Search

ObjectivesDoxycycline is commonly used in medicine for its bacteriostatic antimicrobial properties. Recent studies have reported that doxycycline also has anti-inflammatory effects. Matrix metalloproteinase (MMP)-9 has been found to be involved in the physiological and pathological process of inflammatory airway disease. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, is known to stimulate the expression of MMP and mucin genes in the airway and intestinal epithelial cells. Therefore, the effects and signal pathways of doxycycline on PMA-induced MUC5B expression dependent MMP-9 in human airway epithelial cells were investigated.MethodsIn human NCI-H292 airway epithelial cells, MUC5B and MMP-9 mRNA expression, MUC5B protein expression, and MMP-9 protein activity after the treatment with PMA, MMP-9 or doxycycline were determined by reverse transcriptase-polymerase chain reaction, enzyme immunoassay, gelatin zymography, and Western blot analysis.ResultsPMA increased MMP-9 and MUC5B expression. MMP-9 increased MUC5B expression. Doxycycline inhibited PMA-induced MUC5B expression, and PMA-induced MMP-9 mRNA expression and protein activity. Doxycycline inhibited phosphorylation of p38 induced by PMA and MMP-9.ConclusionThe results of this study suggest that doxycycline inhibited PMA-induced MUC5B mRNA expression and protein production through the MMP-9 and p38 pathways in human NCI-H292 airway epithelial cells.

Background: CD4 and IL-17 markers can be used for separating TH17 lymphocytes from the population of CD4+ lymphocytes. The best technique for detecting TH17 cells in peripheral blood is flow cytometry in which the cells need to first be activated by phorbol 12-myristate 13-acetate (PMA) and ionomycin. Moreover, monensin is used in order to prevent interleukin 17 (IL-17) secretion out of cells. Methods: Peripheral blood mononuclear cells (PBMC) were separated from freshly isolated heparinized venous blood by centrifugation on a Ficoll-Hypaque (Lymphopreb, Sigma, USA) density gradient. Cell suspension was prepared in three concentrations and cultured with three concentrations of PMA, ionomycin and monensin (each concentration was prepared in three series). Then these were incubated for 6, 12, and 20 hours. Findings: The percentage of TH17 cells was detected by culture of 2 × 106 cells/ml of peripheral blood mononuclear cells with 150 ng/ml of phorbol 12-myristate 13-acetate, 1.5 μM of ionomycin, and 500 ng/ml of monensin and incubation at 37oC in a humidified and 5% CO2 atmosphere for 12 hours. Conclusion: Optimum procedures have to be determined based on the instruments and conditions of each laboratory.

Moxifloxacin is useful in the treatment of respiratory infections, including community-acquired pneumonia, and also shows promise in the treatment of tuberculosis, a clinical setting which necessitates extended administration of this agent. Relatively little is known, however, about the effects of this agent on the antimicrobial and proliferative activities of human neutrophils and T-lymphocytes, respectively. In the current study, we have investigated the effects of moxifloxacin at therapeutic concentrations and greater (1–20 µg/mL) on cytosolic Ca2+ fluxes, generation of antimicrobial reactive oxygen species (ROS), and release of the primary granule protease, elastase, following activation of the cells with the chemoattractant, fMLP (1 µM), or the phorbol ester, PMA (25 ng/mL), using radiometric, chemiluminescence, and colourimetric procedures, respectively. The effects of moxifloxacin on mitogen-activated proliferation of T cells and expression of the interleukin-2 (IL-2) receptor (CD25) were measured using radiometric and flow cytometric procedures respectively. With the exception of elastase release, which was significantly increased (P < 0.05) by treatment of the cells with moxifloxacin at 10 and 20 µg/mL, none of the other neutrophil or lymphocyte functions was affected by moxifloxacin. These observations suggest that extended use of this agent is unlikely to compromise the protective functions of neutrophils and T-lymphocytes and may even potentiate neutrophil-mediated antimicrobial activity by increasing the release of elastase.

Abstract Background Fat embolism syndrome (FES) associated with acute lung injury (ALI) is a clinical condition following long bone fracture. We have reported 14 victims due to ALI with FES. Our laboratory has developed an animal model that produced fat emboli (FE). The major purpose of this study was to test whether neutrophil activation with phorbol myristate acetate (PMA) and inhibition with sivelestat (SVT) exert protection on the lung. Methods The lungs of Sprague-Dawley rats were isolated and perfused. FE was produced by addition of corn oil micelles into the lung perfusate. PMA and SVT were given simultaneously with FE. Parameters such as lung weight/body weight ratio, LW gain, exhaled nitric oxide (NO), protein concentration in bronchoalveolar lavage relating to ALI were measured. The neutrophil elastase (NE), myeloperoxidase, malondialdehyde and phopholipase A2 activity were determined. We also measured the nitrate/nitrite, methyl guanidine (MG), and cytokines. Pulmonary arterial pressure and microvascular permeability were assessed. Lung pathology was examined and scored. The inducible and endothelial NO synthase (iNOS and eNOS) were detected. Results FE caused ALI and increased biochemical factors. The challenge also resulted in pulmonary hypertension and increased microvascular permeability. The NE appeared to be the first to reach its peak at 1 hr, followed by other factors. Coadministration with PMA exacerbated the FE-induced changes, while SVT attenuated the effects of FE. Conclusions The FE-induced lung changes were enhanced by PMA, while SVT had the opposite effect. Sivelestat, a neutrophil inhibitor may be a therapeutic choice for patients with acute respiratory distress syndrome (ARDS) following fat embolism.