Graham M. O'Hanlon

Monoclonal antibodies raised against Guillain-Barré syndrome–associated Campylobacter jejuni lipopolysaccharides react with neuronal gangliosides and paralyze muscle-nerve preparations

Guillain-Barré syndrome and its variant, Miller-Fisher syndrome, are acute, postinfectious, autoimmune neuropathies that frequently follow Campylobacter jejuni enteritis. The pathogenesis is believed to involve molecular mimicry between sialylated epitopes on C. jejuni LPSs and neural gangliosides. More than 90% of Miller-Fisher syndrome cases have serum anti-GQ1b and anti-GT1a ganglioside antibodies that may also react with other disialylated gangliosides including GD3 and GD1b. Structural studies on LPS from neuropathy-associated C. jejuni strains have revealed GT1a-like and GD3-like core oligosaccharides. To determine whether this structural mimicry results in pathogenic autoantibodies, we immunized mice with GT1a/GD3-like C. jejuni LPS and then cloned mAbs that reacted with both the immunizing LPS and GQ1b/GT1a/GD3 gangliosides. Immunohistology demonstrated antibody binding to ganglioside-rich sites including motor nerve terminals. In ex vivo electrophysiological studies of nerve terminal function, application of antibodies either ex vivo or in vivo via passive immunization induced massive quantal release of acetylcholine, followed by neurotransmission block. This effect was complement-dependent and associated with extensive deposits of IgM and C3c at nerve terminals. These data provide strong support for the molecular mimicry hypothesis as a mechanism for the induction of cross-reactive pathogenic anti-ganglioside/LPS antibodies in postinfectious neuropathies.

The immunopathogenesis of Miller Fisher syndrome.

Over the past decade, remarkable progress has been made in our understanding of the pathogenesis of Miller Fisher syndrome (MFS), a clinical variant of Guillain Barré syndrome (GBS). MFS comprises the clinical triad of ataxia, areflexia and ophthalmoplegia. It is associated with acute-phase IgG antibodies to GQ1b and GT1a gangliosides in over 90% of cases which are highly disease specific. Like GBS, MFS is a post-infectious syndrome following diverse infections, but particular attention has been paid to its association with Campylobacter jejuni enteritis. Serostrains of C. jejuni isolated from infected patients bear ganglioside-like epitopes in their lipopolysaccharide core oligosaccharides, which elicit humoral immune responses exhibiting molecular mimicry with GQ1b/GT1a gangliosides. These antibodies are believed to be the principal cause of the syndrome and physiological studies aimed at proving this have focused on the motor-nerve terminal as a potential site of pathogenic action. This review describes these findings and formulates a pathogenesis model based on our current state of knowledge.

A somatically mutated human antiganglioside IgM antibody that induces experimental neuropathy in mice is encoded by the variable region heavy chain gene, V1-18.

IgM paraproteins associated with autoimmune peripheral neuropathy and anti-Pr cold agglutinins react with sialic acid epitopes present on disialylated gangliosides including GD1b, GT1b, GQ1b, and GD3. A causal relationship between the paraprotein and the neuropathy has never been proven experimentally. From peripheral blood B cells of an affected patient, we have cloned a human hybridoma secreting an antidisialosyl IgM mAb, termed Ha1, that shows identical structural and functional characteristics to its serum counterpart. Variable region analysis shows Ha1 is encoded by the same VH1 family heavy chain gene, V1-18, as the only other known anti-Pr antibody sequence and is somatically mutated, suggesting that it [correction of is] arose in vivo in response to antigenic stimulation. In the rodent peripheral nervous system, Ha1 immunolocalizes to dorsal root ganglia, motor nerve terminals, muscle spindles, myelinated axons, and nodes of Ranvier. After intraperitoneal injection of affinity-purified antibody into mice for 10 d, electrophysiological recordings from the phrenic nerve-hemidiaphragm preparation demonstrated impairment of nerve excitability and a reduction in quantal release of neurotransmitter. These data unequivocally establish that an antidisialosyl antibody can exert pathophysiological effects on the peripheral nervous system and strongly support the view that the antibody contributes to the associated human disease.

Mapping immunoreactive epitopes in the human peripheral nervous system using human monoclonal anti-GM1 ganglioside antibodies

Abstract A series of monoclonal IgM anti-GM1 ganglioside antibodies has been cloned from peripheral blood lymphocytes of patients with multifocal motor neuropathy and Guillain-Barré syndrome. In solid-phase immunoassay, the antibodies react with GM1, and also in differing degrees to the structurally related glycolipids asialo-GM1 (GA1) and GD1b. Here we describe the binding patterns of six human anti-GM1 antibodies to epitopes within the human nervous system. Antibodies were observed to bind to motor neurons and spinal grey matter, dorsal and ventral spinal roots, dorsal root ganglion neurons, nodes of Ranvier, neuromuscular junctions and skeletal muscle. The distribution of immunoreactive epitopes, which included sensory structures, extended beyond those sites conventionally regarded as pathologically affected in anti-GM1 antibody-associated motor nerve syndromes. This undermines a model of disease pathogenesis based solely on antigen distribution. Factors other than the presence or absence of antigen, such as the local ganglioside topography, antibody penetration into, and pathophysiological vulnerability of a particular site may also influence the clinicopathological outcome of anti-GM1 antibody-mediated autoimmune attack.

Peripheral Neuropathy Associated with Anti-GM2 Ganglioside Antibodies: Clinical and Immunopathological Studies

GM2 ganglioside is a potential peripheral nerve antigen for neuropathy-associated autoantibodies. However little data are available on their pathogenic effects, if any. In this study we have screened both neuropathy-associated and control sera for anti-GM2 antibodies and subsequently used high titre sera for immunohistological and complement mediated cytotoxicity studies. We identified abnormally elevated anti-GM2 antisera in the normal population, as well as in patients with peripheral neuropathies and other neurological diseases. GM2 antibodies were either mono-reactive, cross-reactive with GMla, or cross-reactive with Gal-NAc-GMlb and/or GalNAc-GDla. All GM2 antisera from neuropathy subjects and normal controls bound to, and were capable of complement-mediated lysis of the NSC-34 cell line which expresses high levels of membrane-associated GM2. However, in immunohistological studies on human and rodent peripheral nervous system tissues, no specific binding was seen with GM2 antisera, either cross-reactive with GalNAc-GMlb and GalNAc-GDla, or with GMla. These data indicate that although GM2 antisera can lyse neural membranes containing GM2, this antigen(s) is not detectable by standard immunohistological techniques in human or rodent peripheral nerve. This raises doubts about their pathophysiological significance in human autoimmune neuropathy

Anti-GM1 ganglioside antibodies cloned from autoimmune neuropathy patients show diverse binding patterns in the rodent nervous system

We have recently cloned a panel of monoclonal IgM anti-GMl ganglioside antibodies from peripheral blood lymphocytes of patients with multifocal motor neuropathy and Guillain Barr6 syndrome. In solid-phase immunoassay, the antibodies all reacted with GM1 and also reacted to different degrees with the structurally related glycolipids asialo-GMl and GDlb. These antibodies are being used to study the pathogenesis of anti-GMl antibody-mediated neuropathy in different experimental systems. In the present immunofluorescence study we report the binding patterns of 5 of these antibodies in the rodent nervous system. The antibodies demonstrated highly diverse binding patterns on tissue sections and teased fibers when compared to one another and between different species. The antibodies bound many central and peripheral nervous system structures, including neurons and myelin, motor end plate regions, and muscle spindles. The diversity of binding shown by these antibodies provides evidence that may account for the differing clinical phenotypes, including normality, associated with elevated titers of anti-GMl antibodies.

Anti-disialosyl antibodies mediate selective neuronal or Schwann cell injury at mouse neuromuscular junctions

The human paralytic neuropathy, Miller Fisher syndrome (MFS) is associated with autoantibodies specific for disialosyl epitopes on gangliosides GQ1b, GT1a, and GD3. Since these gangliosides are enriched in synaptic membranes, anti‐ganglioside antibodies may target neuromuscular junctions (NMJs), thereby contributing to disease symptoms. We have shown previously that at murine NMJs, anti‐disialosyl antibodies induce an α‐latrotoxin‐like effect, electrophysiologically characterized by transient massive increase of spontaneous neurotransmitter release followed by block of evoked release, resulting in paralysis of the muscle preparation. Morphologically, motor nerve terminal damage, as well as perisynaptic Schwann cell (pSC) death is observed. The relative contributions of neuronal and pSC injury to the paralytic effect and subsequent repair are unknown. In this study, we have examined the ability of subsets of anti‐disialosyl antibodies to discriminate between the neuronal and glial elements of the NMJ and thereby induce either neuronal injury or pSC death. Most antibodies reactive with GD3 induced pSC death, whereas antibody reactivity with GT1a correlated with the extent of nerve terminal injury. Motor nerve terminal injury resulted in massive uncontrolled exocytosis with paralysis. However, pSC ablation induced no acute (within 1 h) electrophysiological or morphological changes to the underlying nerve terminal. These data suggest that at mammalian NMJs, acute pSC injury or ablation has no major deleterious influence on synapse function. Our studies provide evidence for highly selective targeting of mammalian NMJ membranes, based on ganglioside composition, that can be exploited for examining axonal–glial interactions both in disease states and in normal NMJ homeostasis.

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, α‐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.

Detection and prevalence of α-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 α‐latrotoxin (α‐LTx) and are induced by antibody‐mediated activation of complement. We developed an assay for detection of the α‐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 α‐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.

Complex Gangliosides as Autoantibody Targets at the Neuromuscular Junction in Miller Fisher Syndrome: A Current Perspective*

Glycosphingolipid biology has increasingly interfaced with the field of human autoimmune neuropathy over the last two decades. There are currently over 20 distinct glycolipids that have been identified as autoantibody targets in a wide range of clinical neuropathy syndromes. This review sets out the clinical and experimental background to one interesting example of anti-glycolipid antibody-associated neuropathy termed Miller Fisher syndrome. This syndrome, comprising the triad of ataxia, areflexia, and ophthalmoplegia, correlates highly with the presence of serum anti-GQ1b antibodies, arising through molecular mimicry with microbial oligosaccharides. Anti-GQ1b antibodies mediate neural injury through binding to GQ1b-enriched sites in the peripheral nervous system, including extraocular nerves. Animal experimental evidence, along with a hypothetical background, indicates the motor nerve terminal may be a key site for anti-GQ1b antibody binding with consequent defects in synaptic transmission, as occurs in botulism and other toxinopathies. Our work in recent years on this hypothesis is summarized.