RESUMEN
Microarray technology has become a standard tool for generation of gene expression profiles to explore human disease processes. Being able to start from minute amounts of RNA extends the fields of application to core needle biopsies, laser capture microdissected cells, and flow-sorted cells. Several RNA amplification methods have been developed, but no extensive comparability and concordance studies of gene expression profiles are available. Different amplification methods may produce differences in gene expression patterns. Therefore, we compared profiles processed by a standard microarray protocol with three different types of RNA amplification: (i) two rounds of linear target amplification, (ii) random amplification, and (iii) amplification based on a template switching mechanism. The latter two methods accomplish target amplification in a nonlinear way using PCR technology. Starting from as little as 50 ng of total RNA, the yield of labeled cRNA was sufficient for hybridization to Affymetrix HG-U133A GeneChip array using the respective methods. Replicate experiments were highly reproducible for each method. In comparison with the standard protocol, all three approaches are less sensitive and introduced a minor but clearly detectable bias of the detection call. In conclusion, the three amplification protocols used are applicable for GeneChip analysis of small tissue samples.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Complementario/síntesis química , ADN Complementario/síntesis química , ADN de Neoplasias/química , Humanos , Neoplasias Pulmonares/genética , Análisis por Micromatrices/instrumentación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
IL-12 is indispensable for the control of many intracellular pathogens, but the components of the signaling pathway that are essential for its function in vivo are incompletely understood. Here, we investigated in the Leishmania major mouse model whether Tyk2 kinase is required for the generation of a protective immune response. Unlike C57BL/6 controls, Tyk2(-/-)mice developed severe skin lesions after infection that frequently ulcerated, but ultimately healed. NK cell cytotoxicity was absent in infected Tyk2(-/-) mice, even after IL-12 pretreatment, which correlated with a STAT4 activation defect. IFN-alpha / beta, which was still able to activate STAT1 in Tyk2(-/-) NK cells, reconstituted their cytotoxic activity, but not their IL-12 responsiveness. The IL-12-induced production of IFN-gamma by NK cells and CD8(+) T cells was strongly suppressed in Tyk2(-/-) mice at day 1 of infection, but partly regained during the late phase of infection. Tyk2(-/-) CD4(+) T cells developed into Th1 cells (although in a delayed fashion) and infected Tyk2(-/-) mice expressed normals levels of inducible NO synthase. Thus, Tyk2 is required for the IL-12 response of NK cells and CD8(+) T cells in L. major-infected mice, but not for the generation of Th1 cells and the ultimate control of the disease.
Asunto(s)
Interleucina-12/inmunología , Leishmania major/enzimología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Proteínas Tirosina Quinasas , Proteínas/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Interacciones Huésped-Parásitos , Factor 3 de Genes Estimulados por el Interferón , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Leishmaniasis Cutánea/enzimología , Leishmaniasis Cutánea/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/inmunología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Proteínas/genética , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT4 , Transducción de Señal , TYK2 Quinasa , Células TH1/inmunología , Transactivadores/inmunología , Transactivadores/metabolismo , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
Mature dendritic cells (DCs) are believed to induce T cell immunity, whereas immature DCs induce T cell tolerance. Here we describe that injections of DCs matured with tumor necrosis factor (TNF)-alpha (TNF/DCs) induce antigen-specific protection from experimental autoimmune encephalomyelitis (EAE) in mice. Maturation by TNF-alpha induced high levels of major histocompatibility complex class II and costimulatory molecules on DCs, but they remained weak producers of proinflammatory cytokines. One injection of such TNF/DCs pulsed with auto-antigenic peptide ameliorated the disease score of EAE. This could not be observed with immature DCs or DCs matured with lipopolysaccharide (LPS) plus anti-CD40. Three consecutive injections of peptide-pulsed TNF/DCs derived from wild-type led to the induction of peptide-specific predominantly interleukin (IL)-10-producing CD4(+) T cells and complete protection from EAE. Blocking of IL-10 in vivo could only partially restore the susceptibility to EAE, suggesting an important but not exclusive role of IL-10 for EAE prevention. Notably, the protection was peptide specific, as TNF/DCs pulsed with unrelated peptide could not prevent EAE. In conclusion, this study describes that stimulation by TNF-alpha results in incompletely matured DCs (semi-mature DCs) which induce peptide-specific IL-10-producing T cells in vivo and prevent EAE.