Pseudomonas aeruginosa elastase induces IL-8 production in the lung cells via the epidermal growth factor/extracellular signal-regulated proteins/NFκB pathway

Background: The induction of chemokine secretion by fibroblasts is crucial for the migration of leukocytes into the parenchyma of the injured lung. Several bacterial products activate the lung’s structural as well as immune cells to produce pro inflammatory cytokines and chemokines. We report that elastase from Pseudomonas aeruginosa (PE) evokes IL-8 mRNA expression and protein secretion in nonmalignant culture of human lung fibroblasts by activating the receptor for epidermal growth factor (EGFR) and downstream mitogen-activated protein kinases (MAPK) pathway. Methods: We utilized western blot analysis to detect phosphorylation of EGFR and signal transduction intermediates. Northern blot and ELISA analyses were used to determine IL-8 RNA expression and cytokine secretion. Results: We found that the enzymatically active PE enhances IL-8mRNA and protein secretion but does not increase IL-10 or TNF expression. PE induces phosphorylation of the EGFR and the extracellular signal-regulated proteins (ERK1/2) of the MAPK pathway. Pretreatment of the cells with neutralizing antibody to EGFR or the EGFR-specific tyrphostin AG1478 markedly attenuated the PE-induced ERK1/2 activation. PE-induced IL-8 expression is also abolished in the presence of the MEK inhibitor U0126,indicating the involvement of ERK1/2 in this process. Conclusion: Taken together, the results show PE could modulate lung inflammation by exploiting the EGFR/ERK/NFκB pathway and enhancing IL-8 production by lung fibroblasts

Targeting the PAI-1 Mechanism with a Small Peptide Increases the Efficacy of Alteplase in a Rabbit Model of Chronic Empyema

The incidence of empyema is increasing and associated with a mortality rate of 20% in patients older than 65 years. Since 30% of patients with advanced empyema have contraindications to surgical treatment, novel, low-dose, pharmacological treatments are needed. A Streptococcus pneumoniae-induced rabbit model of chronic empyema recapitulates the progression, loculation, fibrotic repair, and pleural thickening of human disease. Treatment with single chain (sc) urokinase (scuPA) or tissue type (sctPA) plasminogen activators in doses 1.0–4.0 mg/kg were only partially effective in this model. Docking Site Peptide (DSP; 8.0 mg/kg), which decreased the dose of sctPA for successful fibrinolytic therapy in acute empyema model did not improve efficacy in combination with 2.0 mg/kg scuPA or sctPA. However, a two-fold increase in either sctPA or DSP (4.0 and 8.0 mg/kg or 2.0 and 16.0 mg/kg sctPA and DSP, respectively) resulted in 100% effective outcome. Thus, DSP-based Plasminogen Activator Inhibitor 1-Targeted Fibrinolytic Therapy (PAI-1-TFT) of chronic infectious pleural injury in rabbits increases the efficacy of alteplase rendering ineffective doses of sctPA effective. PAI-1-TFT represents a novel, well-tolerated treatment of empyema that is amenable to clinical introduction. The chronic empyema model recapitulates increased resistance of advanced human empyema to fibrinolytic therapy, thus allowing for studies of muti-injection treatments

Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

BACKGROUND:To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA). METHODOLOGY:Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated. RESULTS:The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations--DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold. CONCLUSIONS:Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections

Mechanism of Enhanced Activity of Liposome-Entrapped Aminoglycosides against Resistant Strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of ≥32 versus ≤8 μg/ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa, leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% ± 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% ± 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 ± 2.3 versus 24.2 ± 6.2 gold particles/bacterium, P ≤ 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells

Hip joint torques in type II diabetes with and without neuropathy

Background: Patients with diabetes and peripheral neuropathy demonstrate significantly reduced peak torques at the peripheral joints. Objectives: The aim of this study was to assess isometric and concentric peak torques of the hip joint in people with type II diabetes with and without peripheral neuropathy in comparison with healthy participants. Methods: 27 patients with type II diabetes including 15 patients without peripheral neuropathy, 12 patients with diabetes and peripheral neuropathy and 15 healthy people participated. Isometric and concentric peak torques of hip flexion, extension, adduction and abduction of the non-dominant leg were measured by motorized dynamometer. Results: Peak and average peak concentric torques of the hip extension and abduction in patients with diabetes and peripheral neuropathy were lower than those patients with diabetes and control group. Angle of extension peak torque was significantly greater in patients with diabetes and peripheral neuropathy compared with other groups. Angle of flexion peak torque was lower in the patients with diabetes and peripheral neuropathy. Conclusions: Torque related parameters in patients with type II diabetes with or without peripheral neuropathy, are different from healthy subjects. As a result, patients with diabetes especially with peripheral neuropathy are more susceptible of injury and disability in lower limbs. Keywords: type II diabetes, hip, joint, torques, peripheral neuropath

Bactericidal activity and inhibition of tobramycin by DNA and F-actin.

<p>Bactericidal concentrations of free tobramycin (F-TOB), and liposomal tobramycin (L-TOB) at 2 mg/L were incubated with <i>P. aeruginosa</i> (ATCC 27853), or in presence of DNA/F-actin (125 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal tobramycin was made by ANOVA one-way post <i>t</i>-test, and <i>P</i>-values were considered significant when (***) <i>p</i><0.001.</p