Project description:Acute lung inflammation can alter the pulmonary function of susceptible individuals and exacerbate the pathogenesis of chronic inflammatory lung diseases including chronic obstructive pulmonary disease (COPD), cystic fibrosis and asthma. Exposure to lipopolysaccharide (LPS) or endotoxin, a constituent of outer cell membrane of gram negative bacteria, induces airway inflammation that is primarily characterized by increased polymorphonuclear neutrophils (PMNs) at early time points. Because LPS is present in variety of occupational and home environments and is an active constituent of cigarette smoke it is a risk factor for increasing prevalence and severity of non-occupational COPD, for adult onset of asthma and for wheezing in children. In airway epithelial cells, LPS stimulation increases mucin gene expression and mucous production. Hypersecretion of mucus overwhelms the ciliary clearance and obstructs airways, causing morbidity and mortality in chronic inflammatory respiratory lung diseases. In addition, acute bacterial infection contributes to the exacerbation of chronic airway diseases, specifically in advanced COPD and CF subjects, leading to increased healthcare burden and higher mortality. Bcl-2, a prosurvival protein that inhibits cell death plays a key role in normal cellular homeostasis and regulates the integrity of the mitochondrial and endoplasmic reticulum membranes. Gain- and loss-of-function studies showed that Bcl-2 expression sustains hyperplastic epithelial cells, and Bcl-2 expression is elevated in airway epithelial cells of subjects with cystic fibrosis and asthma. The present study investigated which inflammatory mediators induce mucous cell metaplasia and Bcl-2 expression following LPS exposure. Microarray analyses of mRNA from airway epithelial cells captured by laser microdissection from rat lungs snap-frozen at day 0 and 2 post LPS exposure were analyzed. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection from rat lungs snap-frozen at day 0 and day 2 post LPS exposure was performed to identify inflammatory mediators modulated by LPS exposure. Specific pathogen-free F344/NCrR male rats of 8–10 wk of age (from NCI, Frederick, MD) were exposed to LPS (1000 ug in 0.5 ML Saline) and rats were sacrificed on day 0 and day 2 of LPS exposure. Right lungs were snap-frozen in OCT and airway epithelial cells were captured by laser-dissection microsopy to extract RNA. Gene expression data from nonexposed rats (0d) was used a control.

Project description:The airways of cystic fibrosis patients are chronically colonized by a diverse range of microbial pathogens, the composition of which changes throughout life Alteration to the pulmonary environment caused by inter-microbial interactions and pathogen-host interactions influence the type of microbes that can engage in sustained infection. The opportunistic bacterial pathogen Pseudomonas aeruginosa is the primary cause of morbidity and mortality amongst individuals with cystic fibrosis and it is estimated that 60 – 80 % of cystic fibrosis patients experience chronic P. aeruginosa infection by the age of 20 years Aspergillus fumigatus is the most prevalent fungal pathogen isolated from cystic fibrosis airways, affecting up to 58% of patients. It is the causative agent of allergic bronchopulmonary aspergillosis (ABPA), a hypersensitivity disorder resulting from the inhalation of fungal conidia. Although co-colonization of the cystic fibrosis airways by P. aeruginosa and A. fumigatus is rare (3.1 – 15.8 %) disease prognosis is poor when both are present. However, sequential infection is more common. It has recently been suggested that A. fumigatus is more prevalent in juvenile cystic fibrosis patients that has been initially reported due to inconsistencies in the culture methods used to detect A. fumigatus. Despite the prevalence and persistence of A. fumigatus, P. aeruginosa predominates as the primary pathogen in the cystic fibrosis lung, suggesting that interactions with other pathogens such as A. fumigatus may influence the pathogenicity of P. aeruginosa by altering its virulence. We report here an investigation of the effect of culturing P. aeruginosa in the presence of A. fumigatus by measuring differences in growth rate and the overall proteome of the bacteria. It was hypothesized that A. fumigatus creates an environment that promotes a metabolic-driven increase in P. aeruginosa that results in it outcompeting the fungus. The molecular basis of this increased proliferation was investigated further using Label-free quantitative (LFQ) proteomics to characterise the proteome changes in P. aeruginosa when exposed to the supernatant of i) A. fumigatus alone ii) the supernatant of an A. fumigatus/P. aeruginosa co-culture and iii) P. aeruginosa alone. LFQ proteomics involves the simultaneous identification and quantification of thousands of proteins (the ultimate determinants of phenotype) from a single sample has recently been employed to characterise the P. aeruginosa proteome in response to iron limiting conditions, resolving how how the bacteria survives and proliferates in such environments.

Project description:Acute lung inflammation can alter the pulmonary function of susceptible individuals and exacerbate the pathogenesis of chronic inflammatory lung diseases including chronic obstructive pulmonary disease (COPD), cystic fibrosis and asthma. Exposure to lipopolysaccharide (LPS) or endotoxin, a constituent of outer cell membrane of gram negative bacteria, induces airway inflammation that is primarily characterized by increased polymorphonuclear neutrophils (PMNs) at early time points. Because LPS is present in variety of occupational and home environments and is an active constituent of cigarette smoke it is a risk factor for increasing prevalence and severity of non-occupational COPD, for adult onset of asthma and for wheezing in children. In airway epithelial cells, LPS stimulation increases mucin gene expression and mucous production. Hypersecretion of mucus overwhelms the ciliary clearance and obstructs airways, causing morbidity and mortality in chronic inflammatory respiratory lung diseases. In addition, acute bacterial infection contributes to the exacerbation of chronic airway diseases, specifically in advanced COPD and CF subjects, leading to increased healthcare burden and higher mortality. Bcl-2, a prosurvival protein that inhibits cell death plays a key role in normal cellular homeostasis and regulates the integrity of the mitochondrial and endoplasmic reticulum membranes. Gain- and loss-of-function studies showed that Bcl-2 expression sustains hyperplastic epithelial cells, and Bcl-2 expression is elevated in airway epithelial cells of subjects with cystic fibrosis and asthma. The present study investigated which inflammatory mediators induce mucous cell metaplasia and Bcl-2 expression following LPS exposure. Microarray analyses of mRNA from airway epithelial cells captured by laser microdissection from rat lungs snap-frozen at day 0 and 2 post LPS exposure were analyzed. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection from rat lungs snap-frozen at day 0 and day 2 post LPS exposure was performed to identify inflammatory mediators modulated by LPS exposure.

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Longitudinal Study of PCD: 5-18 Years of Age

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