Functional characterization of a novel 3D model of the epithelial-mesenchymal trophic unit

2017

Background/Aim: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. Materials and Methods: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). Res…

Polmunary epithelial barrier formation on biodegradable poly-L-lactic-acid (PLLA) membrane

2016

Aims: Investigation of epithelial barrier formation using PLLA membranes for application in bioengineering. Background: The development of functional and biocompatible substitutes for damaged tissue or organs is a major challenge in biomedical engineering. The epithelial barrier plays a central role in tissue homeostasis and immunity preventing damage and contamination of the interstitial tissues. Different in vitro models of the lung and intestinal epithelial barriers have been well characterized, however these tend to use non-biodegradable and/or poorly biocompatible scaffolds. Therefore, there is a need for better supports for epithelial cells for future applications in tissue engineerin…

IL-13 receptor α 2: A regulator of IL-13 and IL-4 signal transduction in primary human fibroblasts

2005

Background IL-13 and IL-4 share many functional properties as a result of their use of a common receptor complex comprising IL-13 receptor α 1 (IL-13Rα1) and IL-4 receptor α (IL-4Rα). The nonsignaling receptor IL-13 receptor α 2 (IL-13Rα2) binds IL-13 with high affinity and specificity and is believed to be a decoy receptor for IL-13. Objective We sought to examine the inhibitory effects of soluble and membrane-bound IL-13Rα2 on IL-13– and IL-4–mediated effects. Methods Primary human fibroblasts were grown from endobronchial biopsy specimens obtained from volunteers. Upregulation of IL-13Rα2 mRNA was measured by means of RT-PCR, and the level of surface expression was measured by means of F…

Chronological expression of Ciliated Bronchial Epithelium 1 during pulmonary development

2009

Ciliated Bronchial Epithelium (CBE) 1 is a novel gene, which is expressed in ciliated cells. As cilia are important during embryogenesis, the present authors characterised the murine homologue of CBE1 (Cbe1) and compared its temporal expression during murine and human lung development. Cbe1 cDNA was cloned and characterised using sequencing, standard PCR and Western blotting. Mouse and human embryonic/fetal lungs (HELs) were harvested for mRNA analysis and protein localisation in vivo and in vitro using RT-PCR and immunohistochemistry. The Cbe1 amino acid sequence was >75% identical with CBE1 and its alternative splicing and tissue distribution were highly conserved. Pulmonary expression of…

Cigarette smoke causes caspase-independent apoptosis of bronchial epithelial cells from asthmatic donors

2015

BackgroundEpidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.ObjectivesSince primary bronchial epithelial cells (PBECs) from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.MethodsPBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE); cell survival and apoptosis were assessed by fl…

Medium-term Culture of Normal Human Oral Mucosa: A Novel Three-dimensional Model to Study the Effectiveness of Drugs Administration

2012

Tissue-engineered oral mucosal equivalents have been developed for in vitro studies for a few years now. However, the usefulness of currently available models is still limited by many factors, mainly the lack of a physiological extracellular matrix (ECM) and the use of cell populations that do not reflect the properly differentiated cytotypes of the mucosa of the oral cavity. For this reason, we have developed a novel three-dimensional culture model reflecting the normal architecture of the human oral mucosa, with the main aim of creating a better in vitro model where to test cellular responses to drugs administration. This novel 3D cell culture model (3D outgrowth) was set up using an arti…

Establishment of a pulmonary epithelial barrier on biodegradable poly-L-lactic-acid membranes

2019

Development of biocompatible and functional scaffolds for tissue engineering is a major challenge, especially for development of polarised epithelia that are critical structures in tissue homeostasis. Different in vitro models of the lung epithelial barrier have been characterized using non-degradable polyethylene terephthalate membranes which limits their uses for tissue engineering. Although poly-L-lactic acid (PLLA) membranes are biodegradable, those prepared via conventional Diffusion Induced Phase Separation (DIPS) lack open-porous geometry and show limited permeability compromising their use for epithelial barrier studies. Here we used PLLA membranes prepared via a modification of the…

Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics

2015

The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epith…

Asthmatic Bronchial Epithelium Is More Susceptible to Oxidant-Induced Apoptosis

2002

Abnormal apoptotic mechanisms are associated with disease pathogenesis. Because the asthmatic bronchial epithelium is characteristically damaged with loss of columnar epithelial cells, we postulated that this is due to unscheduled apoptosis. Using an antibody directed toward the caspase cleavage product of poly(ADP-ribose) polymerase, immunohistochemistry applied to endobronchial biopsies showed higher levels of staining in the bronchial epithelium of subjects with asthma as compared with normal control subjects (% epithelial staining [median (range) = 10.5 (1.4-24.5) versus 0.4 (0.0-9.7)]; P < 0.001). Because we were unable to determine whether this difference was due to ongoing inflammati…

Induction of a disintegrin and metalloprotease 33 during embryonic lung development and the influence of IL-13 or maternal allergy.

2009

Background Asthma pathogenesis involves gene and environmental interactions. A disintegrin and metalloprotease 33 (ADAM33)/Adam33 is a susceptibility gene for asthma and bronchial hyperresponsiveness in human beings and mice. ADAM33 is almost exclusively expressed in mesenchymal cells, including mesenchymal progenitors in developing lungs. Objective Because maternal allergy is a risk factor for asthma, we hypothesized that an allergic environment affects ADAM33/Adam33 expression during human and mouse lung development. Methods Human embryonic/fetal lung (HEL) tissues were collected from first-trimester terminations of pregnancy. These were processed immediately or used for explant culture ±…

Epithelial-mesenchymal communication in the pathogenesis of chronic asthma.

2005

Although Th-2-mediated inflammation is a key therapeutic target in asthma, its relationship to altered structure and functions of the airways is largely unknown. In addition to inflammation, asthma is a disorder involving the airway epithelium that is more vulnerable to environmental injury and responds to this by impaired healing. This establishes a chronic wound scenario that is capable of sustaining chronic inflammation as well as remodeling. This response occurs as a consequence of activation of the epithelial-mesenchymal unit, involving reciprocal activities of growth factors belonging to the fibroblast growth factor, epidermal growth factor, and transforming growth factor-beta familie…

Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Pheno…

2016

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 'alveolar' cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII ce…

Exposure of bronchial epithelial cells to Cigarette Smoke Extracts (CSE) induces caspase-independent apoptosis.

2007

ADAM 33 in Embryonic Airways.

2005

Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus

2005

Rhinoviruses are the major trigger of acute asthma exacerbations and asthmatic subjects are more susceptible to these infections. To investigate the underlying mechanisms of this increased susceptibility, we examined virus replication and innate responses to rhinovirus (RV)-16 infection of primary bronchial epithelial cells from asthmatic and healthy control subjects.Viral RNA expression and late virus release into supernatant was increased 50- and 7-fold, respectively in asthmatic cells compared with healthy controls. Virus infection induced late cell lysis in asthmatic cells but not in normal cells. Examination of the early cellular response to infection revealed impairment of virus induc…

CD40 ligation protects bronchial epithelium against oxidant-induced caspase-independent cell death.

2006

KEYWORDS CLASSIFICATION: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide;Antigens,CD40;Apoptosis;Bronchi;cytology;Caspases;Cell Cycle;Cell Death;Cell Line,Transformed;Cell Survival;Cell Transformation,Viral;Cytoprotection;drug effects;Epithelial Cells;Humans;Italy;mechanisms of carcinogenesis;metabolism;Oxidants;pharmacology;physiology;Research;Simian virus 40;toxicity;Transcription Factor AP-1. CD40 and its ligand regulate pleiotropic biological responses, including cell proliferation, differentiation, and apoptosis. In many inflammatory lung diseases, tissue damage by environmental or endogenous oxidants plays a major role in disease pathogenesis. As the epithelial barrier is a major t…

IFN-gamma-induced protein 10 is a novel biomarker of rhinovirus-induced asthma exacerbations

2007

BACKGROUND: Rhinovirus-induced acute asthma is the most frequent trigger for asthma exacerbations. OBJECTIVE: We assessed which inflammatory mediators were released from bronchial epithelial cells (BECs) after infection with rhinovirus and then determined whether they were also present in subjects with acute virus-induced asthma, with the aim to identify a biomarker or biomarkers for acute virus-induced asthma. METHODS: BECs were obtained from bronchial brushings of steroid-naive asthmatic subjects and healthy nonatopic control subjects. Cells were infected with rhinovirus 16. Inflammatory mediators were measured by means of flow cytometry with a cytometric bead array. Subjects with acute a…

Medium-Term Culture of Primary Oral Squamous Cell Carcinoma in a Three- Dimensional Model: Effects on Cell Survival Following Topical 5-Fluororacile …

2012

Since the activity of several conventional anticancer drugs is restricted by resistance mechanisms and dose-limiting side-effects, the design of formulations for local application on malignant lesions seems to be an efficient and promising drug delivery approach. In this study, the effect of locally applied 5-FU on cell death was evaluated both in a SCC4/HEK001 model and in a newly proposed 3D outgrowth model of oral squamous cell carcinoma (OSCC). Initially, the optimal drug dose was established by delivery of solutions containing different amounts of 5-FU. The solution containing 1% (w/v) of 5-FU resulted effective in inducing cell death with complete eradication of cell colonies. Buccal …

Effect of IL-13 receptor α2 levels on the biological activity of IL-13 variant R110Q

2007

Background IL-13 is a key cytokine associated with the asthmatic phenotype. IL-13 signals via its cognate receptor, a complex of IL-13 receptor (IL-13R) α 1 chain with IL-4 receptor α; however, a second protein, IL-13Rα2, also binds IL-13. Recently a polymorphic variant of IL-13 (R110Q) has been shown to be associated with atopy. Objective To investigate the binding properties of this IL-13 variant to its cognate receptors. Methods We used surface plasmon resonance to measure the binding kinetics of R110Q to its receptors. Primary human fibroblasts were grown from endobronchial biopsies obtained from volunteers. Receptor levels were measured by fluorescence-activated cell sorting. Results T…

Exposure of bronchial epithelial cells to Cigarette Smoke Extract (CSE) induces caspase-indipendent apoptosis.

2007

Functional characterization of a novel 3D model of the epithelial-mesenchymal trophic unit

2017

Background/Aim: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. Materials and Methods: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). Res…