Immunobiology of myasthenia gravis, experimental autoimmune myasthenia gravis, and Lambert-Eaton syndrome.
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Animals↗
Biomedical subjects
J Lindstrom
Publications and source records attributed to J Lindstrom.
Suppression of cellular and humoral immunity to T-dependent antigens by calorie restriction.
Calorie restriction is known to preserve the immunologic function and prolong the life span of autoimmune-susceptible mice. In order to determine the influence of calorie restriction on cellular and humoral immunity, we tested lymphocyte proliferative response and antibody response to T-dependent antigens in C57Bl/6 mice maintained on restricted diets. C57Bl/6 mice fed a low-calorie diet demonstrated a marked reduction in T-dependent-antigen-specific lymphocyte proliferation and antibody response when compared to mice fed ad libitum. The depressed lymphocyte response seen in calorie-restricted animals is attributed to a defect in both the macrophages and T cells in antigen processing, presentation, and/or proliferation.
Animals↗
Structural features of the nicotinic acetylcholine receptor revealed by antibodies to synthetic peptides.
Antibodies were raised to the amino- and carboxy-terminal decapeptides of Torpedo californica acetylcholine receptor. Structural studies of the native receptor using the antipeptide antibodies as probes proved the existence of the carboxy terminal sequence in the alpha subunit predicted from its cDNA sequence and supported structural models of the native receptor that place the carboxy termini on the intracellular side. The amino termini of the subunits were not accessible on the surface of native receptor.
Amino Acid Sequence↗
[Oral stratified epithelial cancer].
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Adult↗
Expression of acetylcholine receptor alpha-subunit mRNA during differentiation of the BC3H1 muscle cell line.
The accumulation of translatable acetylcholine receptor alpha-subunit mRNA was examined in the BC3H1 muscle cell line in response to serum and cell growth. Relative amounts of alpha-subunit mRNA were quantitated during differentiation by cell-free translation and immunoprecipitation with an alpha-subunit-specific monoclonal antibody. Logarithmically growing cells do not possess cell surface acetylcholine receptors; however, a significant amount of alpha-subunit mRNA is detectable in cells under these conditions. Furthermore, alpha-subunit is synthesized in growing undifferentiated cells at a rate similar to that of differentiated cultures. Following growth arrest of BC3H1 cells, surface receptors are induced to levels greater than 100-fold above that of growing cells. The relative level of translatable alpha-subunit mRNA in differentiated cells, however, is only approximately 4-fold greater than in growing cultures. Induction of alpha-subunit mRNA appears to be reversible since reinitiation of growth in quiescent differentiated BC3H1 cells results in a reduction in relative abundance of this mRNA species to levels comparable to that of undifferentiated cells and the concomitant loss of surface receptors. These results indicate that receptor expression during differentiation is regulated both post-translationally and at the level of receptor subunit mRNA accumulation.
Animals↗
Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers.
The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.
Animals↗
Monoclonal and anti-idiotypic antibodies as probes for receptor structure and function.
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Animals↗
The acetylcholine receptor channel from Torpedo californica has two open states.
We recorded single channel currents activated by agonist binding to purified acetylcholine receptors from the electric organ of Torpedo californica reconstituted in planar lipid bilayers. Analysis of single channel records indicate that the acetylcholine receptor channel displays two kinetically distinct open states which differ in their mean open times but have similar channel conductances.
Animals↗
Regulation of surface expression of acetylcholine receptors in response to serum and cell growth in the BC3H1 muscle cell line.
The regulation of acetylcholine receptor synthesis, assembly, and cell surface expression in response to serum and cell growth in the BC3H1 muscle-like cell line have been examined. In the presence of media containing low concentrations of serum, BC3H1 cells withdraw from the cell cycle and express high levels of cell surface acetylcholine receptors. Exposure of quiescent, differentiated BC3H1 cells to high concentrations of serum results in re-entry of cells into the cell cycle and a loss of surface acetylcholine receptors by a first order process (t1/2 = 8.6 h) which appears to be initiated within 1 h following serum addition. Under these conditions, the rate of synthesis of acetylcholine receptor alpha-subunits is not significantly reduced; however, the efficiency of assembly of alpha-subunits into functional acetylcholine receptors is significantly less than that observed in quiescent cells. The receptors which are assembled following serum stimulation of quiescent cells are not transported to the cell surface, nor do they accumulate within the cells in a functional form (defined by the ability to bind alpha-bungarotoxin) in the cells. We conclude that reinitiation of cell division in quiescent cultures of BC3H1 cells results in alterations in several post-translational processing steps required for the surface expression of acetylcholine receptors.
Animals↗
Cellular immune response to acetylcholine receptors in murine experimental autoimmune myasthenia gravis: inhibition with monoclonal anti-I-A antibodies.
Gene(s) at the I-A subregion of the murine major histocompatibility complex influence susceptibility to experimental autoimmune myasthenia gravis. C57Bl/6 mice immunized with acetylcholine receptors (AChR) in complete Freund's adjuvant demonstrated cellular and humoral immune responses to AChR. They developed muscle weakness characteristic of myasthenia gravis and demonstrated a reduction in the muscle AChR content. The kinetics of AChR-specific lymphocyte proliferation generally correlate with anti-AChR antibody response. AChR-specific lymphocyte proliferation was also observed in C57Bl/6 splenocytes after secondary immunization with AChR. The in vitro cellular reactivity to AChR in experimental autoimmune myasthenia gravis (EAMG) mice (C57Bl/6) was suppressed by monoclonal anti-I-Ab antibodies directed against private (Ia20) or public (Ia8) specificities, suggesting a critical role for these Ia determinants in the cellular immune response to AChR in murine EAMG.
Animals↗
Deficient T-cell mitogen response in murine experimental autoimmune myasthenia gravis: a defect in the adherent cell population.
T-Lymphocyte number and functions are often reduced, while B-lymphocyte function is often increased in patients with autoimmune disorders. To study the mechanisms responsible for these T-cell malfunctions in autoimmunity we adapted the murine experimental autoimmune myasthenia gravis (EAMG) model. Splenocytes from C57BL/6 mice immunized with acetylcholine receptors (AChR) in complete Freund's adjuvant (CFA) produced approximately half the amount of concanavalin A (Con A)-induced interleukin 2 (IL-2) as did splenocytes of CFA-inoculated controls. Further, AChR plus CFA-immunized splenocytes showed a marked reduction in T-cell proliferative responses induced by Con A or phytohemagglutinin when compared with CFA-inoculated controls. By contrast, lipopolysaccharide-induced B-cell function is preserved. Deficient Con A splenic T-cell response is seen early after secondary inoculation with CFA or AChR in CFA. T-Cell recovery occurs in CFA-inoculated mice but not in AChR plus CFA-inoculated mice. Defective Con A splenic T-cell response seen early after secondary immunization with CFA or AChR in CFA is due to the presence of a defective splenic adherent cell population. Moreover, defective Con A splenic T-cell response seen after established autoimmunity to AChR in EAMG is also due to the presence of a defective splenic adherent cell population.
Animals↗
Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor.
Eleven cloned hybridomas which secrete antibodies to acetylcholine receptors from human muscle have been prepared. All of these monoclonal antibodies to have the same basic specificity as shown by competition for binding to the main immunogenic region on the receptor, but these antibodies differ in fine specificity as shown by reaction with denatured receptor subunits and interspecies cross-reaction.
Animals↗
Single-channel recordings from purified acetylcholine receptors reconstituted in bilayers formed at the tip of patch pipets.
The channel of the purified acetylcholine receptor from Torpedo californica electric organ reconstituted in lipid vesicles was assayed by direct electrical recording using patch-clamp pipets. High-resistance seals were obtained by gentle suction of vesicles into the pipet or after the formation of lipid bilayers from monolayers at the tip of the pipet. Single-channel currents were activated by three cholinergic ligands: acetylcholine, carbamylcholine, and suberyldicholine. The single-channel conductance, gamma, was 40 +/- 5 pS in 0.5 M NaCl, irrespective of the agonist used. The distributions of channel open times were fitted by a sum of two exponentials. The lifetimes of the two exponential components were a factor of 2 longer for suberyldicholine than for acetylcholine or carbamylcholine. At desensitizing concentrations of agonists the single events appeared in paroxysms of channel activity followed by quiescent periods. These results suggest that the full cycle of solubilization, purification, and reconstitution of this membrane receptor can be achieved without impairment of channel function.
Acetylcholine↗
Characterization of the mRNA for mouse muscle acetylcholine receptor alpha subunit by quantitative translation in vitro.
The nicotinic acetylcholine receptor of mammalian skeletal muscle is a multisubunit membrane glycoprotein whose synthesis is regulated by developmental and physiological cues. We report here the identification and characterization of the primary translation product of alpha subunit mRNA. The alpha subunit synthesized in rabbit reticulocyte lysate is approximately 2000 larger in apparent molecular weight than the native alpha subunit polypeptide found in acetylcholine receptor. Evidence from peptide maps and the effect of co-translational incubation with dog pancreas microsomes suggests that the in vitro product differs in two ways from native alpha subunit: 1) it is synthesized with an NH2-terminal signal peptide which is removed in vivo, and 2) the in vitro product is not glycosylated. We have characterized the alpha subunit mRNA activity by using a quantitative the membrane-bound polysome fraction. It is poly(A+) and approximately 2000 nucleotides long. Finally, we have shown that in BC3H-1 cells, alpha subunit mRNA is regulated developmentally. We detected a 10-fold increase in the relative abundance of alpha subunit mRNA in cells which had undergone the transition from log phase growth to differentiated myoblast.
Animals↗
Progress on studies of myasthenia gravis during 1982.
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Antibodies, Monoclonal↗
Assembly in vivo of mouse muscle acetylcholine receptor: identification of an alpha subunit species that may be an assembly intermediate.
We have studied assembly of acetylcholine receptor in vivo using subunit-specific monoclonal antibodies and immunoprecipitation with alpha-bungarotoxin and antitoxin. We have identified three distinct forms of the alpha subunit. The newly synthesized alpha subunit species has a sedimentation coefficient of 5S and is recognized only by antibody specific for SDS-denatured alpha subunit. We have called this species alpha 61. The 5S alpha Tx species is not associated with beta subunits and is probably monomeric. alpha Tx is formed from alpha 61 with a half-time of 15 min and an efficiency of approximately equal to 30%. Formation of alpha Tx involves a conformational change, and we suggest that this conformation is dependent upon or stabilized by disulfide bond formation. The assembly of alpha Tx with beta subunits (and probably gamma and delta) into a 9S complex appears to be an efficient but slow process requiring more than 90 min. Unassembled alpha 61 subunits are degraded rapidly. However, subunit degradation is a result of failure to assemble, rather than its cause.
Animals↗
cDNA clone for the alpha subunit of the acetylcholine receptor from the mouse muscle cell line BC3H-1.
Sequences from a gene coding for mouse acetylcholine receptor alpha subunit have been inserted into a recombinant plasmid and cloned in Escherichia coli. mRNAs for acetylcholine receptors occur in low abundance in vertebrate muscle. To clone the mouse alpha-subunit cDNA, we made use of (i) a cell line, BC3H-1, that overproduces the alpha-subunit mRNA and (ii) a polysome fractionation procedure that results in enrichment of alpha-subunit mRNA. Polyadenylylated RNA was used to construct a cDNA library of 750 recombinant clones. Acetylcholine receptor-specific sequences were identified by hybrid-selected translation, followed by monoclonal antibody precipitation and peptide mapping of the translation product. One clone (pA59) that fit these criteria was found in the first 80 isolates. It had a 700-base-pair insert that was excised with Pst I. Blot-hybridization experiments with nick-translated pA59 DNA showed that BC3H-1 cells contain 100-1,000 times more alpha-subunit mRNA than does newborn or adult mouse muscle. Blot hybridization of restriction digests of mouse liver DNA revealed that pA59 is homologous to a very small number (probably one) of genomic sequences.
Animals↗
Immunohistochemical localization of monoclonal antibodies to the nicotinic acetylcholine receptor in chick midbrain.
We used the indirect immunofluorescence method to determine the crossreactivity of a library of 57 monoclonal antibodies (mAbs) against each of the subunits of the nicotinic acetylcholine receptor (nAcChoR) isolated from Torpedo and Electrophorus electric organs or from fetal calf and human muscle, with specific neural elements in the midbrain of the chick. Out of 17 mAbs that recognized motor end plates on chick muscle, 14 produced a similar pattern of labeling in the midbrain: the neuronal perikarya and dendrites in the lateral spiriform nucleus (SpL) were intensely labeled, and there was moderate labeling of fibers in certain of the deeper layers of the optic tectum, which disappeared after the SpL was destroyed electrolytically. Two lines of evidence suggest that the mAbs may be crossreacting with nAcChoRs in the midbrain. First, all of the mAbs that stained the SpL also stained neuromuscular junctions in skeletal muscle, whereas none of the 40 mAbs that failed to stain end plates crossreacted with the SpL; second, in vitro immunological studies and blocking experiments on tissue sections (in which unlabeled mAbs were used to block the staining of a directly fluorescein-treated mAb) indicated the presence of mAbs specific for unique antigenic determinants on all four of the subunits (alpha, beta, gamma, and delta) from Torpedo nAcChoR in chick midbrain and muscle. On the other hand, the distribution of mAb staining in the optic tectum does not closely parallel that of either acetylcholinesterase staining or of 125I-labeled alpha-bungarotoxin binding; no toxin binding has been observed autoradiographically in the SpL, but the nucleus does contain moderately dense acetylcholinesterase staining. Take together, our observations suggest that there may be a cholinergic input to the SpL and that the projection fibers from the SpL to the optic tectum (which are also stained with an antiserum to [Leu]enkephalin) may contain presynaptic nAcChoRs. It is clear, however, that the distribution of the putative nAcChoRs, alpha-bungarotoxin binding sites, and acetylcholinesterase staining in the avian midbrain are quite different, although they do overlap to some degree in the deeper layers of the optic tectum.
Acetylcholinesterase↗