Journal: PLoS ONE

Article Title: GDF15/MIC1 and MMP9 Cerebrospinal Fluid Levels in Parkinson’s Disease and Lewy Body Dementia

doi: 10.1371/journal.pone.0149349

Figure Lengend Snippet: Demographical, clinical and biochemical data of the included cohorts.

Article Snippet: A mix of 75 μL assay buffer and 25 μL biotinylated goat anti-MMP9 antibody (R&D Systems, cat# BAF911; 50 ng/well, diluted in assay buffer) was then added and incubated for three h at room temperature on a mini orbital shaker.

Techniques:

Journal: PLoS ONE

Article Title: GDF15/MIC1 and MMP9 Cerebrospinal Fluid Levels in Parkinson’s Disease and Lewy Body Dementia

doi: 10.1371/journal.pone.0149349

Figure Lengend Snippet: Correlation coefficients (r 2 ) of CSF GDF15 and MMP9 and demographic, clinical and neurodegenerative parameters.

Article Snippet: A mix of 75 μL assay buffer and 25 μL biotinylated goat anti-MMP9 antibody (R&D Systems, cat# BAF911; 50 ng/well, diluted in assay buffer) was then added and incubated for three h at room temperature on a mini orbital shaker.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The BLT1 Inhibitory Function of α-1 Antitrypsin Augmentation Therapy Disrupts Leukotriene B4 Neutrophil Signaling.

doi: 10.4049/jimmunol.1500038

Figure Lengend Snippet: FIGURE 3. AAT modulates LTB4-induced neutro- phil degranulation. (A–C) Neutrophils (2 3 107cells/ml) isolated from MM healthy controls (n = 4) were stimulated with LTB4 (100 nM) in the absence or presence of increasing concentrations of AAT (1.8– 27.5 mM). Cell-free supernatants were collected at 0, 10, and 20 min and analyzed by Western blotting for MPO, hCAP-18, and MMP-9 as markers of primary, secondary, and tertiary granule release, respectively. (D–F) Neutrophils were isolated from MM controls (open bars) or ZZ-AATD patients (filled bars). Cells remained unstimulated (Con) or were treated with LTB4 in the absence or presence of AAT (27.5 mM). AAT significantly reduced LTB4 induced degranulation of MPO (D) and hCAP-18 (E) (Student t test; n = 4 subjects/group). All measurements are mean 6 SEM.

Article Snippet: Polyvinylidene difluoride membranes were incubated with 1 mg/ml polyclonal rabbit anti-MPO Ab (Abcam, Cambridge, U.K.), 0.2 mg/ml polyclonal goat anti-MMP9 Ab (R&D Systems, Abingdon, U.K.), or 1 mg/ml polyclonal rabbit anti–hCAP-18 Ab (Innovagen, Lund, Sweden).

Techniques: Isolation, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The BLT1 Inhibitory Function of α-1 Antitrypsin Augmentation Therapy Disrupts Leukotriene B4 Neutrophil Signaling.

doi: 10.4049/jimmunol.1500038

Figure Lengend Snippet: FIGURE 6. Purified M-AAT and ZZ-AAT interact with LTB4. (A) Coomassie blue stained gel (left panel) and Western blot analysis of purified M-AAT (right panel), purified ZZ-AAT, and commercially available AAT in lanes 1, 2, and 3, respectively. (B) The triene chromophore structure of LTB4 (solid line) with the addition of M-AAT (broken line) or ZZ-AAT (dotted line). The molar ratio of ligand to protein was 5:1. The following molar absorptivity were calculated; LTB4 alone (38,400 M21 cm21), LTB4–M-AAT (24,800 M21 cm21), and LTB4-ZZ-AAT (20,000 M21 cm21). (C) Area under the curve analysis demonstrate the ability of both MM-AAT and ZZ-AAT to bind LTB4 (p = 0.01 and p = 0.0005, respectively, Student t test; n = 3 independent experiments). (D) Native ZZ-AAT protein (lane 1) and polymerized ZZ-AAT (lane 2) were run under nondenaturing PAGE conditions and visualized using a polyclonal anti-AAT Ab. (E) UV spectra were recorded for LTB4 (solid line) with the addition of ZZ-AAT (broken line) or polymerized ZZ-AAT (dotted line). The molar ratio of ligand to protein was 5:1. The following molar absorptivities were calculated: LTB4 alone (46,000 M21 cm21), LTB4–ZZ-AAT (22,000 –) and LTB4–poly-ZAAT (49,600 M21 cm21). (F) Area under the curve analysis demonstrates the inability of polymerized ZZ-AAT to bind LTB4 (p = NS, Student t test; n = 3 independent experiments). (G) LTB4-coated polystyrene beads (Con) were exposed to ZZ-AAT or polymerized protein (Poly ZZ-AAT) before incubation with 1 mg/ml FITC-labeled goat polyclonal anti-AAT for 1 h at room temperature, and bound protein was measured by flow cytometry. Data are presented as MFU and indicate significantly reduced binding of the polymerized form of ZZ-AAT to LTB4 (p = 0.0005, Student t test; n = 3 independent experiments).

Article Snippet: Polyvinylidene difluoride membranes were incubated with 1 mg/ml polyclonal rabbit anti-MPO Ab (Abcam, Cambridge, U.K.), 0.2 mg/ml polyclonal goat anti-MMP9 Ab (R&D Systems, Abingdon, U.K.), or 1 mg/ml polyclonal rabbit anti–hCAP-18 Ab (Innovagen, Lund, Sweden).

Techniques: Staining, Western Blot, Incubation, Labeling, Cytometry, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The BLT1 Inhibitory Function of α-1 Antitrypsin Augmentation Therapy Disrupts Leukotriene B4 Neutrophil Signaling.

doi: 10.4049/jimmunol.1500038

Figure Lengend Snippet: FIGURE 7. LTB4 and AAT interaction abrogates LTB4 receptor engagement. (A) UV spectra were recorded for LTB4 (solid line) with the addition of AAT (broken line) or BLT1 (dotted line). The molar ratio of ligand to protein was 5:1. The following molar absorptivity were calculated: LTB4 alone (48,000 M21 cm21), LTB4-AAT (25,600 M21 cm21), and LTB4-BLT1 (31,000 M21 cm21). Results are representative of three technical replicates. (B and C) MM neutrophils remained untreated or treated with LTB4 (100 nM) in the presence and absence of AAT (27.5 mM) or U-75302 (1 mM) and were analyzed by flow cytometry employing an FITC-labeled BLT1 Ab. Data are represented as MFU, with untreated cells in green, LTB4 treated (blue), LTB4 and AAT (red), and LTB4 with U-75302 (yellow). Results in MFU demonstrate significantly decreased LTB4/BLT engagement in the presence of AAT (p = 0.001, Student t test; n = 3). (D) Anti-NE activity of AAT alone and in the presence of LTB4 (5:1 lipid/protein ratio) was assessed. LTB4 did not affect the ability of AAT to inhibit NE (n = 3 technical repeats). (E) AAT was incubated with NE in the presence or absence of LTB4 in a molar ratio of 1:0.75:5, respectively. Western blot analysis with a goat polyclonal anti-AAT Ab revealed an AAT/NE complex (band 1), the truncated form of the AAT-NE complex (band 2), uncomplexed AAT (band 3), and the truncated or cleaved form of AAT (band 4) (representative image of n = 4 technical repeats). LTB4 did not impair AAT binding to NE. (F) UV spectra were recorded for LTB4 (solid line) with the addition of AAT (dashed line) or AAT incubated with NE for 5 min (dotted line). The molar ratio of ligand to protein was 5:1. (G) Area under the curve analysis of LTB4 binding to AAT or AAT-NE revealed that the AAT-NE complex cannot interact with LTB4 as determined by the same peak intensity as LTB4 alone (p = NS, Student t test; n = 3 independent experiments).

Article Snippet: Polyvinylidene difluoride membranes were incubated with 1 mg/ml polyclonal rabbit anti-MPO Ab (Abcam, Cambridge, U.K.), 0.2 mg/ml polyclonal goat anti-MMP9 Ab (R&D Systems, Abingdon, U.K.), or 1 mg/ml polyclonal rabbit anti–hCAP-18 Ab (Innovagen, Lund, Sweden).

Techniques: Cytometry, Labeling, Activity Assay, Incubation, Western Blot, Binding Assay