Derivation of ganglioside-specific T cell lines of suppressor or helper phenotype from cerebrospinal fluid of multiple sclerosis patients.

In order to investigate the specificity of activated T cells in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), we have cultured cells in the presence of mitogen-free IL-2 but without any antigen. Two T cell lines have been derived and showed specific reactivity to certain purified gangliosides (GM1, GD1a, GD1b and GQ1b). However, responses to other brain and viral antigens were not seen, and neither were T cell lines from peripheral blood lymphocytes (PBL) of normal, MS or other neurological disease patients stimulated by these gangliosides. Release of IL-2 could be detected after incubation of these CSF lines with specific gangliosides. One line exhibited predominantly helper/inducer (T4+) phenotype whilst the other was suppressor/cytotoxic (T8+), and further analysis indicated it could be of the suppressor phenotype. These data may have implications for T cell-induced demyelination in MS.

Control of immune-mediated disease of the central nervous system with monoclonal (CD4-specific) antibodies.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in Biozzi AB/H (H-2dq1) mice by active sensitization with spinal cord antigens. A single i.p. injection of CD8-depleting (YTS169.4) monoclonal antibody (mAb) failed to affect the clinical course of CREAE when administered prior to and during the onset of both the initial clinical and subsequent relapse phase of the disease. By contrast similar treatment with both CD4-depleting (YTS191.1) or CD4-blocking/non-depleting (YTS177.9) mAb significantly inhibited disease progression. Treatment shortly before the anticipated onset of clinical EAE prevented the subsequent development of disease, although disease could be provoked following antigen-rechallenge. In contrast, treatment with these antibodies during post-acute remission phase mainly served to delay the incidence of relapse. This suggests that, unless tolerance can be re-induced, treatment of ongoing neuroimmunological disease will require 'pulse' therapy and thus potentiate the problems of long-term immunosuppresion. Despite the findings that CD4-specific antibodies can rapidly reverse overt clinical disease shortly after the onset of disease exacerbation, once neurological dysfunction becomes established anti-CD4 treatment fails to improve the animals clinically, possibly due to the inability to rapidly reverse established demyelination. Although this study does not exclude the potential central action of the injected mAb, the failure to significantly dissociate therapeutic benefit between mAb administered directly into the CNS and that given systemically suggests that a major action of these agents is probably by selectively removing T cells in the peripheral T cell pool.

Therapy of chronic relapsing experimental allergic encephalomyelitis and the role of the blood-brain barrier: elucidation by the action of Brequinar sodium.

The immunosuppressive effect of the novel 4-quinoline carboxylic acid derivative Brequinar sodium on the chronic relapsing experimental allergic encephalomyelitis CREAE model in the Biozzi AB/H mouse was investigated. Although Brequinar sodium actively inhibited peripheral immune responses, it showed a limited potential to control an ongoing disease of the central nervous system (CNS). Doses of 25 mg/kg inhibited in vivo induced proliferative response and prevented EAE when treated from day 9 post-inoculation (p.i.). However, when administered from day 12 p.i. or during the post-acute remission phase-limited effects on the course of disease were observed. By comparison, treatment with a single high dose of cyclophosphamide (200 mg/kg) at these time points was significantly effective in controlling disease. As a possible explanation of the observed results it is suggested that for a compound to be effective in treating an ongoing immune response in the CNS, it must be capable of crossing the blood-brain barrier and act on the disease-inducing cells activated within the CNS. This hypothesis is supported by the finding that intracerebral injections of Brequinar sodium on day 12 p.i. significantly inhibited disease progression. This suggests that strategies aimed at controlling immune-mediated disease of the CNS require therapeutic doses of the compounds to be delivered into the CNS.

The evoked release of endogenous amino acids from tissue prisms of human neocortex.

The K+-evoked release of 13 amino acids has been determined from tissue prisms of neocortex from patients of various ages, and from rats. Prisms were prepared from various regions of human neocortex obtained at neurosurgery. Upon depolarization aspartate, glutamate and gamma-aminobutyrate (GABA) were shown to be preferentially released. The efflux of glutamate was calcium-dependent. Prisms prepared from human neocortex obtained shortly after death also exhibited preferential K+-induced release of putative amino acid transmitters. Absolute concentrations released into the media were similar to those found for neurosurgical samples. Comparison of the release data for rat and human samples revealed that the efflux of aspartate, glutamate and GABA occurred to a greater extent from rat brain preparations. The K+-evoked release of glutamate from human samples showed a significant linear increase from 12 to 68 years of age.

Amino acid release from biopsy samples of temporal neocortex from patients with Alzheimer's disease.

Tissue prisms prepared from neurosurgical samples of temporal neocortex of Alzheimer and control patients, upon depolarization preferentially released aspartate, glutamate and gamma-aminobutyrate (GABA). The Alzheimer and control samples did not significantly differ in the pattern of amino acid release, although acetylcholine synthesis by the Alzheimer tissue prisms was greatly reduced. There was no correlation between the efflux of any amino and acetylcholine synthesis. These observations suggest that in Alzheimer's disease there are no major changes in the extracellular concentrations of these putative amino acid transmitters.

Uptake studies of taurine in vivo and its effects on the course of experimental focal epilepsy in rats.

The passage of orally administered taurine across the intestinal wall to the blood plasma and target sites of neuromuscular excitability has been studied. This has been correlated with the effect of repeated oral dosing on the manifestations of cobalt-induced epilepsy in the rat. The blood-brain barrier and the organ distribution of taurine uptake have important implications in testing its effect on hyperexcitability phenomena.

Cellular hypersensitivity to gangliosides and myelin basic protein in multiple sclerosis.

Peripheral blood lymphocytes from patients with multiple sclerosis (MS), other neurological diseases and healthy controls were investigated for the presence of cell-mediated hypersensitivity to brain gangliosides and myelin basic protein using an active E-rosette assay. Sensitivity to myelin basic protein and gangliosides was found in MS patients in acute relapse and with progressive disease, whereas no sensitivity was found in MS patients in remission. Patients with other neurological diseases showed no response to gangliosides, but sensitization to myelin basic protein was found in a patient with leucoencephalopathy and in 4 of 6 stroke patients. Healthy controls did not respond to either antigen. In MS patients a positive correlation was seen between lymphocyte responses to myelin basic protein and to gangliosides. The data suggest that in comparison to gangliosides, myelin basic protein is a weaker stimulator of active rosette-forming cells. Moreover, cellular hypersensitivity to myelin basic protein is not MS-specific and may be present as a consequence of brain damage. However, cellular hypersensitivity to gangliosides appears to be more specific to MS and may be important in the pathogenesis of the disease.

Galactolipid fatty acid composition in adrenoleukodystrophy.

The non-hydroxy and hydroxy fatty acids of the major brain galactosphingolipids, cerebroside and sulphatide, have been isolated from white matter, gray matter and myelin of 2 children with adrenoleukodystrophy and from a corresponding control. In addition, cerebroside fatty acids were recovered from a myelin-related fraction on 1 patient. In comparison to control observations, myelin and the myelin-related fraction cerebroside demonstrate a loss of C24:1 and C24h:o fatty acids from normal and hydroxy fatty acid fractions, respectively. White and gray matter galactolipids isolated from 1 patient indicated a signficant increase in short chain (C16, C18 and C18:1) non-hydroxy fatty acids. In the isolated diseased myelin, the ratio of cerebroside hydroxy to non-hydroxy fatty acids was elevated about 1.5 times above the value for normal myelin, whereas the ratio derived for the myelin-related fraction was about two-thirds the values for the control myelin cerebroside.

Adrenoleukodystrophy brain cholesteryl esters and other neutral lipids.

Further examination of the neutral lipid fractions derived from brain tissue of two patients afflicted with adrenoleukodystrophy has demonstrated the presence not only of free cholesterol and cholesteryl ester, but also of appreciable free fatty acid and triglyceride. Using a gas--liquid chromatographic system normally employed for the analysis of long-chain fatty acids of galactolipids and spingomyelin, it was possible to establish the presence of long-chain (greater than C20) fatty acids in the cholesteryl ester, free fatty acid and triglyceride fractions. Long-chain fatty acids were most abundant in the cholesteryl esters. Fatty acids identified by gas--liquid chromatography and gas chromatography--mass spectroscopy included normal saturated and monounsaturated fatty acids as large as C34. Several unknown fatty acyl compounds, of as yet undetermined structure, were also observed. All investigations thus far would indicate that the pathogenesis of adrenoleukodystrophy is closely related to the aberrant metabolism of these long-chain fatty acids.

Neurochemical findings in adreno-leukodystrophy.

The results of neurochemical examination of brain tissue derived from 2 cases of adreno-leukodystrophy have been presented. Both white and grey matter contained suadanophilic material. Although free fatty acid was also present, cholesteryl ester accounted for the bulk of the sudanophilic material. Total cholesterol, galactolipid and phospholipid content was reduced in both white and grey matter. The lipid loss was particularly severe from white matter indicative of considerable demyelination. Cholesterol was found to be the only major sterol present in white or grey matter. Subcellular fractionation of the diseased white matter resulted in myelin and two related fractions, one of which was very fatty and was rich in steryl ester. Morphological examination of myelin indicated loosely-packed lamellae. All of 3 fractions had adenosine 2',6'-cyclic nucleotide-3'-phosphohydrolase activity. Myein and the fraction not rich in cholesteryl ester had discernable basic protein bands when examined by polyacrylamide gel electrophoresis. Analysis of the fatty acid composition of choline and ethanolamine glycerophospholipids indicated a general increase of saturated fatty acids, relative to control values and a decrease in long-chain fatty acids. Examination of sphingomyelin fatty acids also demonstrated a loss of long-chain fatty acids. The fatty acid composition of the cholesteryl esters from white and grey matter differed. The findings indicate generalized damage to the brain, both of white and grey matter, with the damage to the white matter being much more severe. No abnormal sterol or other lipid was isolated.

Immunocytochemical characterisation of the immune reaction in the central nervous system in multiple sclerosis. Possible role for microglia in lesion growth.

As there is evidence that in multiple sclerosis T-cell activation occurs in the central nervous system rather than outside, the inflammatory lesion may be extended through antigen presentation by cells at the edge of the plaque. In this study we present an immunocytochemical report on CNS tissue from an active case of MS, with an analysis of the distribution of CD4 and CD8 binding T cells and the expression of class I and II MHC determinants in plaques and white matter. Perivascular cuffs of early lesions, as judged by hypercellularity and minimal demyelination, contained activated T (Tac+) cells, which reacted with an anti-IL-2 monoclonal antibody. Thus sufficient T-cell growth factor would appear to be present to fuel the immune reaction in a growing lesion. The preponderance of T cells of the cytotoxic/suppressor (CD8) phenotype in the CNS parenchyma was found in conjunction with widespread staining of class I MHC antigen, a prerequisite for activity of cytotoxic T cells. Potential antigen presenting cells were demonstrated in MS plaques with a monoclonal antibody against the cytoplasmic, invariant chain of class II MHC. Macrophages and astrocytes, contributed to the staining in the hypercellular plaque border while the distribution of class II+ microglia in white matter suggest they may also be of importance in local antigen presentation.

Myelin composition in acute and chronic multiple sclerosis in relation to cerebral lysosomal activity.

The neuropathology of 3 cases of acute multiple sclerosis was correlated with biochemical analyses. Astrocytosis was a characteristic feature of the diffuse demyelinating lesions in one case and lymphocytic cuffing characterized the well-defined plaques present in the white matter of the other two cases. No abnormalities were found in the protein or lipid composition of isolated myelin, despite a wide range of recovery. Nevertheless, the gel electrophoretic protein pattern of white matter adjacent to plaque areas showed selective loss of myelin basic protein. Lysosomal acid proteinase and beta-glucuronidase levels were very significantly increased in all white matter samples in which astrocytosis was a major neuropathological feature. Levels were less markedly raised in samples containing discrete active plaques. Enzyme changes were also found in the apparently normal white matter of 2 of the cases. Acid proteinase activity was in the normal range but the activities of beta-glucuronidase and acetylcholine esterase were elevated. The significance of these results in relation to glial cell activity in the early stages of demyelination is discussed.

Leucocyte proteinase activity and acute multiple sclerosis.

Increased leucocyte neutral proteinase activity is associated with an attack of multiple sclerosis. Raised neutral proteinase is found in other diseases with rapid destruction of neural tissues. Increased enzyme activity may be responsible for removing antigenic protein from the blood.

Immunosuppression by cyclosporin A of experimental allergic encephalomyelitis.

Cyclosporin A (CsA), an immunosuppressant which acts selectively on antigen-responding T cells, was tried in the treatment of experimental allergic encephalomyelitis (EAE). The drug was highly effective in preventing the appearance of clinical and pathological signs of EAE in rats, guinea pigs and monkeys. Treatment of the established disease also reduced the incidence and severity of symptoms, and significantly reduced the number of inflammatory lesions in the central nervous system.

Neurochemical findings in a perinatal sudanophilic leukodystrophy rich in steryl ester.

Neurochemical and neuropathological studies have been made of a 10-day-old child who suffered from a sudanophilic leukodystrophy. The brain white matter contained abundant sudanophilic material. The patient's grey matter total cholesterol content was 30% higher than whole brain tissue derived from a comparable control. White matter cholesterol content was more than double the control value. Nearly 80% of the white matter cholesterol was esterified. Subcellular fractionation of the white matter resulted in a "floating fraction" rich in cholesteryl ester. The steryl ester fatty acid composition was not typical of control tissue or demyelinating tissue. Patient phospholipid fatty acid composition patterns differed from control, but white matter galactolipid fatty acid composition appeared normal. Cholesteryl ester hydrolase activity appeared normal. Myelin and myelin-like fractions, isolated from diseased and normal brain tissue, were of a primitive developing nature but appeared to be comparable. The findings indicate a neonatal sudanophilic leukodystrophy which doubtless began in prenatal life and which was rich in cholesteryl ester. The aetiology of the leukodystrophy is unknown.

Stimulation of acetylcholine synthesis by blockade of presynaptic muscarinic inhibitory autoreceptors: observations in rat and human brain preparations and comparison with the effect of choline.

The central presynaptic muscarinic inhibitory autoreceptor has been monitored by measuring the effects of muscarinic agents on acetylcholine (ACh) synthesis by rat and human neocortical tissue prisms. Quinuclidinyl benzilate (QNB), the antimuscarinic which of 20 tested caused the most marked stimulation of ACh synthesis in rat, significantly increased ACh synthesis in human prisms over a range or concentrations of 0.1 microM-10 microM. This data provides the first evidence that human brain contains presynaptic muscarinic receptors. However, the most marked effect of QNB was to increase synthesis to only 112% of control (value without drug) which was much less than in rat (to 140% of control). ACh synthesis is reduced to 50% of control in neocortex from Alzheimer patients so none of the antimuscarinics tested seem to be potentially capable of appreciably reversing this deficit. A high concentration of choline (10 mM) stimulated synthesis in rat prisms to about the same extent as QNB. Moreover, the ACh precursor was at least as effective in stimulating synthesis in human prisms (including those from a patient with Alzheimer's disease). This suggests that an elevated intracellular concentration of choline is likely to be much more effective than an antimuscarinic agent in stimulating synthesis in Alzheimer brain.

Preparation of 1-acyl-2-succinyl glycero-3-phosphorylcholine and evidence against its involvement in succinate dehydrogenase action.

1-Acyl-2-succinyl glycero-3-phosphorylcholine (GPC) was synthesized and its properties described. Although 1-acyl-2-succinyl GPC is a good substrate for succinate dehydrogenase, experiments on the incorporation of [2,3-14C] succinate into mitochondrial lipids gave no evidence to indicate that it is an intermediate in the enzymic oxidation of succinate to fumerate, as has been suggested earlier.

Effects of Cd2+, Mn2+, and Al3+ on rat brain synaptosomal uptake of noradrenaline and serotonin.

Cd2+, Mn2+, and Al3+ inhibited synaptosomal amine uptake in a concentration-dependent and time-dependent manner. In the absence of Ca2+, the rank order of inhibition of noradrenaline uptake was: Cd2+ (IC50 = 250 microM) greater than Al3+ (IC50 = 430 microM) greater than Mn2+ (IC50 = 1.50 mM), the IC50 being the concentration of metal ions that gave rise to 50% inhibition of uptake. In the presence of 1 mM Ca2+, the rank order of inhibition of uptake was: Al3+ (IC50 = 330 microM) greater than Cd2+ (IC50 = 540 microM) greater than (IC50 = 1.5 mM). The rank order of inhibition of serotonin uptake without Ca2+ was: Al3+ (IC50 = 370 microM) greater than Cd2+ (IC50 = 610 microM) greater than Mn2+ (IC50 = 3.4 mM) and the rank order in the presence of 1 mM Ca2+ was: Al3+ (IC50 = 290 microM) greater than Cd2+ (IC50 = 1.5 mM) greater than Mn2+ (IC50 = 4.0 mM). Ca2+, at 1 mM, definitely antagonized the inhibitory actions of Cd2+ on noradrenaline and serotonin uptake. Al3+ stimulated noradrenaline uptake at concentrations around 20-250 microM but inhibited this uptake at concentrations exceeding 300 microM in a dose-related fashion. Ca2+, at 1 mM, enhanced both the stimulatory and inhibitory effects of Al3+. Ca2+ also enhanced the inhibitory actions of Al3+ on serotonin uptake. These results, in conjunction with those we have previously published, suggest that Cd2+, Mn2+, and Al3+ exert differential and selective effects on the structure and function of synaptosomal membranes.

Selective inhibition of L-glutamate and gammaaminobutyrate transport in nerve ending particles by aluminium, manganese, and cadmium chloride.

AlCl3, MnCl2, and CdCl2 inhibited the rates of accumulation of [14C] L-glutamate and [3H] gammaaminobutyrate (GABA) in purified rat forebrain nerve-ending particles in a dose-dependent fashion. The concentrations that would give 50% inhibition (IC50) of GABA transport were 316 muM, 7.4 mM, and 1.4 mM, respectively. Ca2+ (1 mM) enhanced the inhibitory effect of Al3+ (IC50 decreased to 149 muM) but antagonized that of Mn2+ (IC50 = 10 mM) and Cd2+ (IC50 = 2.1 mM). For glutamate transport 1 mM Ca2+ changed the IC50 values from 299 to 224 micron for Al3+, 7.1 to 10 mM for Mn2+, and 2 to 3 mM for Cd2+. In contrast, the rates of accumulation of [14C] 2-deoxy-glucose and [3H] L-phenylalanine were mostly unaffected by these metal ions. The results indicate that Al3+, Mn2+, and Cd2+ exerted selective and differential effects on the transport systems of neurotransmitter substances in the synaptosomal membrane.

Biosynthesis of myelin and neurotoxic factors in the serum of multiple sclerosis patients.

The in vitro synthesis of myelin proteins has been studied by measuring the incorporation of [3H] lysine in developing rat brain slices. This incorporation system has been used to assay potentially gliotoxic and myelinolytic agents. A reduced incorporation of the labelled amino acid into myelin proteins occurs in the presence of anti-myelin anti-serum and anti-basic protein anti-serum. Diphtheria toxin has been found to inhibit the synthesis of myelin basic and proteolipid protein in the white matter slices of developing rats. Recent experiments with serum samples from multiple sclerosis patients in exacerbation suggest the presence of a factor which interferes with the synthesis of myelin in white matter slices.