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J mice and experimental hepatitis induced by propionibacterium acnes and lipopolysaccharide
Posters/International Hepatology Communications 3 SuppL (1995) $37-8169 D1~}1
PBC LIKE LESION IS INDUCED BY IMMUNIZATION WITH RECOMBINANT PDC-E2/BCKD-E2 HYBRID MOLECULE AND LIPOPOLYSACCHARIDE INJECTION IN N E O N A T A L L Y THYMECTOMIZED MICE. T . M a s a n a g a , Y . W a t a n a b e , P.S.C. L e u n g , M . K a m i y a s u , E . S a n a d a , M . M y o z a k i , T . N a k a n i s h i , G . K a j i y a m a , M.E. Gershwin. 1st Dept. of I n t e r n a l Medicine, Hiroshima Univ. of M e d i c i n e , H i r o s h i m a , Japan., I n t e r n a l Medicine, Univ. of C a l i f o r n i a a t Davis, USA a ".t~Jt
< I n t r o d u c t i o n > T h i s s t u d y w a s d e s i g n e d to t e s t the h y p o t h e s i s t h a t a u t o i m m u n i t y to PDC-E2 or BCKD E2 itself can i n d u c e the p a t h o l o g y of PBC. < Methods >Neonatally thymectomy (NTx) was p e r f o r m e d 2 d a y s a f t e r b i r t h . The mice in g r o u p A w e r e i m m u n i z e d w i t h a m i x t u r e of r e c o m b i n a n t PDC-E2/BCKDE2 h y b r i d molecule (150/zg) and FCA a f t e r NTx. The mice in g r o u p B w e r e i m m u n i z e d w i t h h y b r i d m o l e c u l e a n d FCA w i t h o u t NTx. The mice in g r o u p C were i m m u n i z e d w i t h a m i x t u r e of h y b r i d m o l e c u l e and FCA a f t e r N T x and LPS (0 5/zg) w a s i n j e c t e d i n t r a v e n o u s l y 2 d a y s a f t e r each i m m u n i z a t i o n . < Results > The mice in group C showed positive AMA and a h i g h incidence of n o n s u p p u r a t i v e c h o l a n g i t i s w h i c h is c h a r a c t e r i z e d b y ' t h e mononuclear cell infiltration around the b i l i a r y e p i t h e l i a l cells. .--Conelusion > PBC-like lesion is i n d u c e d b y immuniz a t i o n w i t h PDC-E2 or BCKD-E2 p r o t e i n s , a n d e n d o t o x i n such as l i p o p o l y s a c c h a r i d e m a y a u g m e n t the p a t h o g e n e s i s of PBC.
1~-123 Histopathological Re-evaluation of Interlobular Bile Duct Damage in Patients with Autoimmune Hepatitis (AIH) in Japan Y.Takahashi, S.Maeyama, J.Koike, M.Kishi, Y.Aida, H.Yoshida, T.Takakuwa, T.Uchikoshi 2nd Dept.ot Pathology, St. Marianna University School of Medicine, Kawasaki, Japan Aims : Histopathological characteristics of AIH show severe parenchymal damage with chronic active hepatitis. In Europe and America, the appearance of interlobular bile duct damage in AIH is less frequent than in chronic hepatitis type C(C-CH). We have re-evaluated the histopathologioal changes of interlebular bile duct damage in AIH, and have compared them with chronic hepatitis type B(B-CH) and C-CH. Materials & Methods: Forty-six cases of AIH satisfying the criteria advocated in 1992, showing "/-globulin (more than 2g/dl) and negative for serum anti-HCV antibody were studied in comparison with 242 cases of C-CH and 52 of B-CH. Bile duct damage was histologically considered present if there were swelling and stratification with papillary growth of the epithelial cells, disruption of basement membrane by mononuolear cell infiltrates, vacuolizations of epithelial cells, degeneration or mitotic activity, or a combination of these. Results: 1)The incidence of bile duct damage in AIH was 35 cases(76%), and the frequency is higher than in B-CH (12 cases,23%) and C-CH(86 cases, 36%). 2) The bile duct damage in AIH was not related to lymphtollicle formation. However, damages seen in C-CH were related to formation of lymphfollicles. The damages in B-CH were mild and occurred with the progress of histological changes Conclusion: The criteria of AIH decided at the international meeting in Brighton in 1993 did not include interlobular bite duct damage. The fact that the frequency of bile duct damage in AIH in Japan is higher than that in C-CH and B-CH may be due to the development of cirrhosis in AIH in Europe and America. In Japan, bile duct changes in AIH are thought to be similar to those in autoimmune cholangiopathy (AC), making the histological differentiation between AIH and AC necessary.
$67
D 1")") Z°-L~Z'~ IDENTIFICATION OF IMMUNODOMINANT T CELL EPITOPES OF PYRUVATE DEHYDROGENASE COMPLEXES IN PRIMARY BILIARY CIRRHOSIS S. Shlmoda, Y. Ichiki, M. Nakamura, K. Hayashida, H. lshibash[, Y. Niho 1st Dept. of Internal Medicine, Faculty of medicine, Kyushu Univ. In order to clarify the immunopathogenesis of primary biliary cirrhosis (PBC), we identified immunodominant T cell autoepitopos of mitochondrial antigens in PBC. Peripheral blood T cells purified from 13 patients with PBC were tested for proliferative responses to pyruvate dehydrogenase complexes (PDC). T cells derived from all patients with PBC but none of those from controls showed proliferative responses to PDC. Repeated stimulation ofT cells with PDC generatedl6 T cell lines specific for PDC. Most of the T cell lines specific for PDC proliferated in response to the purified E2 component but not to other components of PDC, indicating that PDC-E2 contains the immunodominant T cell autoepitopes in PBC, In order to investigate further the T cell autoepitopes of PDC-E2, the proliferative response of T cells was studied using 33 different short peptldes covering the full amino acid sequenceof human PDC-E2. Ten out of 13 patients with PBC showed significant T cell proliferative responses to peptides containing 197-216, 216-235, 221-240, 235-254 and 251-270 amino acid residues. Repeated stimulation of T cells with the above 5 peptides generated six T cell lines specific for peptide 216-235 from 4 patients with PBC. The critical amino acids for the recognition of the 14 peptides were estimated to be amino acids (EXDK) by the T cell proliferation experlment using alanine or serine-substituted versions of the original 14 peptides. One of the human PDC-E2 peptide-specificT cell lines cross-reacted with E. coil PDC-E2 peptidcs. We suspect that, in response to an infection such as E. coli, T cells may recognize peptides similar to PDC-E2 of the infecting organism. When these T cells are stimulated by antigens with aberrantly expressed HLA class II on biliary epithelial cells, molecular nfimicry subsequently occurs and this initiates an autoimmune response leading to the destruction of bile ducts.
P-124
INCREMENT OF Vfl4-POSITIVET CELLS IN LIVER OF NEONATALLY THYMECTOMIZED A/J MICE AND EXPERIMENTAL HEPATITIS INDUCED BY PROPIONIBACTERIUM ACNES AND LIPOPOL¥SACCHARIDE E. Sanada, Y. Watanabe, M. Kamiyasu, M. Naiki, M. Myozaki, M. Kitamoto, T. Nakanishi, G. Kajiyama. First Dept. of Internal Medicine, Himshima Univ. of Medicine, Himshima. Institute of Bid-Active Science, Hyogo, Japan. During the healing stage of acute liver injury induced by administration of sublethal doses of heat-killed Pmpionibacterium acnes (P. acnes) and lipopolysaccharide (LPS), neonatally thymectomized (NTx) mice show the pathology like autoimmune hepatitis accompanied by production of autoantibodies to liver specific lipoprotein and antinuclear antibodies and mononuclear cell infiltration in the portal area. We also have showed that both CD4 ~T cells and CD8 ~T cells play a critical role in the inflammatory liver in this model. To clarify the involvement of the specific T cell repertoire in this hepatitis, Vfl usage of liver T cells and spleen T cells was analyzed by flow cytometry using anti-CD3 and the set of mAbs to TCRV,8 such as V,83, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 17. Neonatal thymectomy was performed between 2 and 3 days after birth. Hepatitis was induced in the mice by an intravenous injection of 1.5 rag/20 g body weight of P. aches followed by an intravenous injection of a non-lethal level (0.05 /Zg) of LPS 7 days later. Mice were dissected 2 weeks after the LPS injection. Liver-infiltrating lymphocytes were separated by the method of Abe et al. using a gradient centrifugation. The proportion of T cells expressing various V/3 TCRs were analyzed in spleen cells and infiltrating lymphocytes of liver by three color flow cytometry. In results, V,84 TCRexpressing T cells among liver-infiltrating lymphocytes increased in number in NTx A/J mice with inflammatory liver injury compared with non-thymectomized A/J mice, while there were no difference in the number of that in spleen cells between NTx and non-thymectomized A/J mice. There were no increase in the number of V.83, 5, 7, 8, 9, 10~ l 1, 12, 14, 17 in liver-infiltrating lymphocytes and spleen cells in NTx A/J mice. This suggests that T cells expressing V/?4 TCR are involved in the pathogenesis of this experimental hepatitis in NTx A/J mice.
PBC LIKE LESION IS INDUCED BY IMMUNIZATION WITH RECOMBINANT PDC-E2/BCKD-E2 HYBRID MOLECULE AND LIPOPOLYSACCHARIDE INJECTION IN N E O N A T A L L Y THYMECTOMIZED MICE. T . M a s a n a g a , Y . W a t a n a b e , P.S.C. L e u n g , M . K a m i y a s u , E . S a n a d a , M . M y o z a k i , T . N a k a n i s h i , G . K a j i y a m a , M.E. Gershwin. 1st Dept. of I n t e r n a l Medicine, Hiroshima Univ. of M e d i c i n e , H i r o s h i m a , Japan., I n t e r n a l Medicine, Univ. of C a l i f o r n i a a t Davis, USA a ".t~Jt
< I n t r o d u c t i o n > T h i s s t u d y w a s d e s i g n e d to t e s t the h y p o t h e s i s t h a t a u t o i m m u n i t y to PDC-E2 or BCKD E2 itself can i n d u c e the p a t h o l o g y of PBC. < Methods >Neonatally thymectomy (NTx) was p e r f o r m e d 2 d a y s a f t e r b i r t h . The mice in g r o u p A w e r e i m m u n i z e d w i t h a m i x t u r e of r e c o m b i n a n t PDC-E2/BCKDE2 h y b r i d molecule (150/zg) and FCA a f t e r NTx. The mice in g r o u p B w e r e i m m u n i z e d w i t h h y b r i d m o l e c u l e a n d FCA w i t h o u t NTx. The mice in g r o u p C were i m m u n i z e d w i t h a m i x t u r e of h y b r i d m o l e c u l e and FCA a f t e r N T x and LPS (0 5/zg) w a s i n j e c t e d i n t r a v e n o u s l y 2 d a y s a f t e r each i m m u n i z a t i o n . < Results > The mice in group C showed positive AMA and a h i g h incidence of n o n s u p p u r a t i v e c h o l a n g i t i s w h i c h is c h a r a c t e r i z e d b y ' t h e mononuclear cell infiltration around the b i l i a r y e p i t h e l i a l cells. .--Conelusion > PBC-like lesion is i n d u c e d b y immuniz a t i o n w i t h PDC-E2 or BCKD-E2 p r o t e i n s , a n d e n d o t o x i n such as l i p o p o l y s a c c h a r i d e m a y a u g m e n t the p a t h o g e n e s i s of PBC.
1~-123 Histopathological Re-evaluation of Interlobular Bile Duct Damage in Patients with Autoimmune Hepatitis (AIH) in Japan Y.Takahashi, S.Maeyama, J.Koike, M.Kishi, Y.Aida, H.Yoshida, T.Takakuwa, T.Uchikoshi 2nd Dept.ot Pathology, St. Marianna University School of Medicine, Kawasaki, Japan Aims : Histopathological characteristics of AIH show severe parenchymal damage with chronic active hepatitis. In Europe and America, the appearance of interlobular bile duct damage in AIH is less frequent than in chronic hepatitis type C(C-CH). We have re-evaluated the histopathologioal changes of interlebular bile duct damage in AIH, and have compared them with chronic hepatitis type B(B-CH) and C-CH. Materials & Methods: Forty-six cases of AIH satisfying the criteria advocated in 1992, showing "/-globulin (more than 2g/dl) and negative for serum anti-HCV antibody were studied in comparison with 242 cases of C-CH and 52 of B-CH. Bile duct damage was histologically considered present if there were swelling and stratification with papillary growth of the epithelial cells, disruption of basement membrane by mononuolear cell infiltrates, vacuolizations of epithelial cells, degeneration or mitotic activity, or a combination of these. Results: 1)The incidence of bile duct damage in AIH was 35 cases(76%), and the frequency is higher than in B-CH (12 cases,23%) and C-CH(86 cases, 36%). 2) The bile duct damage in AIH was not related to lymphtollicle formation. However, damages seen in C-CH were related to formation of lymphfollicles. The damages in B-CH were mild and occurred with the progress of histological changes Conclusion: The criteria of AIH decided at the international meeting in Brighton in 1993 did not include interlobular bite duct damage. The fact that the frequency of bile duct damage in AIH in Japan is higher than that in C-CH and B-CH may be due to the development of cirrhosis in AIH in Europe and America. In Japan, bile duct changes in AIH are thought to be similar to those in autoimmune cholangiopathy (AC), making the histological differentiation between AIH and AC necessary.
$67
D 1")") Z°-L~Z'~ IDENTIFICATION OF IMMUNODOMINANT T CELL EPITOPES OF PYRUVATE DEHYDROGENASE COMPLEXES IN PRIMARY BILIARY CIRRHOSIS S. Shlmoda, Y. Ichiki, M. Nakamura, K. Hayashida, H. lshibash[, Y. Niho 1st Dept. of Internal Medicine, Faculty of medicine, Kyushu Univ. In order to clarify the immunopathogenesis of primary biliary cirrhosis (PBC), we identified immunodominant T cell autoepitopos of mitochondrial antigens in PBC. Peripheral blood T cells purified from 13 patients with PBC were tested for proliferative responses to pyruvate dehydrogenase complexes (PDC). T cells derived from all patients with PBC but none of those from controls showed proliferative responses to PDC. Repeated stimulation ofT cells with PDC generatedl6 T cell lines specific for PDC. Most of the T cell lines specific for PDC proliferated in response to the purified E2 component but not to other components of PDC, indicating that PDC-E2 contains the immunodominant T cell autoepitopes in PBC, In order to investigate further the T cell autoepitopes of PDC-E2, the proliferative response of T cells was studied using 33 different short peptldes covering the full amino acid sequenceof human PDC-E2. Ten out of 13 patients with PBC showed significant T cell proliferative responses to peptides containing 197-216, 216-235, 221-240, 235-254 and 251-270 amino acid residues. Repeated stimulation of T cells with the above 5 peptides generated six T cell lines specific for peptide 216-235 from 4 patients with PBC. The critical amino acids for the recognition of the 14 peptides were estimated to be amino acids (EXDK) by the T cell proliferation experlment using alanine or serine-substituted versions of the original 14 peptides. One of the human PDC-E2 peptide-specificT cell lines cross-reacted with E. coil PDC-E2 peptidcs. We suspect that, in response to an infection such as E. coli, T cells may recognize peptides similar to PDC-E2 of the infecting organism. When these T cells are stimulated by antigens with aberrantly expressed HLA class II on biliary epithelial cells, molecular nfimicry subsequently occurs and this initiates an autoimmune response leading to the destruction of bile ducts.
P-124
INCREMENT OF Vfl4-POSITIVET CELLS IN LIVER OF NEONATALLY THYMECTOMIZED A/J MICE AND EXPERIMENTAL HEPATITIS INDUCED BY PROPIONIBACTERIUM ACNES AND LIPOPOL¥SACCHARIDE E. Sanada, Y. Watanabe, M. Kamiyasu, M. Naiki, M. Myozaki, M. Kitamoto, T. Nakanishi, G. Kajiyama. First Dept. of Internal Medicine, Himshima Univ. of Medicine, Himshima. Institute of Bid-Active Science, Hyogo, Japan. During the healing stage of acute liver injury induced by administration of sublethal doses of heat-killed Pmpionibacterium acnes (P. acnes) and lipopolysaccharide (LPS), neonatally thymectomized (NTx) mice show the pathology like autoimmune hepatitis accompanied by production of autoantibodies to liver specific lipoprotein and antinuclear antibodies and mononuclear cell infiltration in the portal area. We also have showed that both CD4 ~T cells and CD8 ~T cells play a critical role in the inflammatory liver in this model. To clarify the involvement of the specific T cell repertoire in this hepatitis, Vfl usage of liver T cells and spleen T cells was analyzed by flow cytometry using anti-CD3 and the set of mAbs to TCRV,8 such as V,83, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 17. Neonatal thymectomy was performed between 2 and 3 days after birth. Hepatitis was induced in the mice by an intravenous injection of 1.5 rag/20 g body weight of P. aches followed by an intravenous injection of a non-lethal level (0.05 /Zg) of LPS 7 days later. Mice were dissected 2 weeks after the LPS injection. Liver-infiltrating lymphocytes were separated by the method of Abe et al. using a gradient centrifugation. The proportion of T cells expressing various V/3 TCRs were analyzed in spleen cells and infiltrating lymphocytes of liver by three color flow cytometry. In results, V,84 TCRexpressing T cells among liver-infiltrating lymphocytes increased in number in NTx A/J mice with inflammatory liver injury compared with non-thymectomized A/J mice, while there were no difference in the number of that in spleen cells between NTx and non-thymectomized A/J mice. There were no increase in the number of V.83, 5, 7, 8, 9, 10~ l 1, 12, 14, 17 in liver-infiltrating lymphocytes and spleen cells in NTx A/J mice. This suggests that T cells expressing V/?4 TCR are involved in the pathogenesis of this experimental hepatitis in NTx A/J mice.