Journal: Scientific Reports

Article Title: Phospholipid transfer protein and a lpha-1 antitrypsin regulate Hck kinase activity during neutrophil degranulation

doi: 10.1038/s41598-018-33851-8

Figure Lengend Snippet: Lung extracellular PLTP activity is enhanced in BALF of AAT deficient subjects on AAT augmentation therapy. ( a ) PLTP activity was measured in lung BALF from age-matched PiMM, PiZZ subjects and PiZZ subjects on AAT augmentation therapy. ( b ) PLTP immunoblot from concentrated (10-fold) BALF from PiMM, PiZZ subjects and PiZZ subjects on AAT augmentation therapy. ( c ) BALF cathepsin G activity levels were determined and corrected to urea concentrations. ( d ) BALF from AAT deficient subjects was incubated with AAT isolated from either PiMM or PiZZ plasma for one hour followed by incubation with recombinant PLTP for 24 hours and PLTP immunoblots were performed and ( e ) PLTP activity recorded. ( f ) Antiprotease activity assays were performed to determine the activity of the PiMM and PiZZ forms of AAT isolated from subjects (n = 6/group). Graphs are represented as mean ± S.E.M. *Denotes a p value < 0.05, when comparing both treatments connected by a line, determined by 2-way ANOVA with Tukey’s post hoc test.

Article Snippet: BALF was concentrated utilizing 3,000 NMWL Centriplus filter devices (Millipore) and PLTP immunoblots were performed to observe PLTP degradation utilizing a rabbit anti-human PLTP polyclonal rabbit antibody (Cat. # NB400-106, Novus Biological, Littleton, CO, USA) that recognizes a partial peptide of human PLTP.

Techniques: Activity Assay, Western Blot, Incubation, Isolation, Clinical Proteomics, Recombinant

Journal: Scientific Reports

Article Title: Phospholipid transfer protein and a lpha-1 antitrypsin regulate Hck kinase activity during neutrophil degranulation

doi: 10.1038/s41598-018-33851-8

Figure Lengend Snippet: PLTP reduces release of neutrophil granules and activation in AAT deficient subjects. Neutrophils from PiMM and PiZZ subjects were exposed to LTB 4 ( a , c – e ) or fMLP ( b ) at 37 °C for 30 minutes, following stimulation with vehicle (albumin) or PLTP protein. Cell-free supernatants were collected and ( a,b ) primary (NE by ELISA and cathepsin G by substrate activity assay), ( c ) secondary (hCAP-18 and lactoferrin by immunoblots) and ( d ) tertiary (MMP9 by zymography) granules were evaluated. NE degranulation was recorded in fMLP and PLTP stimulated neutrophils to determine if PLTP prevent degranulation in more than one stimulus. ( b ) The use of equal cell numbers in each reaction is demonstrated by the identical electrophoretic profile of whole cell lysates in the Coomassie blue stained gels (loading control; bottom panel). Densitometry units (DU) of the expression levels of each protein were quantified. ( e ) A reduced cytochrome c assay was used to determine production of O 2 − by neutrophils. N = 14 subjects per group. Each measurement is the mean ± SEM. *Denotes a p value < 0.05, when comparing both treatments connected by a line, by 2-way ANOVA with Tukey’s post hoc test.

Article Snippet: BALF was concentrated utilizing 3,000 NMWL Centriplus filter devices (Millipore) and PLTP immunoblots were performed to observe PLTP degradation utilizing a rabbit anti-human PLTP polyclonal rabbit antibody (Cat. # NB400-106, Novus Biological, Littleton, CO, USA) that recognizes a partial peptide of human PLTP.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Zymography, Staining, Control, Expressing

Journal: Scientific Reports

Article Title: Phospholipid transfer protein and a lpha-1 antitrypsin regulate Hck kinase activity during neutrophil degranulation

doi: 10.1038/s41598-018-33851-8

Figure Lengend Snippet: Loss of PLTP expression enhances neutrophil degranulation and superoxide production. Neutrophils were collected from C57BL/6 and Pltp −/− mice. Neutrophils were exposed to AAT (PiMM: a,c – e and PiZZ: b ) prior to fMLP at 37 °C for 30 minutes. Cell-free supernatants were collected and ( a , b ) primary (NE by ELISA and cathepsin G by substrate activity assay), ( c ) secondary (hCAP-18 and lactoferrin by immunoblots) and ( d ) tertiary (MMP9 by zymography) granules were evaluated. DU of the expression levels of each protein were quantified. ( b ) NE degranulation was recorded in fMLP and PiZZ AAT stimulated neutrophils to determine if PiZZ prevent degranulation. ( e ) A reduced cytochrome c assay was used to determine production of O 2 − by neutrophils. N = 5 animal per group. Each measurement is the mean ± SEM. *Denotes a p value < 0.05, when comparing both treatments connected by a line, determined by ANOVA with Tukey’s post hoc test.

Article Snippet: BALF was concentrated utilizing 3,000 NMWL Centriplus filter devices (Millipore) and PLTP immunoblots were performed to observe PLTP degradation utilizing a rabbit anti-human PLTP polyclonal rabbit antibody (Cat. # NB400-106, Novus Biological, Littleton, CO, USA) that recognizes a partial peptide of human PLTP.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Zymography

Journal: Scientific Reports

Article Title: Phospholipid transfer protein and a lpha-1 antitrypsin regulate Hck kinase activity during neutrophil degranulation

doi: 10.1038/s41598-018-33851-8

Figure Lengend Snippet: Neutrophils from Pltp deficient mice has elevated tyrosine kinase activity of Hck. Neutrophils were collected from C57BL/6 and Pltp −/− mice. Neutrophils were exposed to AAT prior to fMLP at 37 °C for 5 minutes. Immunoblots were performed for ( a ) phosphorylated Src kinase family (Tyr416) and total Hck, Fgr, Lyn, phosphorylated and total p38, and β-Actin. Densitometry was performed for p-p38 and p-Src. ( b ) HCK, Fgr and Lyn were immunoprecipitated and tyrosine kinases activity assays and immunoblots were performed with the IP product. Data is represented as relative activity, corrected to absorbance for the wild type non-treated neutrophil group. N = 6 animal per group. Each measurement is the mean ± SEM. *Denotes a p value < 0.05, when comparing both treatments connected by a line, determined by 2-way ANOVA with Tukey’s post hoc test.

Article Snippet: BALF was concentrated utilizing 3,000 NMWL Centriplus filter devices (Millipore) and PLTP immunoblots were performed to observe PLTP degradation utilizing a rabbit anti-human PLTP polyclonal rabbit antibody (Cat. # NB400-106, Novus Biological, Littleton, CO, USA) that recognizes a partial peptide of human PLTP.

Techniques: Activity Assay, Western Blot, Immunoprecipitation

Journal: Scientific Reports

Article Title: Phospholipid transfer protein and a lpha-1 antitrypsin regulate Hck kinase activity during neutrophil degranulation

doi: 10.1038/s41598-018-33851-8

Figure Lengend Snippet: Chemical inhibition of Src tyrosine kinase activity subdues neutrophil degranulation in Pltp −/− mice. Neutrophils were collected from C57BL/6 and Pltp −/− mice and were exposed to Src inhibitors, saracatinib or dasatinib, prior to fMLP at 37 °C for ( a ) 5 minutes or (b-cu unit3d) 0 minutes. ( b ) Immunoblots were performed for phosphorylated Src kinase family (Tyr416) and total Hck, Fgr, Lyn, phosphorylated and total p38, and β-Actin. Densitometry was performed for p-p38 and p-Src. ( c ) A reduced cytochrome c assay was used to determine production of O 2 − by neutrophils. ( c ) Cell-free supernatants were collected and primary (NE by ELISA and cathepsin G by substrate activity) granules were evaluated. N = 5 animal per group. Each measurement is the mean ± SEM. *Denotes a p value < 0.05, when comparing both treatments connected by a line, determined by 2-way ANOVA with Tukey’s post hoc test.

Article Snippet: BALF was concentrated utilizing 3,000 NMWL Centriplus filter devices (Millipore) and PLTP immunoblots were performed to observe PLTP degradation utilizing a rabbit anti-human PLTP polyclonal rabbit antibody (Cat. # NB400-106, Novus Biological, Littleton, CO, USA) that recognizes a partial peptide of human PLTP.

Techniques: Inhibition, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Phospholipid transfer protein and a lpha-1 antitrypsin regulate Hck kinase activity during neutrophil degranulation

doi: 10.1038/s41598-018-33851-8

Figure Lengend Snippet: Proposed inhibition of neutrophil degranulation by AAT and PLTP. ( a ) Left panel, functional AAT (PiMM) prevents the cleavage of PLTP by proteases. Both AAT and PLTP prevent Src kinase activation to reduce superoxide production and degranulation. Right panel, AAT deficiency (PiZZ) results in reduced AAT circulation and elevated PLTP cleavage in the lungs. Reduced AAT and cleaved PLTP are less effective at minimizing superoxide production and degranulation in neutrophils. ( b ) Stimulation with fMLP or LTB 4 induces a signaling cascade leading to Hck activation and granule release. PLTP expression, AAT stimulation or Src inhibitors reduces Hck activation and degranulation. Fgr is known to regulate p38 activation but Hck could also contribute. Release of granulates further enhance PLTP cleavage and AAT complexes to serine proteases, which could further enhance degranulation.

Article Snippet: BALF was concentrated utilizing 3,000 NMWL Centriplus filter devices (Millipore) and PLTP immunoblots were performed to observe PLTP degradation utilizing a rabbit anti-human PLTP polyclonal rabbit antibody (Cat. # NB400-106, Novus Biological, Littleton, CO, USA) that recognizes a partial peptide of human PLTP.

Techniques: Inhibition, Functional Assay, Activation Assay, Expressing