Phagocytic cells provide the first line of defense against mycobacteria. We examined the relative mycobacteriostatic contributions of normal human alveolar macrophages (HAM), peripheral blood monocytes (PBM), and polymorphonuclear leukocytes (PMN) in the early time period after infection with mycobacteria (48 h). Cells were infected with Mycobacterium bovis (BCG) or M. tuberculosis H37Ra and their ability to inhibit growth was determined by mycobacterial incorporation of [3H]uracil. HAM inhibited the growth of both mycobacteria (44.2 +/- 7.9 and 37.6 +/- 10.5% inhibition, respectively). Two populations of HAM donors were subsequently defined: inhibitors and noninhibitors. The ability to inhibit growth of H37Ra correlated with that of BCG. In contrast to HAM, PBM and PMN did not inhibit mycobacterial growth. Because nitric oxide (NO) has been proposed to mediate growth inhibition in murine models, we examined whether NO was responsible for the early growth inhibition of mycobacteria by HAM. As expected, in murine peritoneal macrophages (MPM) IFN-gamma (2,500 U/ml) enhanced growth inhibition of BCG; the effect was abolished by the nitric oxide synthase (NOS) inhibitor NMMA. In contrast, IFN-gamma failed to enhance growth inhibition by HAM or PBM and NMMA had no effect. MPM expressed inducible nitric oxide synthase (NOS2) mRNA in response to LPS and IFN-gamma and produced NO. Neither NOS2 mRNA nor NO could be detected in HAM stimulated with LPS and IFN-gamma or mycobacteria. These data demonstrate that HAM, but not PBM or PMN, have NO-independent mycobacteriostatic activity in the early time period after infection with mycobacteria.
BackgroundSelection programs for growth and stress traits in cultured fish are fundamental to the improvement of aquaculture production. The gilthead sea bream (Sparus aurata) is the main aquacultured species in the Mediterranean area and there is considerable interest in the genetic improvement of this species. With the aim of increasing the genomic resources in gilthead sea bream and identifying genes and mechanisms underlying the physiology of the stress response, we developed a cDNA microarray for gilthead sea bream that is enriched by suppression substractive hybridization with stress and immunorelevant genes. This microarray is used to analyze the dynamics of gilthead sea bream liver expression profile after confinement exposure.ResultsGroups of confined and control juvenile fish were sampled at 6, 24, 72 and 120 h post exposure. GeneSpring analyses identified 202 annotated genes that appeared differentially expressed at least at one sampling time (P < 0.05). Gene expression results were validated by quantitative PCR of 10 target genes, and K-means clustering of differently expressed genes identified four major temporal gene expression profiles. Set 1 encompassed a rapid metabolic readjustment with enhanced uptake and intracellular transport of fatty acids as metabolic fuels. Set 2 was associated with a wide variety of tissue repair and remodeling processes that were mostly mediated by the stress response of the endoplasmic reticulum (ER). Sets 3 and 4 encompassed the re-establishment of cellular homeostasis with increased intracellular trafficking and scavenging of reactive oxygen species (ROS), accompanied by a bidirectional regulation of the immune system and a general decline of ROS production.ConclusionsCollectively, these findings show the complex nature of the adaptive stress response with a clear indication that the ER is an important control point for homeostatic adjustments. The study also identifies metabolic pathways which could be analyzed in greater detail to provide new insights regarding the transcriptional regulation of the stress response in fish.
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