New insights into the substrate specificity of macrophage elastase MMP-12.

Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence Pro-X-X↓Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.

Biosafety of DNA vaccines: New generation of DNA vectors and current knowledge on the fate of plasmids after injection.

DNA vaccination has been widely studied to develop new, alternative, efficient and safe vaccines for humans and animals. Many efforts have been made to increase the immunising potential of these vaccines and three veterinary vaccines are now available on the market. Much work is also being dedicated to develop effective DNA vaccines for humans. However, this new vaccination technique raises issues concerning biosafety due to the nature of the vector, i.e. a DNA molecule that contains sequences of prokaryotic origin (e.g. genes for antibiotic resistance). This review describes the development of the new generation of DNA vectors that are partially or completely devoid of elements of prokaryotic origin and outlines the results of studies on the fate of plasmids after their injection in vivo.

In vivo tissue distribution and kinetics of a pseudorabies virus plasmid DNA vaccine after intramuscular injection in swine.

Previous biodistribution studies of plasmids following intramuscular or intradermal injections of DNA vaccines have been performed in mice, rats or rabbits, but not in large mammals. The aim of the present study was to determine the biodistribution of plasmids in swine using the PRV-specific DNA vaccination model consisting of a single intramuscular (i.m.) injection of three plasmids individually encoding glycoproteins gB, gC and gD. The weak bioavailability of the plasmids (less than 10%) after i.m. injection was consistent with the tissue distribution study. Plasmids remained in the injected muscle for at least 4 weeks and were also detected in liver, spleen, kidney, lung, remote muscle, lymph nodes and ovaries for shorter periods. Differences in persistence, apparent elimination half-lives and clearance in blood were observed between the three plasmids. In conclusion, the three plasmids behaved differently and were transiently detected in most of the organs tested. The exact persistence in the injected muscle was not determined but exceeded 4 weeks. To date this is the first published DNA vaccine tissue distribution study in large animals.