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Evolution of HCV-specific T cell responses and in vivo and in vitro TNFα and interferon-γ production in chronic HCV patients treated with high doses of interferon-α
A1026
AASLD ABSTRACTS
• EVOLUTION OF HCV-SPECIFIC T CELL RESPONSES AND IN VIVO AND IN VITRO TNFc( AND INTERFERON-7 PRODUCTION IN CHRONIC HCV PATIENTS TREATED WITH HIGH DOSES OF INTERFERON-c<. C, Alvarado Esquivel 1, J. Philipp4~1, A. Elewaut 1, J. Paradijs1, A.E, Elewaut 1, R. Deleys2, L. Stuyver 2, G. Maertens2, G. Leroux-Roels 1. 1University Hospital, Gent, Belgium and 21nnogenetics NV, Gent, Belgium. Background. We have studied the HCV-specific lymphoproliferation and in vivo and in vitro cytokine production in chronic HCV patients treated with high ~loses of IFN-(~in an attempt to understand the mechanism(s) of action of this drug. Methods. Ten patients with chronic HCV infection, unsuccessfully treated with standard doses of IFN-~, were retreated with IFN-c( after a therapy-free interval of at least 6 months. IFN-c~was given at 10 MU per day for 5 days, followed by 10 MU thrice weekly for 12 weeks, 6 MU thrice weekly for 4 weeks and3 to 6 MU thrice weekly for another 8 weeks. The proliferative response of PBMC was assessed at monthly intervals using 9 pools of partially overlapping peptides representing core, El, E2 of HCVla and recombinant NS3 protein of HCVlb. Tetanus toxoid (TT) was used as a positive control antigen. In vivo serum levels and in vitro induced TNFc~and IFN# were measured on weeks 0, 4, 8 and 12 of IFN-c~therapy. All assays were performed in healthy subjects for comparisod. Results. PBMC from the HCV patients most frequently recognized the COOHterminus of Core (pool 3). The peptide recognition patterns varied from subject to subject, but remained constant over time within one individual. The response of healthy subjects and HCV patients to TT was comparable throughout the study period. This was not the case for several HCV antigens. Before the initiation of the therapy, pool 3 was better recognized by HCV patients and this became more accentuated during therapy. Once therapy was stopped this difference waned. The recognition of HCV antigens was not influenced by the clinical response to IFN-c~. The serum levels of TNFc~ were higher in HCV patients than in healthy control subjects but decreased during the observation period. A significant decrease of IFN-y serum levels was observed from week 4 on and was even more pronounced at weeks 8-t2. The in vitroTNFc~ and IFN-7 production dropped markedly after 4 weeks of therapy, This decrease was most prominent in clinical responders to IFN-c~. Conclusion. In chronic HCV patients treated with high doses of IFN-~ the COOH-terminal part of Core is the most immunogenic region at the T cell level. Significant changes in lymphoproliferative responses and in vivo and in vitro cytokine production occur between week 4 and 12 of therapy. The reduction of TNFo~and IFN-7 production in vivo was most prominent in clinical responders to IFN-~. This phenomenon requires further elucidation.
• THE SPECTRUM OF LIVER DISEASE IN ALEXANDRIA, EGYPT, A NYPERENDEMIC AREA FOR SCHISTOSOMIASIS AND HCV INFECTION. M.An~elico, M.C.Profili, C.Gandin, E.Renganathan, M.Fathy, M.Rapicetta, F.Callea, W.Refai, E.Spada, K.Cristiano, L.Capocaccia, G.Badr and G.Rocchi. Med Res Inst. Alexandria Univ,Egypt, Dpt Publ Elth 2nd Rome Univ, Dpt Pathol Brescia, 2nd GI Unit ist Rome Univ, and Ist Sup San, Rome, Italy. The prevalence of anti-HCV antibodies among Egyptians is 10-30-fold greater than in Western countries, yet the potential pathogenicity of this finding is unclear, as chronic liver disease in Egypt is usually attributed to Schistosoma mansoni infection. We studied the etiology and the clinico-pathological features of chronic liver diseases in 118 consecutive patients (82 M, 36 F) admitted to a major hospital in Alexandria. Patients underwent clinical, biochemical, parasitological and sonographic evaluations, and HCV (Elisa III) and RBV serology studies. RCV-RNA was assayed by nested PCR and positive sera were evaluated for NCV genotyping. All patients had liver biopsy, unless contraindicated. Results: Serological evidence of schistosomiasis (positive anti-SEA antibodies) was found in 94 patients (79.7%), but eggs in stools (active disease) in 29 only (24.6%). Anti-HOW antibodies were found in 73 patients (61.9%), 15 of whom had eggs in stools. Eighteen patients (15.2%) were HBsAg positive. Anti-HCV positive and negative patients did not differ with respect no gender, demographics, history of surgery/transfusions and previous schistosomiasis, but were older (47~9 vs 37~14 yrs, p=O.008) and had more frequently received parenteral therapy for Schistosoma (54.8% vs 33.3, p<0.02). HCV-RNA was detected in 39 (54.9%) anti-HCV positive patients. All but five HCV isolates were classified as genotypes 4a (Simmonds). Viremic patients had higher ALT (p
GASTROENTEROLOGY, VOl. 108, NO. 4
• PROTEIN KINASE A IS INVOLVED IN THE GLUCAGON STIMULATION OF CI'/HC03" EXCHANGER IN ISOLATED RAT HEPATOCYTE COUPLETS (IRHC). D. ALVARO, A.BINI, S. FURFARO, T. LAROSA, C. FRANCIA, L. CAPOCACCIA. I I Dpt. G a s t r o . , Univers. o f Rome, Rome, ITALY. We have previously shown that glucagon stimulates, through cAMP, the activity of the CI'/HC03" exchanger in IRHC (Gastro. I06:A858;1994), a mechanism which could account for the bicarbonate rich choleresis induced by this hormone. This study investigates the role of protein kinase A and protein kinese C on the glucagon stimulation of the CI-/HCO~" exchanger. Intracellular pH (PHi) was measured in IRHC by BCECF-AM (microfluorimetric method). The activity of the CI'/HC03" exchanger was evaluated by measuring p ~ changes induced by acute CI" removal/readmission (J Clin Invest 92:1314;1993). After acute CI" removal (n= 1O), pH i rose to 0.19+0.07 pHU at a maximal rate of 0.067±0.034 pHU/min (H+ flux rates= 3.68fi.85 mM/min) a n d recovered after CI- readmission (0.091+0.043 pHU/min; H ÷ flux rates= 6.36±3.34 mM/min). Glucagon (200 nM) increased (p< 0.02) the net pH i change after CI" removal (0.26±0.05 pHU), the rate of alkalinizatien (0.140±0.045 pHU/min, H÷ flux rates= 7.49 ± 2.44) end the rate of pH~ recovery after CI" readmission (0.151±0.046 pHU/min, H ÷ flux rates= i0.83f3.39), indicating stimulation of CI'/HC03" exchange. The effect of glecagon was associated with a 7-fold increase in cAMP (RIA) i n t r a c e n u l a r levels in isolated hepatocytes. Rp-eAMPS (I0 pM, n= 9), a competitive inhibitor of protein kinase A, did not change the basal activity of the CI-/HC03" exchanger, but did inhibit the stimulatery effect of glucagen by 80% (p< 0.02). Furthermore, glucagen stimulation of CI-/HC03" exchanger was completely inhibited by H-89 (30 pM, n= 9), another protein kinase A inhibitor. H-89 did net change basal and glucagon-stimulated cAMP intracellular levels. The protein kinase C agonist, PMA (10 p M ) , also blocked the glucacen stimulation of CI'/HC03" exchanger (n=13), but this occurred through an inhibition (P< 0.01) of glucagen-induced cAMP accumulation. Conclusions: glucag0n stimulation of CI'/HC03 exchange is blocked by two different protein kinase A inhibiters, indicating a direct involvement of protein kinase A in glucagen-stimulation of bicarbonate excretion. Protein kinase C agonist,PMA, blocks the cAMP production induced by glueagen.
SYNTHESIS OF FIBRONECTIN BY MONONUCLEAR PHAGOCYTES OF NORMAL AND ACUTELY DAMAGED RAT LIVER. Th. Armbrust, G. Ramadori. Dept. of Int. Medicine, Georg-August-University, G6ttingen, FRG Fibronectin (FN) is supposed to be an important glycoprotein involved in pathogenesis of hepatic fibrosis. It is synthesized by hepatocytes, "activated" fat storing cells, and sinusoidal endothelial cells. After acute liver injury, FN gene expression is strongly increased in the areas of damage. It is not known which cells are responsible for the increased FN synthesis. As the number of mononuclear phagocytes (MP) and Kupffer cells (KC) is increased in acutely damaged liver, the aim of this work was to study the contribution of MP of the acutely damaged liver to hepatic FN synthesis. Methods: KC and MP of acutely injured rat liver (single CCI4 gavage) were isolated, characterized by the moabs ED1 and ED2, and separated according to size into small, ED1 + ED2" (unmature) and large, ED1 + ED2 ~ cells (mature macrophages). For comparison, KC (ED1 ÷ ED2 and ED1 + ED2 + ) were isolated from normal rat liver. Protein synthesis was studied by biosynthetic labeling with 35-S-methionine, immunoprecipitation, and SDSPAGE. RNA was extracted and analyzed by Northern Blotting. Results: KC of normal rat liver synthesized FN in hardly detectable amounts: Small KC synthesized trace amounts, while FN was hardly detected in large KC. In contrast, MP synthesized and secreted FN in abundant amounts. Synthesis was higher in cultures of small MP (unmature) than in large (mature macrophages). It was highest when cells were isolated shortly after the injury (36h) and decreased at later stages (60h). In all cultures of the different cell populations, FN synthesis decreased by rising time of culture. Conclusions: The results indicate that MP could be an important source of FN in early stages of liver injury. FN synthesis could be a marker of the differentiation process of unmature MP into KC.
AASLD ABSTRACTS
• EVOLUTION OF HCV-SPECIFIC T CELL RESPONSES AND IN VIVO AND IN VITRO TNFc( AND INTERFERON-7 PRODUCTION IN CHRONIC HCV PATIENTS TREATED WITH HIGH DOSES OF INTERFERON-c<. C, Alvarado Esquivel 1, J. Philipp4~1, A. Elewaut 1, J. Paradijs1, A.E, Elewaut 1, R. Deleys2, L. Stuyver 2, G. Maertens2, G. Leroux-Roels 1. 1University Hospital, Gent, Belgium and 21nnogenetics NV, Gent, Belgium. Background. We have studied the HCV-specific lymphoproliferation and in vivo and in vitro cytokine production in chronic HCV patients treated with high ~loses of IFN-(~in an attempt to understand the mechanism(s) of action of this drug. Methods. Ten patients with chronic HCV infection, unsuccessfully treated with standard doses of IFN-~, were retreated with IFN-c( after a therapy-free interval of at least 6 months. IFN-c~was given at 10 MU per day for 5 days, followed by 10 MU thrice weekly for 12 weeks, 6 MU thrice weekly for 4 weeks and3 to 6 MU thrice weekly for another 8 weeks. The proliferative response of PBMC was assessed at monthly intervals using 9 pools of partially overlapping peptides representing core, El, E2 of HCVla and recombinant NS3 protein of HCVlb. Tetanus toxoid (TT) was used as a positive control antigen. In vivo serum levels and in vitro induced TNFc~and IFN# were measured on weeks 0, 4, 8 and 12 of IFN-c~therapy. All assays were performed in healthy subjects for comparisod. Results. PBMC from the HCV patients most frequently recognized the COOHterminus of Core (pool 3). The peptide recognition patterns varied from subject to subject, but remained constant over time within one individual. The response of healthy subjects and HCV patients to TT was comparable throughout the study period. This was not the case for several HCV antigens. Before the initiation of the therapy, pool 3 was better recognized by HCV patients and this became more accentuated during therapy. Once therapy was stopped this difference waned. The recognition of HCV antigens was not influenced by the clinical response to IFN-c~. The serum levels of TNFc~ were higher in HCV patients than in healthy control subjects but decreased during the observation period. A significant decrease of IFN-y serum levels was observed from week 4 on and was even more pronounced at weeks 8-t2. The in vitroTNFc~ and IFN-7 production dropped markedly after 4 weeks of therapy, This decrease was most prominent in clinical responders to IFN-c~. Conclusion. In chronic HCV patients treated with high doses of IFN-~ the COOH-terminal part of Core is the most immunogenic region at the T cell level. Significant changes in lymphoproliferative responses and in vivo and in vitro cytokine production occur between week 4 and 12 of therapy. The reduction of TNFo~and IFN-7 production in vivo was most prominent in clinical responders to IFN-~. This phenomenon requires further elucidation.
• THE SPECTRUM OF LIVER DISEASE IN ALEXANDRIA, EGYPT, A NYPERENDEMIC AREA FOR SCHISTOSOMIASIS AND HCV INFECTION. M.An~elico, M.C.Profili, C.Gandin, E.Renganathan, M.Fathy, M.Rapicetta, F.Callea, W.Refai, E.Spada, K.Cristiano, L.Capocaccia, G.Badr and G.Rocchi. Med Res Inst. Alexandria Univ,Egypt, Dpt Publ Elth 2nd Rome Univ, Dpt Pathol Brescia, 2nd GI Unit ist Rome Univ, and Ist Sup San, Rome, Italy. The prevalence of anti-HCV antibodies among Egyptians is 10-30-fold greater than in Western countries, yet the potential pathogenicity of this finding is unclear, as chronic liver disease in Egypt is usually attributed to Schistosoma mansoni infection. We studied the etiology and the clinico-pathological features of chronic liver diseases in 118 consecutive patients (82 M, 36 F) admitted to a major hospital in Alexandria. Patients underwent clinical, biochemical, parasitological and sonographic evaluations, and HCV (Elisa III) and RBV serology studies. RCV-RNA was assayed by nested PCR and positive sera were evaluated for NCV genotyping. All patients had liver biopsy, unless contraindicated. Results: Serological evidence of schistosomiasis (positive anti-SEA antibodies) was found in 94 patients (79.7%), but eggs in stools (active disease) in 29 only (24.6%). Anti-HOW antibodies were found in 73 patients (61.9%), 15 of whom had eggs in stools. Eighteen patients (15.2%) were HBsAg positive. Anti-HCV positive and negative patients did not differ with respect no gender, demographics, history of surgery/transfusions and previous schistosomiasis, but were older (47~9 vs 37~14 yrs, p=O.008) and had more frequently received parenteral therapy for Schistosoma (54.8% vs 33.3, p<0.02). HCV-RNA was detected in 39 (54.9%) anti-HCV positive patients. All but five HCV isolates were classified as genotypes 4a (Simmonds). Viremic patients had higher ALT (p
GASTROENTEROLOGY, VOl. 108, NO. 4
• PROTEIN KINASE A IS INVOLVED IN THE GLUCAGON STIMULATION OF CI'/HC03" EXCHANGER IN ISOLATED RAT HEPATOCYTE COUPLETS (IRHC). D. ALVARO, A.BINI, S. FURFARO, T. LAROSA, C. FRANCIA, L. CAPOCACCIA. I I Dpt. G a s t r o . , Univers. o f Rome, Rome, ITALY. We have previously shown that glucagon stimulates, through cAMP, the activity of the CI'/HC03" exchanger in IRHC (Gastro. I06:A858;1994), a mechanism which could account for the bicarbonate rich choleresis induced by this hormone. This study investigates the role of protein kinase A and protein kinese C on the glucagon stimulation of the CI-/HCO~" exchanger. Intracellular pH (PHi) was measured in IRHC by BCECF-AM (microfluorimetric method). The activity of the CI'/HC03" exchanger was evaluated by measuring p ~ changes induced by acute CI" removal/readmission (J Clin Invest 92:1314;1993). After acute CI" removal (n= 1O), pH i rose to 0.19+0.07 pHU at a maximal rate of 0.067±0.034 pHU/min (H+ flux rates= 3.68fi.85 mM/min) a n d recovered after CI- readmission (0.091+0.043 pHU/min; H ÷ flux rates= 6.36±3.34 mM/min). Glucagon (200 nM) increased (p< 0.02) the net pH i change after CI" removal (0.26±0.05 pHU), the rate of alkalinizatien (0.140±0.045 pHU/min, H÷ flux rates= 7.49 ± 2.44) end the rate of pH~ recovery after CI" readmission (0.151±0.046 pHU/min, H ÷ flux rates= i0.83f3.39), indicating stimulation of CI'/HC03" exchange. The effect of glecagon was associated with a 7-fold increase in cAMP (RIA) i n t r a c e n u l a r levels in isolated hepatocytes. Rp-eAMPS (I0 pM, n= 9), a competitive inhibitor of protein kinase A, did not change the basal activity of the CI-/HC03" exchanger, but did inhibit the stimulatery effect of glucagen by 80% (p< 0.02). Furthermore, glucagen stimulation of CI-/HC03" exchanger was completely inhibited by H-89 (30 pM, n= 9), another protein kinase A inhibitor. H-89 did net change basal and glucagon-stimulated cAMP intracellular levels. The protein kinase C agonist, PMA (10 p M ) , also blocked the glucacen stimulation of CI'/HC03" exchanger (n=13), but this occurred through an inhibition (P< 0.01) of glucagen-induced cAMP accumulation. Conclusions: glucag0n stimulation of CI'/HC03 exchange is blocked by two different protein kinase A inhibiters, indicating a direct involvement of protein kinase A in glucagen-stimulation of bicarbonate excretion. Protein kinase C agonist,PMA, blocks the cAMP production induced by glueagen.
SYNTHESIS OF FIBRONECTIN BY MONONUCLEAR PHAGOCYTES OF NORMAL AND ACUTELY DAMAGED RAT LIVER. Th. Armbrust, G. Ramadori. Dept. of Int. Medicine, Georg-August-University, G6ttingen, FRG Fibronectin (FN) is supposed to be an important glycoprotein involved in pathogenesis of hepatic fibrosis. It is synthesized by hepatocytes, "activated" fat storing cells, and sinusoidal endothelial cells. After acute liver injury, FN gene expression is strongly increased in the areas of damage. It is not known which cells are responsible for the increased FN synthesis. As the number of mononuclear phagocytes (MP) and Kupffer cells (KC) is increased in acutely damaged liver, the aim of this work was to study the contribution of MP of the acutely damaged liver to hepatic FN synthesis. Methods: KC and MP of acutely injured rat liver (single CCI4 gavage) were isolated, characterized by the moabs ED1 and ED2, and separated according to size into small, ED1 + ED2" (unmature) and large, ED1 + ED2 ~ cells (mature macrophages). For comparison, KC (ED1 ÷ ED2 and ED1 + ED2 + ) were isolated from normal rat liver. Protein synthesis was studied by biosynthetic labeling with 35-S-methionine, immunoprecipitation, and SDSPAGE. RNA was extracted and analyzed by Northern Blotting. Results: KC of normal rat liver synthesized FN in hardly detectable amounts: Small KC synthesized trace amounts, while FN was hardly detected in large KC. In contrast, MP synthesized and secreted FN in abundant amounts. Synthesis was higher in cultures of small MP (unmature) than in large (mature macrophages). It was highest when cells were isolated shortly after the injury (36h) and decreased at later stages (60h). In all cultures of the different cell populations, FN synthesis decreased by rising time of culture. Conclusions: The results indicate that MP could be an important source of FN in early stages of liver injury. FN synthesis could be a marker of the differentiation process of unmature MP into KC.