lipoteichoic-acid and Cystic-Fibrosis

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).. 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.. 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.. 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.

Topics: Actins; Animals; Anti-Bacterial Agents; Cystic Fibrosis; DNA; Humans; Lipopolysaccharides; Liposomes; Microbial Sensitivity Tests; Polyelectrolytes; Polymers; Pseudomonas aeruginosa; Rabbits; Sputum; Teichoic Acids; Tobramycin

The goal of this study was to evaluate the effects of DNA and F-actin [polyanions present in high concentration in cystic fibrosis (CF) airway fluid] on the antibacterial activities of the cationic steroid antibiotic CSA-13 and the cationic peptides LL37, WLBU2 and HB71.. Light scattering intensity was used to evaluate the aggregation of DNA and F-actin by the cationic antibacterial agents. Bacterial killing assays, atomic force microscopy, determination of MIC values and bacterial load of CF sputa were used to determine the bactericidal activity. Inhibition of nuclear factor-kappaB (NF-kappaB) translocation in human aorta endothelial cells (HAECs) was quantified as an assay of anti-inflammatory action.. CSA-13 is significantly more effective than cationic antibacterial peptides against kanamycin-resistant Pseudomonas aeruginosa and less susceptible to inactivation by DNA or F-actin. The concentration of CSA-13 sufficient to decrease the CF sputa bacteria load by approximately 90% is at least 10 times lower than that at which CSA-13 formed aggregates with DNA or F-actin. Both CSA-13 and LL37 prevent lipopolysaccharide-induced translocation of NF-kappaB in HAEC, thereby suggesting that these antibacterial molecules might prevent systemic inflammation caused by bacterial wall components.. Charge-based interactions that strongly inhibit the antibacterial activity of host cationic antibacterial peptides present in CF sputa have significantly less effect on molecules from the ceragenin family such as CSA-13 due in part to their smaller net charge and distribution of this charge over a hydrophobic scaffold. CSA molecules therefore have potential for the treatment of chronic infections and inflammation that occur in CF airways and other settings in which extracellular polyanions accumulate.

Topics: Actins; Adult; Amino Acid Sequence; Anti-Bacterial Agents; Bacteria; Cell Line; Cystic Fibrosis; Deoxyribonuclease I; DNA; Drug Resistance, Bacterial; Electrolytes; Endothelial Cells; Erythrocytes; Hemolysis; Humans; Lipopolysaccharides; Microbial Sensitivity Tests; Microscopy, Atomic Force; Peptides; Sputum; Steroids; Teichoic Acids

Airway epithelial cells provide an immediate response to bacterial pathogens by producing chemokines and cytokines that recruit polymorphonuclear leukocytes (PMNs) to the site of infection. This response is excessive in patients with cystic fibrosis (CF) who have bacterial contamination of their airways. We postulated that CF airway pathogens, in activating nuclear factor-kappaB-dependent gene transcription in epithelial cells, would promote expression of cytokines that inhibit constitutive apoptosis of recruited PMNs. Epithelial cell culture supernatants from CF (IB-3) and corrected (C-38) epithelial cells stimulated by Staphylococcus aureus or Pseudomonas aeruginosa, increased survival of PMNs by 2- to 5-fold. Enhanced PMN survival was attributed to effects of epithelial granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor expression, which inhibit PMN apoptosis, and was negated by neutralizing antibody to either cytokine. Both CF and normal cells responded to bacteria with increased cytokine production. Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor expression were activated by ligation of asialoGM1, a receptor for P. aeruginosa and S. aureus, and by S. aureus lipoteichoic acid. Lipopolysaccharide was not a potent stimulus of cytokine expression, and P. aeruginosa algC (lipopolysaccharide) and lasR (quorum sensing) mutants were fully capable of activating epithelial cells. Induced expression of cytokines by airway cells repeatedly exposed to bacteria, as occurs in CF, serves not only to recruit and activate PMNs, but also to enhance their survival.

Topics: Antibodies; Apoptosis; Bacterial Proteins; Bronchi; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Cystic Fibrosis; DNA-Binding Proteins; Epithelial Cells; G(M1) Ganglioside; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Lipopolysaccharides; Mutation; Neutrophils; Phosphotransferases (Phosphomutases); Pseudomonas aeruginosa; Reference Values; Staphylococcus aureus; Teichoic Acids; Trans-Activators

Topics: Adolescent; Antibodies, Bacterial; Child; Cystic Fibrosis; Humans; Lipopolysaccharides; Reference Values; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids