1-hydroxyphenazine and Inflammation

The purpose of this study was to discuss the effects of an extract from the culture medium of Pseudomonas aeruginosa (P. aeruginosa) 2016NX1 (chloroform extract of P. aeruginosa, CEPA) and its purified product 1-hydroxyphenazine on RAW264.7 cell inflammation. Cell viability was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. TNF-α production was determined by an ELISA method. The effects of CEPA and its purified product 1-hydroxyphenazine on cell morphology were investigated using an inverted microscope. Quantitative real-time PCR was performed to determine mRNA expression levels. CEPA and 1-hydroxyphenazine had no obvious toxicity to cells when their concentrations were no more than 20 μg ml

Topics: Animals; Anti-Inflammatory Agents; Inflammation; Mice; NF-kappa B; Phenazines; Plant Extracts; Pseudomonas aeruginosa; RAW 264.7 Cells

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

Bacterial supernatants (BS) obtained from broth cultures of Pseudomonas aeruginosa cause bronchoconstriction in sheep, suggesting that BS contain proinflammatory metabolites. In this study we investigated the mechanism(s) responsible for this bronchial effect. BS were obtained from 48 h cultures and sterilized by filtration. Sheep (n = 6) were intubated and swallowed an esophageal balloon for the measurement of specific lung resistance (SRL). Aerosols of BS (3 ml total) immediately increased SRL (541%). Neither aerosolized broth (control) nor inhaled endotoxin in excess of that contained in the BS had an effect. BS challenges were repeated on separate occasions except that the sheep were treated 30 min before challenge with the anticholinergic agent atropine (0.2 mg/kg, intravenously); the anti-allergic agent nedocromil (1 mg/kg, aerosol); the histamine H1 antagonist chlorpheniramine (2 mg/kg); or the bradykinin (BK) B2 receptor antagonists NPC-567 (5 mg/ml, aerosol) or NPC-17761 (1 mg/ml aerosol). The results showed that greater than 90% protection (p < 0.05) was achieved when the animals were pretreated with atropine, nedocromil sodium, or either of the two BK antagonists, but only 27 +/- 21% protection was seen with chlorpheniramine pretreatment. These findings are characteristic of a BK-mediated response. Analysis of bronchoalveolar lavage fluid obtained before and after BS challenge confirmed that i-kinins, but not histamine, increased (p < 0.05) from 61 +/- 7 to 304 +/- 55 pg/ml. Control (broth) challenges produced no such change. To identify the metabolites involved, we tested the effects of aerosolizing two suspected components of BS, 1-hydroxyphenazine (1-HP) and pyocyanine (PYO) in five sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Administration, Inhalation; Aerosols; Airway Resistance; Animals; Atropine; Bradykinin; Bradykinin Receptor Antagonists; Bronchial Diseases; Bronchoalveolar Lavage Fluid; Chlorpheniramine; Constriction, Pathologic; Drug Evaluation, Preclinical; Endotoxins; Female; Inflammation; Kinins; Nedocromil; Phenazines; Premedication; Pseudomonas aeruginosa; Pyocyanine; Sheep

The Pseudomonas aeruginosa-derived phenazine pigments pyocyanin and 1-hydroxyphenazine (1-hp) prime human neutrophils for enhanced, stimulus-activated release of superoxide and myeloperoxidase (MPO), respectively. In the present study, the modulatory potentials of the antimicrobial agents clindamycin, erythromycin, and roxithromycin (10 and 20 micrograms/ml) on the prooxidative interactions of pyocyanin and 1-hp (12.5 microM) with human neutrophils have been investigated. Clindamycin, erythromycin, and especially roxithromycin caused dose-related inhibition of the generation of superoxide by both untreated and pyocyanin-treated neutrophils during activation with either the synthetic chemotactic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or the calcium ionophore A23187. The antimicrobial agents also inhibited the generation of reactive oxidants by the MPO-H2O2-halide system during activation of both untreated and 1-hp-treated neutrophils by FMLP. These effects appeared to be due to drug-related interference with membrane-associated oxidative metabolism, since none of the antimicrobial agents inhibited the release of MPO by activated neutrophils, nor did they possess oxidant-scavenging properties. These data demonstrate that clindamycin, erythromycin, and especially roxithromycin antagonize the proinflammatory interactions of pyocyanin and 1-hp with neutrophils and indicate a possible therapeutic role for these antimicrobial agents in the prevention of tissue damage in diseases characterized by P. aeruginosa infection.

Topics: Clindamycin; Erythromycin; Humans; In Vitro Techniques; Inflammation; Neutrophils; Oxygen Consumption; Peroxidase; Phenazines; Pigments, Biological; Pseudomonas aeruginosa; Pyocyanine; Roxithromycin; Superoxides