A549 Cell Monolayers Research Articles

Evaluation of anti-adenoviral effects of the polyvinyl alcohol iodine ophthalmic solution.

To investigate the virucidal effects of a polyvinyl alcohol iodine, Saniode, against 16 types of human mastadenovirus (HAdV) causing ophthalmic, respiratory, gastrointestinal, urinary, and systemic infections. Laboratory investigation METHODS: Fifty microliters of Saniode were exposed to 10 μL each containing HAdV virus stock solution of 1 × 106copies/μL of HAdV-1, -2, -3, -4, 5, -6, -7, -8, -11, -37, -53, -54, -56, -64, -81, and -85 for 10 s, 30 s, 1 min, and 3 min. After neutralization with 0.5% sodium thiosulfate, the mixture was diluted by ten-fold serial dilution and inoculated into 24 wells containing confluent A549 cell monolayers. Virucidal effects were calculated relative to the positive control on days 7-10 and observed until 30 days post-infection. Saniode satisfied the EN-14476 criterion for virucidal effects (>99.99%) for all HAdV types at all exposure times, including at 10s on days 7 to 10 post-infection. All types of HAdVs that reacted for > 1 min achieved 99.99% reduction, including after 30 days. Saniode displayed virucidal effects against all tested HAdV types. Currently, with no specific medication available for HAdVs in ocular infection, this could be an option to prevent the spread of keratoconjunctivitis.

The role of Pseudomonas aeruginosa DesB in pathogen-host interaction.

Pseudomonas aeruginosa, commonly found in environments, can cause chronic lung disease in immunocompromised patients. In previous study, an aerobic desaturase (DesB) in P. aeruginosa exerted considerable effects on virulence factor production. The objective of this study was to analyze the role of DesB on the virulence traits of P. aeruginosa in the host. For the in vitro experiments, cells and supernatants from wild-type (WT) P. aeruginosa and its desB mutant were collected. The diluted cells were added to the A549 cell monolayer in order to determine cell viability, invasion ability, and/or immune response. For the in vivo experiments, 6-week-old ICR mice were infected with 6-7 log CFU bacterial cells using endotracheal intubation. The ratio of lung weight to body weight and survival rate of each bacterial strain in the lung were measured. The histopathology of lung tissue was also studied. desB mutants exhibited lower cytotoxicity in A549 cells. In addition, more pro-inflammatory cytokines and chemokines were present in desB mutant-treated. In the lungs of mouse model, WT survived longer than desB mutant, and the WT migrated from the lung to the liver and spleen. The results suggest that P. aeruginosa DesB affects the pathogenicity of the organism in the host.

Adherence and Cytotoxicity of Candida spp. to HaCaT and A549 cells.

Candida species are opportunistic fungal pathogens that cause superficial or invasive infections. Recently, the incidence of infection by non-Candida albicans species, especially Candida glabrata, has increased. In this study, we analyzed the adhesion and cytotoxicity of various Candida spp. that are part of the normal human microbiota. C. albicans adheres well to cell culture plates and to cultured cells. C. glabrata selectively adheres to epithelial cells rather than to cell culture plates. Candida parapsilosis insufficiently adheres to confluent monolayers of human lung epithelial A549 and keratinocyte HaCaT cells. We then analyzed the cytotoxicity of C. albicans and C. glabrata, which adhered well to epithelial cells. C. glabrata has been found to cause more damage to A549 cells than to HaCaT cells, suggesting that resident Candida spp. have distinct cytotoxic effects in different tissues. It is important to clarify the properties of Candida spp. as there is evidence that normal microbiota can cause infections. Our data suggest that it is necessary to use appropriate cell lines for characterizing the adherence and cytotoxicity of Candida spp.

Evaluation of permeability alteration and epithelial–mesenchymal transition induced by transforming growth factor-β1 in A549, NCI-H441, and Calu-3 cells: Development of an in vitro model of respiratory epithelial cells in idiopathic pulmonary fibrosis

IntroductionIdiopathic pulmonary fibrosis (IPF) is a lethal lung disease, which is accompanied by changes in lung structure. With regard to treatment, aerosolized drugs administered intrapulmonarily are rapidly distributed into the plasma and do not remain in the lungs due to damage to the alveolar epithelium that occurs from pulmonary fibrosis. In this study, we sought to develop an in vitro model of respiratory epithelial cells in IPF for the evaluation of the intrapulmonary distribution of aerosolized drugs. We investigated transforming growth factor (TGF)-β1-induced epithelial–mesenchymal transition (EMT) and permeability alteration in A549, NCI-H441, and Calu-3 cell monolayers. MethodsAfter TGF-β1 treatment of A549, NCI-H441, and Calu-3 cells, EMT markers including E-cadherin and vimentin and tight junction proteins including claudins-1, -3, and -5 were stained using immunofluorescence methods and detected using immunoblotting methods. Transport experiments were performed using TGF-β1-treated cell monolayers and fluorescein isothiocyanate dextrans (FD; 4.4, 10, and 70kDa). In addition, TGF-β1-induced apoptosis and necrosis were evaluated by flow cytometry using Annexin V and ethidium homodimer III, respectively. ResultsIn NCI-H441 cells, incomplete EMT, destruction of claudins-1 and -3, and enhancement of FD permeability were caused by TGF-β1 treatment. In A549 cells, complete EMT occurred but was not adequate for transport experiments because of low transepithelial electrical resistance. Whereas in Calu-3 cells, no changes were observed. TGF-β1-induced apoptosis and necrosis were not observed in any of the cell lines. DiscussionIncomplete EMT and permeability enhancement were observed in the alveolar epithelium of IPF. Therefore, our results indicate that TGF-β1-treated NCI-H441 cell monolayers may serve as a useful in vitro model of respiratory epithelial cells for IPF.

Improvement of anti-tumor abilities on human non-small cell lung carcinoma by micellization and cross-linking of N-(2-hydroxypropyl) methacrylamide copolymers

Non-small cell lung carcinoma is one of the most frequently occurred cancers with a very high rate of recurrence. Self-assembly N-(2-hydroxypropyl) methacrylamide (HPMA) micelles and cross-linked micelles were developed to improve antitumor ability of linear HPMA copolymer. The characters of HPMA micelles were investigated and compared using human non-small cell lung carcinoma 3-D culture model and nude mice xenograft model. Cross-linked micelles showed highest cytotoxicity on A549 cell monolayers after a short time treatment in vitro. Moreover, both of the two micelles exhibited better in vitro anti-tumor activity on A549 tumor spheroids than linear HPMA conjugates especially the cross-linked micelles. On BALB/c nude mice bearing A549 xenograft tumors, the cross-linked micelles exhibited the greatest tumor accumulation and the best anti-tumor activity due to the highly improved stabilities and the more pronounced enhanced permeability and retention (EPR) effect, which were followed by the non-cross-linked micelles. Meanwhile, neither the two micelles nor the linear HPMA copolymers showed significant toxicity on the main organs of mice while free doxorubicin (DOX) showed obvious cardiac toxicity. All the results suggested that micellization improved the anti-tumor activity of HPMA copolymers on A549 human non-small cell lung carcinoma, furthermore, cross-linked HPMA copolymer micelles with pH-sensitivity and biodegradability showed more excellent anti-tumor activity.

Indirect targeting of IGF receptor signalingin vivoby substrate-selective inhibition of PAPP-A proteolytic activity

The insulin-like growth factor (IGF) signaling pathway is involved in certain human cancers, and the feasibility of directly targeting the IGF receptor has been actively investigated. However, recent evidence from clinical trials suggests that this approach can be problematic. We have developed an alternative strategy to indirectly inhibit the IGF signaling by targeting the metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). PAPP-A associated with the cell surface cleaves IGF binding protein-4 (IGFBP-4), when IGF is bound to IGFBP-4, and thereby increases IGF bioavailability for receptor activation in an autocrine/paracrine manner. We hypothesized that inhibition of PAPP-A would suppress excessive local IGF signaling in tissues where this is caused by increased PAPP-A proteolytic activity. To test this hypothesis, we developed an inhibitory monoclonal antibody, mAb 1/41, which targets a unique substrate-binding exosite of PAPP-A. This inhibitor selectively and specifically inhibits proteolytic cleavage of IGFBP-4 with an inhibitory constant (Ki) of 135 pM. In addition, it inhibited intracellular signaling of the IGF receptor (AKT phosphorylation) in monolayers of A549 cells, an IGF-responsive lung cancer-derived cell line found to express high levels of PAPP-A. We further showed that mAb 1/41 is effective towards PAPP-A bound to cell surfaces, and that it is capable of inhibiting PAPP-A activity in vivo. Using a murine xenograft model of A549 cells, we demonstrated that mAb 1/41 administered intraperitoneally significantly inhibited tumor growth. Analysis of xenograft tumor tissue recovered from treated mice showed penetration of mAb 1/41, reduced IGFBP-4 proteolysis, and reduced AKT phosphorylation. Our study provides proof of concept that IGF signaling can be selectively reduced by targeting a regulatory proteinase that functions extracellularly, upstream of the IGF receptor. PAPP-A targeting thus represents an alternative therapeutic strategy for inhibiting IGF receptor signaling.

Simvastatin prevents proliferation and bone metastases of lung adenocarcinoma in vitro and in vivo

To explore the mechanism about how HMG-CoA reductase (HMGR) inhibitor inhibit proliferation and bone metastases of lung adenocarcinoma in vitro and in vivo. The HMGR inhibitor simvastatin, human lung cancer cell line A549 and Balb/c nude mouse were used in this study. The mice were randomly divided into 2 groups: control group (0.9% NaCl solution, i.v.) and simvastatin group (5mg/kg simvastatin, i.v.). A scratch assay using A549 cell monolayer was also tested. An invasion assay using collagen-coated membrane in trans-wells was applied to evaluate the effect of simvastatin on the metastatic potential of A549 cells in vitro. The expressions of CD44, PUMA, P53, MMP2 and MMP9 were determined by real-time PCR and western blotting; the phosphorylation status of MAPK/ERK signaling parthway was investigated by western blot. . Compared with the control group, the migration of A549 cells in simvastatin-treated group was markedly inhibited (p ≤ 0.01). Untreated A549 cells showed marked invasion, while simvastatin significantly inhibited the invasion of tumor cells (p ≤ 0.001). Incubation of A549 cells with simvastatin significantly reduced the levels of CD44, MMP2 and MMP9 (p <0.01), while significantly increased p53 (p < 0.01). Simvastatin significantly inhibits tumor growth and bone metastasis in lung cancer xenograft mouse model, simvastatin can inhibit the kinase phosphorylation inMAPK/ERK signaling parthway. The HMGR inhibitor simvastatin prevents proliferation and osteolytic bone metastases of lung adenocarcinoma cells in vitro and vivo. Its mechanism may be associated with regulation of CD44, P53, MMP family and inactivation of MAPK/ERK signaling parthway.

Multi-walled carbon nanotube-induced inflammatory response and oxidative stress in a dynamic cell growth environment

BackgroundRapid increase in multi-walled carbon nanotube (MWCNT) production for their industrial and biomedical applications has led to concerns over the effects of MWCNTs on human health and the environment. Both animal and in vitro studies have provided important findings about MWCNT-induced effects on the lung cells or tissues. In vitro studies have provided a considerable amount of fundamental information on MWCNT-induced effects on the specific lung cells. However, the cell culture systems used in those studies were limited by the absence of dynamic nature of lung tissues. We hypothesized that MWCNT-induced cellular responses such as proliferation, inflammation, and oxidative stress under dynamic cell growth environment may differ from those under static cell growth environment.ResultsIn this study, we used a dynamic cell growth condition to mimic mechanically dynamic environment of the lung and characterized interleukin 8 (IL-8), reactive oxygen species (ROS), glutathione (GSH), and cell proliferation for three days following exposure of MWCNTs at different concentrations (5, 10, and 20 μg/ml) to A549 cell monolayer under both static and dynamic cell growth conditions. Our results demonstrated the distinct differences in the levels of inflammatory response and oxidative stress between static and dynamic cell growth conditions.ConclusionsIn conclusion, the dynamic cell growth system used in this study provided important changes in cellular responses that were not found in the static cell growth system and were similar to animal studies. The dynamic cell growth system can be considered as a viable alternative to in vivo test system in combination with existing in vitro static cell growth systems to evaluate the effect of MWCNTs on cellular responses in the respiratory system.

944 Preceding Human Metapneumovirus Infection Increases Adherence of Streptococcus Pneumoniae and Severity of Murine Pneumococcal Pneumonia

Co-infections with respiratory virus and Streptococcus pneumoniae have been frequently reported in epidemiologic studies. Here, we explore how inoculating A549 and Hep-2 cell monolayers with human metapneumovirus (hMPV) affected subsequent...

Elevated intracellular levels of iron in host cells promotes Burkholderia pseudomallei infection

Background: Burkholderia pseudomallei is the causative agent of melioidosis. The disease is endemic in northeast Thailand. Several studies have reported the effect of iron-overloaded environment in promoting bacterial infection. However, little is known about the effect of host cell iron elevation on melioidosis. Objective: We investigated the impact of increased host cell iron levels on B. pseudomallei infection. Methods: HeLa and A549 cell monolayers were supplemented with ferrous sulfate (FeSO 4 ) and ascorbic acid to increase intracellular iron levels. The iron elevated host cells were infected with B. pseudomallei and examined for plaque formation, cell invasion, intracellular survival and multinucleated giant cell (MNGC) formation and compared to bacteria infecting control cells. Results: The ability of B. pseudomallei to form plaques in iron supplemented HeLa cells and to invade iron supplemented A549 cells was significantly higher. Furthermore, the intracellular survival of B. pseudomallei and the ability to induce MNGC formation in iron-supplemented host cells was greater than in infected control cells. Conclusion: Elevation of iron levels in host cells promotes B. pseudomallei infection.

Potassium channels as operators of alveolar epithelial repair

A common feature of inflammatory lung diseases such as acute lung injury and acute respiratory distress syndrome (ARDS) is extensive damage and remodeling of alveolar epithelium. A better understanding of determinants of alveolar regeneration is thus necessary to develop strategies able to restore alveolar integrity. Recent studies have shown that K+ channels (KCh) are key components of cell proliferation and migration processes, necessary for tissues repair. We therefore postulated that KCh play a role in respiratory epithelial repair.Using a wound‐healing assay, we first showed that glibenclamide and clofilium, inhibitors of KATP and KvLQT1 channels, decrease repair rates of A549 and primary alveolar ATII cell monolayers and their inhibitory effects are additive. The role of KvLQT1 and KATP was then confirmed by a molecular approach using specific siRNAs. On the contrary, KvLQT1 and KATP activators significantly stimulate the wound repair. We also evaluated the influence of KCh on cell growth and we showed that clofilium dose‐dependently inhibits alveolar cell growth and induces an accumulation of cells arrested in G0/G1 phase.In conclusion, we demonstrated that KCh, particularly KvLQT1 and KATP, are involved in epithelial repair processes and should be identify as novel targets to promote alveolar repair necessary to ARDS resolution.Project funded by Canadian Institute of Health Research.

A Brazilian lineage of Staphylococcus lugdunensis presenting rough colony morphology may adhere to and invade lung epithelial cells

Staphylococcus lugdunensis is an unusually virulent coagulase-negative species, which causes serious infection similar to S. aureus. We evaluated the expression of virulence factors such as S. lugdunensis synergistic haemolysin (SLUSH), fibrinogen-binding protein (Fbl), biofilm production and biofilm-production-related genes in 23 S. lugdunensis clinical isolates and one type strain that had been previously characterized for their genotypes. In addition, the biofilm composition and the ability of isolates to adhere to and invade human epithelial lung cells were also investigated. The PCR method used detected the presence of slush and intercellular adhesin (ica) virulence genes in all isolates. All isolates produced the Fbl protein and, with the exception of the type strain, all isolates produced the SLUSH haemolysin. Fourteen (60.9 %) isolates produced biofilms. The detachment assay, using sodium metaperiodate or proteolytic enzymes to analyse the biofilm composition, showed protein-mediated biofilms in two representative isolates, one for each colony type (rough and smooth). All strongly biofilm-producing isolates, including three with rough colony morphology, had the same prevalent PFGE pattern. However, among the representative strains tested, only the S. lugdunensis isolate that formed rough colonies was able to adhere to and invade A549 cell monolayers in the same quantities as those observed with S. aureus isolates (P = 1.000). No significant adhesion or invasion was observed for the other isolates in comparison with the S. aureus isolate, independent of biofilm production or clonality. Our results could explain the incredible ability of this pathogen to cause infections that are as aggressive as S. aureus. In addition, the ability of S. lugdunensis to adhere to and invade eukaryotic cells was also noticed for isolates with rough colony morphology, reinforcing the increased virulence in this species.

Competitive inhibition of [14C]‐TEA uptake in A549 cell monolayers by formoterol and salbutamol

Formoterol (For) and salbutamol (Sal), two β2‐receptor agonistic drugs, are positively charged at physiological pH. Here, we investigated, if organic cation/carnitine transporters (OCT/Ns) are involved in the translocation of the drugs in human alveolar epithelial cells (A549). A549 cells were grown to confluent monolayers for 5 days. Investigation of OCT/N‐mediated transport was performed using [14C]‐tetraethylammonium (TEA, 10 μM), [3H]‐acetylcarnitine (5.5 nM, Ac‐Car) and the β2‐agonists (both at 500 μM) in KRB. A549 monolayers were incubated for 30 min (TEA) or 20 min (Ac‐Car) at 37°C. Competitive inhibition studies were also performed in which TEA uptake (0.25 – 2 mM) was studied in the presence and absence of For and Sal for 30 min. The β2‐agonists showed pronounced inhibition of TEA uptake into A549 monolayers. TEA uptake was decreased to 15.4% (For) and 54.5% (Sal) of control, respectively. Neither β2‐receptor agonist had a significant effect on Ac‐Car uptake. Eadie‐Hofstee analysis of TEA uptake revealed linearity and Km values of 858.2 μM and 1050.6 μM in the presence of For and Sal, respectively, which was a 6.5‐fold (For) and 4.5‐fold (Sal) increase compared to control values. No significant change in Vmax was observed. Ki values were 115 μM (For) and 271 μM (Sal). β2‐agonists competitively inhibited TEA uptake via OCTs, but no interaction between the drugs and OCTNs was found.

Nanoparticles attenuate P-glycoprotein/MDR1 function in A549 human alveolar epithelial cells

P-glycoprotein/MDR1 (P-gp) is a well-characterised membrane transporter relevant in drug disposition and multi-drug resistance. In this study, we aimed to investigate how far nanoparticulates impair the function of the P-gp transport system and which particle properties govern these interactions.Expression and function of P-gp was confirmed in A549 cell monolayers. Rhodamine 123 (Rh123) release studies were carried out in the presence of known inhibitors of P-gp function (i.e., cyclosporine A and verapamil), under ATP depletion (NaN3/DOG) and after acute exposure to nanoparticles (NPs) with different surface modifications, ζ-potentials and sizes (plain, carboxylated, and amine- and sulphate-modified). The cytotoxic potential of NPs on A549 monolayers was evaluated by MTT assay. The effects on P-gp protein level, after incubation with NPs, were investigated by Western blot analysis of A549 cell lysate and supernatant.Cellular retention of Rh123 was significantly (P<0.05) increased in the presence of carboxylated (100nm), amine- and sulphate-modified NPs. A slight, but not significant, decrease in Rh123 release was also observed for plain latex and carboxylated (500nm) NPs. The MTT assay demonstrated that most NPs caused only marginal levels of cytotoxicity (78–88% cell viability); the positively charged amine-NPs, however, were considerably more cytotoxic. Western blot showed that NPs did not cause any cell membrane disruption.Our findings suggest that nanomaterials can attenuate membrane transporter function depending on their size and surface properties and hence might influence the disposition of xenobiotics as well as endogenous substrates.

Identification of Burkholderia mallei and Burkholderia pseudomallei adhesins for human respiratory epithelial cells

BackgroundBurkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms.ResultsComparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649) that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells) and A549 (type II pneumocytes), as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures.A second YadA-like gene product highly similar to BoaA (65% identity) was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705). The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to thrive inside J774A.1 murine macrophages, suggesting a possible role for these proteins in survival within professional phagocytic cells.ConclusionsThe boaA and boaB genes specify adhesins that mediate adherence to epithelial cells of the human respiratory tract. The boaA gene product is shared by B. pseudomallei and B. mallei whereas BoaB appears to be a B. pseudomallei-specific adherence factor.

Mechanistic aspects of adenovirus serotype 2 inactivation with free chlorine.

Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery.

Bronchoalveolar lavage fluid from preterm infants with chorioamnionitis inhibits alveolar epithelial repair

BackgroundPreterm infants are highly susceptible to lung injury. While both chorioamnionitis and antenatal steroids induce lung maturation, chorioamnionitis is also associated with adverse lung development. We investigated the ability of bronchoalveolar lavage fluid (BALF) from ventilated preterm infants to restore alveolar epithelial integrity after injury in vitro, depending on whether or not they were exposed to chorioamnionitis or antenatal steroids. For this purpose, a translational model for alveolar epithelial repair was developed and characterised.MethodsBALF was added to mechanically wounded monolayers of A549 cells. Wound closure was quantified over time and compared between preterm infants (gestational age < 32 wks) exposed or not exposed to chorioamnionitis and antenatal steroids (≥ 1 dose). Furthermore, keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) were quantified in BALF, and their ability to induce alveolar epithelial repair was evaluated in the model.ResultsOn day 0/1, BALF from infants exposed to antenatal steroids significantly increased epithelial repair (40.3 ± 35.5 vs. -6.3 ± 75.0% above control/mg protein), while chorioamnionitis decreased wound-healing capacity of BALF (-2.9 ± 87.1 vs. 40.2 ± 36.9% above control/mg protein). BALF from patients with chorioamnionitis contained less KGF (11 (0-27) vs. 0 (0-4) pg/ml) and less detectable VEGF (66 vs. 95%) on day 0. BALF levels of VEGF and KGF correlated with its ability to induce wound repair. Moreover, KGF stimulated epithelial repair dose-dependently, although the low levels in BALF suggest KGF is not a major modulator of BALF-induced wound repair. VEGF also stimulated alveolar epithelial repair, an effect that was blocked by addition of soluble VEGF receptor-1 (sVEGFr1/Flt-1). However, BALF-induced wound repair was not significantly affected by addition of sVEGFr1.ConclusionAntenatal steroids improve the ability of BALF derived from preterm infants to stimulate alveolar epithelial repair in vitro. Conversely, chorioamnionitis is associated with decreased wound-healing capacity of BALF. A definite role for KGF and VEGF in either process could not be established. Decreased ability to induce alveolar epithelial repair after injury may contribute to the association between chorioamnionitis and adverse lung development in mechanically ventilated preterm infants.

Nitric oxide gas phase release in human small airway epithelial cells

BackgroundAsthma is a chronic airway inflammatory disease characterized by an imbalance in both Th1 and Th2 cytokines. Exhaled nitric oxide (NO) is elevated in asthma, and is a potentially useful non-invasive marker of airway inflammation. However, the origin and underlying mechanisms of intersubject variability of exhaled NO are not yet fully understood. We have previously described NO gas phase release from normal human bronchial epithelial cells (NHBEs, tracheal origin). However, smaller airways are the major site of morbidity in asthma. We hypothesized that IL-13 or cytomix (IL-1β, TNF-α, and IFN-γ) stimulation of differentiated small airway epithelial cells (SAECs, generation 10–12) and A549 cells (model cell line of alveolar type II cells) in culture would enhance NO gas phase release.MethodsConfluent monolayers of SAECs and A549 cells were cultured in Transwell plates and SAECs were allowed to differentiate into ciliated and mucus producing cells at an air-liquid interface. The cells were then stimulated with IL-13 (10 ng/mL) or cytomix (10 ng/mL for each cytokine). Gas phase NO release in the headspace air over the cells was measured for 48 hours using a chemiluminescence analyzer.ResultsIn contrast to our previous result in NHBE, baseline NO release from SAECs and A549 is negligible. However, NO release is significantly increased by cytomix (0.51 ± 0.18 and 0.29 ± 0.20 pl.s-1.cm-2, respectively) reaching a peak at approximately 10 hours. iNOS protein expression increases in a consistent pattern both temporally and in magnitude. In contrast, IL-13 only modestly increases NO release in SAECs reaching a peak (0.06 ± 0.03 pl.s-1.cm-2) more slowly (30 to 48 hours), and does not alter NO release in A549 cells.ConclusionWe conclude that the airway epithelium is a probable source of NO in the exhaled breath, and intersubject variability may be due, in part, to variability in the type (Th1 vs Th2) and location (large vs small airway) of inflammation.

Development of plaque assays for adenoviruses 40 and 41

Enteric adenoviruses, important agents of infantile gastroenteritis, are difficult to culture with low titers and limited CPE. Consequently, few plaque assays have been reported and none are used routinely by investigators who may need reproducible quantitative assays for these viruses. CPE in A549 cells (an epithelial lung carcinoma cell line) was induced by isolates of human adenovirus (HAdV) serotypes 40 or 41 that were obtained by prior limited passage in primary cynmolgous monkey kidney (pCMK), human embryonic kidney (HEK), and Graham 293 cells. CPE with HAdV 40 (Dugan strain) and HAdV 41 (Tak strain) inoculated in A549 cells was also observed. Monolayers of A549 cells were inoculated with a low multiplicity of infection (MOI) of the archived stock isolates and harvested at days 10–14 with full CPE. Subsequent passages were harvested in as few as 7 days with 100% CPE to prepare virus stocks for plaque assay. Large individual plaques under agarose overlay were picked prior to staining and clonal stocks prepared. Titers of final stock preparations after six to eight passages in A549 cells were in the range of 5 × 10 7–1 × 10 8 PFU/ml, which provides adequate virus for quantitative recovery studies. The particle to infectivity (P:I) ratios of the early passages of virus stocks were in the range reported previously. The ratio of non-infectious to infectious particles decreased with successive passages of HAdVs 40 and 41 in A549 cells. The specificity of the assay was confirmed by neutralization of plaques with type-specific antisera. Furthermore, sequence analysis of the HAdVs 40 and 41 plaque forming stocks ruled out contamination with any other HAdVs. The plaque assay developed will be useful for evaluation of virus recovery methods from water, food or other environmental matrices, as well as determination of the efficacy of water treatment techniques for inactivation of these viruses.

Transactivation of Src, PDGF receptor, and Akt is involved in IL‐1β‐induced ICAM‐1 expression in A549 cells

In previous study, interleukin-1beta (IL-1beta) has been shown to induce ICAM-1 expression through MAPKs and NF-kappaB in A549 cells. In addition to these pathways, transactivation of non-receptor tyrosine kinase (Src), PDGF receptors (PDGFRs), and phosphatidylinositol 3-kinase (PI3K)/Akt has been implicated in the expression of inflammatory genes. Here, we further investigated whether these different mechanisms participating in IL-1beta-induced ICAM-1 expression in A549 cells. We initially observed that IL-1beta-induced ICAM-1 promoter activity was attenuated by the inhibitors of Src (PP1), PDGFR (AG1296), PI3-K (LY294002 and wortmannin), and Akt (SH-5), revealed by reporter gene assay, Western blotting, and RT-PCR analyses. The involvement of Src and PI3-K/Akt in IL-1beta-induced ICAM-1 expression was significantly attenuated by transfection of A549 cells with dominant negative plasmids of Src, p85 and Akt, respectively. Src, PDGFR, and PI3K/Akt mediated the effects of IL-1beta because pretreatment with PP1, AG1296, and wortmannin also abrogated IL-1beta-stimulated Src, PDGFR, and Akt phosphorylation, respectively. Moreover, pretreatment with p300 inhibitor (curcumin) also blocked ICAM-1 expression. We further confirmed that p300 was associated with ICAM-1 promoter which was dynamically linked to histone H4 acetylation stimulated by IL-1beta, determined by chromatin immunoprecipitation assay. Association of p300 and histone-H4 to ICAM-1 promoter was inhibited by LY294002. Up-regulation of ICAM-1 enhanced the adhesion of neutrophils onto A549 cell monolayer exposed to IL-1beta, which was inhibited by PP1, AG1296, LY294002, wortmannin, and helenalin. These results suggested that Akt phosphorylation mediated through transactivation of Src/PDGFR promotes the transcriptional p300 activity and eventually leads to ICAM-1 expression induced by IL-1beta.