Detection of CSF 14-3-3 protein in Guillain-Barré syndrome.

OBJECTIVE: To search for biologic markers in the Guillain-Barré syndrome (GBS), we studied CSF samples from patients with GBS and neuropathy of various etiologies for the presence of 14-3-3 protein. METHODS: CSF samples from patients with GBS, chronic neuropathies, motor neuron disease (MND), definite sporadic Creutzfeldt-Jakob disease (sCJD), and normal control subjects were analyzed by standard immunoblot assay, using a polyclonal anti-14-3-3 antibody. CSF samples were also tested with antibodies recognizing specific isoforms of 14-3-3 proteins, either after one-dimensional or two-dimensional electrophoretic separation. RESULTS: A positive 14-3-3 assay was observed in 29 of 38 patients with GBS and in 4 patients with MND and other neuropathies, including 2 subjects with vasculitic neuropathy (VN). In GBS, 14-3-3 protein was detected as early as 12 to 48 hours after disease onset and showed an isoform pattern different from that encountered in patients with noninflammatory neuropathies, VN, MND, and sCJD. Immunohistochemical studies performed in archival fatal GBS cases disclosed marked 14-3-3 expression by mononuclear inflammatory infiltrates and Schwann cells. CONCLUSION: CSF 14-3-3 assay may represent a useful biologic marker in patients with Guillain-Barré syndrome.

Long term disability and social status change after Guillain-Barré syndrome.

OBJECTIVE: Even if the majority of patients with Guillain-Barré syndrome (GBS) have a favourable functional outcome some residual motor and sensory signs and symptoms may remain. The aim of this study was to evaluate the long-term effect of GBS on daily life,working activities, hobbies and social status and the presence of residual symptoms. PATIENTS AND METHODS: Seventy patients with GBS enrolled in a case-control study were examined. Information on signs or symptoms during the acute phase of the disease was retrieved from medical records and an ad-hoc questionnaire administered during hospitalization. Patients were interviewed by phone 3 to 5 years after disease onset about residual symptoms and changes in daily living. Disability and handicap were assessed using the Hughes, Rankin and Rotterdam 9-items scale. RESULTS: At follow-up 45 patients (64 %) made a complete functional recovery; 19 patients (27%) had some minor limitations in daily life although they were able to perform all their activities independently while 6 (9 %) needed aid for some hours or continuously during the day. Nineteen patients (27 %) had, however, to make substantial changes in their job, hobbies or social activities. There was no significant correlation between clinical and laboratory features during the acute phase of GBS and outcome. CONCLUSIONS: Although over 90% of our GBS patients had a more or less complete functional recovery, almost 30% of them had to make substantial changes in daily life. These findings indicate that GBS still has a significant impact on patients' life which may go beyond their residual disability or impairment. Treatment of GBS should not be only aimed at improving patients' disability but also at limiting the impact of the disease on their social life.

Anti-GM(2) IgM antibody-induced complement-mediated cytotoxicity in patients with dysimmune neuropathies.

Anti-GM2 IgM antibodies have been reported in some patients with dysimmune neuropathy or lower motor neuron syndrome. To determine whether these antibodies can induce complement-dependent cytolysis we performed a cytotoxicity assay on neuroblastoma cells with sera from seven patients with demyelinating dysimmune neuropathies and high titers of anti-GM2 IgM. As controls we used sera from seven patients with other anti-neural reactivities, six with the same neuropathies but no anti-GM2 or other anti-neural reactivity and from eight normal subjects. Of the seven positive sera tested, six induced complement-mediated cytotoxicity, while none of the controls had any relevant effect on neuroblastoma cells. Preincubation of positive sera with purified GM2 removed cytotoxic activity. Affinity purified anti-GM2 IgM had the same cytotoxic anti-GM2 effect of whole serum while serum or complement alone did not have any effect. In four anti-GM2-positive patients the percentage of cell lysis correlated with anti-GM2 titers and with IgM staining of neuroblastoma cells while in two the cytotoxic effect was higher than expected from antibody titers. Complement-mediated cell lysis induced by anti-GM2 IgM antibodies may be a possible mechanism of neural damage in patients with dysimmune neuropathy and high titers of anti-GM2 IgM antibodies.

Anti-sulfatide IgM antibodies in peripheral neuropathy.

Anti-sulfatide IgM antibodies have been recently associated with neuropathy but the clinical and electrophysiological correlations of this reactivity remains unclear. We reviewed the clinical and electrophysiological features of patients with high anti-sulfatide titers detected in our laboratory from 1991 to 1998. Of the 564 patients with different neurological diagnosis tested by enzyme-linked immunosorbent assay (ELISA), 11 had high anti-sulfatide IgM titers (>1/8000), 26 had titers of 1/8000 while 78 had titers of 1/4000. All patients with high anti-sulfatide IgM titers had a chronic, dysimmune, mostly sensorimotor neuropathy that in seven was associated with IgM monoclonal gammopathy. In most of these patients electrophysiological and morphological studies were consistent with a predominantly demyelinating neuropathy frequently associated with prominent axonal loss. Antibody titers of 1/8000, though always associated with neuropathy, did not correlate with a particular form or cause of neuropathy, while lower titers were equally distributed in patients with different neurological disorders. Our study indicate that high anti-sulfatide IgM titers (>1/8000) are highly predictive for a chronic, dysimmune, mostly demyelinating neuropathy often associated with IgM monoclonal gammopathy, and may therefore have potential diagnostic relevance.

Clinical presentation and outcome of Guillain-Barré and related syndromes in relation to anti-ganglioside antibodies.

We correlated the clinical features of 78 patients with Guillain-Barré syndrome (GBS) or related variants, with the presence of serum antibodies to the gangliosides GM1, GM2, GD1a, GD1b and GQ1b in order to determine whether these antibodies may influence the clinical presentation or outcome of GBS. Sixty-three patients had typical GBS (81%), nine a pure motor form (11%), three a paraparetic form (4%), and three had Miller Fisher syndrome (MFS). IgG or IgM (or both) anti-ganglioside antibodies were found by ELISA in 37% of patients, including 36% with typical, 33% with pure motor and 100% with MFS. Beside the constant occurrence of anti-GQ1b antibodies in patients with MFS (P<0.00001), the other clinical forms were not associated with a specific anti-ganglioside reactivity. Anti-GM1 and anti-GD1a antibodies tended to be associated with a worse disability at 6 month than other or no reactivity and, similarly to anti-GM2 antibodies, with a more frequent respiratory impairment. Anti-GM2 and anti-GD1b antibodies were always associated with typical GBS and, in all but one patient, with a complete recovery; still they were found in only 13 and 3%, respectively, of the patients with this presentation. Anti-GQ1b antibodies, though always associated with ophthalmoplegia and ataxia in both MFS and GBS, were found in only 36 and 26%, respectively, of patients with these symptoms. Even if different anti-ganglioside antibodies tend to be associated with some clinical features possibly suggesting that they may influence the clinical presentation or outcome, with the exception of anti-GQ1b antibodies for ophthalmoplegia and ataxia, they do not permit to predict the clinical presentation or outcome in individual patients.

Anti-myelin-associated glycoprotein antibodies predict the development of neuropathy in asymptomatic patients with IgM monoclonal gammopathy.

We examined 52 asymptomatic patients with IgM monoclonal gammopathy and correlated anti-myelin-associated glycoprotein (anti-MAG) IgM with the presence of subclinical neuropathy and, in 24 of these patients, with the development of symptomatic neuropathy during a follow-up interval of 40 to 144 months (mean, 75.3 months). Three of 6 patients (50%) with high (>1/6,400) anti-MAG IgM had subclinical neuropathy at entry compared with 2 of 46 patients (4.3%) with low or no reactivity. At follow-up, a symptomatic neuropathy occurred in 3 of 4 patients with high reactivity and in 3 of 21 patients with low or no reactivity. The correlation of high anti-MAG IgM with the presence of subclinical neuropathy or the development of symptomatic neuropathy supports its pathogenetic role in the neuropathy.

Anti-GM2 IgM antibodies: clinical correlates and reactivity with a human neuroblastoma cell line.

Anti-GM2 IgM antibodies have been reported in some patients with dysimmune neuropathy or lower motor neuron syndrome, in whom they were often associated with a concomitant reactivity with GM1. To investigate the possible clinical and pathogenetic relevance of these antibodies we measured serum anti-GM2 IgM titers by ELISA in 224 patients with different neuropathies and motor neuron disease and examined their binding to SK-N-SH neuroblastoma cells by indirect immunofluorescence (IIF). High titers of anti-GM2 IgM antibodies were found in eight patients with dysimmune neuropathies including two with multifocal motor neuropathy (MMN), two with purely motor demyelinating neuropathy without conduction block (MN) and four with Guillain-Barré syndrome (GBS). In two MMN patients reactivity with GM2 was associated with anti-GM1 reactivity and in one MN patient with anti-GM1, -GD1a and -GD1b reactivity. All but one patient had a concomitant reactivity with GalNAc-GD1a. Serum IgM from all positive patients intensely stained by IIF the surface of SK-N-SH neuroblastoma cells. This reactivity was blocked by serum pre-incubation with GM2, was not observed with sera from patients without anti-GM2 antibodies including those with high anti-GM1 or other anti-glycolipid antibodies, and correlated with the presence of GM2 in the SK-N-SH neuroblastoma cells. These findings indicate that anti-GM2 antibodies, though infrequent, are strictly associated with dysimmune neuropathies and suggest that SK-N-SH neuroblastoma cells can be a suitable in vitro model to study the functional and biological effects of these antibodies.

Clinical features and anti-neural reactivity in neuropathy associated with IgG monoclonal gammopathy of undetermined significance.

Neuropathy has been frequently reported in patients with IgG monoclonal gammopathy of undetermined significance (MGUS) but it is still unclear whether this association has clinical or pathogenetic relevance. In order to clarify the possible role of IgG MGUS in the neuropathy we correlated the clinical and electrophysiological features of the neuropathy with the duration and anti-neural activity of the M-protein in 17 patients with neuropathy and IgG MGUS. Ten patients (59%) had a chronic demyelinating neuropathy clinically indistinguishable from chronic inflammatory demyelinating polyneuropathy (CIDP) while 7 (41%) had a predominantly sensory axonal or mixed neuropathy. In 80% of patients in the CIDP-like and 28% in the sensory group the IgG M-protein became manifest several months to years after onset of the neuropathy. Antibodies to one or more neural antigens (including tubulin, a 35KD P0-like nerve myelin glycoprotein, GD1a, GM1 and chondrotin sulfate C) were found in 40% of patients with CIDP-like and 43% with sensory neuropathy but also in 37% patients with IgG MGUS without neuropathy. Neuropathy associated with IgG MGUS is probably less heterogeneous than previously considered suggesting that this association may not be merely casual. The evidence for primary pathogenetic role of IgG M-proteins in the neuropathy remains however elusive.

Expression of glycosylated recombinant human myelin-associated glycoprotein on a neuroblastoma cell line and its reactivity with HNK-1 but not human anti-MAG antibodies.

A correctly glycosylated myelin-associated glycoprotein (MAG) must express the carbohydrate epitope HNK-1, which is the target antigen for IgM antibodies in some patients with neuropathy. We transfected a human MAG cDNA clone into the neuroblastoma cell line SK-N-SH and verified by immunoblot the expression of the HNK-1 epitope on the recombinant molecule. By the same method and by indirect immunofluorescence we did not find any reactivity of human anti-MAG IgM antibodies with glycosylated recombinant MAG and transfected neuroblastoma cells. These findings suggest that the mere presence of the HNK-1 epitope is probably not sufficient for MAG to be recognized by human antibodies and that other factors such as the concentration or fine structure of this epitope in MAG, which mostly depend on the cellular context, may be also critical for this reactivity.

Clinical correlate and fine specificity of anti-GQ1b antibodies in peripheral neuropathy.

We studied the frequency, fine specificity and clinical correlate of anti-GQ1b IgG and IgM antibodies in 216 patients with neuropathy including three with Miller Fisher syndrome (MFS), 73 with Guillain-Barré syndrome (GBS), 99 with neuropathy associated with IgM monoclonal gammopathy (PN+IgM) and 41 with other neuropathies, and compared the data with 92 disease or normal controls. We found high (>1/100) anti-GQ1b IgG titers in all three MFS patients and in two GBS patients (2.7%) with ophthalmoplegia and ataxia, while high anti-GQ1b IgM were only found in two patients with a chronic demyelinating sensorimotor neuropathy associated with IgMkappa monoclonal gammopathy (2%). By overlay HPTLC, IgG antibodies in MFS and GBS either selectively reacted with GQ1b or also bound to GD3, and less intensely to GD1b, while IgM antibodies from both patients with PN+IgM also strongly reacted with GD1b and, in one, with GD3 and GT1b. The constant association of anti-GQ1b antibodies with dysimmune neuropathies and the correlation between their isotype, fine specificity and clinical presentation, support a possible pathogenetic link between these antibodies and the neuropathy.

Anti-GD1a ganglioside antibodies in peripheral motor syndromes.

High titers of anti-GD1a antibodies have been found in patients with Guillain-Barre syndrome or motor neuropathy. To determine the possible diagnostic relevance of these antibodies, we measured serum anti-GD1a IgG and IgM antibodies by enzyme-linked immunosorbent assay in 195 patients with different motor syndromes and in 335 control subjects. Moderately high antibody titers (1/1,280-1/5,120) were occasionally found in patients with chronic inflammatory demyelinating polyneuropathy (5%), multifocal motor neuropathy (18%), lower motor neuron disease (3.8%), or amyotrophic lateral sclerosis (1.8%) and in immunological control subjects (1.2%), while titers of 1/20,480 or higher were only found in 2 patients with Guillain-Barre syndrome (IgG in both) and 2 with motor neuropathy and IgM lambda monoclonal gammopathy improving with immunotherapy. In both motor neuropathy patients and the Guillain-Barre syndrome patient who were retested during recovery, anti-GD1a titers decreased concomitantly with clinical improvement. High anti-GD1a antibody titers may be found in several motor syndromes but only markedly increased anti-GD1a titers are strictly associated with potentially treatable dysimmune neuropathies.

Anti-alpha- and beta-tubulin IgM antibodies in dysimmune neuropathies.

An increased frequency of serum IgM antibodies to beta-tubulin has been found by ELISA in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) and the Guillain-Barré syndrome (GBS). We used an immunoblot technique to compare the frequency, titer and specificity of anti-tubulin IgM antibodies in 207 patients with different neuropathies, 109 with other neurological disease (OND) and 110 non-neurological controls. High titers of serum anti-tubulin IgM antibodies (> 1:1600) were present in 2 patients with CIDP (10.5%), 1 with multifocal motor neuropathy (MMN) (11%), 1 with GBS (1.8%), two with IgM monoclonal gammopathy, one with (1.3%) and one without neuropathy (2.1%), and in two with OND (1.8%). Even if the relative binding to alpha- and beta-tubulin differed among positive patients, in all IgM bound to both tubulin subunits. All positive patients had a similarly intense IgM reactivity with tubulin by ELISA that showed high anti-tubulin IgM in 4 additional CIDP patients (total positive by ELISA 30%) and in 7 of 23 normal subjects (30%). Even if high anti-tubulin IgM were slightly more frequent by immunoblot in chronic dysimmune neuropathies than in other diseases, possibly reflecting a secondary response to neural damage during an ongoing immune response, their relatively low frequency in these diseases does not seem to justify their measurement for diagnostic application.

Serum IgG antibodies to a 35-kDa P0-related glycoprotein in motor neuron disease.

Using an immunoblot technique we found a significantly higher frequency of serum IgG antibodies to a 35-kDa peripheral nerve myelin glycoprotein in patients with motor neuron disease (MND) (39% of 70) than in patients with neuropathy (13% of 61), other neurological disease (9% of 32) and normal subjects (5% of 20) (P < 0.005 in all cases), but not with multiple sclerosis (MS) (20% of 30) or non-neural immune diseases (25% of 32). Most positive patients had antibody titers of 1:200 or 1:2000 while higher titers were only found in seven patients with MND, one with chronic inflammatory demyelinating neuropathy, two with MS, two with non-neural immune diseases and one with stroke. The reacting protein had a higher molecular mass than P0 and was only faintly bound by an anti-P0 antiserum, but had the same N-terminal amino acid sequence of P0. The difference in molecular mass between P0 and the 35-kDa protein and the IgG reactivity of one patient's IgG with the 35-kDa protein persisted after its deglycosylation and dephosphorylation. Although there is no evidence that these antibodies are pathogenic, their frequent occurrence in MND and other immune-mediated conditions supports the hypothesis of an activation of the immune system in MND.