RESUMEN
We have used a synthetic-peptide approach to map epitope regions of the Mycobacterium tuberculosis ESAT-6 antigen recognized by human T cells in relation to major histocompatibility complex (MHC) restriction. ESAT-6-specific CD4+ T-cell lines were established by stimulating peripheral blood mononuclear cells from 25 HLA-DR-typed tuberculosis patients with complete antigen in vitro. The established T-cell lines were then screened for proliferation and interferon-gamma (IFN-gamma) secretion in response to eight overlapping 20-mer peptides covering the ESAT-6 sequence. The response of the T-cell lines to ESAT-6 and peptides from a human leucocyte antigen (HLA)-heterogeneous group of donors suggested the presence of multiple epitopes and promiscuous recognition of the antigen. Analysis of antigen and peptide recognition in the presence of anti-HLA class I and class II antibodies suggested that the T-cell lines recognized ESAT-6 in association with HLA-DR and -DQ molecules. Furthermore, testing of selected T-cell lines with ESAT-6 and the peptides in the presence of autologous and allogeneic HLA-DR- and -DQ-typed antigen-presenting cells identified HLA-DR2, -DR52 and -DQ2 amongst the HLA molecules involved in the presentation of ESAT-6 and its peptides to human Th1 cells. In addition, the T-cell lines were cytotoxic for monocytes and macrophages pulsed with ESAT-6 and peptides. In conclusion, the recognition of ESAT-6 by IFN-gamma-secreting and cytotoxic CD4+ T cells in association with frequently expressed HLA class II molecules supports the application of this antigen to either specific diagnosis or subunit vaccine design.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos HLA-DQ/inmunología , Antígenos HLA-DR/inmunología , Mycobacterium tuberculosis/inmunología , Células TH1/inmunología , Secuencia de Aminoácidos , Proteínas Bacterianas , Mapeo Epitopo , Humanos , Interferón gamma/biosíntesis , Datos de Secuencia Molecular , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Antigen 85B (Ag85B/MPT59) is a major secreted protein from Mycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4(+) T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-gamma) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-gamma assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1, DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosis Ag85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.
Asunto(s)
Aciltransferasas , Antígenos Bacterianos , Proteínas Bacterianas/inmunología , Mycobacterium tuberculosis/inmunología , Células TH1/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno , Antígenos Bacterianos/genética , Vacuna BCG/inmunología , Proteínas Bacterianas/genética , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Epítopos/genética , Antígenos HLA , Humanos , Activación de Linfocitos , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Homología de Secuencia de Aminoácido , Tuberculosis Pulmonar/inmunologíaRESUMEN
A brain tumour-associated marker, urokinase (UK), was investigated using rabbit anti-UK polyclonal and murine anti-UK monoclonal antibodies, which were prepared by immunization with low molecular weight UK (LMW-UK) and high molecular weight urokinase (HMW-UK) synthetic peptide respectively. The polyclonal antibody cross-reacted with both LMW-UK and HMW-UK, whereas the murine MAbs were specific for HMW-UK. These immunological probes were used to study urokinase in glioma extracts, tissues, sera and cell lines that had been prepared from primary cultures of freshly dissected gliomas. Radioimmunoassays showed that glioma extracts had much higher level (5- to 44-fold) of UK than normal human brain extracts. This result was confirmed by immunoblotting of electrophoresis gels of glioma and human brain extracts. Immunohistochemical study using anti-UK MAb demonstrated much higher levels of UK in glioma tissue than normal brain tissue. Immunohistochemical study using anti-UK MAbs localized UK on the cell surface of glioma cells. Anti-UK MAbs inhibited the proliferation of AA cell lines and GB cell lines (50% to > 90%) and exerted minor effects (< or = 20%) on normal human liver, intestine and lymphocyte cell lines. Taken together, these results suggest that anti-UK MAbs may have therapeutic potential for human gliomas and cancer metastasis.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Biomarcadores de Tumor/análisis , Neoplasias Encefálicas/enzimología , Activador de Plasminógeno de Tipo Uroquinasa/análisis , Reacciones Antígeno-Anticuerpo , Astrocitoma/enzimología , Astrocitoma/patología , Astrocitoma/radioterapia , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , División Celular/fisiología , División Celular/efectos de la radiación , Membrana Celular/enzimología , Membrana Celular/efectos de la radiación , Supervivencia Celular/inmunología , Técnica del Anticuerpo Fluorescente , Secciones por Congelación , Glioblastoma/enzimología , Glioblastoma/patología , Glioblastoma/radioterapia , Humanos , Timidina/metabolismo , Extractos de Tejidos , Tritio , Células Tumorales CultivadasRESUMEN
Seasonal variations of total lipids, free fatty acids, triglycerides, phospholipids and cholesterol content of the freshwater fish Tilapia nilotica and the marine fish Sparus auratus were investigated. Male fish of S. auratus showed higher muscular and hepatic total lipids and hepatic free fatty acids than those of T. nilotica (P less than 0.05). The mean differences in gonadal male lipids of the two species were not significant. Tilapia nilotica female fish showed a significantly higher content of hepatic free fatty acids, phospholipids and cholesterol (P less than 0.01, 0.01, 0.05 respectively) and gonadal total lipids, triglycerides, and cholesterol (P less than 0.05) than those of S. auratus females. In contrast S. auratus females exhibited higher muscular total lipids, triglycerides, phospholipids and cholesterol content (P less than 0.01, 0.05, 0.02, 0.05, respectively) and gonadal phospholipids (P less than 0.05) than those of the T. nilotica females. In general hepatic and gonadal lipids of freshwater fish T. nilotica were higher than those of the marine fish S. auratus, and in contrast the marine fish contained higher muscular lipids than the freshwater fish.