RESUMEN

Several questions regarding the production and functioning of autoantibodies (AAb) during malaria infection remain open. Here we provide an overview of studies conducted in our laboratory that shed some light on the questions of whether antiphospholipid antibodies (aPL) and other AAb associated with autoimmune diseases (AID) can recognize Plasmodia antigens and exert anti-parasite activity; and whether anti-parasite phospholipid antibodies, produced in response to malaria, can inhibit phospholipid-induced inflammatory responses and protect against the pathogenesis of severe malaria. Our work showed that sera from patients with AID containing AAb against dsDNA, ssDNA, nuclear antigens (ANA), actin, cardiolipin (aCL) and erythrocyte membrane antigens recognize plasmodial antigens and can, similarly to monoclonal AAb of several specificities including phospholipid, inhibit the growth of P. falciparum in vitro. However, we did not detect a relationship between the presence of anti-glycosylphosphatidylinositol (GPI) antibodies in the serum and asymptomatic malaria infection, although we did register a relationship between these antibodies and parasitemia levels in infected individuals. Taken together, these results indicate that autoimmune responses mediated by AAb of different specificities, including phospholipid, may have anti-plasmodial activity and protect against malaria, although it is not clear whether anti-parasite phospholipid antibodies can mediate the same effect. The potential effect of anti-parasite phospholipid antibodies in malarious patients that are prone to the development of systemic lupus erythematosus or antiphospholipid syndrome, as well as the (possibly protective?) role of the (pathogenic) aPL on the malaria symptomatology and severity in these individuals, remain open questions.

Asunto(s)

Autoanticuerpos/sangre , Autoinmunidad , Malaria/inmunología , Glicosilfosfatidilinositoles/inmunología , Humanos , Parasitemia/inmunología , Fosfolípidos/inmunología

RESUMEN

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-19) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-19may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.

Asunto(s)

Antígenos de Protozoos/inmunología , Infecciones Asintomáticas , Glicosilfosfatidilinositoles/inmunología , Malaria Falciparum/inmunología , Proteína 1 de Superficie de Merozoito/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Adolescente , Adulto , Anciano , Angola , Formación de Anticuerpos , Brasil , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Humanos , Malaria Falciparum/sangre , Persona de Mediana Edad , Plasmodium falciparum/química , Adulto Joven

RESUMEN

Infection with the intracellular protozoan parasite Leishmania mexicana causes chronic disease in C57BL/6 mice, in which cutaneous lesions persist for many months with high parasite burdens (10(7)-10(8) parasites). This chronic disease process requires host IL-10 and FcγRIII. When Leishmania amastigotes are released from cells, surface-bound IgG can induce IL-10 and suppress IL-12 production from macrophages. These changes decrease IFN-γ from T cells and nitric oxide production in infected cells, which are both required for Leishmania control. However, antibodies targets and the kinetics of antibody production are unknown. Several groups have been unsuccessful in identifying amastigote surface proteins that bind IgG. We now show that glycoinositol phospholipids (GIPLs) of L. mexicana are recognized by mouse IgG1 by 6 weeks of infection, with a rapid increase between 12 and 16 weeks, consistent with the timing of chronic disease in C57BL/6 mice vs. healing in FcγRIII-deficient mice. A single prominent spot on TLC is recognized by IgG, and the glycolipid is a glycosyl phosphatidylinositol containing a branched mannose structure. We show that the lipid structure of the GIPL (the sn-2 fatty acid) is required for antibody recognition. This GIPL is abundant in L. mexicana amastigotes, rare in stationary-phase promastigotes, and absent in L. major, consistent with a role for antibodies to GIPLs in chronic disease. A mouse monoclonal anti-GIPL IgG recognizes GIPLs on the parasite surface, and induces IL-10 from macrophages. The current work also extends this mouse analysis to humans, finding that L. mexicana-infected humans with localized and diffuse cutaneous leishmaniasis have antibodies that recognize GIPLs, can bind to the surface of amastigotes, and can induce IL-10 from human monocytes. Further characterization of the target glycolipids will have important implications for drug and vaccine development and will elucidate the poorly understood role of glycolipids in the immunology of infections.

Asunto(s)

Anticuerpos Antiprotozoarios/inmunología , Glicosilfosfatidilinositoles/inmunología , Inmunoglobulina G/inmunología , Interleucina-10/metabolismo , Leishmania mexicana/inmunología , Leishmaniasis Cutánea/inmunología , Animales , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/parasitología

RESUMEN

The flatworm Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease that occurs throughout the developing world. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control schistosomiasis is through immunization with an antischistosomiasis vaccine combined with drug treatment. In the search for potential vaccine candidates, numerous tegument antigens have been assessed. As the major interface between parasite and mammalian host, the tegument plays crucial roles in the establishment and further course of schistosomiasis. Herein, we evaluated the potential of a GPI fraction, containing representative molecules located on the outer surface of adult worms, as vaccine candidate. Immunization of mice with GPI-anchored proteins induced a mixed Th1/Th2 type of immune response with production of IFN-γ and TNF-α, and low levels of IL-5 into the supernatant of splenocyte cultures. The protection engendered by this vaccination protocol was confirmed by 42% reduction in worm burden, 45% reduction in eggs per gram of hepatic tissue, 29% reduction in the number of granulomas per area, and 53% reduction in the granuloma fibrosis. Taken together, the data herein support the potential of surface-exposed GPI-anchored antigens from the S. mansoni tegument as vaccine candidate.

Asunto(s)

Glicosilfosfatidilinositoles/inmunología , Proteínas del Helminto/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/prevención & control , Vacunas/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Femenino , Interferón gamma/biosíntesis , Interleucina-5/biosíntesis , Ratones , Ratones Endogámicos C57BL , Esquistosomiasis mansoni/inmunología , Células TH1/inmunología , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Vacunación

RESUMEN

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. CD55 inhibits the formation of C3 convertases, and CD59 prevents the terminal polymerisation of the membrane attack complex. It has been reported that SLE patients seems to have an acquired deficiency of these proteins associated with secondary autoimmune haemolytic anaemia and lymphopenia. The aim of this study was to evaluate the presence of altered CD55 and CD59 expression on peripheral blood cells from SLE patients. Flow cytometric analyses were performed on red and white blood cells from 23 SLE patients and 23 healthy controls. We observed more CD55- and CD59-lymphocytes (p=0.005 and p=0.019, respectively), and CD59-granulocytes (p=0.045) in SLE patients than in controls. These results suggest there is an altered pattern of CD55 and CD59 expression on the peripheral blood cells of SLE patients, and it may play a role in the cytopenias in these patients.

Asunto(s)

Antígenos CD55/sangre , Antígenos CD59/sangre , Lupus Eritematoso Sistémico/sangre , Adulto , Anemia Hemolítica Autoinmune/sangre , Anemia Hemolítica Autoinmune/etiología , Recuento de Células Sanguíneas , Células Sanguíneas/inmunología , Células Sanguíneas/patología , Femenino , Citometría de Flujo , Glicosilfosfatidilinositoles/sangre , Glicosilfosfatidilinositoles/inmunología , Humanos , Inmunofenotipificación , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/inmunología , Linfopenia/sangre , Linfopenia/etiología , Masculino , Persona de Mediana Edad , Adulto Joven

RESUMEN

Toll-like receptors (TLRs) are important to initiate the innate immune response to a wide variety of pathogens. The protective role of TLRs during infection with protozoan parasites has been established. In this regard, malaria represents an exception where activation of TLRs seems to be deleterious to the host. In this article, we review the recent findings indicating the contrasting role of Myeloid Differentiation Primary-Response gene 88 (MyD88) and TLRs during malaria and infection with other protozoa. These findings suggest that MyD88 may represent an Achilles' heel during Plasmodium infection.

Asunto(s)

Citocinas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Malaria/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Infecciones por Protozoos/inmunología , Receptores Toll-Like/metabolismo , Animales , Citocinas/inmunología , Glicosilfosfatidilinositoles/inmunología , Humanos , Malaria/metabolismo , Factor 88 de Diferenciación Mieloide/inmunología , Infecciones por Protozoos/metabolismo , Transducción de Señal , Receptores Toll-Like/agonistas , Receptores Toll-Like/inmunología

RESUMEN

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44%. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.

Asunto(s)

Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Glicosilfosfatidilinositoles/inmunología , Antagonistas de Heparina/inmunología , Protaminas/inmunología , Schistosoma mansoni/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/administración & dosificación , Femenino , Glicosilfosfatidilinositoles/administración & dosificación , Antagonistas de Heparina/administración & dosificación , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Protaminas/administración & dosificación , Esquistosomiasis mansoni/prevención & control , Factores de Tiempo , Vacunas de ADN/administración & dosificación

RESUMEN

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44 percent. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.

Asunto(s)

Animales , Femenino , Ratones , Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Glicosilfosfatidilinositoles/inmunología , Antagonistas de Heparina/inmunología , Protaminas/inmunología , Schistosoma mansoni/inmunología , Vacunas de ADN/inmunología , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/administración & dosificación , Glicosilfosfatidilinositoles/administración & dosificación , Antagonistas de Heparina/administración & dosificación , Inmunoglobulina G/inmunología , Ratones Endogámicos BALB C , Protaminas/administración & dosificación , Esquistosomiasis mansoni/prevención & control , Factores de Tiempo , Vacunas de ADN/administración & dosificación

RESUMEN

Detergent-resistant membranes (DRMs) from Leishmania (Viannia) braziliensis promastigotes, insoluble in 1% Triton X-100 at 4 degrees C, were fractionated by sucrose density gradient ultracentrifugation. They were composed of glycoinositolphospholipids (GIPLs), inositol phosphorylceramide (IPC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and sterols. In contrast, 1% Triton X-100-soluble fraction was composed of PE, phosphatidylcholine, phosphatidylserine, PI, IPC, sterol, and lyso-PI. High-performance thin-layer chromatography (HPTLC) immunostaining using monoclonal antibody SST-1 showed that 85% of GIPLs are present in DRMs, and immunoelectron microscopic analysis showed that SST-1-reactive components are located in patches along the parasite surface. No difference in GIPL pattern was observed by HPTLC between Triton X-100-soluble versus -insoluble fractions at 4 degrees C. Analysis of fatty acid composition in DRMs by GC-MS showed the presence of GIPLs containing an alkylacylglycerol, presenting mainly saturated acyl and alkyl chains. DRMs also contained sterol, IPC with saturated fatty acids, PI with at least one saturated acyl chain, and PE with predominantly oleic acid. Promastigotes treated with methyl-beta-cyclodextrin to disrupt lipid microdomains showed significantly lower macrophage infectivity, suggesting a relationship between lipid microdomains and the infectivity of these parasites.

Asunto(s)

Leishmania braziliensis/citología , Leishmania braziliensis/fisiología , Macrófagos/parasitología , Microdominios de Membrana/química , Animales , Centrifugación por Gradiente de Densidad , Detergentes/química , Detergentes/farmacología , Glicosilfosfatidilinositoles/análisis , Glicosilfosfatidilinositoles/inmunología , Inmunidad Innata , Leishmania braziliensis/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Solubilidad

RESUMEN

A major malaria vaccine candidate, the circumsporozoite (CS) protein of Plasmodium, is a pre-erythrocytic stage antigen that is attached to the surface of the sporozoites through a glycosylphosphatidylinositol (GPI) anchor. However, here we show that the motif that signals for glycosylphosphatidylinositol anchor addition interferes with the immunogenicity of this protein and reduces protection in mice upon immunization with a recombinant adenovirus. The presence of the glycosylphosphatidylinositol-anchoring motif sequentially affected total circumsporozoite protein production, cellular distribution, antigen processing and secretion, leading to less effective antigen presentation. Consistently, vaccination with an adenovirus recombinant carrying the anchoring motif-disrupted circumsporozoite gene, resulted in significant increase of the number of interferon-gamma (IFN-gamma) producing T cells and specific IgG2a isotype antibodies, ensuing more effective vaccination. Given that the anchoring motif is highly conserved among different species of Plasmodium, anti-malaria subunit vaccines encoded by recombinant vectors that aim at the induction of strong cellular immunity could maximize immunogenicity by removing anchoring motifs.

Asunto(s)

Adenoviridae/inmunología , Glicosilfosfatidilinositoles/inmunología , Vacunas contra la Malaria/uso terapéutico , Malaria/prevención & control , Plasmodium yoelii/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Western Blotting , Secuencia Conservada , ADN Viral/genética , ADN Viral/inmunología , Células Dendríticas/inmunología , Técnica del Anticuerpo Fluorescente , Inmunoglobulina G/inmunología , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Fracciones Subcelulares/metabolismo , Células TH1/inmunología , Vacunas Sintéticas/uso terapéutico

RESUMEN

Chagas' disease is a chronic disease affecting millions of people in Latin America. The cell surface of Trypanosoma cruzi, the etiological agent, is covered by a glycocalyx whose components play important roles in parasite survival and infectivity. The most abundant surface component is a glycolipid (glycoinositol phospholipid, GIPL) related in structure to glycosylphosphatidyl inositol anchors. In this review, we describe the biological effects of highly purified native GIPLs and their glycan or lipid moities on cells of the host immune system.

Asunto(s)

Enfermedad de Chagas/inmunología , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/inmunología , Transducción de Señal , Trypanosoma cruzi/inmunología , Animales , Secuencia de Carbohidratos , Humanos , Linfocitos/inmunología , Macrófagos/inmunología , Datos de Secuencia Molecular , Relación Estructura-Actividad , Trypanosoma cruzi/fisiología

RESUMEN

Activation of cells from the innate immune system has an important role in host resistance to early infection with the intracellular protozoan parasite, Trypanosoma cruzi. Here we review the studies that have identified and structurally characterized the glycosylphosphatidylinositol (GPI) anchors, as parasite molecules responsible for the activation of cells from the macrophage lineage. We also cover the studies that have identified the receptor, signaling pathways as well as the array of genes expressed in macrophages that are activated by these glycoconjugates. We discuss the possible implications of such response on the host resistance to T. cruzi infection and the pathogenesis of Chagas disease.

Asunto(s)

Proteínas de Drosophila , Glicoproteínas/inmunología , Glicosilfosfatidilinositoles/inmunología , Macrófagos/inmunología , Transducción de Señal , Trypanosoma cruzi/inmunología , Animales , Secuencia de Carbohidratos , Citocinas/biosíntesis , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , Receptores de Superficie Celular/inmunología , Relación Estructura-Actividad , Receptores Toll-Like , Trypanosoma cruzi/fisiología

Asunto(s)

Enfermedades Parasitarias/inmunología , Animales , Quimiocinas/inmunología , Citocinas/inmunología , Modelos Animales de Enfermedad , Eucariontes/inmunología , Glicosilfosfatidilinositoles/inmunología , Humanos , Inmunidad Innata , Inflamación/etiología , Células Asesinas Naturales/inmunología , Enfermedades Parasitarias/terapia , Linfocitos T/inmunología

RESUMEN

We have characterized glycoinositol phospholipids (GIPLs) from three strains of the trypanosomatid parasites Endotrypanum schaudinni and Endotrypanum monterogeii. Methanolysis of the intact GIPLs liberated methyl esters of tetracosanoic acid, docosanoic acid, octadecanoic acid and hexadecanoic acid and C20 and C21 phytosphingosines. Phosphoinositol oligosaccharides were released from the GIPLs by mild base treatment, and their structures were determined by compositional analysis, fast-atom-bombardment MS and NMR spectroscopy. Similar compounds were detected in all three strains, although their relative proportions varied. The predominant components in E. schaudinni strain LV59 and E. monterogeii LV88 were Galpbeta1-3Galpbeta1-3Manalpha1-3Manalpha1-4G lcNalpha1-6Ins-1-P and Arapbeta1-2Ga lpbeta1-3Galpbeta1-3Manalpha1-3Manalpha1-4Glc Nalpha1-6Ins-1-P, and the major phosphoinositol oligosaccharide in E. schaudinni LV58 was the hybrid-type GIPL Manalpha1-2(EtNP-6)Manalpha1-6(Galpbeta1-3Man alpha1-3)Manalpha1-4GlcN alpha1-6Ins-1-P (where EtNP is ethanolamine phosphate). Several minor oligosaccharides containing additional galactose and/or arabinose residues were also detected.

Asunto(s)

Antígenos de Protozoos/química , Epítopos/biosíntesis , Glicoesfingolípidos/química , Glicosilfosfatidilinositoles/química , Leishmania major/química , Oligosacáridos/química , Trypanosomatina/química , Animales , Antígenos de Protozoos/biosíntesis , Secuencia de Carbohidratos , Epítopos/química , Técnica del Anticuerpo Fluorescente Indirecta , Glicoesfingolípidos/inmunología , Glicosilfosfatidilinositoles/inmunología , Glicosilfosfatidilinositoles/aislamiento & purificación , Leishmania major/inmunología , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Oligosacáridos/inmunología , Espectrometría de Masa Bombardeada por Átomos Veloces , Trypanosomatina/inmunología

RESUMEN

Components of Trypanosoma cruzi able to induce the production of IL-12 and other proinflammatory cytokines by macrophages were identified. Murine inflammatory macrophages were cultured with live parasites or with cellular components from different developmental forms of T. cruzi (i.e., trypomastigotes, amastigotes, metacyclic trypomastigotes, and epimastigotes), and the cytokine levels were measured after 24 and 48 h. Our results indicate that live trypomastigotes or live amastigotes (but not live epimastigotes or live metacyclic trypomastigotes) as well as trypomastigote extracts (but not extracts derived from epimastigotes) induce IL-12 and TNF-alpha synthesis by macrophages. Such biological activity is enhanced in membrane preparations from trypomastigotes. Further enrichment of the trypomastigote-derived monokine-inducing factor was obtained by solvent extraction and hydrophobic-interaction chromatography. The resultant purified molecules are a family of closely related glycoconjugates with predominant species at 70 to 80 and 120 to 200 kDa. These molecules are composed of carbohydrate chains O-linked to a polypeptide backbone that is anchored to the trypomastigote membrane via a glycosylphosphatidylinositol structure. The trypomastigote-derived glycoconjugates are active in inducing cytokine synthesis by macrophages at concentrations of 100 ng/ml. These effects are highly potentiated by IFN-gamma. Mapping of the glycoconjugate molecules to characterize the structural requirements for macrophage activation suggested that nonsaturated acyl fatty acid chains and periodate-sensitive units from the glycosylphosphatidylinositol anchor are important elements for the infective trypomastigote form to initiate cytokine synthesis by macrophages.

Asunto(s)

Glicoproteínas/inmunología , Glicosilfosfatidilinositoles/inmunología , Interleucina-12/biosíntesis , Macrófagos/inmunología , Mucinas/inmunología , Trypanosoma cruzi/inmunología , Animales , Línea Celular , Glicoproteínas/química , Glicosilfosfatidilinositoles/aislamiento & purificación , Interferón gamma/farmacología , Ratones , Ratones Endogámicos C3H , Ratones SCID , Mucinas/química , Trypanosoma cruzi/química

RESUMEN

In the present study, we investigated the role of glycosylphosphatidylinositol-anchored mucin-like glycoproteins (GPI-mucins) from Trypanosoma cruzi trypomastigotes in triggering the synthesis of nitric oxide as well as the microbicidal activity in murine macrophages. Our results show that GPI-mucins isolated from trypomastigote membranes are potent inducers of nitric oxide synthesis by IFN-gamma-primed macrophages, even at concentrations as low as 10 ng/ml. Our data also indicate the important role of glycosylphosphatidylinositol anchors from GPI-mucins as the second signal responsible for induction of nitric oxide synthesis by macrophages. To further investigate the role of these parasite molecules in inducing parasiticidal function, we cultured macrophages in the presence or absence of trypomastigote GPI-mucins and/or IFN-gamma and then infected these cells with either Leishmania spp. or T. cruzi. IFN-gamma was sufficient to induce microbial activity in macrophages infected with T. cruzi trypomastigotes. In contrast, killing of different species of Leishmania was further enhanced when macrophages exposed to IFN-gamma were also costimulated with trypomastigote-derived GPI-mucins. Our results also indicate that different glycolipids obtained from Leishmania major or Leishmania donovani (i.e., lipophosphoglycans or glycoinositolphospholipids) were unable to potentiate nitric oxide synthesis and/or microbicidal activity displayed by IFN-gamma-primed macrophages.

Asunto(s)

Antiprotozoarios/aislamiento & purificación , Glicosilfosfatidilinositoles/fisiología , Interferón gamma/farmacología , Leishmania/inmunología , Macrófagos/metabolismo , Mucinas/fisiología , Óxido Nítrico/biosíntesis , Trypanosoma cruzi/inmunología , Adyuvantes Inmunológicos/fisiología , Animales , Antiprotozoarios/farmacología , Membrana Celular/química , Membrana Celular/inmunología , Glicosilfosfatidilinositoles/inmunología , Glicosilfosfatidilinositoles/aislamiento & purificación , Células L , Leishmania/efectos de los fármacos , Leishmania/crecimiento & desarrollo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/parasitología , Masculino , Ratones , Ratones Endogámicos C3H , Mucinas/inmunología , Mucinas/aislamiento & purificación , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/crecimiento & desarrollo , Factor de Necrosis Tumoral alfa/fisiología

RESUMEN

Sera of patients with chronic Chagas' disease (American trypanosomiasis) contain elevated levels of anti-alpha-galactosyl antibodies that are lytic to Trypanosoma cruzi. The T. cruzi trypomastigote F2/3 antigen complex recognized by these antibodies runs as a broad smear on SDS/PAGE [Almeida, Krautz, Krettli and Travassos (1993) J. Clin. Lab. Anal. 7, 307-316]. Treatment of T. cruzi trypomastigote cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) abolished most of their reactivity to chronic Chagas'-disease ((Chagasic, Ch) anti-alpha-galactosyl antibodies (anti-Gal). The F2/3 antigen complex, purified by solvent extraction and hydrophobic-interaction chromatography, contained 60% carbohydrate by weight and substantial amounts of Thr, Ser, Glx, Asx, Gly, Ala and Pro, but relatively few hydrophobic amino acids. The presence of myoinositol, ethanolamine and 1-O-hexadecylglycerol suggested the presence of glycosyl-phosphatidylinositol membrane anchors. This was confirmed by PI-PLC treatment, which rendered the F2/3 molecules hydrophilic and reactive to anti-(cross-reacting determinant) antibodies. The majority of the GlcNAc content of the F2/3 antigens was found at the reducing termini of oligosaccharides in O-glycosidic linkage to Thr residues. These O-linked oligosaccharides could be released by beta-elimination and by mild hydrazinolysis. The smallest released oligosaccharitol that was reactive with the Ch anti-Gal was Gal alpha 1-3Gal beta 1-4GlcNAcol (where GlcNAcol is N-acetyl-glucosaminitol). Several other Gal-containing oligosaccharitols were observed, most of which were branched and contained 4,6-di-O-substituted GlcNAcol at their reducing termini. About half of the total released oligosaccharitols could bind to immobilized Ch anti-Gal, but none of them bound to the anti-Gal isolated from normal human sera. These data suggest that the specificities of the Ch anti-Gal are quite different from the natural anti-Gal isolated from normal human sera. Therefore, these novel T. cruzi O-linked oligosaccharides are highly immunogenic under the conditions of natural infection and are the targets for lytic Ch anti-Gal.

Asunto(s)

Anticuerpos Antiprotozoarios/inmunología , Enfermedad de Chagas/inmunología , Galactosa/inmunología , Glicoproteínas/inmunología , Glicosilfosfatidilinositoles/inmunología , Mucinas/inmunología , Oligosacáridos/inmunología , Proteínas Protozoarias/inmunología , Trypanosoma cruzi/inmunología , Animales , Anticuerpos Antiprotozoarios/metabolismo , Especificidad de Anticuerpos , Borohidruros/farmacología , Secuencia de Carbohidratos , Galactosa Oxidasa/metabolismo , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Humanos , Hidrazinas/farmacología , Datos de Secuencia Molecular , Mucinas/metabolismo , Oligosacáridos/metabolismo , Proteínas Protozoarias/metabolismo

RESUMEN

Amastigotes of the protozoan parasite Leishmania proliferate in phagolysosomes of macrophages. They abundantly express glycoinositol phospholipids (GIPLs), which are considered necessary for parasite survival by providing a shield at the surface against lysosomal hydrolases and by serving as receptors for the interaction with host cells. The structures of four GIPLs of L. mexicana amastigotes were characterized by a combination of gas-liquid chromatography-mass spectrometry, methylation linkage analysis and enzymatic treatments. They contain the glycan structures Man alpha 1-3Man alpha 1-4GlcN (iM2), Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (iM3), Man alpha 1-2Man alpha 1-6(Man alpha 1-3)-Man alpha 1-4GlcN (iM4) and (NH2-CH2CH2-PO4)Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (EPiM3), which are linked to alkylacyl-phosphatidylinositol. The predominant amastigote GIPL, EPiM3 (approximately 2 x 10(7) molecules/cell), is located at the parasite cell surface, in the flagellar pocket and in lysosomal membranes, but not on host cell structures as shown by immunofluorescence and immunoelectron microscopy. In addition, amastigotes in infected Balb/c mice contain a glycolipid with similar distribution as EPiM3, which has the same characteristics as the Forssman antigen of mammalian cells. In contrast to EPiM3, there is strong evidence that this glycosphingolipid is not synthesized by amastigotes but by macrophages in the lesion. This suggests a mechanism of lipid transfer from the macrophage to the parasite.

Asunto(s)

Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Antígeno de Forssman/inmunología , Glucolípidos/inmunología , Glicoesfingolípidos/inmunología , Glicosilfosfatidilinositoles/inmunología , Leishmania mexicana/inmunología , Macrófagos/inmunología , Fosfolípidos/inmunología , Animales , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/química , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/química , Secuencia de Carbohidratos , Antígeno de Forssman/metabolismo , Glucolípidos/biosíntesis , Glucolípidos/química , Glicoesfingolípidos/metabolismo , Glicosilfosfatidilinositoles/química , Leishmania mexicana/crecimiento & desarrollo , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Fosfatidilinositol Diacilglicerol-Liasa , Hidrolasas Diéster Fosfóricas/metabolismo

RESUMEN

The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form.

Asunto(s)

Proteínas Bacterianas/genética , Glicosilfosfatidilinositoles/genética , Mycobacterium leprae/inmunología , Proteínas de Saccharomyces cerevisiae , Proteínas Bacterianas/inmunología , Proteínas Fúngicas/genética , Genes Fúngicos , Vectores Genéticos , Glicosilfosfatidilinositoles/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Mycobacterium leprae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/inmunología

RESUMEN

The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form

Asunto(s)

Glicosilfosfatidilinositoles/genética , Mycobacterium leprae/inmunología , Proteínas Bacterianas/genética , Genes Fúngicos , Vectores Genéticos , Glicosilfosfatidilinositoles/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Mycobacterium leprae/genética , Proteínas Bacterianas/inmunología , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/inmunología