Clinical and laboratory assessment of distal peripheral nerves in Gulf War veterans and spouses.

BACKGROUND: The prevalence of symptoms suggesting distal symmetric polyneuropathy (DSP) was reported to be higher among deployed veterans (DV) to the Persian Gulf in 1990-1991 than to control non-deployed veterans (NDV). The authors therefore compared the prevalence of DSP by direct examination of DV and their spouses to control NDV and spouses. METHODS: The authors performed standardized neurologic examinations on 1,061 DV and 1,128 NDV selected from a cohort of veterans who previously participated in a national mail and telephone survey. Presence of DSP was evaluated by history, physical examination, and standardized electrophysiologic assessment of motor and sensory nerves. Similar examinations were performed without electrophysiologic tests in 484 DV spouses and 533 NDV spouses. Statistical analyses were performed with appropriate adjustments for the stratified sampling scheme. RESULTS: No differences between adjusted population prevalence of DSP in DV and NDV were found by electrophysiology (3.7% vs 6.3%, p = 0.07), by neurologic examination (3.1% vs 2.6%, p = 0.60), or by the methods combined (6.3% vs 7.3%, p = 0.47). Excluding veterans with non-military service related diseases that may cause DSP did not alter outcomes. DV potentially exposed to neurotoxins from the Khamisiyah ammunition depot explosion did not significantly differ in DSP prevalence compared to non-exposed DV. The prevalence of DSP in DV spouses did not differ from NDV spouses (2.7% vs 3.2%, p = 0.64). CONCLUSIONS: Neither veterans deployed during the Gulf War era nor their spouses had a higher prevalence of DSP compared to NDV and spouses.

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.

Elevated neopterin levels in Guillain-Barré syndrome. Further evidence of immune activation.

Neopterin is a by-product of guanosine triphosphate metabolism and is produced by macrophages in response to lymphocytic activation. We have studied serum neopterin levels in patients with Guillain-Barré syndrome to obtain further evidence of immune activation in this disease. Serum neopterin levels were significantly elevated in patients with Guillain-Barré syndrome compared with patients with other peripheral neuropathies and multiple sclerosis and with healthy control subjects. Serial analysis demonstrated that as neopterin levels fell, the clinical status of the patients with Guillain-Barré syndrome improved and soluble interleukin 2 receptor levels dropped. Thus, lymphocytic and macrophage activation may play a role in the pathogenesis of Guillain-Barré syndrome.

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.

Clinical correlation with serum-soluble interleukin-2 receptor levels in Guillain-Barré syndrome.

In patients with Guillain-Barré syndrome (GBS) soluble interleukin-2 receptor (sIL-2R) levels were elevated compared with those of patients with other neurologic diseases (OND), and of healthy controls. Smaller increases in sIL-2R levels occurred in OND patients compared to healthy subjects. Monitoring of GBS patients clearly demonstrated that decreases in sIL-2R levels correlated with clinical recovery. Thus, T-cell activation may be relevant in the pathogenesis of GBS.

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.

Human alpha tumor necrosis factor does not damage cultures containing rat Schwann cells and sensory neurons.

The effects of recombinant human alpha tumor necrosis factor (alpha-TNF) were compared with those of cytotoxic serum from patients with the acute Guillain-Barré syndrome (GBS) in myelinated cultures containing only rat Schwann cells and dorsal root ganglion neurons. Alpha-TNF did not damage rat peripheral nervous system tissue in culture. These observations suggest that alpha-TNF is not responsible for the cytotoxic activity of acute GBS serum in culture.

Effects of A23187 in cultures containing only rat Schwann cells and sensory neurons.

Serum from approximately 40% of patients with the acute Guillain-Barré syndrome (GBS) selectively destroys myelin and myelin-related Schwann cells in cultures containing only rat dorsal root ganglion neurons and Schwann cells. To determine if the effects of GBS serum on myelin and myelin-related Schwann cells could be mediated through elevations in the intracellular concentration of calcium ions, we compared the effects of cytotoxic serum to A23187, a divalent cation ionophore. Both myelin- and nonmyelin-related Schwann cells were killed along with neurons in the presence of A23187 and extracellular calcium ions. Myelin sheaths also underwent vesicular disruption. The ultrastructural appearance of myelin and myelin-related Schwann cell lysis caused by A23187 were essentially identical to damage produced by GBS serum. These observations suggest that GBS serum factors might damage myelin and myelin-related Schwann cells in culture by an increase in myelin-related Schwann cell permeability to extracellular calcium ions. In contrast, A23187 causes necrosis of Schwann cells and neurons as well as myelin lysis by a nonselective increase in membrane permeability to extracellular calcium ions.

Effects of ethanol on rat Schwann cell proliferation and myelination in culture.

It is possible to treat dissociated embryonic rat dorsal root ganglia in culture to inhibit proliferation of all nonneuronal cells except Schwann cells. Neurons have been shown to produce a mitogenic stimulus for Schwann cells under these conditions. Additionally, myelin-competent neurons induce Schwann cells to elaborate myelin sheaths. Groups of sibling cultures were exposed to various nonlethal concentrations of ethanol (0, 43, 86, or 172 mM) for 4 wk. Cultures were assessed weekly by light microscopy in a blind fashion for evidence of Schwann cell proliferation and myelin formation. Ethanol adversely affected both Schwann cell proliferation and myelin formation in culture. No obvious differences in neuronal morphology were observed among the various groups of cultures by light or electron microscopy. These observations suggest that ethanol might interfere with Schwann cell proliferation and myelin formation in culture by one or both of the following means: a) inhibit neuronal production of signals for Schwann cell proliferation and myelination or b) impede Schwann cell responses to neuronal signals. Investigation of these possibilities in culture may provide insight into neuropathologic mechanisms operative in the fetal alcohol syndrome or alcohol-associated peripheral neuropathy in humans.

Ultrastructural effects of Guillain-Barré serum in cultures containing only rat Schwann cells and dorsal root ganglion neurons.

Serum from patients with the acute form of the Guillain-Barré syndrome was applied to cultures containing only rat dorsal root ganglion neurons and Schwann cells. Serum taken from 4 of 10 patients during the first 1-3 weeks of clinical onset had previously been shown to have significant demyelinating activity in this culture system when observed at the light microscopic level. More detailed assessment made at the ultrastructural level showed that: (1) wide-spread myelin-related Schwann cell lysis occurred in concert with vesicular myelin breakdown; (2) non-myelin-related Schwann cells avidly phagocytized necrotic cell debris and fragments of compact myelin; and (3) neurites and non-myelin-related Schwann cells remained structurally undamaged. Cultures treated with convalescent phase serum from patients whose acute phase serum had cytolytic activity displayed no significant ultrastructural damage to either neurites or Schwann cells. This is the first electron microscopic study to provide direct evidence that acute Guillain-Barré serum can be cytolytic for myelin-related Schwann cells and peripheral myelin in an experimental setting free of leukocytes, lymphocytes and mononuclear phagocytes.

Immunohistochemical study of myelin sheaths formed by oligodendrocytes interacting with dissociated dorsal root ganglion neurons in culture.

The addition of central nervous system (CNS) glial cells to dissociated networks of rat dorsal root ganglion neurons in tissue culture provided a useful system for the study of CNS myelin sheath formation. The CNS myelin basic proteins (BP) and proteolipid protein (PLP) were demonstrable in these cultures by immunoperoxidase techniques. Both BP and PLP were detectable in myelinating oligodendrocytes and CNS myelin sheaths. Anti-BP serum and anti-PLP serum were useful immunohistochemical staining reagents for the identification of myelinating oligodendrocytes and CNS myelin sheaths in tissue culture.

Studies with antisera against peripheral nervous system myelin and myelin basic proteins. I. Effects of antiserum upon living cultures of nervous tissue.

We studied the effects of antiserum against rat peripheral nervous system (PNS) myelin, rat or chicken central nervous system myelin basic protein (BP), or rabbit P2 protein from PNS myelin on myelinated cultures containing only rat dorsal root ganglion neurons and Schwann cells. While anti-PNS myelin serum consistently produced segmental PNS demyelination, anti-BP serum and anti-P2 serum did not. The culture results suggest that the myelin PNS proteins P1 (identical to basic protein from central nervous system myelin) and P2 are not exposed on the extracellular surfaces of myelin-related Schwann cells in tissue culture.

Studies with antisera against peripheral nervous system myelin and myelin basic proteins. II. Immunohistochemical studies in cultures of rat dorsal root ganglion neurons and Schwann cells.

Antiserum against rat peripheral nervous system (PNS) myelin contained immunoglobulins which bound preferentially to the extracellular surfaces of myelin-related Schwann cells in intact cultures of dorsal root ganglion (DRG) neurons and Schwann cells, while antiserum against basic protein (BP) from central nervous system myelin or the PNS basic protein P2 did not. We demonstrate the presence of PNS myelin proteins P1 (identical to BP) and P2 by immunoperoxidase techniques in DRG cultures that had been treated to disrupt cellular membranes. These observations suggest that P1 and P2 are not exposed on the extracellular surfaces of myelin-related Schwann cells in culture. The results also support the hypothesis concerning the possible mechanisms by which anti-PNS myelin serum demyelinates DRG cultures, while anti-BP serum and anti-P2 serum do not.

Expression of the trembler mouse mutation in organotypic cultures of dorsal root ganglia.

Organotypic dorsal root ganglion (DRG) cultures were established from all the embryos of two trembler (Tr/+) female mice mated to normal (+/+) males to determine if the trembler mutation would be expressed in nerve tissue culture. Dorsal root ganglia from normal mice maintained in our culture system exhibit substantial myelination after 6 weeks of growth. This normal pattern was observed in approximately one half of the cultures in the present series. The remaining half of the explants had marked PNS myelin abnormalities readily detectable at the light microscopic level in living cultures; furthermore, the ultrastructural appearance of these Tr/+ cultures was similar to that of adult trembler sciatic nerve. An analysis of unmyelinated nerve fibers in Tr/+ cultures revealed that the number of neurites resident within each non-myelinating Tr/+ Schwann cell was significantly less than the number observed in +/+ cultures. There are distinctive PNS myelin abnormalities which: (1) develop in DRG cultures established from embryos at risk for the trembler mutation; (2) are highly reliable and readily detectable markers for the trembler genotype; and (3) are similar to the trembler PNS defects detectable in vivo.