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255 A Bacterial Serine Protease Inhibitor, Ecotin Inhibits Neutrophil Elastase in Cystic Fibrosis Airway Samples

OBJECTIVES/GOALS: Ecotin is a potent periplasmic serin protease inhibitor that was first described in Escherichia coli (E. coli). Ecotin is known to inhibit a broad range of serine proteases including NE. Therefore, we hypothesized that ecotin can inhibit the NE activity present in CF clinical sputu...

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Autores principales: Fantone, Kayla, Crippen, Clay, Nothaft, Harald, Stecenko, Arlene, Szymanski, Christine, Rada, Balazs
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cambridge University Press 2022
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9209219/
http://dx.doi.org/10.1017/cts.2022.138
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author Fantone, Kayla
Crippen, Clay
Nothaft, Harald
Stecenko, Arlene
Szymanski, Christine
Rada, Balazs
author_facet Fantone, Kayla
Crippen, Clay
Nothaft, Harald
Stecenko, Arlene
Szymanski, Christine
Rada, Balazs
author_sort Fantone, Kayla
collection PubMed
description OBJECTIVES/GOALS: Ecotin is a potent periplasmic serin protease inhibitor that was first described in Escherichia coli (E. coli). Ecotin is known to inhibit a broad range of serine proteases including NE. Therefore, we hypothesized that ecotin can inhibit the NE activity present in CF clinical sputum samples. METHODS/STUDY POPULATION: To investigate the function of ecotin on CF sputum, orthologues from Campylobacter rectus and Campylobacter showae were recombinantly expressed and purified from E. coli to generate C. rectus ecotin, C. showae ecotin, and E. coli ecotin. Ecotin was added to CF sputum supernatant samples at various concentrations and NE enzymatic activity was measured by a fluorometric assay kit. In addition, CF sputum and healthy sputum was added to PMNs isolated from healthy donors for 3.5 hours and NE was measured with immunofluorescence staining. The sputum was washed two times prior to measuring NE released and on the PMNs, and PBMCs was used as a control. RESULTS/ANTICIPATED RESULTS: Our results indicate a clear inhibition of NE activity in CF sputum supernatant. C. showae ecotin showed the greatest inhibitory effect on NE activity in CF sputum supernatant. We also saw that CF sputum supernatant causing healthy PMNs to release more NE suggesting that NE in the CF airway may trigger more NE release from newly incoming PMNs to the lung. Our next steps will be to determine if ecotin can inhibit NE on the PMN surface and released from PMNs. DISCUSSION/SIGNIFICANCE: Neutrophil elastases are produced by PMNs to kill microbial pathogens in the lung, however in CF, the pathogens are unable to be killed by NE and instead causes severe lung damage. PMNs and NE has been shown to be elevated in the sputum of CF patients and there is currently no NE therapeutic inhibitor that has been effective in these patients.
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spelling pubmed-92092192022-07-01 255 A Bacterial Serine Protease Inhibitor, Ecotin Inhibits Neutrophil Elastase in Cystic Fibrosis Airway Samples Fantone, Kayla Crippen, Clay Nothaft, Harald Stecenko, Arlene Szymanski, Christine Rada, Balazs J Clin Transl Sci Valued Approaches OBJECTIVES/GOALS: Ecotin is a potent periplasmic serin protease inhibitor that was first described in Escherichia coli (E. coli). Ecotin is known to inhibit a broad range of serine proteases including NE. Therefore, we hypothesized that ecotin can inhibit the NE activity present in CF clinical sputum samples. METHODS/STUDY POPULATION: To investigate the function of ecotin on CF sputum, orthologues from Campylobacter rectus and Campylobacter showae were recombinantly expressed and purified from E. coli to generate C. rectus ecotin, C. showae ecotin, and E. coli ecotin. Ecotin was added to CF sputum supernatant samples at various concentrations and NE enzymatic activity was measured by a fluorometric assay kit. In addition, CF sputum and healthy sputum was added to PMNs isolated from healthy donors for 3.5 hours and NE was measured with immunofluorescence staining. The sputum was washed two times prior to measuring NE released and on the PMNs, and PBMCs was used as a control. RESULTS/ANTICIPATED RESULTS: Our results indicate a clear inhibition of NE activity in CF sputum supernatant. C. showae ecotin showed the greatest inhibitory effect on NE activity in CF sputum supernatant. We also saw that CF sputum supernatant causing healthy PMNs to release more NE suggesting that NE in the CF airway may trigger more NE release from newly incoming PMNs to the lung. Our next steps will be to determine if ecotin can inhibit NE on the PMN surface and released from PMNs. DISCUSSION/SIGNIFICANCE: Neutrophil elastases are produced by PMNs to kill microbial pathogens in the lung, however in CF, the pathogens are unable to be killed by NE and instead causes severe lung damage. PMNs and NE has been shown to be elevated in the sputum of CF patients and there is currently no NE therapeutic inhibitor that has been effective in these patients. Cambridge University Press 2022-04-19 /pmc/articles/PMC9209219/ http://dx.doi.org/10.1017/cts.2022.138 Text en © The Association for Clinical and Translational Science 2022 https://creativecommons.org/licenses/by-nc-nd/4.0/This is an Open Access article, distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is unaltered and is properly cited. The written permission of Cambridge University Press must be obtained for commercial re-use or in order to create a derivative work.
spellingShingle Valued Approaches
Fantone, Kayla
Crippen, Clay
Nothaft, Harald
Stecenko, Arlene
Szymanski, Christine
Rada, Balazs
255 A Bacterial Serine Protease Inhibitor, Ecotin Inhibits Neutrophil Elastase in Cystic Fibrosis Airway Samples
title 255 A Bacterial Serine Protease Inhibitor, Ecotin Inhibits Neutrophil Elastase in Cystic Fibrosis Airway Samples
title_full 255 A Bacterial Serine Protease Inhibitor, Ecotin Inhibits Neutrophil Elastase in Cystic Fibrosis Airway Samples
title_fullStr 255 A Bacterial Serine Protease Inhibitor, Ecotin Inhibits Neutrophil Elastase in Cystic Fibrosis Airway Samples
title_full_unstemmed 255 A Bacterial Serine Protease Inhibitor, Ecotin Inhibits Neutrophil Elastase in Cystic Fibrosis Airway Samples
title_short 255 A Bacterial Serine Protease Inhibitor, Ecotin Inhibits Neutrophil Elastase in Cystic Fibrosis Airway Samples
title_sort 255 a bacterial serine protease inhibitor, ecotin inhibits neutrophil elastase in cystic fibrosis airway samples
topic Valued Approaches
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9209219/
http://dx.doi.org/10.1017/cts.2022.138
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