Project description:Alveolar macrophages (AMs), the tissue dwelling monocytes of the lungs, are the first to encounter the menagerie of items that pass the mechanical barriers of the nose and throat. AMs must be able to mount an effective immune response against invading pathogens as well as maintain homeostasis in the lungs to avoid excessive inflammatory responses. In vivo data suggests that AMs can take on different phenotypes depending on the conditions of simulation. Limited progress has been made in defining the mechanisms responsible for these phenotypic issues largely due to logistical difficulties in isolating a sufficient number of cells from the lungs that have not been activated by the isolation protocol. To circumvent these logistical issues, an in vitro cell culture model was developed to study AM biology with specific emphasis on the generation of alternatively activated alveolar macrophages (AAAMs). Utilizing the BALB/c AM cell line MH-S it was determined that LPS could be used to generate classically activated AMs (CAAMs) and that IL-4 was effective in producing AAAMs. We examined the full transcriptome of AAAMs and CAAMs using microarray technology in an attempt to confirm previously described AAAM expression patterns and to identify new molecules associated with AM phenotypic change. AAAMs up-regulated an entirely distinct group of transcripts including genes encoding Arg1, Ym1 and a number of repair and remodeling genes whereas CAAMs up-regulated proinflammatory cytokines such as IL-1, IL-6 and IL-12. Additional novel markers are identified in this study to better characterize CAAMs and AAAMs in the lung. Keywords: Characterization of Cell Type Based on Activation Stimulus Overall design: MH-S Cells (ATCC CRL-2019) were stimulated with either 50 ng/ml IL-4 or 1µg/ml LPS and analyzed 12 hours later by whole genome DNA microarray analysis.
Project description:Mycobacteria-induced apoptosis of macrophages plays an important role in modulation of the host immune response involving TNF-alpha as major cytokine. The underlying mechanisms are still ill-defined. Here, we show for the first time that methylglyoxal (MG) and AGEs levels were elevated during mycobacterial infection of macrophages and that their increased levels mediated mycobacteria-induced apoptotic and immune response of macrophages. Moreover, we show that high levels of AGEs were formed at the sites of pulmonary tuberculosis. This observation represents the first evidence of the potential involvement of AGEs in tuberculosis and in infectious diseases in general. Global gene expression profiling of MG-treated macrophages reveals diversified potential roles of MG in cellular processes, including apoptosis, immune response, and growth regulation. The results of this study provide new insights into intervention strategies to develop therapeutic tools against infectious diseases in which MG and AGE production plays critical roles. Experiment Overall Design: MH-S cells (ATCC Number: CRL-2019), an alveolar macrophage cell line, was treated with 0.8 mM MG. At different time points after treatment (30 min, 4 h, and 8 h) the cells were harvested for total RNA preparation. As negative control the cells without treatment were included. RNA preparation was performed using Trizol method. Totally three independent experiments were performed, so that each time point consists of biological triplicates.
Project description:Comparing two subclones (Taiwan clone and Asian-Pacific clone) of CA-MRSA ST59. The Taiwan clone carries the Panton-Valentine leukocidin (PVL) genes, the staphylococcal chromosomal cassette mec (SCCmec) VT and is frequently isolated from patients with severe disease. The Asian-Pacific clone is PVL-negative, carries SCCmec IV, and is a frequent colonizer of healthy children.
Project description:Francisella tularensis LVS was grown in MH broth in the presence or absence of 200uM spermine. RNA was harvasted from overnight (16 hour) cultures and processed for microarray hybridization.