RESUMO
Lipophosphoglycan (LPG) is the major Leishmania surface glycoconjugate having importance during the host-parasite interface. Leishmania (Viannia) braziliensis displays a spectrum of clinical forms including: typical cutaneous leishmaniasis (TL), mucocutaneous (ML), and atypical lesions (AL). Those variations in the immunopathology may be a result of intraspecies polymorphisms in the parasite's virulence factors. In this context, we evaluated the role of LPG of strains originated from patients with different clinical manifestations and the sandfly vector. Six isolates of L. braziliensis were used: M2903, RR051 and RR418 (TL), RR410 (AL), M15991 (ML), and M8401 (vector). LPGs were extracted and purified by hydrophobic interaction. Peritoneal macrophages from C57BL/6 and respective knock-outs (TLR2-/- and TLR-4-/-) were primed with IFN-γ and exposed to different LPGs for nitric oxide (NO) and cytokine production (IL-1ß, IL-6, IL-12, and TNF-α). LPGs differentially activated the production of NO and cytokines via TLR4. In order to ascertain if such functional variations were related to intraspecies polymorphisms in the LPG, the purified glycoconjugates were subjected to western blot with specific LPG antibodies (CA7AE and LT22). Based on antibody reactivity preliminary variations in the repeat units were detected. To confirm these findings, LPGs were depolymerized for purification of repeat units. After thin layer chromatography, intraspecies polymorphisms were confirmed especially in the type and/size of sugars branching-off the repeat units motif. In conclusion, different isolates of L. braziliensis from different clinical forms and hosts possess polymorphisms in their LPGs that functionally affected macrophage responses.
Assuntos
Glicoesfingolipídeos/química , Glicoesfingolipídeos/imunologia , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmaniose Cutânea/imunologia , Ativação de Macrófagos , Receptor 4 Toll-Like/metabolismo , Animais , Citocinas/metabolismo , Técnicas de Inativação de Genes , Glicoesfingolipídeos/isolamento & purificação , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Macrófagos/imunologia , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico , Psychodidae/parasitologia , Receptor 4 Toll-Like/genética , Fatores de VirulênciaRESUMO
The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.
Assuntos
Glicoesfingolipídeos/química , Glicoesfingolipídeos/imunologia , Interações Hospedeiro-Parasita , Leishmania mexicana/química , Macrófagos Peritoneais/imunologia , Psychodidae/parasitologia , Receptor 4 Toll-Like/imunologia , Animais , Brasil , Células CHO , Cricetulus , Citocinas/imunologia , Glicoesfingolipídeos/isolamento & purificação , Interações Hospedeiro-Parasita/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Leishmaniose Cutânea/parasitologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Receptor 4 Toll-Like/genéticaRESUMO
Glycosphingolipids (GSLs) are ubiquitous membrane components and have key roles in biological systems, acting as second messengers or modulators of signal transduction by affecting several events, ranging from cell adhesion, cell growth, cell motility, regulation of apoptosis and cell cycle. Over the last 20 years our laboratory and other research groups determined the glycan and ceramide structures of more than 20 GSLs from several pathogenic/opportunistic fungi, using a combination of gas chromatography, mass spectrometry, nuclear magnetic resonance as well as other immunochemical and biochemical techniques. Fungal GSLs can be divided in two major classes: neutral GSLs, galactosyl- and glucosylceramide (GlcCer), and acidic GSLs, the glycosylinositol-phosphorylceramides (GIPCs). Glycosyl structures in fungal GIPCs exhibited significant structural diversity and distinct composition when compared to mammalian GSLs, e.g., the expression of inositol-mannose and inositol-glucosamine cores and the terminal residue of ß-D-galactofuranose which are absent in mammalian cells. Studies performed by our group demonstrated that GIPC (Galfß 6[Manα3]Manα2InsPCer) elicited in patients with paracoccidioidomycosis an immune response with production of antibodies directed to the terminal residue of ß-D-galactofuranose. Further studies also showed that inhibition of GlcCer biosynthetic pathways affects fungal colony formation, spore germination and hyphal growth, indicating that enzymes involved in GlcCer biosynthesis may represent promising targets for the therapy of fungal infections. Recently, it was shown that GlcCer and GIPCs are preferentially localized in membrane microdomains and monoclonal antibodies directed to these GSLs interfere in several fungal biological processes such as growth and morphological transition. This review focuses on glycan structures carried on sphingolipids of pathogenic/opportunistic fungi, and aspects of their biological significance are discussed.
Assuntos
Fungos/metabolismo , Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Animais , Antifúngicos/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Fungos/classificação , Fungos/efeitos dos fármacos , Fungos/genética , Fungos/imunologia , Glicoesfingolipídeos/isolamento & purificação , Interações Hospedeiro-Patógeno/imunologia , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Estrutura Molecular , Micoses/tratamento farmacológico , Micoses/imunologia , Micoses/microbiologiaRESUMO
BACKGROUND: The dominant, cell surface lipophosphoglycan (LPG) of Leishmania is a multifunctional molecule involved in the interaction with vertebrate and invertebrate hosts. Although the role of LPG on infection has been extensively studied, it is not known if LPG interspecies variations contribute to the different immunopathologies of leishmaniases. To investigate the issue of interspecies polymorphisms, two Leishmania species from the New World that express structural variations of side chains of LPG repeat units were examined. In this context, the procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains, while the L. infantum LPG (strain BH46) has up to three glucoses residues in the repeat units. METHODS: Mice peritoneal macrophages from Balb/c, C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG from both species. Nitric oxide and cytokine production, MAPKs (ERK, p38 and JNK) and NF-kB activation were evaluated. RESULTS: Macrophages stimulated with L. braziliensis LPG, had a higher TNF-α, IL-1ß, IL-6 and NO production than those stimulated with that of L. infantum. Furthermore, the LPGs from the two species resulted in differential kinetics of signaling via MAPK activation. L. infantum LPG exhibited a gradual activation profile, whereas L. braziliensis LPG showed a sharp but transient activation. L. braziliensis LPG was able to activate NF-kB. CONCLUSION: These data suggest that two biochemically distinct LPGs were able to differentially modulate macrophage functions.
Assuntos
Glicoesfingolipídeos/imunologia , Leishmania braziliensis/imunologia , Leishmania infantum/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Visceral/imunologia , Macrófagos Peritoneais/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Glicoesfingolipídeos/química , Glicoesfingolipídeos/isolamento & purificação , Interações Hospedeiro-Parasita , Imunidade Inata , Leishmania braziliensis/metabolismo , Leishmania infantum/metabolismo , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/imunologia , NF-kappa B/metabolismo , Nitritos/imunologia , Nitritos/metabolismoRESUMO
The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2-/- and TLR4-/-) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-ß, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages.
Assuntos
Glicoesfingolipídeos/química , Glicoesfingolipídeos/imunologia , Imunidade Inata , Leishmania braziliensis/química , Leishmania braziliensis/imunologia , Leishmania infantum/química , Leishmania infantum/imunologia , Animais , Carboidratos/análise , Citocinas/biossíntese , Glicoesfingolipídeos/isolamento & purificação , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismoRESUMO
The interaction between Leishmania and sand flies has been demonstrated in many Old and New World species. Besides the morphological differentiation from procyclic to infective metacyclic promastigotes, the parasite undergoes biochemical transformations in its major surface lipophosphoglycan (LPG). An upregulation of beta-glucose residues was previously shown in the LPG repeat units from procyclic to metacyclic phase in Leishmania (Viannia) braziliensis, which has not been reported in any Leishmania species. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the Subgenus Leishmania. These adaptations were explored for the first time in a species from the Subgenus Viannia, L. (V.) braziliensis with its natural vectors Lutzomyia (Nyssomyia) intermedia and Lutzomyia (Nyssomyia) whitmani. Using two in vitro binding techniques, phosphoglycans (PGs) derived from procyclic and metacyclic parasites were able to bind to the insect midgut and inhibit L. braziliensis attachment. Interestingly, L. braziliensis procyclic parasite attachment was approximately 11-fold greater in the midgut of L. whitmani than in L. intermedia. The epidemiological relevance of L. whitmani as a vector of American Cutaneous Leishmaniasis (ACL) in Brazil is discussed.
Assuntos
Leishmania braziliensis/patogenicidade , Psychodidae/parasitologia , Animais , Sistema Digestório/metabolismo , Sistema Digestório/parasitologia , Glicoesfingolipídeos/química , Glicoesfingolipídeos/isolamento & purificação , Glicoesfingolipídeos/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Estágios do Ciclo de Vida , Microscopia de FluorescênciaRESUMO
Toll-like receptors (TLRs) mediate the cellular response to conserved molecular patterns shared by microorganisms. We report that TLR-2 on human NK cells is upregulated and stimulated by Leishmania major lipophosphoglycan (LPG), a phosphoglycan belonging to a family of unique Leishmania glycoconjugates. We found that purified L. major LPG upregulates both mRNA and the membrane expression of TLR-2 in NK cells. Additionally, IFN-gamma and TNF-alpha production and nuclear translocation of NF-kappaB was enhanced. The activation effect was more intense with LPG purified from infectious metacyclic parasites than from noninfectious procyclic Leishmania. Since the difference between the molecules derived from these two stages of the parasite growth cycle lies exclusively in the number of phosphosaccharide repeat domains and in the composition of glycan side chains that branch off these domains, we propose that TLR-2 possibly distinguishes between phosphorylated glycan repeats on LPG molecules. The effect of LPG on cytokine production and on membrane expression of TLR-2 could be blocked with F(ab')2 fragments of the mAb against LPG (WIC 79.3). Confocal microscopy demonstrated the co-localization of LPG and TLR-2 on the NK cell membrane. Binding of LPG to TLR-2 in NK cells was demonstrated by immunoprecipitations done with anti-TLR-2 and anti-LPG mAb followed by immunoblotting with anti-LPG and anti-TLR-2, respectively. Both antibodies recognized the immune complexes. These results suggest that NK cells are capable of recognition of, and activation by, Leishmania LPG through TLR-2, enabling them to participate autonomously in the innate immune system and thereby increasing the effective destruction of the parasite.
Assuntos
Glicoesfingolipídeos/imunologia , Células Matadoras Naturais/imunologia , Leishmania major/química , Leishmania major/imunologia , Ativação Linfocitária , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Antiprotozoários/metabolismo , Antígenos de Protozoários/imunologia , Regulação da Expressão Gênica , Glicoesfingolipídeos/isolamento & purificação , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , RNA Mensageiro/biossíntese , Receptor 2 Toll-Like , Receptores Toll-Like , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Eight glucosylceramides (1-8) were isolated from the water-insoluble lipid fraction of a methylene chloride/methanol/water extract of the Patagonian starfish Anasterias minuta. One of the constituents was identified as a new glucosylceramide, anasterocerebroside A (1), while the known glucosylceramide 7 was isolated and characterized for the first time as a pure compound. The structures of 1 and 7 were established by spectroscopic and chemical methods.
Assuntos
Cerebrosídeos/química , Glucosilceramidas/química , Estrelas-do-Mar/química , Animais , Argentina , Cerebrosídeos/isolamento & purificação , Glucosilceramidas/isolamento & purificação , Glicoesfingolipídeos/química , Glicoesfingolipídeos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Extratos de Tecidos/químicaRESUMO
The effect of purified glycosphingolipids from Leishmania (Leishmania) amazonensis on human lymphoproliferation, on expression of human lymphocyte and monocyte markers (CD3, CD4, CD8, CD14, CD19, and CD45), and on lymphocyte protein kinase C activity was analyzed.
Assuntos
Glicoesfingolipídeos/farmacologia , Leishmania/química , Linfócitos/efeitos dos fármacos , Animais , Antígenos CD/análise , Células Sanguíneas , Glicoesfingolipídeos/isolamento & purificação , Humanos , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/imunologia , Monócitos/imunologia , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismoRESUMO
Magnaporthe grisea is a fungal pathogen that infects rice leaves and causes rice blast, a devastating crop disease. M. grisea produces active elicitors of the hypersensitive response in rice that were previously identified as ceramide monohexosides (CMHs). Using several chromatographic approaches, mass spectrometry, and nuclear magnetic resonance, we identified ceramide mono- and dihexosides (CDH) in purified lipid extracts from M. grisea cells. As described by other authors, CMH consists of a ceramide moiety containing 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecenoic or 2-hydroxyhexadecenoic acids and a carbohydrate segment consisting of one residue of glucose. CDHs, however, contain beta-galactose (1-->4)-linked to beta-glucose as sugar units and phytosphingosine as the long-chain base, bound to a C24 alpha-hydroxylated fatty acid. To our knowledge, this is the first report on the occurrence of CDH in a fungal species and illustrates the existence of an alternative path of ceramide glycosylation in fungal cells.
Assuntos
Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Magnaporthe/metabolismo , Esfingosina/análogos & derivados , Esfingosina/química , Configuração de Carboidratos , Glicoesfingolipídeos/isolamento & purificação , Glicosilação , Magnaporthe/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Esfingosina/metabolismoRESUMO
During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterized by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This glycoconjugate is polymorphic among species with variations in sugars that branch off the conserved Gal(beta 1,4)Man(alpha 1)-PO(4) backbone of repeat units and the oligosaccharide cap. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly. These adaptations were explored in the context of the structure and function of LPG for the first time on a New World species, Leishmania chagasi. The distinguishing feature of LPG of procyclic L. chagasi consisted of beta 1,3-glucose residues that branch off the disaccharide-phosphate repeat units and also are present in the cap. Importantly, metacyclic L. chagasi significantly down-regulate the glucose substitutions in the LPG. The significance of these modifications was demonstrated in the interaction of L. chagasi with its vector Lutzomyia longipalpis. In contrast to procyclic parasites and procyclic LPG, metacyclic parasites and metacyclic LPG were unable to bind to the insect midgut. These results are consistent with the proposal that a New World Leishmania species, similar to Old World species, adapts the expression of terminally exposed sugars of its LPG to mediate parasite-sand fly interactions.
Assuntos
Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Leishmania infantum/crescimento & desenvolvimento , Psychodidae/parasitologia , Animais , Sequência de Carboidratos , Pré-Escolar , Sistema Digestório/parasitologia , Glicoesfingolipídeos/isolamento & purificação , Humanos , Insetos Vetores/parasitologia , Leishmania infantum/metabolismoRESUMO
Despite the immunological changes recognized to be produced during Leishmania infection and the central role played by Langerhans cells, it is not known whether Leishmaina lipophosphoglycan, the most abundant glycolipid on the parasite surface, affects the functions of Langerhans cells. Here, we provide evidence that exposure of Langerhans cells to Leishmaina (L.) major lipophosphoglycan has consequences for the expression of surface receptors. Down-regulation of receptors involved in host cell-parasite interaction are observed after 4 h exposure of Langerhans cells to lipophosphoglycan. Many of the changes are also induced in Langerhans cells incubated with L. major-conditioned medium, indicating that the observed effects may be mediated by soluble factors released by the parasite into the culture, as it is the case for the carbohydrate moiety of lipophosphoglycan. Taken together, these results indicate that the changes in surface molecule expression induced by the exposure of Langerhans cells to lipophosphoglycan might reflect changes in their signalling functions from the infected skin.
Assuntos
Antígenos de Superfície/efeitos dos fármacos , Glicoesfingolipídeos/farmacologia , Células de Langerhans/efeitos dos fármacos , Leishmania major/química , Animais , Antígenos de Superfície/imunologia , Citometria de Fluxo , Glicoesfingolipídeos/isolamento & purificação , Células de Langerhans/imunologia , Células de Langerhans/parasitologia , Leishmania major/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Diabetes mellitus is associated with various structural and functional liver abnormalities that affect the glycogen and lipid metabolisms. The effects of streptozotocin-induced diabetes and of insulin supplementation to Sprague-Dawley diabetic rats on ganglioside patterns in liver were determined. Diabetic livers showed a tendency to hepatomegaly 3 weeks after STZ-induction of diabetes. The concentration of total gangliosides in diabetic and non-diabetic livers was similar, but the concentration of total gangliosides in the liver of insulin-stabilized rats was slightly increased. Bidimensional TLC chromatographic analysis of gangliosides isolated from normal diabetic and insulin-stabilized diabetic livers showed quantitative and qualitative changes. In comparison with normal controls, the densitometric analyses of diabetic liver ganglioside patterns had increased amounts of GM3, GM1, GD1b, and GT1b gangliosides, while GM2 could not be detected. The hepatic ganglioside pattern of insulin-stabilized diabetic rats was partially restored, resembling the profile of normal rats. The activity of GalNAcT, GalT-2 and SialT-4 transferases was measured in liver microsomal fractions of the different groups of animals. Diabetic rats showed an increased activity of GalNAcT and a decrease in the activity of GalT-2 and SialT-4 compared with the controls. The enzymatic activities found in insulin-treated rats showed a tendency to return to the values observed in normal control animals. The results evidenced that streptozotocin-induced diabetes affects the liver ganglioside pattern and the ganglioside synthesis enzyme activity. The alterations found in ganglioside metabolism could represent one of the earliest changes associated with the diabetic pathology.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Gangliosídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Fígado/metabolismo , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Gangliosídeos/isolamento & purificação , Glicoesfingolipídeos/isolamento & purificação , Glicosiltransferases/metabolismo , Insulina/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Neutral glycosphingolipids were isolated from mouse heart muscle cells and their structures were analyzed. The molecular compositions of these glycosphingolipids were examined using column chromatography, HPTLC, GC-MS and fast atom bombardment-mass spectrometry (FAB-MS). Monohexosylceramides are a mixture of glucosyl- and galactosylceramides in a ratio of 1:1, sphingosine as the long chain base and as fatty acyl groups mainly C16, C18 saturated and C22 and C24 hydroxy fatty acids. Dihexosylceramide, identified as lactosylceramide contains C18 sphingosine and C18, C20 and C22 were the major fatty acids. No evidence for the occurrence of hydroxylated fatty acids in this glycolipid could be obtained from the GC-MS data. Our results clearly demonstrated that Trypanosoma cruzi and heart muscle cells have similar glycosphingolipid structures. In addition, heart muscle cells neutral glycosphingolipids have been shown to be immunoreactive. Antibodies reactive with each of the immunogenic glycolipids from heart cells or T. cruzi epimastigotes were present in the sera of human patients with Chagas disease as detected by ELISA. These cross-reactive antigens could be involved in the Chagasic autoimmunity.
Assuntos
Anticorpos Antiprotozoários/imunologia , Reações Cruzadas/imunologia , Glicoesfingolipídeos/análise , Glicoesfingolipídeos/imunologia , Glicoesfingolipídeos/isolamento & purificação , Miocárdio/química , Miocárdio/imunologia , Trypanosoma cruzi/química , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antiprotozoários/análise , Doença de Chagas/sangue , Doença de Chagas/imunologia , Cromatografia , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Ácidos Graxos/análise , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Camundongos , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Esfingosina/análiseRESUMO
The amastigote form of Leishmania mexicana parasites colonizes macrophage phagolysosomes and induces the enlargement of these compartments to form huge parasitophorous vacuoles. We report here that a purified secreted amastigote product, proteophosphoglycan, is a macromolecule which causes vacuolization of peritoneal macrophages in vitro. Secretion of this glycoconjugate by intracellular parasites may contribute to the expansion of phagolysosomal compartments in infected cells.
Assuntos
Glicoesfingolipídeos/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/metabolismo , Macrófagos Peritoneais/parasitologia , Vacúolos/parasitologia , Animais , Glicoesfingolipídeos/isolamento & purificação , Glicoesfingolipídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Peso Molecular , Vacúolos/efeitos dos fármacosRESUMO
Two lipophosphoglycans (LPG) from two Leishmania mexicana (Soberano and Yucatan) strains, isolated in Mexico, were purified by affinity chromatography with the Con A lectin. The LPG from each strain, with a 10 kDa molecular weight, possesses two fractions: one with high mannose concentrations and the other with a more heterogeneous saccharidic composition; the mannose-rich fraction and the heterogeneous fraction from the Yucatan strain show a single band of identity with rabbit IgG against promastigotes from L. mexicana Yucatan. Antigen recognition was abolished by treating the high mannose LPGs with alpha-mannosidase. The presence of non reductor alpha-mannose sequences, as determinant epitopes in L. mexicana Soberano and Yucatan strains, was determined by mass spectrometry analysis and enzymatic cleavage.
Assuntos
Glicoesfingolipídeos/isolamento & purificação , Leishmania mexicana/química , Animais , Cromatografia de Afinidade/métodos , Cromatografia em Gel , Concanavalina A , Feminino , Glicoesfingolipídeos/química , Concentração de Íons de Hidrogênio , Imunodifusão , Espectrometria de Massas , CoelhosAssuntos
Encéfalo/fisiologia , Glicoesfingolipídeos/farmacologia , Interleucina-2/biossíntese , Fígado/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/imunologia , Testículo/fisiologia , Animais , DNA/biossíntese , Glicoesfingolipídeos/isolamento & purificação , Glicoesfingolipídeos/fisiologia , Linfócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Ratos , Ratos Endogâmicos Lew , Baço/imunologiaRESUMO
Glycoinositolphospholipids (GIPLs) were extracted from the trypanosomatid Leishmania adleri by hot phenol extraction and the carbohydrate moieties isolated after base cleavage. Purification of the crude oligosaccharides by high performance anion exchange (HPAE) chromatography yielded four fractions whose structures were determined by a combination of methylation analysis, fast atom bombardment (FAB) mass spectrometry and two-dimensional nuclear magnetic resonance (NMR) spectroscopy
Assuntos
Animais , Glicoesfingolipídeos/química , Oligossacarídeos/química , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Trypanosomatina/química , Sequência de Carboidratos , Glicoesfingolipídeos/isolamento & purificação , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Espectroscopia de Ressonância MagnéticaRESUMO
Nuclear magnetic resonance (NMR) spectroscopy provides an extremely powerful technique to determine the structure of oligosaccharides, particularly when used in conjuction with other physical techniques such as methylation analysis and fast atom bombardment mass spectroscopy (FAB-MS). This brief review describes the application of NMR to the determination of the structure of an oligosaccharide isolated from the glycophosphosphingolipid (GPS) from the monogenetic trypanosomatid Leptomonas samueli. Where ambiguities arise in the NMR interpretation, the use of other data will be discussed
Assuntos
Animais , Glicoesfingolipídeos/química , Oligossacarídeos/química , Espectroscopia de Ressonância Magnética , Trypanosomatina/química , Sequência de Carboidratos , Glicoesfingolipídeos/isolamento & purificação , Fosfatos de Inositol/análise , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Two glycoinositol phospholipids (GIPL A and GIPL B) have been purified from epimastigotes of Trypanosoma cruzi at the logarithmic phase of growth (2 days). The GIPLs differ mainly in the lipid moiety and are similar to the lipopeptidophosphoglycan (LPPG) previously isolated from epimastigotes at the stationary phase (4-5 days). [3H]-palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in GIPL A and into a sphinganine ceramide with palmitic acid and lignoceric acid as the fatty acids in GIPL B. The lipids could be released by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) or glycosylphosphatidylinositol phospholipase D (GPI-PLD) from rat serum. The oligosaccharides share the common core structure of the glycosylphosphatidilinositol (GPI) membrane anchors. Microheterogeneity was demonstrated, as well as substitution by galactose, which is mainly in the furanose configuration as was previously described for the LPPG. However, methylation analysis indicated that 20 percent of the galactose is present as terminal pyranose units. In infective trypomastigotes, [3H]-palmitic acid was incorporated into the anchor of the Tc-85 glycoprotein. The lipid cleaved by phospholipase C digestion was identified as 1-O-hexadecylglycerol and the main oligosaccharide has the structure of the conserved core of all GPI anchors. [3H]-palmitic acid-labelled Tc-85 released into the culture medium as membrane vesicles showed 80 percent resistance to the action of PI-PLC. However, after mild alkaline hydrolysis, part of the radioactivity was released by the enzyme