David P. Richman

Detection of in vivo stimulated cerebrospinal-fluid lymphocytes by flow cytometry in patients with multiple sclerosis.

To examine the immune response in the central nervous system in patients with multiple sclerosis (MS), we characterized the cell-cycle phase of lymphocytes from cerebrospinal fluid. Cells were stained with acridine orange, and both RNA and DNA content were determined by flow cytometry. Although most cells were in the quiescent phase of the cycle, a significant increase was seen in the percentage of cells in the first stage of the proliferative cycle (G1 phase) when 21 samples of cerebrospinal fluid from 17 patients with MS were compared with samples from 21 controls (P < 0.001). Stimulated cells in the first stage of the cycle were seen in all categories of MS: active, progressive, or stable. In addition, increased numbers of cells in the second stage of the cycle (S phase) were seen in six specimens from patients with MS, five of whom had actie disease, but no increases were seen in controls (P < 0.02). These data indicate that stimulated lymphocytes were present in the cerebrospinal fluid durng all phases of MS and that stimulation becomes more intense during an acute exacerbation.

Cellular immunity in myasthenia gravis. Response to purified acetylcholine receptor and autologous thymocytes

To determine the potential importance of an immune response directed against the acetylcholine receptor in myasthenia gravis, we studied cell-mediated immunity to receptor as measured by lymphocyte stimulation in 21 myasthenic patients and 21 controls, including five with amyotrophic lateral sclerosis. The mean (+/- S.E.M.) stimulation index was 5.3 +/- 1.3 for patients and 1.2 +/- 0.3 for controls (P less than 0.005). Fourteen patients had indexes greater than 2.0 (nine of 11 males, five of 10 females, 10 of 11 elderly patients, and five of six with thymoma). Stimulation index correlated with disease activity (rs = 0.71, P less than 0.01). Peripheral blood lymphocytes from one of three young female myasthenic patients responded to autologous thymocytes but not to receptor; peripheral blood lymphocytes from the other two responded to receptor but not to autologous thymocytes. Our findings are further evidence that autoimmunity to the acetylcholine receptor plays a central part in myasthenia gravis.

Transcervical thymectomy for myasthenia gravis

The use of transcervical thymectomy in the treatment of myasthenia gravis remains controversial. We retrospectively reviewed our experience with this procedure to determine its usefulness in the management of myasthenia gravis. Fifty-three selected myasthenic patients without thymoma underwent transcervical thymectomy between 1977 and 1991. The mean age (27.5 +/- 1.5 years), duration of symptoms (2 +/- 1.0 years), and preoperative Osserman classification (13% class I, 53% class IIA, 28% class IIB, 6% class III) were consistent with previous reports. The average hospitalization was 3.0 +/- 0.3 days, but has been 1.6 +/- 0.2 days since 1987 (n = 14). There were no deaths, and no patients required mechanical ventilation for more than 24 hours. Average follow-up was 4.3 +/- 0.4 years with a range of 0 to 13 years. Eighty-one percent of patients are symptom free, and 9 of 21 (43%) are in complete remission at least 5 years postoperatively. One patient required a transsternal exploration for worsening symptoms. Clinical improvement continued over an extended period of time, and a statistically significant decrease in symptoms was evident comparing the first and sixth postoperative years. Patients were more likely to be improved or in remission if thymectomy was performed within the first year of the onset of symptoms (p < 0.05). Osserman classification, thymus histology, and patient age were not prognostic indicators. Transcervical thymectomy is effective surgical therapy for myasthenia gravis in selected patients without thymoma.

Monoclonal antibodies against purified nicotinic acetylcholine receptor.

Abstract Eleven stable monoclonal hybridoma cell lines synthesizing antibodies against purified acetylcholine receptor from Torpedo californica were produced. Spleen cells from a rat immunized with acetylcholine receptor and exhibiting experimental autoimmune myasthenia gravis were fused with a mouse myeloma cell line. Studies of the binding of these antibodies to acetylcholine receptor by use of a passive hemagglutination assay in the presence of various cholinergic ligands revealed four general categories of binding specificities: 1) blockade only by alpha bungarotoxin, 2) partial blockade by all ligands, 3) increased titer in the presence of alpha bungarotoxin and benzoquinonium chloride, 4) absence of effect by any ligand.

Inflammation at the neuromuscular junction in myasthenia gravis

To better define the pathogenic mechanisms in the antibody-mediated autoimmune disease myasthenia gravis (MG), we analyzed the morphology and electrophysiology of the neuromuscular junction in anconeus muscle biopsy specimens from eight patients with MG and seven control subjects. There were inflammatory cells at the neuromuscular junction in seven of the eight biopsies from MG patients. The endplate index (length of the postsynaptic membrane divided by the length of the apposed presynaptic membrane) was abnormally reduced in all the MG patients, and fiber type grouping, suggestive of reinnervation, was present in six of the eight MG patients. Intracellular recording revealed diminished amplitude of miniature endplate potentials and miniature endplate currents in the MG patients compared with the controls. The time constant of decay of miniature endplate currents did not differ from that of controls, suggesting no change in mean channel open time of the acetylcholine receptor. The endplate receptor sensitivity to iontophoretically applied acetylcholine was also decreased in MG patients compared with controls. The quantal content of neurally evoked endplate potentials was reduced in six of the eight MG patients, demonstrating abnormal presynaptic function as well. The presence of inflammatory cells at the neuromuscular junctions of limb muscles in MG reconciles an apparent disparity between the animal model of MG, experimental autoimmune myasthenia gravis, and the human disease. This study also demonstrates a frequent presynaptic component to the abnormal neuromuscular transmission in MG.

A cholinergic receptor site on murine lymphocytes with novel binding characteristics.

To further analyze functionally important cholinergic receptors on lymphocytes, we studied the binding of the muscarinic antagonist Quinuclidinyl benzilate (QNB) to murine splenic lymphocytes. Studies of displacement of [3H]QNB by unlabeled QNB on lymphocytes revealed at least two binding sites. Scatchard analysis of equilibrium binding isotherms also distinguished two sites with apparent Kds of 480 nM and 16 microM. There was greater specific QNB binding to B cell-enriched lymphocyte fractions than to T cell fractions. Lymphocyte binding demonstrated temperature-dependent dissociability, and specific binding occurred on isolated lymphocyte membranes as well. Both muscarinic and nicotinic ligands competed for QNB binding to lymphocytes with low and nearly equal affinity. Therefore, QNB binding sites on lymphocytes appear to be of low affinity and of mixed muscarinic and nicotinic character.

Lymphocyte function and the role of regulator cells in multiple sclerosis.

Although the etiology of multiple sclerosis (MS) remains unknown, evidence continues to accumulate pointing to aberrant immune function in this disease, both when the disease is active and during periods of its seeming quiescence. Classically, the immune system has been considered to be comprised of three major cell types: (1) thymus-derived lymphocytes (T cells), which are the effectors of cell-mediated immune responses; (2) bone-marrow derived lymphocytes (B cells), which act in antibody-mediated immune responses; and (3) cells of the monocyte-macrophage series. The standard classification oversimplifies the in vivo situation: Subclasses of T and B cells can now be delineated and evidence has been advanced for the existence of functional differences between macrophages harvested from different loci. The best studied of immunocyte subclasses are those of the T cells. Broadly, these can be divided into effector and regulator subgroups. Among regulator T cells are helper and suppressor subsets. The suppressor T-cell subset can be further subdivided into specific and nonspecific types. Specific suppressors are antigen triggered and suppress the response to a specific antigen. Nonspecific suppressors depress the immune response in toto. Study of specific suppressor cell function is not feasible in MS since no antigen specific for MS is known. On the other hand, nonspecific suppression can be studied in MS and, as will be shown, is markedly decreased when the disease is active. Helper T cells augment the magnitude of the immune response and suppressor T cells lessen it. At any moment the net immune response in vivo is a function of the balance between these countervailing forces. Thus, an immune response may be abrogated equally well by abolition of helper function or by augmentation of suppressor function. Given these considerations, current enthusiasm for cytotoxic immunosuppressive drugs as therapy in MS may be misdirected. Cytotoxic drugs depress both helper and suppressor cell populations and, if this occurs to a comparable extent in both cell populations, the effect on the net immune response may be zero. In animals, cyclophosphamide has been reported to selectively kill suppressor cells. 1,2 In consequence, use of this drug during flareups of MS might be contraindicated. Since MS is characterized by exacerbations and remissions, study of immune parameters which fluctuate with disease activity might offer clues t o the defect in immune regulation which permits attacks to occur or disease to progress. Manipulation-by chemical or pharmacologic means-of regulator cell functions which are abnormal when disease is active might blunt the severity of attacks of MS even while the fundamental cause of the disease itself remains unknown. The work on this problem currently being undertaken in our laboratories constitutes the theme of this paper.

Cellular immunity to acetylcholine receptor in myasthenia gravis Relationship to histocompatibility type and antigenic site

To debermine the nature of the cellular immune response directed against acetylcholine receptor in myasthenia gravis, we compared lymphocyte stimulation by eel receptor with clinical factors. The mean (k SEM) stimulation index wa.s 4.5 ± 0.9 for 39 myasthenic patients and 0.97 ± 0.18 for 48 controls (p < 0.001). Positive response in patients was associated with disease onset after 50 years (10 of 13 patients) and presence of thymoma (seven of eight patients), but not with HLA type. Stimulation index correlated with disease activity (rs = 0.6 3, < 0.01). Blocking the active portion of the receptor molecule with naja toxin resulted in 68 percent diminution of the response, suggesting that this site plays a significant role in the cellular immune response in myasthenia gravis.

Effects of a monoclonal anti-acetylcholine receptor antibody on the avian end-plate.

1. The effects of anti‐acetylcholine receptor (AChR) monoclonal antibodies (mAbs) 370 and 132A on miniature end‐plate potentials (MEPPs) and end‐plate currents (EPCs) in the posterior latissimus dorsi muscle of adult chickens were investigated. 2. After incubation of the electrophysiological preparation with mAb 370 (5‐50 micrograms/ml), which blocks both agonist (carbamylcholine) and alpha‐bungarotoxin (alpha‐BTX) binding and induces a hyperacute form of experimental autoimmune myasthenia gravis (EAMG), MEPP and EPC amplitudes were irreversibly reduced. 3. This effect was not associated with any significant change in the time constant describing EPC decay (tau EPC), current reversal potential, or the voltage dependence of tau EPC. The tau EPC at ‐80 mV was 5.9 +/‐ 0.6 ms before incubation with mAb 370 (50 micrograms/ml) and 6.0 +/‐ 0.9 ms afterwards. Current reversal potential was ‐3.9 +/‐ 0.4 mV before mAb incubation and ‐4.8 +/‐ 1.5 mV afterwards. The change in membrane potential required to produce an e‐fold change in tau EPC was 128 +/‐ 2.3 mV before antibody incubation compared to 125 +/‐ 6.6 mV after incubation. 4. A second anti‐AChR mAb, 132A (50 micrograms/ml), which is capable of inducing the classically described form of EAMG without blocking agonist or alpha‐BTX binding, or inducing hyperacute EAMG, produced no significant change in MEPP amplitude, EPC amplitude, tau EPC or EPC reversal potentials. 5. The mAb 370 (50 micrograms/ml) induced a partially reversible decrease of the quantal content of the neurally evoked end‐plate potential (EPP). This effect was not observed with mAb 132A, (+)tubocurarine (10(‐7)‐10(‐5) g/ml) or an irrelevant anti‐oestrogen receptor mAb. 6. These data suggest that the rapid onset of weakness observed in chicken hatchlings after the injection of mAb 370 (Gomez & Richman, 1983) can be attributed to a combined effect of a block of acetylcholine (ACh)‐induced ion channel activity in the postsynaptic membrane and a reduction of the neurally evoked release of acetylcholine from the nerve terminal.

Induction of the morphologic changes of both acute and chronic experimental myasthenia by monoclonal antibody directed against acetylcholine receptor

SummaryTo investigate pathogenic mechanisms in experimental autoimmune myasthenia gravis (EAMG) and myasthenia gravis (MG), we studied the acute and chronic effects in rats of injection of rat monoclonal antibodies (MCABs) directed against the acetylcholine receptor (AChR). Animals were severely weak 12 h after a single injection, at which time macrophages were found invading endplate regions of muscle and cholinesterase-stained regions were separted from the underlying muscle fibers. Ultrastructural studies showed findings identical to the acute phase of EAMG: degenerating postsynaptic membranes and invasion and phagocytosis of endplate regions by macrophages. Animals receiving sublethal doses of MCAB recovered clinically by 4–5 days after injection. Recovery was accompanied by a progressive decrease in the number of macrophages associated with endplates and reapposition to the myofibers of the cholinesterasestained regions. Animals injected once, or repeatedly over several months, remained clinically and electromyographically normal after recovery from the initial episode of weakness, but their endplate ultrastructure was highly simplified with blunted or absent synaptic folds and shallow or absent secondary synaptic clefts. These studies demonstrate that anti-AChR MCABs can induce the changes of both acute and chronic EAMG. There is good correlation between the inflammatory changes and the acute clinical disease but poor correlation between morphological and clinical parameters in the chronic syndrome. The latter observation suggests that severe ultrastructural changes, similar to those seen in chronic EAMG and MG, cannot account, at least in rats, for the clinical and electrophysiologic abnormalities of MG.