Induction of experimental ataxic sensory neuronopathy in cats by immunization with purified SGPG.

IgM paraproteins in about 50% of the patients with neuropathy associated with IgM gammopathy react with carbohydrate moieties in myelin-associated glycoprotein (MAG) and in sulfated glucuronic glycolipids (SGGLs) in human peripheral nerves. However, the role of anti-MAG/SGGL antibodies in the pathogenesis of neuropathy remains unclear. In order to induce an animal model of neuropathy associated with anti-MAG/SGGL antibodies, cats were immunized with sulfoglucuronyl paragloboside (SGPG). All four cats immunized with SGPG developed clinical signs of sensory neuronopathy within 11 months after initial immunization, characterized by unsteadiness, falling, hind limb weakness and ataxia. In two cats the ataxia and hind limb paralysis were so severe that the animals had to be euthanized. Pathological examination revealed sensory ganglionitis with inflammatory infiltrates in the dorsal root ganglia. No overt signs of pathology were noted in the examined roots or nerves. High titer anti-SGPG/MAG antibodies were detected in all 4 cats immunized with SGPG but not in 3 control cats. Our data demonstrate that immunization of cats with SGPG induced anti-SGPG antibodies and sensory neuronopathy clinically resembling the sensory ataxia of patients with monoclonal IgM anti-MAG/SGPG antibodies. This study suggests that these anti-MAG/SGPG antibodies play a role in the pathogenesis of this neuropathy.

Immunoglobulin G subclass distribution of autoantibodies to gangliosides in patients with Guillain-Barre syndrome.

IgG anti-ganglioside antibodies are present in a proportion of patients with the Guillain-Barré syndrome (GBS). To determine if antibodies to gangliosides are restricted in IgG subclass distribution, we evaluated IgG subclass antibody responses to gangliosides in sera of patients with GBS. Sera from GBS patients with IgG activity against gangliosides were analyzed for IgG subclass distribution using an enzyme-linked immunosorbent assay. The anti-LM1 antibodies in sera from GBS patients were predominantly of the IgG3 subclass while anti-GM1 and anti-GT1a antibodies were predominantly of the IgG1 and IgG3 subclasses. The results indicate a Th2-dependent antibody response.

Antibodies to GT1a ganglioside in patients with Guillain-Barré syndrome.

Serum antibodies from 8 (13%) of 62 patients with the acute Guillain-Barré syndrome (GBS) and 1 of 3 patients with the Miller Fisher syndrome (MFS) recognized a minor ganglioside in bovine and human brain trisialoganglioside fractions. The ganglioside antigen migrated between GD1a and GD1b on thin-layer chromatograms. The structure of this ganglioside was established to be GT1a by thin-layer chromatography blotting and mass spectrometry. GT1a a ganglioside was also detected in human and bovine peripheral nerves by thin-layer chromatogram immunostaining. Serum from the GBS patients had IgM, IgG, or IgA antibodies against GT1a detectable by enzyme-linked immunosorbent assay (ELISA). Serum from the MFS patient also had elevated levels of IG against GT1a. None of the sera from 43 patients with other neurological diseases or from 24 healthy controls reacted with GT1a. Sera from 6 of 8 GBS patients with anti-Gt1a antibodies also reacted with GQ1b. There was no difference in the incidence of anti-GT1a immunoglobulins in acute GBS patients with or without oculomotor abnormalities. Levels of anti-GT1a antibodies correlated temporally wit clinical symptoms in GBS patients. Although the incidence of dysphagia was slightly higher in GBS patients with anti-GT1a antibodies than in those without, the number of patients studied may have been too small to detect an association between anti-GT1a antibodies and an a specific clinical variant of GBS. Our data demonstrate that a proportion of GBS patients have antibodies against GT1a ganglioside and suggest that these antibodies may play a role in the pathogenesis of neuropathy in GBS.

Serologic evidence of previous Campylobacter jejuni infection in patients with the Guillain-Barré syndrome.

OBJECTIVE: To determine if patients with the Guillain-Barré syndrome are likely to have had Campylobacter jejuni infection before onset of neurologic symptoms. DESIGN: A case-control study. SETTING: Several university medical centers. PATIENTS: Case patients met clinical criteria for the Guillain-Barré syndrome between 1983 and 1990 and had a serum sample collected and frozen within 3 weeks after onset of neurologic symptoms (n = 118). Disease controls were patients with other neurologic illnesses (n = 56); healthy controls were hospital employees or healthy family members of patients (n = 47). MEASUREMENTS: Serum IgA, IgG, and IgM antibodies to C. jejuni were determined by enzyme-linked immunosorbent assays. Assays were done in a blinded manner. RESULTS: Optical density ratios > or = 2 in two or more immunoglobulin classes were seen in 43 (36%) of patients with the Guillain-Barré syndrome and in 10 (10%) of controls (odds ratio, 5.3; 95% CI, 2.4 to 12.5; P < 0.001). Increasing the optical density ratio or the number of immunoglobulin classes necessary to yield a positive result increased the strength of the association. The number of patients with the Guillain-Barré syndrome who had positive serologic responses was greatest from September to November (P = 0.02). Male patients were three times more likely to have serologic evidence of C. jejuni infection (P = 0.009); the proportion of patients with the syndrome who had a positive serologic response increased with age. CONCLUSIONS: Patients with the Guillain-Barré syndrome are more likely than controls to have serologic evidence of C. jejuni infection in the weeks before onset of neurologic symptoms. Campylobacter jejuni may play a role in the initiation of the Guillain-Barré syndrome in many patients.

Effects of Guillain-Barré sera containing antibodies against glycolipids in cultures of rat Schwann cells and sensory neurons.

Serum samples from 52 patients with the acute Guillain-Barré syndrome (GBS), 19 patients with other neurological disorders, and 18 healthy volunteers were tested for cytotoxicity in cultures of rat Schwann cells and dorsal root ganglion neurons. The samples were also examined by enzyme-linked immunosorbent assay for IgG and IgM antibodies against various acidic and neutral glycolipids. Samples from 16 of the 52 (31%) acute GBS patients and from 1 of the 6 patients with chronic inflammatory demyelinating polyneuropathy produced myelin breakdown in culture. Although 10 of the 16 cytotoxic acute GBS serum samples contained anti-glycolipid immunoglobulins, there was no correlation in individual samples between cytotoxic activity and the presence of antibodies against specific glycolipids. While our results do not exclude a role for anti-glycolipid antibodies in the pathogenesis of the acute GBS, the cytotoxic effects of acute GBS serum in cultures of Schwann cells and sensory neurons are probably not due to these antibodies alone.

Anti-MAG IgM paraproteins from some patients with polyneuropathy associated with IgM paraproteinemia also react with sulfatide.

Sera from five of 11 patients with neuropathy associated with IgM paraproteinemia (NAIgMPP) and reactivity against myelin-associated glycoprotein (MAG) also had elevated levels of IgM against sulfatide. Although three patients had anti-sulfatide IgM titers of less than or equal to 1:1000, two patients had titers of greater than or equal to 1:50,000. Absorption of patient serum with sulfatide revealed that anti-MAG IgM paraproteins in two patients with high titer anti-sulfatide IgM crossreacted with sulfatide. Our study is the first to show that some anti-MAG IgM paraproteins crossreact with sulfatide, a major acidic glycolipid of myelin. Moreover, some patients with NAIgMPP have polyclonal anti-sulfatide IgM in addition to anti-MAG IgM paraproteins. Therefore, sulfatide may be a target antigen in some patients with NAIgMPP.

Antibodies to acidic glycolipids in Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy.

Using an enzyme-linked immunosorbent assay and a thin-layer chromatography-immunostaining procedure, we detected serum antibodies against acidic glycolipids in 36 of 53 patients with Guillain-Barré syndrome (GBS) and 8 of 16 patients with chronic inflammatory demyelinating polyneuropathy (CIDP). Although we also found anti-acidic glycolipid antibodies in 4 of 13 patients with other neurological diseases; 2 of 10 patients with multiple sclerosis; 8 of 33 patients with inflammatory, infectious, allergic or autoimmune disorders and 3 of 32 healthy subjects, the levels of antibodies in these controls were much lower than in GBS patients. There were several patterns of reactivity of GBS sera including antibodies to LM1 and HexLM1, GM1 or GD1b or both, various other gangliosides, sulfated glycolipids, and as yet unidentified glycolipids. Sera from 30% of GBS patients had antibodies against two or more antigenically distinct acidic glycolipid antigens. Levels of anti-acidic glycolipid antibodies correlated with clinical symptoms in 9 of 11 GBS patients. While the increased incidence of antibodies to acidic glycolipids in patients with GBS (P less than 0.001) and CIDP (P less than 0.025) compared to controls could be an epiphenomenon, anti-acidic glycolipid antibodies may play a role in nerve injury in some GBS and CIDP patients.

Anti-GM1 IgA antibodies in Guillain-Barré syndrome.

Serum anti-GM1 IgA antibodies were detected in 15 of 53 (28%) patients with the acute Guillain-Barré syndrome (GBS) and in one of 26 (4%) patients with other neurological diseases. Although nine GBS patients had anti-GM1 IgA titers of 1:200 or less, six patients had titers of 1:800 or more. Some GBS patients with anti-GM1 IgA antibodies also had antibodies against GD1b or GM2 or both. The presence of markedly elevated anti-GM1 IgA was associated with a poor clinical outcome at 6 and 12 months following onset of the GBS. The possible pathogenetic role of anti-GM1 IgA antibodies remains to be established.

Antibodies to sulfated glycolipids in Guillain-Barré syndrome.

Sera from 53 patients with acute Guillain-Barré syndrome (GBS), 15 patients with chronic inflammatory demyelinating polyneuropathy (CIDP), 13 patients with other neurological diseases (OND) and 31 healthy controls were tested for IgM and IgG antibodies to sulfoglucuronyl paragloboside (SGPG) and sulfatide by both an ELISA and a thin-layer chromatogram-overlay technique. Although the mean levels of anti-SGPG or anti-sulfatide antibodies in GBS patients were not elevated compared to controls, the occurrence of anti-SGPG antibodies was more frequent in GBS patients than in controls (P less than 0.02). Acute GBS patients with antibodies to SGPG or sulfatide were clinically indistinguishable from other GBS patients. Our data suggest that elevated levels of antibodies to SGPG could be important in the pathogenesis of neuropathy in some GBS patients.

Search for antibodies to neutral glycolipids in sera of patients with Guillain-Barré syndrome.

Sera from 54 patients with Guillain-Barré syndrome (GBS), 34 patients with other neurological diseases (OND) and 32 healthy controls were tested for antibodies to total lipid fractions and higher neutral glycolipid fractions isolated from human and dog nerves, purified Forssman glycolipid and a panel of purified neutral glycolipids by both an enzyme-linked immunosorbent assay (ELISA) and a thin-layer chromatogram (TLC)-overlay technique. IgM and IgG antibodies to total lipid fractions, as well as to galactocerebroside, ceramide dihexoside, ceramide trihexoside, and globoside were not significantly elevated in the sera of GBS patients as compared to controls. High levels of anti-asialo-GM1 IgG antibodies, however, were detected in 6 of 54 (11%) GBS patients and 1 of 30 (3%) OND patients. Intense reactivity with purified Forssman glycolipid and a number of glycolipid antigens in higher neutral glycolipid enriched fractions of human cauda equina and dog sciatic nerves was noted by TLC-immunostaining in many GBS and control sera. Although the levels of anti-Forssman IgM were significantly decreased in GBS sera compared with normal sera (P less than 0.05) and OND sera (P less than 0.02), the levels of anti-Forssman IgG antibodies were not significantly different. With the possible exception of IgG antibodies to asialo-GM1, our results suggest that serum antibodies against Forssman glycolipid and neutral glycolipids are not significantly elevated in GBS patients and, thus, are unlikely to play an important role in the pathogenesis of this disease.

Variability in the structural requirements for binding of human monoclonal anti-myelin-associated glycoprotein immunoglobulin M antibodies and HNK-1 to sphingoglycolipid antigens.

A high proportion of patients with neuropathy have immunoglobulin M (IgM) paraproteins that react with carbohydrate determinants on the myelin-associated glycoprotein (MAG) and two sphingoglycolipids, 3-sulfoglucuronyl paragloboside (SGPG) and 3-sulfoglucuronyl lactosaminyl paragloboside. In order to characterize the fine specificities of these human antibodies further, the binding of 10 anti-MAG paraproteins to several chemically modified derivatives of SGPG was compared with the binding to intact SGPG by both TLC-overlay and enzyme-linked immunosorbent assay. The following derivatives were tested: the desulfated lipid, glucuronyl paragloboside (GPG); the methyl ester of GPG (MeGPG); the methyl ester of SGPG, 3-sulfomethylglucuronyl paragloboside (SMeGPG); and 3-sulfoglucosyl paragloboside (SGlcPG) produced by reduction of the carboxyl group of the glucuronic acid with sodium borohydride. All 10 IgM paraproteins and the related mouse IgM antibody, HNK-1, reacted most strongly with intact SGPG, but variations in the reactivity with the derivatives revealed striking differences in the structural requirements for binding between the antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies to gangliosides and myelin proteins in Guillain-Barré syndrome.

An earlier investigation from our laboratory (Ilyas AA, Wilson HJ, Quarles RH, et al. Serum antibodies to gangliosides in Guillain-Barré syndrome. Ann Neurol 1988;23:440-447) demonstrating the presence of high levels of antiganglioside ganglioside antibodies in the sera of 5 of 26 patients with Guillain-Barré Syndrome (GBS) but not in control sera is summarized. The ganglioside antigens varied among the 5 patients with positive findings, and the antiganglioside antibodies decreased concurrently with clinical improvement in those patients for whom longitudinal samples were available for analysis. The results are discussed in the context of antibodies to acidic glycolipids in other types of neuropathy and other studies on antiglycolipid antibodies in GBS. Data showing the occurrence of lower levels of antibodies to P2 protein, P0 glycoprotein, and myelin-associated glycoprotein in some of the GBS patients are also summarized. The findings from our laboratory combined with the results of others make it unlikely that antiganglioside antibodies have a notable pathogenic effect in most patients with GBS, but the possibility remains that they are of pathogenic importance in some patients with the highest antibody titers.

Molecular cloning of a human immunoglobulin heavy chain variable (VH) region with anti-myelin-associated glycoprotein activity.

A cDNA clone that encodes the heavy chain variable region (VH) of an IgM M-protein with anti-myelin-associated glycoprotein (MAG) activity secreted by chronic lymphocytic leukemia cells (B-C11) from a patient with peripheral neuropathy was cloned and sequenced. The JH region was identical to the germline JH4 sequence except for deletion of a thymidine residue at the site of D-JH recombination, and the D region showed greatest homology to DM2. Sequence analysis of the VH region revealed greatest homology to VH26, a member of the VH3 gene family, but homology was only 83.7% over 326 bases, suggesting that it was derived from as yet an unidentified member of the VH3 gene family.

Anti-GM1 ganglioside antibodies with differing fine specificities in patients with multifocal motor neuropathy.

Antibodies to gangliosides were detected in sera from three of 19 patients with chronic inflammatory polyneuropathy (CIP) by a thin-layer chromatogram overlay technique. All three of the patients fell into a clinical subset of the group that had multifocal motor neuropathy, and in all three patients the antibodies reacted with GM1 ganglioside. However, the fine specificities of the antibodies differed as demonstrated by cross-reactivity with different gangliosides in each of the three patients. The antibodies in patient 1 reacted with GM1, GD1b, and asialo-GM1 suggesting that the terminal Gal(beta 1-3)GalNAc moiety that is common to these three glycolipids is an important part of the epitope(s). This was confirmed by showing reactivity of the antibodies with Gal(beta 1-3)GalNAc conjugated to bovine serum albumin. Patient 2 had antibodies that did not react with GD1b, but cross-reacted with GM2 ganglioside suggesting that the epitope(s) involved the inner portion of the oligosaccharide moiety that is shared between GM1 and GM2. Patient 3 had antibodies that reacted with GM1 and asialo-GM1, but they did not cross-react with either GD1b or GM2. These results provide further evidence for a relationship between motor nerve syndromes and anti-GM1 antibodies and also suggest that GM1 could be a principal target antigen since other reactive gangliosides differed among the patients. However, the possible pathogenic effects of anti-GM1 antibodies on motor nerves remain to be established.

Multiple sclerosis. Oligodendrocyte proliferation and differentiation in fresh lesions.

Fresh lesions in the brain and spinal cord of patients with multiple sclerosis who died shortly after the onset of symptoms were examined immunocytochemically for myelin and oligodendrocyte antigens that are known to be sequentially expressed during normal development. Cells with oligodendrocyte-like morphology that appear in large numbers throughout fresh lesions after acute myelin breakdown and before new myelin formation were found to express galactocerebroside, carbonic anhydrase, and 2',3'-cyclic nucleotide 3'-phosphohydrolase but not myelin-associated glycoprotein or myelin basic protein. They also exhibit intense surface reactivity for a carbohydrate epitope associated with the family of cell adhesion molecules recognized by the monoclonal antibody HNK-1. With the onset of remyelination and the appearance of myelin-associated glycoprotein, myelin basic proteins, CNP, and the HNK-1 epitope is newly formed myelin sheaths, perikaryon CNP and HNK-1 reactivity diminished. A possible oligodendrocyte precursor cell in the form of a large HNK-1 positive glial fibrillary acidic protein negative glial cell was observed among interfascicular oligodendrocytes in white matter bordering these hypercellular plaques. Because a similar progression in the expression of CNP and the HNK-1 epitope occurs during normal oligodendrocyte differentiation, these observations are additional evidence that extensive oligodendrocyte regeneration occurs in some plaques early in the course of the disease. The finding of large numbers of immature oligodendrocytes, presumably expressing many developmentally restricted antigens not normally present in the mature nervous system, in plaques at a particular stage in their evolution may be important in understanding why remyelination eventually fails in multiple sclerosis.

Identification and characterization of gangliosides reacting with IgM paraproteins in three patients with neuropathy associated with biclonal gammopathy.

IgM monoclonal antibodies from three patients with polyneuropathy associated with biclonal gammopathy reacted with monosialoganglioside GM1 on thin-layer chromatograms. An IgM paraprotein in one of the patients with a predominantly motor neuropathy also reacted strongly with the ganglioside GD1b and asialo-GM1. All three of these antigenic lipids have a Gal(beta 1-3)GalNAc moiety in common which would appear to be the antigenic determinant. However, this IgM also cross-reacted weakly with paragloboside which has an N-acetyllactosaminyl [Gal(beta 1-4)GlcNAc] terminal structure. The specificity of the other paraprotein in this patient is not known. The IgM paraproteins reacting with GM1 in both of the other patients exhibited different specificity because they did not react with GD1b and asialo-GM1, but reacted strongly with GM2 ganglioside. The data suggest that the epitope for both of these IgMs is in the GalNAc(beta 1-4)(NeuAc alpha 2-3)Gal(beta 1-4)Glc region of the gangliosides that is common to both GM2 and GM1. The second IgM paraproteins in both of these latter patients react with the myelin-associated glycoprotein (MAG) and two 3-sulfoglucuronyl glycolipids that share antigenic determinants with MAG.

A treatable multifocal motor neuropathy with antibodies to GM1 ganglioside.

We report 2 patients with a treatable, immune-mediated motor polyneuropathy associated with antibodies to defined neural antigens. In these patients asymmetrical weakness developed in one arm and progressed over 2 to 3 years to involve the other arm, legs, and trunk. Both patients were initially diagnosed as having lower motor neuron forms of amyotrophic lateral sclerosis. However, repeated electrophysiological testing eventually showed multifocal conduction blocks in motor but not sensory fibers compatible with patchy selective demyelination. Serum testing by thin-layer chromatography and enzyme-linked immunosorbent assay revealed that both patients had high titers of antibody directed against GM1 and other gangliosides. Initial therapeutic trials of prednisone (100 mg daily for 4 to 6 months) and plasmapheresis were unsuccessful. Treatment with cyclophosphamide, however, was followed by marked improvement in strength in both patients.

Serum antibodies to gangliosides in Guillain-Barré syndrome.

To determine whether antibodies to acidic glycolipids of nervous tissue are present in patients with Guillain-Barré syndrome (GBS), sera from patients with GBS and appropriate control subjects were tested by a thin-layer chromatogram overlay technique. Chromatograms on which the whole ganglioside fractions from peripheral nerve and brain had been separated were overlaid with appropriate dilutions of the patients' sera (1:100 or greater), and antibody binding was revealed with a radiolabeled or peroxidase-labeled second antibody. Antibodies to ganglioside antigens were detected in 5 of 26 patients with GBS. IgG antibodies in 1 patient reacted strongly with LM1 (sialosyl paragloboside), the major ganglioside of human peripheral nervous system myelin, and its hexaose analog (sialosyl lactosaminyl paragloboside), a minor ganglioside of human peripheral nervous system myelin. The antibody titer in this patient fell 8-fold over 6 weeks coincident with clinical improvement. IgG from 2 other patients with GBS reacted with GD1b ganglioside, and the antibody titers in these patients also decreased substantially with clinical improvement. IgM antibodies in the sera from 2 other patients reacted with GD1a and GT1b gangliosides, which have a shared terminal carbohydrate sequence. Antibodies to gangliosides were not detected in the sera from 19 patients with other neurological diseases or from 10 normal subjects, and the frequency with which antiganglioside antibodies occurred in the patients with GBS was significantly greater than that in the combined control subjects (p less than 0.01). The results demonstrate relatively high levels of antibodies to gangliosides in some GBS patients.

Gangliosides GM2, IV4GalNAcGM1b, and IV4GalNAcGC1a as antigens for monoclonal immunoglobulin M in neuropathy associated with gammopathy.

It was previously reported that monoclonal IgM from two patients with gammopathy and neuropathy showed similar specificity by reacting with the same group of unidentified minor components in the ganglioside fractions of human nervous tissues (Ilyas, A. A., Quarles, R. H., Dalakas, M. C., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6697-6700). Enzymatic degradation, ion-exchange chromatography, and immunostaining of purified ganglioside standards on thin-layer chromatograms have now revealed that the antigenic glycolipids recognized by the IgM from these patients are gangliosides GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer(GM2), GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (IV4GalNAcGM1b), and GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4 beta Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1-Cer (IV4GalNAcGD1a). The monoclonal IgM appears to be reacting with the terminal [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-] moiety shared by these three gangliosides and is a useful probe for detecting small amounts of GM2, IV4GalNAcGM1b, IV4GalNAcGD1a, and other gangliosides with the same terminal sugar configuration in tissues. Species distribution studies using the antibody revealed that GM2 is present in the brains and nerves of all species examined, while IV4GalNAcGM1b and IV4GalNAcGD1a exhibit some striking species specificity. GM2, but not IV4GalNAcGD1a, is enriched in purified myelin from human brain.