RESUMO
To explore the molecular basis of the T-cell-mediated immune response to Listeria monocytogenes, we cloned and expressed listerial antigens in Escherichia coli using the lambda-ZAP bacteriophage and Bluescript plasmid vectors. A two-stage screening strategy was implemented to identify T-cell-reactive antigens; the first stage involved antibodies or oligonucleotide probes and the second stage was based on assays for T-cell activation. A library of genomic DNA from L. monocytogenes was generated in lambda-ZAP, and then antigens, were detected in infected cells with a polyclonal rabbit anti-L. monocytogenes antiserum and an L. monocytogenes-specific monoclonal antibody. Also, synthetic oligonucleotide probes corresponding to the structural gene for listeriolysin O (LLO) were used to screen the recombinant DNA library. In each case, positive isolates were evaluated for T-cell antigenicity by measuring antigen-induced interleukin-2 production by polyclonal T cells taken from L. monocytogenes-immune mice. Phage clones were subcloned and expressed in the Bluescript plasmid and tested further for antigenic activity and LLO expression. Using this screening strategy, we successfully identified bacterial clones producing recombinant listerial antigens which activate L. monocytogenes-immune T cells in vitro. Antigens operative in the T-cell response during infection with L. monocytogenes include LLO, 62- and 39-kilodalton proteins, and other poorly defined bacterial surface components. We also found that high concentrations of recombinant LLO inhibited macrophage-mediated antigen presentation. These results are discussed in terms of the multiple functions of LLO as a virulence factor, inhibitor of antigen presentation, and potent antigen in the T-cell response to L. monocytogenes. These studies represent the first step toward a genetic definition of the antigens recognized in immune defense to L. monocytogenes.
Assuntos
Antígenos de Bactérias/genética , Toxinas Bacterianas , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Listeria monocytogenes/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos de Bactérias/imunologia , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/imunologia , Escherichia coli/genética , Feminino , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Implantation of oestradiol into adult rats of both sexes induced different patterns of LH secretion depending on the time at which gonadectomy or testosterone injection were performed. Castration 2 h after birth allowed an LH peak to occur daily at 18.00 h, but its amplitude was lower than that of adult gonadectomized female rats treated with oestradiol. Castration 24 h after birth elicited two kinds of response; a circadian discharge of LH lower than that of male rats gonadectomized 2 h after birth or a steady low level of LH. The LH rhythmicity induced by implantation of oestradiol was not seen after castration at 8 weeks of age. Neonatal administration of testosterone to female rats prevented the LH peak induced by oestradiol that was seen in adult ovariectomized rats. Neonatal or adult ovariectomy did not interfere with the rhythmical response of LH after implantation of oestradiol. Thus, it is concluded that sexual differentiation of the hypothalamus is primarily of masculine origin.
