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Association of multispecific CD4+ response to hepatitis C and severity of recurrence after liver transplantation
April 1998
• L0534 REGULATED RELEASE OF ATP FROM HUMAN HEPATOCYTES PLAYS A KEY ROLE IN CELL VOLUME HOMEOSTASIS. R.M.Roman, A.P.Feranchak, D.K.Guenette, and J.G.Fitz. University of Colorado Health Sciences Center, Denver, CO. Regulated increases in membrane CI- permeability are essential for cell volume homeostasis. Previous investigations in liver cell lines indicate that activation of C1- channels following increases in cell volume is dependent upon cellular release of ATP and autocrine stimulation of membrane P2 receptors. The purpose of these studies was to establish whether similar pathways are operative in primary human hepatocytes. Methods. Studies were performed in human hepatocytes isolated using collagenase perfusion and percoll gradient centrifugation. C1- currents (whole-cell patch clamp techniques), release of ATP from cell monolayers (luciferase-luciferin assay), and cell volume (Coulter Multisizer) were measured within 12 hr of isolation. Results. 1. Compared to basal values in isotonic buffer (116_+22 pA), C1- current amplitude increased significantly following cell swelling during hypotonic exposure (20% less NaC1, 707_+ 125 pA), and purinergic stimulation by extracellular ATP (1,007_+253 pA). Currents were characterized by outward rectification, reversal near 0 mV, and inhibition by the anion channel blocker NPPB. 2. Induction of cell swelling increased cellular ATP release from basal values of 7.8 ± 0.54 ALU (arbitrary light units), to 38.6 -+ 12.8 ALU at 25%, 55.9 ± 13.1 ALU at 33%, and 70.3 -+ 15,21 ALU at 41% decreases in buffer osmolarity, Bioluminescence was eliminated by the ATPDase apyrase (2U/ml). 3. During exposure to hypotonic buffer (40% less NaC1), an increase in mean relative volume to 1.08 ± 0.01 at 2.5 min was followed by gradual volume recovery to 1.02 _+0.01 by 30 min. Volume recovery was abolished by anion channel blockade with NPPB (25uM) and removal of extracellular ATP with apyrase (1U/ml). Summary. In primary human hepatocytes: i) cell volume increases are associated with C1- channel activation and enhanced cellular ATP release; and ii) both channel-mediated CI- efflux and extracellular ATP are critical for cell volume recovery from swelling. Conclusion. Since changes in cell volume directly modulate many liver functions, and P2 receptors are broadly expressed among liver cell types, volume-sensitive release of ATP into the extracellular milieu may represent an important autocrine/paracrine signaling pathway coupling cell volume to membrane ion permeability. • L0535 ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODIES IN PRIMARY SCLEROSING CHOLANGITIS: TARGET ANTIGENS AND CLINICAL ASSOCIATIONS. C. Roozendaal, 1 A.W.M. Van Milligen de Wit, 3 E,B. Haagsma, 2 G. Horst, l C. Schwarze, 4 H.H. Peter,4 J.H. Kleibeuker, 2 J.W. Cohen Tervaert, 1 P.C. Limburg, 1 and C.G.M. Kallenberg. 1 1Dept. of Clinical Immunology and 2Dept. of Gastroenterology and Hepatology, University Hospital Groningen, The Netherlands; 3Dept. of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands; 4Dept. of Rheumatology and Clinical Immunology, University Hospital Freiburg, Germany. The antigenic specificities and clinical significance of anti-neutrophil cytoplasmic autoantibodies (ANCA) in primary sclerosing cholangitis (PSC) are unclear. Aim of this study was to investigate the antigenic specificities and clinical associations of ANCA in 69 patients with PSC. The presence of ANCA was analyzed by indirect immunofluorescence (IIF). Antibodies directed against proteinase 3, myeloperoxidase, elastase, bactericidal /permeability-increasing protein (BPI), cathepsin G, and lactoferrin were identified by ELISA. ANCA were detected by IIF in 67% of PSC patients. In antigen-specific ELISAs, 55% of the serum samples recognized one or more antigens. Fortysix percent contained antibodies to BPI, 23% to cathepsin G, and 22% to lactoferrin. Only 4% had antibodies to proteinase 3, antibodies to myeloperoxidase or elastase were not detected. Frequently, antibodies to different antigens were simultaneously present in a serum sample. The presence of ANCA was not related to the duration of PSC. Also, ANCA as detected by IIF were not associated with the presence of cirrhosis nor with the coexistence of inflammatory bowel disease. However, antibodies to BPI and cathepsin G were both associated with the presence of cirrhosis, and antibodies to lactoferrin were more frequently detected in patients with PSC in conjunction with ulcerative colitis. We conclude from this study that BPI is a major ANCA antigen in PSC. Antibodies to BPI and cathepsin G are related to cirrhosis, and antibodies to lactoferrin in PSC are associated with the concurrentpresence of ulcerative colitis.
AASLD A1329 • L0536 ASSOCIATION OF MULTISPECIFIC CD4+ RESPONSE TO HEPATITIS C AND SEVERITY OF RECURRENCE AFTER LIVER TRANSPLANTATION. HR Rosen, M Blake, M Houghton*, DJ Hinrichs, S Chou, DR Gretch**, HA Bouwer. Dept of Medicine, Molecular Microbiology, and Immunology. Portland VAMC/Oregon Health Sciences University, *Chiron Corp., Emeryville, CA and **Virology Division, University of Washington, Seattle, WA. Indirect evidence suggests that the cellular immunity to HCV is an important determinant of disease recurrence following liver transplantation (Transplantation 1997; 64: 721-26), but to date no study has attempted to define specific defects in the immune response. Aim: To determine if the multispecific, HLA class II-restricted proliferative response to HCV after OLT is associated with different clinical outcomes. Methods: Twenty-four HCV+ OLT recipients with identical initial immunosupprassion (CsA-based) were studied (range of histologic followup: 1 to 8.2 yrs). Group 1 (n = 18) was comprised of pts with no or mild evidence of histologic recurrence; group 2 consisted of 6 pts with severe recurrence (1 died, 2 underwent re-OLT for graft failure). Briefly, PBMC were isolated from freshly heparinized blood by Ficoll-Hypaque density centrifugation. 2 X l05 cells were cultured in 96-well plates and stimulated with 1 and 10 ~g/ml of highly purified recombinant HCV antigens (c33, el00, c200, and NS5 expressed as SOD fusion proteins); tetanus toxoid and CMV antigens (Towne and AD169 strains) were used as controls. Stimulation indexes (SI's) > 3 by 3H-thymidine uptake were considered significant. Results: Peripheral CD4+ T lymphocyte responses to at least one HCV antigen were detectable in 12 of 18 (67%) of group 1 vs. none of the patients in group 2 (p = .01) although all pts in the latter group responded to non-HCV control antigens. Among the 12 patients who responded to HCV Ag's, 11 did so to c33; 4 pts responded to 2 or more Ag's, and 1 pt responded to all 4 HCV Ag's. Conclusions: Following OLT for HCV, vigorous multispecific T cell responses are detectable only in patients with no or mild histological recurrence, and patients with severe HCV recurrence are hyporesponsive to HCV antigens despite responding to non-HCV antigens. Future therapeutic interventions may target specific, defective T cell responses (i.e., adoptive immunotherapy). Supported in part by MRF Grant #9735. • L0537 THE EPIDEMIOLOGY, TREATMENT PATTERNS AND RESOURCE UTILIZATION OF HEPATITIS B PATIENTS IN A LARGE INSURED NEW ENGLAND POPULATION. Daniel M. Rosenberg, Suzanne F. Cook, Susan C, Eaton, William B. Sannders, and Lee L. Lanza,. Glaxo Wellcome Inc., Research Triangle Park, NC; Epidemiology Resources Inc., Newton Lower Falls, MA. The Centers for Disease Control estimates that the incidence of acute HBV is approximately 1.15 per 100,000 in the U.S. and 2-10% of such infections progress to chronic HBV disease. The purpose of this cross-sectional study is to describe the epidemiology, treatment patterns and major health care resources associated with hepatitis B virus (HBV) in a Large Insured New England (LINE) population. The LINE database maintains claims for over 2 million persons; data on patients treated from January 1, 1995 to December 31, 1996 were analyzed. Demographics, type of provider, type of insurance coverage, utilization of outpatient and inpatient services, prescription medications, diagnostic, laboratory, surgical procedures and comorhidities with hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) were examined. Preliminary findings indicate that the prevalence of patients with an HBV diagnosis for whom services were rendered was 60/100,000, HBV/HCV coinfection was 2.8/100,000 and HBV/HIV coinfection was 0.89/100,000. Persons aged 0-9 years had the greatest HBV prevalence (male: 96/100,000 and female: 88/100,000). One percent of patients with only HBV had interferon-alpha therapy. Estimates in this population are subject to underestimation due to the non-inclusion of persons classified with nonspecific viral hepatitis codes, infected persons not presenting for services and institutional differences in diagnostic screening patterns. Further resource utilization and treatment pattern data will be detailed. The prevalence of patients with an HBV diagnosis for whom services were rendered in this LINE population is lower than expected but these data provide important information on treatment patterns and resource utilization. This research was funded by Glaxo WeUcome, Research Triangle Park, NC.
• L0534 REGULATED RELEASE OF ATP FROM HUMAN HEPATOCYTES PLAYS A KEY ROLE IN CELL VOLUME HOMEOSTASIS. R.M.Roman, A.P.Feranchak, D.K.Guenette, and J.G.Fitz. University of Colorado Health Sciences Center, Denver, CO. Regulated increases in membrane CI- permeability are essential for cell volume homeostasis. Previous investigations in liver cell lines indicate that activation of C1- channels following increases in cell volume is dependent upon cellular release of ATP and autocrine stimulation of membrane P2 receptors. The purpose of these studies was to establish whether similar pathways are operative in primary human hepatocytes. Methods. Studies were performed in human hepatocytes isolated using collagenase perfusion and percoll gradient centrifugation. C1- currents (whole-cell patch clamp techniques), release of ATP from cell monolayers (luciferase-luciferin assay), and cell volume (Coulter Multisizer) were measured within 12 hr of isolation. Results. 1. Compared to basal values in isotonic buffer (116_+22 pA), C1- current amplitude increased significantly following cell swelling during hypotonic exposure (20% less NaC1, 707_+ 125 pA), and purinergic stimulation by extracellular ATP (1,007_+253 pA). Currents were characterized by outward rectification, reversal near 0 mV, and inhibition by the anion channel blocker NPPB. 2. Induction of cell swelling increased cellular ATP release from basal values of 7.8 ± 0.54 ALU (arbitrary light units), to 38.6 -+ 12.8 ALU at 25%, 55.9 ± 13.1 ALU at 33%, and 70.3 -+ 15,21 ALU at 41% decreases in buffer osmolarity, Bioluminescence was eliminated by the ATPDase apyrase (2U/ml). 3. During exposure to hypotonic buffer (40% less NaC1), an increase in mean relative volume to 1.08 ± 0.01 at 2.5 min was followed by gradual volume recovery to 1.02 _+0.01 by 30 min. Volume recovery was abolished by anion channel blockade with NPPB (25uM) and removal of extracellular ATP with apyrase (1U/ml). Summary. In primary human hepatocytes: i) cell volume increases are associated with C1- channel activation and enhanced cellular ATP release; and ii) both channel-mediated CI- efflux and extracellular ATP are critical for cell volume recovery from swelling. Conclusion. Since changes in cell volume directly modulate many liver functions, and P2 receptors are broadly expressed among liver cell types, volume-sensitive release of ATP into the extracellular milieu may represent an important autocrine/paracrine signaling pathway coupling cell volume to membrane ion permeability. • L0535 ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODIES IN PRIMARY SCLEROSING CHOLANGITIS: TARGET ANTIGENS AND CLINICAL ASSOCIATIONS. C. Roozendaal, 1 A.W.M. Van Milligen de Wit, 3 E,B. Haagsma, 2 G. Horst, l C. Schwarze, 4 H.H. Peter,4 J.H. Kleibeuker, 2 J.W. Cohen Tervaert, 1 P.C. Limburg, 1 and C.G.M. Kallenberg. 1 1Dept. of Clinical Immunology and 2Dept. of Gastroenterology and Hepatology, University Hospital Groningen, The Netherlands; 3Dept. of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands; 4Dept. of Rheumatology and Clinical Immunology, University Hospital Freiburg, Germany. The antigenic specificities and clinical significance of anti-neutrophil cytoplasmic autoantibodies (ANCA) in primary sclerosing cholangitis (PSC) are unclear. Aim of this study was to investigate the antigenic specificities and clinical associations of ANCA in 69 patients with PSC. The presence of ANCA was analyzed by indirect immunofluorescence (IIF). Antibodies directed against proteinase 3, myeloperoxidase, elastase, bactericidal /permeability-increasing protein (BPI), cathepsin G, and lactoferrin were identified by ELISA. ANCA were detected by IIF in 67% of PSC patients. In antigen-specific ELISAs, 55% of the serum samples recognized one or more antigens. Fortysix percent contained antibodies to BPI, 23% to cathepsin G, and 22% to lactoferrin. Only 4% had antibodies to proteinase 3, antibodies to myeloperoxidase or elastase were not detected. Frequently, antibodies to different antigens were simultaneously present in a serum sample. The presence of ANCA was not related to the duration of PSC. Also, ANCA as detected by IIF were not associated with the presence of cirrhosis nor with the coexistence of inflammatory bowel disease. However, antibodies to BPI and cathepsin G were both associated with the presence of cirrhosis, and antibodies to lactoferrin were more frequently detected in patients with PSC in conjunction with ulcerative colitis. We conclude from this study that BPI is a major ANCA antigen in PSC. Antibodies to BPI and cathepsin G are related to cirrhosis, and antibodies to lactoferrin in PSC are associated with the concurrentpresence of ulcerative colitis.
AASLD A1329 • L0536 ASSOCIATION OF MULTISPECIFIC CD4+ RESPONSE TO HEPATITIS C AND SEVERITY OF RECURRENCE AFTER LIVER TRANSPLANTATION. HR Rosen, M Blake, M Houghton*, DJ Hinrichs, S Chou, DR Gretch**, HA Bouwer. Dept of Medicine, Molecular Microbiology, and Immunology. Portland VAMC/Oregon Health Sciences University, *Chiron Corp., Emeryville, CA and **Virology Division, University of Washington, Seattle, WA. Indirect evidence suggests that the cellular immunity to HCV is an important determinant of disease recurrence following liver transplantation (Transplantation 1997; 64: 721-26), but to date no study has attempted to define specific defects in the immune response. Aim: To determine if the multispecific, HLA class II-restricted proliferative response to HCV after OLT is associated with different clinical outcomes. Methods: Twenty-four HCV+ OLT recipients with identical initial immunosupprassion (CsA-based) were studied (range of histologic followup: 1 to 8.2 yrs). Group 1 (n = 18) was comprised of pts with no or mild evidence of histologic recurrence; group 2 consisted of 6 pts with severe recurrence (1 died, 2 underwent re-OLT for graft failure). Briefly, PBMC were isolated from freshly heparinized blood by Ficoll-Hypaque density centrifugation. 2 X l05 cells were cultured in 96-well plates and stimulated with 1 and 10 ~g/ml of highly purified recombinant HCV antigens (c33, el00, c200, and NS5 expressed as SOD fusion proteins); tetanus toxoid and CMV antigens (Towne and AD169 strains) were used as controls. Stimulation indexes (SI's) > 3 by 3H-thymidine uptake were considered significant. Results: Peripheral CD4+ T lymphocyte responses to at least one HCV antigen were detectable in 12 of 18 (67%) of group 1 vs. none of the patients in group 2 (p = .01) although all pts in the latter group responded to non-HCV control antigens. Among the 12 patients who responded to HCV Ag's, 11 did so to c33; 4 pts responded to 2 or more Ag's, and 1 pt responded to all 4 HCV Ag's. Conclusions: Following OLT for HCV, vigorous multispecific T cell responses are detectable only in patients with no or mild histological recurrence, and patients with severe HCV recurrence are hyporesponsive to HCV antigens despite responding to non-HCV antigens. Future therapeutic interventions may target specific, defective T cell responses (i.e., adoptive immunotherapy). Supported in part by MRF Grant #9735. • L0537 THE EPIDEMIOLOGY, TREATMENT PATTERNS AND RESOURCE UTILIZATION OF HEPATITIS B PATIENTS IN A LARGE INSURED NEW ENGLAND POPULATION. Daniel M. Rosenberg, Suzanne F. Cook, Susan C, Eaton, William B. Sannders, and Lee L. Lanza,. Glaxo Wellcome Inc., Research Triangle Park, NC; Epidemiology Resources Inc., Newton Lower Falls, MA. The Centers for Disease Control estimates that the incidence of acute HBV is approximately 1.15 per 100,000 in the U.S. and 2-10% of such infections progress to chronic HBV disease. The purpose of this cross-sectional study is to describe the epidemiology, treatment patterns and major health care resources associated with hepatitis B virus (HBV) in a Large Insured New England (LINE) population. The LINE database maintains claims for over 2 million persons; data on patients treated from January 1, 1995 to December 31, 1996 were analyzed. Demographics, type of provider, type of insurance coverage, utilization of outpatient and inpatient services, prescription medications, diagnostic, laboratory, surgical procedures and comorhidities with hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) were examined. Preliminary findings indicate that the prevalence of patients with an HBV diagnosis for whom services were rendered was 60/100,000, HBV/HCV coinfection was 2.8/100,000 and HBV/HIV coinfection was 0.89/100,000. Persons aged 0-9 years had the greatest HBV prevalence (male: 96/100,000 and female: 88/100,000). One percent of patients with only HBV had interferon-alpha therapy. Estimates in this population are subject to underestimation due to the non-inclusion of persons classified with nonspecific viral hepatitis codes, infected persons not presenting for services and institutional differences in diagnostic screening patterns. Further resource utilization and treatment pattern data will be detailed. The prevalence of patients with an HBV diagnosis for whom services were rendered in this LINE population is lower than expected but these data provide important information on treatment patterns and resource utilization. This research was funded by Glaxo WeUcome, Research Triangle Park, NC.