Miller Fisher anti-GQ1b antibodies: α-Latrotoxin–like effects on motor end plates

In the Miller Fisher syndrome (MFS) variant of the Guillain-Barré syndrome, weakness is restricted to extraocular muscles and occasionally other craniobulbar muscles. Most MFS patients have serum antibodies against ganglioside type GQ1b of which the pathophysiological relevance is unclear. We examined the in vitro effects of MFS sera, MFS IgG, and a human monoclonal anti-GQ1b IgM antibody on mouse neuromuscular junctions (NMJs). It was found that anti-GQ1b antibodies bind at NMJs where they induce massive quantal release of acetylcholine from nerve terminals and eventually block neuromuscular transmission. This effect closely resembled the effect of the paralytic neurotoxin α-latrotoxin at the mouse NMJs, implying possible involvement of α-latrotoxin receptors or associated downstream pathways. By using complement-deficient sera, the effect of anti-GQ1b antibodies on NMJs was shown to be entirely dependent on activation of complement components. However, neither classical pathway activation nor the formation of membrane attack complex was required, indicating the effects could be due to involvement of the alternative pathway and intermediate complement cascade products. Our findings strongly suggest that anti-GQ1b antibodies in conjunction with activated complement components are the principal pathophysiological mediators of motor symptoms in MFS and that the NMJ is an important site of their action. Ann Neurol 1999;45:189–199     

Neonatal Guillain-Barré syndrome: blocking antibodies transmitted from mother to child.

OBJECTIVE: To investigate the role of blocking antibodies in neonatal Guillain-Barré syndrome (GBS) occurring 12 days postpartum in a child born to a mother with ongoing GBS. METHODS: We studied plasma filtrate, purified IgG, and monovalent Fab fragments from the affected mother and serum from the neonate as well as serum samples after recovery from disease 3 months later. Experiments were performed on the hemidiaphragms of adult mice and neonatal and juvenile rats. Quantal endplate currents were recorded with the perfused macro-patch clamp electrode. RESULTS: A dual effect was seen. Serum from mother and infant depressed quantal content by approximately 90% and reduced the amplitude of postsynaptic currents by 30 to 40%. The antibody nature of the blockade could be confirmed by showing that monovalent Fab fragments were similarly effective as purified immunoglobulin (Ig) G. No IgG antibodies to gangliosides, fetal or adult nicotinic acetylcholine receptor, or voltage-gated calcium channels could be detected, but IgM antibodies to the ganglioside GM1 were present. After recovery from GBS no blocking activity was seen in the sera of mother and infant. To elucidate why neonatal disease onset was delayed we examined the possible influence of early developmental changes in functional properties of the neuromuscular junction and applied the mother's active serum to postnatal rats. Although blockade was present in 23-day-old rats, it was absent in 5-day-old rats. CONCLUSION: Transplacentally transferred blocking antibodies may be specifically directed at epitopes of the mature but not the fetal neuromuscular junction.     

IgG from patients with Guillain-Barré syndrome interact with nicotinic acetylcholine receptor channels

In Guillain-Barré syndrome (GBS), immunoglobulin G (IgG) antibodies block neuromuscular transmission pre- and postsynaptically and thus are of potential pathogenic relevance. We investigated whether IgG from GBS patients has a direct interaction with nicotinic acetylcholine receptor (nAChR) channels. Purified IgG fractions from six GBS patients that blocked neuromuscular transmission in a previous study were analyzed by the patch-clamp technique in combination with an ultrafast system for solution exchange. Sera from three patients with other inflammatory neurological disorders were used as controls. Mouse myotubes expressing native embryonic-type nAChR channels and human embryonic kidney (HEK) 293 cells transiently transfected with recombinant adult-type nAChR channels were used. Repeated 20-ms pulses of acetylcholine (ACh) were applied to outside-out patches in the presence of GBS-IgG. IgG of the patients had a significant reversible blocking action on embryonic- and adult-type nAChR channels with some variability in the magnitude of the block. Activation and desensitization kinetics were not affected when GBS-IgG was applied. None of the control sera blocked the AChR channels. The observed postsynaptic block effect fulfills the criteria of a channel-blocking IgG antibody similar to those seen in autoimmune myasthenia and may contribute to muscle weakness during the acute phase of GBS.     

Mechanism of block of nicotinic acetylcholine receptor channels by purified IgG from seropositive patients with myasthenia gravis

OBJECTIVE: To clarify the mechanism of block of nicotinic receptor channels by myasthenic antibodies. BACKGROUND: Nicotinic acetylcholine receptor (nAChR) channel currents are functionally blocked by purified immunoglobulin G (IgG) of patients with myasthenia gravis (MG). METHODS: The molecular mechanism of block of IgG fractions containing antibodies to nAChR channels was tested with the patch-clamp technique in combination with a system for ultrafast solution exchange. For the experiments, outside-out patches from cultured mouse myotubes that express embryonic-type nAChR channels at their surface were used. RESULTS: Incubation of outside-out patches with purified IgG from four myasthenic patients blocked nAChR channel currents activated by the application of 1.0 mM ACh reversibly. The peak current amplitude and the time course of block of nAChR channels decreased with increasing concentrations of IgG. The block became at least partly irreversible if incubation time of outside-out patches exceeded 2 minutes. For the block of nAChR channel currents with a-bungarotoxin, a similar mechanism of block was found. CONCLUSIONS: The reversibility of functional block of nAChR channel currents by myasthenic IgG depended strongly on the incubation time of the receptors with antibodies. Interaction of myasthenic antibodies with nicotinic receptors may proceed in several stages from a low-affinity reversible to a high-affinity irreversible binding.     

Block of recombinant nicotinic receptor channels by IgG from myasthenic patients

In myasthenia gravis (MG), specific antibodies directed against nicotinic acetylcholine receptor (nAChR) channels are produced. Recently, a distinct blockade of native embryonic-type nAChR channels by purified IgG from MG patients has been demonstrated. In the present study, we investigated the interaction of myasthenic IgG with recombinant embryonic- or adult-type nAChR channels expressed on human embryonic kidney (HEK) 293 cells. For the experiments, the patch clamp technique was applied in combination with an ultra-fast application system for agonists. Repetitive 20-ms pulses of 1 mM acetylcholine (ACh) were applied with or without purified IgG added to the perfusion system. The purified IgG from MG patients blocked currents through both, embryonic- and adult-type nAChR channels to a similar extent. A block by myasthenic IgG of adult-type nAChR channels, which are present exclusively at the postsynaptic membrane of the neuromuscular synapse, was shown to occur. The reversible pharmacological blockade of adult-type nAChR channels by myasthenic IgG may be one factor responsible for rapid fluctuations of weakness or rapid recovery of muscle strength after plasmapheresis in MG.     

Anti-GQ1b antibodies and evoked acetylcholine release at mouse motor endplates

Miller Fisher syndrome (MFS) is clinically characterized by ataxia, areflexia, and ophthalmoplegia, and is associated with serum anti-GQ1b-ganglioside antibodies. We have previously shown that anti-GQ1b antibodies induce complement-dependent, alpha-latrotoxin-like effects at mouse neuromuscular junctions (NMJs) in vitro. This effect comprises a massive increase in spontaneous quantal acetylcholine (ACh) release, accompanied by block of evoked release and muscle paralysis. This mechanism may contribute to the motor features of MFS. Whether the block of evoked ACh release is a primary effect of anti-GQ1b antibodies or occurs secondary to massive complement-dependent spontaneous release is unknown. Using conventional micro-electrode methods, we measured in detail ACh release evoked with low- and high-rate nerve stimulation, and studied the effect on it of a purified MFS IgG and a mouse monoclonal anti-GQ1b IgM (without added complement). We found that evoked transmitter release was unaffected. Control experiments proved binding of anti-GQ1b antibody at the NMJ. We conclude that the block of nerve-evoked ACh release at the NMJ is not a primary effect of anti-GQ1b antibodies, but is dependent on antibody-mediated complement activation. It remains to be determined whether the block of nerve-evoked ACh release is the consequence of massive spontaneous ACh release or occurs as a concomitant event.     

Miller Fisher syndrome is associated with serum antibodies to GQ1b ganglioside.

A recent report described serum anti-GQ1b ganglioside antibodies in Miller Fisher syndrome (MFS), a clinical variant of Guillain-Barré syndrome (GBS). Four consecutive cases of MFS all had high titre anti-GQ1b antibodies which were absent from all control sera including those of patients with GBS.     

Fine specificity of anti-GQ1b IgG and clinical features

Anti-GQ1b IgG frequently is present in sera of patients with Miller Fisher syndrome (MFS), Guillain-Barré syndrome (GBS) with ophthalmoplegia, Bickerstaff's brainstem encephalitis (BBE), and acute ophthalmoparesis (AO) in the acute phase. Why various clinical signs develop under these conditions, however, has yet to be clarified. We investigated the fine specificity of anti-GQ1b IgG and its clinical correlation in sera from 82 patients: 56 with MFS, 11 with GBS, 13 with BBE, and 2 with AO. Anti-GQ1b IgG antibodies were absorbed by GT1a in 80 (98%) of the 82 sera, by GD1b in 11 (13%), and by the other b-series gangliosides GD3, GD2, or GT1b in 24 (29%). The most frequent pattern of fine specificity was the cross-reaction with GT1a alone, seen in 56 (68%) samples. Of the 11 patients with anti-GQ1b IgG, cross-reacting with GD1b, 6 (55%) had impaired deep sense, and the association was significant (p=0.02). This is the first study to show that the fine specificity of anti-GQ1b IgG is heterogeneous and that the difference is correlated with the presence of a particular clinical sign.     

Effects of phospholipids on antiganglioside antibody reactivity in GBS

Serum antibody activities to mixtures of a ganglioside and various phospholipids were compared with those to a ganglioside alone in 30 anti-GM1 IgG-positive GBS patients and 30 anti-GQ1b IgG-positive Miller Fisher syndrome (MFS) patients. Anti-GM1-positive sera had higher antibody reactivities against a mixture of GM1 and several phospholipids including PA, PI and PS, than against GM1 alone. In contrast, in case of anti-GQ1b antibody, no phospholipid provided significant enhancement. Sphingomyelin provided decrease of the activity for both anti-GM1 and anti-GQ1b IgG. The effects of phospholipids must be considered to determine the pathogenetic role of antiganglioside antibodies in GBS and MFS.     

Electrophysiological evidence by single fibre electromyography of neuromuscular transmission impairment in a case of Miller Fisher syndrome

Abstract Miller Fisher syndrome is an autoimmune neuropathy characterised by ataxia, areflexia and ophthalmoplegia, with minimal if any limb weakness, and in the majority of cases by high titres of IgG anti-GQ1b ganglioside antibodies. In vitro electrophysiological experiments have demonstrated that these antibodies induce a transmission blockade at neuromuscular junction either pre- or post-synaptically. We report the case of a 63-year-old man with MFS that shows blood serum negative for anti-GQ1b but presents an impairment of neuromuscular transmission detected by single fibre electromyography. To the best of our knowledge, this represents the first case in the literature using jitter technique and suggests that other antibodies may be involved in the function of motor end plates by bindings to the synaptic membranes.     

Detection and prevalence of alpha-latrotoxin-like effects of serum from patients with Guillain-Barré syndrome

Anti-GQ1b antibodies are associated with the Miller Fisher syndrome (MFS), a variant of the Guillain-Barré syndrome (GBS). In the ex vivo mouse diaphragm, anti-GQ1b-positive MFS serum induces muscle fiber twitching, a temporary dramatic increase of spontaneous quantal acetylcholine release, and transmission blockade at neuromuscular junctions (NMJs). These effects resemble those of alpha-latrotoxin (alpha-LTx) and are induced by antibody-mediated activation of complement. We developed an assay for detection of the alpha-LTx-like effect, using muscle fiber twitching as indicator. We tested 89 serum samples from GBS, MFS, and control subjects, and studied correlations with clinical signs, anti-ganglioside antibodies, micro-electrode physiology, and complement deposition at NMJs. Twitching was observed with 76% of the MFS and 10% of the GBS samples. It was associated with ophthalmoplegia and anti-GQ1b antibodies in patients, and with increased spontaneous acetylcholine release and C3c-deposition at mouse NMJs. This study strongly suggests that antibodies to GQ1b (with cross-reactivity to related gangliosides) are responsible for the alpha-LTx-like activity. The twitching assay is an efficient test for detection of this effect, and allows for screening of large numbers of samples and modifying drugs.     

Miller Fisher syndrome: immunofluorescence and immunoelectron microscopic localization of IgG at the mouse neuromuscular junction

In vitro electrophysiological experiments have demonstrated that IgG antibodies from patients with Miller Fisher syndrome (MFS) impair neuromuscular transmission by a fast and completely reversible combined pre- and postsynaptic blockade. In this study we investigated the cellular and subcellular binding sites of IgG from four MFS patients at the mouse hemidiaphragm by immunofluorescence and immunoelectron microscopy. IgG from all patients produced significant immunostaining at the neuromuscular junction, whereas sera from healthy volunteers or from patients with other neurological diseases did not stain neuromuscular junction. Immunoelectron microscopy revealed that, when living hemidiaphragms were incubated with IgG from MFS patients, labeling was found on both pre- and postsynaptic membranes of the neuromuscular junction, whereas terminal Schwann cells and the basal lamina covering the synaptic membranes were not labeled. These findings demonstrate that IgG from MFS patients binds to synaptic membranes of the neuromuscular junction where it might interfere with the function of both the pre- and postsynaptic activities.     

Features of sensory ataxic neuropathy associated with anti-GD1b IgM antibody

Some reports have called sensory ataxic neuropathy (SAN) associated with IgM antibody against b-series gangliosides a chronic form of Miller Fisher syndrome (MFS), but this has yet to be established. We examined five patients with SAN and eight patients with IgG anti-GQ1b-positive MFS. Only one patient with SAN complained of diplopia, whose ocular movement was not limited. The other four patients had neither diplopia nor limitation of ocular movement. All the SAN patients had severe deep sense impairment, whereas one patient with MFS showed only mild vibratory sense impairment. All sera from the SAN patients had remarkably high IgM antibody titers to the b-series gangliosides GD3, GD2, GD1b, GT1b, GQ1b, GQ1b alpha, fucosyl-GD1b, and alpha galactosyl [alpha fucosyl] GD1b. An absorption study confirmed that the anti-GQ1b antibodies cross-reacted with GD3, GD2, GD1b, and GT1b. In contrast, only two samples from the MFS patients had IgG antibody to GD3, and no sample reacted with GD2, GD1b, or GT1b. SAN has different clinical or serological features from MFS, and therefore is not a chronic form of it.     

IgM anti-GQ1b monoclonal antibody inhibits voltage-dependent calcium current in cerebellar granule cells

Miller–Fisher syndrome (MFS), which is known to be associated with anti-GQ1b antibodies and to cause ataxia, is a variant of an acute inflammatory neuropathy. However, the pathogenic role of anti-GQ1b antibodies remains unclear. In this study, we investigated the effects of mouse IgM anti-GQ1b monoclonal antibody (IgM anti-GQ1b mAb) on the spontaneous muscle action potential of a rat spinal cord-muscle co-culture system and on the voltage-dependent calcium channel (VDCC) current in cerebellar granule cells and Purkinje cells using the whole-cell patch clamp technique. The frequency of spontaneous muscle action potential of the innervated muscle cells was transiently increased by IgM anti-GQ1b mAb and then was blocked completely, which was the same finding as reported previously. Moreover, the cerebellar granule cell VDCC current was decreased by 30.76 ± 7.60% by 5 μg/mL IgM anti-GQ1b mAb, whereas IgM anti-GQ1b mAb did not affect the VDCC current in cerebellar Purkinje cells. In immunocytochemistry, IgM anti-GQ1b mAb stained the whole cell surface of cerebellar granule cells, but not that of Purkinje cells. Therefore, the clinical symptoms of Miller–Fisher syndrome, such as cerebellar-like ataxia, may be explained by the inhibitory effects of anti-GQ1b antibodies on VDCC current in cerebellar granule cells.     

Acute isolated ophthalmoplegia with anti-GQ1b antibodies

Ophthalmoplegia without ataxia, areflexia or both has been designated as atypical Miller Fisher syndrome (MFS) or acute ophthalmoplegia (AO). This entity, first reported by Chiba et al. is associated with anti-GQ1b IgG antibodies.We report a patient with isolated acute ophthalmoplegia with high titer of anti-GQ1b IgG antibody activity in the acute phase in whom treatment with intravenous immunoglobulin (IVIg) led to the clinical recovery and the decrease in antibody titer.     

Anti-GQ1b IgG antibody syndrome: clinical and immunological range

OBJECTIVES: To clarify the nosological relation among Miller Fisher syndrome (MFS), Guillain-Barré syndrome (GBS) with ophthalmoplegia, Bickerstaff's brain stem encephalitis (BBE), and acute ophthalmoparesis without ataxia. Serum samples from patients with each condition often have anti-GQ1b IgG antibody. METHODS: Information on antecedent illness, initial symptoms, neurological signs during the illness, and CSF findings were reviewed in 194 patients with anti-GQ1b IgG. It was determined whether overlapping MFS and GBS (MFS/GBS), as well as overlapping BBE and GBS (BBE/GBS), is explained by the combined action of anti-GQ1b IgG and anti-GM1 or anti-GD1a IgG, serological markers of GBS. RESULTS: Based on the diagnostic criteria, all the patients with acute ophthalmoparesis, MFS, MFS/GBS, BBE/GBS, and BBE had external ophthalmoplegia; all the patients with MFS, MFS/GBS, or GBS had hyporeflexia or areflexia; and all those with MFS and BBE showed ataxia. Tendon reflexes were decreased or absent in 91% of those with BBE/GBS, 67% of those with BBE, and 53% of those with acute ophthalmoparesis. Ataxia was present in 68% of the patients with MFS/GBS and 45% of those with BBE/GBS. Antecedent illness caused by upper respiratory tract infection had occurred in 60% to 80% of these patients, and CSF albuminocytological dissociation in 25% to 75%. Anti-GM1 or anti-GD1a IgG was present in 50% of those with GBS, 35% of those with MFS/GBS, 27% of those with BBE/GBS, 16% of those with MFS, and 8% of those with BBE. CONCLUSIONS: These findings together with the common autoantibody (anti-GQ1b IgG) suggest that a common autoimmune mechanism functions in the pathogenesis of these illnesses. In a larger study, it was confirmed clinically that MFS, GBS, BBE, and acute ophthalmoparesis are closely related, forming a continuous range. This is supported by the immunological findings. The term "anti-GQ1b IgG antibody syndrome" is not intended to be used as a clinical diagnosis, but recognition of this syndrome is useful for understanding the aetiological relation among the various illnesses and for introducing the established treatments of GBS for use with other conditions.     

Anti-GQ1b IgG antibody is associated with ataxia as well as ophthalmoplegia.

Close association between the increase in anti-GQ1b immunoglobulin G (IgG) antibody and ophthalmoplegia in Miller Fisher syndrome (MFS) and Guillain-Barré syndrome (GBS) has been reported. We investigated whether anti-GQ1b IgG antibody also is associated with ataxia, another of the MFS triad. Of 149 patients who had anti-GQ1b IgG antibody without profound weakness, 144 showed ophthalmoplegia (120 showed both ophthalmoplegia and ataxia; 24, ophthalmoplegia without ataxia). In contrast, five showed ataxia without ophthalmoplegia. Some large neurons of the dorsal root ganglia were immunostained with anti-GQ1b monoclonal antibody. Anti-GQ1b IgG antibody may thus be associated with ataxia as well as ophthalmoplegia. Ataxia may be due to its binding to a subset of primary sensory neurons.     

Serum antibody against a peripheral nerve myelin ganglioside, LM1, in Guillain-Barré syndrome.

Serum IgG antibody against LM1, the predominant ganglioside in the human peripheral nerve myelin, was found in 7 out of 140 patients with Guillain-Barré syndrome (GBS) in the acute phase, 1 out of 33 patients with chronic inflammatory demyelinating polyneuropathy (CIDP), and 2 out of 47 patients with Miller Fisher syndrome (MFS). Anti-LM1 IgM antibody was detected only in 2 patients, each with GBS and MFS. The clinical and electrophysiological features of the seven GBS patients with anti-LM1 IgG antibody in the serum were investigated. Six patients recovered to grade 1 within one month of the onset of neuropathy. Electrophysiological studies revealed demyelination in five patients, of which one had axonal damage in addition, whereas sufficient evidence of demyelination or axonal degeneration was not observed in the remaining two. Five had a respiratory tract infection before the onset of neuropathy, and also had serum anti-GQ1b IgG antibody. IgG antibody against LM1 might be involved in the pathogenetic mechanisms of GBS, as a possible demyelinating factor. Presence of both anti-GQ1b and anti-LM1 antibodies may be associated with some infectious agent(s) affecting the respiratory tract.     

Miller Fisher syndrome and Haemophilus influenzae infection

OBJECTIVE: To examine the association between Miller Fisher syndrome (MFS) and antecedent Haemophilus influenzae infection. BACKGROUND: Little is known about agents in prior respiratory tract infection of MFS, whereas antecedent upper respiratory symptoms are frequent. H. influenzae is a major pathogen that can cause human respiratory tract infection. METHODS: The authors used ELISA to detect serum antibody against the bacterium in 70 consecutive patients with MFS and 110 with Guillain-Barré syndrome (GBS). RESULTS: Serum anti-H. influenzae IgG and IgM antibody activities were significantly higher in the MFS group than in age- and sex-matched patients with other neurologic diseases (n = 62) and normal control subjects (n = 82). The GBS group showed no significant increase in any class of antibody activities compared with control groups. Serologic evidence of recent infection was found in five (7%) of the patients with MFS and two (2%) of 110 patients with GBS, all of whom had a history of antecedent respiratory tract infection. They frequently showed ophthalmoplegia, but other neurologic features were not remarkable. Serum anti-GQ1b IgG antibody that had cross-reactivity with GT1a ganglioside was detected in six of these seven patients. Thin-layer chromatography with immunostaining showed that serum IgG from H. influenzae-seropositive patients with high anti-GQ1b and anti-GT1a IgG antibody titers bound to the lipopolysaccharide fraction extracted from the type b H. influenzae serostrain. These bands were also stained by anti-GT1a monoclonal antibody (GMR11), indicating that the lipopolysaccharide bears the GT1a epitope. CONCLUSIONS: These findings point to H. influenzae being an agent associated with MFS. Epitopic overlap between H. influenzae and human nerve tissue may be involved in the development of MFS much as GBS is associated with Campylobacter jejuni enteritis.     

HLA and anti-GQ1b IgG antibody in Miller Fisher syndrome and Guillain-Barré syndrome

We investigated serological human leukocyte antigen (HLA) types in patients with histories of Miller Fisher syndrome (MFS) and Guillain-Barré syndrome (GBS) with ophthalmoplegia, in whom serum anti-GQ1b IgG antibody was present during the acute phase. We examined class I antigens (A, B and C) in 32 patients and class II antigens (DR and DQ) in 30, but found no association. We conclude that particular serologically defined HLA types are not preferred for the immunoresponse of anti-GQ1b IgG antibody in MFS and GBS.