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TE671 cell and solubilized human muscle acetylcholine receptors: Similarities and differences in immunological properties
904
Abstracts
Studies of EAMC using Torpedo AChR as antigen have the drawback that the response against autologous AChR is cross-reactive. To circumvent this problem we have immunised mice with a recombinant polypeptide (r37-4371 representing most of the & -subunit of the human AChR including the main immunogenic region to which the majority of patients’ anti-AChR antibodies bind, C57Bl/6, Balb/c and BKTO mice were immunised with r37-437 purified from E.coli by gel electrophoresis and the response to autologous AChR was foliowed. All of the mice produced antibodies which bound both to the immunogen and to native mouse and human AChRs in precipitation assays. none of the immunised mice developed AChR loss (as Hoyyy, assessed by I-a-bungarotoxin binding to diaphragm muscle) or had antibody complexed with AChRs in vivo. The binding of sera from BKTO mice to human AChR was inhibited by recombinant polypeptides r37-437 (the immunogen) and by t-65-437, but not by r37-346 or r37-281/360-437 suggesting that all the antibodies to native human AChR bound in the region of amino acids 346-359. This was confirmed by assays in which a peptide for the human sequence 351-368 (gift of Prof. S. Fuchs) inhibited binding but the corresponding Since residues 351-368 are known to be ;;;pNNm~quence dtd not. antibodIes produced against this region would not be expected tb have access to this intracellular determinant in vivo. In this study antibodies raised against denatured recombinant human AChR &_ -subunit showed negligible binding to AChR determinants It will be expressed extracellularly at the neuromuscular junction. with AChR subunits purified interesting to see whether immunisation from eukaryotlc expression systems will produce antibodies which can bind to these conformationally dependent epitopes and thus induce disease.
TE671 CELL AND SOLUBILIZED HUMAN MUSCLE ACETYLCHOLINE SIMILARITIES AND DIFFERENCES IN IiWtJNOLOGICAL PROPERTIES RECEPTORS: Evelyne Morel, INSERM U25, CNRS, UA122, Hopital Necker, Sevres, 75015 Paris, France
161 rue de
Evelyne Morel, Ana Cardona, Gideon Goldstein*& Jean-Francois Bach Research Institute, Hopital Necker, Paris, France &*Immmobiology Annandale, NJ, USA
The human medulloblastoma cell line TE67 1 expresses a nicotinic acetylcholine recentor (AChR) on its surface, and extracts of these cells can be used in imm*unoprecipitation assays for anti-AChR antibody determination in sera from mvasthenic (MC3 natients. To evaluate the correlation between the anti-AChR antibody titers obtained with solubilized human muscle AChR (H.AChR) and TE671.AChR we performed experiments in a series of TE671 cultures by evaluating the binding of MG sera to cells preincubated with the specific neurotoxin ligand ubungarotoxin (oBg~) and on solubilized extracts of these cells: Reaction kinetics were studied. Parallel determinations of anti-AChR antibodies with solubilized human muscle and TE671 AChRs were made for MC sera obtained in several clinical types (ocular and generalized idiopathic, transmitted or induced forms) and with different levels of anti-AChR antibody titers. The response of MG sera with negative or limit anti-AChR antibody titers from patients with ocular MG was enhanced in some cases. Other experiments were made using ACh agonists or the thymic hormone, thymopoietin (Tpo), which had been shown to bind similarly either Torpedo or H.AChR . The specificity of Tpo binding to TE671 cells was demonstrated by inhibiting the incorporation of t2%Tpo in culture plates either by nonradioactive Tpo or by aBgt. Tpo binds to TE671 with an apparent affinity similar to that observed on H.AChR.
Abstracts Our results suggest both similarities and differences between cellular and muscular nAChRs with the presence of determinants which would be recognized specifically by MG sera. The determinants would exist either on all or on some types, i.e., TE671 and ocular muscle nAChRs , Therefore use of the TE67 1 cell line would be interesting not only to replace the human muscle antigen, but also in MG diagnosis for particular sera not reactive with H.AChR.
MOUSE T AND B CELL RECOGNITION THE RECOMBINANT HUMAN MUSCLE CHOLINE RECEPTOR C( -SUBUNIT.
EPITOPES ACETYL-
ON
Dr. J. Palace, Neurosciences Group, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford. OX3 9DU Tel No 0865-752327 Jacqueline Palace, A. and J. Newsom-Davis.
Vincent,
D. Beeson, N. Willcox Oxford, England
Antibodies to human muscle acetylcholine receptor (AChR) are of pathogenic importance in the human disease myasthenia gravis (MCI, and are believed to be T cell dependent. Most of them are directed against the o( -subunit. Similar specificities dominate the response in experimental animals. In order to investigate the intrinsic immunogenicity of the o( -subunit, we have immunised inbred strains of mice (CB57Bl/6, BalbC, SWR, SJL), of defined immunogenetic background, with purified human o( -subunit recombinant fragment r37-437, and examined T cell and serum antibody responses. T cell lines were propagated in vitro from lymph nodes in the presence of r37-437 and ConA supernatant, and tested in an in vitro proliferation assay against smaller h-subunit recombinant fragme@. Antisera w;er; testedlAy immunoprecipitation for reactivity with I-(t-37-437) with I- 6(-BuTx-human AChR, and the specificity of the binding examined by inhibition with unlabelled smaller recombinant fragments. Preliminary results show that T cell clones specific for r37-437 can be raised from several strains of mice. Antibody responses to r37-437 are variable and in some strains anti-(r37-437) antibodies cross-react with native human AChR. Identification of the primary amino acid sequences involved in both T and B cell epitopes is in progress,
UCENT STUDIES ON STKUCTtiRE AND FUNCTION OF THE MAIN IMMUNOGENIC H;EGION OF THE ACETYLCHOLINE RECEPTOR
~Socrates J. Hellenic Athens
Pasteur Institute, Tel. 11521, Greece
Tzartos. 12’7, Vas. Sophias Ave., (30-l)-6430044 kordossi, I. Papadouil, Athens, Greece; I. Hadjidakis and C. M.T. Cung and M. Marraud: Nancy. France.
A. D. Sophianos Sakarellos:
, and H. Loutrari: loannina, Greece;
‘The two-thirds of the antibodies to the nicotinic aoetylcholine receptor (AChR) in myasthenia gravis (MG) and in rats immunized with intact, AChR bind to the main immunogenic region (MIR), on the extracellular side of the AChR a-subunit. Anti-MIR monoclonal antibodies
905
Abstracts
Studies of EAMC using Torpedo AChR as antigen have the drawback that the response against autologous AChR is cross-reactive. To circumvent this problem we have immunised mice with a recombinant polypeptide (r37-4371 representing most of the & -subunit of the human AChR including the main immunogenic region to which the majority of patients’ anti-AChR antibodies bind, C57Bl/6, Balb/c and BKTO mice were immunised with r37-437 purified from E.coli by gel electrophoresis and the response to autologous AChR was foliowed. All of the mice produced antibodies which bound both to the immunogen and to native mouse and human AChRs in precipitation assays. none of the immunised mice developed AChR loss (as Hoyyy, assessed by I-a-bungarotoxin binding to diaphragm muscle) or had antibody complexed with AChRs in vivo. The binding of sera from BKTO mice to human AChR was inhibited by recombinant polypeptides r37-437 (the immunogen) and by t-65-437, but not by r37-346 or r37-281/360-437 suggesting that all the antibodies to native human AChR bound in the region of amino acids 346-359. This was confirmed by assays in which a peptide for the human sequence 351-368 (gift of Prof. S. Fuchs) inhibited binding but the corresponding Since residues 351-368 are known to be ;;;pNNm~quence dtd not. antibodIes produced against this region would not be expected tb have access to this intracellular determinant in vivo. In this study antibodies raised against denatured recombinant human AChR &_ -subunit showed negligible binding to AChR determinants It will be expressed extracellularly at the neuromuscular junction. with AChR subunits purified interesting to see whether immunisation from eukaryotlc expression systems will produce antibodies which can bind to these conformationally dependent epitopes and thus induce disease.
TE671 CELL AND SOLUBILIZED HUMAN MUSCLE ACETYLCHOLINE SIMILARITIES AND DIFFERENCES IN IiWtJNOLOGICAL PROPERTIES RECEPTORS: Evelyne Morel, INSERM U25, CNRS, UA122, Hopital Necker, Sevres, 75015 Paris, France
161 rue de
Evelyne Morel, Ana Cardona, Gideon Goldstein*& Jean-Francois Bach Research Institute, Hopital Necker, Paris, France &*Immmobiology Annandale, NJ, USA
The human medulloblastoma cell line TE67 1 expresses a nicotinic acetylcholine recentor (AChR) on its surface, and extracts of these cells can be used in imm*unoprecipitation assays for anti-AChR antibody determination in sera from mvasthenic (MC3 natients. To evaluate the correlation between the anti-AChR antibody titers obtained with solubilized human muscle AChR (H.AChR) and TE671.AChR we performed experiments in a series of TE671 cultures by evaluating the binding of MG sera to cells preincubated with the specific neurotoxin ligand ubungarotoxin (oBg~) and on solubilized extracts of these cells: Reaction kinetics were studied. Parallel determinations of anti-AChR antibodies with solubilized human muscle and TE671 AChRs were made for MC sera obtained in several clinical types (ocular and generalized idiopathic, transmitted or induced forms) and with different levels of anti-AChR antibody titers. The response of MG sera with negative or limit anti-AChR antibody titers from patients with ocular MG was enhanced in some cases. Other experiments were made using ACh agonists or the thymic hormone, thymopoietin (Tpo), which had been shown to bind similarly either Torpedo or H.AChR . The specificity of Tpo binding to TE671 cells was demonstrated by inhibiting the incorporation of t2%Tpo in culture plates either by nonradioactive Tpo or by aBgt. Tpo binds to TE671 with an apparent affinity similar to that observed on H.AChR.
Abstracts Our results suggest both similarities and differences between cellular and muscular nAChRs with the presence of determinants which would be recognized specifically by MG sera. The determinants would exist either on all or on some types, i.e., TE671 and ocular muscle nAChRs , Therefore use of the TE67 1 cell line would be interesting not only to replace the human muscle antigen, but also in MG diagnosis for particular sera not reactive with H.AChR.
MOUSE T AND B CELL RECOGNITION THE RECOMBINANT HUMAN MUSCLE CHOLINE RECEPTOR C( -SUBUNIT.
EPITOPES ACETYL-
ON
Dr. J. Palace, Neurosciences Group, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford. OX3 9DU Tel No 0865-752327 Jacqueline Palace, A. and J. Newsom-Davis.
Vincent,
D. Beeson, N. Willcox Oxford, England
Antibodies to human muscle acetylcholine receptor (AChR) are of pathogenic importance in the human disease myasthenia gravis (MCI, and are believed to be T cell dependent. Most of them are directed against the o( -subunit. Similar specificities dominate the response in experimental animals. In order to investigate the intrinsic immunogenicity of the o( -subunit, we have immunised inbred strains of mice (CB57Bl/6, BalbC, SWR, SJL), of defined immunogenetic background, with purified human o( -subunit recombinant fragment r37-437, and examined T cell and serum antibody responses. T cell lines were propagated in vitro from lymph nodes in the presence of r37-437 and ConA supernatant, and tested in an in vitro proliferation assay against smaller h-subunit recombinant fragme@. Antisera w;er; testedlAy immunoprecipitation for reactivity with I-(t-37-437) with I- 6(-BuTx-human AChR, and the specificity of the binding examined by inhibition with unlabelled smaller recombinant fragments. Preliminary results show that T cell clones specific for r37-437 can be raised from several strains of mice. Antibody responses to r37-437 are variable and in some strains anti-(r37-437) antibodies cross-react with native human AChR. Identification of the primary amino acid sequences involved in both T and B cell epitopes is in progress,
UCENT STUDIES ON STKUCTtiRE AND FUNCTION OF THE MAIN IMMUNOGENIC H;EGION OF THE ACETYLCHOLINE RECEPTOR
~Socrates J. Hellenic Athens
Pasteur Institute, Tel. 11521, Greece
Tzartos. 12’7, Vas. Sophias Ave., (30-l)-6430044 kordossi, I. Papadouil, Athens, Greece; I. Hadjidakis and C. M.T. Cung and M. Marraud: Nancy. France.
A. D. Sophianos Sakarellos:
, and H. Loutrari: loannina, Greece;
‘The two-thirds of the antibodies to the nicotinic aoetylcholine receptor (AChR) in myasthenia gravis (MG) and in rats immunized with intact, AChR bind to the main immunogenic region (MIR), on the extracellular side of the AChR a-subunit. Anti-MIR monoclonal antibodies
905