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1.
J Appl Toxicol ; 43(6): 862-873, 2023 06.
Article in English | MEDLINE | ID: mdl-36594405

ABSTRACT

Cigarette smoke (CS) exposure induces both cytotoxicity and inflammation, and often causes COPD, a growing cause of morbidity and mortality. CS also inhibits the CFTR Cl- channel, leading to airway surface liquid dehydration, which is predicated to impair clearance of inhaled pathogens and toxicants. Numerous in vitro studies have been performed that utilize acute (≤24 h) CS exposures. However, CS exposure is typically chronic. We evaluated the feasibility of using British-American Tobacco (BAT)-designed CS exposure chambers for chronically exposing human bronchial epithelial cultures (HBECs) to CS. HBECs are polarized and contain mucosal and serosal sides. In vivo, inhaled CS interacts with mucosal membranes, and BAT chambers are designed to direct CS to HBEC mucosal surfaces while keeping CS away from serosal surfaces via a perfusion system. We found that serosal perfusion was absolutely required to maintain HBEC viability over time following chronic CS exposure. Indeed, with this system, we found that CS increased inflammation and mucin levels, while decreasing CFTR function. Without this serosal perfusion, CS was extremely toxic within 24 h. We therefore propose that 5- and 10-day CS exposures with serosal perfusion are suitable for measuring chronic CS exposure and can be used for monitoring new and emerging tobacco products.


Subject(s)
Cigarette Smoking , Cystic Fibrosis Transmembrane Conductance Regulator , Humans , Bronchi , Nicotiana/toxicity , Inflammation , Epithelial Cells
2.
Steroids ; 156: 108575, 2020 04.
Article in English | MEDLINE | ID: mdl-31901423

ABSTRACT

There is increasing evidence for sexual dimorphism of estrogen (E2) actions in the exacerbation of lung function, infection and inflammation in females with cystic fibrosis - the so-called "CF gender gap". The effects of estrogen on virulence factors that enhance P. aeruginosa persistence in CF lung epithelium were investigated by phenotypic and chemical assays in various PsA clinical isolates and laboratory strains in isolation or in co-culture with normal (Nuli-1) and CF dPhe508-CFTR (CuFi-1) human bronchial epithelial cell lines. Estrogen (E2, 10 nM) significantly increased secretion of the virulence factor pyocyanin by 80% in PsA early infection isolates from female CF patients and by 280% in late infection PsA isolates. Estrogen also increased the swarming motility by up to 50% in all PsA isolates and strains tested in 0.5% agar. A significant increase of 110% in the twitching motility of all PsA isolates and strains tested was also observed with estrogen treatment. Treatment with E2 increased biofilm formation of P. aeruginosa PsAO1 which became more adherent to, and invasive into, normal and CF bronchial epithelial cells. The selective estrogen receptor modulators (SERMs), Tamoxifen and ICI 182780 inhibited P. aeruginosa motility. The potency of various steroid hormones to stimulate motility of P. aeruginosa was in the order; estradiol â‰« estrone > E3 estriol ≥ testosterone ≥ progesterone â‰« aldosterone, cortisol. Estrogen was also shown to reduce ciliary beat intensity in CF bronchial epithelium which would further exacerbate PsA trapping and virulence in the CF airways. In conclusion, we have demonstrated for the first time that estrogen exacerbates P. aeruginosa virulence factors and enhances bacterial interactions with CF bronchial epithelium which can be inhibited by tamoxifen. Our work suggests that SERMs could be used as an adjuvant treatment to reduce estrogen-induced P. aeruginosa infections and associated lung exacerbations in females with CF.


Subject(s)
Cystic Fibrosis/metabolism , Estrogens/metabolism , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , Sex Characteristics , Cells, Cultured , Child, Preschool , Cystic Fibrosis/microbiology , Female , Humans , Male , Pseudomonas aeruginosa/isolation & purification , Virulence
3.
Antimicrob Agents Chemother ; 60(10): 6003-12, 2016 10.
Article in English | MEDLINE | ID: mdl-27458217

ABSTRACT

The opportunistic bacteria of the Burkholderia cepacia complex (Bcc) are extremely pathogenic to cystic fibrosis (CF) patients, and acquisition of Bcc bacteria is associated with a significant increase in mortality. Treatment of Bcc infections is difficult because the bacteria are multidrug resistant and able to survive in biofilms. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an innate defense protein that is secreted by the upper airways and pharynx. While SPLUNC1 is known to have antimicrobial functions, its effects on Bcc strains are unclear. We therefore tested the hypothesis that SPLUNC1 is able to impair Bcc growth and biofilm formation. We found that SPLUNC1 exerted bacteriostatic effects against several Bcc clinical isolates, including B. cenocepacia strain J2315 (50% inhibitory concentration [IC50] = 0.28 µM), and reduced biofilm formation and attachment (IC50 = 0.11 µM). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and α6 helix did not affect its function. However, deletion of the α4 helix attenuated antimicrobial activity, while the corresponding α4 peptide displayed antimicrobial activity. Chronic neutrophilia is a hallmark of CF lung disease, and neutrophil elastase (NE) cleaves SPLUNC1. However, we found that the ability of SPLUNC1 to disrupt biofilm formation was significantly potentiated by NE pretreatment. While the impact of CF on SPLUNC1-Bcc interactions is not currently known, our data suggest that understanding this interaction may have important implications for CF lung disease.


Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Burkholderia cepacia complex/drug effects , Glycoproteins/pharmacology , Leukocyte Elastase/chemistry , Phosphoproteins/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Binding Sites , Biofilms/growth & development , Burkholderia cepacia complex/growth & development , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Glycoproteins/chemistry , Glycoproteins/genetics , Glycoproteins/metabolism , Humans , Microbial Sensitivity Tests , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Structure-Activity Relationship
4.
Sci Rep ; 6: 28115, 2016 06 15.
Article in English | MEDLINE | ID: mdl-27301427

ABSTRACT

Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications.


Subject(s)
Phage Therapy/methods , Pseudomonas Infections/therapy , Pseudomonas Phages/genetics , Animals , Bacterial Load , Biofilms , Cell Line , Cystic Fibrosis/pathology , Disease Models, Animal , Epithelial Cells/virology , Gentamicins/pharmacology , Humans , Hydrogen-Ion Concentration , Moths/microbiology , Mutation , Pseudomonas Phages/classification , Pseudomonas Phages/isolation & purification , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/pathogenicity , Pseudomonas aeruginosa/virology , Viral Proteins/chemistry
5.
Biochemistry ; 55(21): 2979-91, 2016 05 31.
Article in English | MEDLINE | ID: mdl-27145151

ABSTRACT

SPLUNC1 is an abundantly secreted innate immune protein in the mammalian respiratory tract that exerts bacteriostatic and antibiofilm effects, binds to lipopolysaccharide (LPS), and acts as a fluid-spreading surfactant. Here, we unravel the structural elements essential for the surfactant and antimicrobial functions of human SPLUNC1 (short palate lung nasal epithelial clone 1). A unique α-helix (α4) that extends from the body of SPLUNC1 is required for the bacteriostatic, surfactant, and LPS binding activities of this protein. Indeed, we find that mutation of just four leucine residues within this helical motif to alanine is sufficient to significantly inhibit the fluid spreading abilities of SPLUNC1, as well as its bacteriostatic actions against Gram-negative pathogens Burkholderia cenocepacia and Pseudomonas aeruginosa. Conformational flexibility in the body of SPLUNC1 is also involved in the bacteriostatic, surfactant, and LPS binding functions of the protein as revealed by disulfide mutants introduced into SPLUNC1. In addition, SPLUNC1 exerts antibiofilm effects against Gram-negative bacteria, although α4 is not involved in this activity. Interestingly, though, the introduction of surface electrostatic mutations away from α4 based on the unique dolphin SPLUNC1 sequence, and confirmed by crystal structure, is shown to impart antibiofilm activity against Staphylococcus aureus, the first SPLUNC1-dependent effect against a Gram-positive bacterium reported to date. Together, these data pinpoint SPLUNC1 structural motifs required for the antimicrobial and surfactant actions of this protective human protein.


Subject(s)
Anti-Infective Agents/pharmacology , Bronchi/drug effects , Burkholderia cenocepacia/drug effects , Glycoproteins/chemistry , Glycoproteins/metabolism , Lipopolysaccharides/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Pseudomonas aeruginosa/drug effects , Amino Acid Sequence , Biofilms/drug effects , Bronchi/cytology , Burkholderia cenocepacia/immunology , Cells, Cultured , Crystallization , Crystallography, X-Ray , Glycoproteins/genetics , Humans , Immunity, Innate/drug effects , Phosphoproteins/genetics , Protein Conformation , Pseudomonas aeruginosa/immunology , Pulmonary Surfactants/chemistry , Pulmonary Surfactants/metabolism
6.
Am J Physiol Lung Cell Mol Physiol ; 310(11): L1053-61, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27084849

ABSTRACT

The specialized proresolution lipid mediator lipoxin A4 (LXA4) is abnormally produced in cystic fibrosis (CF) airways. LXA4 increases the CF airway surface liquid height and stimulates airway epithelial repair and tight junction formation. We report here a protective effect of LXA4 (1 nM) against tight junction disruption caused by Pseudomonas aeruginosa bacterial challenge together with a delaying action against bacterial invasion in CF airway epithelial cells from patients with CF and immortalized cell lines. Bacterial invasion and tight junction integrity were measured by gentamicin exclusion assays and confocal fluorescence microscopy in non-CF (NuLi-1) and CF (CuFi-1) bronchial epithelial cell lines and in primary CF cultures, grown under an air/liquid interface, exposed to either a clinical or laboratory strains of P. aeruginosa LXA4 delayed P. aeruginosa invasion and transepithelial migration in CF and normal bronchial epithelial cell cultures. These protective effects of LXA4 were inhibited by the ALX/FPR2 lipoxin receptor antagonist BOC-2. LXA4 prevented the reduction in mRNA biosynthesis and protein abundance of the tight junction protein ZO-1 and reduced tight junction disruption induced by P. aeruginsosa inoculation. In conclusion, LXA4 plays a protective role in bronchial epithelium by stimulating tight junction repair and by delaying and reducing the invasion of CF bronchial epithelial cells by P. aeruginsosa.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cystic Fibrosis/drug therapy , Lipoxins/pharmacology , Pseudomonas Infections/microbiology , Tight Junctions/metabolism , Cell Line , Cystic Fibrosis/microbiology , Cystic Fibrosis/pathology , Drug Evaluation, Preclinical , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Gene Expression , Humans , Pseudomonas Infections/prevention & control , Pseudomonas aeruginosa/physiology , Receptors, Formyl Peptide/metabolism , Receptors, Lipoxin/metabolism , Respiratory Mucosa/microbiology , Tight Junctions/drug effects , Tight Junctions/microbiology , Zonula Occludens-1 Protein/genetics , Zonula Occludens-1 Protein/metabolism
7.
Microbiology (Reading) ; 162(2): 191-205, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26643057

ABSTRACT

Iron acquisition is vital to microbial survival and is implicated in the virulence of many of the pathogens that reside in the cystic fibrosis (CF) lung. The multifaceted nature of iron acquisition by both bacterial and fungal pathogens encompasses a range of conserved and species-specific mechanisms, including secretion of iron-binding siderophores, utilization of siderophores from other species, release of iron from host iron-binding proteins and haemoproteins, and ferrous iron uptake. Pathogens adapt and deploy specific systems depending on iron availability, bioavailability of the iron pool, stage of infection and presence of competing pathogens. Understanding the dynamics of pathogen iron acquisition has the potential to unveil new avenues for therapeutic intervention to treat both acute and chronic CF infections. Here, we examine the range of strategies utilized by the primary CF pathogens to acquire iron and discuss the different approaches to targeting iron acquisition systems as an antimicrobial strategy.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/microbiology , Iron-Binding Proteins/metabolism , Iron/metabolism , Mucociliary Clearance/physiology , Pseudomonas Infections/pathology , Staphylococcal Infections/pathology , Humans , Lung/microbiology , Microbiota , Mucociliary Clearance/genetics , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/pathogenicity , Siderophores/metabolism , Staphylococcal Infections/microbiology , Staphylococcus aureus/pathogenicity
8.
J Biol Chem ; 290(42): 25710-6, 2015 Oct 16.
Article in English | MEDLINE | ID: mdl-26338706

ABSTRACT

Transforming growth factor ß1 (TGF-ß1) is not only elevated in airways of cystic fibrosis (CF) patients, whose airways are characterized by abnormal ion transport and mucociliary clearance, but TGF-ß1 is also associated with worse clinical outcomes. Effective mucociliary clearance depends on adequate airway hydration, governed by ion transport. Apically expressed, large-conductance, Ca(2+)- and voltage-dependent K(+) (BK) channels play an important role in this process. In this study, TGF-ß1 decreased airway surface liquid volume, ciliary beat frequency, and BK activity in fully differentiated CF bronchial epithelial cells by reducing mRNA expression of the BK γ subunit leucine-rich repeat-containing protein 26 (LRRC26) and its function. Although LRRC26 knockdown itself reduced BK activity, LRRC26 overexpression partially reversed TGF-ß1-induced BK dysfunction. TGF-ß1-induced airway surface liquid volume hyper-absorption was reversed by the BK opener mallotoxin and the clinically useful TGF-ß signaling inhibitor pirfenidone. The latter increased BK activity via rescue of LRRC26. Therefore, we propose that TGF-ß1-induced mucociliary dysfunction in CF airways is associated with BK inactivation related to a LRRC26 decrease and is amenable to treatment with clinically useful TGF-ß1 inhibitors.


Subject(s)
Bronchi/pathology , Cystic Fibrosis/metabolism , Potassium Channels, Voltage-Gated/physiology , Pyridones/pharmacology , Transforming Growth Factor beta1/physiology , Adenosine Triphosphate/metabolism , Bronchi/drug effects , Cystic Fibrosis/drug therapy , Cystic Fibrosis/pathology , Gene Knockdown Techniques , Humans , Mucociliary Clearance/drug effects , Neoplasm Proteins/genetics , Pyridones/therapeutic use , Transforming Growth Factor beta1/antagonists & inhibitors
9.
Am J Physiol Lung Cell Mol Physiol ; 308(10): L1068-77, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25795727

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is a growing cause of morbidity and mortality worldwide. Recent studies have shown that cigarette smoke (CS) induces cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, which leads to airway-surface liquid (ASL) dehydration. This in turn contributes to the mucus dehydration and impaired mucociliary clearance that are seen in the chronic bronchitis form of COPD. Roflumilast is a phosphodiesterase 4 inhibitor that may improve lung function and reduce the frequency of exacerbations in patients with COPD. Although roflumilast can affect cAMP metabolism, little is known about the downstream pharmacological effects in the airways. We hypothesized that roflumilast would increase ASL rehydration in human bronchial epithelial cultures (HBECs) after chronic CS exposure. cAMP production was measured by Förster resonance energy transfer in HEK293T cells and by ELISA in HBECs. ASL height was measured by xz-confocal microscopy after air exposure or following HBEC exposure to freshly produced CS. Roflumilast had little effect on cAMP or ASL height when applied on its own; however, roflumilast significantly potentiated adenosine-induced increases in cAMP and ASL height in CS-exposed HBECs. Roflumilast increased the rate of ASL height recovery in cultures after CS exposure compared with controls. In contrast, the ß2-adrenergic receptor agonists isoproterenol and salmeterol failed to increase ASL height after CS exposure. Our data suggest that roflumilast can increase ASL hydration in CS-exposed HBECs, which is predicted to be beneficial for the treatment of mucus dehydration/mucus stasis in patients with COPD chronic bronchitis.


Subject(s)
Adenosine/pharmacology , Aminopyridines/pharmacology , Analgesics/pharmacology , Benzamides/pharmacology , Phosphodiesterase 4 Inhibitors/pharmacology , Pulmonary Disease, Chronic Obstructive/metabolism , Respiratory Mucosa/metabolism , Smoking/metabolism , Adrenergic beta-Agonists/pharmacology , Bronchi/metabolism , Bronchi/pathology , Cyclic AMP/metabolism , Cyclopropanes/pharmacology , HEK293 Cells , Humans , Isoproterenol/pharmacology , Pulmonary Disease, Chronic Obstructive/diet therapy , Pulmonary Disease, Chronic Obstructive/etiology , Pulmonary Disease, Chronic Obstructive/pathology , Respiratory Mucosa/pathology , Smoking/adverse effects , Smoking/pathology
10.
Biochim Biophys Acta ; 1850(6): 1224-32, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25697727

ABSTRACT

BACKGROUND: Cystic fibrosis transmembrane conductance regulator plays a key role in maintenance of lung fluid homeostasis. Cigarette smoke decreases CFTR expression in the lung but neither the mechanisms leading to CFTR loss, nor potential ways to prevent its loss have been identified to date. METHODS: The molecular mechanisms leading to down-regulation of CFTR by cigarette smoke were determined using pharmacologic inhibitors and silencing ribonucleic acids (RNAs). RESULTS: Using human bronchial epithelial cells, here we show that cigarette smoke induces degradation of CFTR that is attenuated by lysosomal inhibitors, but not proteasome inhibitors. Cigarette smoke can activate multiple signaling pathways in airway epithelial cells, including the MEK/Erk1/2 MAPK (MEK: mitogen-activated protein kinase/ERK kinase Erk1/2: extracellular signal-regulated kinase 1/2 MAPK: Mitogen-activated protein kinase) pathway regulating cell survival. Interestingly, pharmacological inhibition of the MEK/Erk1/2 MAPK pathway prevented the loss of plasma membrane CFTR upon cigarette smoke exposure. Similarly, decreased expression of Erk1/2 using silencing RNAs prevented the suppression of CFTR protein by cigarette smoke. Conversely, specific inhibitors of the c-Jun N-terminal kinase (JNK) or p38 MAPK pathways had no effect on CFTR decrease after cigarette smoke exposure. In addition, inhibition of the MEK/Erk1/2 MAPK pathway prevented the reduction of the airway surface liquid observed upon cigarette smoke exposure of primary human airway epithelial cells. Finally, addition of the antioxidant N-acetylcysteine inhibited activation of Erk1/2 by cigarette smoke and precluded the cigarette smoke-induced decrease of CFTR. CONCLUSIONS: These results show that the MEK/Erk1/2 MAPK pathway regulates plasma membrane CFTR in human airway cells. GENERAL SIGNIFICANCE: The MEK/Erk1/2 MAPK pathway should be considered as a target for strategies to maintain/restore CFTR expression in the lung of smokers.


Subject(s)
Bronchi/drug effects , Cystic Fibrosis Transmembrane Conductance Regulator/drug effects , Epithelial Cells/drug effects , MAP Kinase Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Smoke/adverse effects , Smoking/adverse effects , Antioxidants/pharmacology , Bronchi/enzymology , Cell Line , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Down-Regulation , Epithelial Cells/enzymology , Humans , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-cbl/metabolism , RNA Interference , Time Factors , Transfection
11.
J Pharmacovigil ; 2(1): 118, 2014 Mar 01.
Article in English | MEDLINE | ID: mdl-25126589

ABSTRACT

Asthma, Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis (CF) are all pulmonary diseases which are characterized by chronic inflammation and an increase in mucus production. Excess mucus in the airways correlates with pathophysiology such as a decline in lung function and prolonged bacterial infections. New drugs to treat these chronic respiratory diseases are currently being developed and include both inhaled and orally administered compounds. Whilst oral drugs may be easier to administer, they are more prone to side-effects due to higher bioavailability. Inhaled compounds may show reduced bioavailability, but face their own unique challenges. For example, thick mucus in the respiratory tracts of asthma, CF and COPD patients can act as a physical barrier that impedes drug delivery. Mucus also contains a high number of enzymes and proteases that may degrade compounds before they reach their site of action. Furthermore, some classes of drugs are rapidly absorbed across the respiratory epithelia into systemic circulation, which may limit their duration of action and/or cause off-target effects. This review discusses some of the different treatment options that are currently available and the considerations that need to be taken into account to produce new therapies for the treatment of chronic respiratory diseases.

12.
Respir Res ; 15: 69, 2014 Jun 23.
Article in English | MEDLINE | ID: mdl-24957904

ABSTRACT

BACKGROUND: The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation. METHODS: Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts. RESULTS: We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells. CONCLUSIONS: CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Lung/metabolism , Metals, Heavy/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Respiratory Mucosa/metabolism , Aged , Cells, Cultured , Female , Humans , Lung/pathology , Male , Middle Aged , Pulmonary Disease, Chronic Obstructive/diagnosis , Respiratory Mucosa/pathology , Smoke/adverse effects , Tobacco Products/adverse effects
13.
PLoS One ; 8(12): e83065, 2013.
Article in English | MEDLINE | ID: mdl-24349432

ABSTRACT

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient's death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.


Subject(s)
Bacterial Proteins , Burkholderia Infections , Burkholderia cepacia , Pneumonia, Bacterial , Proteomics , Virulence Factors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Burkholderia Infections/genetics , Burkholderia Infections/metabolism , Burkholderia cepacia/genetics , Burkholderia cepacia/metabolism , Burkholderia cepacia/pathogenicity , Female , Humans , Male , Pneumonia, Bacterial/genetics , Pneumonia, Bacterial/metabolism , Virulence Factors/genetics , Virulence Factors/metabolism
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