Respiratory Viral and Bacterial Exacerbations of COPD-The Role of the Airway Epithelium.

COPD is a leading cause of death worldwide, with acute exacerbations being a major contributor to disease morbidity and mortality. Indeed, exacerbations are associated with loss of lung function, and exacerbation frequency predicts poor prognosis. Respiratory infections are important triggers of acute exacerbations of COPD. This review examines the role of bacterial and viral infections, along with co-infections, in the pathogenesis of COPD exacerbations. Because the airway epithelium is the initial site of exposure both to cigarette smoke (or other pollutants) and to inhaled pathogens, we will focus on the role of airway epithelial cell responses in regulating the pathophysiology of exacerbations of COPD. This will include an examination of the interactions of cigarette smoke alone, and in combination with viral and bacterial exposures in modulating epithelial function and inflammatory and host defense pathways in the airways during COPD. Finally, we will briefly examine current and potential medication approaches to treat acute exacerbations of COPD triggered by respiratory infections.

Rhinovirus-Induced Modulation of Epithelial Phenotype: Role in Asthma.

Human rhinoviruses have been linked both to the susceptibility of asthma development and to the triggering of acute exacerbations. Given that the human airway epithelial cell is the primary site of human rhinovirus (HRV) infection and replication, the current review focuses on how HRV-induced modulation of several aspects of epithelial cell phenotype could contribute to the development of asthma or to the induction of exacerbations. Modification of epithelial proinflammatory and antiviral responses are considered, as are alterations in an epithelial barrier function and cell phenotype. The contributions of the epithelium to airway remodeling and to the potential modulation of immune responses are also considered. The potential interactions of each type of HRV-induced epithelial phenotypic changes with allergic sensitization and allergic phenotype are also considered in the context of asthma development and of acute exacerbations.

Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange.

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ∼2 weeks.