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In our search of new biomarkers for multiple sclerosis (MS), we aimed to characterize the immunoglobulin (Ig) free light chains (FLC) in patients' cerebrospinal fluid (CSF) and serum, and to evaluate the diagnostic utility of FLC monomer-dimer patterns for MS. FLC were analyzed by Western blotting and mass spectroscopy. CSF and serum samples were examined for the presence of oligoclonal Ig bands by a conventional laboratory test for MS. Three distinct pathological FLC monomer-dimer patterns, typical of MS but not of other neurological diseases, were revealed. In 31 out 56 MS patients the highly increased CSF levels of κ monomers and dimers were demonstrated. In 18 MS patients, the increased κ-FLC levels were accompanied by highly elevated λ dimers. Five MS cases showed no significant elevation in κ-FLC, but they displayed abnormally high λ dimer levels. The intensity of the immunoreactive FLC bands was measured to account for κ and λ monomer and dimer levels and their ratios in the CSF and serum. Combined usage of different FLC parameters allowed the determination of the appropriate FLC threshold values to diagnose MS. The developed method showed higher sensitivity and specificity (96% and 90%, respectively), as compared to those of the conventional OCB test (82% and 70%, respectively). Our study highlights the role of the differential analysis of monomeric and dimeric κ- and λ-FLC for the precise diagnosis of MS., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A new procedure of free light chain (FLC) analysis was developed to assist the diagnosis of multiple sclerosis (MS). In this procedure, Western blotting technique was used to analyze monomeric and dimeric FLCs in the cerebrospinal fluid (CSF) and serum of patients with MS and other neurological diseases. The intensity of immunoreactive FLC bands was quantified by a specially developed software. Analysis of the obtained monomer/dimer patterns of κ and λ type FLCs allowed the determination of the diagnostically useful FLC parameters. The combined use of three FLC indices accounting for monomeric FLC-κ level and κ/λ ratio values in the CSF and serum was found to be of promising diagnostic importance for differentiation of MS from other non-MS neurological diseases., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Cell-mediated immunity (CMI) to myelin components has been implicated in Multiple Sclerosis (MS) pathogenesis: two targets were suggested, Myelin Basic Protein with controversial results and, more recently, gangliosides. In order to investigate their possible involvement, we have performed Leukocyte Migration inhibition (LMI) tests in the presence of human brain gangliosides. Thirty nine MS patients (twenty four being "definite", according to McDonald and Halliday's classification), twenty nine patients with Other Neurological Diseases (OND), thirty six patients with Inflammatory diseases (ID) and forty healthy controls were tested. MS patients were divided into two groups, depending on the clinical stage of the disease. The mean migration inhibition percentage of the MS-attack group was found to be significantly different from the four others (p less than 0.01) (24.4 +/- 16.2 versus 10.9 +/- 8.5 in MS without attack, 4.4 +/- 12.9 in OND, 3.9 +/- 13.9 in ID and 11.1 +/- 12.1 in healthy subjects). LMI to gangliosides is therefore significantly increased during the attack stage in MS. These results support the notion of a Delayed Type Hypersensitivity to these glycolipids during the active stage of the disease.

Accumulating evidence suggests that the process of demyelination in MS might involve an autoimmune response to one or more myelin components. A combination of myelin basic protein and myelin haptens was considered as possibly enhancing a cellular or humoral autoimmune reaction in MS. In line with this motion we have used an in-vitro E-rosette assay that correlates with in-vivo delayed hypersensitivity to demonstrate specific immunologic sensitivity of lymphocytes from MS patients to polysialogangliosides. A recent report that only lymphocytes from patients in relapse, but not in remission, are primed by gangliosides, underscores the relevance of the antigenic expression of gangliosides during the active pathological phase of the disease. The antigenic capacity of gangliosides to induce upon immunization a neurological disorder featured by demyelination in the CNS was demonstrated in rabbits. This and previous reports on the induction of peripheral demyelination in rabbits immunized with gangliosides will be further analyzed to gain insight on the possible role of these myelin lipid components as targets for an autoimmune mechanism in MS.

The ganglioside concentration in pooled serum from 20 patients with clinically definite multiple sclerosis (MS) was determined and compared with that in pooled serum from a similar number of healthy blood donors. There as a significant increase in the concentration of ganglioside-bound sialic acid from 691 +/- 57 micrograms/100 ml in the control sera to 926 +/-m 83 micrograms 100 ml in MS patients' sera. The profile of individual gangliosides in the two groups was identical, the four main structures being GM3, GD3, and GD1a and GT1b. The ganglioside pattern and concentration in peripheral blood lymphocytes derived from MS patients and controls was identical with the predominant GM3, and small proportions of Gd3. MS lymphocytes also showed a 39% increase in ganglioside content over control lymphocytes. The implication of such pronounced ganglioside increases is discussed with regard to the impaired immunocompetence of lymphocytes reported in MS.

Peripheral blood T lymphocytes from all of 14 patients with clinically definite multiple sclerosis (MS) were significantly stimulated by MS brain gangliosides in the active rosetting of sheep erythrocytes. Fractionated mono- and disialogangliosides were devoid of any stimulating effect on MS lymphocytes whereas the trisialoganglioside GT1 and to a greater extent the tetrasialoganglioside GQ1b were fully effective at a dose as low as 2 x 10(-18) moles. Gangliosides extracted from MS brains or from MS brain myelin were far more effective than gangliosides derived from control human brains or from bovine and mouse brains, suggesting the importance of highly sialylated gangliosides occurring to a greater extent in MS brain as previously reported. Lymphocytes from only 3 out of 24 other neurological patients were stimulated by the slow migrating gangliosides in the same way, but none of 32 healthy subjects responded to these gangliosides in the active E-rosette test. Lymphocytes from 5 to 8 patients with unilateral optic neuritis reacted positively to brain gangliosides by rosette formation, several weeks before a similar reaction to myelin basic protein was evident. Our data are compatible with a release of gangliosides during demyelination or other CNS degenerative processes occurring in multiple sclerosis.

An experimental autoimmune multiple sclerosis-like disease (EAMSD) was induced in rabbits by immunizing them with bovine brain gangliosides. Forebrain myelin was isolated and fractionated on a discontinuous sucrose gradient into light myelin (LM, buoyant density less than or equal to 0.625 M), and heavy myelin (HM, buoyant density greater than 0.625 M). No abnormalities in either protein or lipid composition of EAMSD myelin fractions were observed. However, the EAMSD tissue yielded 31% less light and 39% more heavy myelin compared to the control brains. Thus, the HL/LM ratio was two-fold greater in experimental than in control myelin. This pathological pattern is similar to that which has been observed in myelin obtained from the brains of multiple sclerosis patients and from the optic nerves of rabbits with experimentally-induced demyelination.