Rapid sample pre-treatment prior to GC-MS and GC-MS/MS urinary toxicological screening.

Drug screening is an important issue in clinical and forensic toxicology. Gas chromatography coupled to mass spectrometry (GC-MS) remains the gold standard technique for the screening of unknown compounds in urine samples. However, this technique requires substantial sample preparation, which is time consuming. Moreover, some common drugs such as cannabis cannot be easily detected in urine using general procedures. In this work, a sample preparation protocol for treating 200 μL of urine in less than 30 min is described. The enzymatic hydrolysis of glucuro-conjugates was performed in 5 min thanks to the use of microwaves. The use of a deconvolution software allowed reducing the GC-MS run to 10 min, without impairing the quality of the compound identifications. Comparing the results from 139 authentic urine samples to those obtained using the current routine analysis indicated this method performed well. Moreover, additional 5-min GC-MS/MS programs are described, enabling a very sensitive target screening of 54 drugs, including THC-COOH or buprenorphine, without further sample preparation. These methods appeared as an interesting alternative to immuno-assays based screening. The analytical strategy presented in this article proved to be a promising approach for systematic toxicological analysis (STA) of drugs in urine.

Chemical probes that differentially modulate peroxisome proliferator-activated receptor alpha and BLTR, nuclear and cell surface receptors for leukotriene B(4).

Peroxisome proliferator-activated receptor alpha (PPARalpha)is a nuclear receptor for various fatty acids, eicosanoids, and hypolipidemic drugs. In the presence of ligand, this transcription factor increases expression of target genes that are primarily associated with lipid homeostasis. We have previously reported PPARalpha as a nuclear receptor of the inflammatory mediator leukotriene B(4) (LTB(4)) and demonstrated an anti-inflammatory function for PPARalpha in vivo (Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39-43). LTB(4) also has a cell surface receptor (BLTR) that mediates proinflammatory events, such as chemotaxis and chemokinesis (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624). In this study, we report on chemical probes that differentially modulate activity of these two LTB(4) receptors. The compounds selected were originally characterized as synthetic BLTR effectors, both agonists and antagonists. Here, we evaluate the compounds as effectors of the three PPAR isotypes (alpha, beta, and gamma) by transient transfection assays and also determine whether the compounds are ligands for these nuclear receptors by coactivator-dependent receptor ligand interaction assay, a semifunctional in vitro assay. Because the compounds are PPARalpha selective, we further analyze their potency in a biological assay for the PPARalpha-mediated activity of lipid accumulation. These chemical probes will prove invaluable in dissecting processes that involve nuclear and cell surface LTB(4) receptors and also aid in drug discovery programs.

Photodynamic therapy for enhanced drug delivery to tumor

Photodynamic therapy (PDT) has been used as an adjunct to cytoreductive surgery in patients with malignant pleura mesothelioma (MPM). However, it was associated with substantial side effects and found to be only of modest clinical benefit. In contrast, Visudyne®-mediated low-dose PDT has been shown to selectively increase the concentration of macromolecular cytostatic compounds in various tumors grown subpleurally on rodent lungs. Consequently, it was thought that PDT-assisted enhanced tumor penetration for cytostatic agents might be better suited to achieve additional tumor control after cytoreductive surgery for mesothelioma. This effect seems to be mainly related to PDT-mediated modulations of tumor vessels which improve the distribution of circulating, systemically administered chemotherapeutic macromolecular agents. However, the mechanisms involved and the optimization of this effect for therapeutic implications remain to be solved. By using the dorsal skin fold chamber method we demonstrated that both angiogenesis and microcirculation of human mesothelioma xenografts can be continuously assessed in vivo by intravital microscopy. We described a new, simple, reproducible and reliable scoring system for the assessment of tumor angiogenesis and microcirculation in this model, thereby allowing the quantitative description of the neo-vascular network development while avoiding a complicated technical setup. This method can serve as a useful tool for the assessment of novel vessel-targeted therapies against MPM. We then applied this newly established model so as to elucidate the underlying mechanisms of PDT-induced extravasation of macromolecular compounds across the endothelial barrier in tumors and surrounding normal tissue. We found that low-dose PDT selectively enhanced the uptake of macromolecular compounds in human mesothelioma xenografts compared to surrounding normal tissue. Interestingly, this increase of effective permeability of tumor vasculature was not related to the inflammatory stimuli generated by PDT such as the mobilization of leucocytes and their adhesion and penetration of the injured vessel wall. We then used the model for optimizing the drug-light conditions of low- dose PDT in order to obtain maximal leakage of the macromolecular compounds in the tumor with minimal uptake in normal surrounding tissue and we were able to identify such a therapeutic window. With these optimized PDT treatment conditions, we assessed the therapeutic effect of this new treatment concept in vivo by measuring tumor growth rates on subcutaneously grown mesothelioma xenografts in nude mice after low-dose PDT of the tumors following systemically administered liposomal (macromolecular) cisplatin, a cytostatic compound commonly used in clinical practice. We were able to demonstrate that low-dose PDT with optimized drug-light conditions combined with systemic chemotherapy indeed resulted in a reduction in tumor growth compared to chemotherapy or PDT alone. In conclusion, our work demonstrates that low-dose PDT may selectively enhance the uptake of macromolecular cytostatic drugs in superficially growing tumors such as mesotheliomas and opens new perspectives for the treatment of these diseases. - Les effets cytotoxiques de la thérapie photodynamique (PDT) sur le mésothéliome pleural malin (MPM) n'ont pas apporté de bénéfice clinique significatif. Toutefois, une application innovante non cytotoxique de la PDT serait la bienvenue en supplément des chimiothérapies pour améliorer le contrôle local de la tumeur. Le prétraitement des néovaisseaux tumoraux par une PDT à bas régime, qui améliorerait la distribution d'une chimiothérapie administrée par voie systémique de façon concomitante, a attiré une attention particulière pour de futures applications cliniques. Toutefois, les mécanismes impliqués dans cet événement et les implications thérapeutiques de ces changements physiopathologiques restent non résolus. Dans cette thèse, nous avons observé en premier que l'angiogenèse et la microcirculation dans les xénogreffes de mésothéliomes humains peuvent être observées et analysées in vivo par microscopie intravitale. Le nouveau système de score appliqué pour l'évaluation de l'angiogenèse et de la microcirculation tumorale dans cette étude est une méthode simple, reproductible et fiable servant à décrire de manière quantitative le réseau néo-vasculaire en développement, tout en évitant d'utiliser une installation technique compliquée. Ce modèle sert de nouvel outil pour l'évaluation des thérapies anti-vasculaires dirigées contre le MPM. Le modèle animal nouvellement établi a alors été utilisé pour élucider les mécanismes sous-jacents de Γ extravasation d'agents macromoléculaires induite par PDT dans les vaisseaux tumoraux et normaux. Nous avons trouvé que la PDT à fable dose améliore la distribution ciblée de drogues macromoléculaires dans des greffes de mésothéliome humain, de manière sélective pour la tumeur. La perméabilité vasculaire tumorale n'est pas influencée par les stimuli inflammatoires générés par la PDT, ce qui joue un rôle important dans la sélectivité de notre photodynamic drug delivery. Ensuite, nous avons recherché la fenêtre thérapeutique optimale de la PDT pour obtenir une accumulation sélective du colorant macromoléculaire dans le tissu tumoral ainsi qu'une efficacité de la PDT combinée avec une chimiothérapie macromoléculaire sur la croissance tumorale. Nous avons démontré que la PDT à faible dose combinée avec une administration systémique de cisplatine liposomale mène à un ralentissement de la croissance tumorale dans notre modèle de mésothéliome malin humain. En conclusion, l'utilisation de la PDT comme prétraitement pour améliorer sélectivement la distribution d'agents thérapeutiques dans des tumeurs poussant superficiellement est prometteuse. Cette observation fourni une preuve du concept remarquable et garanti la suite des investigations, éventuellement ayant pour but de développer de nouveaux concepts de thérapie pour les patients atteints de mésothéliome. Une PDT intra cavitaire à faible dose après pleuro- pneumonectomie pourrait améliorer la pénétration des agents cytostatiques administrés de façon concomitante par voie systémique dans les îlots tumoraux résiduels, et ainsi améliorer le contrôle local.

Vectorisation intra-oculaire [Drug delivery to target the posterior segment of the eye].

Retinal diseases are nowadays the most common causes of vision threatening in developed countries. Therapeutic advances in this field are hindered by the difficulty to deliver drugs to the posterior segment of the eye. Due to anatomical barriers, the ocular biodisponibility of systemically administered drugs remains poor, and topical instillation is not adequate to achieve therapeutic concentrations of drugs in the back of the eye. Ocular drug delivery has thus become one of the main challenges of modern ophthalmology. A multidisciplinary research is being conducted worldwide including pharmacology, biomaterials, ophthalmology, pharmaceutics, and biology. New promising fields have been developed such as implantable or injectable slow release intravitreal devices and degradable polymers, dispersed polymeric systems for intraocular drug delivery, and transscleral delivery devices such as iontophoresis, osmotic pumps or intra-scleraly implantable materials. The first clinical applications emerging from this research are now taking place, opening new avenues for the treatment of retinal diseases.

Hierarchical modeling gave plausible estimates of associations between metabolic syndrome and components of antiretroviral therapy.

OBJECTIVE: Hierarchical modeling has been proposed as a solution to the multiple exposure problem. We estimate associations between metabolic syndrome and different components of antiretroviral therapy using both conventional and hierarchical models. STUDY DESIGN AND SETTING: We use discrete time survival analysis to estimate the association between metabolic syndrome and cumulative exposure to 16 antiretrovirals from four drug classes. We fit a hierarchical model where the drug class provides a prior model of the association between metabolic syndrome and exposure to each antiretroviral. RESULTS: One thousand two hundred and eighteen patients were followed for a median of 27 months, with 242 cases of metabolic syndrome (20%) at a rate of 7.5 cases per 100 patient years. Metabolic syndrome was more likely to develop in patients exposed to stavudine, but was less likely to develop in those exposed to atazanavir. The estimate for exposure to atazanavir increased from hazard ratio of 0.06 per 6 months' use in the conventional model to 0.37 in the hierarchical model (or from 0.57 to 0.81 when using spline-based covariate adjustment). CONCLUSION: These results are consistent with trials that show the disadvantage of stavudine and advantage of atazanavir relative to other drugs in their respective classes. The hierarchical model gave more plausible results than the equivalent conventional model.

The nine-year sustained cost-containment impact of swiss pilot physicians-pharmacists quality circles.

BACKGROUND: Six pioneer physicians-pharmacists quality circles (PPQCs) located in the Swiss canton of Fribourg (administratively corresponding to a state in the US) were under the responsibility of 6 trained community pharmacists moderating the prescribing process of 24 general practitioners (GPs). PPQCs are based on a multifaceted collaborative process mediated by community pharmacists for improving compliance with clinical guidelines within GPs' prescribing practices. OBJECTIVE: To assess, over a 9-year period (1999-2007), the cost-containment impact of the PPQCs. METHODS: The key elements of PPQCs are a structured continuous quality improvement and education process; local networking; feedback of comparative and detailed data regarding costs, drug choice, and frequency of prescribed drugs; and structured independent literature review for interdisciplinary continuing education. The data are issued from the community pharmacy invoices to the health insurance companies. The study analyzed the cost-containment impact of the PPQCs in comparison with GPs working in similar conditions of care without particular collaboration with pharmacists, the percentage of generic prescriptions for specific cardiovascular drug classes, and the percentage of drug costs or units prescribed for specific cardiovascular drugs. RESULTS: For the 9-year period, there was a 42% decrease in the drug costs in the PPQC group as compared to the control group, representing a $225,000 (USD) savings per GP only in 2007. These results are explained by better compliance with clinical and pharmacovigilance guidelines, larger distribution of generic drugs, a more balanced attitude toward marketing strategies, and interdisciplinary continuing education on the rational use of drugs. CONCLUSIONS: The PPQC work process has yielded sustainable results, such as significant cost savings, higher penetration of generics and reflection on patient safety, and the place of "new" drugs in therapy. The PPQCs may also constitute a solid basis for implementing more comprehensive collaborative programs, such as medication reviews, adherence-enhancing interventions, or disease management approaches.

EADock : design of a new molecular docking algorithm and some of its applications

3 Summary 3. 1 English The pharmaceutical industry has been facing several challenges during the last years, and the optimization of their drug discovery pipeline is believed to be the only viable solution. High-throughput techniques do participate actively to this optimization, especially when complemented by computational approaches aiming at rationalizing the enormous amount of information that they can produce. In siiico techniques, such as virtual screening or rational drug design, are now routinely used to guide drug discovery. Both heavily rely on the prediction of the molecular interaction (docking) occurring between drug-like molecules and a therapeutically relevant target. Several softwares are available to this end, but despite the very promising picture drawn in most benchmarks, they still hold several hidden weaknesses. As pointed out in several recent reviews, the docking problem is far from being solved, and there is now a need for methods able to identify binding modes with a high accuracy, which is essential to reliably compute the binding free energy of the ligand. This quantity is directly linked to its affinity and can be related to its biological activity. Accurate docking algorithms are thus critical for both the discovery and the rational optimization of new drugs. In this thesis, a new docking software aiming at this goal is presented, EADock. It uses a hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with .the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 R around the center of mass of the ligand position in the crystal structure, and conversely to other benchmarks, our algorithms was fed with optimized ligand positions up to 10 A root mean square deviation 2MSD) from the crystal structure. This validation illustrates the efficiency of our sampling heuristic, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best-ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures in this benchmark could be explained by the presence of crystal contacts in the experimental structure. EADock has been used to understand molecular interactions involved in the regulation of the Na,K ATPase, and in the activation of the nuclear hormone peroxisome proliferatoractivated receptors a (PPARa). It also helped to understand the action of common pollutants (phthalates) on PPARy, and the impact of biotransformations of the anticancer drug Imatinib (Gleevec®) on its binding mode to the Bcr-Abl tyrosine kinase. Finally, a fragment-based rational drug design approach using EADock was developed, and led to the successful design of new peptidic ligands for the a5ß1 integrin, and for the human PPARa. In both cases, the designed peptides presented activities comparable to that of well-established ligands such as the anticancer drug Cilengitide and Wy14,643, respectively. 3.2 French Les récentes difficultés de l'industrie pharmaceutique ne semblent pouvoir se résoudre que par l'optimisation de leur processus de développement de médicaments. Cette dernière implique de plus en plus. de techniques dites "haut-débit", particulièrement efficaces lorsqu'elles sont couplées aux outils informatiques permettant de gérer la masse de données produite. Désormais, les approches in silico telles que le criblage virtuel ou la conception rationnelle de nouvelles molécules sont utilisées couramment. Toutes deux reposent sur la capacité à prédire les détails de l'interaction moléculaire entre une molécule ressemblant à un principe actif (PA) et une protéine cible ayant un intérêt thérapeutique. Les comparatifs de logiciels s'attaquant à cette prédiction sont flatteurs, mais plusieurs problèmes subsistent. La littérature récente tend à remettre en cause leur fiabilité, affirmant l'émergence .d'un besoin pour des approches plus précises du mode d'interaction. Cette précision est essentielle au calcul de l'énergie libre de liaison, qui est directement liée à l'affinité du PA potentiel pour la protéine cible, et indirectement liée à son activité biologique. Une prédiction précise est d'une importance toute particulière pour la découverte et l'optimisation de nouvelles molécules actives. Cette thèse présente un nouveau logiciel, EADock, mettant en avant une telle précision. Cet algorithme évolutionnaire hybride utilise deux pressions de sélections, combinées à une gestion de la diversité sophistiquée. EADock repose sur CHARMM pour les calculs d'énergie et la gestion des coordonnées atomiques. Sa validation a été effectuée sur 37 complexes protéine-ligand cristallisés, incluant 11 protéines différentes. L'espace de recherche a été étendu à une sphère de 151 de rayon autour du centre de masse du ligand cristallisé, et contrairement aux comparatifs habituels, l'algorithme est parti de solutions optimisées présentant un RMSD jusqu'à 10 R par rapport à la structure cristalline. Cette validation a permis de mettre en évidence l'efficacité de notre heuristique de recherche car des modes d'interactions présentant un RMSD inférieur à 2 R par rapport à la structure cristalline ont été classés premier pour 68% des complexes. Lorsque les cinq meilleures solutions sont prises en compte, le taux de succès grimpe à 78%, et 92% lorsque la totalité de la dernière génération est prise en compte. La plupart des erreurs de prédiction sont imputables à la présence de contacts cristallins. Depuis, EADock a été utilisé pour comprendre les mécanismes moléculaires impliqués dans la régulation de la Na,K ATPase et dans l'activation du peroxisome proliferatoractivated receptor a (PPARa). Il a également permis de décrire l'interaction de polluants couramment rencontrés sur PPARy, ainsi que l'influence de la métabolisation de l'Imatinib (PA anticancéreux) sur la fixation à la kinase Bcr-Abl. Une approche basée sur la prédiction des interactions de fragments moléculaires avec protéine cible est également proposée. Elle a permis la découverte de nouveaux ligands peptidiques de PPARa et de l'intégrine a5ß1. Dans les deux cas, l'activité de ces nouveaux peptides est comparable à celles de ligands bien établis, comme le Wy14,643 pour le premier, et le Cilengitide (PA anticancéreux) pour la seconde.

Weed or wheel! FMRI, behavioural, and toxicological investigations of how cannabis smoking affects skills necessary for driving.

Marijuana is the most widely used illicit drug, however its effects on cognitive functions underling safe driving remain mostly unexplored. Our goal was to evaluate the impact of cannabis on the driving ability of occasional smokers, by investigating changes in the brain network involved in a tracking task. The subject characteristics, the percentage of Δ(9)-Tetrahydrocannabinol in the joint, and the inhaled dose were in accordance with real-life conditions. Thirty-one male volunteers were enrolled in this study that includes clinical and toxicological aspects together with functional magnetic resonance imaging of the brain and measurements of psychomotor skills. The fMRI paradigm was based on a visuo-motor tracking task, alternating active tracking blocks with passive tracking viewing and rest condition. We show that cannabis smoking, even at low Δ(9)-Tetrahydrocannabinol blood concentrations, decreases psychomotor skills and alters the activity of the brain networks involved in cognition. The relative decrease of Blood Oxygen Level Dependent response (BOLD) after cannabis smoking in the anterior insula, dorsomedial thalamus, and striatum compared to placebo smoking suggests an alteration of the network involved in saliency detection. In addition, the decrease of BOLD response in the right superior parietal cortex and in the dorsolateral prefrontal cortex indicates the involvement of the Control Executive network known to operate once the saliencies are identified. Furthermore, cannabis increases activity in the rostral anterior cingulate cortex and ventromedial prefrontal cortices, suggesting an increase in self-oriented mental activity. Subjects are more attracted by intrapersonal stimuli ("self") and fail to attend to task performance, leading to an insufficient allocation of task-oriented resources and to sub-optimal performance. These effects correlate with the subjective feeling of confusion rather than with the blood level of Δ(9)-Tetrahydrocannabinol. These findings bolster the zero-tolerance policy adopted in several countries that prohibits the presence of any amount of drugs in blood while driving.

From geriatric service to primary care: a retrospective pharmaceutical analysis of the medical prescription

Introduction The population of elderly persons is increasing andnegative outcomes due to polymedication are frequent. Discrepanciesin information about medication are frequent when older persons aretransitioning from hospital to home, increasing the risk of hospitalreadmission. The aims of this study were a) to determine discrepanciesin medical regimen indicated in two official discharge documents(DS = discharge summary, DP=discharge prescription); b) to characterizethe pharmacotherapy prescribed in older patients dischargedfrom a geriatric service.Materials & Methods Elderly patients (N=230) discharged from thegeriatric service (CHUV, Lausanne) over a 6-month period (January toJune 2009) were selected. Community pharmacists compared DS andDP to identify discrepancies including (a) drugs' name; (b) schedule ofadministration, dosage, frequency, prn prescription, treatment durationand galenic formulation. Beers' criteria were applied to identifypotentially inappropriate drugs and a descriptive analysis of drug costs,prescription profiles and generics were also performed.Results On average, patients were 82 ± 7 years old and stayed23.0 ± 11.6 days in the geriatric service. The delay between the datesof patient's discharge with the DP and the sending of the DS to hisgeneral physician averaged 14.0 ± 7.5 days (range 1-55). The DPhad an average of 10.0 ± 3.3 drugs (range 2-19). 77% of patients hadat least one discrepancy. A drug was missing on the DS in 57.8% ofpatients and 19.6% had a missing prn prescription. Among the 2312drugs prescribed, 3% belonged to Beers' list. They were prescribed to61 patients (26.5%), with 6 patients cumulating two Beers' potentiallyinappropriate drugs in their treatment. Analgesics (85% of thepatients), anticoagulants (80%), mineral supplements (77%), laxatives(52%) and antihypertensives (46%) were the drug classes most frequentlyprescribed. Mean costs of treatment as per DP was160.4 ± 179.4 Euros. Generic prescription represented more than 5%of the costs for 3 therapeutic classes (cholesterol-lowering agents(64%), antihypertensives (50%) and antidepressants (47%)).Discussion & Conclusion The high discrepancy rate between medicationlisted in the DP and the DS highlights a need for safetyimprovement. Potential benefits are expected from reinforced pharmacist-physician collaboration in transition from hospital to primarycare. In addition, even though Beers' criteria are questionable, thedrugs prescribed in this already fragile population, and the potentialopportunities of economical optimizations, are advocating thedevelopment and the scientific evaluation of a structured advancedcollaborative pharmacy practice service. This foresees improvedeffectiveness, safety and efficiency in the medication management ofelderly persons.

The direct effects of tacrolimus and cyclosporin A on isolated human islets: A functional, survival and gene expression study.

The use of immunosuppressive drugs in transplanted patients is associated with the development of diabetes, possibly due to β-cell toxicity. To better understand the mechanisms leading to post-transplant diabetes, we investigated the actions of prolonged exposure of isolated human islets to therapeutical levels of tacrolimus (Tac) or cyclosporin A (CsA). Islets were isolated from the pancreas of multiorgan donors by enzymatic digestion and density gradient centrifugation. Functional, survival and molecular studies were then performed after 4 days of incubation with therapeutical concentrations of Tac or  CsA. Glucose-induced insulin secretion was significantly decreased in Tac, but not in CsA exposed islets, which was associated with a reduction of the amount of insulin granules as shown by electron microscopy. The percentage of apoptotic β-cells was higher in Tac than CsA exposed islets. Microarray experiments followed by Gene Set Enrichment Analysis revealed that gene expression was more markedly affected upon Tac treatment. In conclusion, Tac and CsA affect features of beta-cell differently, with several changes occurring at the molecular level.

Nouveaux traitements de l'hépatite C: aspects pharmacologiques et potentiel d'interaction [New hepatitis C treatments: pharmacological considerations and potential for drug interactions].

Progress in the understanding of the hepatitis C virus life cycle allowed the development of new, very promising antiviral therapies. Although these new drugs have a favourable profile in terms of efficacy, tolerance and interaction potential, their prescription in the setting of comedication and impaired renal or hepatic function remains a challenge. Here, we provide a summary of pharmacological considerations, focusing on sofosbuvir, simeprevir and daclatasvir. A better understanding of their metabolic pathways and transporters may help the prescriber to identify and manage drug interactions especially in patients under immunosuppressive or anti-HIV therapy. Recommendations for the prescription of these drugs in specific situations are also discussed.

Antihypertensive Combination Treatment: State of the Art.

An adequate control of blood pressure is essential to reduce the risk of target organ damages and cardiovascular events in patients with hypertension. Yet, it is well recognized that a substantial proportion of treated patients remain hypertensive despite treatment. Several reasons have been evoked to explain why so many patients are not adequately controlled. Among them, medical inertia, a poor long-term adherence, and the need to prescribe several antihypertensive drugs to reach the target blood pressure have been identified as major limitations to the success of antihypertensive therapy. In this context, the use of single-pill combinations (SPC) containing two or three drugs in one pill has an important role in reducing the impact of some of these issues. Indeed, the use of SPC enables to reduce the pill burden and to improve the treatment efficacy without increasing the incidence of side effects. However, besides their major advantages, SPC have also some limitations such as a possible lack of flexibility or a higher cost. The purpose of this review is to discuss the place of SPC in the actual management of hypertension. The active development of new single-pill combinations in last years can be considered as a significant improvement in the physicians' capacity to treat hypertension effectively.

Poly-N-acetyl glucosamine nanofibers for negative-pressure wound therapies.

The wound healing promoting effect of negative wound pressure therapies (NPWT) takes place at the wound interface. The use of bioactive substances at this site represents a major research area for the development of future NPWT therapies. To assess wound healing kinetics in pressure ulcers treated by NPWT with or without the use of a thin interface membrane consisting of poly-N-acetyl glucosamine nanofibers (sNAG) a prospective randomized clinical trial was performed. The safety of the combination of NPWT and sNAG was also assessed in patients treated with antiplatelet drugs. In the performed study, the combination of NPWT and sNAG in 10 patients compared to NPWT alone in 10 patients promoted wound healing due to an improved contraction of the wound margins (p = 0.05) without a change in wound epithelization. In 6 patients treated with antiplatelet drugs no increased wound bleeding was observed in patients treated by NPWT and sNAG. In conclusion, the application of thin membranes of sNAG nanofibers at the wound interface using NPWT was safe and augmented the action of NPWT leading to improved wound healing due to a stimulation of wound contraction.

Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

Contribution of estrogen and GluN2B subunit to the HtraKO mouse phenotype

Htr1a is one of the most widespread serotonin receptor across the brain, strongly expressed in CAI region of hippocampus. Our laboratory studies the phenotypic alteration in 5HTla- deficient mice (Htr1aK0), characterized an abnormal anxious-like behavior. Our aim is to evaluate the regulation of this cognitive process by understanding the circuitry involved. This phenotype sets up early during development and has durable effect in adulthood. Our laboratory showed that adult Htr1aK0 male mice displaying exuberant dendritic growth of oblique dendrites in a specific layer of a CAI pyramidal neurons, the stratum radiatum. Application of drugs in organotypic cultures and by in vivo injections revealed that GluN2B, a subunit of NMDA receptor highly expressed during development, is responsible for this dendritic exuberance. Immunohistochemistry highlighted in particular a synaptic enrichment of GluN2B in stratum radiatum of Htr1aK0 CAI pyramidal neurons at puberty. Finally, original analysis of Htr1aK0 mouse behavior showed a different response to anxiety between male and female. Htr1a activation down-regulates the CaMKII activity in the CAI pyramidal neurons. CaMKII directly favors the membrane conductance and stability of GluN2B at the synapse. In the context of the Htr1aK0 mouse, GluN2B is the final common pathway of our phenotype. This subunit is well known to regulate the threshold of LTD/LTP and the dendritogenesis during development. In my thesis, I establish a link between the gender differences in the morphology and the physiology in the Htr1aK0 mice during development to understand how these characteristics shape the circuit with prominent cognitive impacts in adulthood. My study highlighted that during development, Htr1aK0 male mice show a constant increase of the dendritic growth of oblique dendrites from early ages until adulthood associated with an increased physiological impact of altered GluN2A/GluN2B ratio. Whereas during puberty, synaptic contribution of GluN2B to NMDA response is higher in Htr1aK0 compared to WT male mice, this ratio comes back to normal values towards adulthood. However, this recovery of the ratio of GluN2A/GluN2B located at the synaptic level is concomitant with the lateral diffusion of excess GluN2B subunits, leading to extrasynaptic enrichment. The main impact was a lowering of the LTP threshold characterized by strong increased potentiation of synaptic strength after 5 Hz low frequency stimulation. Moreover, the extrasynaptic GluN2B overexpression leads to a shift of the maturation phase switch explaining the exuberant morphology. However, Htr1aK0 females characterized during the 3 first weeks of development by an increase of the dendritic growth of oblique dendrites showed starting at puberty that the dendrite arborization returns progressively to WT values. The physiological impact of GluN2B was investigated and directly linked to this morphology, since Htr1aK0 female mice does not show alteration of the synaptic strength during development. These observations show a compensation occurring in Htr1aK0 female, responsible for a rescue of the phenotype morphologically, physiologically and to be tested behaviorally. We highlighted then the biological processes underlying this compensation. During development, sexual hormones such as testosterone and estrogen are responsible to induce sexual differentiation of specific brain regions. I demonstrated that estrogen, but not testosterone, was able to reduce both in vitro and in vivo the dendritic arborization early during development, through activation of GPER-1, a G-coupled protein estrogen receptor, which phenocopy the activation of Htr1a by reducing GluN2B conductance and stability. I then identified a pathway, parallel to Htr1a, able to regulate GluN2B and responsible for the morphological and physiological phenotype in Htr1aK0 female mice. The specific rise of estrogen occurring at puberty in female is responsible for the compensation observed and induces a late rescue of the Htr1aK0 phenotype by activation GPER-1. -- Htr1a est un des récepteurs à la sérotonine les plus répandus dans le cerveau, fortement exprimé dans la région CAI de l'hippocampe. Notre laboratoire étudie les altérations phénotypiques de souris déficientes pour ce récepteur (Htr1aK0), caractérisées par un comportement avec des traits anxieux. Notre objectif est d'évaluer la régulation de ces processus cognitifs en comprenant les connexions nerveuses impliquées. Ce phénotype se met en place tôt au cours du développement et présente un effet durable à l'âge adulte. Notre laboratoire a montré que les souris Htr1aK0 mâles adultes se caractérisent par une croissance exubérante des dendrites obliques dans une couche spécifique des neurones pyramidaux du CAI, le stratum radiatum. L'application de drogues sur cultures organotypiques et par injections in vivo ont révélé que GluN2B, une sous-unité du récepteur NMDA fortement exprimée au cours du développement, est responsable de cette exubérance dendritique. Des expériences d'immunohistochimie ont notamment mis en évidence un enrichissement synaptique de GluN2B durant la puberté dans le stratum radiatum des neurones de la région CAI des souris Htr1aK0. Finalement, l'analyse originale du comportement des souris Htr1aK0 a montré une différence de réponse à l'anxiété entre mâles et femelles. L'activation de Htr1a diminue l'activité de la CaMKII dans les neurones pyramidaux du CAI. La CaMKII favorise directement la conductance et la stabilité de la sous-unité GluN2B à la synapse. Dans le contexte de la souris Htr1aK0, GluN2B est le « médiateur » de notre phénotype. Cette sous-unité est particulièrement connue pour réguler le seuil de LTD-LTP ainsi que la dendritogénèse durant le développement. Dans ma thèse, j'ai établi le lien entre les différences dépendant du genre dans la morphologie et physiologie des souris Htr1aK0 au cours du développement pour comprendre comment ces caractéristiques modulent le circuit accompagnés d'impacts cognitifs visibles à l'âge adulte. Mon étude a mis en évidence que durant le développement, les souris mâles Htr1aK0 montrent une constante augmentation de la croissance des dendrites obliques entre les premières semaines et l'âge adulte associée à une augmentation de l'impact physiologique du ratio GluN2A/GluN2B altéré. Alors que durant la puberté, la contribution synaptique de GluN2B à la réponse NMDA est plus haute chez la souris mâle Htr1aK0 que le WT, ce ratio revient à des valeurs normales à l'âge adulte. Cependant, cette récupération de l'expression du récepteur au niveau synaptique est concomitante avec la diffusion des sous-unités GluN2B excédantes, amenant alors à un enrichissement extrasynaptique. Le principal impact est une diminution du seuil de la LTP caractérisée par une forte potentiation de la plasticité après une stimulation basse fréquence à 5 Hz. De plus, la surexpression des GluN2B extrasynaptiques conduit à un décalage de la bascule à la phase de maturation, expliquant la morphologie dendritique exubérante. Cependant, les femelles Htr1aK0 initialement caractérisées pendant les 3 premières semaines du développement par une augmentation de la croissance des dendrites obliques montrent à partir de la puberté que cette arborisation dendritique retourne à des valeurs WT. L'impact physiologique de GLuN2B a été investigué et mis en lien avec cette morphologie, étant donné que les femelles Htr1aK0 ne montrent pas d'altération de la plasticité durant le développement. Ces observations montrent une compensation se produisant chez la femelle Htr1aK0, responsable d'une récupération du phénotype morphologique, physiologique et peut-être comportemental. Nous avons souligné les processus biologiques sous-jacent à cette compensation. Au cours du développement, les hormones sexuelles telles que la testostérone et l'estrogène sont responsables de la différentiation sexuelle de régions du cerveau spécifiques. J'ai démontré que l'estrogène, mais pas la testostérone, était capable de réduire in vitro et in vivo l'arborisation dendritique tôt dans le développement au travers de l'activation du récepteur GPER-1, un récepteur aux estrogènes couplés à un protéine G, qui phénocopie l'activation de Htr1a en réduisant la conductance et la stabilité de GluN2B à la membrane. J'ai identifié une voie de signalisation parallèle à celle de Htr1a, capable de réguler GluN2B et responsable du phénotype morphologique et physiologique de la souris femelle Htr1aK0. La montée spécifique d'estrogène se déroulant à la puberté chez la femelle est responsable de cette compensation et implique une récupération tardive du phénotype Htr1aK0 par l'activation de GPER-1.