Journal ofNeurology, Neurosurgery, and Psychiatry 1993;56:361-364 361 A characteristic ganglioside antibody pattern in J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.4.361 on 1 April 1993. Downloaded from the CSF of patients with amyotrophic lateral sclerosis Andreas Stevens, Michael Weller, Horst Wietholter Abstract Materials and methods Paired cerebrospinal fluid and serum Thirty five patients with amyotrophic lateral samples of patients with amyotrophic sclerosis, 16 men and 19 women, mean age lateral sclerosis (n = 35) revealed no con- 53-5 (2 4) years, range 25-79 years at diag- sistent abnormalities of CSF cell count, nosis, without a family history of the illness, CSF albumin, CSF IgG, CSF IgM, IgG were included in this study. The diagnosis or IgM index, or oligoclonal immuno- was clinical and based on the presence of globulin band formation in the CSF. both upper and lower motor neuron symp- Determination of IgG and IgM CSF and toms and signs. Eight patients had prominent serum antibodies to gangliosides GM1, bulbar signs, 31 had definite spasticity. For GM2, GM3, AGMI, GDla, GDlb, and inclusion in this study, the onset of symptoms GTlb showed a characteristic pattern had to be insidious and not started before the which allowed the differentiation of age of 20. The clinical course had to be amyotrophic lateral sclerosis from con- progressive. Specific attention was given to trols and from patients with other neuro- alternative diagnoses of cervical spondylosis, logical disorders including multiple neoplastic lesions of cervical spinal cord sclerosis. Specifically, patients with the and brain stem, polymyositis, peripheral disease had elevated CSF IgM antibodies neuropathy, progressive muscle atrophy, to all gangliosides except AGMI. The Charcot-Marie-Tooth disease, spinocerebel- lack of correlation between the CSF lar degeneration and familial spastic paraple- findings and corresponding serum anti- gia. CSF and serum samples were obtained bodies suggests a chronic, compart- after informed consent, centrifuged and mental, intrathecal immune response of stored at - 70°C. low activity in amyotrophic lateral scle- CSF parameters and ganglioside antibodies rosis. Whether this immune response is were estimated as previously described."'"3 primary and of pathogenetic signifi- Briefly, micro-enzyme linked immunosorbent cance, or an epiphenomenon of neuronal assay (ELISA) plates (M129A, Dynatech, degeneration, remains to be investigated. Denkendorf, Germany) were coated with purified ganglioside antigen, GM1, GM2, GM3, AGM1, GDla, GDlb, or GT1b http://jnnp.bmj.com/ (J Neurol Neurosurg Psychiatry 1993;56:361-364) (150 ng per well, Sigma, St Louis, MO, United States) diluted in methanol. Antigen purity was confirmed by thin layer chro- Amyotrophic lateral sclerosis is characterised matography. Protein binding sites on the well by a progressive loss of upper and lower that might cause non-specific binding of motor neurons of unknown aetiology.' immunoglobulins were blocked by incubation Cerebrospinal fluid studies are scarce and with bovine serum albumin (0-1%) in phos- have produced no consistent abnormalities in phate-buffered saline for 24 hours before the on September 29, 2021 by guest. Protected copyright. or serum samples. The Department of patients with this disease.2' Several authors incubation of CSF Neurology, University have postulated that a disturbance of CSF samples were matched to an IgG con- of Tubingen, immunoregulation may play a role in the centration of 5 mg/l. Serum was diluted 1:50 Germany motor neurons in these in saline. No detergents A Stevens degeneration of phosphate-buffered H Wietholter cases.4-7 The detection of polyclonal serum were used throughout the assay. IgG or IgM National Institute of antibodies to GM1 ganglioside in such ganglioside reactive antibodies were labelled Mental Health, patients89 has reinforced the search for by anti-human IgG (Sigma A-5403) or anti- Clinical Neuroscience immunological events in the pathogenesis of human IgM (Sigma A-3914) antisera conju- Branch, Section on Molecular the disease. As the pathological changes are gated to alkaline phosphatase diluted 1:1000 Pharmacology, largely confined to the central nervous system in phosphate-buffered saline. Monoclonality Bethesda, Maryland, and because there is no significant blood- of positive samples was excluded by differen- United States on for K or chains as previously M Weller brain barrier disturbance, studies the CSF tial assays i. light are more likely to establish immune dysfunc- described. 12 Correspondence to: Dr Andreas Stevens, tion than investigation of the systemic Seven internal reference sera obtained Department of Psychiatry, response. This reports an from healthy volunteers or seven CSF sam- University of Tubingen, immune study Osianderstrasse, 7400 analysis of CSF and serum IgG and IgM anti- ples from patients with suspected, but even- Tubingen, Germany bodies to seven different gangliosides in 35 tually disproven, neurological disease served Received 24 October 1991 sclerosis as a control for interassay variability. The and in final revised form patients with amyotrophic lateral 24 July 1992 and examines their correlation with routine interassay correlation calculated for nine runs Accepted 31 July 1992 CSF parameters. was 0 91. The ratio of the optical density of a 362 Stevens, Weller, Wietholter Table I Mean normalised optical density readings (SD) for ganglioside antibodies in sclerosis of chronic progressive (n = 13) and J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.56.4.361 on 1 April 1993. Downloaded from CSF (adjusted to S mg/l IgG) and serum (diluted 1:50) ofpatients with amyotrophic lateral sclerosis and controls with other neurological diseases. relapsing remitting type (n = 35). The statis- tical analysis included testing for normal Controls ALS CSF OND CSF ALS serum OND serum serum distribution, Student's t test, linear correla- tion, discriminant analysis, and analysis of GMl-IgG 1-09 (0-38) 1-00 (0-15) 1-08 (0 28) 1-00 (0 14) variance. GMI-IgM 1-17 (0-17)* 1-06 (0-15) 1 09 (0 34) 1-00 (0 28) GM2-IgG 0-94 (0-14) 1-04 (0-11) 1 16 (0 21)* 1-00 (0 16) GM2-IgM 1 22 (0 25) 1 00 (0 14) 1 12 (0 36) 1 00 (0-28) GM3-IgG 0.91 (0-18) 1 00 (0 11) 1 11 (0-27)* 1 00 (0 19) GM3-IgM 1-30 (0 37)* 1-00 (0 11) 1 13 (0 33) 1 00 (0 25) Results AGMI-IgG 0 94 (0 17) 0.99 (0-14) 1 21 (0Q34)* 1-00 (0 20) The routine laboratory investigations of CSF AGMl-IgM 1 08 (0 16) 0 99 (0 06) 1 10 (0 34) 1-00 (0 21) GD1a-IgG 1-05 (0-17) 1-01 (0-12) 1-06 (0-18) 1-00 (0-15) and serum samples from amyotrophic lateral GDla-IgM 1-22 (0 28)* 0 98 (0 08) 1-00 (0-31) 1-00 (0 27) sclerosis did not consistent abnormali- GDlb-IgG 095 (0-16) 1-00 (012) 106 (018)* 100 (013) yield GDlb-IgM 1-57 (0 49)* 1-00 (0-06) 1 13 (0 39) 1 00 (0 26) ties. None of the patients had a paraprotein GTIb-IgG 1-18 (0 25) 1 00 (0-13) 1.20 (0 20) 1-00 (0 13) on serum immune GTIb-IgM 1-47 (0 40)* 1 00 1 electrophoresis. Patients (0-10) 1 05 (0 32) 00 (0 26) with the disease did not have higher total ALS = amyotrophic lateral sclerosis; OND = other neurological diseases. serum IgG or IgM levels than controls. There *Significantly (p < 0.03) elevated readings in patients with amyotrophic lateral sclerosis compared with those with other neurological diseases. were also no significant differences between patients and controls in CSF cell counts, CSF albumin, CSF IgG, CSF IgM, or IgG or Table 2 Incidence ofpathological CSF and senum ganglioside antibodies (normalised IgM index. Seven patients had elevated CSF optical density readings beyond the third percentile) in patients with amyotrophic lateral sclerosis. IgG, one had elevated CSF albumin, six had mild pleocytosis. Two had CSF IgG oligo- CSF IgG IgM Serum IgG IgM clonal immunoglobulin n % n % n % n % bands detected. CSF IgG and IgG index in these patients were GM1 3 9 5 14 5 14 4 11 GM2 1 3 10 29 5 14 3 9 normal. GM3 2 6 21 60 3 9 5 14 The results of CSF and serum ganglioside AGM1 0 0 9 26 5 14 6 17 GDla 3 9 13 37 2 6 1 3 antibody determination in amyotrophic lat- GDlb 1 3 27 77 5 14 5 14 eral sclerosis and the incidence of patho- GTIb 10 29 24 69 13 37 2 6 logical normalised optical density readings in these patients are provided in tables 1 and 2. The controls with other neurological diseases patient sample over the mean optical density were not different from the internal controls. of the seven internal standards was calcu- CSF IgG, but not age or gender, had an lated, thus arbitrarily assigning the value of influence on ganglioside antibodies in those one to this mean. These normalised optical with other neurological diseases, before density results obtained in amyotrophic later- adjustment. The Pearson coefficients were al sclerosis were then compared with a con- 014 (GM1), 0-56 (GM2), 037 (GM3), 0-23 trol group of patients with other neurological (AGMI), 0-48 (GDla), 0 47 (GDlb) and diseases (n = 24) such as tension headache or 0 35 (GTIb). Among the seven ganglioside normal pressure hydrocephalus. Patients with antibodies examined, there was considerable neuroinflammatory and neuroimmunological intercorrelation in the amyotrophic lateral disorders were excluded. The means of the sclerosis samples (Pearson coefficient http://jnnp.bmj.com/ actual optical density readings in the group 0 3-0-6), suggesting that most of the sera with other neurological diseases at 30 min- contain either antibodies with multiple gan- utes were between 0-072 and 0-079 for the glioside reactivity or multiple antibodies seven CSF IgM ganglioside antibodies, against different gangliosides, a suggestion between 0138 and 0-191 for CSF IgG, that was confirmed by twin layer chromatog- between 0-294 and 0-462 for serum IgM, and raphy.