pressure. Method: All patients in our institution who have genetically confirmed HH, who underwent regular venesection until a target ferritin was reached, were included. Their systolic and diastolic blood pressure which was measured before their first venesection was compared with their blood pressure which was measured before their final venesection. Results: 132 subjects (101 males) with HH were studied. Mean age was 54 years (51.5 for females). Mean blood pressure (BP) on enrolment was 142/80mmHg. 81 patients (58 males; 23 females) had evidence of hypertension (>140/90 mmHg) on enrolment. Mean BP in the hypertensive group was 152/92mmHg. Following venesection therapy for management of their HH, mean BP for the overall group fell significantly (134/78mmHg; p<0.001). Notably, within the hypertensive group, mean BP fell to 139/76mmHg following venesection therapy. When corrected for age, gender, BMI and smoking status, change in ferritin level was significantly associated with fall in BP (p<0.0001). Conclusion: This study confirms, for the first time, that there is an increased incidence of hypertension in the HH population. Furthermore, it also shows that regular and complete phlebotomy results in a significant decrease in both systolic and diastolic blood pressure. Therefore, as well as reducing the risk of developing diabetes mellitus, cirrhosis, clinical hepatic failure and hepatoma, this study shows for the first time, that regular phlebotomy may reduce the overall cardiovascular risk of this population.

AASLD Abstracts

Regulatory CD4+CD25+CD127dim Cells Inhibit CCL3 and CCL19 Chemokine Production in Myeloid Dendritic Cells of Patients With Chronic HCV Infection Angela Dolganiuc, Chidimma I. Okoli, Christopher Marshall, Karen Kodys, Gyongyi Szabo Introduction: Chronic hepatitis C virus (cHCV) infection is characterized by inflammation driven by an inefficient immune response. Recruitment of immune cells into the liver is critical both in liver injury and anti-HCV immunity. CCL3 attracts neutrophils and myeloid cells. CCL19 attracts dendritic cells, antigen-engaged B cells, and effector-memory T cells. STCP-1 targets dendritic cells, activated NK and activated T cells. These chemokines are produced by dendritic cells (DC) and exhibit inflammatory properties. DCs also use surface molecules to engage T cells to coordinate immune responses. Regulatory T cells (Tregs) limit immune responses by suppressing effector (eff) T cells but their effects on DC are largely unknown. Here we hypothesized that Tregs may affect the capacity of DC to produce chemokines. Methods: Monocyte-derived DCs (MDC) were isolated by adhesion and growth in IL-4+GM-CSF media for 7 days; CD4+CD25- (effTcells), and CD4+CD25+CD127dim (Tregs) were purified with magnetic beads from blood of normal controls (n=7) and those with chronic HCV infection (n=7). Chemokines were assessed with ELISA (protein) and real-time PCR (gene). Results: We found elevated serum levels of CCL3 and CCL19 in HCV patients compared to controls; STCP-1 levels were comparable. MDCs, effTcells and Tregs alone showed minimal production of CCL3, CCL19 or STCP-1, regardless of infection status. Co-cultures of MDCs with effTcells triggered production of CCL3, CCL19, and STCP-1 and cHCV MDCs produced more CCL3 and CCL19, but not STCP-1, compared to N-MDC. Addition of Tregs to MDC/effTcells co-cultures inhibited all chemokine production; equal numbers of HCV-Tregs lead to greater inhibition compared to N-Tregs. We identified increased PD1 and CTLA4 expression in HCV co-cultures, compared to controls. Moreover, supplementation of Tregs/MDCs/effTcells co-cultures with anti-PD1 or anti-CTLA4 antibodies partially restored the inhibitory effect of Tregs on CCL3 and CCL19 production in HCV patients, with minimal effects in controls. Neither anti-PD1 nor anti-CTLA4 antibodies influenced STCP-1 production in co-cultures of either controls or HCV patients. In conclusion, HCV MDCs produce higher levels of CCL3 and CCL19 compared to N-MDCs. Despite higher serum chemokine levels, cHCV MDCs are more susceptible to Tregs-induced inhibition of chemokine production, indicating that Tregs could tightly control the local chemokine production in a PD1- and CTLA4- dependent manner. These data suggest that Treg-induced impairment in chemokine production by DCs may regulate recruitment of target cells to the inflammation site in chronic HCV infection. (supported by AA014372)

310 Impaired Bone Morphogenic Protein Signalling Underlies Hepcidin Deficiency in HFE Hereditary Haemochromatosis John D. Ryan, Eleanor Ryan, Aurelie Fabre, Nathan Maher, Matthew W. Lawless, John P. Crowe Hereditary Haemochromatosis (HH) is a common inherited disorder of iron metabolism. Over 90% of patients are homozygous for the C282Y mutation of the HFE gene, resulting in a non-functioning HFE protein. Hepcidin, the central regulator of iron metabolism, is deficient in HH, leading to unchecked iron absorption and subsequent iron overload. The bone morphogenic protein (BMP)/SMAD signalling pathway is central to the regulation of hepcidin, and BMP6 a key inducer of hepcidin expression. How the mutated HFE protein leads to hepcidin deficiency is unknown. Recent data from HH mice models indicate BMP/ SMAD signalling may be defective in the absence of the HFE protein. Aim: To investigate BMP/SMAD signalling in HFE-HH. Methods: Hepatic expression of BMP/SMAD related genes was examined in 20 untreated HFE-HH males with significant iron overload and compared to 7 male HFE wild-type controls using qRT-PCR. BMP6 expression was further assessed by immunohistochemistry. Results: Hepatic expression of BMP6 was appropriately elevated in HFE-HH compared to controls (p=0.02), likely related to hepatic iron overload. Immunohistochemistry revealed diffuse hepatocytic BMP6 staining, compared to a periportal pattern in controls. Despite this, no increased expression of the BMP target genes hepcidin and ID1 was observed, indicating defective BMP signalling occurs in HFE-HH. Expression of the inhibitory SMAD genes, SMAD6&SMAD7 were significantly upregulated in HFE-HH compared to controls (p<0.001 and p=0.018, respectively). Expression of SMAD6, a known inhibitor of BMP signalling, correlated with serum ferritin (r=0.55, p=0.006), degree of hepatic iron staining (r=0.64, p<0.001), and SMAD7 (r=0.58, p=0.002). Discussion/Conclusion: This study represents the first account of hepatic BMP/SMAD signalling in HFE-HH. Taken together, this data reveals that deficient hepcidin production in HFE-HH is due to impaired BMP/SMAD signalling. Moreover, novel potential modifiers of disease pathogenesis and phenotype are identified.

243 Distribution Dynamics of Radixin and NHERF-1 on Regulation of MRP-2 Trafficking in Hepatocytes Jo Suda, Lixin Zhu, Serhan Karvar Radixin is a member of ezrin, radixin, moesin (ERM) protein family that links F-actin to membranes. The NH2- and COOH (N-C)-terminal association domains of ERM proteins participate in interactions with membrane proteins and F-actin, and molecular interactions within ERM. Radixin has been reported to selectively tether mrp2 to the canalicular membrane. Bile canalicular formation is essential for biliary secretion and is disrupted in cholestatic disorders We have examined the dynamic distribution of radixin and NHERF-1 in live hepatocytes using fluorescence-tagged constructs. Cyan fluorescent protein (CFP)-tagged Radixin, yellow fluorescent protein (YFP)-tagged NHERF-1 wild type and mutant adenoviral constructs were used. The molecular (N-C) binding of radixin was visualized using fluorescence resonance energy transfer (FRET). Live fluorescence imaging showed that wild type radixin and NHERF-1 is localized to the canalicular plasma membrane along with the mrp2 in dual infected cells. CFP-Radixin-T564A is a non phosphorylated mutant that does not bind to F-Actin. Similar to endogenous radixin, wild type and CFP-radixin T564A localized heavily to canalicular membrane. Interestingly, mrp-2 was incorporated into the canalicular membranes along with wild type and CFP-radixin T564A mutant radixin. CFP-Radixin T564D mutant, which mimics constant phosphorylation, was more typically localized to the basolateral membrane, often associated with long spikes and fingerlike projections. Mrp2 distribution was detected throughout the cytoplasm in CFP-radixin T564D infected cells. We have also observed cytoplasmic distribution of NHERF-1 F355R (radixin binding site mutated) where as wild type HNERF-1 was localized at the canalicular memebrane. Mrp2 distribution was detected throughout the cytoplasm in NHERF-1 F355R infected cells. FRET was observed in wild type and T564A YFP-radixin-CFP constructs, indicating molecular (N-C)-terminal binding. No significant FRET was detected in T564D YFP-radixin-CFP. In Vitro FRET analysis and actin binding binding assay demonstrated that Thr564 phosphorylation opens the (N-C)-terminal interaction and the opened phosphorylated form of radixin more readily cosediments with F-actin and binds to membrane. In addition, we have characterized of NHERF1 as a binding partner of Mrp2. We identified the presence of radixin and NHERF-1 in hepatocytes and our data strongly implicate that radixin and NHERF-1 are essential for maintaining the polarized targeting and retaining of canalicular transporters and is a critical determinant of the overall structure and function of the canalicular membrane of hepatocytes.

AASLD Abstracts

311 Final Results of a Rollover Study Assessing Telaprevir in Combination With Peginterferon Alfa-2a and Ribavirin in Chronic HCV Patients With WellCharacterized Null Response, Partial Response, Viral Breakthrough, or Relapse After Prior PR Treatment Andrew J. Muir, Fred Poordad, Nathalie Adda, Mitchell L. Shiffman, Thomas Berg, Peter Ferenci, E. Jenny Heathcote, Jean-Michel Pawlotsky, Stefan Zeuzem, Henk W. Reesink, Geoffrey M. Dusheiko, Emily C. Martin, David Alexanderian, Shelley George, John McHutchison Background and Aims: Study 107 is an open-label rollover study of telaprevir (T), PEG interferon alfa-2a (P) and ribavirin (R) in a group of well-characterized genotype 1 HCV patients who did not achieve SVR following PR treatment in telaprevir Phase 2 studies. Methods: Null responders (<1-log10 HCV RNA decrease at week-4 or <2-log10 at week12), partial responders (≥2-log10 decrease at week-12, detectable at week-24), patients with viral breakthrough and relapsers from PROVE1/2/3 PR arms were eligible for treatment. Initially all patients received T 750 mg q8h plus PR at standard doses for 12 weeks, followed by 12 weeks of PR (T12/PR24). The protocol was amended to allow partial responders, viral breakthroughs and relapsers with undetectable HCV RNA at weeks 4 and 12 (eRVR) to receive T12/PR24. Partial responders, viral breakthroughs and relapsers with detectable HCV RNA at week-4 and/or week-12 and null responders received an additional 24 weeks of PR (T12/PR48). Results: 117 patients were included in an ITT analysis, 97 (83%) had baseline HCV RNA ≥800,000 IU/mL, 69 (59%) and 38 (33%) had genotype subtype 1a and 1b, respectively, 44 (38%) had cirrhosis/bridging fibrosis, and 9 (8%) were black. Viral breakthrough and relapse rates occurred in 25%, 23% of prior null responders; 10%, 22% of prior partial responders; 13%, 0% of prior viral breakthroughs; and 0%, 4% of prior

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