New method for rapid and dynamic quantification of elastase activity on sputum neutrophils from patients with cystic fibrosis using flow cytometry

Hagner, Matthias; Frey, Dario L.; Guerra, Matteo; Dittrich, Anna‐Maria; Halls, Victoria S.; Wege, Sabine; Herth, Felix J.F.; Schultz, Carsten; Mall, Marcus

doi:10.1183/13993003.02355-2019

Cited by 6 publications

(5 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is also worth noting that, as has been mentioned, some proteases, including NE, cathepsin G and MMP-12, are not exclusively present as free soluble forms [ 100 , 157 , 158 ], and as such, can be shielded from their inhibitors by remaining membrane-associated or complexed with extracellular molecules [ 91 , 159 ]. Shielded protease localisations are increasingly being recognised in CLDs and assessing these inaccessible forms by, for example, fluorescence resonance energy transfer (FRET)-based assays [ 160 , 161 ], may bring to light a more substantial protease burden than has been appreciated to date, as well as providing more effective targeting strategies for pharmacological inhibitors.…”

Section: Proteases and Mucosal Immunitymentioning

confidence: 99%

Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease

McKelvey

Brown

Ryan

et al. 2021

IJMS

Self Cite

View full text Add to dashboard Cite

Dysregulated protease activity has long been implicated in the pathogenesis of chronic lung diseases and especially in conditions that display mucus obstruction, such as chronic obstructive pulmonary disease, cystic fibrosis, and non-cystic fibrosis bronchiectasis. However, our appreciation of the roles of proteases in various aspects of such diseases continues to grow. Patients with muco-obstructive lung disease experience progressive spirals of inflammation, mucostasis, airway infection and lung function decline. Some therapies exist for the treatment of these symptoms, but they are unable to halt disease progression and patients may benefit from novel adjunct therapies. In this review, we highlight how proteases act as multifunctional enzymes that are vital for normal airway homeostasis but, when their activity becomes immoderate, also directly contribute to airway dysfunction, and impair the processes that could resolve disease. We focus on how proteases regulate the state of mucus at the airway surface, impair mucociliary clearance and ultimately, promote mucostasis. We discuss how, in parallel, proteases are able to promote an inflammatory environment in the airways by mediating proinflammatory signalling, compromising host defence mechanisms and perpetuating their own proteolytic activity causing structural lung damage. Finally, we discuss some possible reasons for the clinical inefficacy of protease inhibitors to date and propose that, especially in a combination therapy approach, proteases represent attractive therapeutic targets for muco-obstructive lung diseases.

show abstract

Section: Proteases and Mucosal Immunitymentioning

confidence: 99%

Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease

McKelvey

Brown

Ryan

et al. 2021

IJMS

Self Cite

View full text Add to dashboard Cite

show abstract

“…This donor/acceptor combination ensured stable readouts and found wide applicability in the past, from flow cytometry to intravital two-photon confocal microscopy. 25,34 After protease addition to a solution containing H-NE and H-CG, respectively, we observed a strong increase in donor fluorescence, indicating cleavage of the reporters and loss of FRET (Figure 1c and Figure S21b). The ratio between the fluorescence signal recorded at the donor and acceptor λ max was used as an indicator of protease activity (Figure S21b, S21c).…”

mentioning

confidence: 98%

“…Remarkably, NET’s multifaceted functional properties originate from the immunological activity of their associated enzymes: ,, neutrophil elastase (NE) and cathepsin G (CG) are neutrophil serine proteases (NSPs), well-known perpetuators of inflammation and promising prognostic biomarkers. The use of protease activity reporters as anti-inflammatory treatment indicators for airway diseases is spreading. − Importantly, once secreted, NE and CG tether to the plasma membrane, where they act undisturbed by antiproteases, as well as to extracellular DNA webs . During NETosis, NE acts intra- and extracellularly.…”

mentioning

confidence: 99%

Protease FRET Reporters Targeting Neutrophil Extracellular Traps

et al. 2020

Self Cite

View full text Add to dashboard Cite

Neutrophil extracellular traps (NETs) consist of DNA released by terminally stimulated neutrophils. They fine-tune inflammation, kill pathogens, activate macrophages, contribute to airway mucus obstruction in cystic fibrosis, and facilitate tumor metastasis after dormancy. Neutrophil proteases such as elastase (NE) and cathepsin G (CG) attach to NETs and contribute to the diverse immune outcome. However, because of the lack of suitable tools, little spatiotemporal information on protease activities on NETs is available in a pathophysiological context to date. Here, we present H-NE and H-CG, two FRET-based reporters armed with a DNA minor groove binder, which monitor DNA-bound NE and CG activity, respectively. The probes revealed that only NE maintains its catalytic ability when localized to DNA. Further, we demonstrated elevated protease activity within the extracellular DNA of sputum from cystic fibrosis patients. Finally, H-NE showed NE activity at single-cell and free DNA resolution within mouse lung slices, a difficult to achieve task with available substrate-based reporters.

show abstract

“…Weber et al, reported the results for 27 patients with TGLP. Accordingly, 14 individuals underwent laryngeal suspension with stitches between the hyoid bone and mandible, and 18 cases underwent palatal prosthesis augmentation [19]. It is reasonable to believe that the palatal augmentation allows the contact between the reconstructive flap and the soft palate, justifying the good swallowing results reported by the author.…”

Section: Figure 4: Different Options For Reconstructive Flap Positioningmentioning

confidence: 97%

Improving the Swallowing in Total Glossectomy With Laryngeal Preservation

Cristalli¹,

Maio²,

Vallati³

et al. 2021

Multidiscip Cancer Investig

View full text Add to dashboard Cite

This study aims to evaluate the parameters that might have an impact on swallowing performance after a total glossectomy with laryngeal preservation (TGLP). Methods: Among 39 patients who underwent surgery with curative intent for squamous cell carcinoma of the tongue, our retrospective analysis focused on 18 patients treated by TGLP and flap reconstruction. The flap was positioned using three points of suspension: mandible (anteriorly), remaining suspensor muscles (cranially), and hyoid bone (inferiorly). Videofluoroscopic swallowing studies were performed after surgery, and the movements of the hyoid bone on the horizontal and vertical plane were measured on a frame-by-frame basis, and the velopharyngeal contact was recorded. Swallowing was studied at consecutive time-intervals using the Gugging Swallowing Screen (GUSS), and patients were categorized into three groups according to their swallowing ability (good, intermediate, and bad). The relationship between categorical and continuous variables and the swallowing ability were investigated using the chi-squared or Fischer exact test and Mann-Whitney test or t-student test respectively. Results: Swallowing ability at 6-8 months was good in 13 patients and intermediate or bad in five patients. Swallowing improved in 1 and 3 patients at 12 and 18 months, respectively. The hyoid bone movement in the y-axis and extension of surgery to the tonsil were statistically associated with swallowing (P=0.002 and P=0.04, respectively). Velopharyngeal contact was obtained in the entire cohort. Conclusions: Flap suspension using three points of attachment, could allow the restoration of an active hyoid movement and the velopharyngeal closure, thereby achieving valid swallowing.

show abstract

New method for rapid and dynamic quantification of elastase activity on sputum neutrophils from patients with cystic fibrosis using flow cytometry

Abstract: Small molecule FRET flow cytometry is a new method that enables rapid and sensitive quantification of surface-bound elastase activity on sputum neutrophils from patients with cystic fibrosis and potentially other neutrophilic airway diseases http://bit.ly/2IegeSB

Cited by 6 publications

References 31 publications

Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease

Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease

Protease FRET Reporters Targeting Neutrophil Extracellular Traps

Improving the Swallowing in Total Glossectomy With Laryngeal Preservation

Contact Info

Product

Resources

About