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628 AUTOANTIBODIES AGAINST CYTOCHROME P4502E1 (CYP2E1) CONFORMATIONAL ANTIGENS IN PATIENTS WITH CHRONIC HEPATITIS C
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628 AUTOANTIBODIES AGAINST CYTOCHROME P4502E1 (CYP2E1) CONFORMATIONAL ANTIGENS IN PATIENTS WITH CHRONIC HEPATITIS C S. Sutti1 , M. Vidali1 , G. Occhino1 , A. Ivaldi1 , E. Potettu1 , R. Serino1 , S. Moia1 , C. Rigamonti2 , M. Sartori3 , E. Albano1 . 1 Department of Medical Sciences, University of East Piedmont Novara, Novara, 2 Division of Gastroenterology and Liver Transplant Unit 1, IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli E Regina Elena, Milan, 3 Gastroenterology Unit, Ospedale Maggiore e Della Carita’, Novara, Italy E-mail: [email protected] Background: Both non-organ and organ-specific auto-antibodies are a common feature of HCV infection. We have recently shown that about 40% of the patients with Chronic Hepatitis C (CHC) have circulating auto-antibodies against ethanol-inducible cytochrome P4502E1 (CYP2E1) (Vidali et al. J Hepatol 2007;46:605–612). Here we now report on the possible clinical significance of these auto-antibodies in relation to their epitope specificity. Methods: Recombinant human CYP2E1 variants generated by sitedirected mutagenesis were used to investigate the antigenic specificity of anti-CYP2E1 IgG in 59 CHC patients with titres of these auto-antibodies in ELISA above the 97th percentile of the control population. Results: Preliminary experiments using ELISA and Western blotting assays revealed that 28 (47%) of the sera investigated specifically recognized only conformational antigens while the remaining had IgG directed towards linear epitopes. Using a molecular model of CYP2E1 structure potential motifs for amino acid exchanges were selected by computer simulation on the surface of the CYP2E1 molecule. Alanine substitution at Glu320, Lys342, Arg344 and Lys420 reduced by more than 50% the reactivity of the CHC sera containing the conformational anti-CYP2E1 IgG, indicating their specificity toward at least one conformational epitope localized between J’ and K” helices. The clinical and biochemical features of the CHC patients with conformational anti-CYP2E1 IgG were not different from those of patients with antibodies against the linear epitopes. However, at the histology the subjects with the conformational antiCYP2E1 IgG had scores for the necro-inflammatory grading and the fibrosis staging significantly higher (p = 0.009 and p = 0.03, respectively) than those with the linear antibodies. Moreover, the presence of conformational antibodies was associated with 7.2 (95 CI 1.2−44.7; p = 0.03) higher risk of diffuse piecemeal necrosis and a five fold (OR = 4.8; 95 CI 1.39−16.6; p = 0.02) increased risk of severe fibrosis (staging 3 according to Ishak’s score system). Conclusions: The development of auto-reactivity towards conformational CYP2E1 epitopes is associated with a more severe liver damage in patients with CHC. The presence of these auto-antibodies might be used as a predictive marker of patients at risk for the progression of disease. 629 IFN-LAMBDA CONTRIBUTES TO DENDRITIC CELLMEDIATED EXPANSION OF REGULATORY T CELLS DURING HCV INFECTION G. Szabo, A. Dolganiuc. Medicine, UMass Medical School, Worcester, MA, USA E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) infection is associated with suppression of HCV-specific immunity, but the mechanisms involved are incompletely understood. An effective immune response involves coordination between dendritic cells and diverse T cell populations. Type III IFN, IFN-lambda, inhibits HCV replication and triggers interferon-stimulated genes. Here we hypothesized that IFN-lambda exerts immunomodulatory effects in HCV infection. Methods: Treatment-naive chronic HCV-infected patients (all genotypes, n = 22) and normal controls (n = 18) were included. Monocyte-derived
myeloid dendritic cells (DC) were generated with IL-4+GM-CSF in the absence or presence of IFN-lambda (IFN-lambda-DC). IFN-lambda RNA was assessed and DCs were tested in a mixed lymphocyte reaction with normal CD4+ T cells. T cells from MLR were analyzed for expression of CD25, Foxp3 and cytokine production. Results: We found that IFN-lambda modulates myeloid dendritic cells (mDC) towards an inhibitory phenotype. Myeloid DCs of HCV patients (HCV-DC) produced higher levels of IFN-lambda RNA at baseline and upon stimulation of Toll-like receptors 3, 4 and 7/8. Further, they expressed elevated levels of IFN-lambda-specific receptors compared to controls. HCV-DCs were poor T cell activators, however their capacity was corrected with supplementation of IL-2 or neutralization of IL-10. Similar to HCVDC, IFNlambda-DC inhibited T cell activation and stimulated proliferation of CD4+CD25+ T cells. The CD4+CD25+ T cells expanded upon contact with IFN-lambda-DC were FoxP3+, produced immunoinhibitory cytokines and inhibited activation of T cells. We observed a higher frequency of CD4+CD25+ T cells in peripheral blood of HCV patients, however, their per cell expression of FoxP3 and their inhibitory capacity were comparable with those observed in CD4+CD25+ T cells in peripheral blood of controls. There was an increase in expression of PD1/PD-L1 co-stimulatory molecules during co-culture of T cell with both HCV-DCs and IFNl-DC compared to controls. Finally, IFN-lambda-DC stimulated the proliferation of existing rather than de novo generation of CD4+CD25+ T cells. Conclusion: These results indicate a role for IFN-lambda in modulation of dendritic cells-T cells interactions and suggest potential therapeutic targets in chronic HCV infection.
630 SERUM BLYS/BAFF LEVELS IN ACUTE HEPATITIS C PREDICT CLINICAL OUTCOME G. Tarantino1 , V. Di Marco1 , P.L. Almasio1 , F. Barbaria1 , A. Ciaccio1 , F. D’Antoni2 , R. Di Stefano2 , A. Iac`o1 , A. Licata1 , S. Petta1 , A. Crax`ı1 . 1 Cattedra ed Unit` a Operativa di Gastroenterologia ed Epatologia, 2 Dipartimento di Igiene e Microbiologia, Sezione di Virologia, University of Palermo, Palermo, Italy E-mail: [email protected] Background: B-Lymphocyte stimulator (BLyS), also known as B activating factor (BAFF), is a TNF-a family cytokine playing a key role in the generation and maintenance of the mature B cells pool. Its expression by macrophages is stimulated by interferon and IL-10. BLyS/BAFF levels are elevated in patients with chronic hepatitis C, thus suggesting its role in chronicity of infection. Acute hepatitis C evolves into chronic infection in 50−80% of cases, but no reliable predictor of HCV clearance is available. Aim: To compare serum levels of BLyS/BAFF in the acute and chronic phases of HCV infection and to correlate baseline values with the disease outcome. Patients and Methods: We evaluated 29 patients (14 males, mean age 58.7±14.6 years) with acute hepatitis C (HCV G1: 55%, G2: 31%, G3: 11%) based upon HCV seroconversion in comparison to 34 patients with chronic hepatitis C (HCV G1: 82.3%, G2: 5.9%, G3: 5.9%, G4: 5.9%) and to 25 age and sex matched healthy blood donors. All patients were HIV negative, and most acute infections were linked to invasive medical exposures. All subjects with acute hepatitis still HCV-RNA positive at 12 weeks were offered IFN therapy and 5 were treated. BLYs levels were assessed by solid phase ELISA (Quantikine, R&D Systems, MN) on baseline samples collected at first observation. Results: Mean BLYs levels were significantly higher in acute (1,966.3±146.2 pg/ml) as compared to chronic hepatitis C patients (1,213.9±560.7 pg/ml) and to healthy subjects (919.7±171.5 pg/ml) (p < 0.001 by ANOVA). BLYs levels were significantly higher in patients with acute infection who evolved into HCV persistence (2,324.5±1,242.1 pg/ml) than in those who had a spontaneous or IFNinduced viral clearance (1,205.2±64.0 pg/ml) (p 0.01 by t Student). The
POSTERS
628 AUTOANTIBODIES AGAINST CYTOCHROME P4502E1 (CYP2E1) CONFORMATIONAL ANTIGENS IN PATIENTS WITH CHRONIC HEPATITIS C S. Sutti1 , M. Vidali1 , G. Occhino1 , A. Ivaldi1 , E. Potettu1 , R. Serino1 , S. Moia1 , C. Rigamonti2 , M. Sartori3 , E. Albano1 . 1 Department of Medical Sciences, University of East Piedmont Novara, Novara, 2 Division of Gastroenterology and Liver Transplant Unit 1, IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli E Regina Elena, Milan, 3 Gastroenterology Unit, Ospedale Maggiore e Della Carita’, Novara, Italy E-mail: [email protected] Background: Both non-organ and organ-specific auto-antibodies are a common feature of HCV infection. We have recently shown that about 40% of the patients with Chronic Hepatitis C (CHC) have circulating auto-antibodies against ethanol-inducible cytochrome P4502E1 (CYP2E1) (Vidali et al. J Hepatol 2007;46:605–612). Here we now report on the possible clinical significance of these auto-antibodies in relation to their epitope specificity. Methods: Recombinant human CYP2E1 variants generated by sitedirected mutagenesis were used to investigate the antigenic specificity of anti-CYP2E1 IgG in 59 CHC patients with titres of these auto-antibodies in ELISA above the 97th percentile of the control population. Results: Preliminary experiments using ELISA and Western blotting assays revealed that 28 (47%) of the sera investigated specifically recognized only conformational antigens while the remaining had IgG directed towards linear epitopes. Using a molecular model of CYP2E1 structure potential motifs for amino acid exchanges were selected by computer simulation on the surface of the CYP2E1 molecule. Alanine substitution at Glu320, Lys342, Arg344 and Lys420 reduced by more than 50% the reactivity of the CHC sera containing the conformational anti-CYP2E1 IgG, indicating their specificity toward at least one conformational epitope localized between J’ and K” helices. The clinical and biochemical features of the CHC patients with conformational anti-CYP2E1 IgG were not different from those of patients with antibodies against the linear epitopes. However, at the histology the subjects with the conformational antiCYP2E1 IgG had scores for the necro-inflammatory grading and the fibrosis staging significantly higher (p = 0.009 and p = 0.03, respectively) than those with the linear antibodies. Moreover, the presence of conformational antibodies was associated with 7.2 (95 CI 1.2−44.7; p = 0.03) higher risk of diffuse piecemeal necrosis and a five fold (OR = 4.8; 95 CI 1.39−16.6; p = 0.02) increased risk of severe fibrosis (staging 3 according to Ishak’s score system). Conclusions: The development of auto-reactivity towards conformational CYP2E1 epitopes is associated with a more severe liver damage in patients with CHC. The presence of these auto-antibodies might be used as a predictive marker of patients at risk for the progression of disease. 629 IFN-LAMBDA CONTRIBUTES TO DENDRITIC CELLMEDIATED EXPANSION OF REGULATORY T CELLS DURING HCV INFECTION G. Szabo, A. Dolganiuc. Medicine, UMass Medical School, Worcester, MA, USA E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) infection is associated with suppression of HCV-specific immunity, but the mechanisms involved are incompletely understood. An effective immune response involves coordination between dendritic cells and diverse T cell populations. Type III IFN, IFN-lambda, inhibits HCV replication and triggers interferon-stimulated genes. Here we hypothesized that IFN-lambda exerts immunomodulatory effects in HCV infection. Methods: Treatment-naive chronic HCV-infected patients (all genotypes, n = 22) and normal controls (n = 18) were included. Monocyte-derived
myeloid dendritic cells (DC) were generated with IL-4+GM-CSF in the absence or presence of IFN-lambda (IFN-lambda-DC). IFN-lambda RNA was assessed and DCs were tested in a mixed lymphocyte reaction with normal CD4+ T cells. T cells from MLR were analyzed for expression of CD25, Foxp3 and cytokine production. Results: We found that IFN-lambda modulates myeloid dendritic cells (mDC) towards an inhibitory phenotype. Myeloid DCs of HCV patients (HCV-DC) produced higher levels of IFN-lambda RNA at baseline and upon stimulation of Toll-like receptors 3, 4 and 7/8. Further, they expressed elevated levels of IFN-lambda-specific receptors compared to controls. HCV-DCs were poor T cell activators, however their capacity was corrected with supplementation of IL-2 or neutralization of IL-10. Similar to HCVDC, IFNlambda-DC inhibited T cell activation and stimulated proliferation of CD4+CD25+ T cells. The CD4+CD25+ T cells expanded upon contact with IFN-lambda-DC were FoxP3+, produced immunoinhibitory cytokines and inhibited activation of T cells. We observed a higher frequency of CD4+CD25+ T cells in peripheral blood of HCV patients, however, their per cell expression of FoxP3 and their inhibitory capacity were comparable with those observed in CD4+CD25+ T cells in peripheral blood of controls. There was an increase in expression of PD1/PD-L1 co-stimulatory molecules during co-culture of T cell with both HCV-DCs and IFNl-DC compared to controls. Finally, IFN-lambda-DC stimulated the proliferation of existing rather than de novo generation of CD4+CD25+ T cells. Conclusion: These results indicate a role for IFN-lambda in modulation of dendritic cells-T cells interactions and suggest potential therapeutic targets in chronic HCV infection.
630 SERUM BLYS/BAFF LEVELS IN ACUTE HEPATITIS C PREDICT CLINICAL OUTCOME G. Tarantino1 , V. Di Marco1 , P.L. Almasio1 , F. Barbaria1 , A. Ciaccio1 , F. D’Antoni2 , R. Di Stefano2 , A. Iac`o1 , A. Licata1 , S. Petta1 , A. Crax`ı1 . 1 Cattedra ed Unit` a Operativa di Gastroenterologia ed Epatologia, 2 Dipartimento di Igiene e Microbiologia, Sezione di Virologia, University of Palermo, Palermo, Italy E-mail: [email protected] Background: B-Lymphocyte stimulator (BLyS), also known as B activating factor (BAFF), is a TNF-a family cytokine playing a key role in the generation and maintenance of the mature B cells pool. Its expression by macrophages is stimulated by interferon and IL-10. BLyS/BAFF levels are elevated in patients with chronic hepatitis C, thus suggesting its role in chronicity of infection. Acute hepatitis C evolves into chronic infection in 50−80% of cases, but no reliable predictor of HCV clearance is available. Aim: To compare serum levels of BLyS/BAFF in the acute and chronic phases of HCV infection and to correlate baseline values with the disease outcome. Patients and Methods: We evaluated 29 patients (14 males, mean age 58.7±14.6 years) with acute hepatitis C (HCV G1: 55%, G2: 31%, G3: 11%) based upon HCV seroconversion in comparison to 34 patients with chronic hepatitis C (HCV G1: 82.3%, G2: 5.9%, G3: 5.9%, G4: 5.9%) and to 25 age and sex matched healthy blood donors. All patients were HIV negative, and most acute infections were linked to invasive medical exposures. All subjects with acute hepatitis still HCV-RNA positive at 12 weeks were offered IFN therapy and 5 were treated. BLYs levels were assessed by solid phase ELISA (Quantikine, R&D Systems, MN) on baseline samples collected at first observation. Results: Mean BLYs levels were significantly higher in acute (1,966.3±146.2 pg/ml) as compared to chronic hepatitis C patients (1,213.9±560.7 pg/ml) and to healthy subjects (919.7±171.5 pg/ml) (p < 0.001 by ANOVA). BLYs levels were significantly higher in patients with acute infection who evolved into HCV persistence (2,324.5±1,242.1 pg/ml) than in those who had a spontaneous or IFNinduced viral clearance (1,205.2±64.0 pg/ml) (p 0.01 by t Student). The