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BackgroundMucin disulfide cross-links mediate pathologic mucus formation in muco-obstructive lung diseases. MUC-031, a novel thiol-modified carbohydrate compound, cleaves disulfides to cause mucolysis. The aim of this study was to determine the mucolytic and therapeutic effects of MUC-031 in sputum from patients with cystic fibrosis (CF) and mice with muco-obstructive lung disease (βENaC-Tg mice).MethodsWe compared the mucolytic efficacy of MUC-031 and existing mucolytics (N-acetylcysteine (NAC) and recombinant human deoxyribonuclease I (rhDNase)) using rheology to measure the elastic modulus (G') of CF sputum, and we tested effects of MUC-031 on airway mucus plugging, inflammation and survival in βENaC-Tg mice to determine its mucolytic efficacy in vivo.ResultsIn CF sputum, compared to the effects of rhDNase and NAC, MUC-031 caused a larger decrease in sputum G', was faster in decreasing sputum G' by 50% and caused mucolysis of a larger proportion of sputum samples within 15 min of drug addition. Compared to vehicle control, three treatments with MUC-031 in 1 day in adult βENaC-Tg mice decreased airway mucus content (16.8±3.2 versus 7.5±1.2 nL·mm-2, p

Trehalose is commonly used as a protein stabilizer in spray dried protein formulations delivered via the pulmonary route. Spray dried trehalose formulations are highly hygroscopic, which makes them prone to deliquescence and recrystallization when exposed to moisture, leading to impairment in aerosolization performance. The main aim of this study was to investigate and compare the effect of hydrophobic amino acids (i.e. L-leucine and L-isoleucine) in enhancing aerosolization performance and in mitigating moisture-induced changes in spray dried trehalose formulations. Trehalose was spray dried with 20-60% w/w of amino acid (i.e. L-leucine or L-isoleucine). The spray dried formulations were stored at 25 °C/50% RH for 28 days. Solid state characterization and in vitro aerosolization performance studies were performed on the spray dried formulations before and after storage. The addition of 20-60% w/w of amino acid (i.e. L-leucine or L-isoleucine) improved the emitted fractions of spray dried trehalose formulations from a dry powder inhaler. However, ≥ 40% w/w of L-leucine/L-isoleucine was needed to prevent recrystallization of trehalose in the formulations when exposed to 25 °C/50% RH for 28 days. X-ray photoelectron spectroscopy (XPS) demonstrated that samples with 40-60% w/w L-isoleucine had more amino acid on the surfaces of the particles compared to their L-leucine counterparts. This may explain the greater ability of the L-isoleucine (40-60% w/w) samples to cope with elevated humidity compared to L-leucine samples of the same concentrations, as observed in the dynamic vapour sorption (DVS) studies. In conclusion, this study demonstrated that both L-leucine and L-isoleucine were effective in enhancing aerosolization performance and mitigating moisture-induced reduction in aerosolization performance in spray dried trehalose formulations. L-isoleucine proved to be superior to L-leucine in terms of its moisture protectant effect when incorporated at the same concentration in the formulations.

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.

The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

Airway mucus in cystic fibrosis (CF) is highly elastic, but themechanism behind this pathology is unclear. We hypothesized that the biophysical properties of CF mucus are altered because of neutrophilic oxidative stress. Using confocal imaging, rheology, and biochemical measures of inflammation and oxidation, we found that CF airway mucus gels have a molecular architecture characterized by a core of mucin covered by a web of DNA and a rheological profile characterized by high elasticity that can be normalized by chemical reduction. We also found that high levels of reactive oxygen species in CF mucus correlated positively and significantly with high concentrations of the oxidized products of cysteine (disulfide cross-links). To directly determine whether oxidation can cross-link mucins to increase mucus elasticity, we exposed induced sputum from healthy subjects to oxidizing stimuli and found a marked and thiol-dependent increase in sputum elasticity. Targeting mucin disulfide cross-links using current thiol-amino structures such as N-acetylcysteine (NAC) requires high drug concentrations to have mucolytic effects. We therefore synthesized a thiol-carbohydrate structure (methyl 6-thio-6-deoxy-α-D-galactopyranoside) and found that it had stronger reducing activity than NAC and more potent and fast-acting mucolytic activity in CF sputum. Thus, oxidation arising from airway inflammation or environmental exposure contributes to pathologic mucus gel formation in the lung, which suggests that it can be targeted by thiol-modified carbohydrates.

Abstract: Zwitterionic capsular polysaccharide extracts, --8 kDa in mass, from Streptococcus pneumoniae type 1 (Spt1) have shown unique T-cell activating properties. Oligomers of the trisaccharide repeating unit of the Spt1 [...]