30 ne inhibitor sivelestat Search Results


ne inhibitor sivelestat  (Ono Pharma)
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Neutrophil elastase (NE) downregulates cytokine gene transcription in macrophages stimulated with TLR agonists. THP-1-derived macrophages were stimulated with HK-Spn D39 or Escherichia coli LPS (100 ng/mL) in the presence or absence of hNE (250–500 mU/mL) and/or SSH (100 µg/mL) for 4 h under serum-free conditions. Real-time PCR was performed to quantify TNF, IL6 , and IL8 mRNA in the macrophages exposed to these stimuli. The relative quantity of these cytokine mRNAs was normalized to the relative quantity of GAPDH mRNA. Data represent the mean ± SD of quadruplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different within the same activation status at P < 0.05. HK-Spn, heat-killed S. pneumoniae ; LPS, lipopolysaccharide; hNE, human neutrophil elastase; SSH, <t>sivelestat</t> sodium hydrate; TLR, toll-like receptor; TNF, tumor necrosis factor.
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Neutrophil elastase (NE) downregulates cytokine gene transcription in macrophages stimulated with TLR agonists. THP-1-derived macrophages were stimulated with HK-Spn D39 or Escherichia coli LPS (100 ng/mL) in the presence or absence of hNE (250–500 mU/mL) and/or SSH (100 µg/mL) for 4 h under serum-free conditions. Real-time PCR was performed to quantify TNF, IL6 , and IL8 mRNA in the macrophages exposed to these stimuli. The relative quantity of these cytokine mRNAs was normalized to the relative quantity of GAPDH mRNA. Data represent the mean ± SD of quadruplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different within the same activation status at P < 0.05. HK-Spn, heat-killed S. pneumoniae ; LPS, lipopolysaccharide; hNE, human neutrophil elastase; SSH, sivelestat sodium hydrate; TLR, toll-like receptor; TNF, tumor necrosis factor.

Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Neutrophil elastase (NE) downregulates cytokine gene transcription in macrophages stimulated with TLR agonists. THP-1-derived macrophages were stimulated with HK-Spn D39 or Escherichia coli LPS (100 ng/mL) in the presence or absence of hNE (250–500 mU/mL) and/or SSH (100 µg/mL) for 4 h under serum-free conditions. Real-time PCR was performed to quantify TNF, IL6 , and IL8 mRNA in the macrophages exposed to these stimuli. The relative quantity of these cytokine mRNAs was normalized to the relative quantity of GAPDH mRNA. Data represent the mean ± SD of quadruplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different within the same activation status at P < 0.05. HK-Spn, heat-killed S. pneumoniae ; LPS, lipopolysaccharide; hNE, human neutrophil elastase; SSH, sivelestat sodium hydrate; TLR, toll-like receptor; TNF, tumor necrosis factor.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Activation Assay

Neutrophil elastase (NE) cleaves TLRs and MD2 on macrophages. (A,B) THP-1-derived macrophages were cultured in serum free RPMI 1640 and exposed to hNE (125–500 mU/mL) for 4 h. (A) Representative fluorescence microscopy images of untreated and hNE-treated (500 mU/mL) macrophages stained for DNA (DAPI; blue) and TLRs (green). (B) TLR2, TLR4, and TLR-related molecules were determined by western blot analysis. (C) Recombinant human (rh) TLR2, rhTLR4, and rhMD2 were exposed to hNE (125–500 mU/mL) in the presence or absence of SSH (100 µg/mL) for 3 h and determined by western blot analysis. MD2, myeloid differentiation factor 2; hNE, human neutrophil elastase; SSH, sivelestat sodium hydrate; TLR, toll-like receptor.

Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Neutrophil elastase (NE) cleaves TLRs and MD2 on macrophages. (A,B) THP-1-derived macrophages were cultured in serum free RPMI 1640 and exposed to hNE (125–500 mU/mL) for 4 h. (A) Representative fluorescence microscopy images of untreated and hNE-treated (500 mU/mL) macrophages stained for DNA (DAPI; blue) and TLRs (green). (B) TLR2, TLR4, and TLR-related molecules were determined by western blot analysis. (C) Recombinant human (rh) TLR2, rhTLR4, and rhMD2 were exposed to hNE (125–500 mU/mL) in the presence or absence of SSH (100 µg/mL) for 3 h and determined by western blot analysis. MD2, myeloid differentiation factor 2; hNE, human neutrophil elastase; SSH, sivelestat sodium hydrate; TLR, toll-like receptor.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Derivative Assay, Cell Culture, Fluorescence, Microscopy, Staining, Western Blot, Recombinant

Neutrophil elastase (NE) degrades TNF, IL-6, and IL-8 (A) . THP-1-derived macrophages were exposed to hNE (125–500 mU/mL) in the presence or absence of HK-Spn or LPS (100 ng/mL) for 4 h. TNF, IL-6, and IL-8 concentrations in the culture supernatants were determined by ELISA. Data represent the mean ± SD of quadruplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different within the same activation status at P < 0.05. (B) rhTNF, rhIL-6, and rhIL-8 were exposed to hNE (125–500 mU/mL) in the presence or absence of SSH (100 µg/mL) for 3 h and determined by western blot analysis. ELISA, enzyme-linked immunosorbent assay; HK-Spn, heat-killed S. pneumoniae ; LPS, lipopolysaccharide; hNE, human neutrophil elastase; rh, recombinant human; SSH, sivelestat sodium hydrate; IL, interleukin; TNF, tumor necrosis factor.

Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Neutrophil elastase (NE) degrades TNF, IL-6, and IL-8 (A) . THP-1-derived macrophages were exposed to hNE (125–500 mU/mL) in the presence or absence of HK-Spn or LPS (100 ng/mL) for 4 h. TNF, IL-6, and IL-8 concentrations in the culture supernatants were determined by ELISA. Data represent the mean ± SD of quadruplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different within the same activation status at P < 0.05. (B) rhTNF, rhIL-6, and rhIL-8 were exposed to hNE (125–500 mU/mL) in the presence or absence of SSH (100 µg/mL) for 3 h and determined by western blot analysis. ELISA, enzyme-linked immunosorbent assay; HK-Spn, heat-killed S. pneumoniae ; LPS, lipopolysaccharide; hNE, human neutrophil elastase; rh, recombinant human; SSH, sivelestat sodium hydrate; IL, interleukin; TNF, tumor necrosis factor.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Recombinant

Multiplex assay reveals that neutrophil elastase (NE) degrades various cytokines. Human cytokine/chemokine cocktail was exposed to hNE (125–500 mU/mL) in the presence or absence of SSH (100 µg/mL) for 3 h. Then, cytokines were detected using a multiplex assay. Data represent the mean ± SD of quadruplicate experiments and were evaluated using two-way ANOVA. *Significantly different from the hNE-untreated control at P < 0.05. † Significantly different from the hNE-treated (500 mU/mL) group in the absence of SSH at P < 0.05. hNE, human neutrophil elastase; SSH, sivelestat sodium hydrate.

Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Multiplex assay reveals that neutrophil elastase (NE) degrades various cytokines. Human cytokine/chemokine cocktail was exposed to hNE (125–500 mU/mL) in the presence or absence of SSH (100 µg/mL) for 3 h. Then, cytokines were detected using a multiplex assay. Data represent the mean ± SD of quadruplicate experiments and were evaluated using two-way ANOVA. *Significantly different from the hNE-untreated control at P < 0.05. † Significantly different from the hNE-treated (500 mU/mL) group in the absence of SSH at P < 0.05. hNE, human neutrophil elastase; SSH, sivelestat sodium hydrate.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Multiplex Assay, Control

Administration of NE inhibitor increases cytokine level and decreases bacterial load in BALF. BALB/c mice (seven mice each) were intratracheally infected with Streptococcus pneumoniae D39 (2 × 10 8 CFU in 50 µL PBS). Unchallenged naive mice (Sham group) were administered PBS only. NE inhibitor (SSH group; 50 mg/kg) or PBS (PBS group) was administrated intraperitoneally to the infected mice every 6 h. (A–C) Groups of animals were sacrificed at 18 h postinfection. (A) BALF samples were plated onto blood-agar plates and cultured aerobically for enumerating recovered CFU. Data represent the mean ± SD and were evaluated using unpaired t tests. *Significantly different from the infected control group at P < 0.05. (B) The levels of TNF and IL-6 in BALF were determined by using ELISA kits. (C) Serum TNF and IL-6 levels were determined. (B,C) Data represent the mean ± SD and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different from the infected control group at P < 0.05. (D) Survival of BALB/c (twenty mice each) mice was monitored following the intratracheal infection with S. pneumoniae (5 × 10 8 CFU) in the presence or absence of intraperitoneal administration of an NE inhibitor. Statistical analysis was performed with log-rank test. *Significantly different from the SSH-untreated control at P < 0.001. BALF, bronchoalveolar lavage fluid; CFUs, colony forming units; ELISA, enzyme-linked immunosorbent assay; NE, neutrophil elastase; PBS, phosphate buffered saline; SSH, sivelestat sodium hydrate; IL, interleukin; TNF, tumor necrosis factor.

Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Administration of NE inhibitor increases cytokine level and decreases bacterial load in BALF. BALB/c mice (seven mice each) were intratracheally infected with Streptococcus pneumoniae D39 (2 × 10 8 CFU in 50 µL PBS). Unchallenged naive mice (Sham group) were administered PBS only. NE inhibitor (SSH group; 50 mg/kg) or PBS (PBS group) was administrated intraperitoneally to the infected mice every 6 h. (A–C) Groups of animals were sacrificed at 18 h postinfection. (A) BALF samples were plated onto blood-agar plates and cultured aerobically for enumerating recovered CFU. Data represent the mean ± SD and were evaluated using unpaired t tests. *Significantly different from the infected control group at P < 0.05. (B) The levels of TNF and IL-6 in BALF were determined by using ELISA kits. (C) Serum TNF and IL-6 levels were determined. (B,C) Data represent the mean ± SD and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different from the infected control group at P < 0.05. (D) Survival of BALB/c (twenty mice each) mice was monitored following the intratracheal infection with S. pneumoniae (5 × 10 8 CFU) in the presence or absence of intraperitoneal administration of an NE inhibitor. Statistical analysis was performed with log-rank test. *Significantly different from the SSH-untreated control at P < 0.001. BALF, bronchoalveolar lavage fluid; CFUs, colony forming units; ELISA, enzyme-linked immunosorbent assay; NE, neutrophil elastase; PBS, phosphate buffered saline; SSH, sivelestat sodium hydrate; IL, interleukin; TNF, tumor necrosis factor.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Infection, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Saline