[A disseminated strongyloidiasis in a non immunocompromised patient]. / Une strongyloïdose disséminée chez un patient immunocompétent.

Strongyloidiasis is a parasitosis mainly located in tropical and subtropical regions. Its European incidence is increasing due to population migration. The symptomatology is variable, largely depending on the host immune status. The diagnosis may be challenging as a specific inquiry is needed. The disseminated status, called hyperinfection syndrome, is a life-threatening disease that might occur in immunosuppressed patients. The patient's chances for a successful outcome depend on a quick and specific management. The treatment is based on anthelminthic agents such as ivermectine or albendazole for which the dosage will vary according to the infection type.

La strongyloïdose ou anguillulose est une parasitose principalement retrouvée dans les régions tropicales ou subtropicales. Son incidence en Europe est en augmentation suite aux flux migratoires. Sa symptomatologie est très variable et dépend fortement du statut immunitaire de l'hôte. Le diagnostic peut être difficile et nécessite une recherche spécifique. La forme disséminée, appelée hyperinfection, est typiquement présente chez les patients immunodéprimés. Elle est mortelle, surtout en l'absence de prise en charge adéquate. Le traitement est basé sur les antihelminthiques tels que l'ivermectine et l'albendazole dont la posologie est à adapter en fonction du type d'infection.

Unravelling the mechanisms of a protein refolding process based on the association of detergents and co-solvents.

A new technique associating the detergent Sodium Dodecyl Sulphate (SDS) and an alcohol-type co-solvent has been set up, showing an unexpected efficiency to refold several types of soluble or membrane proteins. The present contribution deepens the fundamental knowledge on the phenomena underlying this process, considering the refolding of two model peptides featuring the main protein secondary structures: α-helix and ß-sheet. Their refolding was monitored by fluorescence and circular dichroism, and it turns out that: (i) 100% recovery of the folded structure is observed for both peptides, (ii) the highest the SDS concentration, the more co-solvent to be added to recover the peptides' native structures, (iii) a high alcohol concentration is required to alter the SDS denaturing properties, (iv) the co-solvent performance relies on its specific lipophilic-hydrophilic balanced character, (v) the size of the micelle formed by the detergent does not enter the process critical parameters, and (vi) increasing the salt concentration up to 1 M NaCl has a beneficial impact on the process efficiency. These mechanistic aspects will help us to improve the method and extend its application. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis.

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella ß-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-ß-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination.

Effects of hydration on the proton transfer mechanism in the adenine-thymine base pair.

We report the first density functional study of water catalytic effect in the double proton transfer (DPT) taking place in the adenine-thymine (AT) base pair. To gain more insight regarding the accuracy of several theoretical methods, the ability of various functionals and models for describing the geometry of this system has first been checked. According to our results, BP86/6-311++G(d,p) is the best option for describing the solvation effects in AT when applied to a two-water-molecule-featuring model. The two possible mechanisms for DPT in solution are explored: in the first one, water molecules only remain passive elements, whereas in the second one they are directly included in the reaction path. For the noncatalyzed mechanism, the stable structures constitute the canonical form of the base pair and the first proton transfer product. Nevertheless, by involving the two water molecules in the reaction, we found three stable species: canonical base pair, first proton transfer product, and double proton transfer product. Although the thermodynamic analysis confirms that AT does not contribute to spontaneous mutation through proton transfer catalyzed by surrounding water, our results suggest that microhydration may play a crucial role for DPT reaction in others DNA or RNA basis pair.

Intermolecular proton transfer in microhydrated guanine-cytosine base pairs: a new mechanism for spontaneous mutation in DNA.

Accurate calculations of the double proton transfer (DPT) in the adenine-thymine base pair (AT) were presented in a previous work [J. Phys. Chem. A 2009, 113, 7892.] where we demonstrated that the mechanism of the reaction in solution is strongly affected by surrounding water. Here we extend our methodology to the guanine-cytosine base pair (GC), for which it turns out that the proton transfer in the gas phase is a synchronous concerted mechanism. The O(G)-H-N(C) hydrogen bond strength emerges as the key parameter in this process, to the extent that complete transfer takes place by means of this hydrogen bond. Since the main effect of the molecular environment is precisely to weaken this bond, the direct proton transfer is not possible in solution, and thus the tautomeric equilibrium must be assisted by surrounding water molecules in an asynchronous concerted mechanism. This result demonstrates that water plays a crucial role in proton reactions. It does not act as a passive element but actually catalyzes the DPT.

[Trochanteric bursitis, pelvic enthesopathy and giant cell arteritis]. / Bursite trochantérienne, enthésopathie pelvienne et maladie de Horton.

Giant cell arteritis, a large-sized vessel vasculitis, may be associated with musculoskeletal proximal (polymyalgia rheumatica) or distal manifestations. A 68-year-old woman, who had inflammatory pelvic girdle pain, was diagnosed with giant cell arteritis and was successfully treated with corticosteroids. The magnetic resonance imaging and ultrasonography revealed a bilateral bursitis and pelvic girdle enthesopathy. Bursitis is the main anatomic lesion occurring in polymyalgia rheumatica and can be underlined by ultrasonography.

Plasma concentration of insulin-like growth factor I (IGF-I) in growing Ardenner horses suffering from juvenile digital degenerative osteoarthropathy.

Degenerative osteoarthropathy resulting in a reduced active lifespan was observed in Ardenner horses. In the context of joint biology, insulin-like growth factor I (IGF-I) is a potential candidate to affect the anabolism of cartilage matrix molecules. A group of 30 Ardenner horses reared under standardized conditions from weaning were evaluated periodically from 15 to 28 months of age to detect the early manifestations of the disease. At the end of this period, horses were classified in two pathological groups related to the degree of interphalangeal degenerative osteoarthropathy based on clinical and radiographic evaluations: healthy (46.7%) and pathological (53.3%) horses. Seven sequential blood samples were taken from each horse (during the evaluation period) to study the variation of IGF-I plasma concentration. We tested the variations of the IGF-I plasma concentration during growth, and the effect of sex and of pathological classes. Significant variations were observed during the research period, with a maximum value corresponding to spring and a minimum in autumn. A significant reduction of the IGF-I plasma concentration was also observed in the pathological horses (433.5 +/- 19.5 ng/ml) compared to the healthy horses (493.9 +/- 18.2 ng/ml). An alteration in the level of this growth factor could induce a disregulation of the mechanisms involved in the local control of joint and bone tissue development.

Brain stem auditory potentials evoked by clicks in the presence of high-pass filtered noise in dogs.

This study evaluates the effects of a high-frequency hearing loss simulated by the high-pass-noise masking method, on the click-evoked brain stem-evoked potentials (BAEP) characteristics in dogs. BAEP were obtained in response to rarefaction and condensation click stimuli from 60 dB normal hearing level (NHL, corresponding to 89 dB sound pressure level) to wave V threshold, using steps of 5 dB in eleven 58 to 80-day-old Beagle puppies. Responses were added, providing an equivalent to alternate polarity clicks, and subtracted, providing the rarefaction-condensation potential (RCDP). The procedure was repeated while constant level, high-pass filtered (HPF) noise was superposed to the click. Cut-off frequencies of the successively used filters were 8, 4, 2 and 1 kHz. For each condition, wave V and RCDP thresholds, and slope of the wave V latency-intensity curve (LIC) were collected. The intensity range at which RCDP could not be recorded (pre-RCDP range) was calculated. Compared with the no noise condition, the pre-RCDP range significantly diminished and the wave V threshold significantly increased when the superposed HPF noise reached the 4 kHz area. Wave V LIC slope became significantly steeper with the 2 kHz HPF noise. In this non-invasive model of high-frequency hearing loss, impaired hearing of frequencies from 8 kHz and above escaped detection through click BAEP study in dogs. Frequencies above 13 kHz were however not specifically addressed in this study.

Cloning and functional characterization of Δ6 fatty acid desaturase (FADS2) in Eurasian perch (Perca fluviatilis).

The Eurasian perch (Perca fluviatilis) is a freshwater carnivorous species of high interest to diversify inland aquaculture. However, little is known about its ability to bioconvert polyunsaturated fatty acids (PUFAs) from plant oils into long chain polyunsaturated fatty acids (LC-PUFAs). In this study, special attention has been given to the fatty acid desaturase 2 (FADS2) which is commonly described to be a rate-limiting enzyme of the LC-PUFA biosynthesis. This work reports on the cloning, tissue expression and functional characterization of the Eurasian perch fads2, but also on the cloning of two alternative splicing transcripts named fads2-AS1 and fads2-AS2. The fads2 cDNA cloned is composed of an open reading frame (ORF) of 1338 nucleotides (nt) and encodes a protein of 445 amino acids. This deduced amino acid sequence displays the typical structure of microsomal FADS2 including two transmembrane domains and an N-terminal cytochrome b5 domain with the "HPGG" motif. Quantitative real-time PCR assay of fads2, fads2-AS1 and fads2-AS2 expressions revealed that the fads2 transcript was mainly expressed in the liver and intestine and exhibited a typical gene expression pattern of freshwater species while fads2-AS1 and fads2-AS2 genes were highly expressed in the brain, followed by the liver and intestine. Functional characterization of Eurasian perch FADS2 in transgenic yeast showed a fully functional Δ6 desaturation activity toward C18 PUFA substrates, without residual Δ5 and Δ8 desaturase activities.

Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design.

All biological phenomena depend on molecular recognition, which is either intermolecular like in ligand binding to a macromolecule or intramolecular like in protein folding. As a result, understanding the relationship between the structure of proteins and the energetics of their stability and binding with others (bio)molecules is a very interesting point in biochemistry and biotechnology. It is essential to the engineering of stable proteins and to the structure-based design of pharmaceutical ligands. The parameter generally used to characterize the stability of a system (the folded and unfolded state of the protein for example) is the equilibrium constant (K) or the free energy (deltaG(o)), which is the sum of enthalpic (deltaH(o)) and entropic (deltaS(o)) terms. These parameters are temperature dependent through the heat capacity change (deltaCp). The thermodynamic parameters deltaH(o) and deltaCp can be derived from spectroscopic experiments, using the van't Hoff method, or measured directly using calorimetry. Along with isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) is a powerful method, less described than ITC, for measuring directly the thermodynamic parameters which characterize biomolecules. In this article, we summarize the principal thermodynamics parameters, describe the DSC approach and review some systems to which it has been applied. DSC is much used for the study of the stability and the folding of biomolecules, but it can also be applied in order to understand biomolecular interactions and can thus be an interesting technique in the process of drug design.

Factor Xa activates endothelial cells by a receptor cascade between EPR-1 and PAR-2.

In addition to its pivotal role in hemostasis, factor Xa binds to human umbilical vein endothelial cells through the recognition of a protein called effector cell protease receptor (EPR-1). This interaction is associated with signal transduction, generation of intracellular second messengers, and modulation of cytokine gene expression. Inhibitors of factor Xa catalytic activity block these responses, thus indicating that the factor Xa-dependent event of local proteolysis is absolutely required for cell activation. Because EPR-1 does not contain proteolysis-sensitive sites, we investigated the possibility that signal transduction by factor Xa requires proteolytic activation of a member of the protease-activated receptor (PAR) gene family. Catalytic inactivation of factor Xa by DX9065 suppressed factor Xa-induced increase in cytosolic free Ca(2+) in endothelial cells (IC(50)=0.23 micromol/L) but failed to reduce ligand binding to EPR-1. In desensitization experiments, trypsin or the PAR-2-specific activator peptide, SLIGKV, ablated the Ca(2+) signaling response induced by factor Xa. Conversely, pretreatment of endothelial cells with factor Xa blocked the PAR-2-dependent increase in cytosolic Ca(2+) signaling, whereas PAR-1-dependent responses were unaffected. Direct cleavage of PAR-2 by factor Xa on endothelial cells was demonstrated by cleavage of a synthetic peptide duplicating the PAR-2 cleavage site and by immunofluorescence with an antibody to a peptide containing the 40-amino acid PAR-2 extracellular extension. These data suggest that factor Xa induces endothelial cell activation via a novel cascade of receptor activation involving docking to EPR-1 and local proteolytic cleavage of PAR-2.

First isolation of a rabid bat infected with European bat lyssavirus in Luxembourg.

Rabid bats are regularly reported in Europe, especially in countries that have implemented a bat surveillance network. In May 2013, bat rabies was evidenced for the first time in Luxembourg (southern city of Differdange). The rabies virus, an EBLV-1b strain, was diagnosed in a serotine bat that bit a 29-year-old male person while he was asleep. The man received rapidly a post-exposure RABV treatment and was put under strict medical supervision.

Comparative effects of two synthetic oligosaccharides on platelet activation induced by plasma from HIT patients.

Heparin-induced thrombocytopenia (HIT) is a serious secondary event encountered in the clinical use of heparin. HIT results from the consumption of platelets that are immunologically activated by antibodies directed against complexes formed by platelet factor 4 (PF4) and sulfated polysaccharides that activate platelet aggregation, leading to paradoxical, life-threatening thrombosis. There is strong evidence that the ability of heparin and related compounds to induce HIT is closely linked to the structure of the polysaccharide, and particularly to its negative charge and to the length of the molecule. To test this hypothesis, we synthesized two sulfated oligosaccharides: SanOrg123781, a 16-mer, presenting two terminal charged domains separated by a 7-mer neutral linker, and SR121903, a highly sulfated 17-mer. Both of them displayed strong anti-factor (F) Xa and anti-FIIa activities but their affinities for PF4 were markedly different. SR121903 displaced PF4-bound heparin, whereas SanOrg123781 did not, underlining the importance of the charge of the molecule for the interaction with PF4. Platelet studies, in the presence of HIT serum, showed that SR121903 induced the secretion of platelet-dense granules (measured by the release of serotonin) whereas SanOrg123781 did not, a result in accordance with an absence of affinity of this molecule for PF4. These results were confirmed by measurements of platelet activation by flow cytometry (measured by annexin V binding, CD62 detection and activation of the GpIIb-IIIa complexes). In conclusion, we have demonstrated the importance of the charge of the polysaccharides in the HIT-induced platelet reactions measured by diverse methods, of which some are described for this purpose for the first time.

Pharmacokinetics of new synthetic heparin mimetics.

Pharmacokinetics of oligosaccharides displaying various affinities for antithrombin (AT) allowed us to show that there was a close relationship between the plasma half-life of these antithrombotic oligosaccharides and their affinity for AT. Recently, we have described new heparin mimetics comprising an AT binding domain and a thrombin binding domain separated by a neutral methylated saccharide sequence. These compounds displayed strong anti-Xa and antii-IIA activities and, in contrast to heparin, escaped neutralisation by platelet factor 4. The aim of this work was to compare the pharmacokinetics of several of these heparin mimetics in rats. These compounds differed by their length, charge and affinity for AT (AT-binding domain). The results obtained indicate that the prolongation of the AT binding domain did not modify significantly their affinities for AT. However, an increase in the number of charges leads to a decrease in the half-life. When a methylated spacer was added in order to minimise the non-specific interactions to other proteins, half-lives of the heparin mimetic were in the same range than that of the pentasaccharide used as an AT binding domain. In conclusion, this study shows the influence of the charge of the oligosaccharides on their pharmacokinetics and underlinesthe importance of minimising their non-specific binding to plasma proteins in order to obtain compounds with predictive pharmacokinetics.

Structural approach for COX-2 inhibition.

The design of selective COX-2 inhibitors is a new approach to obtain potent, anti-inflammatory drugs but with less side effects. Several families of such inhibitors were reported in literature. In this review, the drug design processes used to understand their binding mode and the origin of selectivity of these compounds are described.

Activity of a novel dual thromboxane A(2)receptor antagonist and thromboxane synthase inhibitor (BM-573) on platelet function and isolated smooth muscles.

The pharmacomodulation of sulfonylureas structurally related to torasemide and characterized by a TXA(2)antagonism led to the synthesis of BM-573. This original molecule showed a high affinity (IC(50)1.3 nM) for the TXA(2)receptor of human platelets in comparison with both reference compounds, SQ-29548 (IC(50)21 nM) and sulotroban (IC(50)930 nM). Moreover, this torasemide derivative was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid (ED(100)=0.13 microM) or by U-46619 (ED(50)=0.24 microM), a TXA(2)agonist. BM-573 relaxed the isolated rat thoracic aorta (ED(50)=28.4 nM) and guinea-pig trachea (ED(50)=17.7 nM) contracted by U-46619. BM-573 (1 microM) completely reduced the platelet production of TXB(2)induced by arachidonic acid. Finally, BM-573 (30 mg/kg, per os) lost the diuretic properties of torasemide in rats.

Evaluation of classical NSAIDs and COX-2 selective inhibitors on purified ovine enzymes and human whole blood.

Cyclooxygenase is the key enzyme in the biosynthesis of prostanoids, biologically active substances involved in several physiological processes and also in pathological conditions such as inflammation. It has been well known for 10 years that this enzyme exists under two forms: a constitutive (COX-1) and an inducible form (COX-2). Both enzymes are sensitive to inhibition by conventional non-steroidal anti-inflammatory drugs (NSAIDs). Observations were made that COX-1 was mainly involved in homeostatic processes, while the COX-2 expression was associated with pathological conditions leading to the development of COX-2 selective inhibitors. Several methods have been reported for the evaluation of the COX-1 and COX-2 inhibitory potency and selectivity of conventional or COX-2 selective NSAIDs. In this study, we evaluated the COXs inhibitory profile of both conventional NSAIDs and COX-2 selective inhibitors using two different in vitro methods: the first test was performed using purified enzymes while the second method consisted of a whole blood assay. The results obtained with reference drugs in these two assays will be discussed and compared in this article.

Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function.

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.

Prevalence of antibody to rotavirus in Moroccan cattle.

A serological survey was carried out in order to determine the prevalence of antirotavirus antibodies in Moroccan cattle under different management conditions. From the 493 serum samples examined, 325 (65.9%) were found positive, using a counter-immunoelectroosmophoresis technique. Animals of indigenous breed coming from farms with rapid turnover or large number of animals, or having frequent contacts with imported cattle, had a higher rate of seropositivity; however, positive sera were also found in cattle from small farms in remote areas, showing that rotavirus infection is ubiquitous in that country. No relationship was found between the prevalence of anti-rotavirus antibodies and the frequence of calf diarrhoea. The percentage of seropositive animals in a herd has to be considered as an epidemiological indicator.

Isosterism among analogues of torasemide: conformational, electronic and lipophilic properties.

The structures, electronic (charges, molecular electrostatic potential, molecular orbitals) and lipophilic properties of three isostere analogues of torasemide were determined and the influence of the replacement of the sulfonyl urea group on the conformation and electronic properties of the molecules is discussed. Lipophilicity of the compounds seems to be the most discriminating property along the series and affects their pharmacological activities.