Anti-ganglioside Autoantibodies Research Articles

Increased serum levels of anti-ganglioside M1 auto-antibodies in autistic children: relation to the disease severity

BackgroundAutoimmunity to the central nervous system (CNS) may play a pathogenic role in a subgroup of patients with autism. This study aimed to investigate the frequency of serum anti-ganglioside M1 auto-antibodies, as indicators of the presence of autoimmunity to CNS, in a group of autistic children. We are the first to measure the relationship between these antibodies and the degree of the severity of autism.MethodsSerum anti-ganglioside M1 antibodies were measured, by ELISA, in 54 autistic children, aged between 4 and 12 years, in comparison to 54 healthy-matched children. Autistic severity was assessed by using the Childhood Autism Rating Scale (CARS).ResultsAutistic children had significantly higher serum levels of anti-ganglioside M1 antibodies than healthy children (P < 0.001). The seropositivity of anti-ganglioside M1 antibodies was found in 74% (40/54) of autistic children. Serum levels of anti-ganglioside M1 antibodies were significantly higher in autistic children with severe autism (63%) than those with mild to moderate autism (37%), P = 0.001. Moreover, serum anti-ganglioside M1 antibodies had significant positive correlations with CARS (P < 0.001).ConclusionsSerum levels of anti-ganglioside M1 antibodies were increased in many autistic children. Also, their levels had significant positive correlations with the degree of the severity of autism. Thus, autism may be, in part, one of the pediatric autoimmune neuropsychiatric disorders. Further wide-scale studies are warranted to shed light on the possible etiopathogenic role of anti-ganglioside M1 auto-antibodies in autism. The role of immunotherapy in autistic patients who have increased serum levels of anti-ganglioside M1 antibodies should also be studied.

Serum antibody profiles against gangliosides and sulfatide in peripheral neuropathies: evaluation of a new immunoassay

We evaluated the analytical performances of a new line immunoassay (LIA) for the simultaneous detection of twelve anti-ganglioside and anti-sulfatide autoantibodies from Generic Assays, in comparison with our dot immunoassay. The LIA detected IgG and/or IgM autoantibodies against human GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GQ1b and sulfatide. Forty sera of patients with IgG autoantibody profiles in acute autoimmune neuropathies and 39sera of patients with IgM autoantibody profiles in chronic autoimmune neuropathies were tested. To have a better sensitivity, 20μL of sera instead of 10μL were used. In these conditions, we observed a good concordance of IgG and IgM autoantibody profiles by both immunoassays. The test including novel gangliosides was easy to perform and superior for identifying autoimmune neuropathies. The data indicate that the test provides reliable simultaneous detection of autoantibodies against a large panel of human gangliosides and sulfatide with a good diagnostic usefulness in combination with clinical and electrophysiological data.

Anti‐ganglioside autoantibodies in type 1 diabetes

Type 1 diabetes is an autoimmune disease that is accompanied by an immune response against pancreatic cells. As gangliosides are expressed in both peripheral nerves and pancreatic cells, we examined the possibility of correlation between type 1 diabetes, anti-ganglioside autoantibodies, and neuropathy. Fifty diabetic patients and 30 controls with other autoimmune diseases underwent neurological examination and search for antibodies to gangliosides, glutamic acid decarboxylase (GAD(65)), and tyrosine phosphatase (IA2). Sixteen (32%) diabetic patients had neuropathy. Twelve diabetic sera were found to have anti-ganglioside autoantibodies. Twenty sera were positive for anti-GAD(65) and nine for anti-IA2 antibody. Sera from three control patients had anti-ganglioside autoantibodies. No significant correlation was found between autoantibodies, neuropathy, and disease duration. Disease duration was shorter in patients with antibodies to GAD(65) and IA2 and longer in neuropathic patients. The higher prevalence of antibodies in diabetic patients compared to controls may reflect the common pattern of antigens shared by peripheral nerve and pancreatic islet cells.

A New Line Immunoassay for the Multiparametric Detection of Antiganglioside Autoantibodies in Patients with Autoimmune Peripheral Neuropathies

A novel line immunoassay for the multiparametric detection of 11 antiganglioside autoantibodies (GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b, and GQ1b) was evaluated by comparing the reactivities in sera of 77 patients with suspected or definite autoimmune peripheral neuropathies (PNP), 60 blood donors, and 30 systemic lupus erythematodes (SLE) patients. At least one antiganglioside autoantibody was detectable in 97.4% of the patients with neuropathies compared to 12.2% in the control group. A broad spectrum of reactivities with more than two antiganglioside autoantibodies including all tested gangliosides except GD2 and GT1b was found in nearly one-third of the patients with neuropathies whereas in the control group autoantibody profiles with more than two reactivities were observed in two SLE patients only. For the first time anti-GM4 IgG and IgM antibodies were shown in PNP including Guillain-Barré syndrome (GBS), Miller-Fisher syndrome (MFS), and multifocal motor neuropathy (MMN). Different autoantibody profiles detectable by this multiparametric assay may help to diagnose different entities within the growing spectrum of autoimmune PNP.

Comprehensive Analysis of Bacterial Risk Factors for the Development of Guillain‐Barré Syndrome afterCampylobacter jejuniEnteritis

Guillain-Barre syndrome (GBS), a postinfectious autoimmune-mediated neuropathy, is a serious complication after Campylobacter jejuni enteritis. To investigate the bacterial risk factors for developing GBS, genotypes, serotypes, and ganglioside mimics on lipo-oligosaccharide (LOS) were analyzed in C. jejuni strains from Japanese patients. Strains from patients with GBS had LOS biosynthesis locus class A more frequently (72/106; 68%) than did strains from patients with enteritis (17/103; 17%). Class A strains predominantly were serotype HS:19 and had the cstII (Thr51) genotype; the latter is responsible for biosynthesis of GM1-like and GD1a-like LOSs. Both anti-GM1 and anti-GD1a monoclonal antibodies regularly bound to class A LOSs, whereas no or either antibody bound to other LOS locus classes. Mass-spectrometric analysis showed that a class A strain carried GD1a-like LOS as well as GM1-like LOS. Logistic regression analysis showed that serotype HS:19 and the class A locus were predictive of the development of GBS. The high frequency of the class A locus in GBS-associated strains, which was recently reported in Europe, provides the first GBS-related C. jejuni characteristic that is common to strains from Asia and Europe. The class A locus and serotype HS:19 seem to be linked to cstII polymorphism, resulting in promotion of both GM1-like and GD1a-like structure synthesis on LOS and, consequently, an increase in the risk of producing antiganglioside autoantibodies and developing GBS.

Subacute demyelinating polyneuropathy in B-cell lymphoma with IgM antibodies against glycolipid GD1b

Peripheral neuropathy associated with IgM monoclonal gammopathy of unknown significance is a common disorder, while the association of paraproteinaemic neuropathies with haematological malignancies is far less frequent. We report a 76-year-old patient with a subacute and rapidly progressive sensorimotor demyelinating polyneuropathy causing sensory ataxia, painful paraesthesias and marked motor and sensory deficit in four limbs. Monoclonal gammopathy of IgM type associated with a rectal low-grade B-cell non-Hodgkin lymphoma was detected. Research for anti-MAG and antiganglioside autoantibodies including anti-GM1 and anti-GQ1b evidenced a high titre of IgM antibodies against the disialosyl group of GD1b. This is the first report on a paraproteinaemic polyneuropathy with IgM autoantibodies against glycolipid GD1b associated with B-cell lymphoma. The IgM type of these autoantibodies suggests that they represent all or part of the paraprotein produced by lymphoma cells.

Human Antiganglioside Autoantibodies: Validation of ELISA

Gangliosides have a hydrophilic sugar chain that contains antigenic determinants and a hydrophobic ceramide. In humans, gangliosides elicit a T-cell independent IgM response; antiganglioside IgM autoantibodies may be pentameric or polymeric. A correlation between specific neuropathies and antiganglioside autoantibodies has been confirmed. Although many neurologists attempt to lower titers of antiganglioside autoantibodies, oncologists are developing strategies to augment production of IgM antibodies that will remove immunosuppressive gangliosides from the circulation of patients and target gangliosides and kill tumor cells. Antiganglioside IgM antibodies can cause leakage of the blood-nerve barrier in a concentration-dependent and complement-independent manner, bind to neuronal gangliosides to create a neuromuscular block and serve as a marker of axonal damage in neuropathies such as multiple sclerosis. They are also a promising biomarker of early prostate cancer. There is a need to validate the protocol for enzyme-linked immunosorbent assay (ELISA) of antiganglioside IgM autoantibodies. This validation must consider the purity of gangliosides from different commercial sources, the coating of gangliosides onto a solid matrix in a manner that maximizes exposure of oligosaccharide epitopes to IgM paratopes, techniques to minimize background noise and eliminate nonspecific antibody binding, and carefully defined positive and negative controls. The validated protocol also must include a simple formula to estimate titers for several replicas. Finally, antibody titers must be converted to natural logs for statistical appraisal.

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(α2–8)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 V H and V L V(D)J 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.

Acute demyelinating sensorimotor polyneuropathy in B‐cell lymphoma with IGM autoantibodies against glycolipid GD1B

We report the case of an acute demyelinating sensorimotor polyneuropathy in a patient with IgM autoantibody against peripheral nerve myelin glycolipid GD1b produced by a B‐cell lymphoma. A 76‐year‐old woman presented with one month history of weight loss, paresthesias at the distal extremities of the four limbs followed by progressive and diffuse motor weakness. Neurological examination showed sensory ataxia, marked motor deficit (4 at MRC scale in proximal and distal muscle districts of limbs), superficial and proprioceptive sensory deficit with a stocking‐glove distribution, areflexia at lower limbs. Electroneurography documented absent sensory action potentials, prolonged distal latencies of motor action potentials, markedly slowed motor conduction velocities and evidence of conduction block and temporal dispersion of motor action potentials. Diagnosis of acute demyelinating sensorimotor polyneuropathy was established. CSF examination showed a slight increase of protein concentration. Haematological screening revealed a monoclonal gammopathy with high serum IgM (1151 mg/dL) and light chain kappa proteinuria. A total body CT scan evidenced a pelvic mass identified as low grade B‐cell lymphoma by rectal biopsy. Research for antiganglioside autoantibodies evidenced the presence of IgM antibodies against glycolipid GD1b. Autoantibodies anti‐Mag, anti‐Hu, anti‐Yo and anti‐Ri were negative. The pathogenetic role of serum autoantibodies against glycolipid GD1b in the development of demyelinization in the peripheral nervous system has already been documented either clinically in post‐infective polyneuropathies or experimentally in animal research. This is the first report of selective positivity of anti‐GD1b in polyneuropathy associated to B‐cell lymphoma supporting the possible paraneoplastic significance of these serum autoantibodies.

Possible role of autoantibodies in the pathophysiology of GM2 gangliosidoses.

Mice containing a disruption of the Hexb gene have provided a useful model system for the study of the human lysosomal storage disorder known as Sandhoff disease (SD). Hexb(-/-) mice rapidly develop a progressive neurologic disease of ganglioside GM2 and GA2 storage. Our study revealed that the disease states in this model are associated with the appearance of antiganglioside autoantibodies. Both elevation of serum antiganglioside autoantibodies and IgG deposition to CNS neurons were found in the advanced stages of the disease in Hexb(-/-) mice; serum transfer from these mice showed IgG binding to neurons. To determine the role of these autoantibodies, the Fc receptor gamma gene (FcR gamma) was additionally disrupted in Hexb(-/-) mice, as it plays a key role in immune complex-mediated autoimmune diseases. Clinical symptoms were improved and life spans were extended in the Hexb(-/-)FcR gamma(-/-) mice; the number of apoptotic cells was also decreased. The level of ganglioside accumulation, however, did not change. IgG deposition was also confirmed in the brain of an autopsied SD patient. Taken together, these findings suggest that the production of autoantibodies plays an important role in the pathogenesis of neuropathy in SD and therefore provides a target for novel therapies.

Tolerance to self gangliosides is the major factor restricting the antibody response to lipopolysaccharide core oligosaccharides in Campylobacter jejuni strains associated with Guillain-Barré syndrome.

Guillain-Barré syndrome following Campylobacter jejuni infection is frequently associated with anti-ganglioside autoantibodies mediated by molecular mimicry with ganglioside-like oligosaccharides on bacterial lipopolysaccharide (LPS). The regulation of antibody responses to these T-cell-independent antigens is poorly understood, and only a minority of Campylobacter-infected individuals develop anti-ganglioside antibodies. This study investigates the response to gangliosides and LPS in strains of mice by using a range of immunization strategies. In normal mice following intraperitoneal immunization, antibody responses to gangliosides and LPS are low level but can be enhanced by the antigen format or coadministration of protein to recruit T-cell help. Class switching from the predominant immunoglobulin M (IgM) response to IgG3 occurs at low levels, suggesting B1-cell involvement. Systemic immunization results in poor responses. In GalNAc transferase knockout mice that lack all complex gangliosides and instead express high levels of GM3 and GD3, generation of anti-ganglioside antibodies upon immunization with either complex gangliosides or ganglioside-mimicking LPS is greatly enhanced and exhibits class switching to T-cell-dependent IgG isotypes and immunological memory, indicating that tolerance to self gangliosides is a major regulatory factor. Responses to GD3 are suppressed in knockout mice compared with wild-type mice, in which responses to GD3 are induced specifically by GD3 and as a result of polyclonal B-cell activation by LPS. The anti-ganglioside response generated in response to LPS is also dependent on the epitope density of the ganglioside mimicked and can be further manipulated by providing secondary signals via lipid A and CD40 ligation.

Complex Gangliosides at the Neuromuscular Junction Are Membrane Receptors for Autoantibodies and Botulinum Neurotoxin But Redundant for Normal Synaptic Function

One specialization of vertebrate presynaptic neuronal membranes is their multifold enrichment in complex gangliosides, suggesting that these sialoglycolipids may play a major functional role in synaptic transmission. We tested this hypothesis directly by studying neuromuscular synapses of mice lacking complex gangliosides attributable to deletion of the gene coding for β1,4 GalNAc-transferase (GM2/GD2 synthase), which catalyzes an early step in ganglioside synthesis. Our studies show that complex gangliosides are surprisingly redundant for regulated neurotransmitter release under normal physiological conditions. In contrast, we show that they are membrane receptors for both the paralytic botulinum neurotoxin type-A and human neuropathy-associated anti-ganglioside autoantibodies that arise through molecular mimicry with microbial structures. These data prove the critical importance of complex gangliosides in mediating pathophysiological events at the neuromuscular synapse.

Complex gangliosides at the neuromuscular junction are membrane receptors for autoantibodies and botulinum neurotoxin but redundant for normal synaptic function.

One specialization of vertebrate presynaptic neuronal membranes is their multifold enrichment in complex gangliosides, suggesting that these sialoglycolipids may play a major functional role in synaptic transmission. We tested this hypothesis directly by studying neuromuscular synapses of mice lacking complex gangliosides attributable to deletion of the gene coding for beta1,4 GalNAc-transferase (GM2/GD2 synthase), which catalyzes an early step in ganglioside synthesis. Our studies show that complex gangliosides are surprisingly redundant for regulated neurotransmitter release under normal physiological conditions. In contrast, we show that they are membrane receptors for both the paralytic botulinum neurotoxin type-A and human neuropathy-associated anti-ganglioside autoantibodies that arise through molecular mimicry with microbial structures. These data prove the critical importance of complex gangliosides in mediating pathophysiological events at the neuromuscular synapse.

Autoantibodies to the GM2-1 Islet Ganglioside and to GAD-65 at Type 1 Diabetes Onset

The GM2-1 islet ganglioside has been sequenced, found to be a novel ganglioside structure with a sialic acid moiety in the terminal position and two residues of non-acetylated galactosamine and also shown to be a target of autoantibodies in a subset of ICA+relatives of type 1 diabetic patients who subsequently progressed to the overt disease. In the present study we determined whether antibodies to GM2-1 or to other pancreatic gangliosides (a) are also expressed at disease onset and (b) are correlated with other diabetes-associated autoantibodies. Pancreatic gangliosides were extracted from human pancreas and purified by thin layer chromatography (TLC). Anti-ganglioside autoantibodies were determined using an indirect immuno-peroxidase technique performed directly on TLC plates in the following groups of patients: (a) newly diagnosed type 1 diabetic subjects before insulin therapy (n=45); all were tested for GAD65 autoantibodies in a fluid-phase RIA using35S-methionine-labelled recombinant human GAD65. Of these patients, 24 were also tested for insulin autoantibodies (IAA) by a competitive fluid phase radioimmunoassay and 21 were tested for GAD67 reactivity. (b) Forty-two age- and sex-matched normal control subjects. Autoantibodies to GM2-1, but not to other pancreatic gangliosides (GM3, GD3, GD1a), were expressed in 31 of 45 new-onset type 1 diabetic subjects and in one of 42 normal controls (P<0.01), while anti-GAD65, IAA and anti-GAD67 were found in 31 of 45, 12 of 24 and three of 21 patients respectively, but not in the control group of subjects. Interestingly, occurrence of GM2-1 autoantibodies was significantly correlated (P<0.005) with positivity for GAD65 autoantibodies, but not for IAA or GAD67 autoantibodies. It is of note that both GAD and gangliosides are mainly expressed in islets and in neuronal tissues and, therefore, type 1 diabetes may be regarded as a neuroendocrine autoimmune disease.

Structure and specificities of anti-ganglioside autoantibodies associated with motor neuropathies.

Autoantibodies that bind to GM1 ganglioside and asialo GM1 (GA1) have been implicated in the pathogenesis of motor neuropathies. To investigate the structure and specificity of these autoantibodies, peripheral blood B cells from patients with motor neuron diseases and from normal individuals were immortalized by EBV, and B cells secreting anti-GM1 or GA1 antibodies were cloned. We report an analysis of the structure and specificities of eight autoantibodies from patients with motor neuropathy, and two from normal individuals. Four antibodies were IgM, six were IgG, and all bound predominantly to GA1. The sequences of V domains of H and L chains were determined by a reverse transcription-polymerase chain reaction procedure. A variety of V genes were used to encode these antibodies: four VH1, two VH3, three VH4, one VH5, two V kappa I, two V kappa II, three V kappa III, and two V lambda II. Most V genes (13/19) exhibited less than 95% similarity to known germ-line genes, which suggests that somatic mutation was required to generate these autoantibodies, or that the relevant germ-line genes have not been identified. The average length of the H chain CDR3 was 16 amino acids, and in three antibodies this segment contained more than 20 amino acids. It was not possible to identify amino acid sequences that were encoded by germ-line D segments by conventional alignment of sequences. Partial analogies could be identified by introducing gaps, allowing mismatches and searching for D-D fusions and inversions. These results indicate that anti-GA1 antibodies can be encoded by a variety of VH-VL pairs, that the antibodies exhibit extensive somatic mutation, and that the CDR3 segments are generated by a number of nonconventional mechanisms.