RESUMEN

OBJECTIVE: To determine whether serum levels of a proliferation-inducing ligand (APRIL) are altered in patients with systemic lupus erythematosus (SLE), and correlate with disease parameters. METHODS: Clinical and biological parameters were analysed for 43 patients that fulfilled American College of Rheumatology (ACR) criteria for SLE classification and were positive for anti-double-stranded DNA (dsDNA) antibodies at least once in their medical records. Tests included measurement of serum levels of the tumour necrosis factor (TNF) family members APRIL and B lymphocyte stimulator (BLyS; a cytokine shown to promote SLE disease). RESULTS: Median APRIL levels were elevated in patients with SLE compared to patients with osteoarthritis and healthy controls, but did not correlate with the SLE Disease Activity Index (SLEDAI). APRIL serum levels showed an inverse correlation with BLyS serum levels (r = -0.339; p = 0.03). For patients with SLE with positive anti-dsDNA titres (>40 arbitrary units (AU)/ml) at inclusion (n = 25), circulating APRIL was inversely correlated with BLyS levels (r = -0.465; p = 0.022) and anti-dsDNA antibody titres (r = -0.411; p = 0.046). In a follow-up study at their second visit, 27 patients showed an inverse correlation of APRIL serum levels with BLyS (r = -0.398; p = 0.03) as well as with anti-dsDNA (r = -0.408; p = 0.03) titres and SLEDAI (r = -0.408; p = 0.01). CONCLUSION: The inverse correlation observed between APRIL and BLyS suggests that APRIL acts as a protective factor. APRIL and BLyS may thus have opposite roles in SLE, which must be considered when defining therapeutic applications of these cytokines.

Asunto(s)

Factor Activador de Células B/sangre , Lupus Eritematoso Sistémico/sangre , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/sangre , Enfermedad Aguda , Adulto , Anticuerpos Antinucleares/sangre , Autoanticuerpos/sangre , Estudios de Casos y Controles , ADN/inmunología , Femenino , Estudios de Seguimiento , Humanos , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Adulto Joven

RESUMEN

APRIL, a proliferation-inducing ligand, is a member of the tumor necrosis factor (TNF) family that is expressed by various types of tumors and influences their growth in vitro and in vivo. Two receptors, transmembrane activator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), bind APRIL, but neither is essential for the tumor-promoting effects, suggesting that a third receptor exists. Here, we report that APRIL specifically binds to heparan sulfate proteoglycans (HSPG) on the surface of tumor cells. This binding is mediated by the heparin sulfate side chains and can be inhibited by heparin. Importantly, BCMA and HSPG do not compete, but can bind APRIL simultaneously, suggesting that different regions in APRIL are critical for either interaction. In agreement, mutation of three lysines in a putative heparin sulfate-binding motif, which is not part of the TNF fold, destroys interaction with HSPG, while binding to BCMA is unaffected. Finally, whereas interaction of APRIL with HSPG does not influence APRIL-induced proliferation of T cells, it is crucial for its tumor growth-promoting activities. We therefore conclude that either HSPG serve as a receptor for APRIL or that HSPG binding allows APRIL to interact with a receptor that promotes tumor growth.

Asunto(s)

Proteoglicanos de Heparán Sulfato/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Humanos , Linfocitos/citología , Linfocitos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Mutación/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genética

RESUMEN

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.

Asunto(s)

Células Presentadoras de Antígenos/inmunología , Antígeno B7-1/inmunología , Antígenos CD40/inmunología , Enfermedad de Chagas/inmunología , Citocinas/metabolismo , Trypanosoma cruzi/inmunología , Animales , Células Presentadoras de Antígenos/metabolismo , Complejo CD3/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Susceptibilidad a Enfermedades , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-2/inmunología , Interleucina-2/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nitritos , Especificidad de la Especie , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

We analyse the effect of Trypanosoma cruzi heat-shock protein-70 (HSP70) on the maturation of murine dendritic cells (DC)generated from bone marrow precursor cells. The results obtained show that HSP70, both alone and fused to the KMP11 antigen, as well as a HSP70 fragment, is capable of maturing murine DC. Mature DC have enhanced expression of IL12, TNF-alpha cytokines, costimulation molecules and activation markers, showing a clear increase in the allostimulatory capacity. These findings suggest that T. cruzi HSP70 may be a very useful vehicle for developing DC-based immunoprophylaxis and therapy against infections.

Asunto(s)

Antígenos de Protozoos/inmunología , Células Dendríticas/inmunología , Proteínas HSP70 de Choque Térmico/farmacología , Glicoproteínas de Membrana/inmunología , Proteínas Protozoarias/inmunología , Trypanosoma cruzi/inmunología , Animales , Antígenos de Protozoos/genética , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Células Madre Hematopoyéticas/fisiología , Glicoproteínas de Membrana/genética , Ratones , Polimixina B/farmacología , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/farmacología

RESUMEN

We developed a method to highly purify CD4+ and CD8+ T cells from murine spleen by negative enrichment strategy. Single-cell suspensions of spleen cells were depleted from erythrocytes by ammonium chloride-mediated lysis. The obtained cell suspension contained approximately 28% CD4+ cells and 14% CD8+ cells. Passing of these cells over a nylon wool column removed up to 75% of all cells, leading to a suspension containing approximately 50% CD4+ and 23% CD8+ cells. These cells were further purified by a single immunomagnetic depletion step using a panel of eight antibodies in combination with MACS magnetic beads and an autoMACS machine. After purification, cells were viable and mostly non-activated based on the expression of activation markers and did not or only minimally respond to polyclonal stimuli such as soluble anti-CD3 antibodies or Concanavalin A. With this method, 19-38% of all CD4 cells and 10-29% of all CD8 cells in a spleen cell suspension were recovered at the mentioned purity. The whole procedure is fast (<4 h of preparation), simple and cost effective, as all antibodies and the magnetic beads have been titrated to the minimal concentration needed for purification. The method is highly reproducible, routinely leading to CD4+ cells with >97% purity (range 97.4-99%) and CD8+ cells with >96% purity (range 95.6-96.7%). The described protocol should facilitate studies aiming at the physiology of "untouched" murine T cells.

Asunto(s)

Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Separación Celular/métodos , Bazo/citología , Bazo/inmunología , Animales , Anticuerpos , Adhesión Celular , Separación Celular/economía , Análisis Costo-Beneficio , Femenino , Citometría de Flujo , Separación Inmunomagnética , Ratones , Ratones Endogámicos BALB C , Nylons

RESUMEN

Cytotoxic T lymphocyte response against Jurkat-A2/K(b) cells expressing the T. cruzi KMP11 protein has been evaluated after immunization of C57BL/6-A2/K(b) transgenic mice with the KMP11 and KMP11-HSP70 recombinant proteins. The results show that mice immunized with KMP11 covalently fused to the T. cruzi HSP70 protein, but not mice immunized with KMP11 alone, elicit a CTL response against the Jurkat-A2/K(b) cells expressing the KMP11 protein. The data also show that spleen cells from mice immunized with the fusion protein and stimulated with the K1 peptide induce lysis of both the Jurkat-A2/K(b) cells transfected with the KMP11 coding gene and the Jurkat-A2-K(b) cells pulsed with the K1 peptide. Splenocytes stimulated with the K3 peptide induce lysis of the Jurkat-A2/K(b) cells loaded with the K3 peptide but they do not recognize the target cells expressing the KMP11 protein. Similar results were obtained using lymph node from mice immunized with the peptides. Thus, we believe there are two cytotoxic T cell epitopes restricted to the A2 molecule (K1(KMP11) (4-12) and K3(KMP11) (41-50)) in the KMP11 protein, and suggest that the K1 peptide could be considered an immunodominant antigen whilst the K3 peptide may be regarded as a cryptic epitope. The fact that the CTL lines induced in B6-A2/K(b) mice recognize human cells expressing KMP11 protein, indicates that the KMP11 antigen fused to HSP70 could be a good candidate for the induction of immunoprotective cytotoxic responses against T. cruzi natural infection.

Asunto(s)

Antígenos de Protozoos/inmunología , Antígenos H-2/inmunología , Antígeno HLA-A2/inmunología , Proteínas HSP70 de Choque Térmico/genética , Linfocitos T Citotóxicos/inmunología , Trypanosoma cruzi/inmunología , Animales , Antígenos de Protozoos/genética , Pruebas Inmunológicas de Citotoxicidad , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/metabolismo , Humanos , Inmunización , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Transfección

RESUMEN

Murine immunization with Trypanosoma cruzi KMP11-HSP70 fused genes but not the KMP11 gene alone elicited both an immunoglobulin G2a long-lasting humoral immune response against KMP11 protein and activation of CD8+ cytotoxic T lymphocytes specific for two KMP11 peptides containing A2 motifs. Moreover, protection against the parasite challenge was observed after immunization with the chimeric gene.

Asunto(s)

Adyuvantes Inmunológicos , Enfermedad de Chagas/prevención & control , ADN Protozoario/inmunología , Proteínas HSP70 de Choque Térmico/genética , Glicoproteínas de Membrana/genética , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Formación de Anticuerpos , Fusión Artificial Génica , Células COS , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Proteínas HSP70 de Choque Térmico/inmunología , Inmunoglobulina G/biosíntesis , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Protozoarias/inmunología , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología , Vacunación

RESUMEN

The high variability among strains and isolates of Trypanosoma cruzi and the existence of shared antigenic determinants with other pathogens, particularly with members of the Leishmania genus make difficult the specific diagnosis of Chagas' disease. The data reported in this paper show that the T. cruzi KMP11 protein is an immunodominant antigen highly recognized by the sera from chagasic and leishmaniasis patients. By the use of amino- and carboxyl-terminal truncated KMP11 recombinant proteins and synthetic peptides, evidence is provided that while the sera from chagasic patients recognize linear peptides the sera from patients with visceral leishmaniasis must be predominantly directed against conformational epitopes. We found that a particular linear determinant, located in the carboxyl-terminal region of the protein, is recognized with high specificity and sensitivity only by sera from Chagas' disease patients, suggesting it could be a good candidate for differential serodiagnosis of Chagas' disease.

Asunto(s)

Anticuerpos Antiprotozoarios/inmunología , Enfermedad de Chagas/inmunología , Mapeo Epitopo , Glicoproteínas de Membrana/inmunología , Mitocondrias/inmunología , Proteínas Protozoarias/inmunología , Adulto , Secuencia de Aminoácidos , Anticuerpos Antiprotozoarios/sangre , Unión Competitiva , Epítopos , Humanos , Leishmaniasis/inmunología , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología

RESUMEN

The Trypanosoma cruzi HSP70 recombinant protein has the capacity to stimulate splenocytes or lymph node cells from naive mice in a non-haplotype-restricted way. The proliferative response is abolished by proteinase K digestion and by specific anti-HSP70 antibodies. The induced stimulation index was maximal after 24 h of incubation with the protein. This stimulation leads to cell death in a Fas-Fas ligand-independent way. The phenotype of the expanded cells was CD3(+) TCRalphass(+) CD4(+). HSP70-responsive cells express a broad range of cytokines including IFN-gamma, IL-2 and tumor necrosis factor-alpha. After 48 h of incubation with HSP70 there was a significant increase in relative intracellular levels of CD3 TCRalphass receptors. The expanded CD4(+) cell population expressed CD25; however, in contrast to concanavalin A-treated culture, delayed CD44 expression was observed.

Asunto(s)

Antígenos de Protozoos/farmacología , Apoptosis , Proteínas HSP70 de Choque Térmico/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Trypanosoma cruzi/inmunología , Animales , Complejo CD3/análisis , Antígenos CD4/análisis , Células Cultivadas , Citocinas/inmunología , Endopeptidasa K/farmacología , Proteína Ligando Fas , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Proteínas Recombinantes/farmacología , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/inmunología , Factores de Tiempo , Receptor fas/metabolismo

RESUMEN

In this paper, we describe the application of electroporation to deliver phage DNA into bacterial cells in order to recover it as phage particles. The methodology represents a quicker and cheaper alternative to the use of packaging extracts to rescue phage clones stored as naked DNAs. Furthermore, our data demonstrate that there were not rearrangements or recombinations between phage DNAs when a mixture of different DNAs was electroporated, suggesting the use of electroporation as a reliable method for construction of gene libraries.

Asunto(s)

Bacteriófago lambda/aislamiento & purificación , ADN Viral/administración & dosificación , Electroporación/métodos , Bacteriófago lambda/genética , ADN Viral/genética , Escherichia coli/genética , Escherichia coli/virología , Biblioteca de Genes , Reordenamiento Génico , Genes Virales , Vectores Genéticos , Recombinación Genética , Transformación Genética