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HEPATOLOGY Vol. 34, NO. 4, Pt. 2, 2001 AASLD ABSTRACTS 483A 1243 INTRAHEPATIC INTERLEUKIN-8 PRODUCTION DURING DISEASE PROGRESSION OF CHRONIC HEPATITIS C. Yoshiya Tachibana, Yasunari Nakamoto, Shuichi Kaneko, Naofumi Mukaida, Kenichi Kobayashi, Kanazawa Univ, Kanazawa Japan Background: Chemokines are not only known to mediate tissue infiltration with inflam- matory cells, but also regulate the balance of helper T lymphocytes (Thl/Th2) and the activation of antigen-presenting dendritic cells. Moreover, ehemokine-mediated cellular responses have been found to be involved in the process of neovascularisation, fibrosis and malignant transformation. Recent reports demonstrated that the hepatitis C virus (HCV) e,~re and NS5A proteins directly enhanced interleukin (IL)-8 gene expression in vitro, suggesting the involvement of chemokines in the progression of liver disease. To under- stand the relative contribution of chemokines in the pathogenesis of chronic HCV infec- tion, we established a sandwich enzyme-linked immunosorbent assay (ELISA) to quauti- tare serum levels of IL-8, monocyte chemoattraetant protein-t (MCP-1) and macrophage inflammatory protein-lalpha (MIP-la), and immunohistochemically analyzed expression of the chemokines in liver tissues in patients exhibiting various degrees of chronic hepatitis C. Methods: Sera were obtained from eighty-nine patients positive for anti-HCV, 30 were histologically diagnosed with chronic hepatitis (CH), 29 with liver cirrhosis (LC), and 30 with cirrhosis with hepatoeenular carcinoma (HCC). 20 sera from individuals without chronic liver disease and negative for anti-HCV served as controls. Serum concentrations of IL-8, MCP-1 and MIP-la were quantitated by a solid-phase sandwich ELISA in which a specific routine monoclonal antibody was used as a capture and the rabbit polyclonal antibodies as the secondary antibodies. Expression of the chemokines in liver tissues was evaluated immunohistoehemicafly using the specific monoclonal antibodies. Differences between groups were analyzed for statistical significance using one-way ANOVA. Factors associated with the progression of liver disease were determined by multivariate logistic regression analysis. Results: The detection limits of this ELISAsystem were 10, 40 and 'tO pg/mL for IL-8, MCP-1 and MIP-la, respectively, In the patients with the escalating sever- ity' of liver disease, serum IL-8 levels increased significantly (CH, 18.75 -+ 2.32; LC, 32.12 -+ 3.80; HCC, 49.13 -+ 11.03; and controls, 17.43 +- 1.11 pg/mL) (P < 0.01 ). Furthermore, in the HCC patients, IL-8 concentrations were positively correlated with the TNM stages (P < 0.01 ), and inversely correlated with the survival periods (P < 0.01). Additionally, using logistic regression analysis, IL-8 concentration was determined to be an independent risk factor for a poor prognosis for the HCC patients, as well as platelet count and serum albumin concentration. Immunohistochemical analysis of liver tissues indi- cated that IL-8 was stronglystained in the cytoplasm of HCC ceils and the weak expression was detected in the cytoplasm of part of hepatocytes in the cirrhotic liver although it was not detectable in hepatocytes of the control tissues. In contrast, there was no correlation between serum MCP-1 or MIP-la concentrations and the progression of chronic hepatitis C. Conclusions: The results demonstrate, first, that IL-8 production is increased with the escalating severity of chronic hepatitis C, suggesting its involvement in the process of disease progression. Second, IL-8 levels are correlated with the tumor stages of the patients with HCC in which IL-8 expression is induced in tumor ceils, suggesting that IL-8 may be expressed with the malignant transformation of hepatocytes in chronic hepatitis C. 1244 A MAJOR PRODUCER OF TYPE I IFN, THE SO-CALLED PLASMACY- TOID DENDRITIC CELLS, MAY BE AN ACTIVE REPLICATION SITE DURING HCV NATURAL INFECTION. Nad~ge Goutagny, Ens, Lyon France; Christine Bain, Biomerieux, Lyon France; Ahmed Fatmi, CNRS, Lyon France; Patrice Couzigou, CHU Bordeaux, Bordeaux France', Genevieve In- chauspe, INSERM, Lyon France Plasmacytoid Dendritic Cells (pDC), a component of peripheral blood mono- nuclear cells (PBMC), is the major producer of type I interferon in response to enveloped viruses. We have investigated the capacity of PBMC from 100 indi- viduals to produce IFNa upon HSV-1 stimulation. Six cohorts of individuals were included: HCV seronegative controls comprising Healthy individuals (Ctrl, n=20) and Non Viral Hepatic Disorders (NVHD, n=20); HCV chronic carriers divided into Non Treated (NT, n=18), Responders (R, n=ll) and Non Responders (NR, n=9) to therapy; and Long Term Responders to antiviral therapy (LTR, n=23). All these groups of patients displayed a similar capacity to secrete IFNa except for the group of NR. The production of IFNa by PBMC of these patients (mean = 122 pg/mL) was dramatically decreased when com- pared with other HCV cohorts (mean=1011 pg/mL, p<0.05), or with the controls (mean=935 pg/mL, p<0.05). As NR are unable to control viral rep- lication, these data suggest a link between the intrinsic capacity of pDC to produce IFNa and the control of HCV viremia. For 24 patients, we were able to purify the PB-DC from large blood samples with a purification rate of about 95%. We searched for the presence of HCV sequences in this subpopulation, as well as in contaminating cells, by RT-PCR. While HCV genomic RNA could be detected in all cell populations for 19/24 patients, a specific detection of HCV sequences in PB-DC could be demonstrated in 5/24 patients. Furthermore, for 3/24 patients, the replicative intermediate was specifically detected in the PB- DC. A longitudinal study was performed for one patient who became a LTR to therapy during follow up, allowed us to demonstrate a loss of HCV genomic sequences in the PB-DC with time. For one other patient, analysis of the quasispecies distribution within the 5'Non Coding Region from PB-DC, plasma, PBMC, B cells or the T, NK, monocyte fraction is currently under investigation. These data strongly suggest that, during natural infection, PB-DC may be a reservoir where active replication of HCV takes place. Such a replication could be responsible for the alteration of the production of IFNa by pDC. In NR, such an alteration may be involved in the lack of control of HCV replication by the immune system, the alteration of the polarization of the HCV specific immune response and the impaired survival of anti-HCV activated T cells. 12;45 EXPRESSION OF "TISSUE" TRANSGLUTAMINASE IN CHRONIC HEPA- TITIS C. Roberta Nardacci, Fabiola Ciccosanti, Laura Falasca, Giorgio Anto- nucci, Istituto Nazionale Malattie Infettive L Spallanzani, Roma Italy; Mauro Piacentini, INMI L Spallanzani and Universita' Tor Vergata, Roma Italy; Gi- nseppe Ippolito, Istituto Nazionale Malattie Infettive L Spallanzani, Roma h- aly; Antonio Craxi', University of Palermo, Palermo Italy; Oreste Lo Iacono, INMI L Spallanzani, Roma and University of Palermo, Palermo Italy Tissue transglutaminase (tTG) catalyses protein crosslinking reactions, which le~td to the establishment of e-T(glutamyl) lysine covalent bonds, tTG-catal- ysed crosslinking, promoting the formation of stable and insoluble protein polymers, plays a role in extracellular matrix organisation both in normal and pathological settings. Aim:To investigate the role of tTG in hepatic fibrogenesis due to HCV infection. Methods: Sixty untreated patients with chronic hepatitis C (median grade 1.5, stage 1.5 by METAVIR) were studied. All were HCV-RNA positive (53 % genotype lb), with a median viremia of 618.040 IU/ml. Five normal surgical liver samples were used as controls. TTG expression was assessed by Western blot, electron microscopy immunolabeling and immuno- histochemistry using a anti-tTG monoclonal IgG (NeoMarkers, USA). Resuhs: Levels of tTG protein were 3-4-fold higher in liver homogenates of HCV pa- tients than in controls. Distribution of tTG in the liver varied according to the stage of disease. In mild, nonfibrotic chronic hepatitis C tTG expression was mainly induced in the hepatocytes in contact with the periportal infiltrate. In patients at a more advanced stage of fibrosis (F2-F3), tTG was also detected in extracellular matrix and in the fibrotic tissue. At the latest stages (F4), tTG was confined to the fibrotic tissue. A statistically significant relationship existed between the amount of tTG expressed, the grade of periportal necrosis (p<0.03) and the stage of fibrosis (p<0.005). Conclusions:These data suggest that tTG is directly involved in fibrogenesis due to HCV infection, and point toward its possible role as a non-invasive marker to follow the course of chronic hepatitis C. 1246 ANTIVIRAL RESPONSE AND TH-1 AND TH-2 CYTOKINE PROFILE RE- SPONSE BY PERIPHERAL BLOOD MONONUCLEAR CELLS OF PA- TIENTS WITH DIFFERENT CLINICAL OUTCOME OF HCV-INFECTION. Pietro Andreone, Annagiulia Gramenzi, Carmela Cursaro, Simona Talarico, Marzia Margotti, Elisabetta Loggi, Simona Romano, Stefania Lorenzini, Fed- erica Porzio, Maurizio Biselli, Francesco Felline, Mauro Bernardi, Universita' di Bologna, Bologna Italy Background: the balance between Thl- and Th2-type cytokines has been suggested to play a role in the outcome of HCV infection. Aim: to evaluate the synthesis of Thl- and Th2- cytokines and of 2,5-oligoadenylate synthetase (2,5-OAS) an interferonqnduced antiviral protein, from peripheral blood mononuclear cells (PB~ICs) of patients with different patterns of HCV infection. Methods: Three groups of 10 HCV-Ab positive patients each were studied: group A) "previous" HCV infection (negative HCV-RNA and normal ALT), group B) "asymptomatic" HCV chronic infection (positive HCV-RNA and persistently normal ALT) and group C) "symptomatic" HCV chronic infection (positive HCV-RNA and abnormal ALT). Ten healthy subjects were also studied as controls. For each patient, 106 PBMCs were isolated and cultured in RPMI both in absence and in presence of Concanava- lin A. Thl-cytokines (IL-2 and IFN-'y), Th2-cytokines (1L-4and 1L-10) and 2,5-OAS were directly assayed in the supernatants by commercial kits. Results: are reported in the table: A significant direct correlation was found between IL-2 and 2 5-OASsynthesis considering either all 40 subjects together (r=0.4; p=0.012) or excluding controls (r=0.4; p=0.03). Considering either group C only or group B and C together, a significant negative corre- lation between IL-2 and ALT serum levels was found (r=-0.78, p<0.001 and r=-0.078, p <0.001, respectively). On the contrary a significant direct correlation was found between IL-4 synthesis and ALT serum levels considering either patients of group A, 13 and C together (r=0.4, p=0.039) or patients of groups B and C only (r=0.74; p<0 001) Con- clusion: The results of this study seem to indicate an imbalance toward Th2-cytokine profile in pts with "symptomatic" HCV-chronie infection. On the contrary, a strong antiviral response and a prevalent Thl-cytokine profile seem to characterize "asymptomatic" HCV infection. These data indicate that Th-lymphocyte polarization may play an important pathophysiologic role in influencing the outcome of HCV infection. GROUP A GROUP B GROUP C CONTROLS 2-50AS* 86 222 '~07 40 (0-308) a (39-558)~,b (0-378) (21-139)b 11.-2"* 89 824 190 87 (38-158) c (241-1317)~,~.~ (0445)d (25.139)~ IFN-y** 2t92 2796 2607 2878 (601-4896) (676-8266) (778-6113) (1567-6738) IL-4** 0 0 72 0 (0-297) t (0-0) tg (0-202)g." (0-83)" IL-IO** 791 602 1113 2666 (395-1484) ~ (141-1884)L" (513-2295)' (439-4It8)~. r" Data are expressed as median (minimum-maximum)and analyzed by Mann Whitney U test; *prnol/mtxlO~cells; "pg/mlxtO ~cells; ~p=O.04; b'"'mp=O.004; c"P=O,O002, dp=O.O012; rp=O.03; gp=O.006;'p=0,013, ~p=0,0t9.

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HEPATOLOGY Vol. 34, NO. 4, Pt. 2, 2001 AASLD ABSTRACTS 4 8 3 A

1243

INTRAHEPATIC INTERLEUKIN-8 PRODUCTION DURING DISEASE PROGRESSION OF CHRONIC HEPATITIS C. Yoshiya Tachibana, Yasunari Nakamoto, Shuichi Kaneko, Naofumi Mukaida, Kenichi Kobayashi, Kanazawa Univ, Kanazawa Japan

Background: Chemokines are not only known to mediate tissue infiltration with inflam- matory cells, but also regulate the balance of helper T lymphocytes (Thl/Th2) and the activation of antigen-presenting dendritic cells. Moreover, ehemokine-mediated cellular responses have been found to be involved in the process of neovascularisation, fibrosis and malignant transformation. Recent reports demonstrated that the hepatitis C virus (HCV) e,~re and NS5A proteins directly enhanced interleukin (IL)-8 gene expression in vitro, suggesting the involvement of chemokines in the progression of liver disease. To under- stand the relative contribution of chemokines in the pathogenesis of chronic HCV infec- tion, we established a sandwich enzyme-linked immunosorbent assay (ELISA) to quauti- tare serum levels of IL-8, monocyte chemoattraetant protein-t (MCP-1) and macrophage inflammatory protein-lalpha (MIP-la), and immunohistochemically analyzed expression of the chemokines in liver tissues in patients exhibiting various degrees of chronic hepatitis C. Methods: Sera were obtained from eighty-nine patients positive for anti-HCV, 30 were histologically diagnosed with chronic hepatitis (CH), 29 with liver cirrhosis (LC), and 30 with cirrhosis with hepatoeenular carcinoma (HCC). 20 sera from individuals without chronic liver disease and negative for anti-HCV served as controls. Serum concentrations of IL-8, MCP-1 and MIP-la were quantitated by a solid-phase sandwich ELISA in which a specific routine monoclonal antibody was used as a capture and the rabbit polyclonal antibodies as the secondary antibodies. Expression of the chemokines in liver tissues was evaluated immunohistoehemicafly using the specific monoclonal antibodies. Differences between groups were analyzed for statistical significance using one-way ANOVA. Factors associated with the progression of liver disease were determined by multivariate logistic regression analysis. Results: The detection limits of this ELISA system were 10, 40 and 'tO pg/mL for IL-8, MCP-1 and MIP-la, respectively, In the patients with the escalating sever- ity' of liver disease, serum IL-8 levels increased significantly (CH, 18.75 -+ 2.32; LC, 32.12 -+ 3.80; HCC, 49.13 -+ 11.03; and controls, 17.43 +- 1.11 pg/mL) (P < 0.01 ). Furthermore, in the HCC patients, IL-8 concentrations were positively correlated with the TNM stages (P < 0.01 ), and inversely correlated with the survival periods (P < 0.01). Additionally, using logistic regression analysis, IL-8 concentration was determined to be an independent risk factor for a poor prognosis for the HCC patients, as well as platelet count and serum albumin concentration. Immunohistochemical analysis of liver tissues indi- cated that IL-8 was strongly stained in the cytoplasm of HCC ceils and the weak expression was detected in the cytoplasm of part of hepatocytes in the cirrhotic liver although it was not detectable in hepatocytes of the control tissues. In contrast, there was no correlation between serum MCP-1 or MIP-la concentrations and the progression of chronic hepatitis C. Conclusions: The results demonstrate, first, that IL-8 production is increased with the escalating severity of chronic hepatitis C, suggesting its involvement in the process of disease progression. Second, IL-8 levels are correlated with the tumor stages of the patients with HCC in which IL-8 expression is induced in tumor ceils, suggesting that IL-8 may be expressed with the malignant transformation of hepatocytes in chronic hepatitis C.

1244

A MAJOR PRODUCER OF TYPE I IFN, THE SO-CALLED PLASMACY- TOID DENDRITIC CELLS, MAY BE AN ACTIVE REPLICATION SITE DURING HCV NATURAL INFECTION. Nad~ge Goutagny, Ens, Lyon France; Christine Bain, Biomerieux, Lyon France; Ahmed Fatmi, CNRS, Lyon France; Patrice Couzigou, CHU Bordeaux, Bordeaux France', Genevieve In- chauspe, INSERM, Lyon France

Plasmacytoid Dendritic Cells (pDC), a component of peripheral blood mono- nuclear cells (PBMC), is the major producer of type I interferon in response to enveloped viruses. We have investigated the capacity of PBMC from 100 indi- viduals to produce IFNa upon HSV-1 stimulation. Six cohorts of individuals were included: HCV seronegative controls comprising Healthy individuals (Ctrl, n=20) and Non Viral Hepatic Disorders (NVHD, n=20) ; HCV chronic carriers divided into Non Treated (NT, n=18) , Responders (R, n = l l ) and Non Responders (NR, n=9) to therapy; and Long Term Responders to antiviral therapy (LTR, n=23) . All these groups of patients displayed a similar capacity to secrete IFNa except for the group of NR. The production of IFNa by PBMC of these patients (mean = 122 pg/mL) was dramatically decreased when com- pared with other HCV cohorts (mean=1011 pg/mL, p<0.05) , or with the controls (mean=935 pg/mL, p<0.05). As NR are unable to control viral rep- lication, these data suggest a l ink between the intrinsic capacity of pDC to produce IFNa and the control of HCV viremia. For 24 patients, we were able to purify the PB-DC from large blood samples with a purification rate of about 95%. We searched for the presence of HCV sequences in this subpopulation, as well as in contaminating cells, by RT-PCR. While HCV genomic RNA could be detected in all cell populations for 19/24 patients, a specific detection of HCV sequences in PB-DC could be demonstrated in 5/24 patients. Furthermore, for 3/24 patients, the replicative intermediate was specifically detected in the PB- DC. A longitudinal study was performed for one patient who became a LTR to therapy during follow up, allowed us to demonstrate a loss of HCV genomic sequences in the PB-DC with time. For one other patient, analysis of the quasispecies distribution within the 5'Non Coding Region from PB-DC, plasma, PBMC, B cells or the T, NK, monocyte fraction is currently under investigation. These data strongly suggest that, during natural infection, PB-DC may be a reservoir where active replication of HCV takes place. Such a replication could be responsible for the alteration of the production of IFNa by pDC. In NR, such an alteration may be involved in the lack of control of HCV replication by the immune system, the alteration of the polarization of the HCV specific immune response and the impaired survival of anti-HCV activated T cells.

12;45

EXPRESSION OF "TISSUE" TRANSGLUTAMINASE IN CHRONIC HEPA- TITIS C. Roberta Nardacci, Fabiola Ciccosanti, Laura Falasca, Giorgio Anto- nucci, Istituto Nazionale Malattie Infettive L Spallanzani, Roma Italy; Mauro Piacentini, INMI L Spallanzani and Universita' Tor Vergata, Roma Italy; Gi- nseppe Ippolito, Istituto Nazionale Malattie Infettive L Spallanzani, Roma h- aly; Antonio Craxi', University of Palermo, Palermo Italy; Oreste Lo Iacono, INMI L Spallanzani, Roma and University of Palermo, Palermo Italy

Tissue transglutaminase (tTG) catalyses protein crosslinking reactions, which le~td to the establishment of e -T(g lu tamyl ) lysine covalent bonds, tTG-catal- ysed crosslinking, promoting the formation of stable and insoluble protein polymers, plays a role in extracellular matrix organisation both in normal and pathological settings. Aim:To investigate the role of tTG in hepatic fibrogenesis due to HCV infection. Methods: Sixty untreated patients with chronic hepatitis C (median grade 1.5, stage 1.5 by METAVIR) were studied. All were HCV-RNA positive (53 % genotype lb) , with a median viremia of 618.040 IU/ml. Five normal surgical liver samples were used as controls. TTG expression was assessed by Western blot, electron microscopy immunolabeling and immuno- histochemistry using a anti-tTG monoclonal IgG (NeoMarkers, USA). Resuhs: Levels of tTG protein were 3-4-fold higher in liver homogenates of HCV pa- tients than in controls. Distribution of tTG in the liver varied according to the stage of disease. In mild, nonfibrotic chronic hepatitis C tTG expression was mainly induced in the hepatocytes in contact with the periportal infiltrate. In patients at a more advanced stage of fibrosis (F2-F3), tTG was also detected in extracellular matrix and in the fibrotic tissue. At the latest stages (F4), tTG was confined to the fibrotic tissue. A statistically significant relationship existed between the amount of tTG expressed, the grade of periportal necrosis (p<0.03) and the stage of fibrosis (p<0.005). Conclusions:These data suggest that tTG is directly involved in fibrogenesis due to HCV infection, and point toward its possible role as a non-invasive marker to follow the course of chronic hepatitis C.

1246

ANTIVIRAL RESPONSE AND TH-1 AND TH-2 CYTOKINE PROFILE RE- SPONSE BY PERIPHERAL BLOOD MONONUCLEAR CELLS OF PA- TIENTS WITH DIFFERENT CLINICAL OUTCOME OF HCV-INFECTION. Pietro Andreone, Annagiulia Gramenzi, Carmela Cursaro, Simona Talarico, Marzia Margotti, Elisabetta Loggi, Simona Romano, Stefania Lorenzini, Fed- erica Porzio, Maurizio Biselli, Francesco Felline, Mauro Bernardi, Universita' di Bologna, Bologna Italy

Background: the balance between Thl- and Th2-type cytokines has been suggested to play a role in the outcome of HCV infection. Aim: to evaluate the synthesis of Thl- and Th2- cytokines and of 2,5-oligoadenylate synthetase (2,5-OAS) an interferonqnduced antiviral protein, from peripheral blood mononuclear cells (PB~ICs) of patients with different patterns of HCV infection. Methods: Three groups of 10 HCV-Ab positive patients each were studied: group A) "previous" HCV infection (negative HCV-RNA and normal ALT), group B) "asymptomatic" HCV chronic infection (positive HCV-RNA and persistently normal ALT) and group C) "symptomatic" HCV chronic infection (positive HCV-RNA and abnormal ALT). Ten healthy subjects were also studied as controls. For each patient, 106 PBMCs were isolated and cultured in RPMI both in absence and in presence of Concanava- lin A. Thl-cytokines (IL-2 and IFN-'y), Th2-cytokines (1L-4 and 1L-10) and 2,5-OAS were directly assayed in the supernatants by commercial kits. Results: are reported in the table: A significant direct correlation was found between IL-2 and 2 5-OAS synthesis considering either all 40 subjects together (r=0.4; p=0.012) or excluding controls (r=0.4; p=0.03). Considering either group C only or group B and C together, a significant negative corre- lation between IL-2 and ALT serum levels was found (r=-0.78, p<0.001 and r=-0.078, p <0.001, respectively). On the contrary a significant direct correlation was found between IL-4 synthesis and ALT serum levels considering either patients of group A, 13 and C together (r=0.4, p=0.039) or patients of groups B and C only (r=0.74; p<0 001) Con- clusion: The results of this study seem to indicate an imbalance toward Th2-cytokine profile in pts with "symptomatic" HCV-chronie infection. On the contrary, a strong antiviral response and a prevalent Thl-cytokine profile seem to characterize "asymptomatic" HCV infection. These data indicate that Th-lymphocyte polarization may play an important pathophysiologic role in influencing the outcome of HCV infection.

GROUP A GROUP B GROUP C CONTROLS 2-50AS* 86 222 '~07 40

(0-308) a (39-558) ~,b (0-378) (21-139)b 11.-2"* 89 824 190 87

(38-158) c (241-1317)~,~.~ (0445)d (25.139)~ IFN-y** 2t92 2796 2607 2878

(601-4896) ( 676 -8266 ) (778-6113) (1567-6738) IL-4** 0 0 72 0

(0-297) t (0-0) tg (0-202)g." (0-83)" IL-IO** 791 602 1113 2666

(395-1484) ~ (141-1884)L" (513-2295) ' (439-4It8)~. r" Data are expressed as median (minimum-maximum) and analyzed by Mann Whitney U test; *prnol/mtxlO~ cells; "pg/mlxtO ~ cells; ~p=O.04; b'"'mp=O.004; c"P=O,O002, dp=O.O012; rp=O.03; gp=O.006; 'p=0,013, ~p=0,0t9.