Motor nerve terminal destruction and regeneration following anti-ganglioside antibody and complement-mediated injury: an in and ex vivo imaging study in the mouse.

Both the neural and glial components of the neuromuscular junction (NMJ) have been identified as potential sites for anti-ganglioside antibody (Ab) binding and complement-mediated injury in murine models for the human peripheral nerve disorder Guillain-Barré syndrome (GBS). Some patients suffering from the acute motor axonal neuropathy (AMAN) forms of GBS recover very rapidly from paralysis; it has been proposed that in these cases the injury was restricted to the distal motor axons and nerve terminals (NTs) which are able to regenerate over a very short time-frame. To test this hypothesis, the ventral neck muscles of mice (n=45) expressing cytosolic fluorescent proteins in their axons (CFP) and Schwann cells (GFP) were subjected to a single topical application of anti-ganglioside Ab followed by a source of complement. Group A (n=15) received Ab that selectively bound to the NTs, group B (n=15) received Abs that bound both to the NTs and the perisynaptic Schwann cells (pSCs) and group C (control animals; n=15) only received complement. Evolution of the injury was documented by in vivo imaging, and following euthanasia the muscles were reimaged ex vivo both quantitatively and qualitatively, either immediately, or after 1, 2, 3 or 5 days of regeneration (each n=3 per group). Within 15 minutes of complement application, a rapid loss of CFP overlying the NMJ could be seen; in group A, the GFP signal remained unchanged, whereas in group B the GFP signal was also lost. In group C no changes to either CFP or GFP were observed. At 24 h, 6% of the superficial NMJs in group A and 12% of the NMJs in group B exhibited CFP. In both groups, CFP returned within the next five days (group A: 93.5%, group B: 94%; p=0.739), with the recovery of CFP being preceded by a return of GFP-positive cells overlying the NMJ in group B. Auxiliary investigations revealed that the loss of CFP at the NMJ correlated with a loss of NT neurofilament immuno-reactivity and a return of CFP at the NMJ was accompanied by a return of neurofilament. In ultrastructural investigations, injured NTs were electron lucent and exhibited damaged mitochondria, a loss of filaments and a loss of synaptic vesicles. The examination of muscles after five days of regeneration revealed physiological NT-profiles. The results described above indicate that following a single anti-ganglioside Ab-mediated and complement-mediated attack, independent of whether there are healthy and mature perisynaptic Schwann cells overlying the NMJ, the murine NT is capable of recovering both its architectural and axolemmal integrity very rapidly. This data supports the notion that an equivalent mechanism may account for the rapid recovery seen in some clinical cases of AMAN.

Glycosphingolipid depletion in PC12 cells using iminosugars protects neuronal membranes from anti-ganglioside antibody mediated injury.

Autoimmune neuropathies are frequently associated with pathogenic anti-ganglioside antibodies targeting ganglioside-rich neuronal and glial membranes. The extent of injury is determined by the concentration of membrane ganglioside and thus reduction might be expected to attenuate disease. In this study, we suppressed ganglioside biosynthesis in PC12 cells with the glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin and observed reduced plasma membrane antibody binding and a major neuroprotective effect in complement-mediated lysis assays. These data demonstrate that iminosugar inhibitors, currently used to treat type 1 Gaucher disease, are also of potential value for depleting antigen and thereby suppressing tissue injury in anti-ganglioside antibody-associated neuropathy.

Sulfatide binding properties of murine and human antiganglioside antibodies.

Antiganglioside antibodies form an important component of the innate and adaptive B cell repertoire, where they provide antimicrobial activity through binding encapsulated bacterial glycans. In an aberrant role, they target peripheral nerve gangliosides to induce autoimmune nerve injury. An important characteristic of antiganglioside antibodies is their ability to selectively recognize highly defined glycan structures. Since sialylated and sulfated glycans often share lectin recognition patterns, we here explored the possibility that certain antiganglioside antibodies might also bind 3-O-sulfo-beta-D-galactosylceramide (sulfatide), an abundant constituent of plasma and peripheral nerve myelin, that could thereby influence any immunoregulatory or autoimmune properties. Out of 25 antiganglioside antibodies screened in solid phase assays, 20 also bound sulfatide (10(-5) to 10(-6) M range) in addition to their favored ganglioside glycan epitope ( approximately 10(-7) M range). Solution inhibition studies demonstrated competition between ganglioside and sulfatide, indicating close proximity or sharing of the antigen binding variable region domain. Sulfatide and 3-O-sulfo-beta-D-galactose were unique in having this property amongst a wide range of sulfated glycans screened, including 4- and 6-O-sulfo-beta-D-galactose analogues. Antiganglioside antibody binding to 3-O-sulfo-beta-D-galactose was highly dependent upon the spatial presentation of the ligand, being completely inhibited by conjugation to protein or polyacrylamide (PAA) matrices. Binding was also absent when sulfatide was incorporated into plasma membranes, including myelin, under conditions in which antibody binding to ganglioside was retained. These data demonstrate that sulfatide binding is a common property of antiganglioside antibodies that may provide functional insights into, and consequences for this component of the innate immune repertoire.

Solid phase immunoadsorption for therapeutic and analytical studies on neuropathy-associated anti-GM1 antibodies.

Autoimmune neuropathies including Guillain-Barré syndrome are frequently associated with anti-GM1 ganglioside antibodies. These are believed to play a pathogenic role and their clearance from the circulation would be predicted to produce therapeutic benefit. This study examines the conditions required for effective immunoadsorption of anti-GM1 antibodies using glycan-conjugated Sepharose as a matrix. In solution inhibition studies using a range of GM1-like saccharides in conjunction with mouse and human anti-GM1 antibodies, the whole GM1 pentasaccharide beta-Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal-(1-4)-beta-Glc was the favored ligand for maximal inhibiton of antibody-GM1 interactions in comparison with monosaccharides, Gal-(1-3)-beta-GalNAc-betaOMe, and synthetic GM1 mimetics. Immunoadsorption studies comparing binding of mouse monoclonal anti-GM1 antibodies to GM1-Sepharose and beta-Gal-(1-3)-beta-GalNAc-Sepharose confirmed the preference seen in solution inhibition studies. GM1-Sepharose columns were then used to adsorb anti-GM1 immunoglobulin G and immunoglobulin M antibodies from human neuropathy sera. Anti-GM1 antibodies subsequently eluted from the columns often showed a striking monoclonal or oligoclonal pattern, indicating that the immune response to GM1 is restricted to a limited number of B-cell clones, even in the absence of a detectable serum paraprotein. These data support the view that immunoadsorption plasmapheresis could potentially be developed for the acute depletion of serum anti-GM1 antibodies in patients with neuropathic disease, and also provide purified human anti-GM1 antibodies for analytical studies.

Characterisation of the immunoglobulin variable region gene usage encoding the murine anti-ganglioside antibody repertoire.

Neuropathogenic murine antibodies reactive with terminal disialylgalactose epitopes are innate and preferentially encoded by the VH7183.3b gene. Here we have studied antibodies reactive with internal galactose-linked disialosyl epitopes and the terminal trisaccharide of GT1b. Antibodies were of moderate affinity and unmutated. Anti-GD1b antibodies were often encoded by the VH10.2b heavy and gj38c light chain genes. Anti-GT1b antibodies with broader glycan binding patterns were encoded by VHQ52 and VHJ558 family genes. These data indicate that the discrete specificities of ganglioside-binding antibodies are dictated by particular patterns of V gene usage residing within the innate B cell repertoire.

Protective efficacy of a recombinant plague vaccine when co-administered with another sub-unit or live attenuated vaccine.

Vaccines against bioterrorism agents offer the prospect of providing high levels of protection against airborne pathogens. However, the diversity of the bioterrorism threat means that it may be necessary to use several vaccines simultaneously. In this study we have investigated whether there are changes to the protective immune response to a recombinant sub-unit plague vaccine when it is co-administered with other sub-unit or live attenuated vaccines. Our results indicate that the co-administration of these vaccines did not influence the protection afforded by the plague vaccine. However, the co-administration of the plague sub-unit vaccine with a live vaccine resulted in markedly increased levels of IgG2a subclass antibodies, and markedly reduced levels of IgG1 subclass antibodies, to the plague sub-unit vaccine. This finding might have implications when considering the co-administration of other vaccine combinations.

Innate murine B cells produce anti-disialosyl antibodies reactive with Campylobacter jejuni LPS and gangliosides that are polyreactive and encoded by a restricted set of unmutated V genes.

In Guillain-Barré syndrome following Campylobacter enteritis, anti-lipopolysaccharide antibodies cross-react with neural gangliosides, thereby precipitating autoimmune neuropathy. We examined the properties of 15 murine anti-LPS/ganglioside mAbs specific for NeuAc(alpha2-8)NeuAc-Gal disialosyl epitopes. Many mAbs displayed features of an innate B cell origin including polyreactivity (13/15), hybridoma CD5 mRNA expression (5/15), predominance of IgM (9/15) or IgG3 (3/6) isotype, low affinity, and utilisation of unmutated VH and VL VDJ rearrangements. Antibody specificity resided in highly selective V gene usage, with 6/15 mAbs being encoded by the VH7183.3b gene. These data indicate that neuropathogenic antiganglioside autoantibodies can arise from the natural autoantibody repertoire.

Synthesis of ganglioside epitopes for oligosaccharide specific immunoadsorption therapy of Guillian-Barré syndrome.

Guillain-Barré syndrome is a postinfectious, autoimmune neuropathy resulting in neuromuscular paralysis. Auto-antibodies, often induced by bacterial infection, bind to human gangliosides possessing monosialoside and diasialoside epitopes and impair the function of nerve junctions, where these ganglioside structures are highly enriched. Truncated gangliosides representive of GD3, GQ1b and GM2 epitopes have been synthesized as methyl glycosides and as a glycosides of an eleven carbon tether. The synthetic oligosaccharide ligands are structural mimics of these highly complex ganglioside epitopes and via their ability to neutralize or remove auto-antibodies have the potential for therapy, either as soluble blocking ligands administered systemically, or as immuno-affinity ligands for use as extracorporeal immunoadsorbents.

Synthetic disialylgalactose immunoadsorbents deplete anti-GQ1b antibodies from autoimmune neuropathy sera.

Acute and chronic autoimmune neuropathies, including Guillain-Barré syndromes (GBS) are often characterized by the presence of autoantibodies that react with neural gangliosides. Evidence from human and animal studies indicates that anti-ganglioside antibodies play a primary neuropathogenic role, and their rapid elimination from the circulation through specific immunoadsorption therapy thus has the potential to ameliorate the course of the disease. Here we have tested this therapeutic principle in the Miller Fisher variant of GBS that is associated serologically with acute phase anti-GQ1b ganglioside immunoglobulin G (IgG) antibodies, and in chronic ataxic neuropathies associated with persistently elevated immunoglobulin M (IgM) antibodies that react with GQ1b, GD3 and other disialylated gangliosides. Human and mouse anti-GQ1b IgG and IgM antibodies may also react with GD3, suggesting the shared terminal disialoside epitope could be involved in antibody binding. We thus synthesized the terminal trisaccharide, NeuAc(alpha2-8)NeuAc(alpha2-3)Gal common to GQ1b and GD3, and conjugated it to bovine serum albumin (BSA). This disialylgalactose glycoconjugate (DSG-BSA) binds anti-GQ1b antibodies in 32/58 (55%) human sera containing IgG or IgM anti-GQ1b antibodies at titres up to 1/130 000; it also binds a wide range of mouse monoclonal anti-GQ1b and -GD3 antibodies. When conjugated to Sepharose as mock therapeutic immmunoaffinity columns, the immobilized trisaccharide (DSG-Sepharose) eliminates anti-GQ1b antibodies from positive sera in proportion to their level of binding to DSG-BSA. Oligosaccharide-specific immunoadsorption therapy thus provides a new therapeutic approach to anti-GQ1b antibody-associated syndromes that could be applied to clinical practice. Furthermore, modification of the immobilized oligosaccharide epitopes to incorporate other glycan structures may allow this approach to be adapted to other forms of autoimmune neuropathy associated with uniform anti-glycolipid antibody profiles.