Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 7. Validation of compound-target interactions through molecular docking and protein-small molecule binding assays. (A) Chemical structure of EGCG and molecular docking models showing its interactions with NOX2 and p47phox. (B) Chemical structure of quercetin and molecular docking models illustrating its interactions with NOX2 and p47phox. (C) An SPR sensorgram displaying the binding affinity of the NOX2-EGCG interaction. (D) An SPR sensorgram showing the binding affinity of the NOX2-quercetin interaction. (E) A BLI sensorgram illustrating the binding affinity of the p47phox-EGCG interaction. (F)A BLI sensorgram depicting the binding affinity of the p47phox-quercetin interaction. Abbreviations: BLI, Bio-layer interferometry; EGCG, epigallocatechin-3-gallate; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; Que, quercetin; SPR, surface plasmon resonance.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: Biomarker Discovery, Binding Assay, SPR Assay

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 8. QKPC and its components, EGCG and quercetin, reduce NOX2-p47phox/ROS signaling in vivo and in vitro. (A) Protein levels of NOX2 and p47phox in human neutrophils were assessed by Western blotting analysis, with relative density quantification shown as the ratio of NOX2 or p47phox to GAPDH (n = 3). (B) Protein levels of NOX2 and p47phox in lung tissues of mice were determined by Western blotting analysis and presented as the ratio of NOX2 or p47phox to GAPDH (n = 3). (C) Cellular ROS levels were measured using the oxidant-sensing probe DCFH-DA, and fluorescence intensity was recorded for each group. EGCG, quercetin, and the positive drugs NAC (20 μM, a ROS scavenger), DPI (50 μM, a general NADPH oxidase inhibitor), and GSK (50 μM, a NOX2-specific inhibitor) effectively suppressed ROS levels elevated by the combined CSE and LPS exposure (n = 6–8). (D) MDA levels in lung tissues were measured with an MDA assay kit. Data are presented as mean ± SD, with statistical significance set at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: CS, cigarette smoke; CSE, cigarette smoke extract; DPI, Diphenyleneiodonium chloride; EGCG, epigallocatechin-3-gallate; GSK, GSK2795039; LPS, lipopolysaccharide; MDA, malondialdehyde; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; Que, quercetin; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: In Vivo, In Vitro, Western Blot, Fluorescence, Multiple Displacement Amplification

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 9. Proposed mechanisms of QKPC action against ECOPD. Abbreviations: COPD, chronic obstructive pulmonary disease; EGCG, epigallocatechin-3-gallate; LPS, lipopolysaccharide; MPO, myeloperoxidase; NE, neutrophil Elastase; NET, neutrophil extracellular trap; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

doi: 10.1084/jem.20082242

Figure Lengend Snippet: KLK5 up-regulates TSLP and proinflammatory molecules in keratinocytes. (A) NHKs were stimulated with 400 nM of recombinant KLK5 for 3 h. TSLP , ICAM1 , IL8 , and TNF -α transcripts are increased by seven-, five-, four-, and fivefold, respectively. No difference was observed for IL-1β , TARC , and MDC ( n = 5). (B) NHK transfected with 60 pmol PAR2 siRNA and stimulated with KLK5 showed PAR2 mRNA down-regulation (by 56%) 4 d after transfection. In PAR2 siRNA-transfected cells, KLK5 is less efficient in inducing TSLP , ICAM1 , IL8 , and TNF-α mRNA (48, 55, 60, and 62% of control, respectively; n = 4). (C) In cells treated with 10 µM NF-κB pathway inhibitor (BAY11-7082), KLK5 efficiency to up-regulate TSLP , ICAM1 , IL8 , and TNF-α mRNA is decreased by 81, 70, 59, and 40%, respectively ( n = 3). For each experiment, the mRNAs of interest were measured by quantitative RT-PCR. Each point is the mean of triplicate amplification for at least three independent experiments ( n ). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Cells were transfected with 60 pmol of human PAR2 siRNA or 20 pmol of human KLK5 siRNA (Santa Cruz Biotechnology, Inc.) using the transfection reagent jetSI-ENDO (Ozyme) in serum-free and antibiotic-free EpiLife.

Techniques: Recombinant, Transfection, Control, Quantitative RT-PCR, Amplification

Journal: The Journal of Experimental Medicine

Article Title: Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

doi: 10.1084/jem.20082242

Figure Lengend Snippet: TSLP and proinflammatory molecules are induced by KLK5 in keratinocytes of NS patients. (A) Quantitative RT-PCR on cultured primary keratinocytes reveals that TSLP is significantly overexpressed by fivefold in three NS patients (NSK) compared with three healthy controls (NHK). ICAM1 , IL8 , TNF-α , TARC , and MDC mRNA are overexpressed in two out of three NSKs but not IL-1β mRNA. Each point represents the mean of two independent experiments for each individual. *, P < 0.05. (B) NHK and NSK were treated with 10 µg/ml brefeldin A, and TSLP was detected by immunofluorescence (green). Blue staining is DAPI. Pictures are representative of two independent brefeldin A treatments, each realized on two NHKs and two NSKs. (C and D) NSKs were transfected with KLK5 siRNA and its efficient down-regulation was confirmed by quantitative RT-PCR, Western blotting (laminin 5 used as loading control), and ELISA, 48 and 72 h after transfection. Results are representative of three independent experiments. (E) KLK5 knockdown induces a decrease of TSLP measured by quantitative RT-PCR. ICAM1 , IL8 , and TNF-α mRNAs were less efficiently reduced. Data are the mean ± SD of one experiment, which is representative of three independent experiments. Bars, 49 µm

Article Snippet: Cells were transfected with 60 pmol of human PAR2 siRNA or 20 pmol of human KLK5 siRNA (Santa Cruz Biotechnology, Inc.) using the transfection reagent jetSI-ENDO (Ozyme) in serum-free and antibiotic-free EpiLife.

Techniques: Quantitative RT-PCR, Cell Culture, Immunofluorescence, Staining, Transfection, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: The Journal of Experimental Medicine

Article Title: Kallikrein 5 induces atopic dermatitis–like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome

doi: 10.1084/jem.20082242

Figure Lengend Snippet: KLK5 triggers proinflammatory and proallergic microenvironment independently of external stimuli. Lack of KLK5 inhibition by LEKTI initiates proinflammatory and proallergic cascades independently of environmental factors. In LEKTI-deficient keratinocytes, hyperactive KLK5 directly induces TSLP, IL8, and TNF-α overexpression through PAR2 and NF-κB pathway activation. Unregulated KLK5 also degrades desmosomes at the interface between the GR and the SC leading to defective SC adhesion and IL-1β, IL8, and TNF-α secretion by mechanically stressed keratinocytes. In addition, all these cytokines could induce TARC and MDC chemokines secretion by keratinocytes and dermal fibroblasts. These proinflammatory mediators trigger eosinophilic and mast cell recruitment and activation. TSLP has been reported to activate resident LCs, which migrate to draining lymph nodes and promote the differentiation of naive T cells (Th0) into Th2 cells. Collectively, activated keratinocytes together with eosinophilic and mast cells induce pro-Th2 microenvironment favoring the development of an AD-like phenotype.

Article Snippet: Cells were transfected with 60 pmol of human PAR2 siRNA or 20 pmol of human KLK5 siRNA (Santa Cruz Biotechnology, Inc.) using the transfection reagent jetSI-ENDO (Ozyme) in serum-free and antibiotic-free EpiLife.

Techniques: Inhibition, Over Expression, Activation Assay

Journal:

Article Title: Secretion of parasite-specific immunoglobulin G by purified blood B lymphocytes from immune individuals after in vitro stimulation with recombinant Plasmodium falciparum merozoite surface protein-1 19 antigen

doi: 10.1046/j.1365-2567.1999.00763.x

Figure Lengend Snippet: Cytokine-dependent in vitro secretion by immune blood B lymphocytes of total IgG (a) and parasite-specific IgG (b). The means±SD of ‘n’ (Table 1) individual cultures are shown. Both sγ+/sγ− and sγ− B-cell populations were cultured with or without MSP119 in the presence of anti-CD40 mAb and cytokines as indicated. Antibody levels are indicated as the ratios of OD values from a given set of culture conditions with or without MSP119 (see the Materials and Methods). The dotted line indicates negative or background values (OD ratio = 1).

Article Snippet: In vitro culture of B lymphocytes CD19 + B cells were adjusted to 10 6 cells/ml, and cultured in 48-well plates (Falcon; Becton-Dickinson, Franklin Lakes, NJ), at a final volume of 0·5 ml of Iscove’s Dulbecco’s modified medium (Sigma, St Louis, MO) with 10% fetal calf serum (Hyclone, Logan, UT), and supplemented as previously described, 7 with or without antigen, mitogenic anti-CD40 mAb (clone ‘89’; 10 μg/ml; a gift from Dr J. Banchereau, Schering-Plough, Dardilly, France), and human recombinant cytokines: interleukin-2 (IL-2; a gift from Sanofi, Labège, France); IL-10 (a gift from Dr F. Brière, Schering-Plough, Dardilly, France); IL-6 (a gift from Dr F. Montero-Julian, Immunotech, Marseille, France); IL-1β (Peprotech, London, UK); and IL-4, obtained from Chinese hamster ovary-transfected cell cultures (a gift from Dr T. B. Nutman, National Institute of Allergy and Infectious Diseases, Bethesda, MD).

Techniques: In Vitro, Cell Culture

Journal:

Article Title: Secretion of parasite-specific immunoglobulin G by purified blood B lymphocytes from immune individuals after in vitro stimulation with recombinant Plasmodium falciparum merozoite surface protein-1 19 antigen

doi: 10.1046/j.1365-2567.1999.00763.x

Figure Lengend Snippet: IgG secretion by total (sγ+/sγ−) blood B cells from P. falciparum-immune individuals is increased by exposition to MSP119 in the absence of exogenous cytokines. Histograms represent the means±SD of cultures derived from seven individuals (sγ− B cells) and 15 individuals (sγ+/sγ− B cells). Dots represent single cultures in each group. Open histograms and filled symbols represent total IgG production, and filled histograms and open symbols represent parasite-specific IgG. Antibody levels are indicated as OD ratios.

Article Snippet: In vitro culture of B lymphocytes CD19 + B cells were adjusted to 10 6 cells/ml, and cultured in 48-well plates (Falcon; Becton-Dickinson, Franklin Lakes, NJ), at a final volume of 0·5 ml of Iscove’s Dulbecco’s modified medium (Sigma, St Louis, MO) with 10% fetal calf serum (Hyclone, Logan, UT), and supplemented as previously described, 7 with or without antigen, mitogenic anti-CD40 mAb (clone ‘89’; 10 μg/ml; a gift from Dr J. Banchereau, Schering-Plough, Dardilly, France), and human recombinant cytokines: interleukin-2 (IL-2; a gift from Sanofi, Labège, France); IL-10 (a gift from Dr F. Brière, Schering-Plough, Dardilly, France); IL-6 (a gift from Dr F. Montero-Julian, Immunotech, Marseille, France); IL-1β (Peprotech, London, UK); and IL-4, obtained from Chinese hamster ovary-transfected cell cultures (a gift from Dr T. B. Nutman, National Institute of Allergy and Infectious Diseases, Bethesda, MD).

Techniques: Derivative Assay

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Scanning electron micrographs of P. aeruginosa incubated with cementoin, elafin, pre-elafin/trappin-2 or magainin 2 . P. aeruginosa (~1 × 10 7 in 500 μL) were incubated 2 h with the indicated peptides before being processed for scanning electron microscopy as described in Methods . CNT; control performed in the absence of peptides, PE; pre-elafin/trappin-2, Cem; cementoin, Ela; elafin, Mag; magainin 2. White arrows point to pore-like structures and white arrowheads to ghost bacterial cells.

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques: Incubation, Electron Microscopy, Control

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Depolarization of P. aeruginosa membranes upon incubation with magainin 2, pre-elafin/trappin-2 or derived peptides . Fluorescence emission (arbitrary units) of the probe NPN inserted into the outer membrane ( top panel ) or the probe DiSC 3 inserted into the inner membrane ( bottom panel ) of P. aeruginosa upon addition of the indicated peptides. The controls were performed in phosphate buffer alone. Pre-elafin/trappin-2 and elafin were used at 20 μM, cementoin at 100 μM and magainin 2 at 10 μM. The arrow indicates the time-point for the addition of the various peptides.

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques: Incubation, Derivative Assay, Fluorescence, Membrane

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Confocal microscopy of P. aeruginosa incubated with fluorescein-labeled pre-elafin/trappin-2 . Mid-logarithmic phase cultures of P. aeruginosa were incubated for 1 h at 37°C with fluorescein-labeled pre-elafin/trappin-2 and observed by confocal microscopy at 400 × magnification. From left to right, consecutive 0.2 μm sections of a fluorescent bacterial cell.

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques: Confocal Microscopy, Incubation, Labeling

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Electrophoretic mobility shift assay of plasmid DNA incubated in the absence or presence of pre-elafin/trappin-2, elafin and cementoin . Plasmid pRS426 (100 ng) was incubated with the indicated ratios of peptide/DNA (w/w) for 1 h and then analyzed by agarose gel electrophoresis followed by staining with ethidium bromide. Above are representative gels from an experiment performed in triplicata. M; 1 kb DNA ladder from Invitrogen.

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques: Electrophoretic Mobility Shift Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining

Journal: BMC Microbiology

Article Title: Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa

doi: 10.1186/1471-2180-10-253

Figure Lengend Snippet: Attenuation of P. aeruginosa virulence factors by pre-elafin/trappin-2, elafin and cementoin

Article Snippet: Recombinant human elafin was purchased from AnaSpec (San Jose, CA, USA).

Techniques: