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Abstract: Respiratory sensitizers are considered as substances of higher risk, at the same level as carcinogens, mutagens and toxic chemicals for reproduction. Presently, there is no validated assay for identifying the respiratory sensitizers. Based on a fully differentiated and functional in vitro cell model of the human airway epithelium, MucilAir™, we attempt to develop such assay. To this end, we invented a novel method, using Dextran as carrier, for applying the water insoluble chemicals to the apical surface of the airway epithelia. Using the Dextran carrier method, we successfully tested some reference chemical compounds known to cause respiratory sensitisation in human beings, including MDI, TMA and HCPt. Interestingly, these chemical sensitizers differentially up-regulated the releases of certain cytokines and chemokines involved in allergic responses. We believe that based on MucilAir™ an in vitro assay could be developed for identification and characterization of the respiratory sensitizers. [Copyright &y& Elsevier]

SummaryTo assess whether gap junctions and connexins change during keratinocyte differentiation, we have studied epidermal equivalents obtained in organotypic cultures of keratinocytes from the outer root sheath of human hair follicles. These reconstituted tissues exhibit a number of differentiation and proliferation markers of human epidermis, including gap junctions, connexins, and K6 and Ki67 proteins. Immunostaining and northern blots showed that gap junctions of the epidermal equivalents were made of Cx26 and Cx43. Cx26 was expressed in all keratinocyte layers, throughout the development of the epidermal equivalents. In contrast, Cx43 was initially observed only in the basal layer of keratinocytes and became detectable in the stratum spinosum and granulosum only after the epidermal equivalents had thickened. The levels of Cx26 and its transcript markedly increased as a function of stratification of the epidermal equivalents, whereas those of Cx43 remained almost constant. Microinjection of Lucifer Yellow into individual keratinocytes showed that gap junctions were similarly permeable at all stages of development of the epidermal equivalents. The data show that epidermal equivalents (i) feature a pattern of connexins typical of an actively renewing human interfollicular epidermis, and (ii) provide a model that reproduces the tridimensional organization of intact epidermis and that is amenable for experimentally testing the function of junctional communication between human keratinocytes. [ABSTRACT FROM AUTHOR]

Respiratory viral infections cause mild to severe diseases, such as common cold, bronchiolitis and pneumonia and are associated with substantial burden for society. To test new molecules for shortening, alleviating the diseases or to develop new therapies, relevant human in vitro models are mandatory. MucilAir™, a human standardized air-liquid interface 3D airway epithelial culture holds in vitro specific mechanisms to counter invaders comparable to the in vivo situation, such as mucus production, mucociliary clearance, and secretion of defensive molecules. The objective of this study was to test the relevance of such a model for the discovery and validation of antiviral drugs. Fully differentiated 3D nasal epithelium cultures were inoculated with picornaviruses, a coronavirus and influenza A viruses in the absence or in the presence of reference antiviral drugs. Results showed that, rupintrivir efficiently inhibits the replication of respiratory picornaviruses in a dose dependent manner and prevents the impairment of the mucociliary clearance. Similarly, oseltamivir reduced the replication of influenza A viruses in a dose dependent manner and prevented the impairment of the epithelial barrier function and cytotoxicity until 4 days of infection. In addition we found that Rhinovirus B14, C15 and influenza A(H1N1) induce significant increase of β Defensins 2 and Cathelicidin release with different time course. These results reveal that a large panel of epithelial functions is modified upon viral infection and validate MucilAir™ as a pertinent tool for pre-clinical antiviral drug testing. [ABSTRACT FROM AUTHOR]

We report here the establishment and characterization of an in vitro human small airway model (SmallAir™). The epithelial cells were isolated from the distal lungs by enzymatic digestion. After amplification, the cells were seeded on the microporous membrane of Transwell inserts. Once confluent, the cultures were switched to air-liquid interface. After 3 weeks of culture, the epithelium became fully differentiated, with morphology of columnar epithelium, and a thickness of 10–15 μm. Most significantly, CC-10, a specific marker of Club cells, was highly expressed in SmallAir™. CC-10 was detected by both immune-cytochemistry and Western Blot. As expected, SmallAir™ contained few Muc5-Ac positive cells (goblet cells). In contrast, CC-10 was not detected in MucilAir™, an in vitro model of the human nasal and bronchial epithelial model. Instead, Muc5-Ac was highly expressed in MucilAir™. However, both MucilAir™ and SmallAir™ contain basal cells and ciliated cells, showing cilia beating and mucociliary clearance. Clearly, MucilAir™ and SmallAir™ are two distinct airway epithelial models. [ABSTRACT FROM AUTHOR]

With more than 1 million deaths worldwide every year, lung cancer remains an area of unmet need. Accessible human in vitro 3D tissue models are required to improve preclinical predictivity. OncoCilAir™ is a new in vitro model of Non Small Cell Lung Cancer which combines a reconstituted human airway epithelium, human lung fibroblasts and lung adenocarcinoma cell lines. Remarkably, we found that in this 3D microenvironment tumour cells expand by forming nodules, mimicking a human lung cancer feature. OncoCilAir™ mutated for KRAS and expressing the green fluorescent protein were used to test the antitumour potential of the investigational MEK inhibitors selumetinib and trametinib. As primary endpoint, changes in tumour size were assessed by fluorescence measurements. Tumours showed a reduced growth in response to the MEK inhibitors, but halting the selumetinib dosing resulted in tumour relapse. Importantly, toxicity study on the normal part of the cultures revealed that the airway epithelium integrity was also affected by anticancer drug treatments. These results highlight the possibility to assess simultaneously drug efficacy, drug side-effect and tumour recurrence within a single culture model. OncoCilAir™ heralds a new generation of integrated in vitro tumour models that should be valuable tools for drug development, while reducing animal testing. [ABSTRACT FROM AUTHOR]

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections and inflammation. We previously reported that tumor necrosis factor (TNF)-α decreases gap junction connectivity in cell lines derived from the airway epithelium of non-cystic fibrosis (non-CF) subjects, a mechanism that was defective in cells derived from CF patients, and identified the tyrosine kinase c-Src as a possible bridge between TNF-α and Cx43. To examine whether this modulation also takes place in primary epithelial cells, the functional expression of Cx43 was studied in non-CF and CF airway cells, obtained from surgical polypectomies and turbinectomies, which were grown either on culture dishes or permeable filters. Expression of Cx43 was detected by immunofluorescence on cells grown under both culture conditions. Non-CF and CF airway cells also showed intercellular diffusion of Lucifer Yellow. Dye coupling was rapidly abolished in non-CF cells in the presence of TNF-α, lipopolysaccharide and lysophosphatidic acid, and could be prevented by tyrphostin47, an inhibitor of Src tyrosine kinases. This down-regulation, however, was not detected in CF airway cells. These data indicate that CFTR dysfunction is associated with altered Src signaling, resulting in the persistence of gap junction connectivity in primary and transformed CF airway cells. [ABSTRACT FROM AUTHOR]