Non‐bronchoscopic sampling and culture of bronchial epithelial cells in children

Doherty, Gerard M.; Christie, Sharon; Skibiński, Grzegorz; Puddicombe, Sarah M.; Warke, T.J.; Courcey, Francine de; Cross, A. L.; Lyons, Jeremy; Ennis, Madeleine; Shields, Michael; Heaney, Liam G.

doi:10.1046/j.1365-2222.2003.01752.x

Cited by 42 publications

(53 citation statements)

References 26 publications

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“…The brush was then withdrawn before removal. PBECs were cultured using methods developed in our laboratory, which resulted in homogenous cultures of basal epithelial cells (6).…”

Section: Subjectsmentioning

confidence: 99%

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A 3-D Well-Differentiated Model of Pediatric Bronchial Epithelium Demonstrates Unstimulated Morphological Differences Between Asthmatic and Nonasthmatic Cells

et al. 2010

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There is a need for reproducible and effective models of pediatric bronchial epithelium to study disease states such as asthma. We aimed to develop, characterize, and differentiate an effective, an efficient, and a reliable three-dimensional model of pediatric bronchial epithelium to test the hypothesis that children with asthma differ in their epithelial morphologic phenotype when compared with nonasthmatic children. Primary cell cultures from both asthmatic and nonasthmatic children were grown and differentiated at the air-liquid interface for 28 d. Tight junction formation, MUC5AC secretion, IL-8, IL-6, prostaglandin E2 production, and the percentage of goblet and ciliated cells in culture were assessed. Well-differentiated, multilayered, columnar epithelium containing both ciliated and goblet cells from asthmatic and nonasthmatic subjects were generated. All cultures demonstrated tight junction formation at the apical surface and exhibited mucus production and secretion. Asthmatic and nonasthmatic cultures secreted similar quantities of IL-8, IL-6, and prostaglandin E2. Cultures developed from asthmatic children contained considerably more goblet cells and fewer ciliated cells compared with those from nonasthmatic children. A well-differentiated model of pediatric epithelium has been developed that will be useful for more in vivo like study of the mechanisms at play during asthma. (Pediatr Res 67: 17-22, 2010)

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“…The brush was then withdrawn before removal. PBECs were cultured using methods developed in our laboratory, which resulted in homogenous cultures of basal epithelial cells (6).…”

Section: Subjectsmentioning

confidence: 99%

“…We have previously described a safe and effective nonbronchoscopic sampling method for obtaining bronchial epithelial cells from pediatric subjects (6). There is limited data on epithelial function in childhood asthma.…”

mentioning

confidence: 99%

A 3-D Well-Differentiated Model of Pediatric Bronchial Epithelium Demonstrates Unstimulated Morphological Differences Between Asthmatic and Nonasthmatic Cells

et al. 2010

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“…They were performed after informed consent and with approval from the Research Ethics Committee of Queen's University of Belfast. Primary bronchial epithelial cells were isolated and cells were cultured as described previously (Doherty et al, 2003). Air-liquid interface cultures for establishment of well-differentiated, mucociliary epithe- lium were produced based on previously described methods (Fulcher et al, 2005;Gray et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus

Ludlow

Rennick

Sarlang

et al. 2009

Journal of General Virology

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The lymphotropic and myelotropic nature of wild-type measles virus (wt-MV) is well recognized, with dendritic cells and lymphocytes expressing the MV receptor CD150 mediating systemic spread of the virus. Infection of respiratory epithelial cells has long been considered crucial for entry of MV into the body. However, the lack of detectable CD150 on these cells raises the issue of their importance in the pathogenesis of measles. This study utilized a combination of in vitro, ex vivo and in vivo model systems to characterize the susceptibility of epithelial cells to wt-MV of proven pathogenicity. Low numbers of MV-infected epithelial cells in close proximity to underlying infected lymphocytes or myeloid cells suggested infection via the basolateral side of the epithelium in the macaque model. In primary cultures of human bronchial epithelial cells, foci of MV-infected cells were only observed following infection via the basolateral cell surface. The extent of infection in primary cells was enhanced both in vitro and in ex vivo cornea rim tissue by disrupting the integrity of the cells prior to the application of virus. This demonstrated that, whilst epithelial cells may not be the primary target cells for wt-MV, areas of epithelium in which tight junctions are disrupted can become infected using high m.o.i. The low numbers of MV-infected epithelial cells observed in vivo in conjunction with the absence of infectious virus release from infected primary cell cultures suggest that epithelial cells have a peripheral role in MV transmission. INTRODUCTIONMeasles is a severe disease contributing to significant morbidity and mortality rates in many parts of the developing world (Grais et al., 2007). It is also of growing concern in some areas of the developed world where the vaccination rate has fallen below the level required to prevent periodic outbreaks of measles (Choi et al., 2008). The disease is characterized by fever and a maculopapular rash, often in conjunction with cough, coryza and/or conjunctivitis, and a generalized immunosuppression. The causative agent, wild-type measles virus (wt-MV) is spread by aerosolized droplets or direct contact (Griffin, 2007). The highly infectious nature of MV not only suggests a very efficient route of entry into the body but also an effective means of dissemination to susceptible individuals following systemic spread of the virus within the body.The tropism of MV in vivo is determined mainly by the distribution of CD150, also known as signalling lymphocyte activation molecule (SLAM), which is the primary cellular receptor for wild-type strains of the virus. The molecule is widely expressed in lymphoid tissues on subsets of B and T lymphocytes, thymocytes, macrophages and dendritic cells (DCs) (Kruse et al., 2001;McQuaid & Cosby, 2002). This is reflected by analysis of MV-infected cell types present at different stages of the disease, which shows that, in all tissues involved, the majority of infected cells are of a lymphoid origin (Hall et al., 1971; Moench et al., 1988). Ano...

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“…PBECs were obtained from healthy children undergoing elective surgery (n = 5) (42). The generation and characterization of WD-PBEC cultures were described previously and are outlined in SI Materials and Methods (21,43).…”

Section: Methodsmentioning

confidence: 99%

In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo

Villenave

Thavagnanam

Sarlang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

166

217

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Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

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Non‐bronchoscopic sampling and culture of bronchial epithelial cells in children

Abstract: We have shown that non-bronchoscopic bronchial brushing is a safe and effective technique for recovering viable bronchial epithelial cells that consistently yield primary cultures. This method will facilitate examination of the role of the epithelium in paediatric disease.

Cited by 42 publications

References 26 publications

A 3-D Well-Differentiated Model of Pediatric Bronchial Epithelium Demonstrates Unstimulated Morphological Differences Between Asthmatic and Nonasthmatic Cells

A 3-D Well-Differentiated Model of Pediatric Bronchial Epithelium Demonstrates Unstimulated Morphological Differences Between Asthmatic and Nonasthmatic Cells

Wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus

In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo

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