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.     

Isolated Bovine Spinal Motoneurons Have Specific Ganglioside Antigens Recognized by Sera from Patients with Motor Neuron Disease and Motor Neuropathy

The gangliosides GM1 and GD1b have recently been reported to be potential target antigens in human motor neuron disease (MND) or motor neuropathy. The mechanism for selective motoneuron and motor nerve impairment by the antibodies directed against these gangliosides, however, is not fully understood. We recently investigated the ganglioside composition of isolated bovine spinal motoneurons and found that the ganglioside pattern of the isolated motoneurons was extremely complex. GM1, GD1a, GD1b, and GT1b, which are major ganglioside components of CNS tissues, were only minor species in motoneurons. Among the various ganglioside species in motoneurons, several were immunoreactive to sera from patients with MND and motor neuropathy. One of these gangliosides was purified from bovine spinal cord and characterized as N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)] by compositional analysis, fast atom bombardment mass spectra, and the use of specific antibodies. Among seven sera with anti-GM1 antibody activities, five sera reacted with GM1(NeuGc) and two did not. Two other gangliosides, which were recognized by another patient's serum, appeared to be specific for motoneurons. We conclude that motoneurons contained, in addition to the known ganglioside antigens GM1 and GD1b, other specific ganglioside antigens that could be recognized by sera from patients with MND and motor neuropathy.     

Visual Detection of Human Antibodies Using Sugar Chain-Immobilized Fluorescent Nanoparticles: Application as a Point of Care Diagnostic Tool for Guillain-Barré Syndrome

Sugar chain binding antibodies have gained substantial attention as biomarkers due to their crucial roles in various disorders. In this study, we developed simple and quick detection method of anti-sugar chain antibodies in sera using our previously developed sugar chain-immobilized fluorescent nanoparticles (SFNPs) for the point-of-care diagnostics. Sugar chain structure on SFNPs was modified with the sugar moieties of the GM1 ganglioside via our original linker molecule to detect anti-GM1 antibodies. The structures and densities of the sugar moieties immobilized on the nanoparticles were evaluated in detail using lectins and sera containing anti-GM1 antibodies from patients with Guillain-Barré syndrome, a neurological disorder, as an example of disease involving anti-sugar chain antibodies. When optimized SFNPs were added to sera from patients with Guillain-Barré syndrome, fluorescent aggregates were able to visually detect under UV light in three hours. The sensitivity of the detection method was equivalent to that of the current ELISA method used for the diagnosis of Guillain-Barré syndrome. These results suggest that our method using SFNPs is suitable for the point-of-care diagnostics of diseases involving anti-sugar chain antibodies.     

Immunoglobulin G-class mouse monoclonal antibodies to major brain gangliosides

Mice genetically engineered to lack complex gangliosides are improved hosts for raising antibodies against those gangliosides. We report the generation and characterization of nine immunoglobulin G (IgG)-class monoclonal antibodies (mAbs) raised against the four major brain gangliosides in mammals. These include (designated as ganglioside specificity-IgG subclass) two anti-GM1 mAbs (GM1-1, GM1-2b), three anti-GD1a mAbs (GD1a-1, GD1a-2a, GD1a-2b), one anti-GD1b mAb (GD1b-1), and three anti-GT1b mAbs (GT1b-1, GT1b-2a, GT1b-2b). Each mAb demonstrated high specificity, with little or no cross-reactivity with other major brain gangliosides. Enzyme-linked immunosorbent assay (ELISA) screening against 14 closely related synthetic and purified gangliosides confirmed the high specificity, with no significant cross-reactivity except that of the anti-GD1a mAbs for the closely related minor ganglioside GT1a alpha. All of the mAbs were useful for ELISA, TLC immunooverlay, and immunocytochemistry. Neural cells from wild-type rats and mice were immunostained to differing levels with the anti-ganglioside antibodies, whereas neural cells from mice engineered to lack complex gangliosides (lacking the ganglioside-specific biosynthetic enzyme UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase) remained unstained, demonstrating that most of the mAbs react only with gangliosides and not with related structures on glycoproteins. These mAbs may provide useful tools for delineation of the expression and function of the major brain gangliosides and for probing the pathology of anti-ganglioside autoimmune diseases.     

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.     

Ganglioside GM1 Antibodies and B-Cholera Toxin Bind Specifically to Embryonic Chick Dorsal Root Ganglion Neurons but Do Not Modulate Neurite Regeneration

Polyclonal antibodies to ganglioside GM1 have been prepared and characterised by direct and competitive enzyme-linked immunoassay. An immunoglobulin fraction was prepared from a rabbit antisera showing high specificity and antibody titre for GM1 relative to the other major brain gangliosides. The anti-GM1 immunoglobulin fraction and B-cholera toxin specifically labelled neurons in primary cultures of embryonic chick dorsal root ganglia and there was a good correlation between the relative increase in binding of anti-GM1 immunoglobulin and B-cholera toxin following neuraminidase treatment of a variety of cell types. At antibody concentrations that show saturable binding to endogenous ganglioside in the neuronal membrane, the anti-GM1 immunoglobulin fraction did not interfere with the nerve growth factor (NGF)-mediated fibre outgrowth and neuronal survival as indexed by measurement of neurofilament protein levels. Similarly, at levels in excess of those shown to stimulate thymocyte proliferation, B-cholera toxin was also without effect. These data are not consistent with GM1 in the neuronal membrane functioning as a receptor molecule for NGF and/or other differentiation factors present in the tissue culture media.     

Biological roles of anti-GM1 antibodies in patients with Guillain–Barré syndrome for nerve growth factor signaling

To reveal the biological and pathological roles of anti-GM1 antibody in Guillain–Barré syndrome (GBS), we examined its effects on nerve growth factor (NGF) induced TrkA autophosphorylation (NGF-TrkA signaling) in PC12 cells, a sympathetic nerve cell line. The NGF-TrkA signaling is enhanced by exogenous GM1 ganglioside and this phenomenon is regarded as one of the functional aspects of GM1. The IgGs purified from patients' sera inhibited the NGF-TrkA signaling in GM1 pre-incubated PC12 cells. The degrees of inhibition by IgGs from patients paralleled their immunological reactivity to GM1. In addition, the IgGs also inhibited the neurite outgrowth of NGF-treated PC12 cells. Immunoglobulins in the rabbit sera, which were immunized by GM1, also caused a similar suppressive phenomenon. These results suggested that the anti-GM1 antibody could play roles in pathophysiology in anti-GM1 antibody positive GBS through interfering with the neurotrophic action of NGF and GM1 mediated signal modulation including NGF-TrkA signaling. It is suggested that the modulation of GM1 function is one important action of antibodies and could be one of the important mechanisms in GBS.     

Biological roles of anti-GM1 antibodies in patients with Guillain-Barré syndrome for nerve growth factor signaling

To reveal the biological and pathological roles of anti-GM1 antibody in Guillain-Barré syndrome (GBS), we examined its effects on nerve growth factor (NGF) induced TrkA autophosphorylation (NGF-TrkA signaling) in PC12 cells, a sympathetic nerve cell line. The NGF-TrkA signaling is enhanced by exogenous GM1 ganglioside and this phenomenon is regarded as one of the functional aspects of GM1. The IgGs purified from patients' sera inhibited the NGF-TrkA signaling in GM1 pre-incubated PC12 cells. The degrees of inhibition by IgGs from patients paralleled their immunological reactivity to GM1. In addition, the IgGs also inhibited the neurite outgrowth of NGF-treated PC12 cells. Immunoglobulins in the rabbit sera, which were immunized by GM1, also caused a similar suppressive phenomenon. These results suggested that the anti-GM1 antibody could play roles in pathophysiology in anti-GM1 antibody positive GBS through interfering with the neurotrophic action of NGF and GM1 mediated signal modulation including NGF-TrkA signaling. It is suggested that the modulation of GM1 function is one important action of antibodies and could be one of the important mechanisms in GBS.     

Glycolipid markers of murine lymphocyte subpopulations.

We have shown previously that purified antibodies to ganglioside GM1 react with peripheral T cells and most thymocytes in several strains of mice, independent of Thy-1 phenotype. GM1 and the Thy-1.2 antigen cap independently on C3H thymocytes, which provides additional evidence that GM1 is not the Thy-1.2 antigen. In C3H and nude mice antibodies to GM1 also react with a population of cells, comprising about 25% of lymphocytes from lymph nodes or spleen, that bear surface immunoglobulin. After removal of immunoglobulin from these cells by digestion with proteolytic enzyme, the GM1+ cells regenerate their surface immunoglobulin during 18 hr in culture, which indicates that these double-labeled cells synthesize their surface immunoglobulin. Protease treatment of lymphocytes reveals receptors for antibodies to GM1 on most cells. These data indicate that T and B cells differ in the accessibility of GM1 to antibody, and not necessarily in their content of GM1. Purified antibodies to asialo GM1 react with mature T cells in all strains of mice tested. In contrast to anti-GM1, these antibodies do not react with most thymocytes, with immunoglobulin-bearing lymphocytes of C3H or nude mice, nor with pronase-treated B cells.     

Anti-GM1 antibodies as a model of the immune response to self-glycans

Glycans are a class of molecules with high structural variability, frequently found in the plasma membrane facing the extracellular space. Because of these characteristics, glycans are often considered as recognition molecules involved in cell social functions, and as targets of pathogenic factors. Induction of anti-glycan antibodies is one of the early events in immunological defense against bacteria that colonize the body. Because of this natural infection, antibodies recognizing a variety of bacterial glycans are found in sera of adult humans and animals. The immune response to glycans is restricted by self-tolerance, and no antibodies to self-glycans should exist in normal subjects. However, antibodies recognizing structures closely related to self-glycans do exist, and can lead to production of harmful anti-self antibodies. Normal human sera contain low-affinity anti-GM1 IgM-antibodies. Similar antibodies with higher affinity or different isotype are found in some neuropathy patients. Two hypotheses have been developed to explain the origin of disease-associated anti-GM1 antibodies. According to the "molecular mimicry" hypothesis, similarity between GM1 and Campylobacter jejuni lipopolysaccharide carrying a GM1-like glycan is the cause of Guillain-Barré syndrome associated with anti-GM1 IgG-antibodies. According to the "binding site drift" hypothesis, IgM-antibodies associated with disease originate through changes in the binding site of normally occurring anti-GM1 antibodies. We now present an "integrated" hypothesis, combining the "mimicry" and "drift" concepts, which satisfactorily explains most of the published data on anti-GM1 antibodies.     

Sialylpentaosylceramide detected with anti-GM2 monoclonal antibody. Structural characterization and complementary expression with GM2 in gastric cancer and normal gastric mucosa

The ganglioside fraction of human gastric mucosa was analyzed with a newly established anti-GM2 monoclonal antibody KM531. Using this antibody, accumulation of GM2 was observed in all of four cases of gastric carcinoma. In all ganglioside fractions extracted from normal gastric mucosa obtained from eight cases of peptic ulcer GM2 itself was not detected, but three kinds of glycolipid showing slower mobility than GM2 on thin-layer plates were detected by immunostaining with KM531. These glycolipids were assigned as NGM-1, -2, and -3. They were completely lost in all carcinoma tissues and in non-cancerous gastric mucosa from two cases of gastric cancer, and they were also not detected in the ganglioside fraction of small or large intestine. Of these glycolipids, the major one, NGM-1, was isolated from the pooled ganglioside fraction of normal gastric mucosa obtained from cases of peptic ulcer. The structure was determined by proton nuclear magnetic resonance, negative ion fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry, and treatment with exoglycosidases and mild acid hydrolysis. The structure was GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4GlcNAc beta 1----3 Gal beta 1----4Glc beta 1----1Cer, which has the same terminal sequence as GM2 but has internal neolacto series structure. This epitope was previously identified as Cad blood group antigen. The decrease of this glycolipid and the increase of GM2 was considered to be a cancer-associated change in gastric mucosa.     

Antibodies against gangliosides: a link between preceding infection and immunopathogenesis of Guillain-Barré syndrome

Autoantibodies against gangliosides GM1 and GQ1b, characteristic cell surface glycolipids of the nervous system, are present in specific clinical types of GuillainBarré syndrome (GBS). Close associations of anti-GM1 with acute motor axonal neuropathy, and of anti-GQ1b with Miller Fisher syndrome, strongly suggest that these antibodies contribute to neuropathy pathogenesis. Immune responses against gangliosides are suspected to originate as a result of molecular mimicry between gangliosides and lipopolysaccharides of Campylobacter jejuni, the most frequent infectious trigger of GBS. Thus, antibodies against gangliosides may link C. jejuni infection with the precipitation of neurological disease.     

Antiganglioside antibodies in motor-neuron diseases and peripheral neuropathies: study by ELISA technique and immunodetection on thin-layer chromatography.

We report here our studies on IgM reactivity towards peripheral nervous system gangliosides, in motor-neuron diseases (MND) without IgM gammopathies, and in peripheral neuropathies with IgM gammopathies. We showed by enzyme linked immunosorbent assay technique, that anti-GM1 IgM antibodies were often present at a low level in normal controls in contrast to anti-GD1b antibodies, which were never detected in control sera. We evidenced that several steps of the ELISA technique were critical such as the nonaddition of detergent in buffer solutions used for dilutions and for washing and the choice of the ELISA plates. We studied 50 cases of motor-neuron diseases, among which 40 typical cases of Amyotrophic Lateral Sclerosis, only a few had high anti-GM1 antibodies levels, which were always confirmed by immunodetection on thin-layer chromatography. These antibodies were generally directed against the oligosaccharide epitope present also in asialoGM1. No correlation has been as yet established in relation to the clinical state of the patients. In a few cases of polyneuropathies associated with IgM gammopathies, antiganglioside antibodies have been reported. We have found anti-GD1b antibodies to be present in a sensory-motor axonal neuropathy; axonal involvement was evidenced by electrophysiological study.     

Binding of choleragen and anti-ganglioside antibodies to gangliosides incorporated into preformed liposomes

Exogenously added gangliosides were taken up and incorporated into liposomes just as they are incorporated into cells. Ganglioside GM1 was rapidly taken up by liposomes containing dimyristoyl- or dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate. When incubated with a wide range of GM1 concentrations for 18 h, the liposomes incorporated about 10% of the added ganglioside. The rate of GM1 uptake by preformed liposomes was both time- and temperature-dependent. The liposomes also incorporated other gangliosides to a similar extent. The GM1 taken up by preformed liposomes was predominantly located on the outer surface of the liposomes and did not appear to be internalized into the inner half of the lipid bilayer. Liposomes containing GM1 added after liposome formation bound as many anti-GM1 antibodies and as much choleragen as liposomes having GM1 added during the formation of the lipid bilayers. Thus, preformed liposomes sensitized by incubation with GM1 are a good model system for studying the interactions of antibodies and toxins with membrane-associated gangliosides.     

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

Biochemical and immunochemical analysis of gangliosides of human small cell lung carcinoma: production of monoclonal antibodies against a unique marker of small cell lung carcinoma, ganglioside Fuc GM1

Immunization of mice with a pure preparation of the ganglioside adsorbed on Salmonella typhimurium and hybridization of splenocytes with myeloma P3-X63-Ag 8.653 have resulted in hybridomas producing monoclonal antibodies against ganglioside Fuc GM1, a marker of human small cell lung carcinoma. Characterization of four hybridomal clones and data on the antigenic specificity of the monoclonal antibodies are given. All four monoclonal antibodies reacted only with Fuc GM1 in an enzyme-linked immunosorbent assay. In radioimmunodetection of the antigen on thin-layer plates, two of the four monoclonal antibodies gave cross-reactions with Fuc GD1b. The obtained monoclonal antibodies have revealed the presence of Fuc GM1 in all seven cases of small cell lung carcinoma we have studied and the absence of Fuc GM1 in the normal human lung tissue and in lung adenocarcinomas.     

Isolation and characterization of extremely minor gangliosides, GM1b and GD1 alpha, in adult bovine brains as developmentally regulated antigens

In addition to ganglioside GM1b, an unusual and extremely minor ganglioside, GD1 alpha, was efficiently isolated from bovine brain by combination of Q-Sepharose and Iatrobeads column chromatographies. In the course of purification steps, the presence of the sialidase-labile ganglioside was proved by a highly sensitive TLC/enzyme-immunostaining method. The structure was characterized by gas-liquid chromatography, permethylation study, sialidase degradation, immunostaining with specific antibodies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry. The content of the ganglioside was very small (0.016%) in the total gangliosides. This finding suggests that a synthetic pathway of asialo GM1----GM1b----GD1 alpha may exist in mammalian brains. A monoclonal antibody NA-6 that was obtained by immunizing mice with purified GM1b reacted specifically with GM1b but showed no cross-reactivity with other structurally related gangliosides such as GM1a, GD1a, and so on. Using the method of TLC/immunostaining with NA-6, GM1b was found to be strongly expressed during embryonic days 14-17 in chick brains. Thus, it is assumed that extremely minor gangliosides like GM1b and GD1 alpha found in adult brains are characterized as embryonic molecules.     

Monosialoganglioside (GM1) immunofluorescence in rat spinal roots studied with a monoclonal antibody

Gangliosides are characteristic glycolipid components of plasma cell membranes, especially enriched in the CNS and PNS. In some diseases involving the PNS, in particular motor neuropathies associated with conduction block, IgM autoantibodies against ganglioside GM1 have been implicated as a pathogenic factor. In order to study the GM1 distribution in peripheral nerves we have investigated its in situ localization using a new anti-GM1 monoclonal antibody, GM1:1. Immunization and production of the monoclonal antibody was made by common protocols and binding specificity was investigated by using structurally related glycolipids and modified GM1-molecules. The result showed that an α2–3 bound sialic acid together with a terminal galactose moiety were essential for GM1:1 binding. In situ localization of GM1 in rat dorsal and ventral spinal roots was investigated by conventional immunomicroscopy. GM1 immunoreactivity was the same in both roots and appeared like a finely granular, in places confluent, material confined to Schmidt-Lanterman’s incisures, to myelin sheath paranodal end segments and to some extent to the abaxonal Schwann cell cytoplasm; all of these structures are likely to be the target for GM1 antibodies in peripheral neuropathies. Nodal gaps and fibre contours showed a weak non-specific fluorescence. The localization of GM1 to the incisures of Schmidt-Lanterman and the paranodal end segments of the myelin sheaths might indicate a role of gangliosides as adhesion molecules.     

The anti-oligosaccharide antibodies present in sera from patients with motor neuron disease and neuropathy recognize theN-glycolylneuraminic acid containing gangliotetrahexosyl oligosaccharide

We found that serum antibodies present in the serum of patients with motor neuron disease and neuropathy, which were previously shown to react with the oligosaccharide chain of ganglioside GM1(Neu5Ac), can be recognized and titred using theN-glycolylneuraminic acid containing monosialo-gangliotetrahexosylceramide, GM1(Neu5Gc), which is not a component of normal human cells. The antibody-antigen reaction was abolished by immunoabsorption with the free oligosaccharide chain. This result, together with the knowledge that these antibodies recognize several glycoconjugates, supports the conviction that these antibodies are non-specific for a gangliosidic structure.