Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells.

BACKGROUND: Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. METHODS: Pediatric pAECs derived from children with CF (pAECCF) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. RESULTS: Data showed that pAECCF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAECCF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. SIGNIFICANCE: The current study demonstrates that the halide assay can be adapted for pediatric pAECCF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations.

Cytological evaluation of tracheal aspirate and broncho-alveolar lavage fluid in comparison to endoscopic assessment of lower airways in horses with recurrent airways obstruction or inflammatory airway disease.

The aim of the present study was to compare the grade of discharge accumulation in the tracheal lumen, area of tracheal bifurcation, main bronchi and the tracheal septum thickness with the cytology of the tracheal aspirate (TA) and broncho-alveolar lavage fluid (BALF) in horses with recurrent airways obstruction and inflammatory airway disease from those horses. This study was conducted on 96 horses with RAO, 139 horses with IAD and 10 control horses. In all the horses, both clinical and endoscopic examinations were performed. During endoscopy, a score of mucus accumulation was estimated in 3/4 lower of the trachea and in the tracheal bifurcation. In addition, thickening of the tracheal septum was also assessed; tracheal aspirates and broncho-alveolar lavage were performed. An estimate of cell percentage was done in TA and BALF samples. In horses suffering from RAO and IAD, there was a positive correlation between the percentage of neutrophils and the accumulation of discharge, and in the IAD group, there was a negative correlation between the percentage of eosinophils and the accumulation of discharge. There was no correlation between tracheal septum thickening and the percentage of neutrophils and/or eosinophils.

Development of an in vitro cytotoxicity model for aerosol exposure using 3D reconstructed human airway tissue; application for assessment of e-cigarette aerosol.

Development of physiologically relevant test methods to analyse potential irritant effects to the respiratory tract caused by e-cigarette aerosols is required. This paper reports the method development and optimisation of an acute in vitro MTT cytotoxicity assay using human 3D reconstructed airway tissues and an aerosol exposure system. The EpiAirway™ tissue is a highly differentiated in vitro human airway culture derived from primary human tracheal/bronchial epithelial cells grown at the air-liquid interface, which can be exposed to aerosols generated by the VITROCELL® smoking robot. Method development was supported by understanding the compatibility of these tissues within the VITROCELL® system, in terms of airflow (L/min), vacuum rate (mL/min) and exposure time. Dosimetry tools (QCM) were used to measure deposited mass, to confirm the provision of e-cigarette aerosol to the tissues. EpiAirway™ tissues were exposed to cigarette smoke and aerosol generated from two commercial e-cigarettes for up to 6 h. Cigarette smoke reduced cell viability in a time dependent manner to 12% at 6 h. E-cigarette aerosol showed no such decrease in cell viability and displayed similar results to that of the untreated air controls. Applicability of the EpiAirway™ model and exposure system was demonstrated, showing little cytotoxicity from e-cigarette aerosol and different aerosol formulations when compared directly with reference cigarette smoke, over the same exposure time.

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells.

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Assessment of airway hyperresponsiveness in murine tracheal rings.

Isolated tracheal rings have often been used to directly measure the contractile output of airway smooth muscle (ASM). Here, we describe the method for excising murine tracheas, mounting tracheal rings in organ baths, and measuring the isometric forces generated by the ASM when stimulated by drug additions or electric field stimulation. The apparatus for the setup and the pathways responsible for stimulation are detailed. Examples of the responses and analyses of two types of ASM stimulation are illustrated: (1) the carbachol concentration-response curve and (2) the frequency-response curve elicited by electric field stimulation.

Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro.

The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.

Morphologic assessment of extracellular matrix scaffolds for patch tracheoplasty in a canine model.

BACKGROUND: The optimal management of benign tracheal stricture remains surgical resection. Resection is not always an option because of the challenges posed by anastomotic tension and a tenuous blood supply. Regenerative medicine approaches, such as extracellular matrix (ECM) scaffold technology, may alleviate some of the limitations to tracheal replacement. ECM scaffolds facilitate site-specific tissue remodeling when used to reconstruct a variety of soft-tissue structures. METHODS: A 1-cm wide x 2-cm-long defect was created in the ventral trachea of 15 dogs and repaired with one of three acellular biologic scaffolds: urinary bladder matrix (UBM), UBM crosslinked with carbodiimide (UBMC), and decellularized tracheal matrix (DTM). The grafts were evaluated periodically using bronchoscopy and by macroscopic and microscopic morphologic examination at either 2 months or 6 months. RESULTS: The UBM, UBMC, and DTM groups showed no evidence of stenosis or tracheomalacia. The UBM, UBMC, and DTM groups all showed deposition of organized collagenous tissue at the site of scaffold placement and an intact epithelial layer. Scattered areas of mucociliary differentiation were present at the edges of the graft site. There was no evidence cartilage observed within the remodeled tissue at 6 months. CONCLUSIONS: ECM scaffolds promote healing of significantly sized tracheal defects without stricture and with some, but not all, of the necessary structures required for tracheal reconstruction, including complete coverage with ciliated epithelium and dense organized collagenous tissue.

Assessment of the pathogenic potential of asbestiform vs. nonasbestiform particulates (cleavage fragments) in in vitro (cell or organ culture) models and bioassays.

Asbestos fibers are highly fibrous silicate fibers that are distinguished by having a large aspect (length to diameter) ratio and are crystallized in an asbestiform habit that causes them to separate into very thin fibers or fibrils. These fibers are distinct from nonasbestiform cleavage fragments and may appear as thick, short fibers which break along cleavage planes without the high strength and flexibility of asbestiform fibers. Since cleavage fragments of respirable dimensions have generally proven nonpathogenic in animal studies, little data exists on assessing well-characterized preparations of cleavage fragments in in vitro models. The available studies show that cleavage fragments are less bioreactive and cytotoxic than asbestiform fibers.

Low molecular weight quaternised chitosan (11): in vitro assessment of absorption enhancing properties.

N-Trimethyl chitosan chloride (TMC; high molecular weight) and N-trimethyl chitosan oligosaccharide (TMO; low molecular weight) with different degrees of quaternisation were synthesised and evaluated for their absorption enhancing properties across mucosal epithelia. These quaternised chitosan derivatives (0.0625% w/v-0.5% w/v) showed a significant decrease in the transepithelial electrical resistance (TEER) of cultured rabbit tracheal epithelial cell monolayers as compared to the control. The degree of quaternisation and concentration of the compounds influenced the extent of the reduction in TEER. Higher degrees of quaternisation and an increase in the concentration of the compound were associated with a more pronounced reduction in the TEER. The TMO derivatives seemed to be more effective in lowering the TEER of tracheal cell monolayers as compared to the TMC polymers. Ciliary beat frequency (CBF) is the main defence mechanism of the respiratory tract and is therefore a useful parameter in evaluating the toxicity of nasally administered drugs and additives. The effect of the synthesised chitosan derivatives on the CBF of human nasal epithelial cells at pH 7.4 was determined by a method based on an analogue contrast enhancement technique. The TMO oligomers exhibited lower inhibition of the CBF of human nasal epithelial cells compared to that of the TMC polymers. It was proposed that this reduced effect on the CBF is due to the lower viscosity and molecular weight of TMO. However, no acute toxicity was found with any of the synthesised chitosan derivatives by means of the CBF tests conducted in this study.

Spatial and temporal traction response in human airway smooth muscle cells.

Tractions that cells exert on their substrates are essential in cell spreading, migration, and contraction. These tractions can be determined by plating the cells on a flexible gel and measuring the deformation of the gel by using fluorescent beads embedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we use the recently developed method of Fourier transform traction cytometry (FTTC). The ICM and FTTC methods are applied to human airway smooth muscle cells during stimulation with the contractile agonist histamine or the relaxing agonist isoproterenol. The overall intensity of the cell contraction (the median traction magnitude, the energy transferred from the cell to the gel, and the net contractile moment) increased after activation with histamine, and decreased after treatment with isoproterenol. Cells exhibited regional differences in the time course of traction during the treatment. Both temporal evolution and magnitude of traction increase induced by histamine varied markedly among different cell protrusions, whereas the nuclear region showed the smallest response. These results suggest that intracellular mediators of cell adhesion and contraction respond to contractile stimuli with different rates and intensities in different regions of the cell.

An autoradiographic assessment of epithelial cell proliferation and post-natal maturation of the tracheal epithelium in infant ferrets.

The tracheal epithelium of infant ferrets undergoes rapid postnatal maturation over the first month of life to achieve the pseudostratified columnar configuration characteristic of the large airways of other mammals. We have used in vivo pulsing with tritiated thymidine ((3)HT) to elicit autoradiographic labeling of cells synthesizing nucleic acids in order to characterize more fully the contribution to development of different cell types comprising the nascent epithelial layer during this period of rapid growth. These studies indicate that two distinct populations of epithelial cells possess proliferative potential and contribute to the establishment of the mature adult epithelial layer. These investigations further confirm the mitotic potential of basal cells during a period of rapid postnatal growth and development of the tracheal epithelial layer. These studies also document the contribution to early airway development by non-ciliated cells, which predominate on the luminal border of the ferret trachea at birth. The temporal and histologic patterns of airway epithelial maturation during post-natal life in the ferret as contained in this study exhibit similarities to those which occur with recovery from injury by infection and irritant exposure in mature airways. Thus, the characterization of epithelial cell compartments having proliferative potential may provide insights into the mechanisms whereby normal airway epithelial organization is established and maintained during development as well as the possible recapitulation of these mechanisms during times of epithelial regeneration following injury.

A re-assessment of the tricellular region of epithelial cell tight junctions in trachea of guinea pig.

The tricellular region of epithelial tight junctions was previously dismissed as a possible avenue of permeability. One reason was that the two parallel vertical fibers, which penetrate the depth of the tight junction, were apparently cross-linked. Another reason was that the tricellular region of the tight junction is deeper than the adjacent bicellular regions. In the course of several freeze-fracture studies of epithelial tight junctions we have made observations which led us to re-assess the tricellular region as an avenue of permeability. We believe that information from ectoplasmic fracture faces is less subject to artifacts and, in ectoplasmic fracture faces of tricellular regions, cross-linking of the vertical furrows has not been observed. In guinea pig tracheal epithelium the tricellular junction is only about 1 micron deep. Following exposure to cigarette smoke, lanthanum ion staining has been observed in some tricellular junctions. It seems that earlier reasons for dismissing tricellular regions of the tight junction as permeability sites may be insufficient and that there is some evidence to support a role in permeability.