Metal-Directed Synthesis of Enantiomerially Pure Dimetallic Lanthanide Luminescent Triple-Stranded Helicates

The synthesis and photophysical evaluation of two enatiomerially pure dimetallic lanthanide luminescent triple-stranded helicates is described. The two systems, formed from the chiral (R,R) ligand 1 and (S,S) ligand 2, were produced as single species in solution, where the excitation of either the naphthalene antennae or the pyridiyl units gave rise to Eu(III) emission in a variety of solvents. Excitation of the antennae also gave rise to circularly polarized Eu(III) luminescence emissions for Eu2:13 and Eu2:23 that were of equal intensity and opposite sign, confirming their enantiomeric nature in solution providing a basis upon which we were able to assign the absolute configurations of Eu2:13 and Eu2:23.

Alpha-1-antitrypsin aerosolized augmentation abrogates neutrophil elastase-induced expression of cathepsin B and matrix metalloprotease 2 in vivo and in vitro

Background: Neutrophil elastase (NE) activity is increased in lung diseases such as a1-antitrypsin (A1AT) deficiency and pneumonia. It has recently been shown to induce expression of cathepsin B and matrix metalloprotease 2 (MMP-2) in vitro and in a mouse model. It is postulated that increased cathepsin B and MMP-2 in acute and chronic lung diseases result from high levels of extracellular NE and that expression of these proteases could be inhibited by A1AT augmentation therapy.

Methods: Cathepsin and MMP activities were assessed in bronchoalveolar lavage (BAL) fluid from patients with A1AT deficiency, pneumonia and control subjects. Macrophages were exposed to BAL fluid rich in free NE from patients with pneumonia following pretreatment with A1AT. MMP-2, cathepsin B, secretory leucoprotease inhibitor (SLPI) and lactoferrin levels were determined in BAL fluid from A1AT-deficient patients before and after aerosolisation of A1AT.

Results: BAL fluid from both patients with pneumonia and those with A1AT deficiency containing free NE had increased cathepsin B and MMP-2 activities compared with BAL fluid from healthy volunteers. The addition of A1AT to BAL fluid from patients with pneumonia greatly reduced NE-induced cathepsin B and MMP-2 expression in macrophages in vitro. A1AT augmentation therapy to A1AT-deficient individuals also reduced cathepsin B and MMP-2 activity in BAL fluid in vivo. Furthermore, A1AT-deficient patients had higher levels of SLPI and lactoferrin after A1AT augmentation therapy.

Conclusion: These findings suggest a novel role for A1AT inhibition of NE-induced upregulation of MMP and cathepsin expression both in vitro and in vivo.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFa and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NF?B was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5—Lys-6 and Val-9—Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6—Gln-57 and Ser-10—Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.

Delivery of rSLPI in a liposomal carrier for inhalation provides protection against cathepsin L degradation

Secretory leukocyte protease inhibitor (SLPI) is an endogenous serine protease inhibitor that protects the lungs from excessive tissue damage caused by leukocyte proteases released during inflammation. Recombinant SLPI (rSLPI) has shown potential as a treatment for inflammatory lung conditions. To date, its clinical application has been limited by rapid enzymatic cleavage by cathepsins and rapid clearance from the lungs after inhalation. In this study, rSLPI was encapsulated in 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] : Cholesterol (DOPS : Chol) liposomes for inhalation. Incubation of rSLPI with cathepsin L leads to complete loss of activity while encapsulation of rSLPI in DOPS : Chol liposomes retained 92.6 of its activity after challenge with cathepsin L. rSLPI-loaded liposomes were aerosolized efficiently using a standard nebulizer with a minimal loss of activity and stability. This formulation was biocompatible and encapsulation did not appear to diminish access to intracellular sites of action in in vitro cell culture studies. Liposome encapsulation of rSLPI therefore improves stability and potentially reduces the level and frequency of dosing required for therapeutic effect after inhalation.