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The MHC class II haplotype DRB1*1302-DRB3*0301-DQA1*0102-DQB1*0501 protects against persistent HBV infection
GENERAL SESSIONS
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GENERAL SESSION 1
THE MHC CLASS II HAPLOTYPE DRB1*1302-DRB3*0301-DQAl*0102DQBI'0501 PROTECTS AGAINST PERSISTENT HBV INFECTION.
L
M. Thursz 1` A. Hill 2` C. Allsopp 2` D. Kwiatkowski2 B. Greenwood3` HC. Thomas 1` 1. AcademicDepadmentof Medicine,SI. Mary'sHospitalMedicalSchool,London.2. Molecular ImmunologyGroup,JohnRadcliffeHospital,Oxford. 3 MRCLaboratories,TheGambia To test the hypothesis that elimination of HBV is associated with MHC phenotype 1503 subjects were recruited to an MHC/disease association study in The Gambia. 1344 were children up to the age of 10 who had been seen for conditions unrelated to HBV. 159 adults were recruited from blood donors. Subjects were divided into three groups: Group A, never exposed to HBV were anti-HBc negative; Group B, previously infected and recovered, were anti-HBc positive and HBsAg negative; Group C, persistently infected, were anti-HBc and HBsAg positive. MHC class I phenotype was determined by rnicrelymphocytotoxicity assay and MHC class II haplotype was assigned by RFLP analysis.The study was conducted in two parts. In the children the distribution of 27 class I phenotypes and 10 class II haplotypes were compared between groups B and C by 72 analysis (table). An association identified in thechildren was then tested as a sin ~othesisin adults. HLA Group B Group C Class II Group B Group C HBsA~]- HBsA~* haplotype HBsAg. HBsAg+ A2 21 0% 234% 4- 4 11.9% 5.9% A23 25.9% 21.9% 7-9 147% 14.1% 138 198% 31.3% 8-5 6.6% 86% 1335 28.4% 313% 10- 1 15.1% 178% 950 0% 7.8% " 13- 2 7.3% 2.7% 1" 1353 185% 14.1% 18- 4 4.6% 3.8% C1 12% 10.9% " 21 - 5 50% 46.5% "p = 0.01. C2 4.9% 14.1% 25-1 269% 16.2% " t"p < O05 Haplotype 25-1 corresponds to the MHC Class II haplotype DRB1'1302-DRB3* 0301-DQA1"0102-DQB1*0501. The frequency of this haplotype in the adult population was 0% in group A and 21.4% in group B (p <0.01). Hence DRB1*1302DRB3*0301-DQAI*0102-DQBI'0501, which is &ommon in The Gambia, is protective against persistent HBV infection. This is in accord with a study in caucasians demonstrating a protective effect of HLA DRw6 which is a serological supertype of DRB1*1302 and DRBl*1301.
NATURAL HEPATITIS B VIRUS VARIANTS ARE T CELL RECEPTOR ANTAGONISTS FOR ANTI-VIRAL CYTOTOXIC T CELLS Antonio Bertoletti 1 Amalia Penna I Alessandro Sette 2, Cristina Cavallo 1 Bruno Capone 1, Massimo Levrero 3, Francis V. Chisari 4, Franco Fiaccadori 1, Carlo Ferraril 1 Cattedra Malattie Infettive, Universit~t di Parma. Italy; 2 Cytel Corporation, La Jolla, CA, USA; 3 I Clinica Medica, Universith "La Sapienza", Roma, Italy; 4 The Scripps Research Institute, La Jolla, CA, USA. It has been suggested that mutations within immunodominant cytotoxic T lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting MHC binding or T cell receptor (TCR) recognition. We now show that hepatitis B virus (HBV) isolates derived from two chronically infected patients display mutations within a HLA-A2 restricted CTL epitope (HBc 18-27) that yield variant peptides acting as natural TCR antagonists with the capacity to inhibit the CTL response to the wild type epitope because unable to deliver a full stimulatory signal though still interacting with the T cell receptor. During natural infection by HBV and perhaps by other viruses that display uncommonly high mutation rates, such as HIV and HCV, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence.
THE DIFFERENTIATION OF SINUSOIDAL ENDOTHELIAL CELLS (SEC) DURING LIVER ORGANOGENESIS IN HUMANS
A Couvelerd. ]-Y Scoazec. M-C Dauee. J-L Benifla. P MadelenaL F PoteL G Feldmann. INSERM U327, Facult6 de M6decinc Xavier Bichat, Service d'Anatomie Pathologique and Service de Gyn~cologie, H6pital Bichat, Paris, Fratlcc. The structme and functional adaptations of liver sinnsoids differ from those of most other capillary vessels. Their discontinuous and fenestrated endothelial lining lacks most of the structural markers of continuous endothelia and is characterized by the expression of specific functional markers. The perisinusoidal matrix is characterized by the lack of laminin and the presence of tenascin. It is assumed that liver sinusoids differentiate between 4 and 12 gestational weeks (GW) from the continuous capillary vessels of septum transversum, progressively surrounded by growing hepatoblests. However, the process of structural and functional differentiation of SEC has not been analyzed. The aim of this work was to determine the differentiation sequence of SEC and its relation to the changes in the constitution of the subendothelial mala'ix. Liver fragments of 37 human fetuses aged from 5 to 40 GW were studied. The expression of the following molecules was evaluated by immunoperoxidase: (a) structural markers of continuous endothelia, absent from adult SEC: PECAM-1, CD34 protein, IF10, (b) functional markers specific for adult SEC: CD4 and CD14 proteins, ICAM-1, receptor IT for the Fc fragment of IgG (FcRII), (c) laminin and tanascin. At $ GW, sinusoids of fetal liver expressed PECAM-I, CD34 protein, and 1F10. The functional markers of adult SEC were tmdetectsble. The subendothelial matrix contained no tanescin and moderate amounts of Iaminin. At 8 WG, PECAM-I, CD34 protein, and IF10 were weakly expressed. The functional markers of adult SEC remained undetectable. Laminin was present in scattered deposits and tanescin was absent. At 10 GW, PECAM1, CD34 protein, and 1FI0 were undetectable. CD4 protein, ICAM-1 and FcRH were expressed by fetal SEC. Laminin and tenascin were absent. At 12 GW, the deposition of tenascin in the subendothelial matzix was first detected. After 12 GW, the characteristics of fetal sinusoids remained unchanged apart from the expression of CD14 protein by SEC, t-wet detected at 40 GW. In conclusion, our study shows that SEC differentiate from continuous endothelial ceils and progressively acquire most of their structural and functional characteristics between the 8th to the 12th GW. This process of terminal differentiation is correlated with concomittant changes in the constitution of the subendothelial matrix, which progressively acquires the characteristics of the adult perisinusoidal matrix.
INFLUENCE OF S-ADENOSYLMETHIONINE (SAMe) ON UPID PEROX]DATION AND UVER RBROGENESIS IN CCI4-1NDUCED CIRRHOSIS.
M. ~ , J. Caballerta, M. Cabrr", F. Corrales-, M. Rubio, R. Deulofeu,A. Gim~lez, A. Parrs, J. Camps', AM. Ballesla, JM. Marc" and J. Rodrs. Liver Unit. Hospital Clinic I Pmvindal. University o1 Barcelona. "Canlm de Recorca Biom~ca` Hospital Sent Joan. Reus. " Instituto de Investlgaclones Blorn&icas. C.S.I.C. Madrid. Spain. Recent studies in a model of CCI4-inducad cirrhosis have demonstrated a relationship between hepatic lipid peroxidation and liverfibmsis. Furthermore, in the same model it was observed that SAMe administration reduces liver fibrosis. The aim of the study was to investigate the influence of SAMe on lipid pemxidation and on liver fibrogenesis. Three groups of rats induced to cirrhosis by repeated injections of CCI4 dudng 9 weeks were studied. The animals received only CCI4, and CCI4 plus SAMe (10 mg/kg, i.m. daily) during 3 and 6 weeks, respectively. Liver samples were used for histological diagnosis, and for determination of collagen content (ColH), prolyl hydmxylase activity (PHase), thiobarbituric acid-reactive substances (-rBARS) and glutathione concentration (GSH). All rats from CCI4 group had cirrhosis at the end of the study. Cirrhosis was also present in 5 of the 6 rats receiving SAMe for 3 wk, but in only one of the 6 rats that received SAMe during 6 wk. There were no differences in either ColH or in PHase between rats that received only CCI4 (ColH: 70.5 -+ 6.6 ug/mg prot; PHase: 177.8 _.+5.5 cpm/mg prot) and those treated with SAMe during 3 wk (ColH: 62.2 _ 5.7 ug/mg prot; PHase: 166.5 + 23.2 cpm /mg prot). Rats receiving SAMe during 6 wk had significantly lower ColH (47.8 _+3.8 ug/mg prot, p<0.05) and PHase activity (114.5 _+ 12.5 cpm/mg prot, p<0.05) than the other two groups. The procollagen type I mRNA was increased 3.5 fold in CCI4 treated rats, whereas only a 2 fold increase was observed in rats treated with CCI4 and SAMe during 6 wk. The hepatic q'BARS were significantly lower in rats treated with CCI4 and SAMe during 6 wk (98 + 5 nmol/g), than in rats treated with CCL4 (134 + 12 nmol/g) and in those treated with CCI4 and SAMe dudng 3 wk (127 4" 13 nmol/g). There was a significant correlation between TBARS and PHase and ColH. The GSH was significantly diminished in CCI4 treated rats and retumad to normal in rats receiving SAMe. In conclusion, the early administration of SAMe in a model of CCI4induced liver injury reduces lipid peroxidation and restores the levels of glutathione resulting in a less advanced liver fibrosis.
$3
GENERAL SESSION 1
THE MHC CLASS II HAPLOTYPE DRB1*1302-DRB3*0301-DQAl*0102DQBI'0501 PROTECTS AGAINST PERSISTENT HBV INFECTION.
L
M. Thursz 1` A. Hill 2` C. Allsopp 2` D. Kwiatkowski2 B. Greenwood3` HC. Thomas 1` 1. AcademicDepadmentof Medicine,SI. Mary'sHospitalMedicalSchool,London.2. Molecular ImmunologyGroup,JohnRadcliffeHospital,Oxford. 3 MRCLaboratories,TheGambia To test the hypothesis that elimination of HBV is associated with MHC phenotype 1503 subjects were recruited to an MHC/disease association study in The Gambia. 1344 were children up to the age of 10 who had been seen for conditions unrelated to HBV. 159 adults were recruited from blood donors. Subjects were divided into three groups: Group A, never exposed to HBV were anti-HBc negative; Group B, previously infected and recovered, were anti-HBc positive and HBsAg negative; Group C, persistently infected, were anti-HBc and HBsAg positive. MHC class I phenotype was determined by rnicrelymphocytotoxicity assay and MHC class II haplotype was assigned by RFLP analysis.The study was conducted in two parts. In the children the distribution of 27 class I phenotypes and 10 class II haplotypes were compared between groups B and C by 72 analysis (table). An association identified in thechildren was then tested as a sin ~othesisin adults. HLA Group B Group C Class II Group B Group C HBsA~]- HBsA~* haplotype HBsAg. HBsAg+ A2 21 0% 234% 4- 4 11.9% 5.9% A23 25.9% 21.9% 7-9 147% 14.1% 138 198% 31.3% 8-5 6.6% 86% 1335 28.4% 313% 10- 1 15.1% 178% 950 0% 7.8% " 13- 2 7.3% 2.7% 1" 1353 185% 14.1% 18- 4 4.6% 3.8% C1 12% 10.9% " 21 - 5 50% 46.5% "p = 0.01. C2 4.9% 14.1% 25-1 269% 16.2% " t"p < O05 Haplotype 25-1 corresponds to the MHC Class II haplotype DRB1'1302-DRB3* 0301-DQA1"0102-DQB1*0501. The frequency of this haplotype in the adult population was 0% in group A and 21.4% in group B (p <0.01). Hence DRB1*1302DRB3*0301-DQAI*0102-DQBI'0501, which is &ommon in The Gambia, is protective against persistent HBV infection. This is in accord with a study in caucasians demonstrating a protective effect of HLA DRw6 which is a serological supertype of DRB1*1302 and DRBl*1301.
NATURAL HEPATITIS B VIRUS VARIANTS ARE T CELL RECEPTOR ANTAGONISTS FOR ANTI-VIRAL CYTOTOXIC T CELLS Antonio Bertoletti 1 Amalia Penna I Alessandro Sette 2, Cristina Cavallo 1 Bruno Capone 1, Massimo Levrero 3, Francis V. Chisari 4, Franco Fiaccadori 1, Carlo Ferraril 1 Cattedra Malattie Infettive, Universit~t di Parma. Italy; 2 Cytel Corporation, La Jolla, CA, USA; 3 I Clinica Medica, Universith "La Sapienza", Roma, Italy; 4 The Scripps Research Institute, La Jolla, CA, USA. It has been suggested that mutations within immunodominant cytotoxic T lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting MHC binding or T cell receptor (TCR) recognition. We now show that hepatitis B virus (HBV) isolates derived from two chronically infected patients display mutations within a HLA-A2 restricted CTL epitope (HBc 18-27) that yield variant peptides acting as natural TCR antagonists with the capacity to inhibit the CTL response to the wild type epitope because unable to deliver a full stimulatory signal though still interacting with the T cell receptor. During natural infection by HBV and perhaps by other viruses that display uncommonly high mutation rates, such as HIV and HCV, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence.
THE DIFFERENTIATION OF SINUSOIDAL ENDOTHELIAL CELLS (SEC) DURING LIVER ORGANOGENESIS IN HUMANS
A Couvelerd. ]-Y Scoazec. M-C Dauee. J-L Benifla. P MadelenaL F PoteL G Feldmann. INSERM U327, Facult6 de M6decinc Xavier Bichat, Service d'Anatomie Pathologique and Service de Gyn~cologie, H6pital Bichat, Paris, Fratlcc. The structme and functional adaptations of liver sinnsoids differ from those of most other capillary vessels. Their discontinuous and fenestrated endothelial lining lacks most of the structural markers of continuous endothelia and is characterized by the expression of specific functional markers. The perisinusoidal matrix is characterized by the lack of laminin and the presence of tenascin. It is assumed that liver sinusoids differentiate between 4 and 12 gestational weeks (GW) from the continuous capillary vessels of septum transversum, progressively surrounded by growing hepatoblests. However, the process of structural and functional differentiation of SEC has not been analyzed. The aim of this work was to determine the differentiation sequence of SEC and its relation to the changes in the constitution of the subendothelial mala'ix. Liver fragments of 37 human fetuses aged from 5 to 40 GW were studied. The expression of the following molecules was evaluated by immunoperoxidase: (a) structural markers of continuous endothelia, absent from adult SEC: PECAM-1, CD34 protein, IF10, (b) functional markers specific for adult SEC: CD4 and CD14 proteins, ICAM-1, receptor IT for the Fc fragment of IgG (FcRII), (c) laminin and tanascin. At $ GW, sinusoids of fetal liver expressed PECAM-I, CD34 protein, and 1F10. The functional markers of adult SEC were tmdetectsble. The subendothelial matrix contained no tanescin and moderate amounts of Iaminin. At 8 WG, PECAM-I, CD34 protein, and IF10 were weakly expressed. The functional markers of adult SEC remained undetectable. Laminin was present in scattered deposits and tanescin was absent. At 10 GW, PECAM1, CD34 protein, and 1FI0 were undetectable. CD4 protein, ICAM-1 and FcRH were expressed by fetal SEC. Laminin and tenascin were absent. At 12 GW, the deposition of tenascin in the subendothelial matzix was first detected. After 12 GW, the characteristics of fetal sinusoids remained unchanged apart from the expression of CD14 protein by SEC, t-wet detected at 40 GW. In conclusion, our study shows that SEC differentiate from continuous endothelial ceils and progressively acquire most of their structural and functional characteristics between the 8th to the 12th GW. This process of terminal differentiation is correlated with concomittant changes in the constitution of the subendothelial matrix, which progressively acquires the characteristics of the adult perisinusoidal matrix.
INFLUENCE OF S-ADENOSYLMETHIONINE (SAMe) ON UPID PEROX]DATION AND UVER RBROGENESIS IN CCI4-1NDUCED CIRRHOSIS.
M. ~ , J. Caballerta, M. Cabrr", F. Corrales-, M. Rubio, R. Deulofeu,A. Gim~lez, A. Parrs, J. Camps', AM. Ballesla, JM. Marc" and J. Rodrs. Liver Unit. Hospital Clinic I Pmvindal. University o1 Barcelona. "Canlm de Recorca Biom~ca` Hospital Sent Joan. Reus. " Instituto de Investlgaclones Blorn&icas. C.S.I.C. Madrid. Spain. Recent studies in a model of CCI4-inducad cirrhosis have demonstrated a relationship between hepatic lipid peroxidation and liverfibmsis. Furthermore, in the same model it was observed that SAMe administration reduces liver fibrosis. The aim of the study was to investigate the influence of SAMe on lipid pemxidation and on liver fibrogenesis. Three groups of rats induced to cirrhosis by repeated injections of CCI4 dudng 9 weeks were studied. The animals received only CCI4, and CCI4 plus SAMe (10 mg/kg, i.m. daily) during 3 and 6 weeks, respectively. Liver samples were used for histological diagnosis, and for determination of collagen content (ColH), prolyl hydmxylase activity (PHase), thiobarbituric acid-reactive substances (-rBARS) and glutathione concentration (GSH). All rats from CCI4 group had cirrhosis at the end of the study. Cirrhosis was also present in 5 of the 6 rats receiving SAMe for 3 wk, but in only one of the 6 rats that received SAMe during 6 wk. There were no differences in either ColH or in PHase between rats that received only CCI4 (ColH: 70.5 -+ 6.6 ug/mg prot; PHase: 177.8 _.+5.5 cpm/mg prot) and those treated with SAMe during 3 wk (ColH: 62.2 _ 5.7 ug/mg prot; PHase: 166.5 + 23.2 cpm /mg prot). Rats receiving SAMe during 6 wk had significantly lower ColH (47.8 _+3.8 ug/mg prot, p<0.05) and PHase activity (114.5 _+ 12.5 cpm/mg prot, p<0.05) than the other two groups. The procollagen type I mRNA was increased 3.5 fold in CCI4 treated rats, whereas only a 2 fold increase was observed in rats treated with CCI4 and SAMe during 6 wk. The hepatic q'BARS were significantly lower in rats treated with CCI4 and SAMe during 6 wk (98 + 5 nmol/g), than in rats treated with CCL4 (134 + 12 nmol/g) and in those treated with CCI4 and SAMe dudng 3 wk (127 4" 13 nmol/g). There was a significant correlation between TBARS and PHase and ColH. The GSH was significantly diminished in CCI4 treated rats and retumad to normal in rats receiving SAMe. In conclusion, the early administration of SAMe in a model of CCI4induced liver injury reduces lipid peroxidation and restores the levels of glutathione resulting in a less advanced liver fibrosis.