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Anti-human leukocyte antigen (HLA) antibodies in non-transfused, nontransplanted patients with chronic hepatitis C
were rarely detected in protein and peptide responsive individuals. The DRB1 '0101 allele was presem in 3/17 individuals in the VC group and 2/24 in the VP group. A muhispeefllc response (->2) to the HCV proteins was observed in 4/5 (80%) DRBI*0101 positive individuals compared with 6/36 (16.7%) of DRBI*OI01 negative individuals (p = 0.008)..5/5 (10096) DRBI*0101 positive individuals demonstrated a muhispecific response to the peptides compared with 12/'36 (33.3%) of DRBI*0101 negative individuals (p = 0.007). Conclusion: The T cell and cytokine response was low in this unique cohort intected with HCV 25 years ago. These results may be related to the long duration of HCV infection. Never-the&ss DRBI*0101 positive individuals were significantly more likely to demonstrate a muhispecific response which may in part explain the anela's associauon with x~ral clearance.
M877 Hepatitis C Virus-Specific Effector Cell-mediated Responses in Transient Virologic Responders Stevan A, Oonzalez, Mohamed Tarek M Shata, Gary Dorante, ira M J a c o b i n , Andrew H Tala/ Background: Vigorous hepatitis C virus (HCV)-specific CD4+ T-ceil responses to HCV antigens dunng therapy are associated with a sustained vm)logic response. Transiem virologic responders taft to sustain this response during therapy. HCV-specific immune responses may glve insight into viral and host determinants of treatment outcome. Aims: To evaluate peripheral HCW-specific, lFN-'y-secreting cellular responses in sustained and transient virologic responders compared wuh nonresponders before initiating treatment with pegylated interferon and nbavmn Methods: HCV-specific immune responses were measured in frozen peripheral blood mononudear cells (PBMC s) fl'om 16 patients with chronic HCV infection obtained pnor to intiation of pegylated interferon (1 5~.g/kg/wk) and riba~arin (13.6mg/kg/ d) Patients were divided into three groups: 13 sustained responders (absence of HCV RNA six months following cessation of IFN/RBV therapy), n = 5; 2) transient responders (HCV RNA detectable in serum within s~x months {bllo~ng cessation of 1FN/RBX/therapy), n = 6; and 3) nonresponders, n = 5. The enzyme-linked immunospot (ELISPOT) assay was utilized to determine the nnmbet.'s of iFN-',/-secreting ceils per 2 xl0 ~ PBMCs following 24hmtr incubation with HCV antigens core, NS3, NS4, and NS5. Staphylococcal enterotoxin B and tetanus toxiod were used as positive controls Human superoxide dismutase was used as a negative control The students t-test was utilized to determine staustical sigruficance. Results: Sixteen patients were evaluated (12 men and 4 women; mean age 4 8 9 years; genot,,~ 1, n = 12; genotype not performed, n = 4). At baseline, HCV-specific responses tended to be increased in virologm responders compared with nonresponders (p = 0 3 ) Transient virologic respouders demonstrated stronger cell-mediated responses compared with nom:esponders. The number ot IFN-y-secreting cells per 2 x lff' PBMCs in response to HC,/antigens core 13131 +/- 256.3, p = 0.04), NS4 (892 +/-117.3, p = 0.06), and NS5 (575 +/- 590, p = 002), were increased compared with nonresponders. The differences between responders and transient responders were nnt statistically significant. Conclusions: Transient virologic responders demonstrate increased HCV-specific elte:ctor cell-mediated responses to HCV antigens compared with nonresponders before initiation of antiviral therapy Viral escape mutations may be n.~sponsible for the fail.ire to clear HCV despite the presence of HCV-specific immune response
Ro, Reiponders to ' 1 proteir~ No. Re~onder~ to =1 proteins No. Nosponders to =1 peptlde~ No, Responders to ' 2 p e ~
VP Group,n=24 1012441,2% 6/24 25% 15J2462,5% 11/24 45,8%
M880 The RANTES (-403 G-->A) Promoter Polymorphism does not affect HCV or HIV/HCV Co-lnfection but is increased in HIV Infection Golo Ahlenstiel, Agatbe Iwan, Jurgen K Rockstroh, Bernd Kupler, Bertfned Matz, Hans H. Brackmann, Tilman Sauerbruch, Ulrich Spengler, Rainer P. Woitas Background: Chemokmes and their receptors are crucial for the immune response in HIV and HCV infection. The natural course of these infections may be altered by a polymorph~sm within the RANTES promoter (-403 G-->A) which leads to up-regulated RANTES transcription and possibly to enhanced antiviral activity. Methods: We therefore determined the frequency of the RANTES-403 mutation using real time PCR and hybridization probes m patients with HIV infection (n = 87), HCV infection (n = 129), HIV/HCV co-infection (n = 121), and 109 healthy blood donors. Each group was stratified into -403 homozygotes, 403/wildtype beterozygotes and wildtype homozygotes, respectively. Finally, resulting sufisets were compared with respect to HIV and HCV loads. Results: The RANTES-403 allele frequency in HIV infected patients (50/124 (28.7%)) differed significantly from HCV (51/ 207 (19.7%), HIV/HCV co-infected patients (37/205 (15.3%) and the healthy control group 133/185 115.1%); p < 005). RANTES-403 homozygosity was tound in 5 (5.7%) of the HIV- infected patients compared to 3 in the HCV (2.3%) and HI\4q-ICV co-infected groups (2.5%) and 1 of the healthy blood donors (0.9%). H1V infected patients carrying the mutant allele had viral loads that were 3 times lower than in the wildtype patients (n.s.), turthermore HCV loads were lower in -403 homozygous patients (n.s.). Conclusions: The K4NTES-403 mutation is significantly more frequent in HIV infection than in our other cohorts. This mutation is associated with reduced HIV Ioads. However, our data do not support an association between hepatitis C and the RANTES-403 polymorphism.
M878 Anti-Human Leukocyte Antigen (HLA) Antibodies in Non-Transfused, NonTransplanted Patients with Chronic Hepatitis C Hods A El Aggan Myriam A Helmy Ahmed B. lvtahmoud Background/Aims: The human leukocyte antigens (HLA) may influence host immune response to hepatitis C virus (HCV) infection..as chronic hepatitis C results in the appearance of a variety of autoantibodies, the aim of dus study was to investigate the possibdity for the development of anti-HL4 antibodies in patients with chronic hepatitis C in relation to disease activity (indicated by elevation of transaminases, the presence of viremia or histologically). Methods: S~xty-seven untreated nmle patients with chronic hepatitis C (anti-HCV antibody and HCV-RNA posinve); 38 with elevated serum alanine aminotransferase (ALT) levels and 29 with persistently normal serum ALT values and 33 normal controls, were enrolled in the study. All patmnts had no history of blood transtusion. Sera were analyzed for immunoglobulin G anti-HLA class I and class ll antibodies by e:~ymedinked immunosorbant assay using Tarasaki trays and for non-organ-specific autoantflmdies (anti-nuclear, anti-smooth muscle, anti-mitochondriaI and anti-hver,%zidney microsomes type 1 antibodies) Results: Anti-HLA class I and class lI antibodies were detected in 15/67 (22.4%) and 11/67 (16.4%) patients with chromc hepatitis C respectivdy but in none of normal controls. The appearance of anti-HLA antibodies was significantly more frequent in patmnts with elevated serum ALT than in those with permtently normal serum ALT values (316% vs. 10.3%, P = 0.0393 and was associated with the presence of non-specihc-organ serum autoantibodies in 12/15 (73.3%) pauents Patients with positive anti-HLA antibodies had significantly' h*gher serum HCV RNA level and necroinflammatory and fibrosis scores in liver biopsy than patients negative for anti-H~ antibodies (P < 00013 Conclusions: The detection of anti-HLA antibodies m patients wifh chronic hepatitis C may be a marker of autoimmune response and may interfere with the host immune responses leading to viral persistence and disease progression
M881 Reduced Expression of STAT-4 Protein in Patients with Chronic Hepatitis C Bettina Heberer, Harms F. Loehr Peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis C wms (HCXr infection showed reduced levels of I1--12 upon stimulation with HCV antigens Ib 12 is required to mount sufficient antiviral Thl-type immune responses by activation of the transcription factors STAT-3 and STAT-4. The aim of this study was to show whether reduced levels of IL-12 influences the outcome of HCV infection by decreased expression of STAT-3 and STAT-4 proteins and increased T-cell apoptosis. PBMC from chronic HO" carriers and controls were stimulated with HCV and control antigens, Then, the expression of STAT4 and STAT-4 proteins was analysed by Western Blot and the number of apoptotic cells was determined by Annexin staining. Flow cytometry analysis of apoptotic cell death revealed no significant differences between healthy controls and patients with HCV infection. However, the expression of STAT-4 but not STAT-3 protein was reduced in HCV patients compared to healthy controls. Reduced expression of STAT-4 transcription factor may lead to deficient T cell development and may contribute to the chmincity of HCV infection
M882 Long-Term Follow-Up Of CD4 Positive T Cell Subsets (Thl, Th2) In Peripheral Blood Of Patients With Chronic Hepatitis C Who Are Sustained Responder And Non-Responder To IFN Koji Ishii, Naoko Takamura, Mie Shinohara, Yasukiyo Sumino, Wataru Yamamur0, Hidenari Nagai, Takashi Kawafune, Motonobu Watanabe
M879 Cellular Immune Responses, Possession of HLA Class II DRBI*0101 allele and Outcome of Hepatitis C Virus Infection in a Unique Cohort Mlchad Sweeney Sharon Barrett, John Crowe
BACKGROUND/AIM: Helper T cell subsets govern the immune response to viral infection The mechanisms that lead to viral persistence or clearance in patients with chronic hepatitis C (CHC) are not well known. The goal of this study was to clarify whether the course of Thl and Th2 ceil subsets of CD4+ T cells in peripheral blood of patients with CHC is related with successful elimination of hepatitis C virus (HCV). PATIENTS AND METHODS: Eighteen (14 males / 4 females, mean age of 47yr) patients with chronic hepatitis C (CHC) were the objects of this study. They received natural IFN alpha or recombinat alpha2b, 510 MU dialy for 2 weeks and trice weekly for 22 weeks, and were tbllowed by 24 weeks after completion of [FN monotherapy. According to the response to the iFN, they" were divided into 2 groups; sustained responder (SR) was defined as undetectable serum HCVRNA at the end of follow-up (Week 48), and non-responder (NR) was any other pattern Before, and 2, 24, and 48 weeks alter IFN administration, serum ALT and HCV-RNA assayed by RT-PCR (Amplicore Ver 2), and intracellular c3,tolgme analysis in CD4 + T cells were evaluated. Flow cytometric determination of IFN gamma and IL-4 m the cytoplasm of peripheral CD4 positive T cells were performed, and percentages of IFN gamma + and II_4- (Thl) and IFN gamma- and IL-4+ erh2) cells was counted by FACS. RESULTS: The percentages of Thl cells in both groups peaked at Week 2, and returned to each baseline
We have prevmusly reported an association between the DRB1 '0101 allele and spontaneous rind clearance ( p c =0.013. The aim of Ibis study was (1) to investigate HCV specific T cell responses in indMduals ",vlth resolved and persistent HCV infection, (2) to determine wbetber the DRBi* 0101 allele influenced these T-cell responses and (3) to determine it the T-cell responses were ThI (1FNDor Th2 (IL-10) medmted Methods: 17 patients with spontaneous v~ralclearauce (VC) and 24 witb persistent HCV intection (VP) were investigated PBMCs were stimulated with recombinant proteins (core, NS3, NS4 and NS5) and 20 HCV peptides, which included published HCV epitopes and putative DRBI*0101-restricted epitopes designed using predictive software and fhe amino acid sequence of HCV genotype l b HLA typing was determined by reverse l'eybridization assay. The cytokmes iFNF and IL-10 were assayed by' ELISA. Results: Table 1 presents the number of individuals demonstrating a proliferative respmlse to the HCV proteins and peptides Ahhough the number of individuals responding to tbe proteins and peptides was similar in both groups (Table 13 response to the NS3 and NS5 proteins was increased in the VC compared to the VP groups. A greater percentage of the VC group also responded to each individual peptide Furfbermore, two individuals m the VC group responded to a broader range of peptides IL-10 and IFNF
AASLD Abstracts
VC Group n=17 9/'17 53% 4!17 23,5% 1211770.6% 7/17 38.9%
A-740
M877 Hepatitis C Virus-Specific Effector Cell-mediated Responses in Transient Virologic Responders Stevan A, Oonzalez, Mohamed Tarek M Shata, Gary Dorante, ira M J a c o b i n , Andrew H Tala/ Background: Vigorous hepatitis C virus (HCV)-specific CD4+ T-ceil responses to HCV antigens dunng therapy are associated with a sustained vm)logic response. Transiem virologic responders taft to sustain this response during therapy. HCV-specific immune responses may glve insight into viral and host determinants of treatment outcome. Aims: To evaluate peripheral HCW-specific, lFN-'y-secreting cellular responses in sustained and transient virologic responders compared wuh nonresponders before initiating treatment with pegylated interferon and nbavmn Methods: HCV-specific immune responses were measured in frozen peripheral blood mononudear cells (PBMC s) fl'om 16 patients with chronic HCV infection obtained pnor to intiation of pegylated interferon (1 5~.g/kg/wk) and riba~arin (13.6mg/kg/ d) Patients were divided into three groups: 13 sustained responders (absence of HCV RNA six months following cessation of IFN/RBV therapy), n = 5; 2) transient responders (HCV RNA detectable in serum within s~x months {bllo~ng cessation of 1FN/RBX/therapy), n = 6; and 3) nonresponders, n = 5. The enzyme-linked immunospot (ELISPOT) assay was utilized to determine the nnmbet.'s of iFN-',/-secreting ceils per 2 xl0 ~ PBMCs following 24hmtr incubation with HCV antigens core, NS3, NS4, and NS5. Staphylococcal enterotoxin B and tetanus toxiod were used as positive controls Human superoxide dismutase was used as a negative control The students t-test was utilized to determine staustical sigruficance. Results: Sixteen patients were evaluated (12 men and 4 women; mean age 4 8 9 years; genot,,~ 1, n = 12; genotype not performed, n = 4). At baseline, HCV-specific responses tended to be increased in virologm responders compared with nonresponders (p = 0 3 ) Transient virologic respouders demonstrated stronger cell-mediated responses compared with nom:esponders. The number ot IFN-y-secreting cells per 2 x lff' PBMCs in response to HC,/antigens core 13131 +/- 256.3, p = 0.04), NS4 (892 +/-117.3, p = 0.06), and NS5 (575 +/- 590, p = 002), were increased compared with nonresponders. The differences between responders and transient responders were nnt statistically significant. Conclusions: Transient virologic responders demonstrate increased HCV-specific elte:ctor cell-mediated responses to HCV antigens compared with nonresponders before initiation of antiviral therapy Viral escape mutations may be n.~sponsible for the fail.ire to clear HCV despite the presence of HCV-specific immune response
Ro, Reiponders to ' 1 proteir~ No. Re~onder~ to =1 proteins No. Nosponders to =1 peptlde~ No, Responders to ' 2 p e ~
VP Group,n=24 1012441,2% 6/24 25% 15J2462,5% 11/24 45,8%
M880 The RANTES (-403 G-->A) Promoter Polymorphism does not affect HCV or HIV/HCV Co-lnfection but is increased in HIV Infection Golo Ahlenstiel, Agatbe Iwan, Jurgen K Rockstroh, Bernd Kupler, Bertfned Matz, Hans H. Brackmann, Tilman Sauerbruch, Ulrich Spengler, Rainer P. Woitas Background: Chemokmes and their receptors are crucial for the immune response in HIV and HCV infection. The natural course of these infections may be altered by a polymorph~sm within the RANTES promoter (-403 G-->A) which leads to up-regulated RANTES transcription and possibly to enhanced antiviral activity. Methods: We therefore determined the frequency of the RANTES-403 mutation using real time PCR and hybridization probes m patients with HIV infection (n = 87), HCV infection (n = 129), HIV/HCV co-infection (n = 121), and 109 healthy blood donors. Each group was stratified into -403 homozygotes, 403/wildtype beterozygotes and wildtype homozygotes, respectively. Finally, resulting sufisets were compared with respect to HIV and HCV loads. Results: The RANTES-403 allele frequency in HIV infected patients (50/124 (28.7%)) differed significantly from HCV (51/ 207 (19.7%), HIV/HCV co-infected patients (37/205 (15.3%) and the healthy control group 133/185 115.1%); p < 005). RANTES-403 homozygosity was tound in 5 (5.7%) of the HIV- infected patients compared to 3 in the HCV (2.3%) and HI\4q-ICV co-infected groups (2.5%) and 1 of the healthy blood donors (0.9%). H1V infected patients carrying the mutant allele had viral loads that were 3 times lower than in the wildtype patients (n.s.), turthermore HCV loads were lower in -403 homozygous patients (n.s.). Conclusions: The K4NTES-403 mutation is significantly more frequent in HIV infection than in our other cohorts. This mutation is associated with reduced HIV Ioads. However, our data do not support an association between hepatitis C and the RANTES-403 polymorphism.
M878 Anti-Human Leukocyte Antigen (HLA) Antibodies in Non-Transfused, NonTransplanted Patients with Chronic Hepatitis C Hods A El Aggan Myriam A Helmy Ahmed B. lvtahmoud Background/Aims: The human leukocyte antigens (HLA) may influence host immune response to hepatitis C virus (HCV) infection..as chronic hepatitis C results in the appearance of a variety of autoantibodies, the aim of dus study was to investigate the possibdity for the development of anti-HL4 antibodies in patients with chronic hepatitis C in relation to disease activity (indicated by elevation of transaminases, the presence of viremia or histologically). Methods: S~xty-seven untreated nmle patients with chronic hepatitis C (anti-HCV antibody and HCV-RNA posinve); 38 with elevated serum alanine aminotransferase (ALT) levels and 29 with persistently normal serum ALT values and 33 normal controls, were enrolled in the study. All patmnts had no history of blood transtusion. Sera were analyzed for immunoglobulin G anti-HLA class I and class ll antibodies by e:~ymedinked immunosorbant assay using Tarasaki trays and for non-organ-specific autoantflmdies (anti-nuclear, anti-smooth muscle, anti-mitochondriaI and anti-hver,%zidney microsomes type 1 antibodies) Results: Anti-HLA class I and class lI antibodies were detected in 15/67 (22.4%) and 11/67 (16.4%) patients with chromc hepatitis C respectivdy but in none of normal controls. The appearance of anti-HLA antibodies was significantly more frequent in patmnts with elevated serum ALT than in those with permtently normal serum ALT values (316% vs. 10.3%, P = 0.0393 and was associated with the presence of non-specihc-organ serum autoantibodies in 12/15 (73.3%) pauents Patients with positive anti-HLA antibodies had significantly' h*gher serum HCV RNA level and necroinflammatory and fibrosis scores in liver biopsy than patients negative for anti-H~ antibodies (P < 00013 Conclusions: The detection of anti-HLA antibodies m patients wifh chronic hepatitis C may be a marker of autoimmune response and may interfere with the host immune responses leading to viral persistence and disease progression
M881 Reduced Expression of STAT-4 Protein in Patients with Chronic Hepatitis C Bettina Heberer, Harms F. Loehr Peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis C wms (HCXr infection showed reduced levels of I1--12 upon stimulation with HCV antigens Ib 12 is required to mount sufficient antiviral Thl-type immune responses by activation of the transcription factors STAT-3 and STAT-4. The aim of this study was to show whether reduced levels of IL-12 influences the outcome of HCV infection by decreased expression of STAT-3 and STAT-4 proteins and increased T-cell apoptosis. PBMC from chronic HO" carriers and controls were stimulated with HCV and control antigens, Then, the expression of STAT4 and STAT-4 proteins was analysed by Western Blot and the number of apoptotic cells was determined by Annexin staining. Flow cytometry analysis of apoptotic cell death revealed no significant differences between healthy controls and patients with HCV infection. However, the expression of STAT-4 but not STAT-3 protein was reduced in HCV patients compared to healthy controls. Reduced expression of STAT-4 transcription factor may lead to deficient T cell development and may contribute to the chmincity of HCV infection
M882 Long-Term Follow-Up Of CD4 Positive T Cell Subsets (Thl, Th2) In Peripheral Blood Of Patients With Chronic Hepatitis C Who Are Sustained Responder And Non-Responder To IFN Koji Ishii, Naoko Takamura, Mie Shinohara, Yasukiyo Sumino, Wataru Yamamur0, Hidenari Nagai, Takashi Kawafune, Motonobu Watanabe
M879 Cellular Immune Responses, Possession of HLA Class II DRBI*0101 allele and Outcome of Hepatitis C Virus Infection in a Unique Cohort Mlchad Sweeney Sharon Barrett, John Crowe
BACKGROUND/AIM: Helper T cell subsets govern the immune response to viral infection The mechanisms that lead to viral persistence or clearance in patients with chronic hepatitis C (CHC) are not well known. The goal of this study was to clarify whether the course of Thl and Th2 ceil subsets of CD4+ T cells in peripheral blood of patients with CHC is related with successful elimination of hepatitis C virus (HCV). PATIENTS AND METHODS: Eighteen (14 males / 4 females, mean age of 47yr) patients with chronic hepatitis C (CHC) were the objects of this study. They received natural IFN alpha or recombinat alpha2b, 510 MU dialy for 2 weeks and trice weekly for 22 weeks, and were tbllowed by 24 weeks after completion of [FN monotherapy. According to the response to the iFN, they" were divided into 2 groups; sustained responder (SR) was defined as undetectable serum HCVRNA at the end of follow-up (Week 48), and non-responder (NR) was any other pattern Before, and 2, 24, and 48 weeks alter IFN administration, serum ALT and HCV-RNA assayed by RT-PCR (Amplicore Ver 2), and intracellular c3,tolgme analysis in CD4 + T cells were evaluated. Flow cytometric determination of IFN gamma and IL-4 m the cytoplasm of peripheral CD4 positive T cells were performed, and percentages of IFN gamma + and II_4- (Thl) and IFN gamma- and IL-4+ erh2) cells was counted by FACS. RESULTS: The percentages of Thl cells in both groups peaked at Week 2, and returned to each baseline
We have prevmusly reported an association between the DRB1 '0101 allele and spontaneous rind clearance ( p c =0.013. The aim of Ibis study was (1) to investigate HCV specific T cell responses in indMduals ",vlth resolved and persistent HCV infection, (2) to determine wbetber the DRBi* 0101 allele influenced these T-cell responses and (3) to determine it the T-cell responses were ThI (1FNDor Th2 (IL-10) medmted Methods: 17 patients with spontaneous v~ralclearauce (VC) and 24 witb persistent HCV intection (VP) were investigated PBMCs were stimulated with recombinant proteins (core, NS3, NS4 and NS5) and 20 HCV peptides, which included published HCV epitopes and putative DRBI*0101-restricted epitopes designed using predictive software and fhe amino acid sequence of HCV genotype l b HLA typing was determined by reverse l'eybridization assay. The cytokmes iFNF and IL-10 were assayed by' ELISA. Results: Table 1 presents the number of individuals demonstrating a proliferative respmlse to the HCV proteins and peptides Ahhough the number of individuals responding to tbe proteins and peptides was similar in both groups (Table 13 response to the NS3 and NS5 proteins was increased in the VC compared to the VP groups. A greater percentage of the VC group also responded to each individual peptide Furfbermore, two individuals m the VC group responded to a broader range of peptides IL-10 and IFNF
AASLD Abstracts
VC Group n=17 9/'17 53% 4!17 23,5% 1211770.6% 7/17 38.9%
A-740