Immunization of rats with polypeptide chains from torpedo acetylcholine receptor causes an autoimmune response to receptors in rat muscle.

Four polypeptide chains were purified from acetylcholine receptor of Torpedo californica electric organ. Their apparent molecular weights were 64,000, 57,000, 49,500, and 38,000. Rats immunized with any of the four chains produced antibodies that crossreacted with rat muscle receptors in vivo. Specificities of anti-chain sera were evaluated in vitro by reaction with native receptor solubilized from electric organs and muscles of several species and by inhibition of this reaction with the purified polypeptide chains. The chains are immunologically distinct from one another. Antigenic determinants comparable to each chain of torpedo receptor are found in receptor from both rat and human muscle. At least part of each of these determinants is exposed on the extracellular surface of the muscle membrane. The most immunogenic determinants on native receptor are lost on denaturation to polypeptide chains. Its component peptides are much less immunogenic than native receptor, and induce antibodies of different specificity. Anti-receptor antibodies of many specificities can cause experimental autoimmune myasthenia gravis.     

Atypical antiinsulin receptor antibodies in a patient with type B insulin resistance and scleroderma

We studied a 23-yr-old woman with scleroderma and type B insulin resistance. The association with autoimmune disease suggested that the insulin resistance resulted from autoantibodies to the insulin receptor. However, in preliminary studies, serum antireceptor antibodies were not detected in an assay that measures the ability of the antibodies to inhibit insulin binding to the insulin receptor. Antireceptor antibodies were subsequently detected by their ability to immunoprecipitate affinity-labeled receptors. After the patient had received immunosuppressive therapy with prednisone and cyclophosphamide for 3 months, her insulin resistance remitted, and she developed hypoglycemia. Simultaneously with the remission of insulin resistance, the titer of serum antireceptor antibody (measured by the immunoprecipitation assay) fell to less than 1% of the previous level. In a series of 21 patients, this is the first patient with antireceptor antibodies that bound to the insulin receptor without inhibiting insulin binding.     

Modulation of experimental myasthenia gravis by IVIg

Myasthenia Gravis (MG) is an autoimmune disease mediated by antibodies directed against the acetylcholine receptor (AChR). Treatment by IVIg is effective in acute forms of myasthenia gravis. In order to determine the in vivo effects of the various fractions of human immunoglobulins, we used an experimental model of myasthenia gravis in SCID mice. To this end, thymic cells from MG patients are transferred to these mice according to a well defined protocol. When establishing of the model, we noticed the appearance of anti-AChR antibodies and the loss of AChR expression at the muscle level. After treatment with IVIgG or IVIgM, the mice displayed a lower anti-AChR antibody titer compared to control mice (albumin treated) and the loss of the AChR number at the muscle was significantly reduced. These results obtained from one MG patient indicate that the human immunoglobulin preparations induce significant effects on pathogenic parameters in the SCID mouse model. Therefore this model is interesting to approach the mechanisms of action of human immunoglobulins and deserves further investigation.     

Myasthenia gravis und myasthene Syndrome

Neuromuscular transmission is compromised in a variety of disorders due to immunological, toxic or congenital mechanisms. Myasthenia gravis (MG) is the most frequent among these disorders. In about 15% of cases, MG is associated with a second autoimmune disorder mainly seen in rheumatologists. Some of the drugs used in rheumatology can exacerbate MG or even trigger immunologically the occurrence of MG. In most MG patients, antibodies to the acetylcholine receptor (AChR) are present, but around 10% have AChR antibodies that are only identified by novel methods, and up to 5% have muscle-specific kinase antibodies which define a different subgroup of myasthenia. Among those MG patients with anti-AChR antibodies, a number of clinical subtypes can be identified including early-onset MG (onset ≤ 40 years), late-onset MG (onset after 40 years) and thymoma-associated MG. Even though less common, it is important to recognize Lambert Eaton myasthenic syndrome (LEMS). The abnormality in LEMS is a presynaptic failure to acetylcholine release caused by antibodies to voltage-gated calcium channels. More than half of LEMS patients have small-cell lung cancer.     

Antigen-specific modulation of experimental myasthenia gravis: Nasal tolerization with recombinant fragments of the human acetylcholine receptor α-subunit

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AcChoR) is the major autoantigen. The immune response in these diseases is heterogeneous and is directed to a wide variety of T and B cell epitopes of AcChoR. Candidate molecules for specific immunotherapy of MG should, therefore, have a broad specificity. We used recombinant fragments of the human AcChoR, encompassing the extracellular domain of the alpha-subunit, or shorter fragments derived from it, in experiments to modulate EAMG. We have demonstrated that intranasal administration of these recombinant fragments, which represent a major portion of epitopes involved in MG, prevents the induction of EAMG in rats and immunosuppresses an ongoing disease, as assessed by clinical symptoms, weight loss, and muscle AcChoR content. These effects on EAMG were accompanied by a marked reduction in the proliferative T-cell response and IL-2 production in response to AcChoR, in reduced anti-self AcChoR antibody titers and in an isotype switch of AcChoR-specific antibodies, from IgG2 to IgG1. We conclude that nasal tolerance induced by appropriate recombinant fragments of human AcChoR is effective in suppressing EAMG and might possibly be considered as a therapeutic modality for MG.     

Syndromes of autoimmunity and hypoglycemia. Autoantibodies directed against insulin and its receptor

Humoral autoimmunity plays an important role in the pathogenesis of two forms of hypoglycemia. In one syndrome, antireceptor autoantibodies bind to the insulin receptor, mimic insulin action, and cause fasting hypoglycemia. In most patients with autoantibodies to the insulin receptor, there is other evidence of autoimmune disease as well. Interpretation of the standard tests used in evaluation of hypoglycemia may be confusing in these patients. For example, antireceptor antibodies may inhibit insulin binding, thereby inhibiting insulin clearance and elevating levels of plasma insulin. Nevertheless, because hypoglycemia suppresses beta-cell secretion, C-peptide levels are usually low. This constellation of data is consistent with surreptitious insulin injection. The most important laboratory test in the differential diagnosis is a direct assay for the presence of antibodies directed against the insulin receptor. Therapy with prednisone appears to alleviate the hypoglycemia rapidly, usually within 24 hours. This effect of prednisone appears to result from antagonism of the effects of antireceptor antibodies without actually lowering their titer. The natural history of this syndrome is that the antireceptor antibodies disappear and the syndrome resolves over a time course of several months to several years. In North America, the presence of anti-insulin antibodies in a hypoglycemic patient most commonly suggests that the patient has been immunized with exogenous insulin. However, some patients--especially in Japan--develop spontaneous autoantibodies directed against insulin. These antibodies can cause hypoglycemia, which is generally reactive in that it occurs several hours after a meal or a glucose challenge rather than in a fasting state. The most effective therapy is frequent small feedings and avoidance of large meals.     

The evolving clinical course of patients with insulin receptor autoantibodies: spontaneous remission or receptor proliferation with hypoglycemia.

Three patients with insulin resistance caused by autoantibodies to the insulin receptor were investigated serially over a 3-yr period. Major changes in carbohydrate metabolism, insulin receptor status, and titer of antireceptor antibodies were observed in each case. In one patient, normalization of glucose tolerance, insulin sensitivity, and receptor binding were associated with a spontaneous fall in the titer of antireceptor antibody. A second, more severely affected patient had two entirely distinct phase to her illness. The first, or hyperglycemic phase, was characterized by insulin resistance, negligible insulin binding to receptors on circulating monocytes, and high titers of circulating antireceptor antibodies. The second phase was characterized by refractory hypoglycemia, in association with proliferation of membrance insulin receptors; these occurred despite persistence of high titers of antireceptor antibody. An unusual heptic lesion, diffuse adenomatosis, was observed during this phase. A third patient showed features of both of the other patients, with spontaneous fall in antibody titer as well as a later phase of receptor proliferation. These studies demonstrate that patients with antibodies to insulin receptors may have a fluctuating clinical course. There may be spontaneous changes in antibody titers as well as independent changes in receptor concentration. Hypoglycemia and hepatic proliferation are newly recognized clinical sequelae in patients with this syndrome.     

Shared antigenic determinant between the Electrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons.

Monoclonal antibodies raised against purified acetylcholine receptor from muscle and electric organ were tested for cross-reaction with surface components on chicken ciliary ganglion neurons. Indirect immunofluorescence indicated that antibodies to a determinant in the "main immunogenic region" of the receptor bind to the neurons in culture. Ultrastructural studies on 16-day embryonic ganglia, using horseradish peroxidase-conjugated monoclonal antibody, revealed that most of the conjugate labeling was associated with synaptic membrane on the neurons. A lesser amount of labeling was associated with the short processes extending from the neuronal somata in the region of preganglionic innervation. The labeling was blocked by coincubation with unlabeled antibodies of appropriate specificity and not by nonimmune serum. The pattern of labeling was clearly different from that previously found for a horseradish peroxidase conjugate of alpha-bungarotoxin: the toxin conjugate bound extensively to the short processes but not to synaptic membrane on the neurons. The synaptic antigen identified here by the cross-reacting antibodies is a candidate for the synaptic acetylcholine receptor on chicken ciliary ganglion neurons.     

Immunoadsorption in Myasthenia Gravis Based on Specific Ligands Mimicking the Immunogenic Sites of the Acetylcholine Receptor

Abstract: A specific system for antibody removal from blood circulation in myasthenia gravis (MG) patients was devised by use of the immunoadsorbent bound to an acetylcholine receptor (AChR) peptide that was synthesized corresponding to the sequence of residues 183‐200 of the AChR alpha‐subunit (alpha 183‐200), antibodies which prevent the binding of ACh to AChR. The alpha 183‐200 peptide was confirmed to be immunogenic for induction of an animal model of the disease and for reactivity with MG autoantibodies. We then made use of these results for immunoadsorption therapy through the antigen‐antibody reaction on the molecular level, having given patients relief from myasthenic weakness. The greatest care was taken for the selection of an antigenic region in the molecular structure among various myasthenogenic domains of AChR and for the antigenic conformation of synthetic peptide as the adsorbent to react with antibodies raised against the native protein.     

Neutrophil and platelet antibodies in autoimmune lymphoproliferative syndrome

BACKGROUND AND OBJECTIVES: Autoimmune lymphoproliferative syndrome (ALPS), is an inherited disorder characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly, accumulation of T-cell receptor (TCR)-alphabeta+ CD4- CD8- T cells (double-negative T cells) and autoimmunity. We investigated the incidence and nature of neutrophil and platelet antibodies in patients with ALPS. MATERIALS AND METHODS: Sera from 26 patients with ALPS were tested for neutrophil antibodies by granulocyte immunofluorescence, granulocyte agglutination and monoclonal antibody immobilization assays of granulocyte antigens, and for platelet antibodies using a solid-phase antibody-detection system. RESULTS: Neutrophil antibodies were detected in 46% of patients with ALPS. Antibody specificity could be defined in eight of the 12 patients with neutrophil antibodies. Among these eight patients, four had antibodies directed against more than one antigen. Overall, 14 antibodies directed to specific antigens were identified: three were directed to the HNA-1a antigen of FcgammaRIIIb; two to the HNA-1b antigen of Fcgamma-RIIIb; two to epitopes common to all FcgammaRIIIb molecules; four to the HNA-2a antigen of the NB1 glycoprotein; and three to neutrophil beta2 integrins. Platelet antibodies were detected in 35% of patients with ALPS. No antibody specificities were identified among the platelet antibodies. There was no association between the detection of neutrophil antibodies and a history of clinical neutropenia, or between the detection of platelet antibodies and a history of clinical thromobocytopenia. CONCLUSIONS: Neutrophil and platelet antibodies are important markers of ALPS, but do not always cause clinical cytopenias. The specificities of neutrophil antibody were similar to those found in children with autoimmune neutropenia but without ALPS.     

Prevention of passively transferred experimental autoimmune myasthenia gravis by a phage library-derived cyclic peptide

Many pathogenic antibodies in myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are directed against the main immunogenic region (MIR) of the acetylcholine receptor (AcChoR). These antibodies are highly conformation dependent; hence, linear peptides derived from native receptor sequences are poor candidates for their immunoneutralization. We employed a phage-epitope library to identify peptide-mimotopes capable of preventing the pathogenicity of the anti-MIR mAb 198. We identified a 15-mer peptide (PMTLPENYFSERPYH) that binds specifically to mAb 198 and inhibits its binding to AcChoR. A 10-fold increase in the affinity of this peptide was achieved by incorporating flanking amino acid residues from the coat protein as present in the original phage library. This extended peptide (AEPMTLPENYFSERPYHPPPP) was constrained by the addition of cysteine residues on both ends of the peptide, thus generating a cyclic peptide that inhibited the binding of mAb 198 to AcChoR with a potency that is three orders of magnitude higher when compared with the parent library peptide. This cyclic peptide inhibited the in vitro binding of mAb 198 to AcChoR and prevented the antigenic modulation of AcChoR caused by mAb 198 in human muscle cell cultures. The cyclic peptide also reacted with several other anti-MIR mAbs and the sera of EAMG rats. In addition, this peptide blocked the ability of mAb 198 to passively transfer EAMG in rats. Further derivatization of the cyclic peptide may aid in the design of suitable synthetic mimotopes for modulation of MG.     

Hypoglycemia due to antiinsulin receptor antibodies in systemic lupus erythematosus

An elderly woman with unexplained episodic fasting hypoglycemia was hospitalized for ascites. Evaluation revealed polyserositis, arthritis and immunologic abnormalities that suggested the diagnosis of systemic lupus erythematosus (SLE). Antibodies to insulin receptor with insulin binding inhibitory activity were detected in her serum. Treatment with prednisone was accompanied by resolution of hypoglycemic episodes and disappearance of the antireceptor antibodies. Autoantibody mediated alterations in serum glucose may be included in the growing list of autoimmune phenomena in SLE. Antiinsulin receptor antibodies should be sought in patients with SLE and idiopathic hypoglycemia.     

Acetylcholine receptors and myasthenia gravis

Recent years have seen considerable progress in understanding the nature of the molecular events involved in neuromuscular transmission. The acetylcholine receptor (AChR) has been purified to homogeneity and acetylcholine-induced ion transport has been reconstituted by incorporation of pure AChR into artificial membranes. Immunization against purified AChR induces a condition, clinically and physiologically similar to the human disease myasthenia gravis, which is due to circulating anti-AChR antibodies. This model, experimental autoimmune myasthenia gravis, is proving useful for investigating the role of genetic factors in determining the immune response to AChRs and for testing various experimental approaches to specific treatment. Myasthenia gravis is an autoimmune disease in which there is loss of acetylcholine receptors at the neuromuscular junction. Anti-AChR antibodies can be detected in the majority of patients and they cause loss of AChR by a variety of mechanisms. Anti-AChR antibody is heterogeneous and not restricted in idiotype. The role of the thymus in MG is still uncertain, but recent experiments implicate the presence of a cell type in MG thymus which may be involved in autosensitization to AChR.     

Characterization of anticytoplasmic antibodies in patients with systemic autoimmune diseases

We characterized the cytoplasmic staining patterns identified by indirect immunofluorescence (IF) using human epithelial (HEp-2) cells as substrates, and identified autoantigens using enzyme-linked immunosorbent assay (ELISA) and cognate RNA immunoprecipitation techniques in cytoplasmic antibody-positive sera (CA(+)) in patients with systemic autoimmune diseases. Twenty-three sera (3.7%) of 630 patients were found to have a cytoplasmic staining pattern by IF on HEp-2 cells. The fine-pattern IF specificities were as follows: 12 diffuse fine speckled; 7 coarse granular filamentous speckled; 2 diffuse coarse speckled; 1 condensed large speckled; 1 cytoskeletal. No relationship was found between the staining patterns and the diseases. Anti-SS-A antibodies and antimitochondrial (M2) antibodies were detected by ELISA in 6 and 4 sera, respectively, and antismooth muscle antibody was detected by IF in 1 serum. In RNA immunoprecipitation assays, 6, 11, 3, and 1 patients had antibodies that recognized aminoacyltransfer RNA (tRNA) synthetases (including 2 EJ, 2 PL-7, 1 PL-12, and 1 unidentified tRNA-related), SS-A, ribosomes, and SRP, respectively. Moreover, several other autoantigens were detected by Western blotting using human epithelial (HEp-2) cell lysates. This study suggests that autoantibodies against tRNA synthetases, SS-A, ribosomes, mitochondria, and other autoantigens are present in CA(+) sera from patients with a variety of systemic autoimmune diseases.     

The multichain interleukin-2 receptor: a target for immunotherapy.

Activation of resting T-lymphocytes induces synthesis of interleukin-2 (IL-2) and expression of cell surface receptors for this lymphokine. In contrast to resting normal T-cells that do not express high-affinity IL-2 receptors (IL-2R), abnormal T-cells of patients with leukemia-lymphoma, certain autoimmune disorders, and individuals rejecting allografts express this receptor. Exploiting this difference in receptor expression, antibodies to the IL-2 receptor have been used effectively to treat patients with leukemia and lymphoma. One approach is to use monoclonal antibodies produced in mice; the disadvantage is that they are highly immunogenic. In an effort to reduce the immunogenicity of the mouse monoclonal antibodies, monoclonal-antibody-mediated therapy has been revolutionized by generating humanized antibodies produced by genetic engineering in which the molecule is human except for the antigen-combining regions, which are retained from the mouse. Further, to increase its cytotoxic effectiveness, the monoclonal antibody has been armed with toxins or radionuclides. Alternatively, IL-2 itself has been linked to a toxin to kill IL-2 receptor-bearing cells. Thus, IL-2 receptor-directed therapy provides a new method for treating certain neoplastic diseases and autoimmune disorders and for preventing allograft rejection.     

Anti-receptor antibodies mimic the effect of insulin to down-regulate insulin receptors in cultured human lymphoblastoid (IM-9) cells

Autoantibodies directed against the insulin receptor mimicked the effect of insulin to down-regulate insulin receptors in IM-9 cells. This down-regulation occurred at 37 C, but not at 15 or 22 C. As with insulin itself, antireceptor antibodies caused down-regulation by accelerating the rate of receptor degradation. Down-regulation induced by antireceptor antibody may play a role in desensitizing target cells to the effects of insulin.     

Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies.

We have obtained four monoclonal antibodies, IB4, OKM1, OKM9, and OKM10, all directed against the C3bi receptor of human monocytes and macrophages (M phi). Two criteria were used to determine the specificity of these antibodies. First, culture surfaces coated with the antireceptor antibodies caused specific down modulation of C3bi receptor activity on M phi adherent to these substrates. Second, receptor protein purified by using IB4 or OKM1 retained the ability to bind selectively to C3bi-coated erythrocytes. Each of the antibodies recognizes a distinct epitope on the C3bi receptor; they do not compete with one another for binding to monocytes. Further, when immobilized on a solid support, each of the antibodies binds a molecule from M phi lysates that can simultaneously bind one of the other monoclonal anti-C3bi receptor antibodies. OKM10 binds and masks the ligand-binding site of the C3bi receptor, while IB4, OKM1, and OKM9 bind to sites remote from the C3bi binding site. All four antibodies immunoprecipitated polypeptides of Mr 185,000 and 105,000 from 125I-surface-labeled M phi. IB4 also precipitates polypeptides of Mr 185,000, 153,000, and 105,000. We conclude that the C3bi receptor of human M phi is a complex composed of two polypeptides, Mr 185,000 and 105,000. We have identified monoclonal antibodies reacting with four distinct antigenic determinants of this complex. The determinant recognized by antibody OKM10 is at or near the ligand-binding site of the receptor. The determinant recognized by antibody IB4 is shared by at least two other leukocyte surface proteins.     

Coexistence of systemic lupus erythematosus and myasthenia gravis: two distinct populations of anti-DNA and anti-acetylcholine receptor antibodies

A woman with a four-year history of systemic lupus erythematosus (SLE) developed myasthenia gravis (MG). The clinical features of lupus disappeared slowly while the myasthenic syndrome became predominant. However, her serum was positive for anti-DNA and anti-acetylcholine receptor antibodies. Cross-reactivity between anti-DNA antibodies and anti-acetylcholine receptor antibodies was not demonstrated, suggesting the presence of two different populations. A cellular immunology profile was normal as expected in MG and in contrast to SLE. Conceivably, SLE and MG might represent two opposite extremes in the spectrum of autoimmune diseases.