GANGLIOSIDE ABNORMALITIES IN MULTIPLE SCLEROSIS

Abstract— Gangliosides were isolated from plaque tissue and normal appearing white matter of multiple sclerosis (MS) brain. All four plaques showed decreased ganglioside concn relative to normal human white matter on a wet wt basis, but significant elevation in terms of dry wt. The wet wt and dry wt concn of MS white matter gangliosides showed smaller but statistically significant decreases below normal. Thin-layer patterns of the plaques showed several departures from normal white matter, including decrease of G₄ and G₅, and complete loss of G₇ (sialosylgalactosylceramide). Most of the plaques had significant elevation of G_2A and G_3A along with increases of the slower-migrating polysialogangliosides. An additional ganglioside was present between G₂ and G_2A which was not seen in normal white matter. The TLC pattern of MS white matter gangliosides was essentially normal. The evidence for a general decrease of acidic lipids within normal appearing white matter is discussed.     

A THIOL PROTEINASE HIGHLY ELEVATED IN AND AROUND THE PLAQUES OF MULTIPLE SCLEROSIS

A thiol dependent proteolytic enzyme (tentatively identified as carboxypeptidase B) which liberates phenylalanine from CBZ-glutamyl-phenylalanine at pH 5.3 was shown, by a sensitive micromethod, to be greatly increased in activity in and around MS plaques. These increases exceeded those of other hydrolases previously measured. Plaque tissue, on the basis of lipid-free dry weight, is up to 3-fold richer in this enzyme than control white matter, and most samples of apparently uninvolved MS white matter already show elevated activities. The enzyme is highly dependent on the presence of dithiothreitol. It is unaffected by diisopropyl fluorophosphate and pepstatin, but inhibited by iodoacetate and leupeptin. Macrophages or lymphocytic infiltrations in the tissue do not appear to be the main source of the enzyme. In conjunction with measurements of DNA, reflecting gliosis, invasion by hematogenom cells and proliferation of phagocytes as well as oligodendrocyte loss, and acid lipase-esterase, indicative of the survival or degeneration of oligodendrocytes, these results are interpreted as probably reflecting predominantly the activity of astrocytes. The incidental finding that most samples of unaffected white matter from MS patients contain more DNA per unit lipid free dry weight than average control white matter is considered significant in pointing to more widespread tissue changes independent of or preceding plaque formation.     

Acid hydrolases and other enzymes in secondary demyelination: a quantitative histochemical study in the wobbler mouse

Abstract— The hereditary motor neuron degeneration found in the wobbler (wr) mouse was studied as a model of secondary demyelination. Lysosomal enzymes (acid phosphatase, acid proteinase, β-glucuronidase and β-galactosidase) were found elevated about three-fold in the white matter of the affected cervical spinal cord as compared with normal controls; but they were either not increased, or increased much less, in the anterior horn. Since gliosis and influx of phagocytic cells are minimal in this model, the high hydrolase levels are believed to arise primarily from (a) the accumulations of axonal dense bodies seen in involved areas, and (b) from indigenous cells engaged in breaking down the myelin fragments. Thus, secondary demyelination may, at least in this case, be initiated by enzymes of local origin. DNA levels per unit weight of tissue in both white and gray matter of wobbler cervical cord were elevated 40-50 per cent over controls. However, this was considered to reflect the stunted growth of wobbler mice rather than proliferation or influx of cells (an altered ratio of DNA to protein was demonstrated in the brain). Wobbler mice had similar levels of lactic dehydrogenase as controls; glucose-6-phosphate dehydrogenase was moderately elevated, and glycerol-3-phosphate dehydrogenase was less active in the anterior horn but more active in the white matter of the spinal cord.     

White Matter Proteins in Multiple Sclerosis

Abstract: The SDS-soluble membrane proteins of plaques and of macroscopically normal white matter from multiple sclerosis brain were investigated by gradient polyacrylamide gel electrophoresis (PAGE). Eleven protein bands were analyzed in detail. The extensive loss of myelin proteins in plaque samples was accompanied by changes in at least three other non-myelin proteins, besides glial fibrillary acidic protein (GFAP), which probably reflect gliosis. Densitometric analysis of the PAGE patterns of membrane fractions from MS and control white matter revealed significant quantitative differences in a number of protein bands. A reduction in myelin basic protein (BP) was associated with an equally significant increase in a high-molecular-weight peptide fragment which may prove to be a breakdown product of BP. Small but highly significant differences in the Wolfgram protein and in one non-myelin protein were also a consistent feature of the normal-appearing white matter samples. The problem of defining normal white matter in multiple sclerosis brain is discussed in relation to the results of the present study, which suggest that one of the early events in the pathogenesis of the disease prior to frank demyelination is an alteration in the protein components of the myelin sheath and possibly of glial cells.     

High Resolution Proton NMR Spectroscopy of Multiple Sclerosis Lesions

Abstract: Tissue from postmortem multiple sclerosis and normal control brains was extracted with perchloric acid and analysed using proton NMR spectroscopy. The content of N -acetyl-derived groups (the sum of N -acetylaspartate, acetate, and N -acetylaspartylglutamate) was decreased in multiple sclerosis plaques compared with normal control white matter (mean, 4.36 vs. 6.64 µmol/g wet weight). In normal appearing white matter adjacent to plaques a corresponding decrease was seen, with no change in white matter distant from plaques. A decrease in the content of total creatine was observed in multiple sclerosis plaques in comparison with normal control white matter (mean, 4.64 vs. 6.56 µmol/g wet weight), which correlated strongly with the decrease in N -acetyl-derived groups. No changes in other metabolites such as total choline or myo -inositol were seen. The decreases in content of N -acetyl-derived groups are in agreement with observations from in vivo proton NMR spectroscopy in multiple sclerosis patients. The decrease in total creatine is in contrast to most of the observations made in vivo where total creatine is assumed to be unchanged and metabolite levels are often expressed as a total creatine ratio. The use of a total creatine ratio in vivo could lead to an underestimation of reductions in N -acetylaspartate and an apparent increase in other metabolites in the multiple sclerosis lesion.     

A comparison of cerebrosides, proteolipid proteins, and cholesterol as indices of myelin in the architecture of rat cerebrum

Abstract— —Serial frozen section sampling and microspectrophotometric assays were used to compare the fidelity of cerebrosides, proteolipid proteins (PLP) and cholesterol as indices of myelinated fibres in the somatosensory region of rat cerebrum. Cerebrosides (including sulphatides) were a satisfactory index regardless of the mode of expression. Per unit dry wt., they were lowest in layer II (4 per cent) and increased with the subpial depth to 16 percent in white matter. Elevations were seen at the locations of: the outer tangential plexus (1); the stripe of Kaes-Bechterew (2); the outer and inner stripes of Baillarger (3,4); and the inner tangential plexus (5), respectively. Expression in terms of residue protein, total lipid or DNA gave somewhat sharper profiles of the myeloarchitecture. PLP per unit dry wt. were lowest in layers I, II and III (2 per cent) and increased to 5·5 per cent in white matter. Peaks occurred near (3), (4) and (5). The PLP per cell and per unit residue protein rose six-fold, from the upper layers of cortex to white matter, and depicted the myeloarchitecture more faithfully. In general outline, PLP paralleled cerebrosides in distribution, in contrast with previous findings in human prefrontal cortex. Human cortex may contain more non-myelin PLP in the upper cortical layers owing to its greater wealth of neuropil and synapses. Cholesterol per unit dry weight was a poor index of the myeloarchitecture; it was lowest in layer II (5 per cent) and increased to 13-5 per cent in white matter without revealing the five horizontal bands of myelinated fibres. Expression of cholesterol in terms of total lipid or DNA improved its value as an index, but it was less satisfactory than cerebrosides.     

Lipid and Fatty Acid Composition of Brain Tissue from Adrenoleukodystrophy Patients

Abstract: White matter and active plaque tissue from adrenoleukodystrophy (ALD) patients were analysed for lipid class and fatty acid compositions and the results compared with white matter from normal brain. ALD white matter was characterized by increased levels of cholesteryl esters and decreased levels of phosphatidylethanola- mine, including phosphatidylethanolamine plasmalogen, in comparison with normal brain white matter. In addition to even higher levels of cholesteryl esters, ALD plaque tissue had reduced levels of cerebrosides as well as phosphati-dylethanolamines. The loss of phosphatidylethanolamine plasmalogen is indicative of early demyelination. Total lipid from ALD white matter and ALD plaque tissue contained nearly five times and seven times, respectively, more 26:0 than total lipid from normal brain white matter. The 26:0 in ALD white matter was elevated in all lipid classes except phosphatidylinositol, but was located mainly in cerebrosides, phosphatidylcholine, sphingomyelin, and sulfatides. Most of the 26:0 in ALD plaque tissue was present in cholesteryl esters, followed by phosphatidylcholine and sphingomyelin, with reduced amounts in cerebrosides as compared with ALD white matter. The results are consistent with an initial accumulation of very-long-chain fatty acids in ALD white matter, primarily in sphingolipids and phosphatidylcholine, and subsequent accumulation of very-long- chain fatty acids in cholesteryl esters during demyelination. In addition, it was notable that the sphingolipids, especially sphingomyelin in ALD brain, had decreased levels of 24:1 and increased levels of 18:0, as well as increased levels of very-long-chain fatty acids. The extent to which the data shed light on mechanisms of demyelination in ALD is discussed.     

PROTEOLYTIC ACTIVITY AND BASIC PROTEIN LOSS IN AND AROUND MULTIPLE SCLEROSIS PLAQUES: COMBINED BIOCHEMICAL AND EIISTOCHEMICAL OBSERVATIONS

Abstract— This combined histochemical and biochemical study has shown that acid proteinase activity (PH 3.5) is increased around histologically-defined active plaques of multiple sclerosis (MS). Biochemical estimation showed that the enzyme is more active in most samples of 'normal' white matter in MS than in controls. A gradient of enzyme activity was observed: control white matter-white matter distant from plaqueclose white matter-edgsplaque. Both electrophoretic and histochemical techniques revealed a reduction or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base-line of thin-layer chromatoplates were shown to be predominantly phosphoinositides combined with encephalitogenic protein     

Distribution and fatty acid composition of phosphoglycerides in normal human brain

A thin-layer chromatographic procedure for the isolation of tissue phospholipids and their subsequent analysis is described. The method has been applied to the determination of the fatty acids of phosphoglycerides in human brain from the early fetal stage to old age. The study shows changes in the distribution and fatty acid composition of each phosphoglyceride in normal brain, although they are quite small after early childhood. A lipid-specific fatty acid pattern for each of the four major phosphoglycerides was found. Besides this, the pronounced differences between fatty acids of the lipids from the cerebral cortex and from the adjacent white matter justify speaking of a tissue-specific fatty acid pattern for brain phosphoglycerides. The phospholipids of cerebral white matter contained more monoenoic acid but much less polyunsaturated fatty acid than those of cerebral cortex. The brain phosphoglycerides also showed an age-dependent fatty acid pattern. With increasing age the concentration of the fatty acids of the linoleate family diminished while that of the linolenate family increased. Brain inositol phosphoglycerides, the fatty acid composition of which has not been studied systematically before, were characterized by a large concentration of arachidonate which was nearly as high for white as for gray matter and showed only small changes with age.     

Acid lipase-esterase (4-methylumbelliferyl oleate hydrolase) of white matter localized in oligodendrocyte cell bodies.

Abstract— The localization of an acid lipase-esterase which cleaves the fluorogenic substrate 4-methyl-umbelliferyl oleate at pH 5 was studied, because previous experiments has shown this activity to be reduced in the plaques of multiple sclerosis. In the human cerebellum, quantitative histochemical methods showed the activity to be relatively low in the molecular layer, compared to the granule cell layer; but the underlying white matter was the most active. In the human spinal cord, anterior horn cell bodies were richest in acid lipase, but white matter was, on a dry weight basis, as active as neuropil. Oligodendrocytes obtained in bulk from bovine white matter according to Poduslo & Norton (1972) had a specific activity up to 20 times greater than the crude myelin fraction. While it remains possible that in myelin the enzyme is in an inactive or inhibited state, the results indicate that the enzyme is localized in oligodendroglial cell bodies and suggest its use as a marker.     

DNA and RNA and the cytoarchitecture of human frontal cortex

DNA and RNA were studied in the layers of human prefrontal cortex by quantitative microchemical analyses on microtome-prepared serial frozen sections. Eleven cortical specimens from six autopsy brains were assayed. The mean total number of cells per mm³ of fresh cortex was estimated from DNA values. The number in layer I (61,000) was falsely high because of the inclusion of cells from the pia mater. From a plateau of 82,000-86,000 in layers II, IIIa and IIIb, the number of cells rose to 91,000 and 113,000 in layers IIIc and IV, respectively. The mean number was slightly lower in layer V, then gradually rose through layer VI to 127,000 cells in white matter. Per unit dry weight, DNA and cells varied much less than per unit volume; values averaged 14 per cent higher in layers II and IV than in neighbouring layers and in white matter were 20 per cent lower than in layer I. Intracortical patterns of RNA and RNA/cell reflected chiefly the distribution of neuronal cell bodies. Per unit fresh volume, RNA roughly paralleled DNA in layers I-V; through layer VI and into white matter RNA declined as DNA rose, reflecting the decline in neurons and increasing predominance of glial cells of lower RNA content. Per unit dry weight, RNA rose 50 per cent from layer I to layer II; a plateau of high values extended through layers II-V, then RNA declined rapidly through layer VI to a level in white matter that was 28 per cent of the value in layer II. Mean RNA/cell in cortex was 9-8 pg, with a maximum in layer IIIb (11.4 pg); in subcortical white matter it was 5.3 pg.     

Distribution of Glial Fibrillary Acidic Protein in Gliosed Human White Matter

Glial fibrillary acidic protein (GFAP) in gliosed white matter from multiple sclerosis plaques and cerebral infarcts was examined by polyacrylamide gel electrophoresis and immunoblotting. Using a monoclonal antibody raised against human GFAP, up to 11 GFAP polypeptide bands of molecular weight 37-49 kilodaltons were identified in particulate and supernatant fractions of CNS tissue homogenates. Soluble GFAP constituted about one-quarter of the total GFAP in normal cerebral white matter. In brain lesions in which reactive astrocytes were observed microscopically, the proportion of soluble GFAP was increased, with a greater representation of the lower-molecular-weight forms. In brain chronic sclerotic plaques, almost all of the GFAP was in the particulate form. Purified particulate GFAP was susceptible to proteolysis at acid but not at neutral pH in the presence of CNS homogenates. In tissue autolysis studies, GFAP was stable in situ for periods well in excess of average CNS postmortem times.     

Characterization of low density lipoprotein-like particle in the human aorta from grossly normal and atherosclerotic regions.

Physical and chemical criteria of lipoproteins containing apolipoprotein B, extracted from human aortic intima, were compared with those of plasma low density lipoproteins (LDL). Homogenates of grossly normal intima and advanced atherosclerotic lesions were subjected to differential ultracentrifugation to isolate a d = 1.006--1.063 g/ml density fraction which was extensively characterized. By electroimmunoassay, over 90% of the recovered apolipoprotein B immunological reactivity was found in isolates from both plaques and normal intima. In isolates of plaque and normal intima, particles of the same size as LDL were found, although a small population of very large structures was also present in plaque fractions. Apolipoprotein composition was similar to that of plasma LDL except for the presence of human serum albumin in aortic isolates. Fractions from aorta demonstrated greater electrophoretic mobilities than LDL. The lipid composition of isolates from normal intima was similar to that of LDL. The lipid composition of plaque fractions showed a significant decrease in the cholesteryl ester to free cholesterol ratio and in the triglyceride content in comparison to LDL and to fractions from normal intima. The fatty acid pattern of the cholesteryl ester fraction from isolates of both normal and plaque aortic homogenates demonstrated a significant decrease in the linoleate to oleate ratio as compared to LDL. Our initial studies suggest that althought aortic fractions are similar to LDL by certain criteria, some differences observed are more pronounced in fractions from lesions than from normal intima.     

Activation of rat liver branched-chain 2-oxo acid dehydrogenase in vivo by glucagon and adrenaline.

The activity of liver branched-chain 2-oxo acid dehydrogenase complex was measured in rats fed on low-protein diets and given adrenaline, glucagon, insulin or dibutyryl cyclic AMP in vivo. Administration of glucagon or adrenaline (200 micrograms/100 g body wt.) resulted in a 4-fold increase in the percentage of active complex. As with glucagon and adrenaline, treatment of rats with cyclic AMP (5 mg/100 g body wt.) resulted in marked activation of branched-chain 2-oxo acid dehydrogenase. Insulin administration (1 unit/100 g body wt.) also resulted in activation of enzyme; however, these effects were less than those observed with glucagon and adrenaline. In contrast with the results obtained with low-protein-fed rats, administration of adrenaline (200 micrograms/100 g body wt.) to rats fed with an adequate amount of protein resulted in only a modest (14%) increase in the activity of the complex. The extent to which these hormones activate branched-chain 2-oxo acid dehydrogenase appears to be correlated with their ability to stimulate amino acid uptake into liver.     

Improvement of dry matter digestibility of water hyacinth by solid state fermentation using white rot fungi

Feeding value of water hyacinth biomass colonized by three species of white rot fungi during solid-state fermentation was investigated. All three organisms proved to be efficient degraders and enhanced dry matter digestibility. Loss of organic matter was maximum (23.6+/-0.1% dry wt) after 48 days by P. ostreatus. C. indica showed maximum cellulose degradation (18.5+/-0.1% dry wt) than other two fungi after 48 days of incubation. In all cases, an extensive removal of hemicellulose at the initial growth period and a delayed degradation of lignin were observed. Hemicellulolysis was maximum (46.3+/-0.1% dry wt) by C. indica, but delignification (14.2+/-0.2% dry wt) by P. sajor-caju after 48 days. The amount of reducing sugar in the degraded biomass decreased at early stages, but increased as degradation progressed in all three cases (maximum 1.1+/-0.05% dry wt after 48 days by C. indica). Soluble nitrogen content increased only during 16-32 days of incubation (highest 1.1+/-0.1% dry wt after 32 days by P. sajor-caju). Crude protein of the bioconverted biomass increased gradually up to 32 days but decreased thereafter (maximum 10.3+/-0.1% dry wt after 32 days by P. sajor - caju). Per cent change in in vitro dry matter digestibility of degraded substrates enhanced gradually after 8 days and reached maximum after 32 days but thereafter decreased (highest + 20.4+/-0.3% dry wt by P. sajor-caju). The results demonstrated the efficient degrading capacity of the test fungi and their potential use in conversion of water hyacinth biomass into mycoprotein-rich ruminant feed, more so by P. sajor-caju.     

High expression of ubiquitin conjugates and NF-κB in unstable human intracranial atherosclerotic plaques

Arteries from the first segment of the middle cerebral and adjacent normal blood vessel specimens were collected from 19 patients who had died from cerebral infarction. According to morphologic examination the atherosclerotic plaque present was classified as stable or unstable. The expression of ubiquitin conjugates and nuclear factor kappa B (NF-κB) was assessed and found significantly higher in plaques than normal tissue and higher in unstable plaques than in stable plaques. Ubiquitin conjugates and NF-κB were positively correlated with each other. It was concluded that the ubiquitin proteasome system and NF-κB possibly play a role in the development of atherosclerotic plaques in intracranial arteries.     

Activities of enzymes of amino acid metabolism in rat brown adipose tissue

There was a nil arginase and serine dehydratase activities in interscapular brown adipose tissue, but the activity of adenylate deaminase, glutamine synthetase, glutamate dehydrogenase and the aspartate, alanine and branched chain amino acid transaminases was higher than those of white adipose tissue; the differences were diminished when expressed per unit of protein weight. Brown adipose tissue enzyme activities were in a range between those of liver and muscle. The high amino acid handling capabilities, together with its physiological role, suggest that brown adipose tissue can metabolize significant amounts of amino acids, its enzyme pattern being different both from white adipose tissue, as well as of liver and muscle.     

Basic fibroblast growth factor in Alzheimer's disease

We have examined the presence of basic fibroblast growth factor (FGF) in normal and in Alzheimer brains, studied the distribution of the mitogen by immunohistochemical techniques, measured the quantities of growth factor in selected areas of the brain (Brodmann areas 10/11 and 20/21), characterized the molecular forms by Western blotting and determined its sites of synthesis by in situ hybridization. Although the same molecular forms of basic FGF are found in control and Alzheimer brains, basic FGF is increased in the brains of Alzheimer's patients. Furthermore, basic FGF is not distributed in an identical fashion to normal and Alzheimer brains, but is found in association with the lesions that characterize this disease. In normal controls (n = 5), basic FGF was found to be widely distributed throughout the three brain regions examined (prefrontal cortex, hippocampus, and hypothalamus). Immunoreactivity was observed within astrocytes in both the grey and white matter, as well as within neuronal perikarya. Brain tissues that were obtained from Alzheimer patients (N = 4) showed a substantial increase in the overall specific staining of astrocytes and neurons, particularly in areas of reactive gliosis. Focal concentration of immunoreactive basic FGF was evident within the neuritic plaques, and could be clearly seen in association with the neurofibrillary tangles present within neuronal perikarya. The possibility that basic FGF expression in the CNS is linked to the pathogenesis of the disease is discussed.     

The 2H4 (CD45R) antigen is selectively decreased in multiple sclerosis lesions

To analyze expression of the 2H4 (CD45R) Ag on inflammatory cells in the central nervous system immune response, immunohistochemical staining with a panel of anti-T cell mAb was performed on central nervous system tissues from 12 patients with multiple sclerosis (MS) and 8 patients with viral encephalitis. Only rare cells were stained with anti-2H4 in MS plaques, plaque edges, and adjacent white matter, whereas 2H4+ cells were more numerous in viral encephalitis (p less than 0.001). By contrast, no quantitative differences were found between MS plaque edges and viral encephalitis with anti-4B4 (helper-inducer function associated), anti-CD4, anti-CD3, and anti-IL-2R mAb, although there were fewer CD8+ cells in MS (p less than 0.01). These data indicate that the 2H4 Ag is selectively decreased and, because it is associated with suppressor-inducer function of CD4+ cells, that there may be a defect in the down-regulation of the in situ immune response in MS.     

Enzyme Activities in Relation to pH and Lactate in Postmortem Brain in Alzheimer-Type and Other Dementias

Phosphate-activated glutaminase, glutamic acid decarboxylase, pyruvate dehydrogenase, succinic dehydrogenase, pH, and lactate were measured in frontal cortex and caudate nucleus of postmortem brains from cases of Alzheimer-type dementia (ATD), Down's syndrome, Huntington's disease, and one case of Pick's disease, as well as from sudden death and agonal controls. Lactate levels were higher and pH, phosphate-activated glutaminase, and glutamic acid decarboxylase levels were lower in the agonal controls than in the sudden death controls. Phosphate-activated glutaminase and glutamic acid decarboxylase were correlated with tissue pH and lactate, and also were reduced by in vitro acidification, suggesting that the low activities of these enzymes in agonal controls were related to decreased pH consequent upon lactate accumulation. Compared with control tissues at the same pH, phosphate-activated glutaminase and glutamic acid decarboxylase were unaltered in ATD and Down's frontal cortex and reduced in Huntington's caudate nucleus, and glutamic acid decarboxylase was reduced in Huntington's frontal cortex. These data suggest that GABAergic neurons are not affected in ATD and confirm the GABAergic defect in Huntington's disease. Pyruvate dehydrogenase and succinic dehydrogenase activities were the same in agonal controls and sudden death controls and were unaffected by acid pH and lactate in vitro, and pyruvate dehydrogenase was not correlated with pH or lactate. Reduced pyruvate dehydrogenase in frontal cortex of individual ATD, Down's, and Pick's cases, and in the caudate nucleus of Huntington's and Down's cases, was accompanied by gliosis/neuron loss. We conclude that decreased pyruvate dehydrogenase reflects neuronal loss.