Clara G. Monferran

Membrane instability induced by purified myelin components. Its possible relevance to experimental allergic encephalomyelitis.

The fusogenic properties of purified myelin components in a system employing chicken erythrocytes were studied. Sulphatides, myelin basic protein and the apoprotein of Folch-Lees proteolipid were capable of individually inducing membrane fusion in the presence of Ca2+. By contrast, cerebrosides or a mixture of sulphatides and myelin basic protein (molar ratio 19 : 1) did not show such effect. The fusogenic ability of sulphatide was correlated to its behaviour in mixed monolayers with phospholipids at the air-water interface. Mixed films of sulphatides with phosphatidylcholine or sphingomyelin but not with phosphatidylethanolamine showed reductions of molecular packing and surface potential similar to those found for other fusogenic compounds. The effects of myelin components described could be of importance in the membrane instability and vesicular disruption of myelin occurring in demyelinative disorders.

Escherichia coli heat-labile enterotoxin preferentially interacts with blood group A-active glycolipids from pig intestinal mucosa and A- and B-active glycolipids from human red cells compared to H-active glycolipids

The capacity of cholera toxin (CT) and of the heat-labile enterotoxin produced by Escherichia coli isolated from humans (LTh) to interact with glycolipids bearing ABO(H) blood group determinants isolated from different sources and separated by thin layer chromatography was studied. Toxin binding to the ABO(H)-related glycolipids depends on the glycolipid source, the type of the blood group activity, and the toxin. LTh and CT were capable of interacting with several blood group-active glycolipids from pig intestinal mucosa and both toxins preferentially recognize glycolipids isolated from animals carrying A-blood group antigenic determinants compared to those isolated from animals lacking these antigens. In contrast, LTh but not CT was able to interact with ABO(H)-active glycolipids from human erythrocytes. LTh preferentially binds to glycolipids isolated from A, B, and AB compared to O red cells. Results from competition experiments between CT and LTh for binding to the blood group-active glycolipids suggest that the carbohydrate structure requirements for the interaction of each toxin are different. The present findings may help to understand the results of clinical studies indicating an association between ABO(H) blood groups and the severity of diarrheal diseases produced by some toxigenic enterobacteria.

Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: Relationship with ABH blood group determinants

The capacity of cholera toxin (CT) and type I heat-labile enterotoxin produced by Escherichia coli isolated from human intestine (LTh) to interact with glycoconjugates bearing ABH blood group determinants from rabbit intestinal brush border membranes (BBM) was studied. On the basis of the type of intestinal compounds related to the human ABH blood group antigens, rabbits were classified as AB or H. Toxin binding to the intestinal glycolipids and glycoproteins depends on the blood group determinant borne by the glycoconjugate and on the analyzed toxin. LTh was capable of interacting preferentially with several blood group A- and B-active BBM glycolipids compared to those isolated from animals lacking these antigens (H rabbits). Also, LTh preferably bound to several BBM glycoproteins from AB rabbit intestines compared to those from H ones. One of these glycoproteins, the sucrase-isomaltase complex (EC 3.2.1.48-10) isolated from AB and H rabbits showed the same differential LTh binding. Conversely, CT practically did not recognize either blood group A-, B-, or H-active glycolipids and glycoproteins. These results may be relevant for carrying out in vivo experiments in rabbits in order to disclose the role of ABH active-glycoconjugates in the secretory response induced by LTh in rabbit intestine.

Myelin basic protein domains involved in the interaction with actin

A fluorescence assay was used to measure the interaction of myelin basic protein (MBP) with monomeric actin labeled with a fluorescent compound (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal dependence on the concentration of MBP and the fluorescence maximum is reached at a MBP:actin molar ratio of 1:20. The fluorescence maximum in absence of Ca2+ and ATP is 4 times lower than that in their presence although it is reached at the same MBP:actin molar ratio. Similar behavior is observed when synapsin replaces MBP, while acetylated MBP and bovine serum albumin fail to induce any fluorescence change. To define possible interacting domains on MBP involved in the actin-MBP interaction, experiments were performed using MBP-derived peptides obtained under controlled proteolysis of the whole molecule. The fluorescence changes induced by the different peptides depend on their location in the native protein and can not be explained simply by a difference in the net charge of the peptides. The results suggest that two sites are involved in the interaction. A Ca2+/ATP-dependent site located in the amino-terminal region (peptide 1-44) and a Ca2+/ATP-independent one near the carboxyl terminus of the MBP molecule. The actin-MBP interaction was also observed using immunoblot and ELISA techniques.

Effect of chemical modifications of myelin basic protein on its interaction with lipid interfaces and cell fusion ability

SummaryThe ability of native and chemically modified myelin basic protein to induce fusion of chicken erythrocytes and to interact with lipids in monolayers at the air-water interface and liposomes was studied. Chemical modifications of myelin basic protein were performed by acetylation and succinylation: the positive charges of the native protein were blocked to an extent of about 90–95%.Cellular aggregation and fusion of erythrocytes into multinucleated cells was induced by the native myelin basic protein. This effect was diminished for both acetylated and succinylated myelin basic protein. Native myelin basic protein penetrated appreciably in sulphatide-containing lipid monolayers while lower penetration occurred in monolayers of neutral lipids. Contrary to this, both chemically modified myelin basic proteins did not show any selectivity to penetrate into interfaces of neutral or negatively charged lipids. The intrinsic fluorescence of the native and chemically modified myelin basic proteins upon interacting with liposomes constituted by dipalmitoylphosphatidycholine, glycosphingolipids, egg phosphatidic acid or dipalmitoylphosphatidyl glycerol was studied. The interaction with liposomes of anionic lipids is accompanied by a blue shift of the maximum of the native protein emission fluorescence spectrum from 346 nm to 335 nm; no shift was observed with liposomes containing neutral lipids. The acetylated and succinylated myelin basic proteins did not show changes of their emission spectra upon interacting with any of the lipids studied. The results obtained in monolayers and the fluorescence shifts indicate a lack of correlation between the ability of the modified proteins to penetrate lipid interfaces and the microenvironment sensed by the tryptophan-containing domain.

Inhibitory role of diazepam on autoimmune inflammation in rats with experimental autoimmune encephalomyelitis.

Glutamate and GABA are the main excitatory and inhibitory neurotransmitters in the CNS, and both may be involved in the neuronal dysfunction in neurodegenerative conditions. We have recently found that glutamate release was decreased in isolated synaptosomes from the rat cerebral cortex during the development of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. In contrast to control animals where GABA induced a decrease in the evoked glutamate release, which was abolished by picrotoxin (a GABA(A) antagonist), synaptosomes from EAE rats showed a loss in the inhibition of the glutamate release mediated by GABA with a concomitant diminution of the flunitrazepam-sensitive GABA(A) receptor density. We have presently further evaluated the relevance of the GABAergic system in EAE by treating rats challenged for the disease with the GABA agonist diazepam. Administration of diazepam during 6 days starting at day 6 or 11 after EAE active induction led to a marked decrease of the disease incidence and histological signs associated with the disease. Cellular reactivity and antibody responses against the encephalitogenic myelin basic protein were also diminished. Beyond the effects of diazepam on the autoimmune, inflammatory response, we report also a positive effect on neurotransmission. Treatment with diazepam inhibited the previously described reduction in glutamate release in the frontal cortex synaptosomes from EAE animals. These data suggest that an endogenous inhibitory GABAergic system within the immune system is involved in the diazepam effect on EAE and indicate that increasing GABAergic activity potently ameliorates EAE.

Functional interaction of Escherichia coli heat‐labile enterotoxin with blood group A‐active glycoconjugates from differentiated HT29 cells

Human colon adenocarcinoma cells (HT29‐ATCC) and the clone HT29‐5F7 were cultured under conditions that differentiate cells to a polarized intestinal phenotype. Differentiated cells showed the presence of junctional complexes and intercellular lumina bordered by microvilli. Intestinal brush border hydrolase activities (sucrase, aminopeptidase N, lactase and maltase) were detected mainly in differentiated HT29‐ATCC cells compared with the differentiated clone, HT29‐5F7. The presence of non‐GM1 receptors of Escherichia coli heat‐labile enterotoxin (LT‐I) on both types of differentiated HT29 cells was indicated by the inability of cholera toxin B subunit to block LT‐I binding to the cells. Binding of LT‐I to cells, when GM1 was blocked by the cholera toxin B subunit, was characterized by an increased number of LT‐I receptors with respect to undifferentiated control cells. Moreover, both types of differentiated cells accumulated higher amounts of cyclic AMP in response to LT‐I than undifferentiated cells. Helix pomatia lectin inhibited the binding of LT‐I to cells and the subsequent production of cyclic AMP. LT‐I recognized blood group A‐active glycosphingolipids as functional receptors in both HT29 cell lines and the active pro‐sucrase form of the glycoprotein carrying A‐blood group activity present in HT29‐ATCC cells. These results strongly suggest that LT‐I can elicit an enhanced functional response using blood group A‐active glycoconjugates as additional receptors on polarized intestinal epithelial cells.

Ability of Blood Group A-active Glycosphingolipids to Act as Escherichia coli Heat-Labile Enterotoxin Receptors in HT-29 Cells

We examined the ability of blood group A-active glycoconjugates to act as receptors for Escherichia coli heat-labile type I enterotoxin (LT-I) in HT-29 cells. These cells contained ~4 times more specific binding sites for LT-I than for cholera toxin (CT). Binding of LT-I could not be blocked by the B subunit of CT (CT-B), indicating the existence of LT-I receptors in addition to the glycosphingolipid GM1. LT-I was able to increase levels of cyclic adenosine monophosphate (AMP), even in the presence of CT-B. Helix pomatia and anti-blood group A antibody caused a dose-dependent inhibition of binding of LT-I to cells and production of cyclic AMP. LT-I recognized several complex blood group A-active glycosphingolipids from cells, and this interaction was also interfered with by H. pomatia. Treatment of cells with D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol diminished surface expression of blood group A-active glycosphingolipids and binding of LT-I to non-GM1 receptors. These observations suggest that blood group A-active glycosphingolipids can function as alternative receptors for LT-I in HT-29 cells.

Protective Effect of a Synapsin Peptide Genetically Fused to the B Subunit of Escherichia coli Heat―Labile Enterotoxin in Rat Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease with similarities to multiple sclerosis that requires the activation of auto reactive T cells that infiltrate the central nervous system. In previous studies we have shown that intraperitoneal administration of synaptosomal antigens could suppress EAE. Herein we examined the effect in this animal model of a fusion protein comprising the C domain of synapsin Ia and the B subunit of Escherichia coli heat‐labile enterotoxin (LTBSC). Oral administration to rats of low amounts of LTBSC induced immunological systemic tolerance to the encephalitogenic myelin basic protein. Treatment with LTBSC prior to EAE induction diminished disease incidence, DTH reaction to myelin basic protein, and central nervous system inflammation. LTBSC treatment also reduced the specific T‐cell proliferative response to myelin basic protein, decreased nitric oxide production, and augmented arginase activity by peritoneal macrophages. All animals challenged for EAE developed antibody response specific for myelin basic protein, but rats treated with LTBSC showed a lower IgG2b/IgG1 ratio, indicating a shift to a Th2‐type milieu. The data presented here suggest that well‐conserved synapsin peptides conjugated to the B subunit of enterotoxins from the cholera toxin family have a protective role and provide a potential therapeutic tool for intervention in EAE as well as in multiple sclerosis.

Central nervous system lipid alterations in rats with experimental allergic encephalomyelitis and its suppression by immunosuppressive drugs

Rats with experimental allergic encephalomyelitis (EAE) induced with myelin or spinal cord show decreases in the content of sulphatides and cerebrosides and increases in the level of esterified cholesterol in the CNS. In this work it is shown that brain sulphatide changes can be obtained by injection of mixtures containing glycosphingolipids. Alterations in the content of cerebrosides occur when the injection mixture contains cerebrosides. The alterations of sulphatides and cholesterol ester induced by injection of spinal cord could be suppressed by treatment with immunosuppressive drugs (dexamethasone, cyclophosphamide and 6-mercaptopurine) able to prevent clinical signs of EAE.