Journal: Journal of medicinal chemistry

Article Title: On the Process of Discovering Leads that Target the Heparin-Binding Site of Neutrophil Elastase in the Sputum of Cystic Fibrosis Patients

doi: 10.1021/acs.jmedchem.9b00379

Figure Lengend Snippet: HNE activity was measured after treatment with varying levels of 32 in the presence of 0.3 mg/mL DNase-1 under both (A) low salt (150 mM NaCl) {##, p<0.009 vs NS control; **, p<0.015 vs 0.3 mg/ml DNase-1} and (B) high salt conditions (400 mM NaCl) {##, p<0.002 vs HS control; **, p<0.087 vs 0.3 mg/ml DNase-1}. Note the difference in basal HNE activity (several orders of magnitude) of sputum with addition of salt and DNase-1. NS or HS represents HNE activity in the absence of both DNase-1 and NSGM 32. Graph represents data from sputum samples from 6 patients.

Article Snippet: Low-binding polypropylene 96-well plates for HNE activity assay were from (Greiner Bio One, Monroe, NC.

Techniques: Activity Assay, Control

Journal: Scientific Reports

Article Title: Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections

doi: 10.1038/s41598-019-39726-w

Figure Lengend Snippet: Airway supernatant (ASN) markers of neutrophils activation analyzed by the absence (n = 9–11) or presence (n = 19–21) of a respiratory bacterial co-infection from tracheal aspirate samples obtained within 24 hours of endotracheal intubation (Day 1 samples). ( A ) Human neutrophil elastase (HNE) activity and ( B) myeloperoxidase levels are elevated in children with a respiratory bacterial co-infection compared to those without a bacterial co-infection. There is no significant difference in ( C ) matrix metalloproteinase 9 (MMP-9) or ( D ) lactoferrin protein levels in children with a respiratory bacterial co-infection compared to those without a bacterial co-infection. The central line is the mean value with whiskers representing the standard deviation. Samples were compared using the Mann-Whitney U test. * p < 0.05.

Article Snippet: Human neutrophil elastase (HNE) activity was measured in ASN and plasma according to the supplier’s protocol (Cayman Chemical, Ann Arbor, MI), with a kit-supplied standard curve.

Techniques: Activation Assay, Infection, Activity Assay, Standard Deviation, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections

doi: 10.1038/s41598-019-39726-w

Figure Lengend Snippet: Characterization of cell surface markers of activation by flow cytometry of donor neutrophils from whole blood (WB) (n = 6) and transmigrated through the small-airways model for 14 hours towards airway supernatant (TM ASN) (n = 6–9). ( A ) CD66b, ( B ) CD63, ( C ) CD16, ( D ) CD181, ( E ) HNE, ( F ) CD88, ( G ) HLA-DR, ( H ) CXCR4 (CD184), ( I ) Arg-1, ( J ) PD-L1. Box plots depict median values, the box edges are the 25 th to 75 th interquartile ranges (IQR), and the whiskers are the 5−95% confidence intervals. Samples were compared using the Mann-Whitney U test. * p < 0.05.

Article Snippet: Human neutrophil elastase (HNE) activity was measured in ASN and plasma according to the supplier’s protocol (Cayman Chemical, Ann Arbor, MI), with a kit-supplied standard curve.

Techniques: Activation Assay, Flow Cytometry, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections

doi: 10.1038/s41598-019-39726-w

Figure Lengend Snippet: Transmigration through the airway model primes healthy donor neutrophils to activate the respiratory burst when stimulated by N- formylmethionyl-leucine-phenylalanine (fMLF) as measured by dihydrorhodamine (DHR) flow cytometry assay. ( A ) Representative flow cytometry histograms depicting healthy donor neutrophil DHR response to fMLF without (blue histogram) and with priming by GM-CSF (100 ng/mL; green histogram) for 30 minutes at 37 °C, 5% CO 2 . PMA-stimulated (100 pg/mL) neutrophils serve as a positive control (orange histogram). Unstimulated, DHR loaded neutrophils are shown as a negation control (red histogram). ( B ) Representative flow cytometry data depicting the DHR fluorescence to fMLF stimulus for transmigrated to LTB4 (red histograms), transmigrated to pooled Day 1 ASN (blue histograms) and neutrophils co-incubated with pooled Day 1 ASN (green histograms) for one donor. PMA (magenta histogram) is included as a maximal respiratory burst positive control. ( C ) Change in the DHR response to fMLF stimulation for neutrophils allowed to transmigrate for 14 hours toward LTB4, ASN with no bacterial coinfection (low HNE), or ASN with bacterial coinfection (high HNE) diluted 1:3 in serum-free media. Data are reported as the mean and standard deviation for three donors and analyzed using ANOVA with a post-hoc Tukey test for multiple comparisons. * p < 0.05. ( D ) Representative flow cytometry histogram from one neutrophil donor transmigrated as described in C).

Article Snippet: Human neutrophil elastase (HNE) activity was measured in ASN and plasma according to the supplier’s protocol (Cayman Chemical, Ann Arbor, MI), with a kit-supplied standard curve.

Techniques: Transmigration Assay, Flow Cytometry, Positive Control, Control, Fluorescence, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections

doi: 10.1038/s41598-019-39726-w

Figure Lengend Snippet: Percent bacterial killing of ( A ) Haemophilus influenzae or ( B ) Staphylococcus aureus by neutrophils from 3 donors allowed to transmigrate to LTB4, or ASN with no bacterial coinfection (low HNE) or ASN with bacterial coinfection (high HNE) for 14 hours. Samples were compared using ANOVA with a post-hoc Tukey test for multiple comparisons. * p < 0.05.

Article Snippet: Human neutrophil elastase (HNE) activity was measured in ASN and plasma according to the supplier’s protocol (Cayman Chemical, Ann Arbor, MI), with a kit-supplied standard curve.

Techniques: