Selective Replacement of Cholesterol with Cationic Amphiphilic Drugs Enables the Design of Lipid Nanoparticles with Improved RNA Delivery.

The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

Effect of malate on the activity of ciprofloxacin against Pseudomonas aeruginosa in different in vivo and in vivo-like infection models.

The clinical significance of Pseudomonas aeruginosa infections and the tolerance of this opportunistic pathogen to antibiotic therapy makes the development of novel antimicrobial strategies an urgent need. We previously found that D,L-malic acid potentiates the activity of ciprofloxacin against P. aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium by increasing metabolic activity and tricarboxylic acid cycle activity. This suggested a potential new strategy to improve antibiotic therapy in P. aeruginosa infections. Considering the importance of the microenvironment on microbial antibiotic susceptibility, the present study aims to further investigate the effect of D,L-malate on ciprofloxacin activity against P. aeruginosa in physiologically relevant infection models, aiming to mimic the infection environment more closely. We used Caenorhabditis elegans nematodes, Galleria mellonella larvae, and a 3-D lung epithelial cell model to assess the effect of D,L-malate on ciprofloxacin activity against P. aeruginosa. D,L-malate was able to significantly enhance ciprofloxacin activity against P. aeruginosa in both G. mellonella larvae and the 3-D lung epithelial cell model. In addition, ciprofloxacin combined with D,L-malate significantly improved the survival of infected 3-D cells compared to ciprofloxacin alone. No significant effect of D,L-malate on ciprofloxacin activity against P. aeruginosa in C. elegans nematodes was observed. Overall, these data indicate that the outcome of the experiment is influenced by the model system used which emphasizes the importance of using models that reflect the in vivo environment as closely as possible. Nevertheless, this study confirms the potential of D,L-malate to enhance ciprofloxacin activity against P. aeruginosa-associated infections.

R othia mucilaginosa is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease.

BACKGROUND: Chronic airway inflammation is the main driver of pathogenesis in respiratory diseases such as severe asthma, chronic obstructive pulmonary disease, cystic fibrosis (CF) and bronchiectasis. While the role of common pathogens in airway inflammation is widely recognised, the influence of other microbiota members is still poorly understood. METHODS: We hypothesised that the lung microbiota contains bacteria with immunomodulatory activity which modulate net levels of immune activation by key respiratory pathogens. Therefore, we assessed the immunomodulatory effect of several members of the lung microbiota frequently reported as present in CF lower respiratory tract samples. RESULTS: We show that Rothia mucilaginosa, a common resident of the oral cavity that is also often detectable in the lower airways in chronic disease, has an inhibitory effect on pathogen- or lipopolysaccharide-induced pro-inflammatory responses, in vitro (three-dimensional cell culture model) and in vivo (mouse model). Furthermore, in a cohort of adults with bronchiectasis, the abundance of Rothia species was negatively correlated with pro-inflammatory markers (interleukin (IL)-8 and IL-1ß) and matrix metalloproteinase (MMP)-1, MMP-8 and MMP-9 in sputum. Mechanistic studies revealed that R. mucilaginosa inhibits NF-κB pathway activation by reducing the phosphorylation of IκBα and consequently the expression of NF-κB target genes. CONCLUSIONS: These findings indicate that the presence of R. mucilaginosa in the lower airways potentially mitigates inflammation, which could in turn influence the severity and progression of chronic respiratory disorders.

The Quorum-Sensing Inhibitor Furanone C-30 Rapidly Loses Its Tobramycin-Potentiating Activity against Pseudomonas aeruginosa Biofilms during Experimental Evolution.

The use of quorum-sensing inhibitors (QSI) has been proposed as an alternative strategy to combat antibiotic resistance. QSI reduce the virulence of a pathogen without killing it and it is claimed that resistance to such compounds is less likely to develop, although there is a lack of experimental data supporting this hypothesis. Additionally, such studies are often carried out in conditions that do not mimic the in vivo situation. In the present study, we evaluated whether a combination of the QSI furanone C-30 and the aminoglycoside antibiotic tobramycin would be "evolution-proof" when used to eradicate Pseudomonas aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium. We found that the biofilm-eradicating activity of the tobramycin/furanone C-30 combination already decreased after 5 treatment cycles. The antimicrobial susceptibility of P. aeruginosa to tobramycin decreased 8-fold after 16 cycles of treatment with the tobramycin/furanone C-30 combination. Furthermore, microcalorimetry revealed changes in the metabolic activity of P. aeruginosa exposed to furanone C-30, tobramycin, and the combination. Whole-genome sequencing analysis of the evolved strains exposed to the combination identified mutations in mexT, fusA1, and parS, genes known to be involved in antibiotic resistance. In P. aeruginosa treated with furanone C-30 alone, a deletion in mexT was also observed. Our data indicate that furanone C-30 is not "evolution-proof" and quickly becomes ineffective as a tobramycin potentiator.

Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics.

Antibiotic susceptibility of bacterial pathogens is typically evaluated using in vitro assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy in vitro and in vivo, with some antibiotics being effective in vitro but not in vivo or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen Pseudomonas aeruginosa, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an in vivo-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against P. aeruginosa, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics in vitro and in vivo may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity.

Model system parameters influence the sodium hypochlorite susceptibility of endodontic biofilms.

AIM: To evaluate in a laboratory setting the influence of several model system parameters on the sodium hypochlorite (NaOCl) susceptibility of endodontic biofilms. Based on these findings, a relevant in vitro endodontic biofilm model is proposed. METHODOLOGY: In vitro biofilms were cultured, varying the following experimental model parameters: biofilm composition (monospecies Enterococcus faecalis and a multispecies biofilm including E. faecalis, Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis), incubation time (24 h or 11 days), incubation atmosphere (aerobically or anaerobically) and biofilm substrate (polystyrene microtiter plate wells, hydroxyapatite or dentine). Biofilms were subjected to treatment with NaOCl (0.025%, 0.1%, 0.5%, 2.5%) for 1 min, control groups included treatment with purified water. Biofilms were harvested and the number of surviving cells was determined by plate counting using general (monospecies biofilms) or selective (multispecies biofilms) media. A two-way ANOVA was used to explore the effect of the model parameters on biofilm eradication. Finally, the most physiologically relevant biofilm model (11-day-old multispecies biofilm grown anaerobically on dentine discs) was characterized by selective media plate counting, NaOCl susceptibility testing, scanning and transmission electron microscopy. RESULTS: There was no difference in NaOCl eradication between the anaerobically and aerobically grown E. faecalis biofilms. One-day-old biofilms of E. faecalis were more susceptible to most tested NaOCl concentrations than 11-day-old biofilms (p < .05). When grown in a multispecies biofilm, E. faecalis was significantly less susceptible to NaOCl treatment than in a monospecies biofilm (p < .05). E. faecalis in a multispecies biofilm grown in a MTP was more susceptible to NaOCl (0.025% and 0.1%) than when grown on hydroxyapatite or dentine. No difference in biofilm NaOCl susceptibility was seen between hydroxyapatite and dentine. The multispecies biofilm proved to be a reproducible model with high NaOCl resistance, complex structure and organization. CONCLUSION: The parameters biofilm age, biofilm composition and substrate had a significant influence on the NaOCl susceptibility of E. faecalis biofilms. Older biofilms, multispecies biofilms and biofilms grown on dentine and hydroxyapatite had reduced NaOCl susceptibility. These findings emphasize the importance of selecting relevant parameters when designing a laboratory biofilm model system for the evaluation of antimicrobial treatments.

Antimicrobial Treatment Provides a Competitive Advantage to Mycobacterium abscessus in a Dual-Species Biofilm with Pseudomonas aeruginosa.

The physiological factors that contribute to Mycobacterium abscessus lung infections remain unclear. We determined whether antibiotic treatment targeting a major cystic fibrosis pathogen (i.e., Pseudomonas aeruginosa) could provide the ideal conditions for the establishment of M. abscessus infection. Our data showed that P. aeruginosa inhibited M. abscessus biofilm formation under control conditions and that antimicrobial therapy selectively targeting P. aeruginosa diminished this competitive interaction, thereby increasing M. abscessus survival.

Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms.

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB+BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms.

Modeling Host-Pathogen Interactions in the Context of the Microenvironment: Three-Dimensional Cell Culture Comes of Age.

Tissues and organs provide the structural and biochemical landscapes upon which microbial pathogens and commensals function to regulate health and disease. While flat two-dimensional (2-D) monolayers composed of a single cell type have provided important insight into understanding host-pathogen interactions and infectious disease mechanisms, these reductionist models lack many essential features present in the native host microenvironment that are known to regulate infection, including three-dimensional (3-D) architecture, multicellular complexity, commensal microbiota, gas exchange and nutrient gradients, and physiologically relevant biomechanical forces (e.g., fluid shear, stretch, compression). A major challenge in tissue engineering for infectious disease research is recreating this dynamic 3-D microenvironment (biological, chemical, and physical/mechanical) to more accurately model the initiation and progression of host-pathogen interactions in the laboratory. Here we review selected 3-D models of human intestinal mucosa, which represent a major portal of entry for infectious pathogens and an important niche for commensal microbiota. We highlight seminal studies that have used these models to interrogate host-pathogen interactions and infectious disease mechanisms, and we present this literature in the appropriate historical context. Models discussed include 3-D organotypic cultures engineered in the rotating wall vessel (RWV) bioreactor, extracellular matrix (ECM)-embedded/organoid models, and organ-on-a-chip (OAC) models. Collectively, these technologies provide a more physiologically relevant and predictive framework for investigating infectious disease mechanisms and antimicrobial therapies at the intersection of the host, microbe, and their local microenvironments.

Decreased susceptibility of Streptococcus anginosus to vancomycin in a multispecies biofilm is due to increased thickness of the cell wall.

Background: Streptococcus anginosus, Pseudomonas aeruginosa and Staphylococcus aureus are often co-isolated from the sputum of cystic fibrosis patients. It was recently shown that S. anginosus is protected from the activity of vancomycin when it grows in a multispecies biofilm with P. aeruginosa and S. aureus. Objectives: Elucidating the underlying cause of the reduced susceptibility of S. anginosus to vancomycin when growing in a multispecies biofilm with P. aeruginosa and S. aureus. Methods: The transcriptome of S. anginosus growing in a multispecies biofilm was compared with that of a S. anginosus monospecies biofilm. Subsequently, transmission electron microscopy was performed to investigate changes in cell wall morphology in S. anginosus and S. aureus in response to growth in multispecies biofilm and to vancomycin treatment. Results: S. anginosus responds to growth in a multispecies biofilm with induction of genes involved in cell envelope biogenesis. Cell walls of S. anginosus cultured in a multispecies biofilm were thicker than in a monospecies biofilm, without antibiotic challenge. S. aureus, when cultured in a multispecies biofilm, does not respond to vancomycin treatment with cell wall thickening. Conclusions: Growth in multispecies biofilms can have an impact on the expression of genes related to cell wall synthesis and on the cell wall thickness of S. anginosus.

Intrapulmonary percussive ventilation improves lung function in cystic fibrosis patients chronically colonized with Pseudomonas aeruginosa: a pilot cross-over study.

High levels of shear stress can prevent and disrupt Pseudomonas aeruginosa biofilm formation in vitro. Intrapulmonary percussive ventilation (IPV) could be used to introduce shear stress into the lungs of cystic fibrosis (CF) patients to disrupt biofilms in vivo. We performed a first-of-its-kind pilot clinical study to evaluate short-term IPV therapy at medium (200 bursts per minute, bpm) and high frequency (400 bpm) as compared to autogenic drainage (AD) on lung function and the behavior of P. aeruginosa in the CF lung in four patients who are chronically colonized by P. aeruginosa. A significant difference between the three treatment groups was observed for both the forced expiratory volume in 1 s (FEV1) and the forced vital capacity (FVC) (p < 0.05). More specifically, IPV at high frequency significantly increased FEV1 and FVC compared to AD (p < 0.05) and IPV at medium frequency (p < 0.001). IPV at high frequency enhanced the expression levels of P. aeruginosa planktonic marker genes, which was less pronounced with IPV at medium frequency or AD. In conclusion, IPV at high frequency could potentially alter the behavior of P. aeruginosa in the CF lung and improve lung function. TRIAL REGISTRATION: The trail was retrospectively registered at the ISRCTN registry on 6 June 2013, under trial registration number ISRCTN75391385.

Community Composition Determines Activity of Antibiotics against Multispecies Biofilms.

In young cystic fibrosis (CF) patients, Staphylococcus aureus is typically the most prevalent organism, while in adults, Pseudomonas aeruginosa is the major pathogen. More recently, it was observed that also Streptococcus anginosus plays an important role in exacerbations of respiratory symptoms. These species are often coisolated from CF lungs, yet little is known about whether antibiotic killing of one species is influenced by the presence of others. In the present study, we compared the activities of various antibiotics against S. anginosus, S. aureus, and P. aeruginosa when grown in monospecies biofilms with the activity observed in a multispecies biofilm. Our results show that differences in antibiotic activity against species grown in mono- and multispecies biofilms are species and antibiotic dependent. Fewer S. anginosus cells are killed by antibiotics that interfere with cell wall synthesis (amoxicillin plus sulbactam, cefepime, imipenem, meropenem, and vancomycin) in the presence of S. aureus and P. aeruginosa, while for ciprofloxacin, levofloxacin, and tobramycin, no difference was observed. In addition, we observed that the cell-free supernatant of S. aureus, but not that of P. aeruginosa biofilms, also caused this decrease in killing. Overall, S. aureus was more affected by antibiotic treatment in a multispecies biofilm, while for P. aeruginosa, no differences were observed between growth in mono- or multispecies biofilms. The results of the present study suggest that it is important to take the community composition into account when evaluating the effect of antimicrobial treatments against certain species in mixed biofilms.

Genome-scale model of Rothia mucilaginosa predicts gene essentialities and reveals metabolic capabilities.

Cystic fibrosis (CF), an inherited genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, results in sticky and thick mucosal fluids. This environment facilitates the colonization of various microorganisms, some of which can cause acute and chronic lung infections, while others may positively impact the disease. Rothia mucilaginosa, an oral commensal, is relatively abundant in the lungs of CF patients. Recent studies have unveiled its anti-inflammatory properties using in vitro three-dimensional lung epithelial cell cultures and in vivo mouse models relevant to chronic lung diseases. Apart from this, R. mucilaginosa has been associated with severe infections. However, its metabolic capabilities and genotype-phenotype relationships remain largely unknown. To gain insights into its cellular metabolism and genetic content, we developed the first manually curated genome-scale metabolic model, iRM23NL. Through growth kinetics and high-throughput phenotypic microarray testings, we defined its complete catabolic phenome. Subsequently, we assessed the model's effectiveness in accurately predicting growth behaviors and utilizing multiple substrates. We used constraint-based modeling techniques to formulate novel hypotheses that could expedite the development of antimicrobial strategies. More specifically, we detected putative essential genes and assessed their effect on metabolism under varying nutritional conditions. These predictions could offer novel potential antimicrobial targets without laborious large-scale screening of knockouts and mutant transposon libraries. Overall, iRM23NL demonstrates a solid capability to predict cellular phenotypes and holds immense potential as a valuable resource for accurate predictions in advancing antimicrobial therapies. Moreover, it can guide metabolic engineering to tailor R. mucilaginosa's metabolism for desired performance.IMPORTANCECystic fibrosis (CF) is a genetic disorder characterized by thick mucosal secretions, leading to chronic lung infections. Rothia mucilaginosa is a common bacterium found in various parts of the human body, acting as a normal part of the flora. In people with weakened immune systems, it can become an opportunistic pathogen, while it is prevalent and active in CF airways. Recent studies have highlighted its anti-inflammatory properties in the lower pulmonary system, indicating the intricate relationship between microbes and human health. Herein, we have developed the first manually curated metabolic model of R. mucilaginosa. Our study examined the previously unknown relationships between the bacterium's genotype and phenotype and identified essential genes that impact the metabolism under various conditions. With this, we opt for paving the way for developing new strategies in antimicrobial therapy and metabolic engineering, leading to enhanced therapeutic outcomes in cystic fibrosis and related conditions.

Combining phages and antibiotic to enhance antibiofilm efficacy against an in vitro dual species wound biofilm.

Chronic wound management is extremely challenging because of the persistence of biofilm-forming pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which are the prevailing bacterial species that co-infect chronic wounds. Phage therapy has gained an increased interest to treat biofilm-associated infections, namely when combined with antibiotics. Here, we tested the effect of gentamicin as a co-adjuvant of phages in a dual species-biofilm wound model formed on artificial dermis. The biofilm-killing capacity of the tested treatments was significantly increased when phages were combined with gentamicin and applied multiple times as multiple dose (three doses, every 8 h). Our results suggest that gentamycin is an effective adjuvant of phage therapy particularly when applied simultaneously with phages and in three consecutive doses. The multiple and simultaneous dose treatment seems to be essential to avoid bacterial resistance development to each of the antimicrobial agents.

High throughput determination of the biofilm prevention concentration for Pseudomonas aeruginosa biofilms using a synthetic cystic fibrosis sputum medium.

The presence of Pseudomonas aeruginosa biofilms in cystic fibrosis (CF) patients suffering from chronic lung infections contributes to the failure of antimicrobial therapy. Conventionally, the minimal inhibitory concentration (MIC) is determined to assess the antimicrobial susceptibility of a pathogen, however this parameter fails to predict success in treating biofilm-associated infections. In the present study we developed a high throughput method to determine the antimicrobial concentration required to prevent P. aeruginosa biofilm formation, using a synthetic cystic fibrosis sputum medium (SCFM2). Biofilms were grown in SCFM2 for 24 h in the presence of antibiotics (tobramycin, ciprofloxacin or colistin), whereafter biofilms were disrupted and a resazurin staining was used to quantify the number of surviving metabolically active cells. In parallel, the content of all wells was plated to determine the number of colony forming units (CFU). Biofilm preventing concentrations (BPCs) were compared to MICs and minimal bactericidal concentrations (MBCs) determined according to EUCAST guidelines. Correlations between the resazurin-derived fluorescence and CFU counts were assessed with Kendall's Tau Rank tests. A significant correlation between fluorescence and CFU counts was observed for 9 out of 10 strains investigated, suggesting the fluorometric assay is a reliable alternative to plating for most P. aeruginosa isolates to determine biofilm susceptibility in relevant conditions. For all isolates a clear difference between MICs and BPCs of all three antibiotics was observed, with the BPCs being consistently higher than the MICs. Additionally, the extent of this difference appeared to be antibiotic-dependent. Our findings suggest that this high throughput assay could be a valuable addition to evaluate the antimicrobial susceptibility in P. aeruginosa biofilms in the context of CF.

The development and characterization of in vivo-like three-dimensional models of bronchial epithelial cell lines.

In vitro models of differentiated respiratory epithelium that allow high-throughput screening are an important tool to explore new therapeutics for chronic respiratory diseases. In the present study, we developed in vivo-like three-dimensional (3-D) models of bronchial epithelial cell lines that are commonly used to study chronic lung disease (16HBE14o-, CFBE41o- and CFBE41o- 6.2 WT-CFTR). To this end, cells were cultured on porous microcarrier beads in the rotating wall vessel (RWV) bioreactor, an optimized suspension culture method that allows higher throughput experimentation than other physiologically relevant models. Cell differentiation was compared to conventional two-dimensional (2-D) monolayer cultures and to the current gold standard in the respiratory field, i.e. air-liquid interface (ALI) cultures. Cellular differentiation was assessed in the three model systems by evaluating the expression and localization of markers that reflect the formation of tight junctions (zonula occludens 1), cell polarity (intercellular adhesion molecule 1 at the apical side and collagen IV expression at the basal cell side), multicellular complexity (acetylated α-tubulin for ciliated cells, CC10 for club cells, keratin-5 for basal cells) and mucus production (MUC5AC) through immunostaining and confocal laser scanning microscopy. Results were validated using Western Blot analysis. We found that tight junctions were expressed in 2-D monolayers, ALI cultures and 3-D models for all three cell lines. All tested bronchial epithelial cell lines showed polarization in ALI and 3-D cultures, but not in 2-D monolayers. Mucus secreting goblet-like cells were present in ALI and 3-D cultures of CFBE41o- and CFBE41o- 6.2 WT-CFTR cells, but not in 16HBE14o- cells. For all cell lines, there were no ciliated cells, basal cells, or club cells found in any of the model systems. In conclusion, we developed RWV-derived 3-D models of commonly used bronchial epithelial cell lines and showed that these models are a valuable alternative to ALI cultures, as they recapitulate similar key aspects of the in vivo parental tissue.