Retrival of the rat canalicular conjugate export pump MRP2 is associated with a rearrangement of microfilaments and radixin in taurochenodeoxycholate-induced cholestasis

Category 7: Transport, biliary disease, gallstones Methods: CCK-8 uptake was measured in X. laevis oocytes expressing the organic anion transporting polypeptides of rat (Oatpl, Oatp2, Oatp3 or Oatp4) and human liver (OATP-A, OATP-B, OATP-C or OATP8). Results: CCK-8 uptake in oocytes expressing rat Oatp4 and human OATP8 was stimulated 25fold and 8fold, respectively, compared to water injected control oocytes. No CCK-8 transport was observed in oocytes expressing rat Oatpl, Oatp2 or Oatp3, or human OATP-A, OATP-B or OATP-C. The Km values of CCK-8 uptake were 14.9 4- 2.9 uM for rat Oatp4 and 11.1 -t- 2.9 uM for human OATP8, indicating an identical affinity of the two transporters for CCK-8. Conclusion." CCK-8 is selectively transported by rat Oatp4 and human OATP8, both of which are exclusively expressed at the basolateral membrane of hepatocytes. These two transporters are the first and probably the predominant hepatic uptake systems for CCK-8 and may be critical for the rapid clearance of this hormone from the circulation.

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MINIMAL CHANGE CHOLANGIOPATHY (MCC)

A. Sonzogni 1, G. Colloredo 2, L. Roffi 3, G. Bovo 4 , P. Del Poggio 5 , B. Paris 6, M. Andreoletti 7, B. Brugnetti s, M. Pozzi 3, L. Fabris 6, M. Strazzabosco 6. 1 Pathology dpt; 6 Gastroenterology Unit, Bergamo

H.; 2 Liver Disease Unit, Seriate H (BG); 3 Internal Medicine; 4 Pathology dpt Monza H; 5 Infectious disease Unit, Treviglio H. (BG); 7Internal Medicine, Cernusco sul Naviglio H. (MI); 8Laboratory dpt, Ponte San Pietro H. (BG), Italy Proliferation of duct ceils is a common finding during routine examination of liver biopsies. Occasionally, marginal ductular proliferation is the only histopathological change. We describe the clinical and biochemical features of a group of patients in which the peculiar histological feature was a cryptogenic marginal ductular reaction without significant fibrosis and/or necroinflammation and/or paucity of interlobular biliary ducts (minimal change cholangiopathy, or MCC). Materials and Methods: We retrospectively reviewed 1235 percutaneous liver biopsies performed for suspected chronic liver disease during 4 years in five northern italian centres. Only liver specimen including at least 6 portal tracts were considered representative and included in the study. Definition of cryptogenic MCC was based on a careful exclusion of any condition associated to biliary tract diseases such as immune-mediated conditions, acute infections, drug induced hepatitis, genetic and developmental syndromes, neoplastic disease, gallstone disease. Use of drugs/herbal medicine/alcohol/exposure to xenobiotic agents and celiac disease were also excluded. Liver specimens meeting the clinical/pathological diagnosis of cryptogenic MCC were stained for Epithelial Membrane Antigen (EMA, a marker of mature differentiated cholangiocytes), neural adhesion molecules (NCAM, a marker for immature atypical ductules) and Ki67 (a marker of cell proliferation. Results: Among 157 patients that were negative for HBV, HCV and HDV, 16 cases (10%) satisfied the clinical and histological criteria for cryptogenetic MCC (M/F: 10/6, mean age 38.2 4- 11 years). The mean duration of the disease was 6.9 years (range 1-17), All patients were asymptomatic and biochemistry showed only mild unspecific abnormalities of liver function tests, except for a two-three fold increase in y-GT serum levels. Inununohistochemistry demonstrated that reactive ductules were well differentiated (EMA+ve and N C A M - v e ) and without significant proliferative activity (Ki67-ve). The condition was not progressive since in 5 patients, biochemical abnormalities had been present for up to 10 years. Among biopsies performed in patients with viral liver diseases changes similar to MCC were present in 2% of HBV+ve (3/145) and 2.8% of HCV+ve (25/894). In this case, no relationship with HCV-genotype distribution was observed, and in four patients that achieved sustained virological response to IFN, MCC persisted in the liver more than one year after IFN withdrawal, suggesting no pathogenetic relationship with viral infection. Conclusions: Minimal Change Cholangiopathy is a newly documented

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cryptogenic condition characterised by hyperplastic ductular structures with mature phenotype in the absence of portal fibrosis/inflammation. The condition is not infrequent and likely non-evolutive, but long-term studies are needed to define its natural history.

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BILE ACIDS POTENTIATE CYCLIC AMP DEPENDENT SECRETORY PATHWAYIN HUMAN BILIARY EPITHELIUM

N. Chignard, M. Mergey, D. Veissiere, J. Capeeau, R. Poupon, A. Paul, C. Housset. U402, INSERM, France Fluid secretion across the biliary epithelium prevents the stagnation of toxic bile acids both in the intrahepatic ducts and in the gallbladder. The secretory activity is stimulated mainly by adenosine 3', 5'-cyclic monophosphate (cAMP) in response to gastro-intestinal peptides stimulating adenylyl cyclase activity via G protein coupled receptors. Other regulating signals may also arise from non-peptide molecules such as bile acids. Aims of the study were to investigate the possible crosstalk between non-peptide molecules (tauroursodeoxycholate, TUDC; taurochenodeoxycholate, TCDC) and cAMP dependent pathway in gallbladder derived biliary epithelial cells (BEC). Neither TCDC, nor TUDC, elicited any increase in BEC cAMP content (n > 6; p > 0.2 vs control) as ascertained by a RIA dosage. However, when TCDC, or TUDC, were used in combination with forskolin, they potentiated the increase induced by this adenylyl cyclase diterpene activator (150 4- 24 and 146 -4- 20 in % of forskolin-induced cAMP elevation, respectively; n = 11; p < 0.04). Furthermore, bile acids effect was reproduced by phorbol-12-myristate-13-acetate (PMA), and PKC inhibition experiments showed that PKC activation was necessary for bile acids to exert their effects on forskolin-induced cAMP increase. Determination of adenylyl cyclase expression pattern by RT-PCR, showed that four isoforms present in BEC respond to forskolin, and are regulated by calcium pathway. These results provide evidence that bile acids may participate to BEC secretory functions by potentiating, via a PKC pathway, gastrointestinal hormone induced cAMP elevation.

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RETRIVAL OF THE RAT CANALICULAR CONJUGATE EXPORT PUMP MRP2 IS ASSOCIATED WITH A REARRANGEMENT OF MICROFILAMENTS AND RADIXlN IN TAUROCHENODEOXYCHOLATE-IN DUCED CHOLESTASlS

D. Rost, W. Stremmel, A. Stiehl. Department of Gastroenterology and

Hepatology, Universit~itsklinik Heidelberg, Germany Infusion of Taurochenodeoxycholate (TCDCA) may induce cholestasis in rat. The events leading to cholestasis are incompletely understood. The canalicular conjugate export pump Mrp2 is the major driving force for the bile salt-independent bile flow. Redistribution of Mrp2 has been suggested to cause reduction in bile flow in others models of acute cholestasis (i.e. endotoxin, phalloidin, GSH-depletion). Aim: We have studied the effects of TCDCA on the distribution of Mrp2 and P-glycoproteins with respect to changes in the actin cytoskeleton and actin associated proteins radixin and ZO1. Methods: Bile duct cannulated rats were infused with TCDCA (0.1 and 0.4 /zmol/min/100 g body weight) and bile flow was measured. Afters 60 rain livers were removed and distribution of Mrp2, P-glycoproteins, actin, actin-associated radixin and ZO1 were studied by immunofluorescence analysis. Results: TCDCA at subcholestatic amounts (0.1 #mol/min/100 g body weight) led to distortion and dilation of the canaliculi which affected actin, ZO1 and the Mrp2 fluorescence. Administration of higher amounts of TCDCA (0.4/zmol/min/100 g body weight) led to a reduction of bile flow to 39% of control bile flow. Radixin, which localized strictly to the plasmamembrane in controls, was detected in intracellular structures partially colocalizing with actin aggregates especially at the sinusoidal membranes as visualized by double-immunofluores-

Poster Sessions

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cence staining. Mrp2 appeared in pericanaiicular membrane structures in cholestatic animals whereas P-glycoproteins reamained unchanged under these conditions. Conclusions: TCDCA-induced cholestasis is associated with changes of the actin cytoskeleton and actin binding proteins and a retrival of the canalicular export pump Mrp2.

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HEPATOCELLULARCARCINOMA (HCC) AND EXTRAHEPATIC MALIGNANCIES IN PRIMARY BILIARY CIRRHOSIS (PBC)

M. Deutsch, G.V. Fapatheodoridis, A. Tzakou, S.J. Hadziyannis.

Academic Dept. of Medicine, Hippokration Hospital, Athens, Greece Background/Aim: The incidence of HCC has been reported to be relatively low in patients (pts) with PBC, while an increased incidence of extrahepatic malignancies in PBC has been observed. We evaluated the incidence of HCC and extrahepatic malignancies in a large cohort of PBC pts followed for a median of 5 (1-23) years. Methods: 199 PBC pts (181 F/18 M, mean age: 54 -4- 10 years) with a follow up of _>1 year were included. All pts were followed with clinical examination and LFI's including aFP at least every 6 months; pts with stage IV PBC also had liver U/S. Baseline liver biopsy showed stage I PBC in 19 (12.7%), stage II in 44 (29.5%), stage III in 31 (20.7%) and stage IV in 55 (36.8%) out of 149 pts; histologic stage IV developed in another 32 pts during follow up. Median follow up after development of stage IV was 4 (1-17) years. Results: Maligancies developed in 20 (10.1%) pts (6 HCC, 14 extrahepatic). The cumulative probability of development of maligancy in all pts as well as in the 87 pts with stage IV PBC is shown below: Years

HCC % All pts Stage IV

1

0

0

3 5 10 15

0 0 1.3 9.8

1.6 8.1 13.9

Extrahepatic % All pts Stage IV 1.1 2.2 4.1 3.6 5.0 3.6 12.9 7.6 12.9

All malignancies % All pts Stage IV 1.1 2.2 4.1 4.1 5.0 10.9 14.1 18.1 21.4

Baseline age, gender, Mayo-risk score or treatment with UDCA were not significantly associated with development of malignancy (Cox regression analysis). All 6 HCCs developed in pts with stage IV PBC. Stage IV compared to stages I-III PBC pts bad a significantly higher probability for HCC development (p = 0.027 log.rank) but similar probability for extrahepatic malignancies. Conclusion: There is a significant risk of HCC development only in histologic stage IV PBC pts reaching 14% after 10 years, while the incidence of extrahepatic malignancies does not differ between early and advanced histologic stages of disease.

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identified. 2 changes: E297G and D482G have been found in 25 and 16 European families and 1 of these 2 is present in 30% of European families. 11 changes occur at mutation hot spots and have been found in 21 patients. These all create or abolish restriction endonuclease sites and represent a rapid initial screen in the diagnosis of BSEP deficiency. 25 additional mutations: 8 1-2bp deletions, 2 rearrangements, 1 gene deletion, 14 point changes have been identified. Linkage analysis is possible in consanguineous/multiaffected families using flanking and newly developed intragenic microsatellite markers. Of 34 families examined 12 (35%) are consistent with linkage to chromosome 2, 9 (26%) to chromosome 18 and 3 are uninformative. 10 (29%) do not link to either and present the possibility of a third locus for low y-GT PFIC

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ASSOCIATION BETWEEN KNOWN ABCB4 MUTATIONS AND ICP IN PATIENTS FROM WEST SWEDEN

H.-U. Marschall 1, A. Glantz 2, L.-]k. Mattsson 2, F. Lammert 3.

1Huddinge University Hospital, S-14186 Stockholm," 2 Sahlgren's University Hospital, East, S-41685 GOteborg, Sweden," 3RWTH University Hospital, D-52057 Aachen, Germany Objective: Recently mutations in the ABCB4 (MDR3) gene encoding the hepatic canalicular phosphatidylcholine translocator have been described in five women with intrahepatic cholestasis of pregnancy (ICP), in a French consanguinous family as well as in one British patient. We now assessed whether these mutations account for ICP in 22 patients from a large randomised ICP trial, UDCA vs. dexamethasone vs. placebo. Methods: Diagnosis of ICP was based on otherwise unexplained pruritus and elevated bile acids (> I0 #mol/1) occurring in the third trimester. Genomic DNA was extracted from serum and exon 14 was amplified by (nested) PCR with gene-specific primers. Following amplification, products were gel-purified and both strands were sequenced directly. Results:

Age years 25.9 -4- 7.0

Bile Acids < 10/~mol/1 55.1 4- 49.2

Bilirubin <26/zmol/l 13.7 4- 6.2

ALAT <0.80/zkat/1 3.9 4- 2.5

y-GT <0.80/tkat/1 0.6 4- 0.5

ALAT normal, n = 1; elevated bilirubin (30), n = 1; elevated F-GT (0.97-2.11), n=3. DNA sequence analyses revealed no polymorphisms in exon 14 of the

ABCB4 gene for all patients tested. In particular, the previously described C to A transversion in codon 546 (A546D) and the single-nucleotide deletion starting in codon 571 (1712delT) were not detected. Conclusions: ICP usually features elevated ALAT but normal bilirubin. Genetic screening of the first 10% of women with ICP included in the West Swedish clinical trial did not detect known MDR3 mutations. Further screening for genetic defects will include other candidate genes, e.g. ABCB11 encoding the hepatic canalicular bile salt export pump.

PROGRESSIVE FAMILIAL INTRAHEPATIC CHOLESSTASIS: MUTATION ANALYSIS AND EVIDENCE FOR A THIRD LOCUS

S.S. Strautnieks, J. Byrne, E. Soler, A. Knisely, L. Bull, I. Jankowska, J. Pawlowska, N. Freimer, E. Sokal, G. Mieli-Vergani, R. Thompson.

Institute of Liver Studies, Kings College Hospital, UK; UCSF, USA; CMH, Poland; Louvain, Belgium PFIC is a heterogeneous group of conditions that present in early childhood with progressive cholestatic liver disease. The 2 most common forms are caused by mutations in ABCBll and ATP8B1, the genes encoding the bile salt export pump (BSEP) and FIC proteins respectively. Both are characterised by normal levels of y-glutamyl transpeptidase (GT). BSEP deficiency is diagnosed genetically and 197 patients have entered the screening program. 13 common/recurring mutations have been

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ADULT ONSET CHOLANGIOPATHY-AMA-ve PBC - DUE TO MUTATIONS IN ABCB4

R.J. Thompson, S. Strautnieks, S. Gerred, A. Knisely, B. Portmann, A. Bomford, J. O'Grady. GKT School of Medicine, King's College

Hospital, London, UK Cholangiopathy in adults is a non-specific feature of a variety of conditions. In most cases a diagnosis is made on the basis of other features, others lack diagnostic features and are labelled as " A M A - v e PBC", "Small duct PSC", or simply idiopathic cholangitis/ductopenia. We investigated a family containing 3 such patients. Two sisters presented in pregnancy with cholestasis and marked cholangiopathy, for which no explanation could be found. One has required liver transplantation. Their