Olafur Baldursson

Novel Effects of Azithromycin on Tight Junction Proteins in Human Airway Epithelia

ABSTRACT The macrolide antibiotic azithromycin improves lung function and prognosis among patients with cystic fibrosis or diffuse panbronchiolitis, independently of bacterial eradication. Anti-inflammatory effects have been implicated, but data from in vivo studies are scarce, and the link between abnormal electrolyte content in airway surface liquid and bronchial infections remains uncertain. In the present study, we treated human airway epithelia on filter supports with azithromycin and monitored transepithelial electrical resistance. We found that azithromycin increased transepithelial electrical resistance of airway epithelia in a dose-dependent manner. Immunocytochemistry and Western blotting showed that addition of azithromycin changed the locations of proteins in cell cultures and induced processing of the tight junction proteins claudin-1 and claudin-4, occludin, and junctional adhesion molecule-A. These effects were reversible, and no effect was seen when cells were treated with penicillin or erythromycin. The data indicate that azithromycin increases the transepithelial electrical resistance of human airway epithelia by changing the processing of tight junction proteins. The results are novel and may help explain the beneficial effects of azithromycin in patients with cystic fibrosis, diffuse panbronchiolitis, and community-acquired pneumonia.

Airway branching morphogenesis in three dimensional culture

BackgroundLungs develop from the fetal digestive tract where epithelium invades the vascular rich stroma in a process called branching morphogenesis. In organogenesis, endothelial cells have been shown to be important for morphogenesis and the maintenance of organ structure. The aim of this study was to recapitulate human lung morphogenesis in vitro by establishing a three dimensional (3D) co-culture model where lung epithelial cells were cultured in endothelial-rich stroma.MethodsWe used a human bronchial epithelial cell line (VA10) recently developed in our laboratory. This cell line cell line maintains a predominant basal cell phenotype, expressing p63 and other basal markers such as cytokeratin-5 and -14. Here, we cultured VA10 with human umbilical vein endothelial cells (HUVECs), to mimic the close interaction between these cell types during lung development. Morphogenesis and differentiation was monitored by phase contrast microscopy, immunostainings and confocal imaging.ResultsWe found that in co-culture with endothelial cells, the VA10 cells generated bronchioalveolar like structures, suggesting that lung epithelial branching is facilitated by the presence of endothelial cells. The VA10 derived epithelial structures display various complex patterns of branching and show partial alveolar type-II differentiation with pro-Surfactant-C expression. The epithelial origin of the branching VA10 colonies was confirmed by immunostaining. These bronchioalveolar-like structures were polarized with respect to integrin expression at the cell-matrix interface. The endothelial-induced branching was mediated by soluble factors. Furthermore, fibroblast growth factor receptor-2 (FGFR-2) and sprouty-2 were expressed at the growing tips of the branching structures and the branching was inhibited by the FGFR-small molecule inhibitor SU5402.DiscussionIn this study we show that a human lung epithelial cell line can be induced by endothelial cells to form branching bronchioalveolar-like structures in 3-D culture. This novel model of human airway morphogenesis can be used to study critical events in human lung development and suggests a supportive role for the endothelium in promoting branching of airway epithelium.

Azithromycin Maintains Airway Epithelial Integrity during Pseudomonas aeruginosa Infection

Tight junctions (TJs) play a key role in maintaining bronchial epithelial integrity, including apical-basolateral polarity and paracellular trafficking. Patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) often suffer from chronic infections by the opportunistic Gram-negative bacterium Pseudomonas aeruginosa, which produces multiple virulence factors, including rhamnolipids. The macrolide antibiotic azithromycin (azm) has been shown to improve lung function in patients with CF without reducing the bacterial count within the lung. However, the mechanism of this effect is still debated. It has previously been shown that azm increased transepithelial electrical resistance (TER) in a bronchial epithelial cell line. In this study we used an air-liquid interface model of human airway epithelia and measured TER, changes in TJ expression and architecture after exposure to live P. aeruginosa PAO1, and PAO1-Deltarhl which is a PAO1 mutant lacking rhlA and rhlB, which encode key enzymes for rhamnolipid production. In addition, the cells were challenged with bacterial culture medium conditioned by these strains, purified rhamnolipids, or synthetic 3O-C(12)-HSL. Virulence factors secreted by P. aeruginosa reduced TER and caused TJ rearrangement in the bronchial epithelium, exposing the epithelium to further bacterial infiltration. Pretreatment of the bronchial epithelium with azm attenuated this effect and facilitated epithelial recovery. These data suggest that azm protects the bronchial epithelium during P. aeruginosa infection independent of antimicrobial activity, and could explain in part the beneficial results seen in clinical trials of patients with CF.

Conditions Associated with the Cystic Fibrosis Defect Promote Chronic Pseudomonas aeruginosa Infection

RATIONALE Progress has been made in understanding how the cystic fibrosis (CF) basic defect produces lung infection susceptibility. However, it remains unclear why CF exclusively leads to chronic infections that are noninvasive and highly resistant to eradication. Although biofilm formation has been suggested as a mechanism, recent work raises questions about the role of biofilms in CF. OBJECTIVES To learn how airway conditions attributed to CF transmembrane regulator dysfunction could lead to chronic infection, and to determine if biofilm-inhibiting genetic adaptations that are common in CF isolates affect the capacity of Pseudomonas aeruginosa to develop chronic infection phenotypes. METHODS We studied P. aeruginosa isolates grown in agar and mucus gels containing sputum from patients with CF and measured their susceptibility to killing by antibiotics and host defenses. We also measured the invasive virulence of P. aeruginosa grown in sputum gels using airway epithelial cells and a murine infection model. MEASUREMENTS AND MAIN RESULTS We found that conditions likely to result from increased mucus density, hyperinflammation, and defective bacterial killing could all cause P. aeruginosa to grow in bacterial aggregates. Aggregated growth markedly increased the resistance of bacteria to killing by host defenses and antibiotics, and reduced their invasiveness. In addition, we found that biofilm-inhibiting mutations do not impede aggregate formation in gel growth environments. CONCLUSIONS Our findings suggest that conditions associated with several CF pathogenesis hypotheses could cause the noninvasive and resistant infection phenotype, independently of the bacterial functions needed for biofilm formation.

Challenges in evaluation of chitosan and trimethylated chitosan (TMC) as mucosal permeation enhancers: From synthesis to in vitro application

The polysaccharide chitosan and the water soluble chitosan derivative N,N,N-trimethyl chitosan (TMC) have been widely investigated as permeation enhancersof mucosal surfaces with numerous papers published over the last two decades. Although both chitosan and TMC increase permeation of markers through mucosal membranes, such as the intestinal and airway epitheliumas well as in in vivo models,these investigations have not led to their use in marketed drug formulations. In this review,the reported extent of the permeation enhancement and cell viability after chitosan or TMC treatment in intestinal and airway models is critically evaluated and concluded that the apparent discrepancies can be explained by differences in polymer structure, experimental conditions and in vitro models. Additionally, aspects regarding the synthesis of TMC and its structural characterization are described, focusing on new synthetic strategies implemented to reduce O-methylation. Finally recommendations are provided on how studies can be conducted to improve understanding of the structure-activity relationship and elucidate possible mechanism of action.

deltaNp63 Has a Role in Maintaining Epithelial Integrity in Airway Epithelium

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

Regioselective fluorescent labeling of N,N,N-trimethyl chitosan via oxime formation.

Fluorescent labeling of chitosan and its derivatives is widely used for in vitro visualization and is accomplished by random introduction of the fluorophore to the polymer backbone, conceivably altering the bioactivity of the polymer. Here, we report for the first time the regioselective conjugation of a fluorophore to the reducing end of a fully N,N,N-trimethylated chitosan (TMC) by oxime formation. End-labeled conjugation of 5-(2-((aminooxyacetyl)amino)ethylamino)naphthalene-1-sulfonic acid (EDANS-O-NH(2)) fluorophore to TMC to form TMC-oxime-EDANS (f-TMC) was confirmed by (1)H NMR and fluorescence spectroscopy. Average molecular weight calculations of f-TMC with (1)H NMR and fluorescence spectroscopy gave similar results or ∼7.7kDa. f-TMC in human bronchial epithelial cells was both cell membrane bound as well as intracellularly localized. This demonstrates the proof-of-concept for selective oxime formation at the reducing end of a chitosan derivative, which can be used for tracking chitosan in gene and drug delivery purposes and gives rise to further modifications with other functional groups.

N-alkylation of highly quaternized chitosan derivatives affects the paracellular permeation enhancement in bronchial epithelia in vitro.

This study describes the structure-activity relationship for carefully characterized N-alkyl-N-quaternary chitosan derivatives as permeation enhancers for drugs that are mainly absorbed through the paracellular pathway, such as macromolecular drugs and hydrophilic drugs, in a well defined bronchial epithelial cell line. The O-methyl free derivatives used in the study were fully trimethylated (100%) N,N,N-trimethyl chitosan (TMC) and N-propyl-(QuatPropyl), N-butyl-(QuatButyl) and N-hexyl (QuatHexyl)-N,N-dimethyl chitosan, with 85-91% degree of quaternization. The fully trimethylated TMC, from 0.25mg/ml, decreased transepithelial electrical resistance (TER) in a reversible manner and enhanced the permeation of the macromolecule FITC-dextran 4kDa (FD4) 2-5 fold. TMC did not cause any alterations in the tight junction (TJ) protein claudin-4 or in F-actin architecture. QuatHexyl was the most effective polymer to produce enhanced permeation and decreased TER from 0.016mg/ml. Nevertheless, this enhanced permeation was accompanied by reduced viability and dissociation of F-actin and claudin-4 proteins. The structure-activity relationship suggests that more lipophilic derivatives show more permeation enhancement, TJ disassembly, and less viability in the order of hexyl≈butyl>propyl>methyl and demonstrates that the permeation effect is not only mediated by permanent positive charge but also by the extent of N-alkylation. These results are relevant to elucidate the structural factors contributing to the permeation enhancement of chitosan derivatives and for potential use in pulmonary applications.

Severity of Influenza A 2009 (H1N1) Pneumonia Is Underestimated by Routine Prediction Rules. Results from a Prospective, Population-Based Study

Background Characteristics of patients with community-acquired pneumonia (CAP) due to pandemic influenza A 2009 (H1N1) have been inadequately compared to CAP caused by other respiratory pathogens. The performance of prediction rules for CAP during an epidemic with a new infectious agent are unknown. Methods Prospective, population-based study from November 2008–November 2009, in centers representing 70% of hospital beds in Iceland. Patients admitted with CAP underwent evaluation and etiologic testing, including polymerase chain reaction (PCR) for influenza. Data on influenza-like illness in the community and overall hospital admissions were collected. Clinical and laboratory data, including pneumonia severity index (PSI) and CURB-65 of patients with CAP due to H1N1 were compared to those caused by other agents. Results Of 338 consecutive and eligible patients 313 (93%) were enrolled. During the pandemic peak, influenza A 2009 (H1N1) patients constituted 38% of admissions due to CAP. These patients were younger, more dyspnoeic and more frequently reported hemoptysis. They had significantly lower severity scores than other patients with CAP (1.23 vs. 1.61, P = .02 for CURB-65, 2.05 vs. 2.87 for PSI, P<.001) and were more likely to require intensive care admission (41% vs. 5%, P<.001) and receive mechanical ventilation (14% vs. 2%, P = .01). Bacterial co-infection was detected in 23% of influenza A 2009 (H1N1) patients with CAP. Conclusions Clinical characteristics of CAP caused by influenza A 2009 (H1N1) differ markedly from CAP caused by other etiologic agents. Commonly used CAP prediction rules often failed to predict admissions to intensive care or need for assisted ventilation in CAP caused by the influenza A 2009 (H1N1) virus, underscoring the importance of clinical acumen under these circumstances.

Mortality in healthcare-associated pneumonia in a low resistance setting: a prospective observational study.

Abstract Background: The classification of pneumonia as community-acquired pneumonia (CAP) or healthcare-associated pneumonia (HCAP) has implications for selection of initial antimicrobial therapy. HCAP has been associated with an increased prevalence of multidrug-resistant (MDR) pathogens and with high mortality leading to recommendations for broad empiric therapy. Methods: We performed a prospective, population-based study on consecutive adults (≥ 18 years) admitted for pneumonia over 1 calendar year. Patients were classified by pneumonia type and severity. Microbial etiologic testing was performed on all patients. Treatment, length of stay, and mortality rates were compared. Results: A total of 373 admissions were included, 94% of all eligible patients. They were classified as CAP (n = 236, 63%) or HCAP (n = 137, 37%). Chronic underlying disease was more commonly found among patients with HCAP compared with CAP (74% vs 51%, p < 0.001). Mycoplasma pneumoniae was more common among CAP patients (p < 0.01), while gram-negative bacteria were more often found among HCAP patients (p = 0.02). No MDR pathogens were detected, and rates of Staphylococcus aureus were similar in the two groups. HCAP patients were not more likely to receive ineffective initial antimicrobial therapy. HCAP patients had worse prognostic scores on admission and higher in-house mortality than CAP patients (10% vs 1%, respectively, p < 0.01). Conclusions: Even in a low resistance setting, patients with HCAP have increased mortality compared with patients with CAP. This is most likely explained by a higher prevalence of co-morbidities. Our data do not support broad-spectrum empiric antibiotic therapy for HCAP.