RESUMO
Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are crucially dependent on the invasion of activated autoreactive lymphocytes and blood macrophages into the central nervous system (CNS). Proinflammatory mononuclear cells and activated local microglia mediate inflammation, demyelination and axonal damage at the target organ. Previously, we observed that the administration of a hybrid between the synapsin ABC domains and the B subunit of Escherichia coli heat labile-enterotoxin (LTBABC) to rats with EAE ameliorated disease by modulating the peripheral Th1 response to myelin basic protein (MBP). In the present study, we investigated the effect of LTBABC administration on proinflammatory cell frequency in the CNS of rats with EAE. Treatment with the hybrid in the inductive phase of EAE attenuated disease severity and diminished histological inflammatory infiltrates and demyelination in the spinal cord of rats with acute EAE. Lower frequencies of infiltrating and local macrophages as well as CD4+ T cells that produce the proinflammatory cytokines interferon-gamma (IFN-γ) and interleukin (IL)-17 were found at the target organ. Concomitantly, low levels of INF-γ and IL-17 and increased levels of IL-10 were measured in cultures of CNS infiltrating cells and spinal cord tissue. An increased frequency of CD4+CD25+Foxp3 cells was observed at the disease peak and at the beginning of the recovery stage. These results provide further evidence for the immunomodulatory properties of the fusion protein LTBABC in autoimmune demyelinating disease affecting the central nervous system.
Assuntos
Toxinas Bacterianas/administração & dosagem , Encefalomielite Autoimune Experimental/tratamento farmacológico , Enterotoxinas/administração & dosagem , Proteínas de Escherichia coli/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Medula Espinal/efeitos dos fármacos , Sinapsinas/administração & dosagem , Animais , Toxinas Bacterianas/genética , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Bovinos , Células Cultivadas , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Enterotoxinas/genética , Escherichia coli , Proteínas de Escherichia coli/genética , Feminino , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , Ratos Wistar , Proteínas Recombinantes de Fusão/administração & dosagem , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Sinapsinas/genéticaRESUMO
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.
Assuntos
Autoimunidade/efeitos dos fármacos , Diazepam/farmacologia , Encefalomielite Autoimune Experimental/imunologia , Agonistas GABAérgicos/farmacologia , Inflamação/imunologia , Animais , Autoimunidade/imunologia , Encefalomielite Autoimune Experimental/patologia , Inflamação/patologia , Ratos , Ratos Wistar , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/imunologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/imunologia , Sinaptossomos/patologiaRESUMO
Several authors have demonstrated the presence in normal sera of antibodies that inhibit binding of a variety of autoantibodies. These inhibitory or blocking antibodies are generally considered to play a role in humoral self-tolerance. We examined sera from normal rabbits and from rabbits with experimental autoimmune encephalomyelitis (EAE), in search for antibodies capable to inhibit reactivity of autoantibodies directed to myelin basic protein (MBP). Rabbits injected with bovine myelin in complete Freund's adjuvant (EAE rabbits) or with adjuvant alone (control rabbits) were bled at various intervals post-injection. Sera were subjected to chomatography on a protein A-Sepharose column, retained and nonretained fractions were collected, and ability of these fractions to block reactivity of affinity-purified anti-MBP IgG-antibodies was analyzed by immunoblot technique. Protein A nonretained fraction from control rabbits inhibited anti-MBP IgG reactivity to the same degree at all intervals tested, whereas the same fraction from EAE animals showed an increase in inhibitory activity after induction of the disease. This inhibitory activity declined with the onset of clinical symptoms, and remained low in rabbits that did not recover from the disease. In contrast, the inhibitory activity remained at maximum value in EAE rabbits with spontaneous remission of clinical symptoms. We showed that the inhibitory activity is due to IgM-antibodies, and discussed the role of these antibodies in the development of EAE.
Assuntos
Anticorpos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Proteína Básica da Mielina/imunologia , Animais , Fenômenos Fisiológicos Sanguíneos , Bovinos , Masculino , Coelhos , Ratos , Valores de Referência , Fatores de TempoRESUMO
The ability of membrane ABH blood group-active glycoconjugates to act as receptors of the heat-labile enterotoxin of Escherichia coli (LTh) was studied in vitro and in vivo when GM1 was blocked by the cholera toxin B subunit. Rabbits were classified as AB or H based on intestinal ABH-antigenic activities. Brush border membranes from AB rabbits contained 4 times more LTh binding sites than the H ones. LTh interaction could be inhibited by lectins that recognize ABH determinants. LTh induced a similar dose-dependent secretory response in ligated ileal loops of both types of animals. Anti-AB antibodies and Ulex europaeus I lectin could significantly reduce the fluid accumulation in AB and H rabbits, respectively. LTh caused adenylate cyclase activation even when GM1 was blocked, and this effect was abolished by the addition of specific ABH ligands. These results suggest that ABH glycoconjugates are involved in the host secretory response to LTh in rabbit intestine.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli , Escherichia coli , Glicoconjugados/metabolismo , Microvilosidades/metabolismo , Lectinas de Plantas , Adenilil Ciclases/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Toxina da Cólera/metabolismo , Relação Dose-Resposta a Droga , Enterotoxinas/toxicidade , Gangliosídeo G(M1)/metabolismo , Íleo/metabolismo , Lectinas/metabolismo , Ligantes , CoelhosRESUMO
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.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Toxinas Bacterianas/metabolismo , Toxina da Cólera/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli , Glicoconjugados/metabolismo , Mucosa Intestinal/metabolismo , Animais , Humanos , Microvilosidades/metabolismo , Coelhos , Complexo Sacarase-Isomaltase/metabolismoRESUMO
The ability of glycoproteins from pig intestinal brush border membranes (BBM) to bind cholera toxin (CT) or heat-labile toxins from strains of Escherichia coli isolated from human (LTh) or pig (LTp) intestines was studied. Glycoproteins capable of binding the toxins are also recognized by antibodies or lectins specific for ABO(H) blood group and related antigens. Pigs expressing A, H, or I antigenic determinants were used for comparison. The toxin-binding capacity of a glycoprotein depends on the toxin type and the blood group epitope borne by the glycoprotein. LTh and LTp preferably bound to several blood group A-active glycoproteins rather than H-active glycoproteins. By contrast, CT practically did not recognize either blood group A- or blood group H-active glycoproteins, while glycoproteins from pigs expressing I antigenic determinants were able to interact with LTh, LTp, and CT. LTh, LTp, or CT glycoprotein binding was selectively inhibited by specific lectins or monosaccharides. Affinity purification of the toxin binding brush border glycoproteins on the basis of their blood group reactivity suggests that such glycoproteins are hydrolytic enzymes. BBM from A+ pigs contain about 27 times more LTh binding sites, in addition to those recognized by CT, than an equivalent membrane preparation from H+ pigs. The present findings may help clarify some previous unclear results on LTh binding to intestinal BBM glycoproteins obtained by use of animals not typed by their ABO(H) blood group phenotype.
Assuntos
Sistema ABO de Grupos Sanguíneos , Toxinas Bacterianas/metabolismo , Toxina da Cólera/metabolismo , Enterotoxinas/metabolismo , Epitopos , Proteínas de Escherichia coli , Escherichia coli/patogenicidade , Glicoproteínas/metabolismo , Mucosa Intestinal/metabolismo , Animais , Sequência de Carboidratos , Microvilosidades/metabolismo , Dados de Sequência Molecular , SuínosRESUMO
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.
Assuntos
Actinas/metabolismo , Proteína Básica da Mielina/metabolismo , Actinas/química , Animais , Sítios de Ligação , Bovinos , Corantes Fluorescentes , Soros Imunes , Músculos/metabolismo , Proteína Básica da Mielina/química , Naftalenossulfonatos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Coelhos , Espectrometria de Fluorescência , Medula Espinal/metabolismo , Sinapsinas/metabolismoRESUMO
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.
Assuntos
Sistema ABO de Grupos Sanguíneos/metabolismo , Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Eritrócitos/efeitos dos fármacos , Proteínas de Escherichia coli , Glicolipídeos/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Suínos/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Sequência de Carboidratos , Toxina da Cólera/metabolismo , Toxina da Cólera/farmacologia , Enterotoxinas/farmacologia , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Ligação Proteica , Especificidade da Espécie , Especificidade por SubstratoRESUMO
The capacity of pig gastric mucin-derived glycopeptides to interfere with the binding of cholera toxin (CT) to membrane receptors was studied. Two types of glycopeptide preparations with or without human blood group A antigenic activity were assayed for comparison in a system in which the target for the toxin was rat erythrocyte ghosts. Blood group A-active glycopeptides (A+ glycopeptides) were more potent inhibitors for the toxin binding than those lacking group A activity (A- glycopeptides). The mean values of the 50% inhibitory dose revealed that the A+ glycopeptide preparations were 6.6-fold-more potent inhibitors than the A- ones (P less than 0.001). The inhibitory capacity of the different A+ glycopeptide preparations was not directly proportional to the group A antigenic titer. The A+ glycopeptides showed a higher capacity than the A- glycopeptides to interact with the toxin as revealed by CT-glycopeptide complex formation, which could be detected by Sephacryl S-400 chromatography. This result suggests that glycopeptide inhibition of CT binding to the erythrocyte ghosts is mediated by a competition between the GM1 receptors and the glycopeptides for the toxin. The differential effect between both types of glycoconjugates was independent of the way of measuring the amount of glycopeptides used (dry weight, carbohydrate or protein content). The existence in the gastrointestinal tract of mucins not carrying or carrying different ABO blood group determinants, which could behave as more or less potent inhibitors of CT binding to membrane receptors, may help to explain the relationship between ABO blood groups and severity of cholera.
Assuntos
Sistema ABO de Grupos Sanguíneos , Toxina da Cólera/metabolismo , Epitopos , Mucosa Gástrica/imunologia , Glicopeptídeos/farmacologia , Glicoproteínas/farmacologia , Mucinas/farmacologia , Receptores Imunológicos/metabolismo , Animais , Membrana Celular/metabolismo , Glicopeptídeos/imunologia , Mucinas/imunologia , SuínosRESUMO
A search for compounds from intestinal mucosa of pigs carrying and not carrying blood group A-active substances (A+ and A- pigs, respectively) capable of binding cholera toxin (CT) was performed. Glycolipid extracts from a pool of pig intestinal mucosa resolved in thin-layer chromatography (TLC) revealed the presence of six to eight compounds capable of binding 125I-CT, two of them running as the ganglioside standards GM1 and GD1b. When intestinal mucosa glycolipids from single pigs were assayed by TLC for CT-binding capacity, two different patterns of labeling were observed. The main difference was at the level of compounds running below GD1b. The A+ pigs but not the A- pigs showed CT binding at this level. The major CT-binding compound detected only in A+ pigs was purified and some properties were determined. After TLC developed with different solvent systems, the purified compound bound CT and also immunoreacted with anti-A and anti-AB antisera but not with anti-B antiserum. The compound was also able to inhibit the hemagglutination of human A erythrocytes caused by anti-A antiserum, but inhibition was not observed with the B-anti-B or O (H)-Ulex europaeus lectin systems. A partial chemical characterization indicated that the active compound is a neutral glycosphingolipid containing glucose, fucose, galactose, and hexosamine. The existence of a blood group-active substance(s) able to interact with CT may help to explain the relationship between ABO blood groups and the diarrheal disease caused by infection with Vibrio cholerae.