Abstracts Poster Abstracts - Dr. Falk Pharma GmbH

Falk Workshop
Immunology and
Liver Disease
October 15 – 16, 2009
Maritim Airport Hotel
Hannover
Abstracts
Poster Poster Abstracts

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©2009 Falk Foundation e.V.
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Abstracts of Invited Lectures
Poster Abstracts
Falk Workshop
IMMUNOLOGY AND LIVER DISEASE
Hannover (Germany)
October 15 – 16, 2009
Scientific Organization:
M.E. Gershwin, Davis (USA)
A.W. Lohse, Hamburg (Germany)
M.P. Manns, Hannover (Germany)
D. Vergani, London (Great Britain)

CONTENTS
Session I
Liver immunology I
Session II
Chair:
V. Desmet, Leuven
S.W. Schalm, Rotterdam
Keynote lecture
HCV – 20 years after discovery (No abstract)
M. Houghton, San Francisco
Page
Immune responses mediated by endothelial cells
P.A. Knolle, Bonn 17
NKT cells within the liver
M.G. Swain, Calgary 18
Regulatory T cells induced within the liver
J. Herkel, Hamburg 19
Suicidal emperipolesis mediates deletion of T cells
undergoing primary activation in the liver
P. Bertolino, Sydney 20 – 21
Liver immunology II
Chair:
E. Schrumpf, Oslo
H.C. Thomas, London
Role of serotonin and iNOS in LCMV-induced hepatitis
K.S. Lang, Düsseldorf 25
Lymphocyte homing to the liver
D.H. Adams, Birmingham 26
Monitoring immunosuppression after transplantation
S. Meuer, C. Sommerer, M. Zeier, T. Giese, Heidelberg 27
3

Session III
4
Cancer immunity and cancer immunotherapy
A. Knuth, Zurich 28
Pathology of immune-mediated liver injury
H.P. Dienes, U. Drebber, Cologne 29 – 30
Autoimmunity I
Session IV
Chair:
G. Ramadori, Göttingen
H. Ring-Larsen, Copenhagen
Adaptive immunity in autoimmune hepatitis
D. Vergani, G. Mieli-Vergani, London 33 – 34
Old and new animal models of AIH
E. Jaeckel, Hannover 35
Cytochrome P450 2D6 as a model autoantigen
U. Christen, Frankfurt 36 – 37
Spontaneous chronic liver inflammation in a new transgenic
mouse model of autoimmune hepatitis: Impact and regulation
of autoreactive CD8 + T cells
M. Zierden, Cologne 38
Induction of tolerance in a mouse model of inflammatory
liver disease
G. Tiegs, Hamburg 39
Autoimmunity II
Chair:
G. Gerken, Essen
A.W. Lohse, Hamburg
The role of the liver for the induction of oral tolerance in
presence of naïve and antigen-experienced T cells
N. Kruse, K. Neumann, A. Schrage, K. Eulenburg,
M. Zeitz, A. Hamann, K. Klugewitz, Berlin 43
Therapeutic options to treat AIH in 2009
C.P. Strassburg, Hannover 44 – 48

Session V
New insights into autoimmune cholangitis by animal models
M. Trauner, Graz 49 – 50
Etiological and molecular issues in primary biliary cirrhosis
M.E. Gershwin, Davis 51 – 52
Immunology of viral hepatitis I
Session VI
Chair:
A. Alberti, Padua
C. Trautwein, Aachen
Occult hepatitis B virus infection: Diagnosis and
significance
W.H. Gerlich, Giessen 55
Innate and adaptive immune responses in HBV infection
M.K. Maini, London 56
Hepatitis Delta: Immunopathogenesis and clinical challenges
H. Wedemeyer, J. Grabowski, Hannover 57
Clinical spectrum and therapeutic options in HDV infections
M. Rizzetto, Torino 58
Immunology of viral hepatitis II
Chair:
J. Schölmerich, Regensburg
C. Trépo, Lyon
Apoptosis in immune-mediated liver diseases
A. Kahraman, G. Gerken, A. Canbay, Essen 61
Immune response in HCV infection
C. Ferrari, Parma 62
Immunotherapy of hepatocellular carcinoma
T.F. Greten, Hannover 63 – 64
List of Chairpersons, Speakers and Scientific Organizers 65 – 68
5

Poster Abstracts
NAFLD and NASH
1. Nonalcoholic fatty liver disease in autopsy of children and adolescents
F. Yüksel, D. Ergün, I. Yüksel, S. Kara, E. Samdanci, N. Celik (Ankara, TR)
2. Non-invasive evaluation of liver fibrosis in patients with NAFLD (comparison
between transient elastography and BARD score)
A. Tudora, O.P. Ciof, E. Dragan, D. Rill, I. Sporea, M. Scurtu-Martin
(Timisoara, RO)
3. Assessment of liver fibrosis by using transient elastography (TE) in patients with
nonalcoholic fatty liver disease (NAFLD)
O.P. Ciof, A. Tudora, E. Dragan, D. Rill, I. Sporea, M. Scurtu-Martin
(Timisoara, RO)
4. HFE gene mutations in patients with biopsy-proven NAFLD
E. Stachowska, J. Raszeja-Wyszomirska, J. Suchy, M. Lawniczak, I. Zawada
(Szczecin, PL)
5. Validation of BARD scoring system among Polish patients with NAFLD
J. Raszeja-Wyszomirska, M. Lawniczak, B. Szymanik, P. Milkiewicz, M. Hartleb
(Szczecin, Katowice, PL)
6. High rate of undetected arterial hypertension in patients with NASH
M. Demir, S. Schulte, S. Ubben, S.M. Lang, T. Goeser, U. Töx, H.-M. Steffen
(Cologne, D)
7. Fatty liver disease in children and adolescents
V. Tzaneva, S. Galcheva, D. Baleva (Varna, BG)
8. Effects of dietary chromium picolinate in the treatment of the experimental
nonalcoholic steatohepatitis
N. Kuzu, I.H. Bahcecioglu, A. Gencaslan, K. Metin, B. Üstündag, I.H. Özercan,
K. Sahin (Izmir, Elazig, TR)
9. In what manner does the metabolic syndrome modify the viral and alcoholic
hepatic cirrhosis course?
N.N. Silivontchik, E.I. Adamenko (Minsk, WR)
10. Assessment of liver fibrosis by TE (FibroScan ® ) in patients with metabolic
syndrome
M. Scurtu-Martin, A. Tudora, O.P. Ciof, E. Dragan, D. Rill, I. Sporea
(Timisoara, RO)
7

8
Hepatitis A and B
11. Investigation on cytokines at viral hepatitis A
A. Petrov, N. Vatev (Plovdiv, BG)
12. Blocking Bim-mediated attrition of T cells in patients with chronic HBV infection
A. Schurich, P. Khanna, A. Ross Lopes, G. Nebbia, G.M. Dusheiko, M.K. Maini
(London, GB)
13. Chronic hepatitis HBV in children treated with interferon-α – Prognostic value of
serum interleukin 6 and interleukin 12
M. Gora-Gebka, A. Liberek, G. Sikorska-Wisniewska, J. Golebiewski,
B. Kaminska (Gdansk, PL)
14. High frequency of HBV-specific T cell responses in delta hepatitis
J. Grabowski, P.V. Suneetha, J. Jaroszewicz, V. Schlaphoff, B. Bremer,
M.P. Manns, M. Cornberg, H. Wedemeyer (Hannover, D; Bialystok, PL)
15. Hepatitis B prevalence and flare in Asian patients with rheumatoid arthritis
treated with disease modifying anti-rheumatic drug (DMARD) therapy
O. Bebb, V.J. Appleby, P. Southern, P. Helliwell, S. Moreea (Bradford, GB)
16. Interferon-α treatment fails to induce activation of interferon responsive genes in
HBV chronically infected human chimeric uPA/SCID mice
M. Lütgehetmann, T. Volz, A.W. Lohse, J.H. Bockmann, J. Petersen, M. Dandri
(Hamburg, D)
17. Relationship between liver fibrosis and topographic distribution of alpha-smooth
muscle actin staining in chronic hepatitis B
O. Kosseva, E. Pophristova, D.G. Adjarov, D. Jelev, Z. Krastev (Sofia, BG)
18. Is any role of hepatic steatosis in chronic virus B hepatitis?
C. Mihai, C. Cijevschi, G. Stefanescu, V.L. Drug, M. Dragna, L. Graur, B. Mihai
(Iasi, RO)
19. The effects of chronic B and C hepatitis infections on bone mineral density
C. Serban, L.M. Susan, A. Pacurari, L. Copaceanu, C. Banciu, G. Savoiu,
C. Borza, R. Mateescu, I. Romosan (Timisoara, RO)
20. TGF-β-driven hepatic progenitor cell expansion and ductular reaction
contributes to HBV, but not schistosomiasis, associated liver fibrogenesis
H.-L. Weng, L. Ciuclan, Y. Liu, C. Zhu, E. Wiercinska, I. Ilkavets, J. Dzieran,
T. Huang, C. Meyer, R. Gebhardt, R. Heuchel, P. ten Dijke, J.-L. Chen,
M.V. Singer, P.R. Mertens, S. Dooley (Mannheim, Magdeburg, Leipzig, D;
Leiden, NL; Ningbo, Jiaxing, RC; Uppsala, Huddinge, S)
21. Is there a link between the viral load value and liver stiffness for the
asymptomatic virus B carriers?
A. Tudora, O.P. Ciof, M. Scurtu-Martin, I. Sporea, E. Dragan, D. Rill
(Timisoara, RO)

Hepatitis C
22. Evaluating antiinflammatory and antiviral effect due to the treatment with statins
in patients with chronic virus C hepatitis
O. Andreescu, L. Nedelcu, C. Scarneciu, D. Paul, I. Pantea (Brasov, RO)
23. Variants of the innate immune response genes in patients with chronic
HCV-infection
A.O. Romanov, T.V. Belyaeva, E.V. Esaulenko (St. Petersburg, R)
24. Expression and effect of the NK cell receptor 2B4 (CD244) on virus-specific
CD8+ T cells – Another player in the control of viral hepatitis?
V. Schlaphoff, J. Jaroszewicz, S.V. Pothakamuri, J. Grabowski, K. Stegmann,
M.P. Manns, H. Wedemeyer, M. Cornberg (Hannover, D)
25. Ethanol impairs MHC class I-restricted antigen presentation on HCV-infected
liver cells
N.A. Osna, R. White, S. Weinman, T. Donohue (Omaha, Kansas City, USA)
26. Impairment of interferon-gamma (IFN-γ) signaling in hepatocytes by HCV
proteins and ethanol
L. Austin, R. White, K. Kharbanda, M. Beard, N.A. Osna
(Omaha, USA; Adelaide, AUS)
27. The platelet-derived chemokine CXCL4 (PF4) is a mediator of HCV-induced
and experimental liver fibrosis
M. Moreno Zaldivar, K. Pauels, M.-L. Berres, P. Schmitz, A. Kowalska,
R. Weiskirchen, C. Trautwein, H.E. Wasmuth (Aachen, D)
28. Interleukin 17 pathway is suppressed in chronic hepatitis C
K. Gutkowski, T. Kacperek-Hartleb, J. Musialik, G. Boryczka, M. Kajor,
M. Hartleb, E. Kaminska-Kiszka (Katowice, Rzeszow, PL)
29. Allelic expression of rs2569190/C-159T CD14 gene variants in PBMC and liver
samples from chronic hepatitis C patients
R. Bregadze, G. Ramadori, S. Mihm (Göttingen, D)
30. The content of sphingolipids in the liver is associated with the severity of chronic
hepatitis C
M. Dabrowska, A. Panasiuk, P. Zabielski, J. Gorski, R. Flisiak (Bialystok, PL)
31. Ribavirin-induced anaemia during hepatitis C treatment is related to baseline
haemoglobin leven and drug erythrocyte concentration
M. Delle Monache, M. Carbone, L. Nosotti, L.R. Conti, I. Lenci, F. De Leonardis,
M. Angelico, L. Baiocchi (Rome, I)
32. Sylibin-vitamin E phospholipid complex reduces increased ferritin levels during
antiviral treatment for chronic hepatitis C
R. Cecere, L. Baiocchi, I. Lenci, M. Carbone, F. De Leonardis,
M. Delle Monache (Rome, I)
9

33. Thyroid function in patients with chronic hepatitis C virus infection under
interferon therapy
M. Azmy, Y. Ahmad, A. AbouNar, M. El-Hamamsy (Cairo, ET)
34. The prevalence and impact of serum autoantibodies among chronic hepatitis C
genotype 4 (HCV) patients attending a national center of HCV treatment
program in Egypt
M. Sharaf-Eldin, H. El-Batae (Tanta, ET)
35. Absence of a correlation between endotoxin receptor CD14 rs2569190/C-159T
polymorphism and liver disease progression in chronic hepatitis C
E. Askar, M. Odenthal, H.P. Dienes, G. Ramadori, S. Mihm
(Göttingen, Cologne, D)
36. The metabolic control and antiviral treatment response in diabetic patients with
chronic hepatitis C
B. Mihai, C. Mihai, C. Cijevschi, V.L. Drug, M. Dragna, L. Graur (Iasi, RO)
37. The correlation between erythropoietin and pro- and anti-inflammatory cytokines
and the decreasing of the viral C hepatitis RNA level under the treatment with
statins
R.G. Mihaila, R. Mihaila, O. Fratila, E.C. Rezi, A.V. Zaharie, L. Mocanu,
M. Deac, A. Olteanu, L. Draghila, M. Boitan (Sibiu, Oradea, RO)
38. Increased CD14 transcription initiation in T allele carriers of the rs2569190/
C-159T promoter polymorphism in healthy individuals and chronic hepatitis C
patients
J. Mertens, G. Ramadori, S. Mihm (Göttingen, D)
10
Autoimmune hepatitis
39. Familial occurrence of autoimmune hepatitis
M. Wozniak, M. Woynarowski, J. Socha (Warsaw, PL)
40. Liver sinusoidal endothelial cells induce anti-inflammatory CD4 + T cells
suppressing murine autoimmune hepatitis
N. Kruse, K. Neumann, K. Derkow, A.A. Kühl, A. Hamann, K. Klugewitz
(Berlin, D)
41. Frequency of autoimmune disorders and therapy in patients with autoimmune
hepatitis
A. Genunche-Dumitrescu, P. Mitrut, D. Badea, M. Badea (Craiova, RO)
42. The prediction of the recrudescence risk in autoimmune hepatitis (AIH)
P. Mitrut, A. Genunche-Dumitrescu, A.O. Mitrut, L. Nita, D. Badea
(Craiova, RO)

43. Immune disruptions at the mycotoxin fumonisin-induced autoimmune hepatitis
in young and old mice
E.A. Martinova (Moscow, R)
44. Emerging importance of autoimmune hepatitis in Bulgaria, an endemic area for
viral hepatitis
K. Kalinova, L.M. Pekova, P. Chakarova, P. Stefanova, K. Georgiev
(Stara Zagora, BG)
Cholestatic liver diseases (PBC/PSC)
45. Non-invasive assessment of liver fibrosis by transient elastography in patients
with cholestatic liver diseases (PBC and PSC)
D. Rill, A. Tudora, O.P. Ciof, E. Dragan, I. Sporea (Timisoara, RO)
46. A meta-analysis regarding the effects of ursodeoxycholic acid in patients with
primary sclerosing cholangitis
E.C. Rezi, R.G. Mihaila, M. Deac (Sibiu, RO)
47. The long-term budesonide-UDCA combined therapy in patients with primary
biliary cirrhosis
A. Genunche-Dumitrescu, P. Mitrut, M. Badea, D. Badea (Craiova, RO)
Malignant and benign neoplasm
48. Value of transient elastography (FibroScan ® ) in the evaluation of patients with
hepatocellular carcinoma (HCC)
O.P. Ciof, A. Tudora, E. Dragan, D. Rill, M. Martin (Timisoara, RO)
49. Constitutive active gp130 in the liver and its connection to chronic inflammation
and carcinoma development
A. Schütt, J. Scheller, S. Rose-John (Kiel, D)
50. Increased intrahepatic infiltration with CD4+CD25+FOXP3+ regulatory T cells
correlates with increased tissue levels of TGF-beta and IL-10 in patients with
primary or metastatic liver cancers
I.M. Manolova, M.V. Gulubova, D. Kyurkchiev, J. Ananiev, I.P. Altunkova,
A. Julianov (Stara Zagora, Sofia, BG)
51. TGF-beta occurrence and macrophage infiltration in liver metastasis
J. Ananiev, M.V. Gulubova, I.M. Manolova, A. Matev (Stara Zagora, Sofia, BG)
52. TGF-beta inhibits dendritic cell infiltration in liver metastasis
M.V. Gulubova, I.M. Manolova, J. Ananiev, Y. Yovchev (Stara Zagora, BG)
53. Focal nodular hyperplasia of the liver within our experience
H. Cichoz-Lach, B. Prozorow-Król, J. Swatek, K. Celinski, M. Slomka, L. Buk,
E. Korobowicz, E. Lis (Lublin, PL)
11

12
Basic immunology
54. Liver sinusoidal endothelial cells induce TGF-β dependent conversion of
CD4+Foxp3+ regulatory T cells from conventional CD4+CD25- T cells
A. Carambia, S. Huber, S. Lüth, D. Schwinge, C. Frenzel, C. Schramm,
A.W. Lohse, J. Herkel (Hamburg, D)
55. Alcohol-metabolizing enzyme gene polymorphisms in alcohol liver cirrhosis
among Polish men
H. Cichoz-Lach, E. Lis, K. Celinski, M. Slomka (Lublin, PL)
56. Conversion of naïve T cells into regulatory T cells after tolerance induction in
the murine liver
B. Claaß, A. Erhardt, G. Tiegs (Hamburg, D)
57. Impact of IL-6-transsignaling on liver damage and regeneration after CCI4
J. Gewiese, C. Drucker, J. Scheller, S. Rose-John (Kiel, D)
58. CXCL10 plays a key role in the development of hepatic fibrosis
E. Hintermann, M. Bayer, A. Luster, U. Christen
(Frankfurt, D; Charlestown, USA)
59. Epitope mapping of human cytochrome P450 2D6 (CYP2D6) in patients with
autoimmune hepatitis and in the CYP2D6 mouse model
E. Hintermann, M. Holdener, M. Bayer, M.P. Manns, U. Christen
(Frankfurt, Hannover, D)
60. The role of the hepatic asialoglycoprotein receptor in inflammatory-induced liver
injury and accumulation of intrahepatic lymphocytes
B.L. McVicker, D.J. Tuma, G.M. Thiele, C.A. Casey, N.A. Osna (Omaha, USA)
61. Common variable immunodeficiency-associated granulomatous disease and
nodular regenerative hyperplasia of the liver
H. Varnholt, T. Rubinas (Chapel Hill, USA)
62. Aggravation of liver damage and loss of immunological tolerance in CXCR3deficient
mice in the murine model of concanavalin A-induced liver injury
C. Wegscheid, A. Erhardt, U. Panzer, G. Tiegs (Hamburg, D)
63. Metallothioneins (MT I and MT II) and their gene expression in some chronic
liver diseases
M. Sharaf-Eldin, I. Saber, U. Negm, H. Hamouda, A.A. Hameed (Tanta, ET)
64. Endothelial chemokine presentation as therapeutic target in T-cell mediated
hepatitis
K. Neumann, K. Wechsung, N. Kruse, M. Schumann, A.A. Kühl, A. Hamann,
K. Klugewitz (Berlin, D)

Hepatic vascular disorders, drugs and toxicity, epidemiology and
endoscopy posters
65. Hemostatic disturbances in patients with portal thrombosis
E. Lukina, E. Sysoeva, E. Jakovleva, E. Kitsenko, G. Sukhanova, S. Vasijev
(Moscow, R)
66. False iron deficiency in patients with prehepatic portal hypertension
E. Lukina, E. Sysoeva, A. Levina, Y. Mamukova, E. Voronkova, E. Kitsenko
(Moscow, R)
67. Diagnostics improvement for liver chronic diseases in Turkmenistan
O.B. Nepesova (Ashgabat, XTU)
68. Epidemiology of liver diseases in a pediatric population. Snapshot data from
selected centers in Poland
M. Woynarowski, M. Wozniak, W. Chlebcewicz-Szuba, T. Chmurska Motyka,
A. Gorczyca, B. Korczowski, A. Krzywicka, D.M. Lebensztejn, A. Liberek,
E. Majda-Stanislawska, M. Rokitka, E. Strawinska, S. Wiecek, I. Zaleska,
A. Zdanowska-Ruskan, J. Socha (Warsaw, PL)
69. Alcohol withdrawal treatment audit March 2009, Northern General Hospital,
Sheffield, UK
A. Al-Joudeh, H. Morton, S. Heikal, K. Basu (Sheffield, GB)
70. Effect of different degrees of hepatic dysfunction on the pharmacokinetics of
telmisartan
M. El-Hamamsy, S. Hegazy, W. Awara (Cairo, ET)
71. Risk perception and use of herbal remedies in patients with liver/biliary tract
disorders: An Italian study
M. Marignani, S. Gallina, M. Di Fonzo, I. Deli, P. Begini, F. Attilia, E. Gigante,
M. Epifani, S. Angeletti, G. Delle Fave (Rome, I)
72. Hepatotoxicity induced by antituberculosis drugs
M. González, E. Aravena, C. Peña, E. Astrosa, J. Lyon, G. Serrano, P. Escobar
(Santiago de Chile, RCH)
73. Treatment with Ursofalk ® of bile reflux gastritis
D. Pestroiu, M. Ilie, D. Brooks, C. Vladut (Bucharest, RO)
74. Incidence and therapy of colonic perforations: How feasible and effective is
endoscopic closure?
K. Mönkemüller, H. Neumann, P. Malfertheiner, H.-U. Schulz, L.C. Fry
(Bottrop, Magdeburg, D)
13

Session I
Liver immunology I
15

Immune responses mediated by endothelial cells
Percy A. Knolle, MD
Institute of Molecular Medicine, Bonn, Germany
The liver is an immune organ favouring induction of immune tolerance rather than
T cell immunity. Both, organ-resident cell populations as well as the unique hepatic
microenvironment contribute to skewing of immune responses towards induction of
tolerance.
Endothelial cells of the liver, in particular liver sinusoidal endothelial cells (LSEC)
constitute one these organ-resident cell populations with important immune functions.
LSEC play a central role in local immune regulation in the liver, as they scavenge
most efficiently soluble circulating antigens for cross-presentation on MHC I
molecules to CD8 T cells. Cross-presentation to naïve CD8 T cells by LSEC in vitro
and in vivo leads to induction of a tolerant T cell phenotype characterized by lack of
cytokine expression and cytotoxic effector function upon restimulation. Mechanistically,
mutually tolerogenic signaling through the co-inhibitory molecules B7H1
and PD1 on LSEC and naïve CD8 T cells, respectively, is required for tolerance
induction. Moreover, LSEC function as veto cells to impede antigen-presentation by
dendritic cells to naïve T cells while they reside in the liver and thereby further skew
local hepatic immune responses towards tolerance induction. Importantly, LSEC play
an important role in the effector phase of CD8 T cell immune responses in the liver.
Antigens released from virus-infected hepatocytes are cross-presented by LSEC to
effector CD8 T cells and initiate virus-specific immunity even in the absence of direct
antigen-recognition on hepatocytes, thus demonstrating that cross-presentation is
important also during the execution phase of the immune response in the target
tissue.
Given their strategic localization in the hepatic sinusoid separating hepatocytes from
circulating lymphocytes LSEC play an important role both in skewing of immune
responses towards tolerance induction and immune surveillance during viral
infection.
17

NKT cells within the liver
M.G. Swain, MD, MSc, FRCP
Professor of Medicine, University of Calgary, Calgary, AB, Canada
NKT cells are distinct innate immune T cells which play key roles in the pathogenesis
of various immune-mediated liver diseases. NKT cells are traditionally defined as
cells that co-express the T cell receptor (TCR) and certain natural killer cell surface
markers (e.g. NK1.1 in some mouse strains). Based on CD1d restriction, they are
broadly classified as invariant (iNKT) and non-invariant NKT cells. Both NKT cell
types are abundant within the liver and, mainly through the study of animal models of
immune-mediated liver injury, the role of NKT cells in the context of liver inflammation
is becoming better understood. iNKT cells are the NKT cell subtype which has been
best characterized and therefore this presentation will focus mainly on iNKT cells.
NKT cells recognize both host and microbial glycolipid antigens, presented in the
context of the CD1d molecule, via their TCR. Upon activation NKT cells rapidly
release large quantities of T helper type 1 (Th1; e.g. IFNγ, TNFα), Th2 (e.g. IL-4,
IL-10), and Th17 (e.g. IL-17, IL-21) type cytokines with diverse immunoregulatory
and immunomodulatory effects. Moreover, through their release of these cytokines
NKT cells can drive subsequent downstream immune responses in tissues such as
the liver, including the upregulation of adhesion molecule expression and increased
chemokine production. By regulating immune cell adhesion pathways and chemokine
secretion, NKT cells can direct both proinflammatory (e.g. Th1 cell) and anti-inflammatory
(e.g. regulatory T cell) cell infiltration into the liver. Through this mechanism
NKT cells orchestrate a balance between pro- and anti-inflammatory responses
within the liver during immune mediated liver damage. In this presentation I will
highlight the diverse roles played by iNKT cells as master regulators of immunemediated
liver injury.
18

Regulatory T cells induced within the liver
Johannes Herkel
Department of Medicine I, University Medical Centre Hamburg-Eppendorf, Hamburg,
Germany
CD4 + CD25 + Foxp3 + regulatory T cells (Treg) cells are important mediators of immune
tolerance to self-antigens, and their inactivation may cause autoimmune disease.
Therefore, immunotherapy for autoimmune diseases may aim at restoring or
expanding autoantigen-specific Treg activity. We reported recently that Tregmediated
suppression of autoimmune disease could be achieved in vivo, by taking
advantage of the liver’s ability to promote immune tolerance (Lüth et al. J Clin Invest
2008; 118: 3403–3410). Ectopic expression of the neural autoantigen myelin basic
protein (MBP) in the liver, which was accomplished constitutively in liver-specific
MBP-transgenic mice, or transiently after gene transfer to liver cells in vivo, induced
protection from autoimmune neuroinflammation in a mouse model for human multiple
sclerosis. Protection from autoimmunity was mediated by MBP-specific
CD4 + CD25 + Foxp3 + Treg cells, as demonstrated by adoptive transfer to nontransgenic
mice. The protective Treg were MBP-specific, since they efficiently
suppressed conventional CD4 + CD25 - T cells after antigen-specific stimulation with
MBP in vitro. The generation of these MBP-specific Treg cells in vivo depended on
expression of MBP in the liver, but not in the skin, and occurred by TGFβ-dependent
peripheral conversion from conventional CD4 T cells to CD4 + CD25 + Foxp3 + Treg. To
further address the mechanism of Treg generation in the liver, we analyzed which
liver cell type could mediate the conversion to Treg, and found that the liver
sinusoidal endothelial cells (LSEC) seem to be efficient converters. Indeed, the rate
of TGFβ-dependent conversion of non-Treg into Treg induced by LSEC was
significantly greater than that induced by dendritic cells. These findings indicated that
autoantigen expression in the liver may generate autoantigen-specific Treg cells and
that hepatic Treg generation seems to be an important component of hepatic
tolerance.
19

Suicidal emperipolesis mediates deletion of T cells undergoing
primary activation in the liver
Patrick Bertolino
Centenary Institute of Cancer Medicine and Cell Biology, Sydney, Australia
E-Mail: p.bertolino@centenary.usyd.edu.au
Despite being a non-lymphoid organ, the liver displays immunological properties
distinct from other solid organs and is associated with the induction of T cell
tolerance. This property has been demonstrated in several clinical settings including
transplantation, and hepatotropic viral infections, such as those induced by hepatitis
B and C viruses (HBV and HCV).
Many models have been proposed to explain the “liver tolerance effect”, but the
molecular and cellular mechanism/s mediating this phenomenon remain unknown.
We have previously shown that the liver is the only non-lymphoid organ able to retain
and activate naïve CD8 + T cells independently of lymphoid tissues in an antigenspecific
manner. These findings, confirmed by other groups, have opened new
possibilities to explain the remarkable property of the liver to induce antigen-specific
tolerance in transplantation and following infection by hepatotropic viruses such as
HCV and HBV.
To investigate the mechanisms by which the liver induces antigen-specific tolerance,
we have studied the fate of T cell activated intrahepatically in a model in which most
T cells are retained and activated in this organ. Naïve transgenic Des-TCR CD8 +
T cells specific for the MHC class I molecule H-2K b plus self peptide were adoptively
transferred into mice expressing the antigen ubiquitously (C57BL/6 or C57BL/6x C3H
F1) or into syngeneic mice with no antigen expression (B10.BR). Des-TCR cells were
either radio-labelled with 51 Cr or labelled with the fluorochrome CFSE before transfer.
Within 1 hour, most T cells were retained in the liver of C57BL/6 mice and
upregulated the early activation marker CD69 within 30 min after adoptive transfer.
Despite accumulation of potentially autoreactive T cells, no clinical signs of dermatitis
(rash), colitis (diarrhea) or serological hepatitis (ALT levels) were observed in
C57BL/6 mice injected with CD8 + Des-TCR T cells. Flow cytometry and histology
confirmed that lower numbers of CFSE-labelled Des-TCR T cells were detected in
the liver of C57BL/6 mice within 22 hours after adoptive transfer suggesting that
T cells were physically eliminated in the recipient animals in an antigen-specific
manner.
To determine whether the fallen T cell numbers recovered from the liver was due to
migration to other sites, donor cells were radio-labelled prior to transfer, and organs
harvested at 1 and 22h. Consistent with CFSE data, up to 70% of the total
radioactivity recovered from B6 and F1 animals was contained within the liver at 1h.
Surprisingly, in contrast to the CFSE studies, radioactivity distribution remained
almost unchanged at 22h suggesting that the donor cell content was still retained
within the liver, despite failure to recover viable transferred autoreactive cells. Few
apoptotic cells were detected in the livers of recipient B6 mice during the first 22h
and neither macrophages nor exposure of phosphatidylserine on the T cell surface
following intrahepatic activation were critical for rapid T cell clearance, as inhibition of
20

these pathways failed to prevent deletion. This deletion was not mediated by NK cells
or allogeneic host T cells and was not due to cell phagocytosis by Kupffer cells.
Instead, T cells actively invaded hepatocytes and were found into Lamp-1 coated
vesicles suggesting that T cells are degraded into lysosomes.
In summay, we identify a novel mechanism of peripheral deletion in which the
majority of naïve autoreactive CD8 + T cells is rapidly eliminated in the liver following
intrahepatic activation. T cells actively invade hepatocytes, enter endosomal/lysosomal
compartments where they are degraded by a non-apoptotic mechanism.
Invasion of a cell into another cell is known as emperipolesis, a long-observed
phenomenon for which a physiological role has not been previously demonstrated.
We propose that T cell suicidal emperipolesis is a novel mechanism of autoreactive T
cell deletion, a process critical for the maintenance of tolerance.
21

Session II
Liver immunology II
23

Role of serotonin and iNOS in LCMV-induced hepatitis
Dr. K.S. Lang
Humboldt Research Group, Department of Gastroenterology, Hepatology and
Infectiology, Heinrich Heine University Düsseldorf, Germany
More than 500 million people worldwide are persistently infected with hepatitis B
(HBV) or hepatitis C virus (HCV). Although both viruses are poorly cytopathic,
persistence of either virus carries a risk of chronic liver inflammation potentially
resulting in liver-steatosis, liver cirrhosis, end stage liver failure or hepatocellular
carcinoma. Virus-specific T cells are a major determinant of the outcome of hepatitis
since these cells contribute to the early control of chronic hepatitis viruses, however
they also mediate immunopathology during persistent virus infection.
We have analyzed the role of the platelet-derived vasoactive serotonin during virusinduced
CD8 + T-cell dependent immunopathological hepatitis in mice infected with
the noncytopathic lymphocytic choriomeningitis virus (LCMV). Following virus
infection, platelets were recruited to the liver and their activation correlated with
severely reduced sinusoidal microcirculation, delayed virus elimination and increased
immunopathological liver cell damage. Lack of platelet-derived serotonin in serotonin
deficient mice normalized hepatic microcirculatory dysfunction accelerated virus
clearance in the liver and reduced CD8 + T-cell dependent liver cell damage. In
keeping with these observations, serotonin treatment of infected mice delayed entry
of activated CD8 + T-cells into the liver, delayed virus control and aggravated
immunopathological hepatitis.
Next we analyzed the role of dendritic cells in virus infected livers. We found that
dendritic cells were recruited into the liver during infection with lymphocytic
choriomeningitis virus. Liver-infiltrating CD11c + cells expressed high levels of MHC II
and iNOS. Interestingly, in the liver, iNOS expression in dendritic cells enhanced
T cell mediated IFN-γ production. Conversely, IFN-γ production by T cells enhanced
iNOS expression. Reduced IFN-γ production led to enhanced infection of
hepatocytes followed by severe immunopathology.
In conclusion, Serotonin and iNOS influence CD8 T cell function in the liver, which
influences T cell mediated immunopathology during virus infection.
25

Lymphocyte homing to the liver
David H. Adams
Centre for Liver Research, 5th Floor IBR, The University of Birmingham Medical
School, Wolfson Drive, Edgbaston, Birmingham, B15 2TT, UK
In chronic liver disease leucocyte recruitment from blood drives inflammation, fibrosis
and cirrhosis. The liver has a dual blood supply from the portal vein and hepatic
artery the terminal branches of which supply the parenchyma via hepatic sinusoids
lined by endothelial cells intimately associated with pericytes called hepatic stellate
cells. Blood percolates slowly through the sinusoids before re-entering the circulation
via hepatic veins. Sinusoidal endothelium is morphologically and functionally distinct
from endothelium in other tissues, lacking both tight junctions and a basement
membrane. These unique features suggest that lymphocyte recruitment via the
sinusoids will involve distinct mechanisms. I will review how specific combinations of
chemokines and adhesive events regulate lymphocyte recruitment in the context of
this unique microanatomical environment. The effect of low flow rates in sinusoids on
the molecular requirements for capture and the consequences of the discontinuous
nature of sinusoidal endothelium on the dynamics of transendothelial migration will
be discussed. I will also present evidence to show how all of these processes are
modulated by paracrine signals from adjacent cells in the sinusoid including epithelial
cells (hepatocytes), fibroblasts (stellate cells) and haematopoietic cells (Kupffer
cells). Under basal conditions these paracrine signals maintain sinusoidal endothelial
phenotype and function and in response to inflammation prime endothelium to recruit
effector cells thereby driving the development and maintenance of chronic
inflammation.
26

Monitoring immunosuppression after transplantation
Stefan Meuer, Claudia Sommerer, Martin Zeier and Thomas Giese
University Hospital Heidelberg, Germany
With the introduction of calcineurin inhibitors (CNI) long-term allograft function has
significantly improved. The problem of limited therapeutic margins and the toxicity of
CNI remain unsolved. The recently reported quantitative assessment of inhibition of
NFAT-regulated gene expression 2 hr after Cyclosporine A (CsA) intake represents a
novel approach to evaluate the biological effectiveness of CsA therapy and provides
means to enable individualized immunosuppressive regimens.
In more than 200 patients carrying kidney and in 55 patients with heart allografts we
compared the degree of inhibition of IL-2, IFN-γ and GM-CSF gene expression with
the peak blood concentration of CsA. Functional immunosuppression as assessed by
qRT-PCR varies considerably among CsA treated individuals with stable graft
function. Given the relatively constant level of inhibition over a broad range of drug
concentrations, we felt that a considerable group of patients with unnecessarily high
CsA doses might benefit from a reduced dosing of the drug without compromising the
efficacy of the immunosuppressive therapy. Importantly, we could demonstrate that
patients with infectious episodes during the period of observation had a significant
stronger NFAT-inhibition than patients without complications, indicating that this
pharmacodynamic assay can better predict the risk of infection due to excessive
immunosuppression of transplanted patients in comparison to the conventional
C2-monitoring of CsA. In several patients the doses of CsA could be safely reduced
without significantly changing the level of immunosuppression.
In conclusion, patients treated with CsA might benefit from a reduced dosage of the
drug, if they respond to the drug with a strong inhibition of NFAT-regulated gene
expression.
27

Cancer immunity and cancer immunotherapy
Alexander Knuth
Department of Oncology, University Hospital of Zurich, Zurich, Switzerland
Nearly 35 years ago a new strategy was introduced for the discovery of human
cancer antigens called ‘autologous typing’. Cancer reactive serum antibodies were
tested for their specificity in reacting with autologous tumor cells in vitro. The concept
of autologous typing was then extended to the study of T-lymphocyte clones (CTL)
with testing their specificity for autologous cancer cells in vitro. Combined with
molecular cloning techniques, CTL clones were employed to identify the first human
cancer specific antigen family, the MAGE gene family in a patient MZ-2 from the
University Hospital in Mainz, Germany. MAGE became the prototype of what now is
known as the cancer testis family of antigens (CT-Ag). CT antigens have a unique
expression pattern, i.e. predominantly in cancer cells of different tissue origins and in
germ cells but not in any other normal tissues. The molecular function of these
intracellular antigens is largely unknown, although 10% of the genes encoded on the
X-chromosome appear to be CT antigens. Their immunogenicity in vivo is indicated
by spontaneous and often strong immune responses in cancer patients. The CT
antigen NY-ESO-1 is the most immunogenic human cancer antigen known to date,
often leading to integrated immune responses with NY-ESO-1 specific T cell
responses as well as antibodies. The presence of NY-ESO-1 specific antibodies
indicates the presence of disease and of specific T cells in approximately 50% of
patients. CT-antigen specific immune interventions may induce T cells and antibodies
or may augment pre-existent CT-antigen specific immunity. The cellular and
molecular networks shaping these immune responses are studied with respect to
regulatory T cell subsets and inhibitory receptors like CTLA-4 and other agents to
influence immune reactions in cancer patients. In combination with new antigen
formulations and delivery technologies, adjuvants and regulators of specific immune
responses, cancer immunotherapy will become a valid modality in cancer treatment.
28

Pathology of immune-mediated liver injury
H.P. Dienes, U. Drebber
Institute of Pathology, University of Cologne, Cologne, Germany
The liver is a special lymphoid organ with its own defense mechanisms and is prone
to chronic viral and autoimmune diseases. All constituent liver cells are involved in
immune mechanisms against foreign toxins and agents: hepatocytes, Kupffer cells,
endothelial cells, hepatic stellate cells (HSC) and liver resident lymphocytes as NK,
NKT and dendritic cells. The first line of defense is build up by innate immune system
where as the second line and more antigen specific immune barrier is the adaptive
immune response. Even in drug injury of the liver the immune system is involved. In
the following contribution I just concentrate on some major issues of various
etiologies and the corresponding immune reaction with underscoring the histopathology
of immune mediated lesions in the liver.
In drug induced liver injury mediated by idiosyncratic mechanisms the innate immune
system is activated and can lead to hepatocellular necrosis especially in zone 3.
There is an activation of NK and NKT cells and liver cell damage is induced mainly
by the Fas/FasL pathway, a mechanism that has been investigated for the side
effects in acetaminophen and dihydralazine damage.
In viral hepatitis the pathway of liver injury is mainly immune mediated since almost
none of the hepatitis viruses have turned out to be cytopathic by itself.
In hepatitis A the viral agents are orally transmitted and invade the liver by the portal
vein blood. The predominant liver lesions are therefore in the portal and periportal
area with cytotoxic lymphocytes being the main effectors cells. In HBV infection
about 5 to 10 percent of the individuals run a chronic course and the severity of liver
damage is completely dependent on the presence of an efficient immune response to
the virus with CDS antigen specific cytotoxic lymphocytes. In hepatitis C virus
infection the percentage of a chronic course is much higher than in HBV infection.
Several mechanisms have been explored, that could show, that the viral components
interfere with intracellular signals of interferon production but also at the level of
cellular immune response. We could show that the percentage of inhibitory Tregs is
higher in chronic hepatitis C than in chronic hepatitis B. The fraction of T-cells
displaying PD1, FoxpS and the number of dendritic cells with IDO are significant
larger in hepatitis C than in hepatitis B.
In hepatitis D there are almost identical histopathology and immune mechanisms as
in hepatitis B, but they are on average more severe than in hepatitis B. Also HEV is
regarded as a non cytopathic viral agent and the immune mechanisms and also
histopathology are similar to hepatitis A. In EBV infection liver cell injury seems not to
be due to direct antigen specific CTL attack since hepatocytes are not permissive for
EBV replication and hepatocytes do not harbour viral proteins. Instead the injury is
induced by so called 'bystander reaction' from activated lymphocytes in the sinusoids
of the liver. They seem to be involved Kupffer cell activated T-cell, NK and NKT cells
with various kinds of cytotoxic cytokines and chemokines. So an infection with EBV
mechanisms different from antigen specific immune response seems to be effective.
29

Autoimmune hepatitis is paradigm of immune mediated liver injury and has a special
but not pathognomonic histopathology. Necroinflammatory activity starts from the
portal and periportal area and moves on to the other zones of the lobule. By histopathology
autoimmune hepatitis is a rather progressive disease with cell necrosis
and apoptosis linked to the presence of effector lymphocytes that show the
phenomenon of emperipolesis. Periportal hepatocytes undergo resetting and the
presence of active interface hepatitis is a salient feature of autoimmune hepatitis.
In conclusion the manifestation of immune mediated liver injury is mainly determined
by the dominant mechanism if it is innate immune response, adaptive immune
answer or 'bystander reaction'. Histopathology reflects different patterns of the
etiologic agent and pathways of defense mechanisms.
30

Session III
Autoimmunity I
31

Adaptive immunity in autoimmune hepatitis
Diego Vergani, MD, PhD, FRCPath, FRCP; Giorgina Mieli-Vergani, MD, PhD,
FRCPCH, FRCP
Institute of Liver Studies, King’s College London School of Medicine at King’s College
Hospital, Denmark Hill, London SE5 9RS, UK
The histological lesion of interface hepatitis, with its striking infiltrate of lymphocytes,
plasma cells, and monocytes/macrophages was the first to suggest an autoaggressive
cellular immune attack in the pathogenesis of autoimmune hepatitis (AIH).
Immunohistochemical studies, focused on the phenotype of the inflammatory cells
infiltrating the liver parenchyma in AIH, have revealed a predominance of alpha/beta
T-cells. Amongst these cells, a majority was found to be positive for the CD4
helper/inducer phenotype and a sizeable minority for the CD8 cytotoxic/suppressor
phenotype. Lymphocytes of non-T-cell lineage included NK cells, monocytes/macrophages
and B-lymphocytes.
Tr
APC
APC
Peptide
Co-stimuli
TGF-β
Class II
Th0
IL-4
IL-12
Th2
Liver
cell
Class II
Th1
IL-4
Class I
IL-2
IL-10
IFN-γ
IFN-γ
IL-13
IL-6
CTL Tc
T Tc C
B
M
IL-1
NK
P
Th17 Ts
Liver damage is believed to initiate with the presentation of a self-antigenic peptide to
the T-cell receptor (TCR) of an uncommitted T helper (TH0) lymphocyte by professional
antigen presenting cells (APCs), such as macrophages, dendritic cells and
B-lymphocytes, in the presence of co-stimulating signals, induced by the interaction
of CD28 on TH0 and CD80 on APC. Once exposed to the antigen embraced by an
HLA class II molecule on the APC, the TH0 lymphocytes become activated and,
according to the presence in the milieu of interleukin 12 (IL-12) or IL-4, they
differentiate into TH1 or TH2 cells and initiate a series of immune reactions
determined by the cytokines they produce. TH1 cells predominantly secrete IL-2 and
IFN-gamma, the latter being the main orchestrator of tissue damage because of its
ability to stimulate cytotoxic T-cells (CTL), to enhance the expression of HLA class I
molecules on APCs and of HLA class II molecules on hepatocytes and activate
TNF-α
IL-17
IL-17
33

monocytes/macrophages, which in turn release IL-1 and TNF-alpha. The expression
of HLA class II on hepatocytes, that normally do not express it, enables them to
present the autoantigen to TH1 lymphocytes and hence the perpetuation of the
autoimmune process. The function of TH1 cells is counterbalanced by that of TH2
cells, arising in the presence of IL-4 and mainly producing IL-4, IL-10 and IL-13.
These cytokines induce also the maturation of B-cells into plasma cells, with
consequent production of autoantibodies. As regulatory cells in AIH are numerically
and functionally deficient, effector responses are perpetuated with ensuing persistent
liver cell destruction by the direct action of CTL, cytokines released by TH1 cells and
monocytes/macrophages, complement activation or engagement of natural killer
(NK) cells by the autoantibodies bound to the hepatocyte surface. The role of the
recently described TH17 cells, which arise in the presence of transforming growth
factor beta (TGF-beta) and IL-6, is under investigation.
34

Old and new animal models of AIH
E. Jaeckel, M.D.
Department of Gastroenterology, Hepatology, Endocrinology, Diabetology, Hannover
Medical School, Hannover, Germany
Autoimmune hepatitis (AIH) is difficult to study in humans because of the late
diagnosis, the heterogenic genetic background and the lack of tissue specimens.
Therefore, animal models have been used for more than 90 years to study liverspecific
immune regulation. Early studies of experimental AIH established the
importance of T cells for causing the liver injury and how regulatory pathways were
detected. Studies in transgenic models demonstrated that T cells can be tolerant to
liver antigens by ignorance, deletion, anergy, and receptor downregulation. They
furthermore defined functions in antigen presentation for hepatocytes and sinusoidal
endothelial cells. Studies in cytokine-induced models demonstrated common effector
cascades resulting from T cell activation and the interaction of innate and adaptive
immune response. In the past approaches for breaking tolerance against liver
antigens in animal models were just able to induce transient and self-limiting immune
responses. Here we have developed a strategy to break tolerance of a negatively
selected, wild-type T cell repertoire by combining a self limited infection providing a
danger signal together with modified liver self-antigens into mice. To this end we
generated replication-deficient adenoviral constructs expressing common
autoantigens of human AIH showing homology up to 80% with the murine proteins.
Following an acute phase of liver destruction with increasing levels of transaminases
two weeks after intravenous injection of the virus, we could show chronic evolving
hepatic autoimmune reactions after twelve weeks. Chronic liver disease was
confirmed by consistent leukocyte infiltrates and antigen-specific autoantibodies
within the sera. Immunofluorescent staining of liver sections revealed that CD4 T
cells are more abundant than CD8 T cells in these liver infiltrates. Control
experiments with expression vectors devoid of viral components did not result in any
hepatic reaction after the acute viral infection had been cleared. Furthermore chronic
AIH was not induced in FVB mice. However we detected autoimmunity within the
liver of non-obese diabetic (NOD) mice which received viral constructs expressing
FTCD, an important auto-antigen in AIH type II. Surprisingly the more important AIH
type II auto-antigen CYP2D6 as well as the type III soluble liver antigen (SLA) did not
result in any chronic hepatic immune reaction. This strain and antigen-specificity of
murine AIH supports the notion that autoimmunity develops in genetically
predisposed individuals. To identify the leukocyte populations involved in hepatitis
within our model we isolated liver-specific leukocytes of hepatitis-bearing mice and
analysed these by flow cytometry as well as by a new technique of microimmunology
called iterative Slide Based Cytometry (iSBC). Taken together, this murine model of
chronic AIH will be significant in the study of liver-specific autoimmunity particularly
with regard to the liver-specific immune regulation by different T cell populations.
Moreover the novel model might open new therapeutic options to treat the disease.
35

Cytochrome P450 2D6 as a model autoantigen
Urs Christen, PhD
Klinikum der Johann Wolfgang Goethe-Universität Frankfurt, Pharmazentrum
Frankfurt/ZAFES, Theodor-Stern Kai 10, Frankfurt am Main, Germany
The etiology of autoimmune hepatitis (AIH) is poorly understood although the major
autoantigen, cytochrome P450 2D6 (CYP2D6), has been identified and immunodominant
epitopes mapped. One reason might be the lack of an appropriate animal
model that reflects the pathogenic processes occurring in patients suffering from AIH.
Often experimental hepatitis was reported to be only transient and many current
models for autoimmune liver disease depend on a rather complex disease induction
protocol. Therefore, we generated a mouse model for human AIH using the natural
human autoantigen CYP2D6 as a triggering molecule for autoimmunity. We infected
wildtype FVB mice with an adenovirus expressing the human CYP2D6 (Ad-2D6) to
break self-tolerance to the mouse CYP2D6 homologues. Ad-2D6-infected mice
showed several persistent features characteristic for liver damage associated with
AIH. These features included massive hepatic fibrosis, ‘fused’ liver lobules,
disorganized architecture, cellular infiltrations and focal to confluent necrosis.
Further, all Ad-2D6-infected mice generated high titers of anti-CYP2D6 antibodies.
Epitope mapping revealed that such anti-CYP2D6 antibodies predominantly
recognized the identical immunodominant linear epitope WDPAQPPRD that is
recognized by LKM-1 antibodies from AIH patients. In contrast, mice infected with a
control adenovirus (Ad-C) did neither develop liver damage nor generated anti-
CYP2D6 antibodies. Interestingly, Ad-2D6-infection of transgenic mice expressing
the human CYP2D6 in the liver (CYP2D6 mice) resulted in a delayed kinetics and a
reduced severity of liver damage. Antibody formation was only moderately reduced in
transgenic CYP2D6 mice compared to wildtype FVB mice. Further, anti-CYP2D6
antibodies generated in CYP2D6 mice did react to the major B-cell epitope
WDPAQPPRD indicating that B cell tolerance induction does not account for the
observed differences in disease kinetics and severity between wildtype and CYP2D6transgenic
mice.
We analyzed the T cell mediated immune response by stimulating lymphocytes with
61 overlapping 20-mer peptides covering the entire human CYP2D6. T cells
produced IFNγ after stimulation with peptides only if isolated from Ad-2D6-infected
mice. The frequency of CYP2D6-specific T cells was more then three times higher in
lymphocytes isolated from the liver then from the spleen indicating either an organ
specific proliferation or a selective retention process in the liver. Importantly, the
frequency of CYP2D6-specific CD4 as well as CD8 cells was dramatically decreased
in transgenic CYP2D6 mice indicating the presence of a strong T cell tolerance to
human CYP2D6 established in transgenic CYP2D6 expressing the identical target
antigen compared to wildtype FVB mice expressing the mouse homologues only.
T cell epitope mapping revealed that CYP2D6-specific T cells reacted to human
CYP2D6 peptides with intermediate homology to the mouse homologues but not to
those with high homology or identity. Our data indicate that molecular mimicry rather
than molecular identity breaks tolerance on both the B cell and T cells level
subsequently causing severe and persistent autoimmune liver damage.
36

In summary, the CYP2D6 mouse model for autoimmune hepatitis uses a true human
autoantigen, which may be important for establishing a model that comes close to the
immunopathogenesis of autoimmune liver disease as observed in patients with AIH.
The CYP2D6 model should therefore provide a platform to investigate mechanisms
involved in the immunopathogenesis of autoimmune mediated chronic hepatic injury
as seen in human AIH and to evaluate possible ways of therapeutic interference.
37

Spontaneous chronic liver inflammation in a new transgenic
mouse model of autoimmune hepatitis: Impact and regulation
of autoreactive CD8 + T cells
Dr. Mario Zierden
Institute for Pathology, University Hospital of Cologne, Kerpener Str. 62,
50924 Cologne, Germany
Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease of unknown
etiology. Autoreactive T cells are believed to initiate and orchestrate the destruction
of hepatocytes but the pathogenesis of AIH remains poorly understood.
To investigate liver-specific CD8 + T-cell responses as well as the impact and
regulation of autoreactive CD8 + T cells in the pathogenesis of AIH, we have
generated transgenic mice that express the influenza virus hemagglutinin (HA) model
autoantigen under the control of albumin regulatory elements exclusively in the liver
(Alb-HA mice), affording presentation of the neoself-antigen by hepatocytes and by
professional APCs. Adoptive transfer of HA-specific naïve CD8 + T cells into Alb-HA
mice leads to an activation of autoreactive CD8 + T cells and elicits transient hepatitis.
Alb-HA/CL4-TCR double transgenic mice, in which the majority of CD8 + T cells
express a HA-specific TCR, spontaneously develop persistent autoimmune-mediated
hepatitis characterized by moderate liver inflammation and elevated alanine
aminotransferase level. Liver infiltrates are dominated by HA-specific CD8 + T cells
that display diminished TCR expression, no particular signs of apoptosis induction,
reduced proliferative capabilities and only little effector function upon restimulation
with HA peptide, compatible with the state of T-cell anergy. Moreover, liver infiltrates
are enriched for CD4 + /CD25 + /FoxP3 + regulatory T cells that are potent suppressors
of CD8 + T-cell activation in vitro, suggesting that regulatory CD4 + T cells contribute to
the suppression of a broad HA-specific autoimmune response.
Our results show that even if many autoreactive liver-specific CD8 + T cells are
constantly generated that elicit chronic hepatitis, peripheral tolerance mechanisms
like the induction of T-cell anergy and the accumulation of regulatory CD4 + T cells
protect the liver under normal conditions from fatal injury.
38

Induction of tolerance in a mouse model of inflammatory liver
disease
Gisa Tiegs
Division of Experimental Immunology and Hepatology, University Medical Center
Hamburg-Eppendorf, Hamburg
We have recently observed in a model of immune-mediated liver injury inducible by
the T cell mitogenic lectin concanavalin A (ConA) that C57BL/6 mice developed long
term tolerance towards a second ConA injection within 8 days after the first
application which lasted for at least 6 weeks. Immune-mediated liver injury in this
model depends on activation of CD4 + T cells, NKT cells, and Kupffer cells and is
mediated by T cell derived IFNγ and Kupffer cell derived TNFα whereas IL-10 is
protective. The immunopathology of the ConA model shares certain features of
autoimmune hepatitis, i.e. relevance of CD4 + T cells, genetic prevalence regarding
haplotype-specificity and susceptibility, good responsiveness to immunosuppressive
drugs, and immunosuppression (tolerance) in state of remission.
Induction of tolerance in the liver was characterized by significantly reduced serum
transaminase activities and improvement of liver histopathology as well as by an antiinflammatory
cytokine milieu, i.e. down-modulation of IFNγ, TNFα, IL-6, IL-17 and
IL-2 production and a concomitant increase of IL-10 release. CD4 + CD25 + Tregs as
well as Kupffer cells contributed to IL-10 release in tolerant mice, whereby the
Kupffer cells might have been differentiated into alternatively activated macrophages.
Experiments using IL-10 -/- mice argued for a critical role of IL-10 for induction of
tolerance. The tolerogenic effect was reversed upon in vivo depletion of
CD4 + CD25 + FoxP3 + Tregs in DEREG (“depletion of regulatory T cell“) mice prior to restimulation,
and CD4 + CD25 + FoxP3 + Tregs from ConA-tolerant mice produced more
IL-10 and displayed a higher immunosuppressive potential in vitro and following
adoptive transfer in vivo compared to those from non-pretreated animals.
Frequencies of PD-1 + CCR5 + CXCR3 + Treg cells where higher and those of CD62L +
Tregs were lower in adoptively transferred Tregs from tolerant mice compared to
naïve Tregs from control animals. Tolerance was not inducible in PD-1 -/- or CCR5 -/- or
CXCR3 -/- mice. Interestingly, Tregs from ConA pretreated CCR5 -/- and CXCR3 -/- mice
still retained their immunosuppressive activity in an in vitro suppression assay. These
results suggest that ConA tolerance is mediated by PD-1 expressing Tregs trafficking
into the liver in dependence of the IFNγ-inducible chemokine receptors CCR5 and
CXCR3. Hence, blockade of receptors by low molecular weight antagonists might
have detrimental consequences with respect to regulation of an inflammatory
response in the liver.
39

Session IV
Autoimmunity II
41

The role of the liver for the induction of oral tolerance in
presence of naïve and antigen-experienced T cells
Nils Kruse, Katrin Neumann, Arnhild Schrage, Katharina Eulenburg, Martin Zeitz,
Alf Hamann and Katja Klugewitz
Medizinische Klinik I und Research Center Immunosciences, Charité Campus
Benjamin Franklin, Berlin
Experimentelle Rheumatologie, Charité Campus Mitte, Berlin
Within the liver the local immune balance is shifted to tolerance rather than immunity,
although the organ can also build up a sufficient immune reaction against for
example viral hepatitis. Under physiological conditions, the portal blood flow
transports various food-derived or bacterial antigens from the intestine into the liver.
The observation that oral antigen uptake can induce antigen-specific unresponsiveness,
described already many years ago as “oral tolerance”, has raised the question
whether the liver might also participate in the induction of oral tolerance. The finding
that a portocaval shunt can abrogate oral tolerance in an animal model has enforced
this assumption.
In the healthy liver several cell populations, such as liver dendritic cells, Kupffer cells,
Ito cells and liver sinusoidal endothelial cells (LSEC) constitutively express MHCII,
suggesting the potential to function as antigen-presenting cells and, thus, contribute
to the induction of antigen-specific tolerance. In particular LSEC are believed to shift
the hepatic immune balance towards systemic unresponsiveness: LSEC can crosspresent
MHCI antigens, thereby inducing anergy in naïve CD8 + T cells and oral
tolerance.
Oral antigens are predominantly MHCII-restricted raising the question as to the
intrahepatic modulation of CD4 + T cells upon antigen feeding. Following oral antigen
application, naïve CD4 + T cells undergo apoptosis or acquire a regulatory phenotype
within the liver. However, under physiological conditions only very few naïve CD4 + T
cells enter the liver and, secondly, mesenteric lymph nodes have been demonstrated
to be obligatory for the induction of oral tolerance creating doubts whether tolerance
is locally imposed. To exclude that CD4 + T cells modulated elsewhere are recruited
into the liver, liver-restricted antigen presentation models are required. In a bone
marrow transplantation model that ensures MHCII-restricted antigen presentation
exclusively by LSEC local antigen presentation was not sufficient to establish oral
tolerance in the presence of naïve CD4 + T cells whereas Th1 effector cells that are
predominantly recruited into the liver became deleted.
In conclusion, all these findings point to a role of the liver for oral tolerance by
modulation of CD8 + and antigen-experienced CD4 + T cells populations whereas
under healthy conditions the mesenteric lymph nodes or at least professional liver
APC seem to be mandatory for the (tolerogenic) priming of naïve CD4 + T cells.
43

Therapeutic options to treat AIH in 2009
Christian P. Strassburg
Clinic for Gastroenterology, Hepatology and Endocrinology, Hannover Medical
School, Hannover, Germany
The indication for treatment of AIH is based on inflammatory activity and not so much
on the presence of cirrhosis. In the absence of inflammatory activity immunosuppressive
treatment has only limited effects. An indication for treatment is present
when aminotransferase levels are elvated and thus hepatic inflammation has to be
assumed. Usually IgG is also elevated. Liver biopsy should be performed in every
patient who is considered for therapy and the presence of interface hepatitis
underscores the decision for treatment. Biopsies will also help for the determination
of differential diagnoses and for grading and staging
Independent of clinically or immunoserologically defined type of AIH, treatment is
usually implemented with predniso(lo)ne alone or in combination with azathioprine.
Both strategies are equally effective and this strategy is based on trials that were
performed more tha 2 decades ago and has since then essentially remained
unchanged. The use of prednisone or its metabolite prednisolone is equally effective
since chronic liver disease does not seem to have an effect on the synthesis of
prednisolone from prednisone. Important is the exact differentiation between virus
infection and autoimmune hepatitis. Treatment of replicative viral hepatitis with
corticosteroids must be prevented as well as administration of interferon in AIH, in
which it can lead to dramatic disease exacerbation.
Therapy is usually administered over the course of 2 years. The decision between
monotherapy and combination therapy is guided by principle considerations: Long
term steroid therapy leads to cushingoid side effects. Especially cosmetic side effects
decrease patient compliance considerably. Serious complications such as steroid
diabetes, osteopenia, aseptic bone necrosis, psychiatric symptoms, and
hypertension and cataract formation also have to be anticipated in long term
treatment. Side effects are present in 44% of patients after 12 months and in 80% of
patients after 24 months of treatment. However, predniso(lo)ne montherapy is
possible in pregnant patients. Azathioprine, on the other hand, leads to a decreased
dose of prednisone. It bears a theoretical risk of teratogenicity. In addition, abdominal
discomforts, nausea, cholestatic hepatitis, rashes and leukopenia can be
encountered. These side effects are seen in 10% of patients receiving a dose of 50
mg per day. The side effect profile suggests that there is a need for less side effect
prone future strategies for maintenance therapy.
The consequent administration of immunosuppression is essential since most cases
of relapse are the result of erratic changes of medication and/or dose. Dose
reduction is aimed at finding the maintenance dose. Since histology lags 3 to 6
months behind the normalization of serum parameters therapy has to be continued
beyond the normalization of aminotransferase levels. Usually, maintenance doses of
prednisone range between 10 to 2.5 mg. After 12–24 months of therapy
predniso(lo)ne can be tapered over the course of 4–6 weeks to test whether a
sustained remission has been achieved. Tapering regimens should be attempted
with great caution and only after obtaining a liver biopsy which demonstrates a
complete resolution of inflammatory activity. Relapse of AIH and the risk of
progression to fibrosis is almost universal when immunosuppression is tapered in the
presence of residual histological inflammation.
44

Outcomes can be classified into four categories: remission, relapse, treatment failure
and stabilization.
Remission is a complete normalization of all inflammatory parameters including
histology. This is achieved in 65% of patients after 24 months of treatment.
Remission can be sustained with azathioprine monotherapy of 2 mg/kg bodyweight.
This prevents cushingoid side effects. However, side effects such as arthralgia
(53%), myalgia (14%), lymphopenia (57%) and myelosuppression (6%) have been
observed.
Relapse is characterized by a renewed increase of aminotransferase levels and the
re-occurrence of clinical symptoms. Relapse is present in 50% of patients within
6 months of treatment withdrawal and in 80% after 3 years. Relapse is associated
with progression to cirrhosis in 38% and liver failure in 14%. Occurrence of a relapse
calls for re-initiation of standard therapy and perhaps a long term maintenance dose
with predniso(lo)ne or azathioprine monotherapy.
Treatment failure characterizes a progression of clinical, serological and histological
parameters during standard therapy. This is seen in about 10% of patients. In these
cases the diagnosis of AIH has to be carefully reconsidered to exclude other
etiologies of chronic hepatitis. In these patients experimental regimens can be
administered or ultimately liver transplantation becomes necessary.
Stabilization is the achievement of a partial remission. Since 90% of patients reach
remission within 3 years, the benefit of standard therapy has to be re-evaluated in
this subgroup of patients. Ultimately, liver transplantation provides a definitive
treatment option.
If standard treatment fails or drug intolerance occurs, alternative therapies such as
cyclosporine, tacrolimus, cyclophosphamide, mycophenolat mofetil, rapamycin,
UDCA, and budendoside can be considered. The efficacy of these options has not
yet been definitively decided.
Deflazacort has been proposed as an alternative corticosteroid for immunosuppression
with fewer side effects than conventional glucocorticoids. In a recent
study, 15 patients with AIH type I were treated with Deflazacort, which previously had
been treated with prednisone with or without azathioprine until biochemical remission
was obtained. Remission was sustained during 2 years of follow-up. However, the
long-term role of second generation corticosteroids to sustain remission in AIH
patients with reduced treatment related side effects requires further controlled
studies.
Cyclosporine A (CyA) is a lipophylic cyclic peptide of 11 residues produced by
Tolypocladium inflatum that acts on calcium dependent signaling and inhibits T cell
function via the interleukin 2 gene. Of all alternative agents, the greatest experience
to date has been with CyA. In these studies CyA was successfully used for the
treatment of AIH and was well tolerated. The principal difficulty in advocating
widespread use of the drug as first-line therapy relates to its toxicity profile,
particularly with long-term use (increased risk of hypertension, renal insufficiency,
hyperlipidemia, hirsutism, infection, and malignancy)
45

Tacrolimus is a macrolide lactone compound with immunosuppressive capabilities
exceeding those of cyA. The mechanism of action is similar to that of CyA but it binds
to a different immunophilin. The application of Tacrolimus in 21 patients treated for
1 year lead to an improvement of aminotransferase and bilirubin levels with a minor
increase in serum BUN and creatinine levels. Although Tacrolimus represents a
promising immunosuppressive candidate drug, larger randomized trials are required
to assess its role in the therapy of AIH.
Mycophenolate has attracted attention as a transplant immunosuppressant with an
important role in the steroid free immunosuppressive therapy of patients transplanted
for chronic hepatitis C infection. Mycophenolate is a noncompetitive inhibitor of
inosine monophosphate dehydrogenase, which blocks the rate-limiting enzymatic
step in de novo purine synthesis. Mycophenolate has a selective action on
lymphocyte activation, with marked reduction of both T and B lymphocyte
proliferation. In a recent pilot study 7 patients with AIH type 1 who either did not
tolerate azathioprine or did not respond to standard therapy with a complete
normalization of aminotransferase levels were treated with Mycophenolate in addition
to steroids. In 5 out of 7 patients normalization of aminotransferase levels was
achieved within 3 months. These preliminary data suggest that Mycophenolate may
represent another promising treatment strategy of AIH. However, a recent
retrospective study on mycophenolate showed that many patients do not reach
remission and is less optimistic regarding the role of mycophenolate.
The induction of remission with 1–1.5 mg per kg per day of cyclophosphamide in
combination with steroids has been reported. However the dependency of continued
application of cyclophosphamide with its potentially severe hematological side effects
renders it a highly experimental treatment option.
Ursodeoxycholic acid is a hydrophilic bile acid with putative immunomodulatory
capabilities. It is presumed to alter HLA class I antigen expression on cellular
surfaces and to suppress immunoglobulin production. Uncontrolled trials have shown
a reduction in histological abnormalities, clinical and biochemical improvement but
not a reduction of fibrosis in 4 patients with AIH type 1. However, its role in AIH
therapy or in combination with immunosuppressive therapy is still unclear.
Budesonide is a synthetic steroid with high first-pass metabolism in the liver, which
should limit systemic side effects compared to conventional steroids. Recently the
first multicenter randomized trial testing its efficacy in inducing and maintaining
remission in non-cirrhotic patients with AIH was completed. This study was
undertaken based on previous reports of budesonide in AIH. In a study treating
13 AIH patients with budesonide over a period of 9 months the drug was well
tolerated and aminotransferase levels were normalized. Our own experiences have
confirmed that budesonide is effective but does not offer an advantage over
conventional steroids when cirrhosis and porto-systemic shunts are present and that
induction of remission can be reached in treatment naïve patients with AIH. However,
another study studying difficult to treat AIH patients with previous therapy showed
that budesonide therapy was associated with a low frequency of remission and high
occurrence of side effects. The main advantage of budesonide for the future
treatment of autoimmune hepatitis may be to replace prednisone in long-term
maintenance therapy to reduce steroid side effects. This was now shown in the
46

aforementioned large trial with 207 patients from 30 centers in Europe and Israel.
Budesonide in combination with azathioprine was able to induce complete remission
and was tested against prednisone in combination with azathioprine in a 6 month
treatment schedule followed by an open label phase in which prednisone patients
were switched to budesonide. In this study a stringent definition of remission was
employed that required a complete normalization of aminotransferase activities in
addition to the absence of steroid-specific side effects. Complete responses were
reached in the budesonide arm at 12 months in 60.2% (prednisone 49.4%) and
biochemical remission in 68% (prednisone 50.6%). In patients who were switched
from prednisone to budesonide steroid-specific side effects were reduced from
40.2% to 18.4%. These data show in one of the largest randomized placebo
controlled trials that budesonide is effective, leads to less side effects and represents
an alternative for the future of treatment of AIH.
Liver Transplantation in AIH
In approximately 10% of AIH patients liver transplantation remains as the only life
saving option. The indication for liver transplantation in AIH is similar to that in other
chronic liver diseases and includes clinical deterioration, development of cirrhosis,
bleeding esophageal varices and coagulation abnormalities despite adequate
immunosuppressive therapy. There is no single indicator or predictor for the
necessity of liver transplantation. Candidates for liver transplantation are usually
patients who do not reach remission within 4 years of continuous therapy. Indicators
of a high mortality associated with liver failure are histological evidence of
multilobular necrosis and progressive hyperbilirubinemia. In Europe, 4% of liver
transplants are for AIH. The long term results of liver transplantation for AIH are
excellent. The 5-year survival is up to 92% and well within the range of other
indications for liver transplantation.
The potential of AIH to recur after liver transplantation has been a matter of
controversial debate but are now generally accepted. It is a major reason for graft
dysfunction and loss after OLT. The first case of recurrent AIH after liver transplantation
was reported in 1984, and was based upon serum biochemistry, biopsy
findings and steroid reduction. Studies published during the past years indicate that
the rate of recurrence of AIH ranges between 10–35%, and that the risk of AIH
recurrence is perhaps as high as 68% after 5 years of follow-up. It is important to
consider the criteria upon which the diagnosis of recurrent AIH is based. When
transaminitis is chosen as a practical selection parameter many patients with mild
histological evidence of recurrent AIH may be missed. It is therefore suggested that
all patients with suspected recurrence of autoimmune hepatitis receive a liver biopsy,
biochemical analyses of aminotransferases as well as a determination of immunoglobulins
and autoantibody titers. Significant risk factors for the recurrence of AIH
have not yet been identified although it appeared that the presence of fulminant
hepatic failure before transplantation protected against the development of recurrent
disease. An attractive risk factor for the development of recurrent AIH is the presence
of specific HLA antigens that may predispose toward a more severe immunoreactivity.
In two studies recurrence of AIH appeared to occur more frequently in HLA
DR3 positive patients receiving HLA DR3 negative grafts. However, this association
was not confirmed in all studies. Interestingly, there have not been conclusive data to
support the hypothesis that a specific immunosuppressive regimen represents a risk
47

factor for the development of recurrent AIH. However, data indicate, that patients
transplanted for AIH require continued steroids in 64% versus 17% of patients
receiving liver transplants for other conditions. Based on these results and other
studies it would appear that maintenance of steroid medication in AIH patients is
indicated to prevent not only cellular rejection but also graft threatening recurrence of
AIH but this is controversial. In addition to AIH recurrence the development of de
novo autoimmune hepatitis after liver transplantation has been reported.
48

New insights into autoimmune cholangitis by animal models
Michael Trauner, MD
Division of Gastroenterology and Hepatology, Laboratory of Experimental and
Molecular Hepatology, Department of Internal Medicine, Medical University of Graz,
8036 Graz, Austria; E-Mail: michael.trauner@meduni-graz.at
Improving our understanding of the pathogenesis of chronic immune-mediated
cholangiopathies such primary biliary cirrhosis (PBC) and primary sclerosing
cholangitis (PSC), as well as the development of novel diagnostic, prognostic and
therapeutic tools for these disorders critically depends on easily reproducible animal
models. Recently, several spontaneous mouse models for PBC (not requiring
previous manipulations for breakdown of immunotolerance to pyruvate dehydrogenase
(PDC)-E2 protein) have been reported including NOD.c3c4 and NOD.c3c4derived
mice, IL-2Rα -/- mice, dominant negative TGF-β receptor II mice and Ae2a,b -/mice.
Interestingly, a common feature of all “spontaneous” PBC mouse models
appears to be a relative reduction of circulating naturally T regs, indicating that
disturbed T reg function may play a key role in the pathogenesis of autoimmune
diseases such as PBC through loss of self tolerance. Ae2a,b -/- mice show
immunologic, and, at least in part, biochemical and hepatobiliary morphological
features resembling PBC in humans. The animals show a significantly reduced
CD4 + /CD8 + ratio as a consequence of CD8 expansion and a reduced number of
natural T regs. Moreover, most mice spontaneously developed increased serum IgG,
IgM, and AMA These finding suggest that Ae2 deficiency in mice alters intracellular
pH homeostasis in immunocytes and cholangiocytes consequently leading to
characteristic immunologic and hepatobiliary changes also observed in PBC patients.
Notably genetic AE2 (SLC4A2) variants may determine disease susceptibility,
progression and/or response to therapy in PBC patients. Several studies found
reduced expression and function of AE2 (a Cl - /HCO3 exchanger) in PBC, which may
contribute to reduced bile flow and cholestasis. Moreover, decreased AE2 expression
in salivary and lacrimal glands could explain the frequently associated sicca
syndrome in these patients and pointed towards a more generalized “glandular
failure” in PBC. UDCA restores impaired AE2 function in patients with PBC and
recent findings suggest that a synergistic effects with glucocorticoids.
To date, no animal model has been developed that exhibits all of the attributes of
PSC. Rodent models induced by bacterial cell components or colitis may help to
explain the strong association between PSC and inflammatory bowel disease (IBD).
Other models include direct injury to biliary epithelia, peribiliary vascular endothelia or
portal venous endothelia. Mice with targeted disruption of the Mdr2 (Abcb4) gene
encoding a canalicular phospholipid flippase (Mdr2 -/- mice) spontaneously develop
sclerosing cholangitis with macroscopic and microscopic features of human PSC.
Bile duct injury in these mice is linked to defective biliary phospholipid secretion
resulting in an increased concentration of free non-micellar bile acids which
subsequently cause bile duct epithelial cell (cholangiocyte) injury, pericholangitis,
periductal fibrosis with ductular proliferation and finally sclerosing cholangitis.
In analogy to the Mdr2 -/- mouse model of sclerosing cholangitis, MDR3/ABCB4
(the human orthologue of rodent Mdr2/Abcb4) defects could play a role in the pathogenesis
of various cholangiopathies in humans. MDR3 variants could play a role as
49

modifyer gene in the pathogenesis of various cholangiopathies such as PSC, PBC
and adulthood idiopathic ductopenia/biliary fibrosis.
Another example for a transport defect associated with sclerosing cholangitis is cystic
fibrosis (CF) due to mutations of the cystic fibrosis transmembrane conductance
regulator (CFTR/ABCC7) gene resulting in impaired hydration and alkalinization of
bile, inspissated bile and bile duct injury with sclerosing cholangitis in 4 to 18% of
adult CF patients. Cftr -/- mice develop progressive liver disease with hepatosteatosis,
focal cholangitis, inspissated secretions, bile duct proliferation and progression to
focal biliary cirrhosis after 1 year of age. Interestingly, induction of colitis in Cftr -/- mice
aggravates bile duct injury. Colonic inflammation may impair cftr expression via
impairment of PPARα expression, which has also been implicated in the transcriptional
regulation of Mdr2. Also, patients with IBD who are (heterozygous) carriers of
CFTR mutations may be at increased risk of developing PSC. Recent studies
however suggest that CFTR variants may protect (instead of predispose) to
sclerosing cholangitis via modulation of pathogen internalization resulting in reduced
for inflammation and damage to cholangiocytes.
Xenobiotics and drugs may also lead to bile duct injury and biliary fibrosis via direct
toixc and indirect immune-mediated injury. Toxic drug metabolites excreted into bile
may cause a drug-induced vanishing bile duct syndrome and eventually biliary
cirrhosis. Feeding of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to
increased biliary porphyrin secretion, induction of VCAM, osteopontin, and TNF-α
expression, a pronounced pericholangitis with significantly increased number of
CD11b-positive cells, ductular reaction and activation of periductal myofibroblasts
leading to a biliary fibrosis. Lithocholic acid (LCA) causes bile duct injury, since LCA
per se is highly hydrophobic and toxic. As such, LCA leads to segmental bile duct
obstruction, destructive cholangitis, periductal fibrosis, all features of sclerosing
cholangitis.
In summary, major progress has been made with the identification of spontanous
cholangiopathy models. These novel models should enhance our understanding of
the pathogenesis of PBC and PSC and will hopefully result in improved treatment of
these disorders.
50

Etiological and molecular issues in primary biliary cirrhosis
M. Eric Gershwin, MD
Division of Rheumatology, Allergy and Clinical Immunology, University of California
at Davis School of Medicine, 451 Health Sciences Drive, Suite 6510, Davis, CA
95616, USA; Telephone: 530-752-2884; Fax: 530-752-4669
E-Mail: megershwin@ucdavis.edu
There have been significant advances both in humans and experimental models that
relate to the etiopathogenesis of primary biliary cirrhosis. Many of these advances
are based on the rigorous definition of the antimitochondrial response, the serologic
signature of PBC. First, it is well established that AMA are directed against members
of the 2-oxoacid dehydrogenase complexes (2-OADC), among which the major
epitopes are within the lipoylated domains of the E2 subunit of the pyruvate
dehydrogenase complex (PDC-E2). Second, autoreactive CD4 + and CD8 + T cells
can be detected in PBC peripheral blood, regardless of the AMA status, and the
infiltration of autoreactive T cells in the liver and periductular spaces is one of the
most prominent immune features. Autoreactive T cells of both subtypes recognize
PDC-E2 sequences overlapping with the AMA epitopes. An increase in cytotoxic
T cell precursors in the blood in the early stages of the disease compared to the
advanced ones and a 10-fold increase of specific liver CD8 + T cells compared to
peripheral blood have been demonstrated. Third, additional data on the immunobiology
components of PBC autoimmunity has been recently obtained in
CD4 + CD25 high natural regulatory T cells which appear to be numerically reduced in
PBC. PBC bile duct cells manifest unique features during apoptosis while co-culture
experiments do not support a direct role for these cells in determining their immunemediated
injury. Apoptotic cells are phagocytosed by BECs and consequently are an
exogenous source of autoantigens in cholangiocytes, possibly through anti-CD16. As
a result, the impact of putative changes in apoptosis and autophagy specific to BEC
remains to be fully determined in PBC. Fifth, the innate immune compartment has
been recently investigated in PBC with promising results. PBC monocytes manifest
an increased response to pathogen associated stimuli, as indicated by higher levels
of pro-inflammatory cytokines. Further, the hyper-IgM associated with PBC is
secondary to an aberrant innate immune response, potentially induced by stimulation
of toll like receptor 9 by bacterial CpG-B.
The female preponderance may hold an important key to PBC etiology. X-linked
genes determine gender-related characteristics at different levels while also
regulating the immune function, particulalry to maintain tolerance. Major X chromosome
defects such as those leading to Turner’s syndrome or premature ovarian
failure are commonly characterized by autoimmune comorbidities (particularly thyroid
disease) and, less frequently, cholestasis. Our group first determined a significantly
higher frequency of monosomy of the X chromosome in peripheral leukocytes
(particularly those of the adaptive immune response, i.e. T and B cells) in women
PBC compared to age-matched control women. Monosomy frequency correlated with
age in all three groups, as expected but monosomic cells were not microchimeric
cells. We further demonstrated that the X loss in PBC affected was not random but
affected more frequently one parentally-inherited chromosome.
51

Several key animal models of autoimmune cholangitis have now been described.
First, a genomic variant of the non obese diabetic (NOD) mouse (NOD.c3c4) has
been observed to manifest autoimmune cholestasis with AMA and ANA positivities in
50–60% and 80–90%, respectively. Liver histology demonstrated portal lymphocyte
infiltration with chronic non-suppurative cholangitis and PBC-like granulomas.
Second, a dominant negative form of trasforming growth factor β (TGFβ) receptor II
(dnTGFβRII) mouse develop serum AMA in 100% of mice. The TGFβ receptor II
regulates lymphocyte activation and the appearance of PBC in this model suggests a
specific condition of T cells with impaired TGFβ signaling in the presence or absence
of B cells is involved. Third, the knockout of interleukin 2 receptor α leads to a murine
phenotype with 100% serum AMA positivity, 80% serum ANA positivity, and portal
lymphocyte infiltration and vanishing bile ducts. This model is of particular interest
based on the report of autoimmune cholangitis in a pediatric case of IL2Rα
deficiency. Fourth, Ae2a,b also develop autoimmune phenomenon and a PBC-like
disease. Finally, immunization of mice with chemical xenobiotics has also been
shown to lead to a PBC-like disease.
These data and observations will be put in the context of the key mechanisms that
lead to autoimmune cholangitis in patients.
52

Session V
Immunology of viral hepatitis I
53

Occult hepatitis B virus infection: Diagnosis and significance
Wolfram H. Gerlich
Institute of Medical Virology, Justus-Liebig University Giessen, Germany
An expert group defined March 2008 at Taormina "Occult hepatitis B virus infection"
(OBI) as: "Presence of HBV DNA in the liver (with detectable or undetectable HBV
DNA in the serum) of individuals testing HBsAg negative with currently available
assays. When detectable, the amount of HBV DNA in the serum is usually very low
(< 200 IU/ml)". This definition implies OBI-like phases even in HBsAg positive cases,
because the early “window phase” of acute HBV infection may last several weeks
before HBsAg becomes detectable. We identified two blood donors whose donations
were HBV DNA negative (< 12.5 IU/ml) but transmitted HBV to the recipient. Both
developed later HBsAg acute hepatitis. However, OBI without HBsAg positive phase
occurs also. We followed an acute OBI which showed a peak viremia of 15,000 IU/ml
HBV DNA and sub-borderline HBsAg-levels suggesting a ratio of virions to subviral
particles of 1:10 whereas "normal" cases show at peak viremia 1:1000 or less. In that
case a rapid immune response had down-regulated the viral gene expression.
It is known since long that anti-HBc positive persons without HBsAg may reactivate
HBV infection if they experience immunosuppression and that livers from HBsAg
negative donors may transmit HBV to transplant recipients. Studies from Italy show
that the majority of anti-HBc positive healthy individuals have HBV DNA in the liver.
But one third with HBV DNA had no anti-HBc. On that basis one may estimate that
ca. 7% of the German population may have OBI.
Controversial data exist on the impact of persistent OBI on development of cirrhosis
or HCC and on the course and therapy of HCV- or HIV-coinfection. Consensus exists
that OBI may reactivate under severe immunosuppressiuon and should be searched
for before starting this therapy. Furthermore, blood donors with OBI may transmit
HBV. We studied 5 blood donors with OBI and 55 of their recipients. In 22 cases,
transmission was probably but the recipients remained healthy. However, in 3 cases
who were immunosuppressed at the time of transfusion, fatal fulminant hepatitis B
developed. OBI leads often to selection of HBsAg escape mutations. Inversely,
infection of vaccinated individuals favors development of OBI which may be difficult
to recognise in blood donors. Thanks to HB vaccination, the problem of persistent
overt HBV infection may eventually fade away but the problem of persistent OBI will
possibly remain for longer.
55

Innate and adaptive immune responses in HBV infection
Mala K. Maini
Division of Infection and Immunity, University College of London, London, UK
The hepatotropic hepatitis B virus (HBV) is non-cytopathic; liver disease resulting
from this infection is therefore thought to be immune-mediated. In order to develop
immunotherapeutic strategies for improving the treatment of HBV, there is a pressing
need to dissect out the immune components contributing to viral control versus
disease pathogenesis.
Defects in many aspects of the coordinated innate and adaptive immune response
have been described in patients failing to control HBV infection, but one of the most
profound and critical is depletion of the virus-specific CD8 T cell response. We have
identified Bim-mediated apoptosis as one of the processes accounting for the failure
of HBV-specific CD8 T cells to persist in the face of high antigen load [Lopes et al.,
JCI 2008]. We are investigating whether T cell tolerance through induction of Bim
may be imposed by inappropriate co-stimulation during intrahepatic antigen
presentation and whether blocking this pathway may reconstitute more effective
responses in vivo. We have also characterized signaling defects attributable to the
liver microenvironment (local depletion of arginine), that bias the effector function of
CD8 T cells in chronic HBV infection [Das et al., JEM 2008].
Data from humans and HBV transgenic mice have pointed to the non-virus-specific
lymphocytes infiltrating the liver in the presence of uncontrolled HBV replication as a
major contributor to the ensuing damage. We have identified a pathway whereby NK
cells, which account for 30–40% of intrahepatic lymphocytes, can mediate
hepatocyte death and have shown that this pathway can be stimulated by cytokines
induced during flares of HBV-related liver disease [Dunn et al., JEM 2007]. We have
defined a role for CXCR6/CXCL16 chemokine/chemokine receptor interactions in the
homing of NK cells with pathogenic potential to the HBV-infected liver. These NK
cells have an impaired capacity to produce the antiviral cytokine IFN-γ, which can be
reversed by blocking IL-10 signals. We find that the immunosuppressive cytokine
IL-10 is induced in the early stages of acute HBV infection [Dunn et al., Gastro in
press] and also in flares of chronic infection, and that it is capable of suppressing
both innate and adaptive immune responses.
In summary, features of the intrahepatic cytokine and nutrient microenvironment,
together with aberrant co-regulatory signals, combine with the prolonged high HBV
load to deviate innate and adaptive immune responses towards a pathogenic rather
than antiviral role.
56

Hepatitis Delta: Immunopathogenesis and clinical challenges
Heiner Wedemeyer, Jan Grabowski
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical
School, Carl-Neuberg Str. 1, 30625 Hannover, Germany, Tel.: +49 511 532-6814,
Tel.: +49 511 532-8662
Website: www.mh-hannover.de/ag-wedemeyer.html
E-Mail: wedemeyer.heiner@mh-hannover.de
Hepatitis Delta is caused by infection with the hepatitis D virus (HDV) and is
considered as the most severe form of viral hepatitis in humans. Hepatitis Delta
occurs only in HBsAg-positive individuals as HDV is a defective RNA virus which
requires the hepatitis B virus (HBV) surface antigen (HBsAg) for complete replication
and transmission. At least seven different HDV genotypes have been described with
specific geographic distributions and distinct clinical courses. HDV infection affects
mainly immigrants in Central Europe with about one third of patients born in states of
the former Soviet Union and another third born in Turkey. HDV/HBV coinfection can
be associated with complex and dynamic viral dominance patterns. While HDV is
frequently the dominating virus not only in HBV/HDV coinfection but also in
HBV/HCV/HDV triple-infected patients, fluctuating courses HDV and HBV viremia
can be observed in other patients. Chronic HDV infection leads to more severe liver
disease than HBV mono-infection with accelerated fibrosis progression, earlier
hepatic decompensation and an increased risk for the development of hepatocellular
carcinoma. However and in contrast to HCV infection, hepatic decompensation rather
than development of liver cancer is the first clinical endpoint that develops during the
course of infection.
Few studies have investigated immune responses against HDV and HBV in humans.
Our findings suggest that HDV-specific T cell responses are detectable in the
majority of patients, however, so far we were not able to find a clear-cut association
with the stage or grade of liver disease. Of note, HBV-specific can be detected
similarly at a high frequency. So far, only interferon alpha treatment has proven
antiviral activity against HDV in humans and has been linked to improved long-term
outcome. Recent studies on the use of pegylated interferon showed a sustained
virological response concerning HDV-RNA in about one quarter of patients.
HDV-specific immune responses might be associated with the response to treatment.
Novel alternative treatment options including prenylation inhibitors are still awaiting
clinical development for delta hepatitis.
57

Clinical spectrum and therapeutic options in HDV infections
M. Rizzetto
University of Torino, Department of Gastroenterology, Torino, Italy
Clinical experience has confirmed that chronic hepatitis D (CDH) most usually runs a
severe and progressive course; as HBV is inhibited by the HBV, the diagnostic
harbinger to an underlying HDV disease is the discrepancy between a florid HBsAg
positive disease and lack of HBV-DNA in serum.
HDV is divided into eight genotypes differing as much as 40% in nucleotide
sequence. Genotype I is the most frequent world-wide and has variable
pathogenicity. Genotypes II and IV were found in East Asia causing relatively mild
disease. Genotype III was associated with fulminant hepatitis in South America.
Since the 1990s the circulation of HDV has declined significantly in Europe,
decreasing in Italy in chronic HBsAg disease from a rate of 24.6% in 1983 to 8.3% in
1997. The clinical scenario of hepatitis D has also changed; while the majority of
hepatitis D collected in Italy in the 1980s had a chronic active hepatitis, by the end of
the 1990s the proportion of cirrhosis residual to burnt-out inflammation had increased
to 70%.
However, hepatitis D has not disappeared in Europe but is now being restored
through immigration; the reservoir of HDV in Europe is currently sustained by the
residual ageing domestic pool that survived the brunt of the hepatitis D epidemic in
the 1970–1980s and by a population of young patients with recent HDV infections
migrating to Europe from areas where HDV remains endemic.
α-Interferon was first used in CHD 25 years ago and still remains the only available
treatment. Results, however, have been limited.
Peg-IFN may be more beneficial; in a study of CHD treated with Peg-IFN-α2b
1.5 µg/kg weekly for 12 months, 6 of 14 (43%) achieved a sustained clearance of
serum HDV-RNA.
Famciclovir, Lamivudine and Adefovir, inactive against HDV but active against the
helper HBV, were used in monotherapy or in combination with IFN, but they were not
efficacious.
The problem confronting HDV therapy is that there is no specific viral function to
target; HDV depends on the HBsAg and not on HBV replication, therefore its
synthesis is not influenced by the level of HBV-DNA in serum. A novel strategy
involves disruption of prenylation to prevent the binding of the HD-Ag to HBsAg;
in-vitro inhibition of HDAg prenylation by the prenylation inhibitor BZA-5B and in-vivo
by the inhibitors FTI – 277 and FTI – 2153 were effective at clearing HD viremia in
mice.
58

Session VI
Immunology of viral hepatitis II
59

Apoptosis in immune-mediated liver diseases
Alisan Kahraman, Guido Gerken and Ali Canbay
Division of Gastroenterology and Hepatology, University Hospital Essen, Germany
Autoimmune diseases of the liver are chronic inflammatory processes leading to
immune-mediated injury of hepato- and cholangiocytes. Cell death by apoptosis is a
prominent feature in a variety of liver diseases. It is likely that apoptosis is the initial
cellular response to liver and biliary injury and may thus initiate several cellular and
cytokine cascades that culminate in tissue injury with subsequent fibrosis and finally,
result in cirrhosis. Obviously, this cascade of events within the apoptotic machinery is
of particular importance. However, Fas ligand and granzyme B significantly
contribute to apoptosis observed in autoimmune liver diseases. This overview is
focused on the role of apoptosis in immune-mediated liver diseases, especially in
primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and
autoimmune hepatitis (AIH), respectively. Recently, also soluble forms of major
histocompatibility complex (MHC) class I-related chains A and closely related B (MIC
A and B) were reported to be increased in the sera of patients with autoimmune liver
diseases. MIC A and B are cell surface glycoproteins that function as indicators for
cellular stress by displaying peptides derived from proteins degraded in the cytosol
on the cell surface and thus facilitate the recognition of intracellular antigens by
circulating cytotoxic NK cells leading to enhanced cell death. Nowadays rationalbased
strategies are being developed to suppress apoptotic cell death as a novel
therapeutic option for the treatment of these liver diseases.
Keywords: apoptosis, autoantibodies, cholestatic liver disease, stress-induced
ligands
Short title: Apoptosis in autoimmune liver disease
Abbreviations: AIH, autoimmune hepatitis; AMA, antimitochondrial antibody; ALT,
alanine aminotransferase; AST, aspartate aminotransferase; MIC A/B, major histocompatibility
complex class I-related chains A/B; PBC, primary biliary cirrhosis; PSC,
primary sclerosing cholangitis
Correspondence to:
Ali Canbay, M.D.
Associate Professor of Medicine
Division of Gastroenterology and Hepatology
University Hospital Essen
Hufelandstr. 55
45122 Essen
Germany
Tel: +49 201 723-84713
Fax: +49 201 723-5719
E-Mail: ali.canbay@uni-due.de
61

Immune response in HCV infection
Carlo Ferrari
Unità Operativa di Malattie Infettive ed Epatologia, Laboratorio di Immunopatologia
Virale, Azienda Ospedaliero-Universitaria di Parma, Italy
HCV is able to induce efficiently innate immune response genes in the liver of
HCV-infected chimpanzees in the first weeks of infection and is sensitive to IFNs in
vitro. Yet, HCV seems to ignore early innate defense mechanisms, as it replicates
almost immediately after penetration into target cells, suggesting that the innate
immunity does not significantly contribute to control virus infection. This is the likely
consequence of multiple complementary strategies that HCV has developed to
interfere with the antiviral effect of the different components of the innate immune
system, including NK cells and the intracellular signaling cascade which leads to
type I IFN expression following recognition of specific HCV associated motifs and to
IFN-stimulated gene expression following cellular exposure to IFN-α and IFN-β. As a
result of this interference, the physiologic priming of adaptive HCV-specific
responses may be inadequately promoted and supported by the innate immunity.
Evasion from innate responses and subsequent spread of the virus may set in motion
a series of additional mechanisms of escape from immune surveillance. They include
T cell exhaustion due to the rapid viral spread in the infected host causing an early
and persistent exposure of T cells to high virus and antigen loads, direct functional T
cell inhibition by HCV proteins, emergence of T and B cell escape mutations, hyperactivity
of suppressive regulatory pathways (PD-1/PD-L1, CD4 + CD25 + FoxP3 positive
cells, regulatory cytokines). These mechanisms can all contribute to delay and impair
priming and maturation virus-specific adaptive responses at the early stages of
infection when an efficient helper function of the CD4 T cell subset seems to be
crucial for subsequent control of infection, allowing HCV-specific CD8 cells to
differentiate into functionally efficient antiviral effector and memory cells. In contrast,
inadequate CD4 help probably precludes appropriate maturation of CD8 antiviral
effector and memory functions, favoring persistence of the virus. This leads in turn to
a progressively more severe impairment of the overall antiviral T cell function, which
represents the common feature of chronic HCV infection. In this scenario, the role of
the neutralizing antibody response in HCV pathogenesis is still largely undefined,
because neutralizing antibodies have been detected at high titers not only in
resolving acute hepatitis, but also in patients with a long-lasting condition of chronic
HCV infection. In chronic patients these antibodies can fail to neutralize co-existing
autologous HCV glycoprotein, while good neutralizing activity can be observed
against earlier viral strains. Sequential emergence of viral mutations able to abrogate
B cell recognition of the viral envelope glycoprotein sequence may explain why high
titers of neutralizing antibodies are detectable in chronic infections but are unable to
control the coexisting viral strains.
Thus, available data indicate that HCV is able to acquire an early survival advantage
over the immune system by virtue of its rapid start of replication and its ability to
inhibit innate responses. The subsequent spread of the virus can then set in motion
additional mechanisms of evasion from immune control allowing HCV to outpace the
protective immune response and to maintain a persistent advantage over antiviral
immunity.
62

Immunotherapy of hepatocellular carcinoma
Tim F. Greten
Department of Gastroenterology, Hepatology and Endocrinology, Medical School
Hannover, Germany
No systemic cytotoxic chemotherapy has proven to be safe and efficient for the
treatment of patients with hepatocellular carcinoma (HCC) (1). Therefore, new
treatment options are urgently needed. Recently, the multi-tyrosine kinase inhibitor
sorafenib was shown to enhance overall survival in patients with advanced disease
(2). However, the median survival for patients with HCC remains below 12 months. In
addition, there is also accumulating evidence that immunotherapy might become a
potent therapeutic option for patients with HCC (3) for several reasons: First, it has
been shown that patient’s survival directly depends on the type and number of tumor
infiltrating immune cells (4) (5) (6), indicating that immune responses have a direct
effect on the clinical course of the disease. Second, HCC has been shown to be the
first cancer, against which a potent vaccine is available. Vaccination against hepatitis
B infection has been shown to effectively prevent the development of HCC (7). Third,
local-ablative treatment options, which cause physical destruction of tumors, are
associated with the release of high antigen load, which could induce anti-tumor
immune responses. In addition, more than 80% of all patients with HCC have
significant liver cirrhosis. Cohort studies indicate that HCC has become the major
cause of liver-related death in patients with compensated cirrhosis (8). Therefore, this
patient population might represent an ideal group for a “preventive cancer vaccine”.
Interestingly we have been able to demonstrate that HCC patients mount
spontaneous tumor-specific immune responses (9) indicating that immunesuppressor
mechanisms might counter-balance these immune responses. Indeed,
we have been able to detect different types of immune suppressor mechanisms in
HCC patients such as regulatory T cells and myeloid derived suppressor cells (10)
(11). Recently we have performed a clinical trial with the aim to specifically deplete
immune suppressor cells such as regulatory T cells which could potentially augment
the effect of either spontaneous or vaccination induced T cell responses in HCC
patients. The results from these studies will be presented.
References:
1. Lopez, P.M., A. Villanueva, and J.M. Llovet. 2006. Systematic review: evidencebased
management of hepatocellular carcinoma – an updated analysis of
randomized controlled trials. Aliment Pharmacol Ther 23: 1535–1547.
2. Llovet, J.M., S. Ricci, V. Mazzaferro, P. Hilgard, E. Gane, J.F. Blanc,
A.C. de Oliveira, A. Santoro, J.L. Raoul, A. Forner, M. Schwartz, C. Porta,
S. Zeuzem, L. Bolondi, T.F. Greten, P.R. Galle, J.F. Seitz, I. Borbath,
D. Häussinger, T. Giannaris, M. Shan, M. Moscovici, D. Voliotis, and J. Bruix.
2008. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359:
378–390.
63

3. Greten, T.F., M.P. Manns, and F. Korangy. 2006. Immunotherapy of hepatocellular
carcinoma. J Hepatol 45: 868–878.
4. Wada, Y., O. Nakashima, R. Kutami, O. Yamamoto, and M. Kojiro. 1998.
Clinicopathological study on hepatocellular carcinoma with lymphocytic infiltration.
Hepatology (Baltimore, Md) 27: 407–414.
5. Unitt, E., A. Marshall, W. Gelson, S.M. Rushbrook, S. Davies, S.L. Vowler,
L.S. Morris, N. Coleman, and G.J. Alexander. 2006. Tumour lymphocytic
infiltrate and recurrence of hepatocellular carcinoma following liver transplanttation.
J Hepatol 45: 246–253.
6. Gao, Q., S.J. Qiu, J. Fan, J. Zhou, X.Y. Wang, Y.S. Xiao, Y. Xu, Y.W. Li, and
Z.Y. Tang. 2007. Intratumoral balance of regulatory and cytotoxic T cells is
associated with prognosis of hepatocellular carcinoma after resection. J Clin
Oncol 25: 2586–2593.
7. Chang, M.H., C.J. Chen, M.S. Lai, H.M. Hsu, T.C. Wu, M.S. Kong, D.C. Liang,
W.Y. Shau, and D.S. Chen. 1997. Universal hepatitis B vaccination in Taiwan
and the incidence of hepatocellular carcinoma in children. Taiwan Childhood
Hepatoma Study Group. N Engl J Med 336: 1855–1859.
8. Fattovich, G., T. Stroffolini, I. Zagni, and F. Donato. 2004. Hepatocellular
carcinoma in cirrhosis: incidence and risk factors. Gastroenterology 127:
S35–50.
9. Korangy, F., L.A. Ormandy, J.S. Bleck, J. Klempnauer, L. Wilkens, M.P. Manns,
and T.F. Greten. 2004. Spontaneous Tumor-Specific Humoral and Cellular
Immune Responses to NY-ESO-1 in Hepatocellular Carcinoma. Clin Cancer
Res 10: 4332–4341.
10. Ormandy, L.A., T. Hillemann, H. Wedemeyer, M.P. Manns, T.F. Greten, and
F. Korangy. 2005. Increased populations of regulatory T cells in peripheral
blood of patients with hepatocellular carcinoma. Cancer Res 65: 2457–2464.
11. Hoechst, B., L.A. Ormandy, M. Ballmaier, F. Lehner, C. Kruger, M.P. Manns,
T.F. Greten, and F. Korangy. 2008. A new population of myeloid-derived
suppressor cells in hepatocellular carcinoma patients induces
CD4 + CD25 + Foxp3 + T cells. Gastroenterology 135: 234–243.
64

List of Chairpersons, Speakers and Scientific Organizers
Prof. Dr. D.H. Adams
Queen Elizabeth Hospital
Institute of Clinical Sciences
Liver Research Laboratories
Birmingham B15 2TH
Great Britain
Prof. Dr. A. Alberti
Università di Padova
Clinica Medica 2
Patologia Medica
Via Giustiniani 2
35128 Padova
Italy
Dr. P. Bertolino
Centenary Institute of
Cancer Medicine and Cell Biology
Locked Bag No. 6
Newtown, Sydney, NSW 2042
Australia
C. Büttner, M.D.
Assistant Professor of Medicine
Mount Sinai School of Medicine
Endocrinology, Diabetes
& Bone Disease
One Gustave L. Levy Place
New York, NY 10029-6574
USA
S.H. Caldwell, M.D.
Associate Professor of Medicine
University of Virginia
School of Medicine
GI & Hepatology Division
Hospital West, P.O. Box 800708
Charlottesville, VA 22908
USA
Dr. A. Canbay
Gastroenterologie/Hepatologie
Universitätsklinikum Essen
Hufelandstr. 55
45147 Essen
Germany
Dr. U. Christen
Allgemeine Pharmakologie
Klinikum der Johann Wolfgang
Goethe-Universität Frankfurt
Theodor-Stern-Kai 7
60596 Frankfurt
Germany
Prof. Dr. V.J. Desmet
Catholic University of Leuven
University Hospital St. Rafael
Department of Pathology
Minderbroederstraat 12
3000 Leuven
Belgium
A.M. Diehl, M.D.
Professor of Medicine
Duke University Medical Center
Gastroenterology Division
Box 3256, Snyderman/GSRB-1
595 LaSalle Street
Durham, NC 27710
USA
Prof. Dr. H.P. Dienes
Pathologie
Klinikum der Universität zu Köln
Kerpener Str. 62
50924 Köln
Germany
Prof. Dr. C. Ferrari
Università di Parma
Azienda Ospedaliera di Parma
Laboratorio di Immunopatologia V
Via Gramsci 14
43100 Parma
Italy
Prof. Dr. G. Gerken
Gastroenterologie/Hepatologie
Universitätsklinikum Essen
Hufelandstr. 55
45147 Essen
Germany
65

Prof. Dr. W.H. Gerlich
Medizinische Mikrobiologie
Universitätsklinikum
Gießen und Marburg
Frankfurter Str. 107
35392 Gießen
Germany
M.E. Gershwin, M.D.
Professor of Medicine
University of California
School of Medicine
Rheumatology & Allergy
One Shields Ave., Tupper Hall
Davis, CA 95616
USA
Prof. Dr. T.F. Greten
Klinik für Gastroenterologie,
Hepatologie und Endokrinologie
Medizinische Hochschule Hannover
Carl-Neuberg-Str. 1
30625 Hannover
Germany
PD Dr. J. Herkel
Medizinische Klinik I
Universitätsklinikum Eppendorf
Martinistr. 52
20251 Hamburg
Germany
Prof. Dr. H. Holzmann
Institut für Virologie
Medizinische Universität Wien
Kinderspitalgasse 15
1095 Wien
Austria
M. Houghton, Ph.D.
Epiphany Biosciences, Inc.
Suite 2800
One California Street
San Francisco, CA 94111
USA
66
Dr. E. Jaeckel
Klinik für Gastroenterologie,
Hepatologie und Endokrinologie
Medizinische Hochschule Hannover
Carl-Neuberg-Str. 1
30625 Hannover
Germany
PD Dr. K. Klugewitz
Gastroenterologie
Charité Universitätsmedizin
Campus Benjamin Franklin (CBF)
Hindenburgdamm 30
12203 Berlin
Germany
Prof. Dr. P.A. Knolle
Molekulare Medizin
Universitätsklinikum Bonn
Sigmund-Freud-Str. 25
53127 Bonn
Germany
Prof. Dr. A. Knuth
Medizinische Poliklinik
Rämistr. 100
8091 Zürich
Switzerland
Dr. K. Lang
Gastroenterologie/Hepatologie
Universitätsklinikum Düsseldorf
Moorenstr. 5
40225 Düsseldorf
Germany
Prof. Dr. A.W. Lohse
Medizinische Klinik I
Universitätsklinikum Eppendorf
Martinistr. 52
20251 Hamburg
Germany
Dr. M.K. Maini
University College London
Department of Immunology
& Molecular Pathology
46 Cleveland Street
London W1T 4JF
Great Britain

Dr. A. Mallat
Hôpital Henri Mondor
51, Av. Mal de Lattre de Tassigny
94010 Creteil
France
Prof. Dr. M.P. Manns
Klinik für Gastroenterologie,
Hepatologie und Endokrinologie
Medizinische Hochschule Hannover
Carl-Neuberg-Str. 1
30625 Hannover
Germany
Prof. Dr. S. Matern
Höfchensweg 113
52066 Aachen
Germany
Prof. Dr. S. Meuer
Immunologie
Universitätsklinikum Heidelberg
Im Neuenheimer Feld 305
69120 Heidelberg
Germany
Prof. Dr. Dr. Dr.
K.-H. Meyer zum Büschenfelde
Trabener Str. 8
14193 Berlin
Germany
Prof. Dr. S. Mihm
Gastroenterologie
Universitätskliniken Göttingen
Robert-Koch-Str. 40
37075 Göttingen
Germany
Dr. E. Powell
Royal Prince Charles Hospital
137 Central Avenue
St. Lucia, Brisbane, QLD 4067
Australia
Prof. Dr. Dr. h.c. G. Ramadori
Gastroenterologie
Universitätskliniken Göttingen
Robert-Koch-Str. 40
37075 Göttingen
Germany
Dr. H. Ring-Larsen
University of Copenhagen
Faculty of Pharmaceutical Scienc
Department of Pharmacology
& Pharmacotherapy
Universitetsparken 2
2100 Copenhagen
Denmark
Prof. Dr. M. Rizzetto
Azienda Ospedaliera
S. Giovanni Battista di Torino
Divisione di Gastroenterologia
Corso Bramante 88
10126 Torino
Italy
A.J. Sanyal, M.D.
Assistant Professor of Medicine
Medical College of Virginia
Gastroenterology & Hepatology
Box 980711
Richmond, VA 23298
USA
Prof. Dr. S.W. Schalm
LiverDoc B.V.
Veerkade 8d
3016 DE Rotterdam
The Netherlands
Dr. J. Schattenberg
Innere Medizin I
Klinikum der Universität
Langenbeckstr. 1
55131 Mainz
Germany
Prof. Dr. J. Schölmerich
Klinik für Innere Medizin I
Klinikum der Universität Regensburg
93042 Regensburg
Germany
67

Prof. Dr. E. Schrumpf
University of Oslo
Rikshospitalet
Section of Gastroenterology
& Hepatology
0027 Oslo
Norway
Prof. Dr. C.P. Strassburg
Klinik für Gastroenterologie,
Hepatologie und Endokrinologie
Medizinische Hochschule Hannover
Carl-Neuberg-Str. 1
30625 Hannover
Germany
Dr. M.G. Swain
University of Calgary
Health Sciences Centre
3330 Hospital Drive N.W.
Calgary, AB T2N 4N1
Canada
Prof. Dr. H.C. Thomas
St. Mary's Hospital
Medical School
Department of Medicine
Praed Street
London W2 1NY
Great Britain
Prof. Dr. G. Tiegs
Experimentelle Immunologie
Universitätsklinikum Eppendorf
Martinistr. 52
20251 Hamburg
Germany
Prof. Dr. H. Tilg
Bezirkskrankenhaus
Hall i.T.
Interne Abteilung
Milserstr. 10
6060 Hall/Tirol
Austria
68
Prof. Dr. M. Trauner
Medizinische Universität Graz
Klinische Abteilung für
Gastroenterologie & Hepatologie
Auenbruggerplatz 15
8036 Graz
Austria
Prof. Dr. C. Trautwein
Medizinische Klinik III
Universitätsklinikum Aachen
Pauwelsstr. 30
52074 Aachen
Germany
Prof. Dr. C. Trépo
Inserm U. 271
151, Cours Albert Thomas
69003 Lyon
France
Prof. Dr. D. Vergani
King's College Hospital
Institute of Liver Studies
Denmark Hill
London SE5 9RS
Great Britain
PD Dr. H. Wedemeyer
Klinik für Gastroenterologie,
Hepatologie und Endokrinologie
Medizinische Hochschule Hannover
Carl-Neuberg-Str. 1
30625 Hannover
Germany
Dr. F.T. Wunderlich
Genetisches Institut
Universität Köln
Zülpicher Straße 47
50674 Köln
Germany
Dr. M. Zierden
Pathologie
Klinikum der Universität zu Köln
Kerpener Str. 62
50924 Köln
Germany

POSTER ABSTRACTS
Poster Numbers
NAFLD and NASH 1 – 10
Hepatitis A and B 11 – 21
Hepatitis C 22 – 38
Autoimmune hepatitis 39 – 44
Cholestatic liver diseases (PBC/PSC) 45 – 47
Malignant and benign neoplasm 48 – 53
Basic immunology 54 – 64
Hepatic vascular disorders, drugs and toxicity,
epidemiology and endoscopy posters 65 – 74
Author Index to Poster Abstracts

Nonalcoholic fatty liver disease in autopsy of children and
adolescents
Fadime Yüksel 1 , Dilhan Ergün 2 , İlhami Yüksel 3 , Sema Kara 4 , Emine Şamdancı 5 ,
Nurullah Çelik 6
1
Pediatric Specialist, Ankara Forensic Group, Ankara, Turkey
2
Pathology Specialist, Ankara Forensic Group, Ankara, Turkey
3
Department of Gastroenterology, Dışkapı Yıldırım Beyazıt Education and Research
Hospital, Ankara, Turkey
4
Department of Neonatology, Fatih University School of Medicine, Ankara, Turkey
5
Department of Pathology, İnönü University School of Medicine, Malatya, Turkey
6
Department of Endocrinology, Fatih University School of Medicine, Anakara, Turkey
Objective: Nonalcoholic fatty liver disease (NAFLD) is probably the most common
cause of liver disease in the childhood and adolescent age groups. Although there
are few population-based data available and there are limitations in the populationbased
approach as previously noted, the available data suggest an overall
prevalence of at least 3% for suspected fatty liver disease among children and
adolescents. Liver biopsy cannot be used as a screening tool in population studies;
however, autopsy series are another means of estimating prevalence based on a
histological diagnosis. We conducted our study to determine the prevalence of fatty
liver by histology in autopsy of children and adolescents.
Material and methods: We included all children aged 2 through 20 years at the time
of death who had an autopsy performed by the medical examiner between 2006 and
2008. We included a minimum age of 2 years, because this is the youngest age
reported for biopsy-proven steatohepatitis. Missing clinical data or the absence of a
liver slide for review and identification of a factor that may influence liver histology
(alcohol or drug use) were exclusion criteria. Fatty liver was defined as 5% of
hepatocytes containing macrovesicular fat. Steatohepatitis were subclassified as
Type 1, Type 2 or overlap.
Results: 405 children aged 2 to 20 years had an autopsy performed by the Ankara
Forensic Group medical examiner. Of these, 10 subjects were excluded because of
insufficient data: no available liver tissue (n = 1) or missing clinical data (n = 2).
7 subjects were excluded because of factors that may influence liver histology;
alcohol-use (n = 2), positive drug toxicology (n = 5). The remaining 395 subjects were
included to study.
Motor vehicles were involved in 4 of all deaths in cases with NAFLD. Other accidents
(drowning, electric shock and falls) accounted for an additional 4 deaths. Remaining
2 cases have died because of other reasons. The prevalence of fatty liver was found
5.1% in children and adolescents. Fatty liver was present in 9 boys and 11 girls. Fatty
liver prevalence increased with age ranging from 3/20 for ages 2 to 4 years up to
13/20 for ages 15 to 20 years. Children and adolescents with fatty liver were
significantly older (p < 0.05). There was no significant difference between sexes.
1

BMI in subject with fatty liver was higher than in subjects with nonfatty liver, but this
finding was not statistically significant (p > 0.05). Overweight children accounted for
50% of all of the cases of fatty liver. Simple steatosis was detected in 7 subjects.
Steatohepatitis was characterized as Type 1 in 5 subjects, Type 2 in 7 subjects, and
as overlap in 1 subject.
Discussion: The prevalence of fatty liver was found 5.1% in children aged 2 to
20 years in Ankara, Turkey. These findings showed that fatty liver is an important
health problem in our country. Our findings showed that 50% of fatty liver were
overweight children, consistent with the literature. The risk factors identified may be
useful in the development of protocols designed to screen at-risk children and
adolescents. Following studies about NAFLD in the children and adolescent, will
contribute to our knowledge for both diagnosis and treatment.

Non-invasive evaluation of liver fibrosis in patients with NAFLD
(comparison between transient elastography and BARD score)
Adriana Tudora, O. Ciof, E. Dragan, Diana Rill, Sporea Ioan, Scurtu-Martin Mihaela
Department of Gastroenterology and Hepatology, University of Medicine and
Pharmacy “Victor Babes”, Timisoara, Romania
Introduction: Assessment of the degree of fibrosis, in patients with liver steatosis
(LS) is important for the clinical outcome of the patient, and the progression to
cirrhosis. Therefore, non-invasive tests were developed in order to provide useful
information to discriminate NAFLD from NASH, quantifying the degree of liver
fibrosis. LSMs using transient elastograpy (TE) has intrinsic limitation of subcutaneous
adipose tissue making the method difficult to perform in obese subjects. Seric
markers such as BARD score share the benefits of non-invasive scores, due to the
fact that it was developed in obese subjects and to its simplicity. We aimed to
analyze the correlation between assessment of LF in patient with NAFLD by TE
(FibroScan ® , Echosense, Paris) and BARD score.
Methods: We evaluated 226 patients with NAFLD: 62 women (27.4%) and 164 men
(72.6%), mean age 44.3 ± 3 years. In all patients LSMs by TE was performed and
Bard Score was calculated (Body mass index + AST/ALT ratio + Diabetes mellitus).
Liver biopsy (LB) was carried out in 64% (144) patients.
Results: Mean BMI was 29.9 kg/m 2 (13% patients had normal BMI), mean AAR was
0.93 and diabetes mellitus was present in 27% patients (resulting a mean BARD
score of 2.5 ± 0.7). Mean fibrosis score at LB (METAVIR) was 1.4. Mean value of
LSMs was 7.1 ± 1.2 kPa (vs. 4.8 ± 1.3 kPa in normal subjects). In 77 (36.2%) cases,
LS was ≤ 5.9 kPa (equivalent to F = 0); in 45 (21.1%) patients, LS varied between
6 and 6.8 kPa (equivalent to F = 1); and in the 91 (42.7%) remaining patients, LSMs
were ≥ 6.9 kPa (equivalent to significant fibrosis). In these patients mean BARD
score was 2.9 ± 1.1 vs. 1.2 ± 0.2 (p < 0.001-ES) in F0F1patients. Patients with F ≥ 2
at LB (n = 86, 38%) had a mean BARD score of 3.1 ± 0.8 compared to those with
F < 2 (p < 0.001-ES).
Discussion/Conclusion: In our study, both TE and BARD score were statistically
significant correlated with advanced fibrosis (F ≥ 2).
2

3
Assessment of liver fibrosis by using transient elastography
(TE) in patients with nonalcoholic fatty liver disease (NAFLD)
Octavian Ciof, Adriana Tudora, E. Dragan, Diana Rill, I. Sporea, M. Scurtu-Martin
Department of Gastroenterology and Hepatology, University of Medicine and
Pharmacy “Victor Babes”, Timisoara, Romania
Introduction: Determination of the extent of liver fibrosis in NAFLD subjects is of
great clinical importance due to progression to cirrhosis. Until recently, liver biopsy
(LB) was the only method for evaluating liver fibrosis in these patients. Transient
elastography (FibroScan ® , Echosense, Paris) is a new technique that allows rapid,
non-invasive measurement of mean tissue stiffness, which was evaluated in
estimating liver fibrosis in patients with chronic hepatitis C. We carried out a study to
determine the value of liver stiffness measurements (LSMs) in patients with NAFLD.
Methods: Out of 3459 TE evaluations for hepatopathies of different etiologies
performed in the Department of Gastroenterology and Hepatology Timisoara
between 2008–2009 NAFLD was present in 226 patients: 62 women (27.4%) and
164 men (72.6%), mean age 44.3 ± 3 years. Liver biopsy was performed in
140 patients (62%).
Results: Mean value of LS values was 7.1 ± 1.2 kPa (compared to 4.8 ± 1.3 kPa in
normal subjects) (p < 0.0001-ES). Valid measurements could not be obtained in 7%
(13) cases. In the 213 remaining patients, the following values were obtained: in
77 (36.2%) cases, LS was ≤ 5.9 kPa (equivalent to F = 0); in 45 (21.1%) patients, LS
varied between 6 and 6.8 kPa (equivalent to F = 1); and in the 91 (42.7%) remaining
patients LS was ≥ 6.9 kPa (equivalent to significant fibrosis). When referred to
morphological assessment, mean LF (METAVIR score) was 1.4 ± 1.2, and
furthermore, we noticed a stepwise increase in liver stiffness with increasing
histological severity of hepatic fibrosis (p < 0.0001). The AUROC were 0.78, 0.81,
0.89, and 0.94, for F1, F2, F3 and F4, respectively.
Discussion/Conclusion: TE using FibroScan ® allows a rapid and non-invasive
estimation of the stage of fibrosis in NAFLD patients, especially NASH patients. The
results of our study showed a significant positive correlation between liver stiffness
and the severity of liver fibrosis in patients with NAFLD.

HFE gene mutations in patients with biopsy-proven NAFLD
E. Stachowska 1 , J. Raszeja-Wyszomirska 2 , J. Suchy 3 , M. Ławniczak 4 , I. Zawada 4
1
Department of Biochemistry and Human Nutrition, Pomeranian Medical University,
Szczecin, Poland
2
Liver Unit, Pomeranian Medical University, Szczecin, Poland
3
International Hereditary Cancer Center, Department of Genetics and Pathology,
Pomeranian Medical University, Szczecin, Poland
4
Department of Gastroenterology, Pomeranian Medical University, Szczecin, Poland
Background: The role of iron deposition in the pathogenesis of NAFLD has raised
general interest. Primary hepatic iron overload is associated with clinical features of
insulin resistance. Iron can directly cause lipid peroxidation, leading to activation of
stellate cells and increase collagen production. The results of prevalence of HFE
gene mutations in NAFLD patients are still scanty and controversial.
Aim of the study: To evaluate the frequency of HFE gene mutations in patients with
biopsy-proven NAFLD and to compare it to the frequency in general population and
in patients with ALD.
Materials and methods: 50 consecutive Caucasian patients with biopsy-proven
NAFLD were included into the study. The protocol was approved by local Bioethics
Board.
To evaluate C282Y and H63D HFE gene mutations PCR-RFLP was performed,
according to methodology previously described. The control groups comprised of
119 patients with ALD and 1517 DNA samples obtained from either cord bloods or
healthy subjects from family medicine practices.
Results: The frequency of HFE gene mutations in all analyzed groups was
comparable and statistically not significant. These data are summarized in table 1.
Mutation of Healthy controls ALD
NAFLD
HFE gene
(n = 1517)
(n = 119)
(n = 50)
C282Y Homo 2 (0.13%) 1 (0.63%) 1 (2%)
C282Y Hetero 117 (7.8%) 8 (5%) 1 (2%)
H63D Homo 38 (2.5%) 8 (5%) 2 (4%)
H63D Hetero 380 (25%) 40 (25%) 11 (22%)
Discussion/Conclusion: The results of our study support the hypothesis that HFE
gene mutations do not play a critical role in the development of NAFLD. However
these findings have to be verified in bigger cohorts of patients.
This paper was supported by grant from State Committee for Scientific Research, in
years 2006–2009, No N 402 099 31/3037.
4

5
Validation of BARD scoring system among Polish patients with
NAFLD
J. Raszeja-Wyszomirska 1 , M. Ławniczak 2 , B. Szymanik 3 , P. Milkiewicz 1 , M. Hartleb 4
1 Liver Unit, Pomeranian Medical University, Szczecin, Poland
2 Department of Gastroenterology, Pomeranian Medical University, Szczecin, Poland
3 West-Pomeranian University of Technology, Electric Faculty, Department of
Theoretic Electrotechnics and Computer Science, Szczecin, Poland
4 Department of Gastroenterology and Hepatology, Silesian Medical University,
Katowice, Poland
Introduction: Nonalcoholic fatty liver disease (NAFLD) includes a wide spectrum of
liver diseases, ranging from pure steatosis to nonalcoholic steatohepatitis (NASH),
and eventually to liver cirrhosis with its complications. Identifying patients with more
advanced fibrosis at diagnosis is crucial for their prognosis and possible therapeutic
intervention. Harrison et al. (Gut 2008; 57: 1441–7) proposed a novel easy to
conduct in everyday clinical practice scoring system perfectly identifying NAFLD
patients without significant fibrosis (BARD score).
Aim of the study: To validate clinical utility of BARD scoring system among Polish
patients with NAFLD.
Methods: Group of 101 Caucasians with biopsy-proven NAFLD were included into
the study. Liver biopsies were assessed according to Scheurer score (Clin Liver Dis
2002; 6: 335–47) or Histological Scoring System for Nonalcoholic Fatty Liver
Disease. BARD scoring system was assessed according to Harrison et al.:
BMI ≥ 28 = 1 point, AST/ALT ratio (AAR) ≥ 0.8 = 2 points, type 2 diabetes
mellitus = 1 point. Statistic analyses were performed with Chi2 and ANOVA, using
SPSS 15.0.
Results: None of BARD component showed strong association with advanced
fibrosis, however, summarized BARD score of 2–4 points was associated with F3 or
F4 stages of fibrosis with an odds ratio of 15.5 (95% Cl: 3.18–75.5) and negative
predictive value of 97%.
Discussion/Conclusion: Our results show that BARD scoring system holds a
universal value in non-invasive diagnosis of advanced fibrosis in NAFLD patients.
This paper was supported by grant from State Committee for Scientific Research, in
years 2006–2009, No N 402 099 31/3037.

High rate of undetected arterial hypertension in patients with
NASH
M. Demir, S. Schulte, S. Ubben, S.M. Lang, T. Goeser, U. Töx, H.-M. Steffen
Department of Gastroenterology and Hepatology, University of Cologne, Cologne,
Germany
Introduction: Non-alcoholic fatty liver disease (NAFLD) is an emerging metabolicrelated
disorder characterized by fatty infiltration of the liver in the absence of alcohol
consumption. NAFLD ranges from simple steatosis to non-alcoholic steatohepatitis
(NASH), which might progress to end-stage liver disease. This progression is related
to insulin resistance, which is strongly linked to the metabolic syndrome consisting of
central obesity, dyslipidemia, and hypertension. Each of these abnormalities carries a
risk for cardiovascular disease. The aim of this study was to determine the
prevalence of arterial hypertension and to identify the rate of undetected
hypertension in patients with NAFLD and NASH.
Methods: Patient records from 2006 to 2008 of the in- and outpatient department of
the Clinic of Gastroenterology and Hepatology at the Abdominal Center of the
University of Cologne with biopsy proven NAFLD (n = 16) or NASH (n = 30) were
retrospectively reviewed for the prevalence of arterial hypertension (blood pressure
≥ RR140/90 mmHg). Patients with a history of hypertension or concomitant therapy
with blood pressure lowering agents were categorized as known hypertensives.
Patients with chronic hepatitis C virus infection served as a control group (n = 122).
Results: The number of known hypertensives was 13/122 (10.7%) in the HCV group,
3/16 (18.7%) in the NAFLD group and 5/30 (16.7%) in the NASH group. The number
of previously undetected hypertensives was 25/122 (20.5%) in the HCV group,
2/16 (12.5%) in the NAFLD group and 9/30 (30%) in the NASH group. The
prevalence of arterial hypertension was 31.1% in the HCV group, 31.2% in the
NAFLD group and 46.7% in the NASH group.
Discussion/Conclusion: We observed a high rate of undetected and untreated
arterial hypertension in patients with NASH. Since the incidence of NAFLD/NASH is
expected to rise in Western countries gastroenterologists and hepatologists should
be encouraged to take the opportunity to measure blood pressure routinely and
recommend adjustment of antihypertensive therapy during specialized care for this
high risk population.
6

7
Fatty liver disease in children and adolescents
V. Tzaneva, S. Galcheva, D. Baleva
Department of Pediatric Endocrinology, Department of Radiology 1 , University
Hospital “St. Marine”, Varna, Bulgaria
Introduction: Fatty liver disease (FLD) is a liver condition with increasing frequency.
FLD is associated with diabetes, insulin resistance, hypercholesterolemia, hypertriglyceridemia
and obesity. Until recently it is an important childhood liver disease,
especially because childhood obesity is much more common. Aim was to determine
the prevalence of FLD among children with diabetes mellitus (DM) type 1 and with
obesity, its clinical features and treatment.
Methods: In 1992–2008, 15 children were diagnosed with FLD among these with
DM (258) and 44 children among these with obesity (135). The diagnosis was
determined by elevated serum aminotransferases and abnormal hepatic sonogram.
Test for Wilson’s disease and chronic hepatitis B and C were made as well as a lipid
profile (patients with FLD). In 35 children a therapy was made: vitamin E 200–400
mg/23 h, for 3–6 months or ursodeoxycholic acid (10 mg/kg/24 h) for 3 months.
Results: Among 258 peditatric patients with DM type 1, 15 children were diagnosed
with FLD (10 boys and 5 girls, from 7 to 18 years, median range 13.81 years), it was
5.81%. Among 135 obese children, 44 were with FLD (23 boys and 21 girls, from 4 to
18 years, median range 7.45 years). It was 32.59%. Among obesity grade IV
(19 patients) 10 were with FLD (52.63%). All patients had hepatomegaly and only 2
had splenomegaly. All children had abnormal sonogram suggestive of fatty
infiltration. Abnormal aminotransferases (steatohepatitis) were found in 2.7% among
DM type 1. Abnormal aminotransferases (steatohepatitis) were found in 6.67%
among obese children DM type 1 steatohepatitis. Evaluated children were negative
for Wilson’s disease and viral hepatitis. Lipid profile was abnormal in 28 cases:
15 with hypercholesterolemia and 13 with hypertriglyceridemia. The effect of
treatment was controlled clinically, biochemically, ultrasonographically after
3 months. The effect was better when the control of DM was better and when there
was a reduction in the weight of the obese children.
Discussion/Conclusion: FLD occurs in children with DM type 1 and children with
obesity. It is not a rare condition. The essential basis of the treatment is the adequate
control of DM and body weight.

Effects of dietary chromium picolinate in the treatment of the
experimental nonalcoholic steatohepatitis
N. Kuzu 1 , İ.H. Bahçecioğlu 2 , A. Gençaslan 3 , K. Metin 4 , B. Üstündağ 4 , İ.H. Özercan 5 ,
K. Şahin 6
1
Ege University, Faculty of Medicine, Division of Gastroenterology, Izmir, Turkey
2
Firat University, Faculty of Medicine, Division of Gastroenterology, Elazig, Turkey
3
Firat University, Faculty of Medicine, Department of Internal Medicine, Elazig,
Turkey
4
Firat University, Faculty of Medicine, Department of Biochemistry, Elazig, Turkey
5
Firat University, Faculty of Medicine, Department of Pathology, Elazig, Turkey
6
Firat University, Faculty of Veterinary, Department of Nutrition, Elazig, Turkey
Introduction: The development of nonalcoholic steatohepatitis (NASH) is strongly
associated with the metabolic syndrome and insulin resistance. Cr-picolinate
supplementation facilitates normal protein, fat, and carbohydrate metabolism. Cr-picolinate
is often used as dietary supplement to improve insulin sensitivity and to
correct dyslipidemia. The aim of this study was to investigate to the therapeutic effect
of dietary Cr-picolinate in the experimental nonalcoholic steatohepatitis induced by
high fat diet (HFD).
Methods: Twenty-seven male Sprague-Dawley rats were divided into three equal
groups randomly. First group received only standard rat diet (control group), while
groups 2, and 3 were given HFD ad libitum. HFD was purchased from Dyets
(Benthlehem, P.A.). After the 12 weeks, Cr-picolinate (200 microg Cr/kg BW) was
administered by orogastric gavages daily for 4 weeks to rats in group 3. After
16 weeks all rats killed.
Serum biochemistry, TNF-alpha, plasma and liver tissue malondialdehyde (MDA),
glutathione levels were analyzed. Insulin resistance was assessed by Homa-R
method. Histopathological signs of steatosis, inflammation, and fibrosis were scored
semi-quantitatively and were modified from Brunt’s criteria. Immunohistochemical
staining was done for CYP2E1 and a sMA expression.
Results: Mean body weight, oxidative stress, CYP 2E1 expression and serum
TNF-alpha levels were increased in rats with steatohepatitis. Cr-picolinate was
improved insulin sensivity.
Cr-picolinate was effective in reducing hepatic steatosis and inflammation. Balloning
degeneration and a sMA expression were decreased by supplemantation of Cr-picolinate.
CYP 2E1 expression, liver malondialdehyde level and serum TNF-alpha level were
decreased by supplementation of Cri-picolinate (p < 0.05). Plasma cholesterol and
triglyceride concentrations were lower in the rats with supplementaton of Cr-picolinate.
Discussion/Conclusion: These results indicate that Cr-picolinate attenuates
steatohepatitis inducued by HFD in in the rats. This effect was mediated in part by
reducing body wieght and improving insulin sensivity.
Key words: steatohepatitis, high-fat diet, chromium picolinate
8

9
In what manner does the metabolic syndrome modify the viral
and alcoholic hepatic cirrhosis course?
N.N. Silivontchik, E.I. Adamenko
Educational Establishment “Belarusian Medical Academy of Post-Graduate
Education”, Minsk, Belarus
Introduction: Metabolic syndrome (MS) is an independent cause of liver disease
and the problem has an increasing interest.
Aim: Clinical peculiarities of viral (HCV) and alcoholic hepatic cirrhosis (HC)
associated with MS were analyzed.
Methods: Clinical study was carried out in 623 hospitalized patients with viral and
alcoholic HC including 41 with MS (6.1% had Child A, 51.1% – B, 42.3% – C). HC
was proved by biopsy or simply derived from clinical and laboratory data. MS was
diagnosed according to ATP (2001). All patients suffer from diabetes (28 received
oral hypoglycemic agents, 13 insulin).
Results: MS patients were older than others (54.41 ± 10.36 versus 50.28 ± 11.94
years, p = 0.029), had longer history of HC (Me = 16 versus 12 months, p = 0.045),
and didn’t differ in sex. Contrary to expectations HC associated with MS didn’t
exceed HC without MS in the severity Child’s score (Me = 9 versus 10 points,
p = 0.06) and the majority of characteristics (only cholelithiasis was reliably more
frequent in MS patients: 26.3 versus 10.3%, p < 0.001). Mortality rate (hepatic and
nonhepatic) was equal (χ 2 = 0.338, p = 0.501).
Discussion/Conclusion: MS isn’t a risk factor of alcoholic and HCV HC aggravation
and clinical course worsening. The explanation may be: 1) satisfactory MS patients’
nutrition status, 2) more strict treatment control because of diabetes, 3) decreasing
cardiovascular complication risk.

10
Assessment of liver fibrosis by TE (FibroScan ® ) in patients with
metabolic syndrome
Scurtu-Martin Mihaela, Adriana Tudora, O. Ciof, E. Dragan, Diana Rill, Sporea Ioan
Department of Gastroenterology and Hepatology, University of Medicine and
Pharmacy “Victor Babes”, Timisoara, Romania
Introduction: The aim of this study was to investigate the accuracy of liver stiffness
measurements (LSMs) by transient elastography (TE) (FibroScan ® , Echosense,
Paris, France) as a noninvasive test, in the diagnosis of hepatic fibrosis, and to
determine whether it can replace liver biopsy in patients with metabolic syndrome
without other cause of liver disease.
Methods: 3459 TE evaluations were performed between 2007–2009 in the
Department of Gastroenterology and Hepatology, Timisoara. MS (defined using
NCEP definition as the presence of at least 3 of the following criteria: waist
circumference > 102 cm in men (M) and 88 in women (W), triglycerides
> 1.69 mmol/l, HDL-cholesterol < 1.04 mmol/l (M) and 1.29 (W), blood pressure
> 130/85 mmHg, fasting glucose > 6.1 mmol/l) was present in 10.5% (n = 363 cases,
99 women, 27,4% and 264 men, 72,6%, mean age 44.3 ± 3 years). All subjects had
a complete medical examination and laboratory tests additionally to LSM performed
the same day by a single operator unaware of clinical and biological data.
Results: Valid measurements could not be obtained in 7% (25) cases. In the
338 remaining patients, mean LSMs in patients with MS values were 7.1 ± 1.2 kPa
significantly higher compared to normal subjects (4.8 ± 1.3 kPa), (p < 0.0001). The
following values were obtained: in 77 (36.2%) cases LS was ≤ 5.9 kPa (equivalent to
F = 0); in 45 (21.1%) patients, LS varied between 6 and 6.8 kPa (equivalent to F = 1);
and in the 91 (42.7%) remaining patients, LS was ≥ 6.9 kPa (equivalent to significant
fibrosis). LSM was also higher in obese (BMI > 30 kg/m 2 ) compared to overweight or
normal weight subjects (p = 0.003).
Discussion/Conclusion: MS was positively associated with liver stiffness and liver
fibrosis, thus LSMs using TE proved to be a reliable non-invasive method in
evaluating LF in these patients.

11
Investigation on cytokines at viral hepatitis A
A. Petrov, N. Vatev
Medical University Plovdiv, Department of Infectious Diseases, Parasitology and
Tropical Medicine, University Hospital “S. George”, Clinic of Infectious Diseases,
Plovdiv, Bulgaria
Introduction: The harm mechanisms of the hepatocytes in viral hepatitis A are not
fully cleared. The cytolysis is given mainly to immunopathogenic answer of the
contaminated cells, than to the direct effect of the virus. Non-specific immune
response is fulfilled largely by NK cells and cytokines. They cooperate on the
communication between the cells and reveal various and important role in the liver in
this manner. The cytokines act against virus infections in two main ways: direct – by
inhibition of viral replication and indirectly – by determination of the dominate model
of human's immune answer.
Methods: We investigated Th1 cytokines: IL-1beta, IL-2, TNF-alpha, IL-6 and
IFN-gamma of 83 patients with viral hepatitis A. In 29 patients surveys were done as
of heat of the disease, so as well at early reconvalescence. The cytokines were
determined by the enzyme immunoassay.
Results: Our surveys showed increased amounts of the investigated cytokines in
heat to the viral hepatitis A: IL-1beta – 3.35 pg/ml, IL-2 – 8.54 pg/ml, TNF-alpha –
13.92, IL-6 – 1.92 pg/ml. The average values were significant demoted in the
beginning to the reconvalescence: 1.68 pg/ml, 8.54 pg/ml, 6.63 and 1.42 pg/ml
respectively. Only with three patients the serum level of IFN-gamma were increasing
considerably. Nonparametric correlation test of Spearman showed correlation of
values of the cytokines by clinical heaviness of viral hepatitis A.
Discussion/Conclusion: Proinflammatory cytokines – IL-1beta, IL-2, TNF-alpha,
IL-6 and IFN-gamma at viral hepatitis A are investigated in Bulgaria for the first time.
Positive correlation is discovered between Тh1 cytokines values and the heaviness of
the hepatitis which is contribution to the pathogenesis of the disease.

12
Blocking Bim-mediated attrition of T cells in patients with
chronic HBV infection
Anna Schurich 1 , Pooja Khanna 1,2 , A. Ross Lopes 1 , Gaia Nebbia 1 ,
Geoffrey Dusheiko 2 , Mala K. Maini 1,2
1 Division of Infection and Immunity, UCL, London, UK
2 Centre for Hepatology, Royal Free Campus, University College London Medical
School, London, UK
Introduction: We recently identified a role for Bim-mediated apoptosis in the
profound attrition of HBV-specific CD8 T-cells characteristic of patients with chronic
HBV infection (CHB). Interruption of this pathway rescued functional, multispecific
CD8 responses directly ex-vivo (Lopes et al., JCI 2008). We postulate that
intrahepatic antigen-presentation, shown to induce T cell expression of Bim (Holz et
al., Gastro 2008), imposes this mechanism of T cell tolerance through an
unfavourable balance of positive and negative co-regulation. Here we explore
therapeutic options for reversing this pro-apoptotic phenotype to reconstitute effective
antiviral responses in-vivo.
Methods: HBV-specific CD8 were identified by ICS for IFN-γ following stimulation
with peptides covering HLA-A2 restricted epitopes or spanning the HBV genome.
Intracellular Bim levels were quantified by flow-cytometry before and after blocking
upstream and downstream targets.
Results: Cross-sectional comparison of CHB patients with/without antiviral therapy
showed no differences in intracellular Bim levels in HBV-specific CD8. A longitudinal
sub-study monitoring patients before and during antiviral therapy showed no
reduction in Bim expression during the short-lived recovery of HBV-specific CD8
upon suppression of viral load. In-vitro cyclosporin A treatment of CD8 blocked Bim
expression and we are now investigating the impact of manipulating co-inhibitory
(e.g. CTLA-4) or co-stimulatory (e.g. 41BB) signals on the pro-apoptotic tendency of
HBV-specific CD8 in CHB.
Discussion/Conclusion: HBV-specific CD8 T-cell responses reconstituted with
potent antiviral therapy in CHB are transient and remain highly susceptible to
apoptosis. We propose that specific measures to block Bim-mediated attrition are
necessary to recover durable off-treatment antiviral responses.

13
Chronic hepatitis HBV in children treated with interferon-α –
Prognostic value of serum interleukin 6 and interleukin 12
Gora-Gebka Magdalena, Liberek Anna, Sikorska-Wisniewska Grazyna,
Gołebiewski Jacek, Kaminska Barbara
Department of Paediatrics, Paediatric Gastroenterology, Hepatology and Nutrition,
Medical University of Gdańsk, Poland
Introduction: The course of HBV infection and the outcome of IFN-α of patients with
chronic hepatitis B are determined by the antiviral immune response of the host.
Serum cytokine profile could enable the selection a group of patients prone to
positive response to IFN-α therapy. It appears especially important in pediatric
patients with immature immunological system in whom exogenous IFN-α may cause
distinct, serious side effects.
The aim of the study was 1) to investigate the correlation between IL-6 and IL-12
serum levels and biochemical and histopathological changes in children with chronic
hepatitis B, 2) to evaluate the predictive value of the pre-treatment serum levels of
these cytokines for response to treatment with IFN-α.
Methods: Serum levels of IL-6, IL-12 (heterodimer p70) and IL-12 (heterodimer p70
and p40 subunit) were determined by specific ELISA in 57 children (aged 5.9–17.1
years) with chronic hepatitis B on the first day of IFN-α therapy.
Results: Serum IL-6 levels before IFN-α treatment were 0.5–1.7 pg/ml (median
0.9 pg/ml). Serum levels of IL-12 (p70) were 13.9–81.1 pg/ml (median 22.9 pg/ml)
and IL-12 (p70 and p40) were 89.9–451.7 pg/ml (median 292.8 pg/ml) and they
showed no correlation with biochemical and histopathological changes. There was no
significant difference between the pre-treatment serum levels of these cytokines in
the group of patients who seroconverted to anti-HBe (36%) and those who did not
respond to IFN-α therapy (68%).
Conclusion: Serum levels of IL-6 and IL-12 do not correlate with biochemical and
histopathological markers of hepatitis and seem to be of poor prognostic value for
positive response to the IFN-α therapy in children with chronic hepatitis B.

14
High frequency of HBV-specific T cell responses in delta
hepatitis
J. Grabowski 1 , P.V. Suneetha 1 , J. Jaroszewicz 1,2 , V. Schlaphoff 1 , B. Bremer 1 ,
M.P. Manns 1 , M. Cornberg 1 , H. Wedemeyer 1
1
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical
School, Hannover, Germany
2
Department of Infectious Diseases and Hepatology, Medical University in Bialystok,
Bialystok, Poland
Introduction: Infection with hepatitis D virus (HDV) can cause severe acute liver
disease and rapid progression of chronic hepatitis. However, not all patients develop
liver cirrhosis and immune correlates associated with different outcomes are poorly
defined. The aim of this study was to investigate both HDV-specific and HBV-specific
T cell responses as well as in-vitro and in-vivo cytokine levels in correlation with
virological and clinical parameters.
Methods: In this study, 23 individuals with HBV/HDV infection were included. Ten
HBV monoinfected patients and ten healthy individuals served as controls.
PBMC were stimulated by overlapping peptides spanning the entire HDV genome,
HBV surface, HBcore and HBV polymerase proteins, respectively.
T-cell-proliferation was investigated by 3 H-Thymidin-incorporation. Production of
interleukin-2, interleukin-10, interferon-gamma and interferon-gamma-inducedprotein-10
was evaluated by CBA in cell culture supernatants, serum samples of
HDV patients additionally were tested for interleukin-17A and interleukin-21.
Results: HDV-specific proliferative responses were detected less often than HBVspecific
T-cell-responses in patients with delta hepatitis. Only 2 patients displayed
both HBV- and HDV-specific responses. HDV-specific T-cell-responses were
targeted mainly against amino acids 95–181 while HBcore-specific responses
dominated. HDV patients with HBV-specific T-cell-responses were significantly
younger than individuals with HDV-specific immune responses or nonresponder
patients and had higher HBsAg levels. Furthermore, HBV-DNA-levels were
significantly lower in patients with HDV-specific responses than in patients with
HBV-responses.
Discussion/Conclusion: HDV patients show a rather high frequency of HBVspecific
T-cell-responses despite chronic HBV/HDV infection which may correlate
with distinct virological characteristics. Ongoing work is investigating possible
mechanisms how HDV-coinfection interferes with HBV-specific immunity.

15
Hepatitis B prevalence and flare in Asian patients with
rheumatoid arthritis treated with disease modifying antirheumatic
drug (DMARD) therapy
Owen Bebb, Victoria Appleby, Paul Southern, Philip Helliwell, Sulleman Moreea
Bradford Royal Infirmary, Bradford, UK
Introduction: The prevalence of hepatitis B (HBV) in our large South Asian
population is around 3–4%. HBV can flare during immunosuppressant therapy,
including the use of DMARD therapy. We aimed to analyse the characteristics of and
determine the incidence and outcome of HBV flare in our Asian patients with
rheumatoid arthritis (RA) on DMARD therapy.
Methods: Our RA database and results system were analysed to obtain the
following: demographics, ethnicity, treatment with DMARD therapy, HBV serology/virology
and biochemical results
Results: There were 2861 patients on the RA database: 934 males (32.7%) mean
age 55.6 y; 1927 females (67.3%) mean age 56.0 y. 505 (17.7%) were of Asian
origin – 114 M (20.5% of Asians) mean age 46.3 y; 391 F (79.5% of Asians), mean
age 48.6 y. 103 of all Asian patients (24.3%) had a HBsAg test. 2/103 patients (1 M
[51], 1 F [52]) were HBV positive, prevalence rate 1.94%.
There were 424 Asians on DMARD therapy (84.0% of all Asians and 14.8% of the
population) consisting of 89 M mean age 44.9 y and 335 F mean age 47.6 y (ratio
M:F = 1:4). 206/424 (48.6%) had an ALT flare: 51 M mean age 48.7 y, 155 F mean
age 48.1 y (ratio 1:3). 103 (24.3%) 26 M (mean age 42.1 y)and 77 F mean age
43.8 y) were tested for HBV and 2 patients (1 M [51], 1 F [52]) had a HBV flare. They
were both eAg negative patients with viral breakthrough. They were treated successfully
with lamivudine with no mortality. In the 305 patients (62 M mean age 53.3 y,
243 F mean age 52.7 y) on DMARD therapy without HBV status, 105 (34.4%) had a
rise in ALT during treatment.
Discussion/Conclusion: Our figures suggest a prevalence rate of 1.94% HBV in the
local Asian population. This study has highlighted the ad-hoc HBV testing in our
population. We suggest serological testing for all RA patients on DMARD therapy
before the start of treatment but particularly in high risk patients originating from
South Asia.

16
Interferon-α treatment fails to induce activation of interferon
responsive genes in HBV chronically infected human chimeric
uPA/SCID mice
Marc Lütgehetmann 1 , Tassilo Volz 1 , Ansgar Lohse 1 , Jan-Hendrick Bockmann 1 ,
Jörg Petersen 2 and Maura Dandri 1
1
Department of Internal Medicine, University Medical Hospital Hamburg-Eppendorf,
Germany
2
IFI, Institute for Interdisciplinary Medicine at Asclepius Clinic St. Georg, Hamburg,
Germany
Introduction: Interactions between HBV and the interferon system of the host are
still poorly defined. Experimental infection of chimps showed that the interferon
system is not induced during acute HBV infection, suggesting that HBV may have
evolved mechanisms to avoid activation of the IFN signaling cascade.
Aim of the study was to investigate whether HBV can directly interfere with the
hepatocellular IFN-α response in chronically infected human hepatocytes.
Methods: We used primary human hepatocytes isolated from the same donor to
repopulate the livers of uPA/SCID mice and analysed changes in the expression
levels of IFN responsive genes (IRGs) using primers specific for human transcripts
and not cross-reacting with murine genes. Chimeric mice were then infected with
HBV-positive mouse serum (genotype D) and after establishment of infection
(median viremia 3 x 10 7 copies/ml) mice received 1350 IU/g IFN-α daily (n = 5) or
PBS (n = 4) for 5 days. To determine responsiveness of transplanted human
hepatocytes to IFN treatment, uninfected human chimeric mice were treated for
5 days either with IFN-α (n = 4) or with PBS (n = 7). All animals were sacrified 8 h
after the last injection.
Results: HBV infection of human hepatocytes in chimeric animals did not increase
expression levels of human IRGs like OAS and MXA, though significant activation of
these genes could be demonstrated in the livers of IFN-treated uninfected chimeric
mice (median 3-fold OAS and 10-fold MXA). Treatment with IFN-α induced only a
moderate reduction of HBV-DNA titres (median 0.75 log) as well as intrahepatic
rcDNA and pgRNA amounts compared to untreated control animals. Strikingly, IFN
treatment was not able to increase expression levels of OAS and MXA in HBV-infected
hepatocytes repopulating the uPA mouse livers, while expression levels of
IFNA2 receptor remained unchanged regardless of infection status in treated mice.
Discussion/Conclusion: These results suggest that HBV can specifically hinder the
activation of the IFN signaling in vivo, in chronically infected primary human
hepatocytes.

17
Relationship between liver fibrosis and topographic
distribution of alpha-smooth muscle actin staining in chronic
hepatitis B
O. Kosseva, E. Pophristova, D. Adjarov, D. Jelev, Z. Krastev
University Hospital St. Ivan Rilski, Sofia, Bulgaria
Introduction: Observations have led to the description of distinct patterns of fibrosis,
related to the underlying disorders causing fibrosis. It can therefore be expected that
in this different patterns of fibrogenic evolution, different myofibroblast like subpopulation
participate and distinct topographic localization of activated myofibroblasts
preferentially contribute to the formation of fibrotic tissue.
Aim: To evaluate the relationship between liver fibrotic stage in patients with chronic
viral hepatitis B and the topographic distribution of alpha-smooth muscle actin (alpha-
SMA)-positive myofibroblasts, divided in four compartments: portal/septal and in
lobular zones 1, 2 and 3.
Methods: Liver biopsies from 50 adult patients with HBV chronic hepatitis were
assessed according to Metavir and immunohistochemical staining for alpha-SMA
was performed on paraffin-embedded tissue sections. The number of a smooth
muscle-positive cells in the lobules was scored using the system developed by
Schmitt-Graff et al. Portal/septal expression of alpha-SMA was estimated by score
proposed by us:
0 alpha-SMA with expression in smooth cells within portal blood vessels only.
1 A few alpha-SMA-positive cells beside vascular smooth muscle cells.
2 alpha-SMA-positive expansion of most portal areas with short and thin septa of
alpha-SMA-positive staining.
3 One or few positive septa for alpha-SMA.
4 alpha-SMA staining forms numerous septa.
5 Pseudonodules embraced by alpha-SMA positive staining.
Statistical analyses was done by Mann-Whitney and Spearman correlation test.
Results: Strongest positive correlation was found between fibrotic stage and
portal/septal expression of alpha-SMA (r = 0.757; p < 0.01), modest correlation was
detected with periportal myofibroblast score (r = 0.427), while alpha-SMA staining in
zone 2 showed week relationship (r = 0.256; p < 0.05). Expression of alpha-SMA in
the pericentral part of the lobule did not shown any correlation with fibrotic stage.
Discussion/Conclusion: It should be emphasized that counting the total number of
myofibroblast, without taking in to account their topographic localization could blur
the estimation of fibrogenic potential.

Is any role of hepatic steatosis in chronic virus B hepatitis?
18
Cătălina Mihai 1 , Cristina Cijevschi 1 , Gabriela Ştefănescu 1 , Vasile Drug 1 ,
Mihaela Dragna 1 , Lidia Graur 2 , Bogdan Mihai 2
1
Institute of Gastroenterology and Hepatology, University of Medicine and Pharmacy
“Gr. T. Popa”, Iasi, Romania
2
Clinical Centre of Diabetes and Metabolic Diseases, University of Medicine and
Pharmacy “Gr. T. Popa”, Iasi, Romania
Introduction: It is well-known the influence of hepatic steatosis over fibrosis and the
response to antiviral treatment in chronic hepatitis C, but limited data are available in
chronic hepatitis B. The aim of this study was to evaluate the hepatic steatosis in
patients with chronic hepatitis B and to correlate the steatosis with clinical, biological,
virusological and histological factors.
Methods: We studied 75 patients with chronic hepatitis B who were evaluated for
antiviral treatment by liver biopsy between 1st January 2006 and 31st December
2008. In group A there were no hepatic steatosis and in group B the hepatic steatosis
was over 5%. We studied in these two groups the following parameters: age, sex,
body mass index (BMI), glycaemia, lipidic profile, liver enzymes, the presence/absence
of HBe antigen, co infection with D virus, viremia, the degree of necroinflammation
and fibrosis.
Results: There were 52 patients in group A (69%) and 23 (31%) in group B. The
parameters which were correlated with the presence of hepatic steatosis (p < 0.05)
were: age (mean value 48.6 in group B and 36.5 in group A), BMI (29.7 kg/m 2 in
group B and 24.1 kg/m 2 in group A), glycaemia (109.6 mg% compared to 88.5 mg%),
triglycerides (137.2 mg% compared to 87.4 mg%), necro-inflammatory score (> 8 –
Metavir score – at 56.5% patients in group B and 36.5% in group A) and fibrosis
(F3–F4 – Metavir score – at 39.13% patients in group B and 15.38% in group A). In
group B were more male patients, more elevated liver enzymes and cholesterol, but
without statistical significance. There were no differences between the two groups
regarding the presence of HBe antigen (38.46% in group A and 43.47% in group B),
the presence of virus D (7.69% in group A vs. 8.69% in group B), the level of viremia
(mean value 767,000 U/ml in group A vs. 643,000 U/ml in group B).
Discussion/Conclusion: In chronic hepatitis B the steatosis is present in 1/3 patients,
related with metabolic and non viral factors. It seems to be a positive correlation
between hepatic steatosis and severity of liver injury (necroinflammation and
fibrosis). The complex therapeutic approach in these patients (metabolic and antiviral
treatment) may improve the outcome of chronic hepatitis B.

19
The effects of chronic B and C hepatitis infections on bone
mineral density
Corina Serban, Lelia Susan, Alina Pacurari, Loredana Copaceanu, Christian Banciu,
Germaine Savoiu, Claudia Borza, Rodica Mateescu, Ioan Romosan
UMF Timisoara, Romania
Introduction: The importance of osteoporosis as a complication of liver disease is
well known. The aim of this study was to evaluate bone mineral density in patients
with chronic B and C hepatitis, hospitalised in one year period in The IVth Medical
Clinic of University of Medicine and Pharmacy “Victor Babes”, Timisoara.
Methods: Clinical data, hepatitis B and C status, and markers of bone metabolism
were determined in 120 patients (39 men and 81 women; mean age 52 years). Bone
mineral density was assessed in all patients at the lumbar spine (LS; L1–L4) and
femoral neck (FN) region using X-ray absorbtiometry. Osteoporosis and osteopenia
were defined by WHO criteria. The values were expressed as the T and Z score.
Results: 86 of the patients were with hepatitis C (71.66%) and 34 of them with
hepatitis B (28.34%). 43 patients were with osteoporosis (35.83%), 55 of them
presented osteopenia (45.83%) and 22 of the patients were with normal BMD
(18.33%). Mean T-score value was lower in patients with chronic hepatitis C (lumbar
spine T = -1.15 ± 1.12, femoral neck T = -1.62 ± 1.01) compared with chronic
hepatitis B (lumbar spine T = -0.75 ± 1.31, and femoral neck T = -1.35 ± 1.03). The
values of serum and urine biochemical markers were normally in both groups.
Discussion/Conclusion: This study reveals a high prevalence of osteoporosis in
chronic hepatitis B or C patients. These secondary effects of chronic viral hepatitis
should be further investigated.

20
TGF-β-driven hepatic progenitor cell expansion and ductular
reaction contributes to HBV, but not schistosomiasis,
associated liver fibrogenesis
Hong-Lei Weng 1 , Loredana Ciuclan 1 , Yan Liu 1 , Cheng Zhu 2 , Eliza Wiercinska 3 ,
Iryna Ilkavets 1 , Johanna Dzieran 1 , Tong Huang 4 , Christoph Meyer 1 , Rolf Gebhardt 5 ,
Rainer Heuchel 6,7 , Peter ten Dijke 3 , Jia-Lin Chen 8 , Manfred V. Singer 1 ,
Peter R. Mertens 2 , Steven Dooley 1
1 Molecular Hepatology – Alcohol-dependent Diseases, II. Medical Clinic Faculty of
Medicine Mannheim, University of Heidelberg, Mannheim, Germany
2 Department of Nephrology and Hypertension, Otto-von-Guericke-University,
Magdeburg, Germany
3 Department of Molecular Cell Biology and Center for Biomedical Genetics, Leiden
University Medical Center, RC Leiden, The Netherlands
4 Department of Cardiac Vascular Medicine, Affiliated Hospital, Medical School,
Ningbo University, Ningbo, China
5 Institute for Biochemistry, University of Leipzig, Leipzig, Germany
6 Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden
7 Department of Clinical Science, Intervention and Technology, Karolinska University
Hospital, Huddinge, Sweden
8 Department of Pathology, First Hospital of Jianxing, College of Jiaxing, Jiaxing,
China
Introduction: In HCV- and NASH-associated liver diseases, hepatic progenitor cells
(HPCs) represent the major source of hepatocyte regeneration. Further, HPCs
promote a periportal ductular reaction (DR) and contribute to periportal fibrogenesis.
However, the factor(s) that mediate HPC activation are presently unknown. We
hypothesize that TGF-β may drive HPC activation/DR in patients with chronic HBV
infection.
Methods: 110 biopsied liver tissues with chronic HBV infection were stained with
cytokeratin-7 to quantify HPCs and DR, and with p21 to assess hepatocyte
replication arrest.
Results: Cytokeratin-7 showed significant correlation with inflammatory grades
(r = 0.47, p < 0.001), fibrotic stages (r = 0.53, p < 0.001) and phopsho-Smad2
staining, a marker of active TGF-β signaling (r = 0.24, p < 0.01). p21 positive
hepatocytes were only found in 10 advanced fibrotic liver tissues, indicating that
hepatocyte proliferation arrest is not required for HPC activation/DR in this disease.
9-month IFN-γ treatment, which attenuated TGF-β signaling, significantly decreased
HPCs in 13/18 patients with chronic HBV infection. Confocol microscopy analysis
revealed co-localisation of phospho-Smad2/3 and HPC marker, indicating activated
TGF-β signalling in these cells. Compared with wild type mice, M(2)-isozyme of
pyruvate kinase, a marker for oval cells (HPCs in rodents) increased in TGF-β
transgenic mice. The numbers of oval cells and p21 positive hepatocytes were
significantly increased in Smad7 exon-1 deleted mice after 4-weeks of CCl4 treatment
and were decreased in Smad7 transgenic mice after 2-week of bile duct ligation
compared to wild types. In contrast to HBV patients, HPC activation was not
associated with fibrotic degree in 42 Schistosoma japonicum infected patients.

Discussion/Conclusion: TGF-β signaling plays a crucial role in HPC activation/DR,
which contributes to fibrogenesis in patients with chronic hepatitis B, but not
schistosomiasis, suggesting etiology dependent differences in the mechanism of liver
damage.

21
Is there a link between the viral load value and liver stiffness
for the asymptomatic virus B carriers?
Adriana Tudora, O. Ciof, Mihaela Scurtu-Martin, I. Sporea, E. Dragan, Diana Rill
Department of Gastroenterology and Hepatology, University of Medicine and
Pharmacy “Victor Babes”, Timisoara, Romania
Introduction: To evaluate the role of transient elastography (FibroScan ® Echosense,
Paris), as a noninvasive method to exclude significant liver fibrosis in nonreplicative
virus B carriers compared to those presenting detectable viral load yet below
10,000 copies/ml.
Methods: 687 consecutive patients with viral B infection were evaluated (261 women
– 38%, and 426 men – 62%), average age 43.4 ± 14.2 years. Out of this group,
138 were defined as asymptomatic carriers based on normal ASAT, ALAT levels and
viral load

22
Evaluating antiinflammatory and antiviral effect due to the
treatment with statins in patients with chronic virus C hepatitis
Oana Andreescu 1 , Laurentiu Nedelcu 1 , Camelia Scarneciu 1 , Daniela Paul 2 ,
Ileana Pantea 1
1 Faculty of Medicine, University of Brasov, Romania
2 Clinic Emergency Hospital of Brasov, Romania
Introduction: Lately has been shown that statins can have more beneficial role than
lowering lipid levels. Our study aimed to reveal antiinflammatory as well as antiviral
effect due to the treatment with statins.
Methods: The study was performed on 47 patients (31 women and 16 men aged
between 40–82 years) diagnosed with chronic hepatitis with C virus who were nonresponders
or without antiviral treatment at the beginning of the study. Patients were
divided into 2 groups that received as treatment lovastatin and fluvastatin separately.
All patients underwent clinical examination, viremia level and biochemical tests
including a series of cytokines (IL-6, IL-8, IL-10, TNF-α, erythropoietin) at the
beginning of the study and after 1 month.
Results: Both groups registered especially lower level of TNF-α (56% cases that
received lovastatin and 65% cases that received fluvastatin). The majority of the
study group associated also lower levels of viremia (70% cases that received
lovastatin and 83% cases that received fluvastatin)
Discussion/Conclusion: Our study has demonstrated that the statins could have
both antiinflammatory and antiviral effect which seems to be more important in case
of administrating fluvastatin.

23
Variants of the innate immune response genes in patients with
chronic HCV-infection
A.O. Romanov, T.V. Belyaeva, E.V. Esaulenko
Pavlov State Medical University, Saint-Petersburg, Russia
Introduction: The 2-5 oligoadenylate synthetase (OAS)-ribonuclease L (RNase L)
pathway is a critical component of the immune response to viruses that constitutively
presents in human cell. Double-stranded RNA structure within nontranslated regions
HCV RNA activates OAS resulting in the activation of RNase L that cleaves viral
RNA. The A allele at single nucleotide polymorphism (SNP) rs10774671 of the OAS1
gene has been reported to be associated with low enzyme activity. Furthermore,
1385G/A RNaseL variant showed a 3-fold decrease in RNase L activity. The aim of
this study was to evaluate whether these functional polymorphisms are associated
with the natural outcome of hepatitis C virus infection.
Methods: Genotyping was performed in 76 patients with hepatitis C virus-induced
cirrhosis, in 88 non-cirrhotic patients infected with HCV and in 122 healthy controls.
The A/G OAS1 SNP rs10774671 was determined by PCR-RFLP assay using AluI
restriction enzyme. The polymorphism +1385G/A in the RNaseL gene was detected
by PCR-RFLP assay using a Msp20I site introduced into the forward primer next to
the G to A transition.
Results: There were no significant differences in the distributions of genotype and
allele frequencies of A/G OAS1 SNP rs10774671 or +1385G/A RNASEL gene
polymorphism between HCV patients with or without cirrhosis and between patient
groups and healthy controls. Nevertheless, composite genotypes rs10774671 (AA or
AG)/+1385(AG or AA) RNaseL had increased risk for cirrhosis among HCV-infected
patients (OR 5.51; 95% CI 1.74–17.17).
Discussion/Conclusion: The results of the study suggest that combinations of the
variant genotypes of A/G OAS1 SNP rs10774671 and +1385 G/A RNaseL might
affect the development of cirrhosis in persons with chronic HCV infection.

24
Expression and effect of the NK cell receptor 2B4 (CD244) on
virus-specific CD8+ T cells – Another player in the control of
viral hepatitis?
V. Schlaphoff, J. Jaroszewicz, S.V. Pothakamuri, J. Grabowski, K. Stegmann,
M.P. Manns, H. Wedemeyer, M. Cornberg
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical
School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
This study aimed to investigate expression patterns and function of the costimulatory
NK-cell receptor 2B4 on CD8+ T-cells and to elucidate a possible role in viral
hepatitis.
Expression of 2B4 was analyzed on lymphocytes and virus-specific CD8+ T-cells in
healthy individuals by FACS. 2B4 was found on CD8+ T-cells (mean 35.6%, range
11.9–52.5%), CD56+ NK-cells (100%) and on monocytes (100%), but was only rarely
present on CD4+ T-cells (mean 0.92%, range 0.18–2.1%). A clear activation and
memory phenotype could be seen identifying 2B4+CD8+ T-cells as effector or
effector memory cells. Isolated 2B4+CD8+ cells showed a decreased survival
in-vitro, whereas they displayed strong degranulation upon CD3/28 stimulation in
contrast to 2B4-CD8+ cells.
Expression of other inhibitory molecules PD-1 or CTLA-4 correlated with expression
of 2B4. Similarly, CD161-, CD38- or CD57-positive CD8+ T-cells uniformly expressed
2B4. In contrast, not all 2B4+CD8+ T-cells showed expression of these markers.
2B4 expression was further investigated on PBMCs from patients with chronic HCV
(n = 60) or HBV (n = 26), acute HCV or HBV infection (n = 6 and n = 10) and patients
with AIH, PBC or PSC (n = 15). 2B4+CD8+ T-cells ex-vivo frequency was higher in
patients with chronic HCV, HBV or AIH as compared with healthy individuals (46.9%,
52.8% and 50.6% vs. 35.6%, p > 0.02).
Analyzing virus-specific T-cells for 2B4, CMV- and EBV-specific CD8+ T-cells
showed high expression of 2B4, while Influenza-A-specific CD8+ T-cells were mostly
negative for 2B4. HCV- and HBV-specific CD8+ T-cells were mainly 2B4-positive
during acute infection, while in chronic HCV different patterns of 2B4 on virus-specific
CD8+ T-cells were observed.
In conclusion, 2B4 expression correlates with an end-stage effector type T-cell and
distinct early activation phenotype. Higher frequencies of 2B4+CD8+ T-cells during
acute and persistent viral infection suggest a regulatory role during antigen exposure.
We propose that besides PD-1, CTLA-4 and other receptors the costimulatory
molecule 2B4 is involved in the immune control of hepatitis C and B.

25
Ethanol impairs MHC class I-restricted antigen presentation on
HCV-infected liver cells
Natalia Osna 1 , Ronda White 1 , Steven Weinman 2 , Terrence Donohue 1
1 University of Nebraska Medical Center, Omaha, NE, USA
2 University of Kansas Medical Center, Kansas City, MO, USA
Introduction: Elimination of HCV-infected hepatocytes by cytotoxic T-cells is
restricted to efficient MHC class I-restricted antigen presentation of viral peptides
generated by proteasome. Previously, we have shown that in cultured hepatoma
cells, the combination of HCV core protein and ethanol suppresses proteasome
activity (Gastroenterology, 2008). This study seeks to determine the level ethanolmediated
proteasome suppression in liver cells of HCV core-expressing mice and its
link to impaired antigen presentation in hepatocytes.
Methods: Hepatocytes isolated from livers of HCV core positive C57Bl/6 mice were
exposed overnight to 50 mM ethanol in the presence or absence of IFN-γ and then
C-extended SIINFEKL peptide was delivered to cytoplasm. After processing by
proteasome, SIINFEKL-H2Kb complex was presented on hepatocytes and visualized
with specific antibody by flow cytometry
Results: Ethanol exposure suppressed SIINFEKL-H2Kb presentation on HCV core
positive hepatocytes and these effects were reversed in the presence of N-acetyl
cysteine (NAC), indicating the involvement of oxidative stress. High level of oxidative
stress in HCV-expressing ethanol-exposed hepatocytes was confirmed by 5-fold
induction of ROS. Furthermore, SIINFEKL-H2Kb presentation was abrogated by
treatment with the proteasome inhibitor, Mg132, demonstrating the critical role of this
enzyme for SIINFEKL processing. Proteasome activity was also suppressed in
hepatocytes by ethanol exposure in ethanol metabolism-dependent manner.
Discussion/Conclusion: We conclude that the combination of HCV core protein and
ethanol creates high oxidative stress, leading to the reduction of proteasome activity
and inability of the enzyme to efficiently generate peptides for MHC class I-restricted
antigen presentation in hepatocytes

26
Impairment of interferon-gamma (IFN-γ) signaling in hepatocytes
by HCV proteins and ethanol
Lee Austin 1 , Ronda White 1 , Kusum Kharbanda 1 , Michael Beard 2 , Natalia Osna 1
1 University of Nebraska Medical Center, Omaha, NE, USA
2 University of Adelaide, Adelaide, Australia
Background: In HCV patients, alcohol consumption aggravates the course of
hepatitis and enhances the viral load. IFN-γ signaling in liver cells regulates the main
stages in antigen presentation, where HCV-infected hepatocytes serve as the targets
for immune cells. This study is aimed to determine IFN-γ signaling in HCV-expressing
liver cells exposed to ethanol.
Methods: C5B cells are replicon Huh7-based cells that express full genome of HCV
proteins and an ethanol metabolizing enzyme, CYP2E1. After cell exposure to
ethanol, STAT1 phoshorylation was induced by IFN-γ and measured differentially in
cells, which express HCV proteins, NS5B or core, and in non- or low-expressing cells
by Phoshoflow. In addition, the ratio of pSTAT1/STAT1 was measured by Western
blot in lysates of IFN-γ-exposed C5B and CYP2E1 + parental Huh7 cells.
Results: IFN-γ-induced pSTAT1 was lower in HCV + C5B cells than in Huh7 cells.
Ethanol exposure further suppressed pSTAT1. Importantly, STAT1 phoshorylation
was depended on expression of HCV protein expression in C5B cells: It was
abrogated in HCV-expressing C5B cells, while in non- or low-expressing C5B cells
the level of pSTAT1 was decreased by ethanol treatment. However, these effects of
ethanol were reversed in the presence of ethanol metabolism inhibitor, 4 methyl
pyrazole or N-acetyl cystein, indicating that ethanol metabolism-induced oxidative
stress was responsible for the pSTAT1 reduction.
Discussion/Conclusion: We conclude that HCV protein expression and ethanol
metabolism both suppress IFN-γ signaling in hepatocytes, providing an implication
for the impaired antigen presentation in liver cells of HCV + alcohol drinkers.

27
The platelet-derived chemokine CXCL4 (PF4) is a mediator of
HCV-induced and experimental liver fibrosis
Mirko Moreno Zaldivar, Katrin Pauels, Marie-Luise Berres, Petra Schmitz,
M. Anna Kowalska*, Ralf Weiskirchen, Christian Trautwein, Hermann E. Wasmuth
Medical Department III, University Hospital of Aachen, Aachen, Germany
*University of Pennsylvania School of Medicine, Philadelphia, PA, USA
Background: Liver fibrosis is a major cause of mortality and morbidity worldwide.
Platelets are involved in liver damage but the underlying molecular mechanisms
remain elusive. We here investigate the platelet chemokine CXCL4 (platelet factor 4)
as a molecular mediator of fibrotic liver damage.
Methods: Serum concentrations and intrahepatic mRNA of CXCL4 were measured
in patients with hepatitis C. Platelet infiltration in the liver was visualized by electron
microscopy in humans. Cxcl4 -/- and wild-type mice were subjected to two models of
chronic liver injury (CCl4 and TAA). The fibrotic phenotype of these mice was
analysed by histological, biochemical and molecular analyses. Intrahepatic infiltration
of immune cells was investigated by FACS and immunohistochemistry. Isolated
myofibroblasts were stimulated with recombinant Cxcl4.
Results: Patients with advanced HCV-induced fibrosis had increased PF4 serum
levels and intrahepatic CXCL4 mRNA concentrations. CCl4 and TAA treatment led to
an increase of hepatic Cxcl4 levels and activation of platelets in the peripheral blood
in mice. Accordingly, genetic deletion of Cxcl4 in mice significantly reduced
histological and biochemical damage in vivo. Amelioration of liver damage in Cxcl4 -/mice
was functionally associated with changed expression of fibrosis-related genes
(Timp-1, Mmp9, Tgf-β, IL-10). Functionally, Cxcl4 -/- mice had a strongly decreased
infiltration of neutrophils (Ly6G) and CD8 + T-cells into the liver. In vitro, recombinant
murine Cxcl4 stimulates the proliferation of isolated hepatic myofibroblasts.
Conclusions: These results functionally underscore the important role of the platelet
derived chemokine CXCL4 in chronic liver damage and imply a new pathway for antifibrotic
therapies.

28
Interleukin 17 pathway is suppressed in chronic hepatitis C
Gutkowski Krzysztof 1 , Kacperek-Hartleb Teresa 1 , Musialik Joanna 1 ,
Boryczka Grzegorz 1 , Kajor Maciej 2 , Hartleb Marek 1 , Kamińska-Kiszka Ewelina 3
1
Department of Gastroenterology and Hepatology, Medical University of Silesia,
Katowice, Poland
2
Department of Pathomorphology, Medical University of Silesia, Katowice, Poland
3
Department of Biotechnology, University of Rzeszow, Poland
Introduction: Immune dysregulations in hepatitis C are still unclear, especially
regarding inflammatory pathways. Recent studies showed that TGF-β alone
promoted the generation of anti-inflammatory regulatory T lymphocytes (T-reg) from
naive T CD4 + cells, whereas TGF-β in the presence of IL-6 promoted Th17 cells.
Materials and methods: We studied IL-17 serum levels and its activators (IL-6,
TGF-β) in 36 chronic hepatitis C patients and 33 age and gender-matched healthy
subjects. The results in HCV patients were related to liver histology. Cytokine levels
were assessed by enzyme-linked immunosorbent assays (ELISA).
Results:
Cytokines
Healthy subjects
(n = 33)
Hepatitis C patients
(n = 36)
Mean ± SD Range Mean ± SD Range p
IL-17 [pg/ml] 16.5 ± 6.6 9.6–32.6 12.5 ± 12.2 0.0–48.2 0.0214
IL-6 [pg/ml] 1.7 ± 1.2 0.5–6.6 16.7 ± 7.2 8.4–39.7 0.3732
TGF-β [ng/ml] 30.9 ± 7.4 16.8–46.2 47.9 ± 23.1 18.2–116.0 0.0008
Mann-Whitney test
Serum levels of IL-6 did not differ between healthy subjects and HCV patients but
positively correlated in hepatitis C cohort with stage of fibrosis (r = 0.45; p = 0.005).
Conclusions:
1. Chronic hepatitis C down-regulates Th17 response.
2. TGF-β level is increased in HCV patients and may be responsible for suppressing
of IL-17 production by Th17 cells.

29
Allelic expression of rs2569190/C-159T CD14 gene variants in
PBMC and liver samples from chronic hepatitis C patients
Rusudan Bregadze, Giuliano Ramadori, Sabine Mihm
Department of Gastroenterology and Endocrinology, University Medical Center
Göttingen, Germany
Introduction: The single nucleotide polymorphism (SNP) rs2569190 within the
endotoxin receptor CD14 gene has been described to be associated with the
progression of alcoholic liver disease. This association was suggested to be due to
sensitization of the liver for gut-derived endogenous LPS via an enhanced T allele
mediated hepatic CD14 expression. In parallel to association studies on this SNP
and disease progression in chronic hepatitis C, this study aimed at investigating
allelic expression of CD14 transcripts in PBMC and liver samples from chronically
hepatitis C virus (HCV) infected patients.
Methods: Genotyping was performed by allelic discrimination in 5’-nuclease
reactions. Allelic expression was investigated by allele specific transcript quantification
(ASTQ) using a reporter SNP within the transcript to discriminate mRNAs
originating from rs2569190 C or T allele gene variants in peripheral blood
mononuclear cell (PBMC) and liver biopsy samples from rs2569190- and reporter
SNP-heterozygous hepatitis C patients (n = 3). Samples from rs2569190homozygous
(but reporter SNP-heterozygous) patients served as controls (n = 5).
Results: In 2 of 3 PBMC samples a preferential expression of the T allele gene
variant less than 2-fold could be seen. In liver biopsy specimens a slight overexpression
of C and T allele gene variants could be observed in 2 samples, respecttively.
Among the 5 homozygous controls, however, allelic imbalances in gene
expression to a similar extent were found.
Discussion/Conclusion: ASTQ data on SNP rs2569190 thus support the absence
of an association between rs2569190 genotype and liver fibrosis progression in
patients with chronic hepatitis C.

30
The content of sphingolipids in the liver is associated with the
severity of chronic hepatitis C
M. Dabrowska 1 , A. Panasiuk 1 , P. Zabielski 2 , J. Górski 2 , R. Flisiak 1
1
Department of Infectious Diseases and Hepatology, Medical University of Bialystok,
Bialystok, Poland
2
Department of Physiology, Medical University of Bialystok, Bialystok, Poland
Background and aims: Sphinganine, ceramide and sphingosine are known as
proapoptotic factors, whereas sphingosine-1-phosphate augments cell proliferation
and suppresses apoptosis. The aim of study was to assess the association between
liver sphingolipids content and the activity of chronic HCV infection.
Methods: The study was performed in 38 HCV (+) treatment naïve patients
(11 female and 27 male, aged from 19 to 62 years). Plasma HCV RNA was
evaluated using RT-PCR. The inflammatory activity and fibrosis were graded
according to Scheuer histological classification. Sphinganine, ceramide, sphingosine
and sphingosine-1-phosphate contents as well as neutral and acid ceramidases, and
neutral and acid sphingomyelinases activities were measured in liver tissues. The
significance of differences was calculated by non-parametric U Mann-Whitney and
Kruskal-Wallis test. For correlation analysis, the Spearman nonparametric correlation
was used.
Results: We observed the significant increase in sphinganine content which was
associated with severity of portal/periportal inflammation (14.7 ± 9.0 vs. 21.0 ± 10.0
pmol/mg of protein, p = 0.015) and fibrosis advancement (17.0 ± 10.0 vs. 19.5 ± 6.9
pmol/mg of protein, p < 0.05). Moreover, the decrease in sphingosine-1-phosphate
content was related to inflammatory activity. The positive correlation between
sphinganine and sphingosine and biochemical markers of liver injury (for ALT:
r = 0.52 and r = 0.41 respectively, p < 0.01) were noticed. Additionally, the positive
correlation between spinganine and sphingosine content was observed (r = 0.71,
p < 0.001). Plasma HCV RNA was negatively correlated with ceramide (r = -0.59,
p = 0.02).
Conclusions: Sphinganine and sphingosine could play an important function in
immune mediated liver injury and fibrosis progression during chronic HCV infection.

31
Ribavirin-induced anaemia during hepatitis C treatment is
related to baseline haemoglobin level and drug erythrocyte
concentration
M. Delle Monache 1 , M. Carbone 2 , L. Nosotti 3 , L.R. Conti 4 , I. Lenci 2 , F. De Leonardis 2 ,
M. Angelico 2 and L. Baiocchi 2
1
Hepatology Service, ASL RMG, Palestrina, Rome, Italy
2
Hepatology Unit, Department of Internal Medicine, University of Rome “Tor Vergata”,
Rome, Italy
3
Department of Preventive Medicine S. Gallicano Hospital (IRCCS), Rome, Italy
4
Hepatology Service, S. Spirito Hospital, Rome, Italy
Introduction: Ribavirin-induced haemolytic anaemia is a frequent adverse event
during treatment of chronic hepatitis C but the mechanisms are not fully understood.
Few human studies have evaluated the factors influencing haemolysis in patients
undergoing standard antiviral therapy for hepatitis C, providing conflicting results.
Plasma and erythrocyte ribavirin levels were seldom assessed, with inconclusive
findings possibly due to the small number and the non-homogeneity of the patients
studied. We evaluated plasma and erythrocyte ribavirin levels and other factors
potentially associated with haemolytic anaemia in patients undergoing treatment with
Peginterferon alfa 2a (Peg-IFN) plus ribavirin for hepatitis C.
Methods: Thirty patients (12/18; mean age 44.6 ± 13; 26 with genotype 1 or 4)
undergoing a standard treatment schedule for hepatitis C (Peg-IFN 180 µg weekly +
ribavirin 1000 or 1200 mg daily according to body weight) were included in the study.
Plasma and erythrocyte ribavirin levels were evaluated between week 6 and 8 of
treatment (when a plateau in the drug levels has been consistently reported).
Ribavirin concentration was measured by high performance liquid chromatography
after solid phase extraction and employing 3-methyl-cytidine as internal standard.
Results: The mean daily ribavirin administered dose (14.9 ± 2.2 mg/kg) was
unrelated to the plasma (7.7 ± 4.6 µm/l) and erythrocyte (1592 ± 826 µm/l) ribavirin
concentration, nor to the extent of haemoglobin drop during treatment, which
averaged 3.4 ± 1.4 g/dl. Plasma ribavirin levels were significantly correlated with drug
erythrocyte concentrations (r = 0.4; p = 0.03). High haemoglobin baseline levels and
high erythrocyte ribavirin concentrations were both independently associated with a
greater extent of haemolysis (p = 0.001 and p = 0.01, respectively) at multiple
regression analysis.
Discussion/Conclusion: The extent of ribavirin-induced anaemia during antiviral
treatment for hepatitis C is correlated to the baseline haemoglobin level and to the
drug erythrocyte concentrations rather than to the administered dose.

32
Sylibin-vitamin E phospholipid complex reduces increased
ferritin levels during antiviral treatment for chronic hepatis C
R. Cecere 1 , L. Baiocchi 2 , I. Lenci 2 , M. Carbone 2 , F. De Leonardis 2 , and
M. Delle Monache 1
1 Hepatology Service ASL RMG, Palestrina, Rome, Italy
2 Hepatology Unit, Department of Internal Medicine, University “Tor Vergata”, Rome,
Italy
Introduction: A common finding during antiviral therapy for chronic hepatitis C is an
increase in serum ferritin levels, likely determined by ribavirin-induced haemolysis or
necro-inflammatory response to Peg-IFN. A persistent hepatic iron overload, in this
setting, may increase liver injury and progression toward cirrhosis. Aim of this study
is to investigate the role of iron overload during antiviral therapy for chronic hepatitis
C and the effect of sylibin-vitamin E complex on this.
Methods: Fifty-five consecutive patients underwent treatment with Peg-IFN plus
ribavirin. Patients with ferritin > 500 µg/l during antiviral therapy were treated with
sylibin-vitamin E phospholipid complex (SVEP) 188 mg daily for 3 months.
Afterwards patients with persistently high ferritin level, transferrin saturation > 45%
and raised ALT were treated with desferroxamine 30 mg/kg, iv daily, 5 days a week
plus 250 mg vitamin C for additional 2 months. Biochemical and virologic data were
analyzed before, during and after therapy.
Results: The clinical data of 55 patients (M/F 36/19, mean age 50 ± 10) were
reviewed. Ten patients (18%) had high basal level of ferritin (> 300 µg/l). During
antiviral therapy a consistent increase in serum ferritin was present in thirty out of
55 patients (54.5%), while haemoglobin level significantly decreased (-17%, p < 0.05)
in the whole sample. In all these subjects SVEP was administered. There was a
significant decrease in serum ferritin from beginning to end of treatment with SVEP
( -25.6%, p < 0.05). Only four patients maintained ferritin levels > 500 µg/l, transferrin
saturation > 45%, associated with a flare of ALT (x 4–6 ULN). HCV-RNA was
undetectable in these patients during ALT flare. In these patients desferroxamine
was added. Two patients did not respond to treatment: they were male, infected by
GT3 HCV and heterozygote for H63D mutation of HFE gene.
Discussion/Conclusion: Increase of ferritin level during antiviral therapy for HCV is
a frequent finding and may be a concomitant expression of ribavirin-induced
haemolysis and necro-inflammatory activity, or response to Peg-IFN. Therapy with
SVEP is associated with a significant reduction in serum ferritin. When levels are
very high (≥ 500), associated with ALT flare, and transferrin saturation > 45%, liver
injury by iron overload should be considered, as well as association with chelating
therapy. In this case, screening for HFE gene mutations is to advice.

33
Thyroid function in patients with chronic hepatitis C virus
infection under interferon therapy
Muhammad Azmy*, Youssef Ahmad*, Abedelraouf AbouNar**, and
Manal El-Hamamsy***
Departments of Internal Medicine*, Clinical Pathology** and Clinical Pharmacy***,
Faculty of Medicine, Al-Azhar University and Faculty of Pharmacy, Ain Shams
University, Egypt
Introduction: The aim of the present study was to investigate the effect of interferon
therapy on the thyroid function on Egyptian patients with HCV infection.
Methods: The study included 60 HCV infected patients with normal baseline levels
of TSH. The patients received a subcutaneous pegylated interferon alfa-2b weekly in
addition to oral ribavirin. Before the start of interferon therapy, serum TSH,
thyroglobulin-Ab (TG-Ab) and antiperoxidase antibodies (TPO-Ab) were measured.
Three months after interferon therapy serum levels of TSH were performed to all
patients, patients with abnormal TSH were suspected to the measurements of FT3,
FT4, TPO-Ab, TG-Ab and thyroid stimulating immunoglobulin levels (TSI).
Results: After 3 months of interferon therapy, 48 patients (80%) had normal TSH
and 12 patients (20%) had abnormal TSH. Out of 12 patients had abnormal TSH,
10 patients (16.6%) had high serum levels of TSH (hypothyroidisms), while the
remaining 2 patients (3.4%) had low serum levels of TSH (hyperthyroidism). All
patients with abnormal TSH had significant higher levels of TG-Ab, TPO-Ab and STI
(in cases with hyperthyroidism only) than the patients with normal levels of TSH. The
levels of TPO-Ab of patients with abnormal TSH were above the normal reference
range. In our study 40 patients (66.6%) were responder to interferon therapy (-ve
PCR for HCV), while 20 patients (33.3%) were non-responder.
Discussion/Conclusion: The incidence of thyroid dysfunction during pegylated
interferon therapy in patients with HCV is 20%; hypothyroids was more common than
hyperthyroidism. The patients most at risk for thyroid dysfunctions are female sex
and people with preexisting TPO-Abs. The numbers of non-responder patients were
significantly higher in patients with abnormal TSH than those with normal TSH.
Patients with HCV infection under pegylated interferon and ribavirin therapy should
be screened for thyroid dysfunction before and during treatment.

34
The prevalence and impact of serum autoantibodies among
chronic hepatitis C genotype 4 (HCV) patients attending a
national center of HCV treatment program in Egypt
Mohamed Sharaf-Eldin, and Hassan El-Batae
Tropical Medicine and Infectious Diseases Department, Faculty of Medicine, Tanta
University, Egypt
Introduction: Non-organ-specific autoantibodies (NOSAs) are commonly detected in
HCV. The aim was to determine the prevalence of anti-nuclear (ANA), anti-smooth
muscle (ASMA) and anti-liver kidney microsomes type 1 (anti-LKM1) antibodies in
HCV and assess their impact on the response to antiviral therapy.
Methods: One thousand and sixty two HCV patients were reviewed retrospectively
for autoantibodies and response to treatment. All patients received combined antiviral
therapy in the form of pegylated interferon-alpha 2 (a or b) plus ribavirin (1000–1200
mg/day) for 48 weeks.
Results: Non-organ-specific autoantibodies (NOSAs) were observed in 308 patients
(29%): ANA in 195 (18.36%), ASMA in 142 (13.37%) and anti-LKM1 was the rarest
occurring in 16 (1.51%). Concomitant positivity for ANA and ASMA was observed in
57 of these 308 cases (5.37%). The presence of NOSAs was associated with higher
aspartate transaminase (AST), alanine transaminase (ALT) and γ-globulin. In
contrast no differences were observed regarding age, gender and viral load. End
treatment response in HCV patients with NOSAs was 59.47% while it was 64.57% in
patients negative to NOSAs, and this difference was non significant.
Discussion/Conclusion: ANA was the commonest while anti-LKM1 was the rarest
among HCV patients. NOSAs were associated with more necroinflammatory grades.
Autoantibodies did not predict any change in response to HCV treatment.

35
Absence of a correlation between endotoxin receptor CD14
rs2569190/C-159T polymorphism and liver disease progression
in chronic hepatitis C
Eva Askar 1 , Margarete Odenthal 2 , Hans-Peter Dienes 2 , Giuliano Ramadori 1 ,
Sabine Mihm 1
1
Department of Gastroenterology and Endocrinology, University Medical Center
Göttingen, Germany
2
Institute of Pathology, University of Cologne, Germany
Introduction: Chronic hepatitis C is characterized by a mostly mild hepatic inflammatory
activity, which holds, however, a significant risk to proceed into liver cirrhosis
and hepatocellular carcinoma. The liver is constantly exposed to gut-derived bacterial
lipopolysaccharides (endotoxin), which are suggested to be cofactors in alcoholic
liver disease (ALD) through exacerbating ongoing injury. Recently, the T allele of a
single nucleotide polymorphism (SNP) in the endotoxin receptor CD14 gene has
been reported to be related to susceptibility to ALD-related cirrhosis. This study
aimed at analyzing the correlation between rs2569190/CD14 C-159T and disease
progression in chronic hepatitis C.
Methods: Liver biopsy specimens from a total of 349 chronic hepatitis C patients,
(201 men, mean age 45.8 ± 13.5 years), provided by the German Network of
Competence for Hepatitis (Hep-Net), were evaluated with respect to necroinflammatory
activity (grading) and architectural changes (staging). Samples of genomic
DNA were genotyped for the respective SNP by 5’-nuclease assays using fluorescent
dye-labelled allele-specific probes.
Results: The genotype distribution (CC:CT:TT) of 109:170:70, respectively, followed
Hardy-Weinberg equilibrium (p = 0.828), and was close to that given for Caucasians
in public data bases. Demographic analysis revealed no significant relationship
between genotypes and gender or age. Severe hepatitis activity was found in 14.9%
of patients, and cirrhosis in 6.3% of them. Histopathological analysis showed no
relationship between rs2569190/C-159T CD14 genotypes and hepatitis activity
(p = 0.513) or fibrosis progression (p = 0.928).
Discussion/Conclusion: Association data argue against a possible effect of CD14
rs2569190/C-159T polymorphism on promoting hepatitis activity or fibrosis
progression in chronic hepatitis C.

36
The metabolic control and antiviral treatment response in
diabetic patients with chronic hepatitis C
Bogdan Mihai 1 , Catalina Mihai 2 , Cristina Cijevschi 2 , Vasile Drug 2 , Mihaela Dragna 2 ,
Lidia Graur 1
1 Clinical Centre of Diabetes and Metabolic Diseases, University of Medicine and
Pharmacy “Gr. T. Popa”, Iasi, Romania
2 Institute of Gastroenterology and Hepatology, University of Medicine and Pharmacy
“Gr. T. Popa”, Iasi, Romania
Introduction: Diabetes mellitus (DM) represents a poor prognostic factor for antiviral
therapy response in chronic hepatitis C patients. The aim was to study the diabetic
factors which can influence treatment response in patients with chronic hepatitis C
and DM.
Methods: We studied 50 patients with DM who underwent antiviral therapy with
pegylated interferon and ribavirin for chronic hepatitis C in the Institute of
Gastroenterology and Hepatology Iasi, between 1st January 2007 and 31st
December 2008. We analyzed the following parameters: type of DM, glycated
hemoglobin (HbA1c) determined at the beginning of antiviral therapy, lasting of DM,
macro- and microangiopathic complications. The “cut off” value of HbA1c was
considered 7%. A sustained response was defined as the absence of detectable
hepatitis C virus RNA 6 months after treatment was stopped.
Results: There were 29 females and 21 men, aged between 21 and 60 years (mean
age 38.7 years). 20 patients had Type 1 DM and 30 Type 2 DM. The global rate of
response was 60%. In Type 1 DM were 35% non-responders, and in Type 2 –
43.33%. Abnormal HbA1c was found in 23.33% responders and in 80% nonresponders.
Macroangiopathic complications were found in 30% responders and
40% non-responders. Microangiopathic complications were found in 26.66%
responders and 35% non-responders. There were 11 patients (36.66%) with more
than 10 years of DM in responders and 9 patients (45%) in non-responders. HbA1c
was the only statistical significant factor (p < 0.05) between the responders vs. nonresponders.
Discussion/Conclusion: HbA1c is an important predictive factor to antiviral therapy
response in diabetic patients with chronic hepatitis C. There are more nonresponders
patients with Type 2 DM comparative with Type 1, probably due to
insulin-resistance. All patients with DM and chronic hepatitis C need a good glycemic
control before starting antiviral therapy.

37
The correlation between erythropoietin and pro- and antiinflammatory
cytokines and the decreasing of the viral C
hepatitis RNA level under the treatment with statins
R. Mihăilă 1 , R. Mihăilă 2 , O. Frăţilă 3 , E.C. Rezi 4 , A. Zaharie 4 , L. Mocanu 4 , M. Deac 2 ,
A. Olteanu 4 , L. Draghila 4 , M. Boitan 2
1 Public Health Authority, Sibiu, Romania
2 Lucian Blaga University Sibiu, Medical Faculty, Sibiu, Romania
3 University of Oradea, Medical Faculty, Oradea, Romania
4 Emergency Clinical County Hospital, Sibiu, Romania
Introduction: Some statins inhibit the RNA replication and the assemblage of the
replicative hepatitis C virus complex.
Aim: Our aim was to study if there is any correlation between erythropoietin, pro- and
anti-inflammatory cytokines and the answer of the C virus viral load at the treatment
with fluvastatin.
Methods: We have studied a group of 37 consecutive patients with viral C chronic
hepatitis, who were not under treatment with interferon, to whom we have determined
the serum levels of IL-6, IL-8, TNF-alpha, IL-10, erythropeietin, the liver biochemical
tests and the viral load (determined by real time PCR), before and after a month of
treatment with fluvastatin 40 mg/day. We have compared the results of the biological
parameters with the initial and final viral load and with its variations and we have
statistically analyzed the results (average, standard deviation, Pearson’s test and t
student test).
Results: The medium age of the studied group was 49.26 ± 10 years. The gender
repartition was 25 women and 12 men. In the whole studied group, the viral load
decreased, in average with 1,592,050 ± 3,654,745 UI/ml (p = 0.016). There was a
direct linear correlation between the final IL-6 level and the initial viral load
(r = 0.254). The viral load variation correlated directly with the initial level of IL-6
(r = 0.250) and inversely with the initial level of erythropoietin (r = -0.280). The final
viral load correlated directly with the initial level of erythropoietin (r = 0.437) and
inversely with the initial level of IL-8 (r = -0.286). Also, the initial IL-6 level correlated
directly with the initial AST level (r = 0.424) and inversely with the initial cholesterol
level (r = -0.288). There was an inverse linear correlation between the initial IL-8 level
and the initial level of creatinine (r = -0.322).
Discussion/Conclusion: The initial level of IL-6, a pro-inflammatory cytokine, is
directly correlating with the AST level and inversely correlating with the initial level of
cholesterol and it is a favoring prognostic factor for the decreasing of the viral load
during the treatment with fluvastatin. The higher initial IL-8 level is the lower final viral
load is. The higher initial erythropoietin level is the lower is the decreasing viral load
under the treatment with fluvastatin.
Acknowledgments: This study belongs to a complex research grant (179/2006),
financed by the Research and Education Ministry from Romania, through the
Academy of Medical Science and VIASAN, to whom we are deeply gratefully.

38
Increased CD14 transcription initiation in T allele carriers of the
rs2569190/C-159T promoter polymorphism in healthy individuals
and chronic hepatitis C patients
Jasmin Mertens, Giuliano Ramadori, Sabine Mihm
Department of Gastroenterology and Endocrinology, University Medical Center
Göttingen, Germany
Introduction: Lipopolysaccharide, a main component of the outer cell wall of Gramnegative
bacteria, is recognized by an endotoxin receptor complex of Toll-like
receptor 4, MD2 and CD14. The single nucleotide polymorphism (SNP)
rs2569190/C-159T in the CD14 gene in relation to liver disease progression has
been investigated in various association studies. Furthermore, in vitro experiments
have shown an enhanced promoter activity for T allele reporter gene constructs. This
study aimed at analysing a possible relationship between this SNP and CD14
transcription initiation in healthy individuals and in chronic hepatitis C patients in a
genomic context in vivo.
Methods: Peripheral blood mononuclear cells (PBMC) from healthy (n = 5) and HCV
infected (n = 5) heterozygous individuals were subjected to haplotype-specific
chromatin immunoprecipitation (HaploChIP) using an antibody directed against
serine 5 phosphorylated RNA polymerase II and an IgG-mock control. With this
specifically captured material (output), non-immunoprecipitated material (input) and
the mock control quantitative restriction fragment length polymorphism (RFLP)
analyses were performed.
Results: The input and mock material was always found to contain about the same
amount of C and T allele fragments, while the output material from healthy individuals
and hepatitis C patients contained about twice the number (T:C ratio = 1.65–2.83) or
even more T allele fragments (T:C ratio = 3.08–5.62), respectively.
Discussion/Conclusion: Our data argue for an effect of the rs2569190/C-159T SNP
on CD14 transcription initiation, i.e. on a preferential binding of serine 5 phosphorylated
RNA polymerase II to T allele fragments, in PBMC from healthy and HCV
infected individuals. These data are quite concordant with in vitro findings by others.
A possible impact on the ongoing CD14 mRNA synthesis, however, is going to be
determined.

Familial occurrence of autoimmune hepatitis
39
Woźniak Małgorzata, Woynarowski Marek, Socha Jerzy
Department of Gastroenterology Hepatology and Immunology, Children’s Health
Memorial Institute, Warsaw, Poland
Introduction: It is known that presence of DR3 or DR4 and several autoimmune
diseases are risk factors for autoimmune hepatitis (AIH). The aim of our study was to
detect the families with more than one child affected with AIH.
Methods: We retrospectively reviewed case records of 189 children with AIH treated
at our institution between 1989 and 2006.
Results: Three pairs of siblings (9–14 years of age) were found. The time window
between the diagnosis varied from 4 months to 9 years. Jaundice was the first
symptom of the disease in one child. The remaining children did not present any
characteristic symptoms of liver disease and the diagnosis was based on typical
laboratory and pathological abnormalities. At diagnosis all children presented with
moderate or severe liver fibrosis. All children received standard prednisone and
azathioprine therapy. The treatment was successfully discontinued in one child. Four
subject continue the therapy and one child died due to liver function decompensation.
Discussion/Conclusion: More than one child with AIH was found in 1.6% of the
families affected with this disease. Our data show that the presentation, clinical
course and treatment outcome of AIH in siblings are the same as in isolated cases of
the disease. The presence of AIH in siblings may justify the implementation of
screening liver function tests in the families of AIH patients.

40
Liver sinusoidal endothelial cells induce anti-inflammatory
CD4 + T cells suppressing murine autoimmune hepatitis
Nils Kruse 1 , Katrin Neumann 1 , Katja Derkow 3 , Anja Kühl 2 , Alf Hamann 4 , and
Katja Klugewitz 1
1
Medizinische Klinik I, Charité Campus Benjamin Franklin, Berlin, Germany
2
Research Center Immunosciences, Charité Campus Benjamin Franklin, Berlin,
Germany
3
Medizinische Klinik m. S. Hepatologie und Gastroenterologie, Charité Campus
Virchow-Klinikum, Berlin, Germany
4
Experimentelle Rheumatologie, Charité Campus Mitte, Berlin, Germany
Introduction: Cellular mechanisms that maintain the intrahepatic immune balance
are crucial for inflammatory liver diseases. For naive CD8 + T cells, liver sinusoidal
endothelial cells (LSEC) have been shown to act as non-professional antigen
presenting cells and thus induce tolerance by inhibition of cytotoxicity. In this study
we investigated consequences of CD4 + T cell priming by LSEC.
Methods: We studied the cytokine expression of LSEC primed CD4 + T cells (TLSEC)
and determined the stability of their phenotype in vivo by adoptive transfer into
congenic mice and immunogenic antigen application. Investigating suppressive
capacities of TLSEC we performed an in vitro suppression assay. The ability of TLSEC to
influence proinflammatory reactions in vivo was analyzed in a model of T cellmediated
autoimmune hepatitis. Hepatic inflammation was monitored by ALT levels
and histologic analyses.
Results: We demonstrated that LSEC induce proliferation of naive CD4 + T cells in
vitro. LSEC primed CD4 + T cells (TLSEC) acquired a CD45RB low phenotype without
effector cytokine expression. This phenotype remained stable in vivo. Interestingly,
TLSEC negative for CD25 and Foxp3, suppressed the proliferation of naive CD4 + T
cells in vitro. They did neither support a DTH reaction nor a hepatic inflammation and
were even able to suppress hepatitis.
Discussion/Conclusion: Priming of naive CD4 + T cells by LSEC leads to a
suppressive phenotype here referred to as TLSEC. Thus liver sinusoidal endothelia
may directly contribute to limiting hepatic inflammation and supporting local tolerance
towards exogenous antigens or endogenous self-antigens.

41
Frequency of autoimmune disorders and therapy in patients
with autoimmune hepatitis
Amelia Genunche-Dumitrescu, P. Mitrut, Daniela Badea, M. Badea
University of Medicine and Pharmacy, Clinical Hospital of Emergency, Craiova,
Romania
Introduction: The aim was to assess the frequency of concurrent autoimmune
disorders (CAD) in patients with AIH types 1 and 2 and to evaluate the clinical and
biological response of treatment.
Methods: We studied 88 patients (62 females/26 males, mean ages 43.2 years) with
autoimmune hepatitis (AIH). The diagnosis of AIH was based on international criteria,
including biochemical tests, autoantibodies and liver tissue morphology.
Results: AIH type I was present in 52 patients and type II in 36 patients. CAD was
present at 32 patients with AIH type I and at 24 patients with type II. The incidence of
CAD in AIH type I was: autoimmune thyroiditis (15.62%), rheumatoid arthritis
(18.75%), type I diabetes mellitus (37.5%), Sjogren’s syndrome (9.37%), vitiligo
(12.5%), polyarteritis nodosa (6.26%). At AIH type II was observed: thyroiditis
(20.84%), rheumatoid arthritis (16.67%), type I diabetes mellitus (33.33%), vitiligo
(8.33%), vasculitis (12.5%), colitis ulcerosa (8.33%). Type I diabetes was diagnosed
before AIH type I in 4 cases and in 8 cases it developed after diagnosis was
established. In AIH type II all the cases of diabetes was diagnosed after AIH.
Most patients with AIH type I were treated by only corticoids (40 mg/day with a
reduction of 10 mg every 5 days) and 23 patients follow up associated treatment:
corticoids and azathioprine. After diabetes was diagnosed the steroid doses were
lowered and subsequently stopped.
After 12 months, 42 patients had normal liver biochemistry, 6 had partial response
and 4 no response. In AIH type II, 32 patients had therapeutic success and 4 had
partial response. In AIH type I, after treatment, 3 patients developed mild anemia,
4 developed osteoporosis and 3 severe leukopenia. In AIH type II there were no
serious side-effects.
Discussion/Conclusion: The frequency of autoimmune disorders in AIH was
relatively high. Azathioprine and prednisone associated therapy was effective and
safe.

42
The prediction of the recrudescence risk in autoimmune
hepatitis (AIH)
P. Mitrut, A. Genunche-Dumitrescu, A.O. Mitrut, L. Nita*, D. Badea
University of Medicine and Pharmacy, Craiova, Romania
*Emergency District Hospital, Craiova, Romania
Introduction: There are not yet established initial factors to predict the treatment
response in autoimmune hepatitis (AIH). Immunosuppressive therapy with
corticosteroids, often in combination with azathioprine remains the “golden standard”
for induction and maintains remissions in AIH.
Aim: To evaluate these predictive factors for therapeutic response in AIH patients.
Methods: A prospective study was carried out, including 156 patients who were
diagnosed with AIH in our Medical Clinic beginning with January 1999 to January
2009. To apply this diagnosis we have used the standard score system of
International Group of Study AIH. In our protocol study there were mentioned
demographical and clinical data, cholestatic tests, virological and immunological
tests, hepatic biopsy with histological results, ERCP exams or RMN and therapeutic
response.
Results: According International AIH Group criteria they were 72 (46.1%) patients
with “definite” (> 15 points) and 84 (53.9%) with “probable” diagnosis of AIH (10–15
points) pretreatment. We noted a complete response in 92 patients (58.9%) and
recrudescence in 64 patients (41.1%). Recrudescence risk was correlated with
therapy duration, age at diagnosis and immunity profile. A long lasting period of
therapy, at least 4 years, was associated with a low risk of recrudescence. The
presence of anti-LKM1 and ANCA antibodies was correlated with frequent
recrudescence and the presence of ANA and SMA antibodies was correlated with
long lasting remissions. In overlap syndromes (AIH/PBC or AIH/PSC) the response
of immunosuppressive therapy was insufficient in 48% cases.
Conclusions: The absence of response is not infrequent in AIH. Age at diagnosis
< 30 years, a low therapy duration and the presence of anti-LKM1 and ANCA
antibodies were the parameters independently associated with the absence of
complete response to combined therapy.

43
Immune disruptions at the mycotoxin fumonisin-induced
autoimmune hepatitis in young and old mice
Elena A. Martinova
Institute of Nutrition, Russian Academy of Medical Sciences, Moscow, Russian
Federation
Introduction: Previously, we have shown our data on an experimental autoimmune
hepatitis induced in mice fed by mycotoxin Fumonisin B1 (FB1) produced by fungi
Fusarium moniliforme. FB1 contaminates corn all round the world and it is the
carcinogen for some animals and people. Our new results elucidate the immune
disruptions in liver of old and young mice exposed to FB1.
Methods: FB1 (0.5 mM–20 mM) was fed to mice C57Bl/6 (10 weeks old and
18 months old) in the different regimes. Animal were killed by guillotine. Removed
liver was separated and one part was frozen. Samples were stained and
photographed. Liver cells were stained with monoclonal antibodies (mAb) followed by
flow cytometry.
Results: FB1 causes the autoimmune reaction in liver tissue that was confirmed by
staining to Ig+ cells and nuclear antigens. Reaction of old mice versus young animals
to FB1 was much more marked. Liver tissues of old mice were filled by B-cells
surrounding by cytotoxic lymphocytes. There was clear difference in expression of
such proteins as kinase mTOR, receptors GLUT1, GLUT2, GLUT4, IRβ, heat shock
proteins 25, 60, 70, 90 as well as TNF-R2 and markers of activation, on liver
lymphocytes and hepatocytes from old and young mice. The energy potential of liver
cells was disrupted more significant in ageing mice. Activation of lymphocytes in liver
may be regulated by simple sphingolipids (sphingosine, sphinganine, C2-ceramide)
that cross-talks with signalling initiated by FB1.
Discussion/Conclusion: Animal model of autoimmune hepatitis induced by
mycotoxin Fumonisin B1 discloses the mechanism of immune disruptions and shows
the pathways to regulate the activation of immune cells in liver.

44
Emerging importance of autoimmune hepatitis in children in
Bulgaria, an endemic area for viral hepatitis
Krasimira Kalinova PhD, L. Pekova PhD, P. Chakarova PhD, P. Stefanova PhD,
K. Georgiev
University Hospital Stara Zagora, University Hospital Plovdiv, Bulgaria
Introduction: Untreated autoimmune hepatitis (AIH) can develop into liver cirrhosis,
with potentially fatal outcomes. Viral hepatitis in children was endemic in Bulgaria
before universal hepatitis B vaccination, but AIH has rarely been reported in
Bulgarian children. We performed this retrospective study to characterize the clinical
features of AIH in Bulgarian children.
Methods: We enrolled children with AIH, based on the revised scoring system of the
International Autoimmune Hepatitis Group (IAIHG) from 81 children hospitalized with
hepatitis from January 2000 to April 2009. Other etiologies of hepatitis were
excluded.
Results: There were three definite and six probable AIH cases. The incidence of AIH
among children hospitalized with hepatitis was 2.3%. Ten children had other
autoimmune diseases, including systemic lupus erythematosus (SLE) (6), discoid
lupus erythematosus (DLE) (1), and autoimmune polyendocrinopathy syndrome type
1 (1). Another had biliary atresia, and AIH developed after cadaveric liver
transplantation. Antinuclear antibodies ranged from 1:160–1:2560. Peak alanine
aminotransferase (ALT) values were 346 ± 188 U/l (mean ± SD). Jaundice occurred
in four patients. Liver histology in the three definite AIH patients showed chronic
hepatitis with predominantly lymphoplasmacytic infiltrates in portal areas, with
prominent interface activity. Treatment included prednisolone, azathioprine, and/or
cyclosporine. All patients survived. ALT fell to < 60 U/l after treatment. Hepatitis
relapse occurred in one patient.
Discussion/Conclusion: AIH in Bulgarian children is commoner than previously
thought. It is associated with other autoimmune diseases and may occur before,
simultaneously with, or after other autoimmune diseases. Children with liver
transplants are also at risk of AIH.

45
Non-invasive assessment of liver fibrosis by transient elastography
in patients with cholestatic liver diseases (PBC and PSC)
Diana Rill, Adriana Tudora, O. Ciof, E. Dragan, Ioan Sporea
Department of Gastroenterology and Hepatology, University of Medicine and
Pharmacy “Victor Babes”, Timisoara, Romania
Introduction: Liver stiffness measurement (LSMs) by means of transient elastograpy
(TE) (FibroScan ® , Echosense, France) has been shown to be correlated to liver
fibrosis in patients with liver diseases of different etiologies, including cholestatic
diseases. Non-invasive assessment of liver stiffness measurement (LSMs) is a
reliable tool in the detection and follow up of significant fibrosis in these patients. We
aimed to study role of TE evaluation in patients with cholestatic liver diseases a well
as correlation to fibrosis stages.
Methods: Out of 3459 TE evaluations carried out in the Department of Gastroenterology
and Hepatology, Timisoara, 54 were performed for cholestatic liver
diseases – 44 for primary biliary cirrhosis (PBC) and 10 for primary sclerosing
cholangitis (PSC). Liver biopsy was performed in 39 (72%) patients. 31 (56.9%)
patients were had the diagnosis of cirrhosis (esophageal varices or other
complications).
Results: Overall, mean LSMs was 17.3 ± 11.9 kPa (ranging from 2.8 to 70.1 kPa). In
patients in pre-cirrhotic stage the LSMs average was 7.6 ± 3.7 kPa, and in cirrhotic
patients 27.3 ± 20.2 kPa (p = 0.00014 ES) LSM correlated to both fibrosis (r = 0.8,
p < 0.0001) and histological (0.70, p < 0.0001) stages, correlations that persisted
when PBC and PSC patients were analyzed separately. Comparing the patients with
significant fibrosis (F ≥ 2) (n = 23) in the pre-cirrhosis stage with those having mild
fibrosis (F < 2), we noticed significant statistical differences (p < 0.0001 ES). Optimal
cut-off value for significant fibrosis (F ≥ 2) was 13.5 kPa and for F < 2 – 6.3 kPa.
Discussion/Conclusion: TE is a simple and reliable non-invasive method for
assessing billiary fibrosis suitable in excluding advanced fibrosis using a threshold of
13.5 kPa.

46
A meta-analysis regarding the effects of ursodeoxycholic acid
in patients with primary sclerosing cholangitis
E.C. Rezi 1 , R. Mihaila 2 , M. Deac 2
1 Second Medical Department, Emergency Clinical County Hospital, Sibiu, Romania
2 Lucian Blaga University, Medical Faculty, Sibiu, Romania
Introduction: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver
disease characterized by inflammation and progressive bile duct fibrosis. Therapy
with ursodeoxycholic acid (UDCA) has been reported to be associated with
improvements in both clinical symptoms and abnormal serum biochemical liver tests
in patients with primary sclerosing cholangitis.
Methods: In order to evaluate the effects of UDCA on patients with primary
sclerosing cholangitis, we have performed a meta-analysis study. In a Medline
search we identified 12 clinical trials which studied the efficacy and safety of
treatment with UDCA in PSC, with a particular analyse of the biochemical liver tests
(serum alkaline phosphatase [AP], gamma-glutamyl transpeptidase [GGT], alanine
aminotransferases [ALAT], aspartate aminotransferases [ASAT], total bilirubin level
[Bt]) and of the fibrosis score and histological aspects.
Results: A total number of 282 patients were admitted in these studies. A high
percentage (57%) of the patients with PSC also presented inflammatory bowel
diseases. The average age of the patients was 21 ± 16.46 years. The average
treatment period was 1.68 ± 0.79 years. The medium dosage of UDCA which was
used was 16.18 ± 5.29 mg/kg per day. Three studies used high-dosage of UDCA
(20–30 mg/kg per day). The ASAT and ALAT levels decreased significantly in
72.72% (p = 0.001), respectively 81.81% of the patients (p = 0.02). AP decreased at
81% (p = 0.01) and GGT decreased at 72% of the patients (p = 0.0055). The bilirubin
level decreased in 36% of the patients (p = 0.01). Two studies found a significant
improvement in hepatic inflammation, fibrosis and histological stage of the disease.
No side effects were observed during the period of treatment and follow-up.
Discussion/Conclusion: The treatment with standard-dosage (8–15 mg/kg/day) or
high-dosage (20–30 mg/kg/day) of UDCA generally improves liver chemistries and
sometimes liver histology in patients with PSC. Because UDCA treatment improves
but does not cure cholestatic liver diseases, permanent treatment seems to be
necessary. Such prolonged treatment with UDCA may be recommended because,
until now, no side effects have been reported.

47
The long-term budesonide-UDCA combined therapy in patients
with primary biliary cirrhosis
Amelia Genunche-Dumitrescu, P. Mitrut, M. Badea, D. Badea
University of Medicine and Pharmacy, Clinical Hospital of Emercency, Craiova,
Romania
Introduction: Aim was to evaluate the efficacy and safety of two years combined
therapy (UDCA plus budesonide) and compared with UDCA alone therapy, in
patients with primary biliary cirrhosis (PBC).
Methods: We studied 33 patients with PBC, at stages I to III. A comparative study
was performed on two groups: A group composed 20 patients which received UDCA
(10–15 mg/kg/day) and B group (13 patients) treated with UDCA and budesonide
(6 mg daily divided in 3 doses). We evaluated liver histology, serum levels of
aminotransferase, Bb, AP at 6, 12 and 24 months.
Results: In A group, clinical symptoms significant improved in 25% of cases after
6 month, in 60.7% after 12 months and 89.28% after 24 months. The mean value of
serum bilirubin concentration was reduced from 6.7 ± 2.5 mg%, at baseline, to
2.8 ± 1.3 mg% at 6 months and to 1.7 ± 0.7 mg% at 12 months. Aminotransferase
values were reduced more quickly comparative with bilirubin and AP levels: with
44.6% at 6 months and 63.2% at 12 months. In B group, aminotransferase values
reduced more slowly, but significant decrease AP activity after one year (p = 0.001).
Inflammatory activity was significantly reduced in the combined therapy (6 cases,
46.15%) and in 4 cases (20%) with monotherapy. Fibrosis decreased in group B in
5 cases, but in A group only in one case. After 24 months, histological stage of
disease improved only in B group (3 cases). In A group, we observed side-effect at
one patient (diarrhea) and in B group 2 patients presented hyperglycemia, 2 mild
hirsutism and 4 osteoporosis. Most of the side effects appeared in patients with stage
III PBC and only in two patients we reduced the budesonide dose.
Discussion/Conclusion: UDCA combined with budesonide improved liver histology
and liver enzymes, where as the effect of UDCA mono-therapy was mainly on liver
function tests.

48
Value of transient elastography (FibroScan ® ) in the evaluation
of patients with hepatocellular carcinoma (HCC)
1. Ciof Octavian Paul, MD, Resident Gastroenterology, Department of
Gastroenterology and Hepatology, Emergency Clinical County Hospital Timisoara;
E-Mail: ciof_o_p@yahoo.com; Phone: +40747025062; Address: Aleea Sanatatii
Nr 18, Bl 1, Sc A, Ap 9, City: Timisoara, Romania
2. Adriana Tudora, MD, Department of Gastroenterology and Hepatology,
Emergency Clinical County Hospital Timisoara; E-Mail:
adrianatudora@yahoo.com: Phone: +40721523258, Address: Str. Tapia Nr 3,
City: Timisoara, Romania
3. Emil Dragan, MD, Resident Internal Medicine, E-Mail: emil_dragan@yahoo.com,
Phone: +40724386118, Address: Str. Paul Iorgovici, Nr 6, City: Timisoara,
Romania
4. Rill Diana, MD, Resident Internal Medicine, E-Mail: diana.rill@yahoo.com, Phone:
+40721410775, Address: Str Electronicii Nr 26, City: Timisoara, Romania
5. Martin Mihaela, MD, E-Mail: miha_ella@yahoo.com, Phone: +40745518371,
Address: Str. Magura Nr 6, City: Timisoara, Romania
Introduction: Aim: Main risk factors in the occurrence of hepatocellular carcinoma
(HCC) are represented by liver cirrhosis (LC) of different ethiologies (HBV, HCV or
alcoholic, hemochromatosis, or unknown ethiologies). Thus, the follow up of risk
group patients for developing HCC is mandatory. Transient elastography (TE)
represents a novel, user-friendly, non-invasive method, appropriate to use in
outpatients method for the evaluation of liver fibrosis in patients with chronic liver
diseases of different etiologies. We aimed to establish the value of TE in the
evaluation of patients with LC and HCC.
Methods: Out of 1668 patients with LC, mean age 58.42 ± 11.57 years (January
2000–December 2006). 104 patient with chronic liver diseases (chronic hepatitis or
LC) and HCC were evaluated by TE.
Results: Liver stiffness measurements (LSMs) in patients with chronic liver diseases
ranged between 4.10 and 75 kPa (mean LSM value for patients with chronic hepatitis
was 9.76 ±4.77 kPa and for patients with LC 34.97 ± 20.25 kPa). Mean LSMs values
for patients with LC and HCC was 36.65 ± 20.49 kPa (AUROC 0.94 ± 0.02, ranging
between 0.89–0.98). Considering a cut-off value of LSMs for LC of 14.5 kPa, our
sensibility was 90% and specificity 85%, with a PPV 92% and NPV 78%. No
statistical differences were noticed between LSMs in patients with LC with or without
HCC (p = 0.1123-NS).
Discussion/Conclusion: Despite some other studies in literature, in our study we
could not evidence no significant predictive value of LSMs by TE in which concerns
the differentiation between LC and HCC.

49
Constitutive active gp130 in the liver and its connection to
chronic inflammation and carcinoma development
Antje Schütt, Jürgen Scheller, Stefan Rose-John
Department of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany
Corresponding author: aschuett@biochemie.uni-kiel.de
Introduction: Glycoprotein130 (gp130) is the common signaling receptor subunit for
the cytokines of the IL-6 family. Activation of gp130 leads to activation of the STAT3
and MAP/AKT signaling pathway. By replacing the extracellular part of gp130 with
the leucin-zipper of the c-jun protein, we have recently constructed a dimerized
version of gp130 (L-gp130), which leads to constitutive activation of gp130 signaling.
We have shown earlier in mice that massive gp130 lead to adenoma formation in the
liver. These data were recently confirmed in patients with liver adenomas where
somatic activating mutations in gp130 have been detected.
Methods: We generated a bidirectional expression plasmid in which L-gp130 and
luciferase is placed under the transcriptional control of a tetracycline responsive
promotor element (TRE). In the presence of a tetracycline transactivator protein
(tTA), L-gp130 and luciferase expression can be regulated by the tetracycline
analogue doxycycline. Based on this system we plan to analyze chronic liver
inflammation and carcinoma development in the liver.
Results: L-gp130 and luciferase were shown to be tightly regulated by doxycycline in
human hepatoma cells (HepG2). The expression L-gp130/luciferase expression
plasmid was used to generate transgenic mice, in which expression of L-gp130 and
luciferase can be regulated in tissues, which express the tTA protein, in a cellautonomous
manner.
Discussion/Conclusion: First results on the cellular expression of L-gp130 and on
the generation of liver specific transgenic mice will be presented and the advantages
of an animal model of cell-autonomous gp130 activation will be discussed.

50
Increased intrahepatic infiltration with CD4+CD25+FOXP3+
regulatory T cells correlates with increased tissue levels of
TGF-beta and IL-10 in patients with primary or metastatic liver
cancers
Manolova I 1 , Gulubova M 2 , Kyurkchiev D 3 , Ananiev J 2 , Altunkova I 3 , Julianov A 4
1 Laboratory of Clinical Immunology, University Hospital, 2 Department of General and
Clinical Pathology,
4 Department of General Surgery, Medical Faculty, Trakia
University, Armeiska str. 11, Stara Zagora 6000, Bulgaria; 3 Laboratory of Clinical
Immunology, University Hospital “St Ivan Rilski”, Sofia, Bulgaria
E-Mail: imanolova@mf.uni-sz.bg
Introduction: CD4+CD25+FOXP3+ regulatory T cells (Treg) have been
characterized as a critical population of immunosuppressive cells that maintain
peripheral tolerance to self antigen and also inhibit anti-tumor immune response. The
suppression mechanism also requires interleukin 10 (IL-10) and transforming growth
factor-beta 1 (TGF-beta1) in the local environment. In the present study in patients
with primary or metastatic livers cancers, we examine the relationship between
intrahepatic Treg and the levels of immunosuppressive cytokines IL-10 and
TGF-beta1.
Methods: 17 patients (11 males and 6 females) undergone liver resection for a
primary or metastatic tumor and 6 patients (2 males and 4 females) undergone
surgery for primary gastrointestinal tumors were included in the study. Liver samples
were obtained after surgical resection of metastatic or liver tissue for curative or
diagnostic purpose. Intrahepatic Treg were identified and characterized as
CD4+CD25+FOXP3+ using flow cytometry. Tissue levels of TGF-beta1 and IL-10
were measured by enzyme-linked immunosorbent assay in the supernatants of tumor
homogenate and in normal liver tissue.
Results: Flow cytometry revealed increased proportions of Treg in metastatic liver
compared to normal liver (1.35 ± 1.17 vs. 14.39 ± 10.96%, p = 0.001). TGF-beta1
was detected in supernatants from homogenized normal liver tissue (mean
22.5 ng/100 mg wet tissue, n = 6) and from tumor tissue (45.04 ng/100 mg wet
tissue, n = 17, p = 0.107). We found a trend towards increased IL-10 levels in
metastatic tissue (mean = 115.4 pg/100 mg wet tissue, n = 17) in comparison with
normal liver tissue (mean = 34.6 pg/100 mg wet tissue, n = 6, p = 0.093). There was
a significant, positive correlation between the proportions of CD4+CD25+FOXP3+
Treg and the levels of IL-10 (r = 0.49, p = 0.028) and TGF-beta1 (r = 0.602,
p = 0.005).
Conclusion: Collectively, these data demonstrate the immunosuppressive environment
in the liver bearing primary or metastatic tumors.

51
TGF-beta occurrence and macrophage infiltration in liver
metastasis
Ananiev J. 1 , Gulubova M. 1 , Manolova M. 2 , Matev A. 3
1 Department of General and Clinical Pathology, 2 Laboratory of Clinical Immunology,
University Hospital,
3 Department of General Surgery, Medical Faculty, Trakia
University, Armeiska str. 11, Stara Zagora 6000, Bulgaria; 3 Laboratory of Clinical
Immunology, University Hospital “St Ivan Rilski”, Sofia, Bulgaria
E-Mail: operation@abv.bg
Introduction: TGF-beta1 signaling pathway is implicated in the regulation of cancer.
TGF-beta1 has immune suppressive function. It regulates tumor environment
together with some other cytokines and so modulates tumor cell viability and spread.
The aim of present study was to evaluate the occurrence of TGF-beta1 pathway
components in liver metastasis and to assess its relation with tumor associated
macrophages (TAMs) infiltration.
Methods: 48 patients (30 males and 18 females) with liver metastasis from colorectal
and gastric cancer were investigated. The immunohistochemistry was done with anti-
TGF-beta1, anti-SMAD4, anti-SMAD7, anti-TGF-beta RII, and anti-CD68 antibodies.
Results: Elevated TGF-beta1 expression was detected in tumor cytoplasm in
47.82% of metastasis. SMAD4 was observed mainly in tumor cell cytoplasm in
91.3% of the samples. SMAD7 was observed also in tumor cell cytoplasm in 65.22%
of metastasis. TGF-beta RII was found on tumor cell membranes in 65.22% of
tumors.
Statistically significant correlation was observed between TGF-beta1 expression and
decreased CD68 numbers in tumor stroma (χ 2 = 7.0, p = 0.008) and in tumor border
(χ 2 = 4.33, p = 0.037).
There was a tendency for correlation between low numbers of CD68-positive TAMs
with high expression of TGF-beta RII (χ 2 = 3.582, p = 0.058) and with high
expression of SMAD7 in tumor cells (χ 2 = 3.549, p = 0.06).
Discussion/Conclusion: In liver metastasis TGF-beta1 is located mainly in tumor
cell cytoplasm. Its receptor TGF-beta RII marked with variable intensity the tumor cell
membranes and SMAD4 and SMAD7 had mainly cytoplasmic expression. Elevated
TGF-beta1, TGF-beta RII and SMAD7 expression correlated with decreased TAMs
infiltration.

52
TGF-beta inhibits dendritic cell infiltration in liver metastasis
Gulubova M. 1 , Manolova I. 2 , Ananiev J. 1 , Yovchev Y. 3
1 2
Department of General and Clinical Pathology, Laboratory of Clinical Immunology,
3
University Hospital, Department of General Surgery, Medical Faculty, Trakia
University, Armeiska str. 11, Stara Zagora 6000, Bulgaria
E-Mail: mgulubova@hotmail.com
Introduction: TGF-beta can utilize various programs to promote cancer metastasis
through its effects on the tumor microenvironment, enhanced invasive properties and
inhibition of immune cell function. The aim of the study is to assess TGF-beta1, its
receptor TGF-beta RII and the signaling proteins Smad4 and Smad7 expression in
liver metastasis and their relation to dendritic cell infiltration.
Methods: We investigated 46 samples from liver metastasis from primary colorectal
and gastric cancer. The patients were 28 males and 18 females with median age of
64 years, range from 38 to 86 years. Paraffin sections (7 µm thick) were processed
for immunohistochemistry with anti-TGF-beta1, anti-TGF-beta RII, anti-Smad4, anti-
Smad7, anti-CD1a and anti-CD83 antibodies.
Results: TGF-beta1 expression in tumor cytoplasm correlated with the low infiltration
with CD1a (χ 2 = 8.27, p = 0.004) and CD83 (χ 2 = 28.83, p = 0.000) in tumor border
and in tumor stroma (for CD1a χ 2 = 3.18, p = 0.074 and for CD83 χ 2 = 17.84,
p = 0.000). TGF-beta1 and Smad4 were observed also in some stellate cells at
metastasis border.
The low CD1a and CD83 infiltration in tumor border correlated with higher SMAD4
expression in tumor cells (χ 2 = 3.062, p = 0.08, χ 2 = 0.23, p = 0.045 respectively).
The higher membranous expression of TGF-beta RII in tumor cells correlated with
low numbers of CD1a and CD83 cells in tumor border (χ 2 = 7.659, p = 0.006 and
χ 2 = 11.016, p = 0.001 respectively).
Tissue levels of TGF-beta1 were measured by enzyme-linked immunosorbent assay
in the supernatant of metastasis homogenate and in normal liver tissue. Normal
hepatic TGF-beta1 was detected at 22.5 ng/100 mg wet tissue, whereas in
metastatic tissue TGF-beta1 was detected at 45.04 ng/100 mg wet tissue.
Discussion/Conclusion: Our data show that TGF-beta1, its receptor and the
signaling Smad proteins in tumor metastasis correlated with inhibition of immune cell
function.

Focal nodular hyperplasia of the liver within our experience
53
Halina Cichoż-Lach 1 , Beata Prozorow-Król 1 , Jarosław Swatek 3 , Krzysztof Celiński 1 ,
Maria Słomka 1 , Leszek Buk 2 , Elżbieta Korobowicz 3 , Emilia Lis 1
1 Department of Gastroenterology, Medical University of Lublin, Poland
2 1st Department of Radiology, Medical University of Lublin, Poland
3 Department of Clinical Pathomorphology, Medical University of Lublin, Poland
Introduction: Focal nodular hyperplasia (FNH) is a tumor-like lesion of the liver. The
purpose of this study was to find out if there were any hints from the medical histories
or additional investigations which would suggest FNH later confirmed by liver biopsy.
Methods: The clinical data of 28 patients with FNH hospitalized in the Department of
Gastroenterology, MU of Lublin in the period 1st January, 2002–31st October, 2007
were analyzed retrospectively.
Results: Liver biopsy diagnosed FNH in 28 patients (92.86% women) of total 667
hospitalized in the Gastroenterology Department, Medical University of Lublin for
focal lesions in the liver. In the group of 26 women 16 patients were on oral
contraceptives, 5 were taking hypotensives, men did not receive any medication. All
28 showed normal values of ALT, AST and AFP while each having a single focal
lesion 24–121 mm in diameter found in USG. Their medical histories revealed that
the FNH was asymptomatic and incidentally detected in USG. A central scar had
been detected in 9 of 28 patients on USG and in 14 of 23 patients on CT.
Discussion/Conclusion: In our study FNH was relatively rare pathology and
constituted 4.20% of all cases with focal liver lesion. FNH prevailed in women
(particularly who were oral contraceptives users), did not differentiate in laboratory
results. It manifested as a single focus being incidentally detected by imaging
techniques with a central scar visualized in 32–60% of patients depending on the
techniques applied. Scar absence accounts for the lack of features specific of FNH
and then biopsy is the best option to diagnose the lesion.

54
Liver sinusoidal endothelial cells induce TGF-β dependent
conversion of CD4+Foxp3+ regulatory T cells from conventional
CD4+CD25- T cells
Antonella Carambia, Samuel Huber, Stefan Lüth, Dorothee Schwinge,
Christian Frenzel, Christoph Schramm, Ansgar W. Lohse, Johannes Herkel
Department of Medicine I, University Medical Centre Hamburg-Eppendorf, Germany
Introduction: Recently, it has been demonstrated that ectopic expression of an
autoantigen in the liver can induce antigen-specific CD4+CD25+Foxp3+ regulatory T
cell (Treg) differentiation and protection from autoimmune disease (Lüth et al. 2008.
J Clin Invest. 2008; 118: 3403–10). However, the underlying cellular and molecular
mechanisms remain undefined.
Methods: To test the capacity of liver sinusoidal endothelial cells (LSEC) to induce
Treg from CD4+CD25- conventional T cells, we cultured LSEC (1 x 10 5 ) or splenic
dendritic cells (DC, 5 x 10 4 ) of C57BL/6 mice together with CD4+CD25- T cells
(5 x 10 5 ) and solute antibody to CD3 (1 µg/ml) in the presence or absence of
exogenous TGF-β (0–2 ng/ml).
Results: Upon stimulation by DC, conversion of CD4+CD25- T cells to Treg was
negligible (< 1%); T cell stimulation by LSEC, in contrast, induced significant levels of
Foxp3 expressing Treg (up to 8%). Addition of exogenous TGF-β led to a dosedependent
increase of Treg upon T cell stimulation by LSEC (up to 35%), but
considerably less by DC (up to 7%). These findings were confirmed when T cells
were primed antigen-specifically in the presence of myelin basic protein (MBP;
5 ng/ml) and exogenous TGF-β (2 ng/ml). Of note, cultures of LSEC or DC together
with CD4+CD25- T cells from transgenic mice with impaired TGF-β signaling showed
greatly diminished conversion rates (LSEC-stimulated < 8,5%, DC-stimulated
< 0,6%).
Discussion/Conclusion: In conclusion, we identified LSEC as potent inducers of
TGF-β dependent Treg conversion, indicating a major role of LSEC in the intrahepatic
generation of Treg.

55
Alcohol-metabolizing enzyme gene polymorphisms in alcohol
liver cirrhosis among Polish men
Halina Cichoż-Lach, Emilia Lis, Krzysztof Celiński, Maria Słomka
Department of Gastroenterology Medical University of Lublin, Poland
Introduction: Alcohol liver disease occurs in 77% individuals who abuse alcohol
consumption. Genetic polymorphism of enzymes involved in alcohol metabolism
plays a relevant role in etiopathogenesis of alcohol liver disease. The aim of the
study was to find in the Polish population the ADH1B, ADH1C and ALDH2
genotypes, which are likely to be responsible for higher susceptibility to alcohol liver
cirrhosis.
Methods: The ADH1B, ADH1C and ALDH2 genotype and alleles frequencies were
examined in 202 men: 77 with alcoholic liver cirrhosis, 64 alcoholics without damage
to gastrointestinal organs and 61 nondrinkers (a control group). Genotyping of the
ADH, ALDH2 was performed using PCR-RFLP method on white cell DNA.
Results: The genotype ADH1C*1/*1 and allele ADH1C*1, ADH1B*1 were found to
be significantly more frequent in alcohol abusers compared to non-drinkers.
Frequency of ADH1C*1 allele in alcohol liver cirrhosis group was 62.8%, and
ADH1C*1/*1 genotype was observed in 45.4% and was significantly higher than in
the controls. The differences between of the group of patients who abuse alcohol
were not statistically significant. In the group of nondrinkers ADH1B*2 and ADH1C*2
alleles were more frequent in comparison to the alcohol liver cirrhosis patients and
alcohol addicts. All examined patients were ALDH2*1/*1 homozygotic.
Discussion/Conclusion: In the Polish population examined ADH1C*1, ADH1B*1
allele and ADH1C*1/*1 genotype favor developing alcoholism and alcohol liver
cirrhosis. However ADH1B*2 and ADH1C*2 allele are likely to protect against them.
Genetic polymorphism of ALDH2 shows no correlation with alcohol addiction or
alcohol cirrhosis, Polish is monomorphic ALDH2*1.

56
Conversion of naive T cells into regulatory T cells after
tolerance induction in the murine liver
Benjamin Claaß, Annette Erhardt, Gisa Tiegs
Experimental Immunology and Hepatology, Center of Internal Medicine, University
Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
Introduction: Recently, we have demonstrated that sublethal doses of concanavalin
A (ConA) induce tolerance towards ConA hepatitis in mice within one week.
Tolerance is mediated by IL-10-producing regulatory T cells (Tregs). In vivo and in
vitro data suggested that highly suppressive ConA-primed Foxp3+ Tregs might
convert naive CD4+ T cells into Foxp3+ Tcells with immunosuppressive function.
Hence, this study was intended to identify the relevance of “infectious tolerance” as
one mechanism of ConA tolerance.
Methods: Splenic CD4+ T cells from DEREG (“depletion of regulatory T cells”) mice
were co-cultivated with splenic Tregs from saline- or ConA-pretreated C57BL/6 wt
mice. Moreover, liver non-parenchymal cells (NPCs) or whole splenocytes containing
antigen-presenting cells (APCs) were isolated from DEREG mice. Cells were
stimulated with anti-CD3 mAb for 72hrs. Foxp3 expression correlating with GFP
expression and expression of surface markers with suppressive/regulatory function
were quantified by FACS analysis.
Results: Neither Tregs from ConA- nor from saline-treated mice could induce Foxp3
expression in the co-culture system. However, a transient FoxP3/GFP expression
was detectable in cultures of whole splenocytes and liver NPCs, respectively,
suggesting the requirement of APCs in Treg conversion. PD1, CCR5, and CXCR3
expression were upregulated on Tregs from ConA-tolerant mice, whereas CD45RB
and CD62L were downregulated.
Discussion/Conclusion: In conclusion, different migration patterns and surface
expression profiles seem to characterize the highly suppressive ConA-primed Tregs.
Moreover, conversion of naive responder cells into Foxp3+ T cells by Tregs alone
might be of minor importance for controlling ConA-induced inflammation; however,
differentiation of IL-10 secreting FoxP3-negative Tr1 cells might be induced by Tregs
in an infectious manner. Further studies will identify the mechanism (expansion/proliferation
or conversion) of the transiently increased FoxP3 expression upon
ConA challenge in vivo and the potential of liver-specific APCs, e.g. LSECs and
hepatocytes to convert naive intrahepatic T cells into Tregs.

57
Impact of IL-6-transsignaling on liver damage and regeneration
after CCl4
Jessica Gewiese, Claudia Drucker, Jürgen Scheller, and Stefan Rose-John
Institute of Biochemistry, Medical Faculty, Christian-Albrechts-University Kiel,
Germany
Corresponding author: jgewiese@biochem.uni-kiel.de
Introduction: The role of interleukin-6 (IL-6) in liver regeneration has been
documented. There are two different pathways described for IL-6. In addition to the
classical IL-6 pathway in which IL-6 binds to a membrane bound receptor (IL-6R),
there also exists IL-6 transsignaling via a soluble IL-6R (sIL-6R). sIL-6R binds to IL-6
and activates cells which do not express membrane bound IL-6R.
Methods: To investigate IL-6 transsignaling during of CCl4 liver damage, we blocked
IL-6 transsignaling with soluble gp130Fc protein (sgp130Fc).
Results: We show that blockade of IL-6 transsignaling enhanced CCl4 liver damage.
sgp130Fc pre-treated mice exhibited higher levels of alanine aminotransferase (ALT)
and aspartate aminotransferase (AST). Higher uric acid and potassium concentations
in the sera of these mice were indications for liver cell damage. These differences
neither depended on the bioactivation of CCl4 via Cyp450 2E1, nor on the infiltration
of neutrophils. Liver regeneration is induced by IL-6 transsignaling. Quantification of
regeneration via BrdU shows that regeneration rates are slower when IL-6 transsignaling
is blocked.
Discussion/Conclusion: We focused on IL-6 transsignaling and its impact on liver
damage and regeneration. We showed that IL-6 transsignaling is important since
blockade enhanced liver damage. In view of the fact that a neutralizing IL-6R
antibody that globally blocks IL-6 signaling is already used in the clinic, we will further
investigate whether specific blockade of transsignaling in the liver is superior to
global IL-6 blockade.

58
CXCL10 plays a key role in the development of hepatic fibrosis
Edith Hintermann 1 , Monika Bayer 1 , Andrew Luster 2 , and Urs Christen 1
1 Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-
Universität, Frankfurt am Main, Germany
2 Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA
Introduction: Hepatic fibrosis in chronic liver inflammation as occurring during
chronic hepatitis C infection or autoimmune hepatitis is one of the major concerns
associated with chronic liver disease. Hepatic stellate cells (HSC) are the main
drivers of hepatic fibrosis generating extracellular matrix proteins, such as collagen I.
Here we asked how HSC and infiltrating lymphocytes are activated and recruited to
the site of chronic inflammation.
Methods: We treated wildtype C57Bl/6 and CXCL10 KO mice with carbon tetrachloride
(CCl4) for up to 8 weeks, isolated RNA from whole liver homogenates and
purified HSC. Hepatic fibrosis was assessed by Sirius Red staining. Analysis of HSC
activation was by staining of liver section and isolated HSCs for alpha-smooth
muscle actin (aSMA).
Results: We found a significant elevation in the expression of the chemokine
CXCL10 RNA in HSC. Thus, we used CXCL10 KO mice to investigate the role of
CXCL10 in hepatic fibrosis. Interestingly, CXCL10 KO mice had much lower numbers
of infiltrating lymphocytes in the liver and significantly reduced hepatic fibrosis.
Similarly, blockade of CXCL10 with a neutralizing antibody resulted in diminished
fibrosis and hepatitis. Mechanistically we found that HSC are activated by CCl4
treatment as demonstrated by staining for aSMA. Activated HSC isolated from CCl4treated
mice express CXCR3 and migrated towards CXCL10 in a Transwell migration
assay. Further, activated HSC responded to CXCL10 stimulation by actin
polymerization.
Discussion/Conclusion: In summary, CXCL10 plays an important role in the
development of hepatic fibrosis influencing HSC migration, lymphocyte attraction and
inflammation.

59
Epitope mapping of human cytochrome P450 2D6 (CYP2D6) in
patients with autoimmune hepatitis and in the CYP2D6 mouse
model
Edith Hintermann 1 , Martin Holdener 1 , Monika Bayer 1 , Michael Manns 2 , and
Urs Christen 1
1 Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-
Universität, Frankfurt am Main, Germany
2 Medizinische Hochschule Hannover, Hannover, Germany
Introduction: Cytochrome P450 2D6 (CYP2D6) is the major autoantigen recognized
by LKM-1 antibodies of autoimmune hepatitis (AIH) type 2 patients. We recently
developed a novel mouse model for AIH in which mice are infected with an
adenovirus expressing the human CYP2D6 (Ad-2D6). Mice develop chronic hepatitis
with massive lymphocyte infiltration and subcapsular/periportal fibrosis. Just like AIH
patients, Ad-2D6-infected mice generate high-titer anti-CYP2D6 antibodies.
Methods: Epitope mapping was performed using SPOTs membranes containing
162 peptides (15 aa length, 3 aa offset) covering the entire human CYP2D6
molecule.
Results: Here we report the CYP2D6-epitopes recognized by sera of AIH patients
and Ad-CYP2D6-infected mice and CYP2D6 epitope spreading over time. We found
considerable variation in epitope recognition in between individual AIH patients.
However, the immunodominant epitope (aa262-270: WDPAQPPRD) was found in
most patients as well as in all Ad-2D6 infected mice. This immunodomiant epitope
was already present at time of AIH-diagnosis and was recognized throughout the
whole timeframe analyzed. There is limited intramolecular spreading over time in
both AIH patients and Ad-2D6-infected mice. However, we have identified some
novel epitopes that are only recognized at specific times.
Discussion/Conclusion: Such extraordinary epitopes might provide a basis for
finding possible environmental initiation factors, such as pathogens with structural
similarities conferring molecular mimicry to human CYP2D6. Indeed we found
sequence homology of such CYP2D6 epitopes to human pathogens such as
Hepatitis C virus, Herpes simplex virus, and Legionella pneumophila.

60
The role of the hepatic asialoglycoprotein receptor in inflammatory-induced
liver injury and accumulation of intrahepatic
lymphocytes
Benita L. McVicker, Dean J. Tuma, Geoffrey M. Thiele, Carol A. Casey, and
Natalia A. Osna
University of Nebraska Medical Center and Department of Veterans Affairs, Omaha,
NE, USA
Introduction: We have shown that alcohol impairs hepatic protein trafficking, and
that receptor-mediated endocytosis via the abundant hepatocyte asialoglycoprotein
(ASGP) receptor is especially affected. To further clarify the role of ASGP receptors
in liver disease, we hypothesize that the receptor is involved in T lymphocyte binding
to hepatocytes resulting in the triggering of T-cell clearance mechanisms.
Methods: To examine our hypothesis, we injected wild-type (WT) and ASGP
receptor deficient (RD) mice with T cell-activating anti-CD3 monoclonal antibodies
(which increases T-lymphocyte activation and homing to the liver) followed by
assessment of lymphocyte accumulation and liver injury.
Results: Our results indicate that following anti-CD3 treatment in WT mice, we
observed a two-fold elevation of AST and TNF-alpha expression in the liver within
24 hours which was maintained up to 3 days post injection. Caspase 3 activity was
only slightly increased after 24 hours, but was significantly higher (65% increase,
p < 0.05) two days after anti-CD3 injection in the WT mice. In RD mice, all of the
parameters of liver injury (AST, TNF and caspase activation) were enhanced at least
two-fold compared to WT animals. Additionally, the number of CD8 + /CD45R +
intrahepatic T cells (the proposed cell type involved in hepatocyte-lymphocyte
interactions) were found to be enhanced in the RD animals. Specifically, FACS
analysis demonstrated that following anti-CD3 administration, the percentage of
CD8 + /CD45R + lymphocytes in the liver rose from 3% to 34% in WT animals to 49% in
RD mice.
Discussion/Conclusion: These data support our hypothesis that functional ASGP
receptors play an important role in the regulation of CD8 + T cells in the liver.
Therefore, altered ASGP receptor function in an injured liver can contribute to liver
lymphocyte accumulation and thus may be related to the damaging effects
associated with T cells in the development and/or progression of alcoholic liver
disease.

61
Common variable immunodeficiency-associated granulomatous
disease and nodular regenerative hyperplasia of the liver
H. Varnholt, T. Rubinas
Department of Pathology, University of North Carolina, Chapel Hill, USA
Introduction: Common variable immunodeficiency (CVID) is a primary immunodeficiency
disorder characterized by reduced serum immunoglobulins and heterogeneous
clinical features. Histopathologic findings of liver biopsies obtained for
elevated liver enzymes or hepatosplenomegaly remain ill defined.
Methods: A database search at the University of North Carolina from 1996–2009
yielded two patients with known CVID who had undergone liver biopsies for elevated
liver enzymes.
Results: Patient A is a 16 year-old Caucasian male who was first diagnosed with
CVID one year ago when he had fatigue and underwent splenectomy for splenomegaly
(940 g). Current labs: AST 83 U/l, ALT 101 U/l, Alk Phos 330 U/l, IgA 5 mg/dl,
IgG normal. Liver histology shows numerous nonnecrotizing granulomata with
negative AFB stains and mild lobular lymphocytic inflammation without plasma cells
or fibrosis. Patient B is a 15 month-old African-American female infant with recurrent
respiratory infections, agammaglobulinemia and slightly elevated ALT with normal
other liver function tests. Liver biopsies demonstrate small hyperplastic nodules
without fibrous septae consistent with nodular regenerative hyperplasia (NRH).
Discussion/Conclusions: These cases highlight two common hepatic findings in
CVID: Granulomatous disease and NRH. Although sarcoidosis has been observed
as a cause of multi-organ granulomatous disease, we postulate that in this case it
may have been due to T-cell deficiencies or imbalanced production of cytokines
leading to abnormal sequestration of antigens. NRH, seen in > 80% of cases, may be
caused by variations in blood flow to different areas of hepatic parenchyma or
circulating immune complexes or light chain deposits in the sinusoidal walls.

62
Aggravation of liver damage and loss of immunological
tolerance in CXCR3-deficient mice in the murine model of
concanavalin A-induced liver injury
Claudia Wegscheid 1 , Annette Erhardt 1 , Ulf Panzer 2 , Gisa Tiegs 1
1 Experimental Immunology and Hepatology, Center of Internal Medicine, University
Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
2 Medical Clinic III, Center of Internal Medicine, University Medical Center Hamburg-
Eppendorf, 20246 Hamburg, Germany
Introduction: Injection of the plant lectin concanavalin A (ConA) induces an acute
immune-mediated liver injury in mice. Animals developed tolerance against ConA
rechallenge within one week. Tolerance is mediated by IL-10 producing
CD4+CD25+FoxP3+ regulatory T cells (Tregs). The chemokine receptor CXCR3,
which is expressed by both polarized Th1 cells and Tregs, and its ligands CXCL9-11
have been shown to be involved in several autoimmune diseases. Hence, ConA
hepatitis and tolerance were investigated in CXCR3 KO mice. The results might be
useful to evaluate novel therapeutic approaches in human liver disorders.
Methods: C57/BL6 wildtype and CXCR3-deficient mice were injected with ConA or
saline, respectively, and restimulated with ConA after 8 days. Plasma transaminase
activities were measured and expression of IL-2, IL-10, IL-6, IL-17, TNF-alpha and
IFN-gamma were determined by ELISA and RT-PCR 8 hours after ConA
rechallenge. FACS analyses were performed to characterize the intrahepatic cell
composition and migration of lymphocytes upon ConA challenge.
Results: The expression of CXCL9-11 was significantly upregulated in wt mice upon
ConA challenge correlating with increased IFN-gamma levels. Interestingly, CXCR3deficient
mice exhibited aggravated liver damage and failed to develop tolerance
against ConA indicated by elevated plasma transaminase activity and a proinflammatory
cytokine profile. The frequency of NKT cells was increased in the liver
of CXCR3-deficient mice, whereas the number of Tregs was decreased.
Discussion/Conclusion: Aggravation of liver damage and loss of tolerance in
CXCR3-deficient mice suggests a protective role of CXCR3 during immune-mediated
liver diseases. In contrast CXCR3-deficient mice developed developed less severe
nephritis in a Th1-mediated nephritis model and administration of a blocking anti-
CXCR3 antibody induced prolonged survival of cardiac and islet allografts. Further
studies are intended to identify the role of CXCR3 for migration, conversion and
immunosuppressive capacity of Tregs in immune-mediated liver disorders, since
recruitment of CXCR3-positive Tregs seems to be liver-specific.

63
Metallothioneins (MT I and MT II) and their gene expression in
some chronic liver diseases
Mohamed Sharaf-Eldin*, Saber Ismail*, Usama Negm*, Hala Hamouda**, and
Amal Abdel Hameed***
Departments of Tropical Medicine*, Medical Biochemistery**, and Clinical
Pathology***, Faculty of Medicine, Tanta University, Egypt
Introduction: Much attention was paid to the association between metallothioneins
and chronic liver diseases including hepatocellular carcinoma (HCC).
The aim of the work was to study the role of metallothioneins in chronic liver diseases
and to clarify the role of metallothioneins I and II mRNA expression in hepatocellular
carcinoma.
Methods: The study was carried out on 45 patients with liver diseases (15 patients
with chronic hepatitis, 15 patients with liver cirrhosis and 15 patients with HCC) as
well as 15 healthy individuals as a control group. All patients and controls were
subjected to estimation of metallothioneins levels; also their tissue levels were
estimated in all patient groups. Metallothioneins (MT I and MT II) mRNA expression
by RT-PCR were done for all cases.
Results: The results of the present study showed a significant decrease in serum
and tissue levels of metallothioneins in patients groups. More changes were
demonstrated in HCC patients. Concerning the PCR results of MT genes expression,
there was a significant decrease in MT I and MT II mRNA expression in HCC patients
when compared to the other groups. They also decreased in patients with liver
cirrhosis when compared to the control group and patients with chronic hepatitis. In
contrast their expression does not show significant difference in chronic hepatitis
when compared to the control group.
Discussion/Conclusion: It could be concluded that metallothioneins levels may be
used as a non-invasive biochemical markers for early detection of the progression of
chronic liver diseases. Moreover, the progressive decrease in MT I and MT II gene
expression may play an important role in carcinogenesis of HCC.

64
Endothelial chemokine presentation as therapeutic target in
T-cell mediated hepatitis
Katrin Neumann 1 , Katja Wechsung 1 , Nils Kruse 1 , Michael Schumann 1 , Anja Kühl 2 ,
Alf Hamann 3 , and Katja Klugewitz 1
1 Medizinische Klinik I, Charité, Universitätsmedizin Berlin, Germany
2 Medizinische Klinik I, Research Center ImmunoSciences (RCIS), Charité,
Universitätsmedizin Berlin, Germany
3 Experimentelle Rheumatologie, Charité, Universitätsmedizin Berlin, Germany
Introduction: Transmigration across liver endothelium maintains the homeostatic
balance of intrahepatic T cells and is a central process in liver inflammation. This
study focuses on chemokine-dependent interactions between liver sinusoidal endothelial
cells (LSEC) and lymphocytes and the influence of these processes in T-cell
transmigration into hepatic parenchyma during autoimmune hepatitis.
Methods: Transmigration analysis of CD4 + T cells across LSEC was performed by
the transwell-assay system. Confocal laser scanning microscopy was used for
visualizing chemokine uptake by LSEC. For induction of T-cell mediated hepatitis
Concanavalin A (ConA) was transferred into mice and chlorpromazine was
administered during hepatitis.
Results: LSEC pre-incubated with CXCL12 actively took up the chemokine and
presented it to CD4 + T cells leading to enhanced transmigration of these cells across
the endothelium. Pre-incubation of LSEC with the clathrin-pathway inhibitor
chlorpromazine blocked CXCL12 uptake and significantly reduced chemokine-driven
CD4 + T cell transmigration. Administration of chlorpromazine during ConA-induced
hepatitis reduced T-cell infiltration into hepatic parenchyma counteracting
development of necrosis. Assessing the mode of chemokine uptake, we found that
inhibition of CXCR4 by AMD3100 resulted in the inability of LSEC to take up CXCL12
and subsequently reduced chemotaxis.
Discussion/Conclusion: Our data provide a model for actively enhanced CXCL12driven
transmigration of CD4 + T cells through LSEC layers, where the chemokines
are taken up and transcytosed by chemokine receptors and clathrin-coated vesicles.
Chlorpromazine, an already approved therapeutic compound, inhibits the process in
vitro and in vivo and might offer new approaches for the inhibition of lymphocyte
infiltration into an inflamed liver.

Hemostatic disturbances in patients with portal thrombosis
65
E. Lukina, E. Sysoeva, E. Jakovleva, E. Kitsenko*, G. Sukhanova, S. Vasijev
National Research Center for Hematology, *National Research Center for Surgery,
Moscow, Russia
Introduction: The prehepatic portal hypertension (PPH) due to portal thrombosis
was believed to be a rare condition (about 10–20% of all cases of portal hypertension).
However, during the last 5 years we observed more than 100 patients with
portal thrombosis, who developed PPH.
The aim of the investigation: To study hemostasis in patients with prehepatic portal
hypertension (PPH) due to portal thrombosis.
Materials and methods: We studied 58 patients (median age 42.8 years) with PPH
due to portal thrombosis confirmed by Doppler sonography. The period from the first
manifestation of portal hypertension (splenomegaly, varicose dilatation of esophageal
veins) to examination in our Center varied from 6 to 480 months (M = 92 months).
26 patients (mean age = 45 years) had blood picture compatible with diagnosis of
chronic myeloproliferative disorders (MPD). Other 32 patients (mean age = 41 years)
had normal pattern of bone marrow, normal blood picture or cytopenias. We studied
the basic laboratory parameters concerning the hemostatic status and some
additional parameters: von Willebrand factor, Lupus anticoagulant (LA) homocysteine,
endothelin, soluble thrombomodulin (ELISA).
Results: The majority of patients (pts) had normal rates of fibrinogen, antithrombin III
and protein C. Prothrombin level was decreased in 62% of patients. The level of von
Willebrand factor was increased in 55% of patients, median = 175 (normal rate
< 150). LA was found in 46% of patients. The antiphospholipid antibodies (APL-Abs)
to membranes phospholipids and β2-glycoprotein I were studied in 37 patients. APL-
Abs were found in 22 patients LA+ and in 15 patients LA-, most frequently in patients
with cytopenias. Hyperhomocysteinemia (mild or moderate) was revealed in 54% of
patients. Concentration of soluble thrombomodulin was decreased in 70% of
patients.
Discussion/Conclusion: Thus, patients with PPH due to portal thrombosis had
signs of endothelial damage. The presence of LA and APL-Abs was the typical
feature of PPH in patients with blood cytopenias.

66
False iron deficiency in patients with prehepatic portal
hypertension
E. Lukina, E. Sysoeva, A. Levina, J. Mamukova, E. Voronkova, E. Kitsenko*
National Research Center for Haematology, Moscow, Russia
*National Research Center for Surgery, Moscow, Russia
Background: Patients with prehepatic portal hypertension caused by portal
thrombosis often have recurrent bleedings from esophageal and gastric varices.
Аnemia in these patients is considered to be posthemorrhagic iron deficiency
anemia. Therefore, patients often receive iron treatment.
The aim of the investigation: To study the iron metabolism in patients with
prehepatic portal hypertension due to portal thrombosis.
Materials: We studied 60 patients (22 male and 38 female, median age 43 years)
with prehepatic portal hypertension (PPH) caused by portal thrombosis. 37 (62%)
patients had bleedings from esophageal and gastric varices. The period from the first
manifestation of portal hypertension (splenomegaly, varicose dilatation of esophageal
veins) to examination in our Center varied from 4 to 480 months (median =
72 months). 32 patients had hypo- or normochromic anemia, 28 patients had normal
hemoglobin level.
Methods: We investigated the serum indices of iron metabolism (iron concentration,
total iron binding capacity, transferrin iron saturation, ferritin, transferrin). Hepcidin
level and urine iron excretion were examined in part of the patient group. The control
group consisted of 35 healthy volunteers.
Results: All patients had low or normal levels of serum iron (median = 9.9 mmol/l),
ferritin (m = 39 mkg/l, normal = 40–200) and transferrin iron saturation (TIS,
m = 17.4%, normal = 25–35%). At the same time, serum transferrin level was low or
normal in 64% of patients (median 2.65 g/l, normal 2.65 ± 0.05) and serum indices of
soluble transferrin receptors was not increased in majority of patients. Positive
desferal test revealed increased urine iron excretion in 33 from 35 examined patients.
We also did not find any differences in iron metabolism indices in patients with and
without anemia.
Conclusion: Iron deficiency in patients with portal thrombosis and PPH may
represent the failure of iron metabolism regulation in liver cells caused by ischemic
injury of the liver.
Correspondence to:
Prof. Elena Lukina, National Research Center for Haematology, Noviy Zykovsky pr.
4, 125167, Moscow, Russia, Tel/Fax: 7 (095) 612-0923, E-Mail: lukina@blood.ru

67
Diagnostics improvement for liver chronic diseases in
Turkmenistan
O.B. Nepesova
Turkmen State Medical Institute, Internal Medicine, Ashgabat, Turkmenistan
The study aims to improve diagnostics for liver chronic diseases in Turkmenistan.
Materials and methods: 184 patients at age of 17–77 (averagely 32.5 years) with
various diffuse liver diseases were examined, covering 82 males and 102 females.
All the patients have undergone a liver ultrasonic examination and blood biochemical
indexes as follows have been identified: a transaminase activity, bilirubin and
cholesterol level, a number of lymphocytes and serum protein fractions. The virus
hepatitis markers as follows have also been identified with an immune-enzyme
method. The PCR examinations for DNA HBV and RNA HCV have been held at
serum-positive patients.
Results: The immunological markers of B, C, and D virus hepatitis were found out at
130 patients (70.7%), and no immunological markers were found at 54 patients
(29.3%). Laboratory tests demonstrated a HCV monoinfection at 78 patients (60.0%),
a HBV monoinfection at 18 patients (13.8%) and a mix-virus infection at 34 patients
(26.2%) in seropositive group of patients under observation: B+C hepatitis viruses
were found out at 8 persons, B+C+D at 2 ones, B+D at 24 ones. 1 genotype of the
present virus was found out at 62 patients with a chronic C virus hepatitis, which is
specific for more severe treatment and requires a longer therapy.
Conclusion: Up-to-date laboratory study methods allow to timely and efficiently
select an antivirus therapy required for patient, to monitor efficiency and to forecast a
clinical course.

68
Epidemiology of liver diseases in a pediatric population.
Snapshot data from selected centers in Poland
Woynarowski Marek, Woźniak Małgorzata, Chlebcewicz-Szuba Walentyna,
Chmurska Motyka Teresa, Gorczyca Anna, Korczowski Bartosz, Krzywicka Anna,
Lebensztejn Dariusz, Liberek Anna, Majda-Stanisławska Ewa, Rokitka Maria,
Strawińska Elżbieta, Więcek Sabina, Zaleska Izabella, Zdanowska-Ruskań Anna,
Socha Jerzy
Polish Autoimmune Hepatitis Study Group (PEGAZ), Warsaw, Poland
Introduction: There are no large cross-sectional studies on epidemiology of liver
diseases in pediatric population in Poland. The collaborative group for pediatric liver
diseases (PEGAZ) was established to describe the pediatric hepatology problems in
Poland.
Methods: The questionnaire survey was sent to the hospital involved in liver disease
therapy in children. 13 centers responded to the questionnaire.
Results: Six responding centers are infectious disease hospitals. 6 centers defined
themselves as pediatric hospitals and one site is a gastroenterology, hepatology and
liver transplant center. All centers provide medical care on inpatient and outpatient
basis for children aged 0–18 years. The number of patients treated at every site
varies from 900 to 7000 per year (total around 36,000). Liver disease was present in
3054 (8%). Viral liver disease was present in 1449 subjects (HBV – 1033, HCV – 356
and HBV +HCV in 60). Nonviral liver disease was present in 1605 children (Gilbert
syndrome – 596, liver transplant recipients – 230, cholestasis – 201, autoimmune
hepatitis – 187, Wilson disease – 117, A-1-ATD – 113, cholelithiasis – 83 and other
diseases in 78 children). Combined viral and nonviral liver disease was present in
98 children. The highest rate of hepatotropic viruses coinfection was observed in liver
transplant recipients (17%), autoimmune hepatitis (13.4%) and A-1-ATD (8.8%). The
lowest rate of viral infections was noted in patients with Gilbert syndrome (1.5%).
Discussion/Conclusion: Our data show that the viral infections (especially HBV
infection) remain the first cause of liver problems in Polish pediatric population.

69
Alcohol withdrawal treatment Audit March 2009, Northern
General Hospital, Sheffield, UK
Dr. A. Al-Joudeh, Dr. H. Morton, Dr. S. Heikal, Dr. K. Basu
Sheffield University Teaching Hospitals Foundation NHS Trust, Sheffield, UK
Introduction: In the UK there are approximately 208,000 hospital admissions
(2007/8) with primary or secondary diagnosis of alcohol misuse. 13.3/100,000 deaths
in the UK are alcohol related.
Objectives: To evaluate our current practice in treating alcohol withdrawal with
reference to the local guidelines. Review guidelines and promote the role of
psychiatry liaison team.
Methods: This was a retrospective audit of case notes and prescription charts
between (01/08/2008–31/12/2008). Data collected with a performa (designed against
the Sheffield Teaching Hospitals guidelines).
Inclusion criteria: Alcohol excess > 10 units/day in acute medical admissions.
Results: 183 cases identified, 100 available and 51 patients fulfilled the inclusion
criteria. 69% were male, median age 44 years. 90% of the patients had alcohol
intake recorded. 84% had benzodiazepines prescribed for alcohol withdrawal.
Chlordiazepoxide was prescribed in 53%, this was appropriate in 37%. Lorazepam
was prescribed in 31%, this was appropriate in 50%. 22% had evidence of
hallucinations, 27% of them had haloperidol prescribed. 27% had fits, only 29% of
them had lorazepam prescribed correctly. 45% had Pabrinex prescribed, 44% of
them had Pabrinex prescribed according to guidelines. 61% had Thiamine
prescribed. 75% had vitamin B Co Strong prescribed. 33% of the patients were
referred to psychiatry, 40% of them were followed by alcohol dependence services
after discharge.
Discussion/Conclusion: Unsatisfactory results in the management of alcohol withdrawal
syndrome.
Recommendations: Staff to receive teaching on the subject. Clarify the use and
doses of vitamins and increase the dose of benzodiazepine in the new guidelines. To
formulate a pathway streamlines the management of alcohol withdrawal syndrome in
hospital and follows it up by alcohol dependence services.
Updated local guidelines have taken with the recommendations.

70
Effect of different degrees of hepatic dysfunction on the
pharmacokinetics of telmisartan
Manal El-Hamamsy 1 , Sahar Hegazy 2 , Wageh Awara 3
Faculties of Pharmacy, Ain Shams University 1 and Tanta University 2,3 , Egypt
Introduction: Telmisartan is a nonpeptide angiotensin II receptor antagonist, used
for the treatment of hypertension.
Objective: To describe the pharmacokinetics and safety of telmisartan in healthy
volunteers and in subjects with different degree of hepatic impairment.
Methods: Prospective study. Single oral dose of telmisartan 80 mg was given to
10 healthy subjects served as a control group (group 1), and 20 subjects with hepatic
impairment; divided into two groups; group 2 (10 patients): patients with mild
hepatospleenomegaly and group 3 (10 patients): patients with cirrhosis and ascites.
Plasma samples were collected and analyzed with high performance liquid
chromatography (HPLC).
Results: The pharmacokinetic profile of telmisartan was characterized by rapid
absorption kinetics and a slow terminal elimination phase with mean half life of
25 hours.
The maximum plasma concentration and area under the telmisartan plasma
concentration-time curve (AUC) increased in all hepatically impaired subjects
compared with healthy volunteers. There was a significant increase in the Cmax and
AUC of the cirrhotic patients and non significant increase in the patients with mild
hepatosplenomegaly compared with the healthy volunteers. There was no change in
the half life of telmisartan in all the patients and no adverse events were noticed in
the patients during the study indicating the good tolerability of the drug in
hypertensive patients with hepatic dysfunction.
Discussion/Conclusion: Lower doses of telmisartan should be considered in
cirrhotic patients as well as in the patients with mild hepatitis.

71
Risk perception and use of herbal remedies in patients with
liver/biliary tract disorders: An Italian study
Massimo Marignani, Sara Gallina, Michela Di Fonzo, Ilaria Deli, Paola Begini,
Fabio Attilia, Elia Gigante, Marcella Epifani, Stefano Angeletti, Gianfranco Delle Fave
Digestive and Liver Disease Department, Second Medical Faculty University
“Sapienza” at S. Andrea Hospital, Rome, Italy
Introduction: Use of herbal remedies (HR) has increased in the general population,
particularly among patients with various chronic diseases/conditions. Pharmacovigilance
and regulations regarding HR are still incomplete, and HR-related adverse
reactions are increasingly reported. Nevertheless, studies assessing prevalence of
HR use among patients with liver/biliary tract disorders are limited and no data are
available in Italy. The aims of the study were to assess the prevalence and the risk
perception of HR use in the population attending our outpatient Liver/Biliary Tract
Disorders Clinic. Therefore, to asses the main clinical and demographic characteristics
of patients using HR.
Methods: From October 2007 to April 2008, 231 consecutive patients (119 M, 112 F)
were interviewed, using an ad hoc developed questionnaire. Face to face
questionnaire addressed the following: body mass index (BMI), presence of chronic
conditions, use of conventional therapy, HR-use and perceptions regarding potential
harmful HR-drug interactions. Data were expressed as mean (± SD) or number/total,
and evaluated by student-t and Fisher tests as appropriate. Multivariate logistic
regression (MLR) was also performed.
Results: Prevalence of HR use was 35.5% and 72% of patients using HR had never
considered possible, potentially harmful HR-drug interactions. Therefore 67% of
users used HR in addition to conventional therapy. At MLR use of HR was more
common in women (p = 0.01), and in patients practicing regular sports activity
(p = 0.03). Users were more affected by chronic conditions than non users
(p = 0.0002).
Discussion/Conclusion: More than a third of patients attending Liver/Biliary
Disorders Clinic use HR. Misconceptions about the risk of HR use are widespread
among them. This issue should be specifically considered and addressed with
patients and considered in their management.

72
Hepatotoxicity induced by antituberculosis drugs
M. González, E. Aravena, C. Peña, E. Astrosa, J. Lyon, G. Serrano, P. Escobar
San Borja Arriarán Hospital, Finis Terrae University Santiago, Chile
Introduction: Tuberculosis treatment requires combination of several medicines for
long time, which facilitates emergence of adverse reactions. Liver is involved in drugs
biotransformation, so it develops hepatotoxicity. Its presence demands therapy
suspension and it has mortality, though it is necessary to review the clinical
presentation profile, liver chemistry test pattern, evolution and risk factors associated.
Methods: We reviewed patients’ clinical histories with hepatotoxicity induced by
antituberculosis drugs registered at the Metropolitan Central Health Service of
Santiago of Chile between January 1998 and April 2009. We consigned onset time,
liver chemistry tests alteration pattern, epidemiologic data and their evolution.
Results: In 2676 TBC cases, we identified 53 patients who developed liver toxicity
by drugs (2%). The average age is 50.3±18.4 years (interval 22–81). Gender
proportion: 29 men (57%) and 24 women. Antecedents: 2 VIH patients, 6 cirrhotic
patients, 12 were using multiple drugs. Out of 38 patients consigned, just 8
consumed alcohol. In 34 cases, 29 debuted during the daily dose and 5 during the
twice weekly phase. Liver test pattern: 14 hepatocellular damage (47%), 9 cholestatic
(30%) and 7 mixed (23%). The majority improved with halting and adoptation of a
new anti-TBC schedule, except two patients (3.8%) who developed acute liver
failure, just one survived requiring a liver transplant. The one who died had cirrhosis.
Discussion/Conclusion: In hepatotoxicity induced by antituberculosis drugs, it
emphasizes the antecedents of concomitant ingest of multiple medicines, alcohol
consume and cirrhosis presence. Most of cases present at the beginning of
treatment, highlighting a hepatocellular damage pattern. Patients can develop acute
liver failure.

Treatment with Ursofalk ® of bile reflux gastritis
Dr. Dorina Pestroiu, Dr. Madalina Ilie, Dr. Dana Brooks, Dr. Cosmina Vladut
Clinical Emergency Hospital Bucharest, Romania
73
Introduction: Bile acids refluated in the stomach produce cell destruction,
immunologic modifications or/and inflammatory effects.
Recent studies have shown that administration of UDCA increases its concentration
in the gastric reflux inducing the decrease of the cell destruction effects; it has antiinflammatory
and immune-modulator effect leading to the reduction of the symptoms.
Also, UDCA has a role in normalizing the intestinal motility, decreasing the
duodenogastric and gastroesophageal reflux.
Methods: 45 patients (12 female and 33 male) were studied, with average age of 47,
from which 15 had stomach resection, 12 had cholecystectomy, the rest having the
disease in motility disturbances.
There were included in the study patients whose Helicobacter pylori infection and
AINS consumption were excluded and to whom a gastric mucosa biopsy was
performed which showed mucosa edema with moderate lymphoplasmocyte
infiltration.
UDCA was administrated in dose of 10 mg/kg, for 14 days a month, 3 consecutive
months, in unique dose in the evening.
Results: The patients were examined after 2 weeks and the reduction of clinical
symptoms was observed at 50% of the patients.
At 3 months after beginning the treatment were performed: clinical exam, upper
digestive endoscopy with biopsies taken. At 75% of the patients, the remission of
clinical, endoscopic and histological manifestations was obtained.
Discussion/Conclusion: Adding UDCA in bile reflux gastritis provides a marked
improvement of symptoms after 3 months of treatment with 10 mg/kg, for 14 days a
month.

74
Incidence and therapy of colonic perforations: How feasible
and effective is endoscopic closure?
Klaus Mönkemüller, MD, PhD, FASGE 1,2 , Helmut Neumann, MD,
Peter Malfertheiner, MD, PhD, H.-U. Schulz, MD, PhD, Lucia C. Fry, MD 1,2
1 Department of Internal Medicine, Gastroenterology, Hepatology and Infectious
Diseases, Marienhospital, Bottrop, Germany
2 Divsion of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-
Guericke-University, Magdeburg, Germany
Introduction: Colon perforation is one of the most dreaded complications of
colonoscopy. Traditionally, patients with colon perforation have been treated
surgically. Although there are several case reports documenting the usefulness of
endoscopic closure of colon perforations, there are few current data evaluating the
current management of colon perforations.
The aim of the study was to assess the incidence of colon perforations and the utility
of immediate endoscopic closure of the defect in a tertiary endoscopy unit.
Methods: All patients who underwent colonoscopy at our institution from June 2001
to December 2008 were identified. Data were obtained by searching our
prospectively collected electronic database (Medos, Langensebold, Germany).
Results: During the study period, a total of 8601 colonoscopies were performed
(2472 therapeutic interventions, 28.7%). A total of 12 iatrogenic colonic perforations
occurred, yielding an incidence of 0.14%. Five (41.7%) occurred during a diagnostic
colonoscopy (perforation rate of 0.08%). The remaining 7 perforations (58.3%)
occurred as the result of a therapeutic intervention, perforation rate, 0.28% (p < 0.02,
as compared to perforations occurring during a diagnostic colonoscopy). These
included: polypectomy, n = 4, dilation of stenosis, n = 2, decompression tube
placement, n = 1. Successful endoscopic closure of the perforation site was possible
in 5 patients (42%). Seven patients were treated surgically (large defects, n = 3,
difficult scope position, n = 2, stool contamination, n = 2).
Discussion/Conclusion: The perforation rate of colonoscopy was 0.14%.
Immediate endoscopic closure of the perforation was possible in 42% of patients.
Patients with defects larger than 2 cm and those located in angulated areas or the
presence of stool contamination should undergo immediate laparotomy.

Author Index to Poster Abstracts
(Name - Poster Number)
AbouNar, A. 33
Adamenko, E.I. 9
Adjarov, D.G. 17
Ahmad, Y. 33
Al-Joudeh, A. 69
Altunkova, I.P. 50
Ananiev, J. 50, 51, 52
Andreescu, O. 22
Angeletti, S. 71
Angelico, M. 31
Appleby, V.J. 15
Aravena, E. 72
Askar, E. 35
Astrosa, E. 72
Attilia, F. 71
Austin, L. 26
Awara, W. 70
Azmy, M. 33
Badea, D. 41, 42, 47
Badea, M. 41, 47
Bahcecioglu, I.H. 8
Baiocchi, L. 31, 32
Baleva, D. 7
Banciu, C. 19
Basu, K. 69
Bayer, M. 58, 59
Beard, M. 26
Bebb, O. 15
Begini, P. 71
Belyaeva, T.V. 23
Berres, M.-L. 27
Bockmann, J.H. 16
Boitan, M. 37
Boryczka, G. 28
Borza, C. 19
Bregadze, R. 29
Bremer, B. 14
Brooks, D. 73
Buk, L. 53
Carambia, A. 54
Carbone, M. 31, 32
Casey, C.A. 60
Cecere, R. 32
Celik, N. 1
Celinski, K. 53, 55
Chakarova, P. 44
Chen, J.L. 20
Chlebcewicz-Szuba, W. 68
Chmurska Motyka, T. 68
Christen, U. 58, 59
Cichoz-Lach, H. 53, 55
Cijevschi Prelipcean, C. 18, 36
Ciof, O.P. 2, 3, 10, 21,
45, 48
Ciuclan, L. 20
Claaß, B. 56
Conti, L.R. 31
Copaceanu, L. 19
Cornberg, M. 14, 24
Dabrowska, M. 30
Dandri, M. 16
De Leonardis, F. 31, 32
Deac, M. 37, 46
Deli, I. 71
Delle Fave, G. 71
Delle Monache, M. 31, 32
Demir, M. 6
Derkow, K. 40
Di Fonzo, M. 71
Dienes, H.-P. 35
Donohue, T. 25
Dooley, S. 20
Dragan, E. 2, 3, 10, 21,
45, 48
Draghila, L. 37
Dragna, M. 18, 36
Drucker, C. 57
Drug, V.L. 18, 36
Dusheiko, G.M. 12
Dzieran, J. 20
El-Batae, H. 34
El-Hamamsy, M. 33, 70
Epifani, M. 71
Ergün, D. 1
Erhardt, A. 56, 62
Esaulenko, E.V. 23
Escobar, P. 72
Flisiak, R. 30
Fratila, O. 37

Frenzel, C. 54
Fry, L.C. 74
Galcheva, S. 7
Gallina, S. 71
Gebhardt, R. 20
Gencaslan, A. 8
Genunche-Dumitrescu, A. 41, 42, 47
Georgiev, K. 44
Gewiese, J. 57
Gigante, E. 71
Goeser, T. 6
Golebiewski, J. 13
González, M. 72
Gora-Gebka, M. 13
Gorczyca, A. 68
Gorski, J. 30
Grabowski, J. 14, 24
Graur, L. 18, 36
Gulubova, M.V. 50, 51, 52
Gutkowski, K. 28
Hamann, A. 40, 64
Hameed, A.A. 63
Hamouda, H. 63
Hartleb, M. 5, 28
Hegazy, S. 70
Heikal, S. 69
Helliwell, P. 15
Herkel, J. 54
Heuchel, R. 20
Hintermann, E. 58, 59
Holdener, M. 59
Huang, T. 20
Huber, S. 54
Ilie, M. 73
Ilkavets, I. 20
Jakovleva, E. 65
Jaroszewicz, J. 14
Jaroszewicz, J. 24
Jelev, D. 17
Julianov, A. 50
Kacperek-Hartleb, T. 28
Kajor, M. 28
Kalinova, K. 44
Kaminska, B. 13
Kaminska-Kiszka, E. 28
Kara, S. 1
Khanna, P. 12
Kharbanda, K. 26
Kitsenko, E. 65, 66
Klugewitz, K. 40, 64
Korczowski, B. 68
Korobowicz, E. 53
Kosseva, O. 17
Kowalska, A. 27
Krastev, Z. 17
Kruse, N. 40, 64
Krzywicka, A. 68
Kühl, A.A. 40, 64
Kuzu, N. 8
Kyurkchiev, D. 50
Lang, S.M. 6
Lawniczak, M. 4, 5
Lebensztejn, D.M. 68
Lenci, I. 31, 32
Levina, A. 66
Liberek, A. 13, 68
Lis, E. 53, 55
Liu, Y. 20
Lohse, A.W. 16, 54
Lukina, E. 65, 66
Luster, A. 58
Lütgehetmann, M. 16
Lüth, S. 54
Lyon, J. 72
Maini, M. 12
Majda-Stanislawska, E. 68
Malfertheiner, P. 74
Mamukova, Y. 66
Manns, M.P. 14, 24, 59
Manolova, I.M. 50, 51, 52
Marignani, M. 71
Martin, M. 48
Martinova, E.A. 43
Mateescu, R. 19
Matev, A. 51
McVicker, B.L. 60
Mertens, J. 38
Mertens, P.R. 20
Metin, K. 8
Meyer, C. 20
Mihai, B. 18, 36
Mihai, C. 18, 36
Mihaila, R. 37
Mihaila, R.G. 37, 46
Mihm, S. 29, 35, 38

Milkiewicz, P. 5
Mitrut, A.O. 42
Mitrut, P. 41, 42, 47
Mocanu, L. 37
Mönkemüller, K. 74
Moreea, S. 15
Moreno Zaldivar, M. 27
Morton, H. 69
Musialik, J. 28
Nebbia, G. 12
Nedelcu, L. 22
Negm, U. 63
Nepesova, O.B. 67
Neumann, H. 74
Neumann, K. 40, 64
Nita, L. 42
Nosotti, L. 31
Odenthal, M. 35
Olteanu, A. 37
Osna, N.A. 25, 26, 60
Özercan, I.H. 8
Pacurari, A. 19
Panasiuk, A. 30
Pantea, I. 22
Panzer, U. 62
Pauels, K. 27
Paul, D. 22
Pekova, L.M. 44
Peña, C. 72
Pestroiu, D. 73
Petersen, J. 16
Petrov, A. 11
Pophristova, E. 17
Pothakamuri, S.V. 24
Prozorow-Król, B. 53
Ramadori, G. 29, 35, 38
Raszeja-Wyszomirska, J. 4, 5
Rezi, E.C. 37, 46
Rill, D. 2, 3, 10, 21,
45, 48
Rokitka, M. 68
Romanov, A.O. 23
Romosan, I. 19
Rose-John, S. 49, 57
Ross Lopes, A. 12
Rubinas, T. 61
Saber, I. 63
Sahin, K. 8
Samdanci, E. 1
Savoiu, G. 19
Scarneciu, C. 22
Scheller, J. 49, 57
Schlaphoff, V. 14, 24
Schmitz, P. 27
Schramm, C. 54
Schulte, S. 6
Schulz, H.-U. 74
Schumann, M. 64
Schurich, A. 12
Schütt, A. 49
Schwinge, D. 54
Scurtu-Martin, M. 2, 3, 10, 21
Serban, C. 19
Serrano, G. 72
Sharaf-Eldin, M. 34, 63
Sikorska-Wisniewska, G. 13
Silivontchik, N.N. 9
Singer, M.V. 20
Slomka, M. 53, 55
Socha, J. 39, 68
Southern, P. 15
Sporea, I. 2, 3, 10, 21,
45
Stachowska, E. 4
Stefanescu, G. 18
Stefanova, P. 44
Steffen, H.-M. 6
Stegmann, K. 24
Strawinska, E. 68
Suchy, J. 4
Sukhanova, G. 65
Suneetha, P.V. 14
Susan, L.M. 19
Swatek, J. 53
Sysoeva, E. 65, 66
Szymanik, B. 5
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Thiele, G.M. 60
Tiegs, G. 56, 62
Töx, U. 6
Trautwein, C. 27
Tudora, A. 2, 3, 10, 21,
45, 48
Tuma, D.J. 60
Tzaneva, V. 7

Ubben, S. 6
Üstündag, B. 8
Varnholt, H. 61
Vasijev, S. 65
Vatev, N. 11
Vladut, C. 73
Volz, T. 16
Voronkova, E. 66
Wasmuth, H.E. 27
Wechsung, K. 64
Wedemeyer, H. 14, 24
Wegscheid, C. 62
Weinman, S. 25
Weiskirchen, R. 27
Weng, H.-L. 20
White, R. 25, 26
Wiecek, S. 68
Wiercinska, E. 20
Woynarowski, M. 39, 68
Wozniak, M. 39, 68
Yovchev, Y. 52
Yüksel, F. 1
Yüksel, I. 1
Zabielski, P. 30
Zaharie, A.V. 37
Zaleska, I. 68
Zawada, I. 4
Zdanowska-Ruskan, A. 68
Zhu, C. 20

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