Renee Gaspard

Murine macrophage transcriptional responses to Bacillus anthracis infection and intoxication.

ABSTRACT Interactions between Bacillus anthracis and host macrophages represent critical early events in anthrax pathogenesis, but their details are not clearly understood. Here we report the first genomewide characterization of the transcriptional changes within macrophages infected with B. anthracis and the identification of several hundred host genes that were differentially expressed during this intracellular stage of infection. These loci included both genes that are known to be regulated differentially in response to many other bacterial pathogens and those that appear to be differentially regulated in response to B. anthracis but not other bacterial species that have been tested. These data provide a transcriptional basis for a variety of physiological changes observed during infection, including the induction of apoptosis caused by the infecting bacteria. The expression patterns underlying B. anthracis-induced apoptosis led us to test further the importance of one very highly induced macrophage gene, that for ornithine decarboxylase. Our data show that this enzyme plays an important and previously unrecognized role in suppressing apoptosis in B. anthracis-infected cells. We have also characterized the transcriptional response to anthrax lethal toxin in activated macrophages and found that, following toxin treatment, many of the host inflammatory response pathways are dampened. These data provide insights into B. anthracis pathogenesis as well as potential leads for the development of new diagnostic and therapeutic options.

The limits of log-ratios

BackgroundDNA microarray assays typically compare two biological samples and present the results of those comparisons gene-by-gene as the logarithm base two of the ratio of the measured expression levels for the two samples.ResultsBecause of the fixed dynamic range of fluorescence and other detection systems, there is a limit to the range of comparisons that can be made using any array technology, and this must be taken into account when interpreting the results of any such analysis.ConclusionsThe dynamic range of microarray data collection systems results in limits in the comparative analyses that can be derived from such measurements and suggests that optimal results can be obtained by making measurements that avoid the boundaries of that dynamic range.

Microarray analysis of the chondrocytic cell line T/C-28a2 under dynamic fluid shear

The behavior of the chondrocytic cell line T/C-28a2 under shear flow was examined using a 32,448 element microarray. A parallel plate flow chamber was used to generate a shear stress level of 20 dyn/cm/sup 2/ for 1.5 or 24 hours (h), after which gene regulation was measured. Microarray analysis revealed differentially regulated genes affecting proliferation/differentiation, extracellular matrix/cytoskeleton, and inflammation at both time points. A ribonuclease protection assay was performed on a subset of genes to confirm the data obtained from the microarray. However, the cyclooxygenase-2 (COX-2) gene, which plays a role in the prostaglandin production in inflamed tissues and the synovium of rheumatoid arthritis (RA) patients, was studied further. Western hybridization revealed that COX-2 protein is present at 24 h, but not at 6 or 12 h. Also, immunofluorescence microscopy shows that COX-2 protein localizes in the cytosol after 24 h of shear, and is not present after 1.5 h. By examining the overall gene expression profiles of chondrocytes under different conditions of dynamic fluid shear, new insights on the pathogenesis of cartilage related diseases such as rheumatoid arthritis might be generated.