<p>Strains N402 (WT; black bars), NW283 (<i>ΔcreA;</i> grey bars), and JS014 (<i>ΔrhaR</i>; open bars) were used. Relative transcription levels were measured by RT-qPCR in samples obtained 2 hours after... more
<p>Strains N402 (WT; black bars), NW283 (<i>ΔcreA;</i> grey bars), and JS014 (<i>ΔrhaR</i>; open bars) were used. Relative transcription levels were measured by RT-qPCR in samples obtained 2 hours after mycelium transfer to 5 mM L-rhamnose or 5 mM L-rhamnose plus 50 mM D-glucose. Relative transcript levels of <i>rhtA</i> and <i>rhaB</i> were calculated using the pre-culture condition of each strain (D-sorbitol 100 mM; t = 18h), sampled prior to the mycelium transfer to inducing and inducing/repressing conditions, as reference (*). Results are given as relative transcript ratios in logarithmic scale (lg(10)). The values provided in the figures correspond to two biological replicates per culture condition. Error bars are means of three technical replicates.</p
Toxicity associated with systemic administration of adenoviral vectors occurs in three phases: acute, intermediate and chronic. Chronic toxicity has largely been abolished with the application of helper-dependent vectors (HDV). However,... more
Toxicity associated with systemic administration of adenoviral vectors occurs in three phases: acute, intermediate and chronic. Chronic toxicity has largely been abolished with the application of helper-dependent vectors (HDV). However, the acute toxicity induced by both early generation and helper-dependent adenoviral vectors continues to be a major obstacle for clinical application in humans. We are utilizing microarray technology to determine alterations in gene expression patterns that are brought about in vitro and in vivo by infection with both first generation and helper-dependent adenoviral vectors. Signaling pathways that are activated by Ad infection may contribute to the host innate immune response. These same pathways would be targets for pharmacologic manipulations aimed at decreasing acute toxicity and improving the therapeutic index of HDVs. We have performed preliminary in vitro analyses using RNA collected 24 hours after infection of Human Umbilical Vein Endothelial Cells (HUVEC) with PBS, First Generation or Helper-Dependent adenoviral vectors. This RNA was reverse transcribed and hybridized to either Affymetrix oligo arrays or NFκB pathway arrays. HUVECs represent a relevant target cell type based on substantial endothelial cell transduction following intravenous adenoviral vector administration observed in nonhuman primates, while the NFκB pathway has been implicated in host innate immune response to adenoviral vector by our lab and several others. The Affymetrix chips offer a genome-wide analysis of expression pattern alterations, while the NFκB targeted array has been specifically designed to include 112 genes associated with the NFκB signaling network. Data from the NFκB pathway array suggests that of the 112 spotted genes, approximately 40 genes undergo at least a two-fold induction in helper-dependent vector-infected HUVECs as compared to mock-infected HUVECs. These genes include several TLRs, TRAFs and interferons alpha and beta. On first pass, these data support modulation of NFκB signaling in target cells as a potential approach for addressing acute toxicity of Ad vectors. Current in vitro experiments are addressing additional cell types including hepatocytes and dendritic cells from both mouse and human.
