RESUMEN
Here we present the work of the multidisciplinary consortium IRAS (Development of Genetic Markers for Immune Defence and Resistance in the Porcine Respiratory Tract) which includes different commercial and research institutions and was formed as a response to the call "Functional Genome Analysis in the Animal Organism (FUGATO)" by the German Ministry of Education and Research. IRAS started work in the fall of 2005 and--using the experimental infection of pigs with Actinobacillus pleuropneumoniae as model pathogen--aims at i) characterizing the course of infection by clinical as well as advanced laboratory tools (phenotypic-genetic approach) and ii) defining the diversity and distribution of allels known to be associated with immune defence in mouse and man (homolog-genetic approach). The intention is to identify genetic markers for increased resistance to infection thereby providing additional tools for the estimation of breeding values to the pig industry.
Asunto(s)
Infecciones por Actinobacillus/veterinaria , Actinobacillus pleuropneumoniae , Inmunidad Innata/genética , Infecciones del Sistema Respiratorio/veterinaria , Enfermedades de los Porcinos/inmunología , Infecciones por Actinobacillus/genética , Infecciones por Actinobacillus/inmunología , Infecciones por Actinobacillus/patología , Animales , Cruzamiento , Marcadores Genéticos/inmunología , Genotipo , Inmunidad Innata/inmunología , Pulmón/microbiología , Pulmón/patología , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/patología , Índice de Severidad de la Enfermedad , Porcinos , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/patología , Factores de TiempoRESUMEN
Microbial HSPs (heat-shock proteins) are implicated in the induction of the innate and adaptive arms of the immune response. We set out to determine whether peptides complexed with HSP70 generate efficient CTL (cytolytic T-lymphocyte) responses. Human dendritic cells pulsed with peptide-loaded microbial HSP70 complexes generate potent antigen-specific CTL responses. Using fluorescence anisotropy, we have calculated the peptide-binding affinity of mycobacterial HSP70 (K(D)=14 microM) and show that 120 pM HSP70-bound peptide is sufficient to generate a peptide-specific CTL response that is four orders of magnitude more efficient than the peptide alone. Through the generation of mycobacterial HSP70 truncations, we find that the minimal 136 amino acid, mycobacterial HSP70 peptide-binding domain is sufficient to generate CTL responses. The design of an HSP70 mutant, in which the peptide-binding site of HSP70 is filled with a bulky hydrophobic residue, leads to a large decrease in the peptide-binding affinity. This mutant HSP70 retains stimulatory capacity but is unable to generate CTL and has separated antigen delivery from immunostimulation of dendritic cells.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Péptidos/metabolismo , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Proteínas HSP70 de Choque Térmico/fisiología , Humanos , Péptidos/fisiología , Unión ProteicaRESUMEN
The 70 kDa mycobacterial heat shock protein (Mtb HSP70) stimulates mononuclear cells to release CC-chemokines. We now show that this function of Mtb HSP70, but not human HSP70, is dependent on the cell surface expression of CD40. Deletion of the CD40 cytoplasmic tail abolished, and CD40 antibody inhibited, Mtb HSP70 stimulation of CC-chemokine release. Mtb HSP70 stimulated THP1, KG1 cells, and monocyte-derived dendritic cells to produce RANTES. Specific binding of CD40-transfected HEK 293 cells to Mtb HSP70 was demonstrated by surface plasmon resonance. Coimmunoprecipitation of Mtb HSP70 with CD40 indicates a physical association between these molecules. The results suggest that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production.