Excess Mucin Impairs Subglottic Epithelial Host Defense in Mechanically Ventilated Patients

Powell, Jason; Garnett, James P.; Mather, Michael; Cooles, Faye A H; Nelson, Andrew; Verdon, Bernard; Scott, Jonathan; Jiwa, Kasim; Ruchaud-Sparagano, Marie-Hélène; Cummings, Stephen; Perry, John D.; Wright, Stephen E.; Wilson, Janet A.; Pearson, Jeffrey P.; Ward, Chris; Simpson, A. John

doi:10.1164/rccm.201709-1819oc

Cited by 24 publications

(34 citation statements)

References 46 publications

(23 reference statements)

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“…Exclusion criteria included patients with current or previous head and neck malignancy or with structural subglottic pathology. Subglottic mucosal brushings were obtained under direct microscopic vision in the operating room via a sheathed cytology brush (BC‐202D‐5010, Olympus, Japan), as previously described . The cytology brush was placed in a sterile container containing tissue culture medium (Roswell Park Memorial Institute‐1640, Sigma, Gillingham, UK) for immediate transport to the laboratory on ice.…”

Section: Methodsmentioning

confidence: 99%

“…Epithelial cell culture and passage were performed using methods previously described . In brief, samples were separated from the brush head, pelleted, and then cultured on collagen‐coated flasks in bronchial epithelial growth medium (BEGM) (CC‐4175, Lonza, Basel, Switzerland), supplemented with penicillin/streptomycin 100 U/mL (Sigma, Gillingham, UK), incubated at 37°C in a 5% CO 2 incubator.…”

Section: Methodsmentioning

confidence: 99%

“…Epithelial morphology was confirmed on light microscopy. Comparison of immortalized cells was made with nonimmortalized primary subglottic epithelial cells, which have been extensively characterized by Powell et al…”

Section: Methodsmentioning

confidence: 99%

“…Although rare, subglottic malignancy is nonetheless associated with late presentation and a poor prognosis . The subglottis also has a crucial relationship with the lower airway in terms of infection . This importance is epitomized in intubated and mechanically ventilated patients at high risk of ventilator‐associated pneumonia for whom removal of infected secretions from the subglottis can reduce the incidence of pneumonia …”

Section: Introductionmentioning

confidence: 99%

“…Previous researchers have used animal models or human tissue from other airway locations, such as the nose or lung; however, this is unlikely to fully recapitulate the subglottic airway in humans, which is a distinct respiratory niche . We have previously reported the first description of primary human subglottic epithelial cell culture . Although establishing techniques for the sampling and culture of these primary cells is important, not all researchers will have access to these human samples.…”

Section: Introductionmentioning

confidence: 99%

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Establishment of an immortalized human subglottic epithelial cell line

et al. 2019

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Objective: Translational research into subglottic disease is restricted by the availability of primary human tissue originating from this subsite. Primary epithelial cells are also limited by their inability to survive beyond several divisions in culture outside of the body. Specific subglottic cell lines, useful for in vitro studies, have not yet been described. We therefore demonstrate what we believe to be the first immortalized subglottic epithelial cell line.Methods: Subglottic tissue was derived from a single adult patient's neoplasia-free human subglottic brushing specimen. Cells were immortalized using a lentiviral vector expressing simian virus 40 T antigen. Karyotyping was performed on the transformed cells using single nucleotide polymorphism array comparative genomic hybridization. Transformed cells were phenotypically characterized by light microscopy, immunohistochemistry, and electrophysiology studies.Results: The immortalized subglottic cell line (SG01) was able to divide successfully beyond 20 passages. Karyotyping demonstrated no significant genomic imbalance after immortalization. The cells demonstrated normal epithelial morphology and cytokeratin expression throughout. SG01 cells were also successfully cultured at air-liquid interface (ALI). At ALI cells demonstrated cilia, mucus production, and relevant ion channel expression.Conclusion: The novel SG01 subglottic epithelial cell line has been established. This cell line provides a unique resource for researchers to investigate subglottic diseases, such as subglottic stenosis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Establishment of an immortalized human subglottic epithelial cell line

et al. 2019

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The Microbiome in Acute Lung Injury and ARDS

Kitsios

Franz

McVerry

2022

The Microbiome in Respiratory Disease

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Impact of the Epithelial Lining Fluid Milieu on Amikacin Pharmacodynamics Against Pseudomonas aeruginosa

et al. 2021

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Background Even though nebulised administration of amikacin can achieve high epithelial lining fluid concentrations, this has not translated into improved patient outcomes in clinical trials. One possible reason is that the cellular and chemical composition of the epithelial lining fluid may inhibit amikacin-mediated bacterial killing. Objective The objective of this study was to identify whether the epithelial lining fluid components inhibit amikacin-mediated bacterial killing. Methods Two amikacin-susceptible (minimum inhibitory concentrations of 2 and 8 mg/L) Pseudomonas aeruginosa isolates were exposed in vitro to amikacin concentrations up to 976 mg/L in the presence of an acidic pH, mucin and/or surfactant as a means of simulating the epithelial lining fluid, the site of bacterial infection in pneumonia. Pharmacodynamic modelling was used to describe associations between amikacin concentrations, bacterial killing and emergence of resistance. Results In the presence of broth alone, there was rapid and extensive (> 6 − log 10 ) bacterial killing, with emergence of resistance identified in amikacin concentrations < 976 mg/L. In contrast, the rate and extent of bacterial killing was reduced (≤ 5 − log 10 ) when exposed to an acidic pH and mucin. Surfactant did not appreciably impact the bacterial killing or resistance emergence when compared with broth alone for either isolate. The combination of mucin and an acidic pH further reduced the rate of bacterial killing, with the maximal bacterial killing occurring 24 h following initial exposure compared with approximately 4-8 h for either mucin or an acidic pH alone. Conclusions Our findings indicate that simulating the epithelial lining fluid antagonises amikacin-mediated killing of P. aeruginosa, even at the high concentrations achieved following nebulised administration.

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Excess Mucin Impairs Subglottic Epithelial Host Defense in Mechanically Ventilated Patients

Cited by 24 publications

References 46 publications

Establishment of an immortalized human subglottic epithelial cell line

Establishment of an immortalized human subglottic epithelial cell line

The Microbiome in Acute Lung Injury and ARDS

Impact of the Epithelial Lining Fluid Milieu on Amikacin Pharmacodynamics Against Pseudomonas aeruginosa

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