Ozone induces clear cellular and molecular responses in the mouse lung independently of the transcription-coupled repair status.

The oxidant ozone is a well-known air pollutant, inhalation of which is associated with respiratory tract inflammation and functional alterations of the lung. It is well established as an inducer of intracellular oxidative stress. We investigated whether Cockayne syndrome B, transcription-coupled, repair-deficient mice (Csb(-/-)), known to be sensitive to oxidative stressors, respond differently to ozone than repair-proficient controls (Csb(+/-)). Mice were exposed to 0.8 parts/million ozone for 8 h, and we examined a wide range of biological parameters in the lung at the gene expression, protein, and cellular level 4 h after the ozone exposure. Relevant biological responses to ozone for both repair-deficient Csb(-/-) and repair-proficient Csb(+/-) mice, as determined by biochemical analysis of bronchoalveolar lavage fluid (e.g., increases of polymorphonuclear neutrophils, alkaline phosphatase, macrophage-inflammatory protein-2, and tumor necrosis factor-alpha), pathological examinations, and gene expression (upregulation of oxidative-stress-related genes) analyses were observed. The bronchoalveolar lavage fluid showed significantly more tumor necrosis factor-alpha in repair-deficient Csb(-/-) mice than in repair-proficient Csb(+/-) mice after ozone exposure. In addition, a clear trend was observed toward fewer differentially expressed genes with a lower fold ratio in repair-deficient Csb(-/-) mice than in repair-proficient Csb(+/-) mice. However, repair-deficient Csb(-/-) mice do not respond significantly more sensitively to ozone compared with repair-proficient Csb(+/-) mice at the level of gene expression. We conclude that, under the conditions employed here, although small differences at the transcriptional level exist between repair-proficient Csb(+/-) mice and transcription-coupled repair defective Csb(-/-) mice, these do not have a significant effect on the ozone-induced lung injury.

A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR.

The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by E1-specific quantitative PCR. The detection limit of this assay is 3 RCAs in 1 x 10(10) vector particles per 70 ml of microcarrier cell culture. The main advantage of the combination of cell culture and PCR detection is that replicated virus can be detected long before cytopathic effects become visible and therefore, it is much faster than conventional cell culture-based assays. This assay was validated by spiking replication-incompetent adenoviral vectors with wild-type adenovirus serotype 5 (wt Ad5) as a positive control for RCA. It was found that the replication of wt Ad5 is hampered above a vector particle per cell ratio of 50. However, if microcarrier beads are used, many cells can be grown in a small suspension culture and consequently a large number of vector particles can be tested for contamination with RCA.