Immunological studies of the pathogenesis of multiple sclerosis Cell-mediated cytotoxicity by peripheral blood lymphocytes against basic protein of myelin, encephalitogenic peptide, cerebrosides and gangliosides

Cell-mediated cytotoxic reactions against myelin basic protein (MBP), encephalitogenic peptide, cerebrosides and gangliosides were studied in 165 MS cases. The cytotoxicity, which is detectable only in the autologous system, is mediated by T lymphocytes bearing antigens reactive with antibody OKT8. The cytotoxicity is highest during active MS regardless of whether it is relapsing or chronic progressive. During inactive MS cytotoxicity is significantly less and remains virtually unchanged at a low level over long periods. The degree of cytotoxicity is thus, predominantly, dependent on the activity stage of the disease. Further, there is a correlation with the severity of the clinical deficiencies. The pathogenetic significance of cytotoxic reactions can be recognized by the close correlation between the stage and course of MS, which can be demonstrated both statistically and individually.     

Gangliosides can activate human alternative complement pathway

The alternative complement pathway (ACP) in vertebrates is knownto be important in inflammatory reactions, and to be activatedby foreign substances such as bacterial lipopolysaccharide (LPS)and zymosan, although to date no intrinsic activators have beenidentified except complement receptor type 2. From the pointof the structural similarity of LPS to ganglioside, we haveinvestigated gangliosides which are abundantly present in animalcells for their activity on the human ACP. All of seven gangliosidestested were found to activate this pathway in a manner dependingon the number of sialic acids and neutral sugars contained inthe molecules. A dose - response study suggested a correlationof the threshold in ganglioside concentration with its criticalmicelle concentration. Gangliosides may thus serve as an intrinsicactivator for ACP in animals, thereby leading to inflammation.The possibility of the participation of sialidase in complementactivation is also discussed.     

Expression of GD3 ganglioside by developing rat cerebellar Purkinje cells in situ

GD3 is a major ganglioside of the immature vertebrate CNS, and its expression is suggested to be characteristic of immature neuroectodermal cells. Using immunocytochemistry on cryostat sections of developing rat cerebellum with a monoclonal antibody specific for GD3, we have found that GD3 begins to be expressed on the plasma membrane of Purkinje cell bodies and dendrites beginning at postnatal day 7. Staining became brighter as the dendritic tree of the cells enlarged. As the Purkinje cells began to mature in different folia, they became GD3+, until by 15 days postnatal all Purkinje cells were GD3+. Positive staining of the dendritic tree was still present in the adult cerebellum. Using a monoclonal antibody 7-8D2, which recognizes cerebellar granule cells and their axons (the parallel fibres), and polyclonal antibodies against a synaptic vesicle component synaptophysin, double-immunofluorescence staining together with anti-GD3 antibodies suggested that the appearance of GD3 immunoreactivity did not correlate either with the ingrowth of parallel fibres or the presence of their synapses on Purkinje cell dendrites. However, comparison with earlier morphological studies showed that the appearance of GD3 immunoreactivity correlated well with the formation of climbing fibre synapses on Purkinje cell dendrites and the onset of the rapid expansion of the dendritic tree. These results are in keeping with the idea that elevated GD3 concentrations are found in certain cell types during periods of rapid growth or high metabolic activity but also show that this is not only restricted to immature cells.     

Reversibility of ganglioside effects on astrocyte morphology

The B-subunit of cholera toxin (BCT) induces a morphological change in cultured rat cerebral astrocytes from flat (epithelioid) to stellate (process-bearing). This stellation is reversed by the gangliosides GM1 and GD1a at concentrations of 10 microM or higher. Upon changing to a ganglioside-free medium, the flat astrocytes reacquire the stellate morphology within 3 hr, indicating that the antistellation effect of gangliosides is reversible. The possibility that this reversibility was due to a loss of exogenously acquired gangliosides from the cell membrane can be ruled out since pretreatment with GM1, but not GD1a, which does not bind BCT, results in an increased responsiveness to BCT, which was identical whether measured immediately after withdrawal of the ganglioside or 3 hr later. Asialo-GM1, which neither binds BCT nor reverses BCT-induced stellation by itself, prevents the return to stellation after withdrawal of the gangliosides. These data suggest that while gangliosides remain associated with the cell, their effect on astrocytes can change from opposing to permitting the stellate morphology.     

Human monoclonal IgM with autoantibody activity against two gangliosides (GM1 and GD1b) in a patient with motor neuron syndrome.

Small amounts of oligoclonal immunoglobulins were detected by Western blotting in the serum from a patient with motor neuron syndrome. The prominent one, a monoclonal IgM lambda, reacted strongly with the gangliosides GM1 and GD1b and more weakly with asialo GM1, as shown by immunoenzymatic staining of thin-layer chromatograms of gangliosides, ELISA on purified glycolipid coats and immunoadsorption with purified GM1. Affinity-chromatography with purified GM1 resulted in the purification of monoclonal IgM lambda. This purified IgM and its Fab fragments showed the same pattern of reactivity with gangliosides as that observed with whole serum. Such monoclonal IgM could be responsible for motor neuron diseases in some patients with overt or barely detectable monoclonal gammopathies.     

Ganglioside–basic protein interaction: Protection of gangliosides against neuraminidase action

The ability of acidic phospho- and sphingolipids to interact with basic proteins was studied by double diffusion analysis. The phospholipids, tri- and diphosphoinositide, and the sphingolipid, sulfatide, interacted with myelin basic protein as evidenced by precipitin line formation. Of the sialoglycosphingolipids (gangliosides) tested, only the myelin-specific monosialoganglioside, G_M4

1 The ganglioside nomenclature used here is according to the system of Svennerholm [1963]. The gangliosides are designated as follows: G_M4 = I³NeuAc? GalCer; G_M3 = II³NeuAc? LacCer; G_M1 = II³NeuAc? GgOse₄Cer; G_D1a = IV³ NeuAc, II³NeuAc? GgOse₄Cer; G_D1b = II³(NeuAc)₂? GgOse₄Cer; and G_T1b = IV³NeuAc, II³(NeuAc)₂? GgOse₄Cer.

, formed a precipitin line with myelin basic protein. In addition, myelin basic protein retarded the activity of Clostridium perfringens neuraminidase against G_M4 and the disialoganglioside, G_D1b. Examination of purified rat brain myelin suggested the presence of a neuraminidase activity intrinsic to myelin. This finding, in concert with ganglioside-myelin basic protein complexes which selectively protect against neuraminidase, may provide a physiological explanation for the simplified ganglioside pattern found in myelin.     

The immunobiology of Guillain-Barré syndromes

This presentation highlights aspects of the immunobiology of the Guillain-Barré syndromes (GBS), the world's leading cause of acute autoimmune neuromuscular paralysis. Understanding the key pathophysiological pathways of GBS and developing rational, specific immunotherapies are essential steps towards improving the clinical outcome of this devastating disorder. Much of the research into GBS over the last decade has focused on the forms mediated by anti-ganglioside antibodies, and we have made substantial progress in our understanding in several related areas. Particular highlights include (a) the emerging correlations between anti-ganglioside antibodies and specific clinical phenotypes, notably between anti-GM1/anti-GD1a antibodies and the acute motor axonal variant and anti-GQ1b/anti-GT1a antibodies and the Miller Fisher syndrome; (b) the identification of molecular mimicry between GBS-associated Campylobacter jejuni oligosaccharides and GM1, GD1a, and GT1a gangliosides as a mechanism for anti-ganglioside antibody induction; (c) the development of rodent models of GBS with sensory ataxic or motor phenotypes induced by immunisation with GD1b or GM1 gangliosides, respectively. Our work has particularly studied the motor nerve terminal as a model site of injury, and through combined active and passive immunisation paradigms, we have developed murine neuropathy phenotypes mediated by anti-ganglioside antibodies. This has been achieved through use of glycosyltransferase and complement regulator knock-out mice, both for cloning anti-ganglioside antibodies and inducing disease. Through such studies, we have proven a neuropathogenic role for murine anti-ganglioside antibodies and human GBS-associated antisera and identified several determinants that influence disease expression including (a) the level of immunological tolerance to microbial glycans that mimic self-gangliosides; (b) the ganglioside density in target tissue; (c) the level of complement activation and the neuroprotective effects of endogenous complement regulators; and (d) the role of calcium influx through complement pores in mediating axonal injury. Such studies provide us with clear information on an antibody-mediated pathogenesis model for GBS and should lead to rational therapeutic testing of agents that are potentially suitable for use in humans.     

Ganglioside composition and histology of a spontaneous metastatic brain tumour in the VM mouse.

Glycosphingolipid abnormalities have long been implicated in tumour malignancy and metastasis. Gangliosides are a family of sialic acid-containing glycosphingolipids that modulate cell-cell and cell-matrix interactions. Histology and ganglioside composition were examined in a natural brain tumour of the VM mouse strain. The tumour is distinguished from other metastatic tumour models because it arose spontaneously and metastasizes to several organs including brain and spinal cord after subcutaneous inoculation of tumour tissue in the flank. By electron microscopy, the tumour consisted of cells (15 to 20 microm in diameter) that had slightly indented nuclei and scant cytoplasm. The presence of smooth membranes with an absence of junctional complexes was a characteristic ultrastructural feature. No positive immunostaining was found for glial or neuronal markers. The total ganglioside sialic acid content of the subcutaneously grown tumour was low (12.6 +/- 0.9 microg per 100 mg dry wt, n = 6 separate tumours) and about 70% of this was in the form of N-glycolylneuraminic acid. In contrast, the ganglioside content of the cultured VM tumour cells was high (248.4 +/- 4.4 microg, n = 3) and consisted almost exclusively of N-acetylneuraminic acid. The ganglioside pattern of the tumour grown subcutaneously was complex, while GM3, GM2, GM1, and GD1a were the major gangliosides in the cultured tumour cells. This tumour will be a useful natural model for evaluating the role of gangliosides and other glycolipids in tumour cell invasion and metastasis.