Experimental fetal endoscopic tracheal occlusion in rhesus and cynomolgus monkeys: nonhuman primate models.

OBJECTIVE: The monkey model is the best model to investigate some physiological response to the fetal transitory tracheal occlusion but it has never been described in Macaca monkeys. The aim of this study was to evaluate the feasibility of fetal endoscopic tracheal occlusion (FETO) in a non-human primate model. METHODS: Pregnant rhesus monkeys and cynomolgus were tested as a potential experimental model for FETO in the third trimester of pregnancy, by performing fetal tracheoscopies with and without tracheal occlusion. RESULTS: A total of 22 pregnancies were followed in 16 monkeys and underwent fetal surgery. Percutaneous endoscopic access to the uterine cavity was possible in 20 cases (91%). Of these 20 pregnant monkeys, fetal tracheoscopy could be achieved in 15 cases (75%). In rhesus monkeys, the time between the onset of endoscopy and tracheal penetration decreases as operator experience increases. Neither maternal morbidity nor mortality was related to surgery. Two fetal losses were possibly due to the procedure. CONCLUSION: FETO is feasible in the non-human primate, which closely reflects procedures in humans. The non-human primate model for FETO, specially the rhesus monkeys, may be useful for future studies concerning the mechanisms related to the lung growth after transitory fetal tracheal occlusion.

Lead optimization of antifungal peptides with 3D NMR structures analysis.

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystine-rich antimicrobial peptides with the CS alpha beta scaffold (an alpha-helix linked to a beta-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CS alpha beta peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD-135, and ETD-151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD-135 and ETD-151 were engineered by site-directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD-135 and ETD-151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.

Thanatin activity on multidrug resistant clinical isolates of Enterobacter aerogenes and Klebsiella pneumoniae.

Efflux pumps protect bacterial cells by ejecting intracellular toxic molecules such as antibiotics, detergents and defensins that have penetrated the cell envelope. Some of these efflux pumps recognise structurally unrelated compounds (mdr pump) and account for the resistance of some organisms to two or more agents. It would be of interest to identify molecules that are able to circumvent the problems created by multidrug resistance phenotypes during antibiotic therapy. We have studied the activity of thanatin, a 21-residue cationic antimicrobial peptide produced by an insect, against three bacterial species. The antibacterial effect depended on the size of lipopolysaccharide side chains. In clinically resistant isolates of Enterobacter aerogenes and Klebsiella pneumoniae, the biological activity of thanatin is independent of the membrane permeability, possibly controlled by one or more porins, and/or the expression of drug efflux pumps, two mechanisms which confer high level antibiotic resistance. In addition, thanatin was able to improve the activity of structurally unrelated antibiotics (norfloxacin, chloramphenicol, tetracycline) on a multidrug- resistant E. aerogenes clinical isolate.

Solution structure of Alo-3: a new knottin-type antifungal peptide from the insect Acrocinus longimanus.

Insect peptides are key elements of the innate immunity against bacteria and fungi. These molecules offer remarkable properties: high efficacy, a low probability of resistance, limited toxicity, and immunogenicity. In this context, we are investigating several classes of peptides, and we have been successful in identifying biologically important classes of peptides and small molecules that will provide a stream of drug candidates for treating severe, life-threatening, hospital-acquired infections and other pathologies of high medical need. Recently, we have isolated a new class of antifungal peptides from the coleopteran Acrocinus longimanus. Three homologous peptides, Alo-1, Alo-2, and Alo-3, with sequence identity above 80% and active against the Candida glabrata yeast strain were identified. Alo-3 displayed the highest activity against Candida glabrata and was thus chosen for structure determination using NMR spectroscopy and molecular modeling. Alo-3 contains six cysteine residues forming three disulfide bridges. The pairing of the cysteines was assessed using ambiguous disulfide restraints within the ARIA software, allowing us to establish that Alo-3 belongs to the inhibitor cystine-knot family. It exhibits all the structural features characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first strand to the second strand and a series of turns. To our knowledge, Alo-3 is the first peptide from insects with antimicrobial activity adopting the knottin fold. Alo-3 shows a level of activity significantly higher against C. glabrata than Alo-1 or Alo-2. It has no negatively charged residues and displays on its surface a cationic pole that may account for its antifungal activity. This finding is validated by the comparison of the structure of Alo-3 with the structure of other structurally related peptides from other sources also showing antifungal activity.

Pherokine-2 and -3.

Drosophila is a powerful model system to study the regulatory and effector mechanisms of innate immunity. To identify molecules induced in the course of viral infection in this insect, we have developed a model based on intrathoracic injection of the picorna-like Drosophila C virus (DCV). We have used MALDI-TOF mass spectrometry to compare the hemolymph of DCV infected flies and control flies. By contrast with the strong humoral response triggered by injection of bacteria or fungal spores, we have identified only one molecule induced in the hemolymph of virus infected flies. This molecule, pherokine-2 (Phk-2), is related to OS-D/A10 (Phk-1), which was previously characterized as a putative odor/pheromone binding protein specifically expressed in antennae. The virus-induced molecule is also similar to the product of the gene CG9358 (Phk-3), which is induced by septic injury. Both Phk-2 and Phk-3 are strongly expressed during metamorphosis, suggesting that they may participate in tissue-remodeling.