POSTERS

Results: Circulating monocytes are significantly expanded in

patients with chronic liver diseases with a marked increase of

the non-classical CD14+CD16+ subset that shows an activated

phenotype in patients and correlates with proinflammatory

cytokines, chemokines and clinical progression. Correspondingly,

CD14+CD16+ monocytes/macrophages massively accumulate in the

fibrotic/cirrhotic liver and account for about 50% of all liver

macrophages in cirrhotic in comparison to 10% in normal or early

fibrotic tissue. Monocyte-related chemokine pathways (i.e. CCL2,

CCL3, CCL4, CCL5, CX3CL1) are differentially activated in patients

in the circulation and liver, alongside up-regulated chemokine

receptors on monocytes. Functionally, non-classical CD14+CD16+

monocytes release abundant proinflammatory cytokines upon

isolation, whereas the classical CD14++CD16− subset primarily

provides the chemokine CCL2 (MCP-1) and anti-inflammatory IL10.

Co-culture experiments of primary HSC and monocyte subsets

demonstrated that only classical CD14++CD16− monocytes are able

to exert direct fibrogenic stimuli on HSC by inducing collagen-1

and acta2 mRNA transcripts. In turn, HSC differentially affect the

phenotype of monocyte subsets upon co-culture by regulating the

expression of activation markers and chemokine receptors.

Conclusions: Our data thereby reveal differential functional

contributions of the non-classical and classical human monocyte

subset for the perpetuation of intrahepatic inflammation and

profibrogenic HSC activation, respectively. The modulation of

monocyte-subset recruitment into the liver and their subsequent

differentiation may offer novel opportunities for therapeutic

interventions in human liver fibrosis.

1000

ROLE OF CALCIUM-CALMODULIN PATHWAY IN ESTRADIOL-

17b-GLUCURONIDE INDUCED IMPAIRMENT OF CANALICULAR

SECRETION IN ISOLATED RAT HEPATOCYTE COUPLETS

A. Zucchetti, F.D. Toledo, I.R. Barosso, E.J. Ochoa, F.A. Crocenzi,

E.J. Sanchez Pozzi. Instituto de Fisiologıa Experimental, Rosario,

Argentina

E-mail: zucchetti@ifise-conicet.gov.ar

The endogenous estradiol metabolite, estradiol 17b-D-glucuronide(E17G), induces an acute cholestasis in rat liver due in part to

retrieval of canalicular transporters such as the bile salt export

pump (Bsep, Abcc11) and the multidrug resistance associated

protein 2 (Mrp2, Abcc2), in a process that involves estrogen receptor

(J. Hepatol 50: s103, 2009). Calmodulin interacts with estrogen

receptor and potentiates estrogen activation of the receptor (Cell

Signal 19: 439–443, 2007). The aim of this study was to evaluate

the involvement of calcium-calmodulin pathway in E17G-induced

changes in canalicular excretion.

Methods: Canalicular transport activities: Isolated rat hepatocytes

couplets were cultured for 5h, exposed to verapamyl (V, calcium

channel blocker, 10mM), trifluoroperazin (T, Calcium-calmodulin

inhibitor, 10mM) or W7 (W, Calcium-calmodulin kinase II [CaMKII]

inhibitor, 100mM) for 15 min and then incubated with E17G

(100mM) for 20 min. Finally, all preparations were incubated

with cholyl-lysylfluorescein (CLF, fluorescent bile salt substrate

of Bsep) or CMFDA (intracellularly converted in gluthation-

methylfluorescein [GMF], fluorescent substrate of Mrp2). Couplets

accumulating CLF or GMF in their vacuole were counted in a

fluorescent microscope and informed as a percentage (%AC).

CaMKII activation: Isolated hepatocyte were cultivated in collagen

sandwich for 5 days, exposed to E17G (200mM) for 15 and 30 min

and then lysed. Western blot of the samples were performed using

antibodies against total and phosphorylated CaMKII. The ratio of

the densitometry of phosphorylated/total CaMKII bands was used as

estimation of kinase activation. Results were expressed as mean±SD

and compared by ANOVA followed by Student-Newman-Keuls test.

Results (n =3): See the tables.

Canalicular transport activities

Control E17G E17G+V E17G+T E17G+W

CLF %AC 100±0 66±4a 84±4a,b 90±11b 97±13b

GMF %AC 100±0 65±4a 81±11a,b 95±6b 94±1b

asignificantly different from Control (p < 0.01); bsignificantly different fromE17G (p < 0.05). V, T and W did not affect %AC of the substrates.

CaMKII activation

Control E17G

15 min 30 min

phosphorylated/total CaMKII (arbitrary units) 100±0 126±25 172±34a

asignificantly different from Control (p < 0.05).

Conclusions: E17G activates calcium-calmodulin pathway as

demonstrated by CaMKII activation, and this pathway participates

in estrogen-induced alteration in canalicular secretion.

05e. VIRAL HEPATITIS: e. HEPATITIS B – CLINICAL(THERAPY NEW COMPOUNDS, RESISTANCE)

1001

PRAGMATIC ASSESSMENT OF LIVER FIBROSIS DURING

METHOTREXATE THERAPY: COMPARISON OF PATIENTS WITH

PSORIASIS, RHEUMATOID ARTHRITIS OR CROHN’S DISEASE

B. Alby-Lepresle1, J.-P. Cervoni1,2, E. Monnet1, F. Aubin3,

D. Wendling4, E. Toussirot4, I. Mermet3, M. Nachury5, E. Bertolini4,

F. Carbonnel5, V. Bague2, P. Cales6, V. Di Martino1. 1Service

d’Hepatologie, CHU Jean Minjoz, 2CIC-BT, CHU Besancon, 3Service

de Dermatologie, CHU Saint-Jacques, 4Service de Rhumatologie,5Service de Gastroenterologie, CHU Jean Minjoz, Besancon, 6Service

d’Hepato-Gastroenterologie, CHU Angers, Angers, France

E-mail: blandinealby@yahoo.fr

Background: Reported hepatotoxicity limits the use of

methotrexate and underlines the need for of a non-invasive

sequential and reliable evaluation of liver fibrosis.

Aims:

1. To estimate by non-invasive methods the prevalence

of significant liver fibrosis among patients treated with

methotrexate for psoriasis, rheumatoid arthritis (RA) or Crohn’s

disease (CD);

2. To assess the factors associated with significant fibrosis (≥F2).

Methods: Prospective observation of consecutive patients followed

in our institution between February 2008 and August 2009 for

Psoriasis, PR, or CD. 189 variables were collected (questionnaire,

clinical examination, Fibroscan®, blood sample, abdominal

ultrasound), focused on risk factors for chronic liver disease or

metabolic syndrome. Fibrosis was evaluated by 9 non-invasive tests

(Fibroscan®, Forns, APRI, FPI Hepascore, FIB4, Fibrotest®, Angulo’s

score, FibroMeters). Diagnosis reference of significant fibrosis was

developed without liver biopsy by defining a “specific method”

(concordance of tests) and “sensitive method” (at least one test

indicating ≥F2). Multivariate analyses were performed to assess

the factors associated with fibrosis ≥F2 or numeric values of

Angulo’s score, FibroMeters-S, and Fibroscan®. The performance

of 24 variables including the result of each test for the diagnosis of

significant fibrosis was assessed using ROC curves.

Results: 89 patients (41 psoriasis, 23 PR, 25 MCR) were enrolled

including 57 receiving methotrexate. The prevalence of fibrosis ≥F2

according to the specific or the sensitive definitions was 7% and

S386 Journal of Hepatology 2010 vol. 52 | S319–S457