A glycopolymer improves vascoelasticity and mucociliary transport of abnormal cystic fibrosis mucus.

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

In Vitro Activity of a Novel Glycopolymer against Biofilms of Burkholderia cepacia Complex Cystic Fibrosis Clinical Isolates.

Burkholderia cepacia complex (Bcc) lung infections in cystic fibrosis (CF) patients are often associated with a steady decline in lung function and death. The formation of biofilms and inherent multidrug resistance are virulence factors associated with Bcc infection and contribute to increased risk of mortality in CF patients. New therapeutic strategies targeting bacterial biofilms are anticipated to enhance antibiotic penetration and facilitate resolution of infection. Poly (acetyl, arginyl) glucosamine (PAAG) is a cationic glycopolymer therapeutic being developed to directly target biofilm integrity. In this study, 13 isolates from 7 species were examined, including Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia gladioli, Burkholderia dolosa, Burkholderia vietnamiensis, and B. cepacia These isolates were selected for their resistance to standard clinical antibiotics and their ability to form biofilms in vitro Biofilm biomass was quantitated using static tissue culture plate (TCP) biofilm methods and a minimum biofilm eradication concentration (MBEC) assay. Confocal laser scanning microscopy (CLSM) visualized biofilm removal by PAAG during treatment. Both TCP and MBEC methods demonstrated a significant dose-dependent relationship with regard to biofilm removal by 50 to 200 µg/ml PAAG following a 1-h treatment (P < 0.01). A significant reduction in biofilm thickness was observed following a 10-min treatment of Bcc biofilms with PAAG compared to that with vehicle control (P < 0.001) in TCP, MBEC, and CLSM analyses. PAAG also rapidly permeabilizes bacteria within the first 10 min of treatment. Glycopolymers, such as PAAG, are a new class of large-molecule therapeutics that support the treatment of recalcitrant Bcc biofilm.

Novel glycopolymer sensitizes Burkholderia cepacia complex isolates from cystic fibrosis patients to tobramycin and meropenem.

Burkholderia cepacia complex (Bcc) infection, associated with cystic fibrosis (CF) is intrinsically multidrug resistant to antibiotic treatment making eradication from the CF lung virtually impossible. Infection with Bcc leads to a rapid decline in lung function and is often a contraindication for lung transplant, significantly influencing morbidity and mortality associated with CF disease. Standard treatment frequently involves antibiotic combination therapy. However, no formal strategy has been adopted in clinical practice to guide successful eradication. A new class of direct-acting, large molecule polycationic glycopolymers, derivatives of a natural polysaccharide poly-N-acetyl-glucosamine (PAAG), are in development as an alternative to traditional antibiotic strategies. During treatment, PAAG rapidly targets the anionic structural composition of bacterial outer membranes. PAAG was observed to permeabilize bacterial membranes upon contact to facilitate potentiation of antibiotic activity. Three-dimensional checkerboard synergy analyses were used to test the susceptibility of eight Bcc strains (seven CF clinical isolates) to antibiotic combinations with PAAG or ceftazidime. Potentiation of tobramycin and meropenem activity was observed in combination with 8-128 µg/mL PAAG. Treatment with PAAG reduced the minimum inhibitory concentration (MIC) of tobramycin and meropenem below their clinical sensitivity breakpoints (≤4 µg/mL), demonstrating the ability of PAAG to sensitize antibiotic resistant Bcc clinical isolates. Fractional inhibitory concentration (FIC) calculations showed PAAG was able to significantly potentiate antibacterial synergy with these antibiotics toward all Bcc species tested. These preliminary studies suggest PAAG facilitates a broad synergistic activity that may result in more positive therapeutic outcomes and supports further development of safe, polycationic glycopolymers for inhaled combination antibiotic therapy, particularly for CF-associated Bcc infections.

Characterization of circulating monocytes expressing HLA-DR or CD71 and related soluble factors for 2 weeks after severe, non-thermal injury.

BACKGROUND: Severe injury is associated with changes in monocytes that may contribute to poor outcomes. Longitudinal characterization of monocyte response patterns after trauma may provide added insight into these immunological alterations. METHODS: Venous blood obtained seven times during post-injury days 1 through 13 from 61 patients with an injury severity score >20 was assessed by flow cytometry for monocytes (CD14+) expressing HLA-DR or CD71 (transferrin receptor) and for circulating levels of interleukin (IL) 1alpha, IL-1beta, IL-6, soluble CD14 (sCD14), tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), and endotoxin. Urine neopterin was measured by high-pressure liquid chromatography, expressed as a neopterin-creatinine ratio. RESULTS: Trauma patients had leucocytosis days 1 through 13, monocytosis days 5 through 13, reduced proportions of CD14+HLA-DR+ cells days 2 through 5, and elevated proportions of CD14+CD71+ cells days 1 through 13. Neopterin was elevated all days, peaking on day 10. sCD14 was elevated days 2 through 13, and there were sporadic elevations of IL-1alpha, IL-1beta, IL-6, TNF-alpha, PGE(2), TXB(2), and endotoxin. Sepsis syndrome patients (n = 6) had larger and more prolonged reductions in CD14+HLA-DR+ cells and higher neopterin values, in comparison with uneventful patient outcomes. CONCLUSIONS: Altered proportions of monocytes expressing HLA-DR and CD71 and elevated sCD14 and urine neopterin levels, for up to 2 weeks after severe injury, underscores an extended period of profound immunological effects. Additional studies to more fully assess temporal monocyte response patterns after severe injury, including activation, may be warranted.