A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry.

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients' follow-up. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 100 microL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1-104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3-9.4%), accurate (-9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.

Esophageal and pharyngeal strictures: report on 1,862 endoscopic dilatations using the Savary-Gilliard technique

Treatment of symptomatic pharyngeal and esophageal strictures requires endoscopic dilatation. The Savary-Gilliard bougienage was developed by our department and has been used since 1980 for this purpose. We report our experience using this technique. The records of patients seen from January 1, 1963 to December 31, 2005, who had pharyngeal and esophageal strictures needing dilatation, were reviewed. The prevalence of different etiologies, and the incidence of complications using the Savary-Gilliard dilators were assessed. Efficiency of dilatation was assessed over a 17-year segment of this period, using number of dilatations and time intervals between dilatations until resolution of symptoms as outcome measures. Of the 2,652 pharyngeal and esophageal strictures reviewed, 90% were of organic origin (45% benign and 55% malignant stenoses), and 10% were of functional etiology. The most common etiologies were peptic strictures before the era of proton pump inhibitors, and postoperative anastomotic strictures thereafter. A total of 1,862 dilatations using the Savary-Gilliard technique were analyzed. Complication and mortality rates were 0.18 and 0.09% for benign and 4.58 and 0.81% for malignant etiologies, respectively. The number of dilatations per stricture and the time interval between different sessions were dependent on the type of strictures, varying from 1 to 23 dilatations and 7 days to 16 years, respectively. Pharyngeal and esophageal dilatations using the Savary-Gilliard technique were safe when used together with fluoroscopy. Overall, the efficiency of the dilatation procedure was good, but some types of strictures (e.g., caustic, post-surgical and/or post radiotherapy) were refractory to treatment and required repeated dilatations.

When several queens reign: how polygyny affects social life in ants

Astract: The aim of this thesis was to investigate how the presence of multiple queens (polygyny) affects social organization in colonies of the ant Formica exsecta. This is important because polygyny results in reduced relatedness among colony members and therefore reflects a potential paradox for altruistic cooperation being explained by inclusive fitness theory. The reason for this is that workers in polygynous colonies rear no longer only their siblings (high inclusive fitness gain) but also more distantly ox even unrelated brood (low or no inclusive fitness gain). All research projects conducted in this thesis are novel and significant contributions to the understanding of the social evolution of insect societies. We used a mixture of experimental and observational methodologies in laboratory and field colonies of F. exsecta to examine four important aspects of social life that are impacted by polygyny. First, we investigated the influence of queen number on colony sex allocation and found that the number of queens present in a colony significantly affects colony sex ratio investment. The data were consistent with the queen-replenishment hypothesis, which is based on the observation that newly mated queens are often recruited back to their parental nest. According to this theory, colonies containing many queens should only produce males due to local resource competition (i.e. related queens compete for common resources), whereas colonies hosting few queens benefit most from producing new queens to ensure colony survival. Second, we examined how reproduction is partitioned among nestmate queens. We detected a novel pattern of reproductive partitioning whereby a high proportion of queens were completely specialized in the production of only a subset of offspring classes produced within a colony, which might translate into great differences in reproductive success between queens. Third, we could demonstrate that F. exsecta workers indiscriminately reared highly related and unrelated brood although such nepotistic behaviour (preferential rearing of relatives) would be predicted by inclusive fitness theory. The absence of nepotism is probably best explained by its negative effects on overall colony efficiency. Finally, we conducted a detailed population genetic analysis, which revealed that the genetic population structure is different for queens and workers. Our data were best explained with queens forming family-based groups (multicolonial population structure), whereas workers from several nests seemed to be grouped into larger unites (unicolonial population structure) with workers moving freely between neighbouring nests. Altogether, the presented work significantly increased our understanding of the complex organization of polygynous social insect colonies and shows how an important life history trait such as queen number affects social organization at various levels. Résumé: Le but de cette thèse était d'étudier comment la présence de plusieurs reines par colonie (polygynie) influence la vie sociale chez la fourmi Formica exsecta. Ce sujet est important parce que la polygynie chez les insectes sociaux présente un passible paradoxe au niveau de la théorie du "fitness inclusive". Ce paradoxe est basé sur le fait que les ouvrières n'élèvent plus uniquement leurs frères et soeurs (gain de "fitness inclusive" maximale), mais également des individus moins ou pas du tout apparentés (gain de "fitness inclusive" réduit ou absent). Tous les projets de recherche présentés au cours de cette thèse apportent une meilleure compréhension et connaissance au niveau de l'organisation des colonies chez les insectes sociaux. Nous avons employé des méthodes d'observation et de laboratoire afin de mettre en évidence des aspects importants de la vie sociale chez les fourmis influencés par la polygynie. Quatre aspects ont été caractérisés : (1) l'influence du nombre de reines sur le sexe ratio produit par la colonie. Nous avons démontré que les colonies contenant beaucoup de reines produisaient rarement des reines tandis que les colonies contenant peu de reines souvent investissaient beaucoup de ressources dans la production des reines. Ces résultats sont en accord avec la "queen-replenishment hypothesis" qui est basé sur l'observation que les nouvelles reines sont recrutées dans la colonie où elles étaient nées. Cette hypothèse postule que la production des reines est défavorable dans les colonies contenant beaucoup de reines, parce que ces reines apparentées, rentrent en compétition pour des ressources communes. Au contraire, la production des reines est favorable dans des colonies contenant peu de reines afin d'assurer la survie de la colonie ; (2) comment les reines dans une colonie répartissent leur reproduction. Nous avons mis en évidence un nouveau pattern de cette répartition où une grande proportion de reines est complètement spécialisée dans la production d'un seul type de couvain ce qui probablement aboutit à des différences significatives entre reines dans le succès reproducteur ; (3) la capacité des ouvrières à discriminer un couvain de soeur d'un couvain non apparenté. Les résultats ont montré que les ouvrières ne font pas de discrimination entre le couvain de soeur et le couvain non apparenté ce qui n'est pas en accord avec la théorie de la "fitness inclusive". Cette absence de discrimination est probablement due à des effets négatifs comme par exemple la diminution de la production du couvain; (4) la structure génétique d'une population de F. exsecta. Nous avons mis en évidence que la structure génétique entre des groupes de reines est significativement différente de la structure génétique entre des groupes d'ouvrières. Les données suggèrent que les reines forment des groupes basés sur une structure familiale tandis que les ouvrières sont groupées dans des unités plus grandes.

A spatially explicit life cycle inventory of the global textile chain

Life cycle analyses (LCA) approaches require adaptation to reflect the increasing delocalization of production to emerging countries. This work addresses this challenge by establishing a country-level, spatially explicit life cycle inventory (LCI). This study comprises three separate dimensions. The first dimension is spatial: processes and emissions are allocated to the country in which they take place and modeled to take into account local factors. Emerging economies China and India are the location of production, the consumption occurs in Germany, an Organisation for Economic Cooperation and Development country. The second dimension is the product level: we consider two distinct textile garments, a cotton T-shirt and a polyester jacket, in order to highlight potential differences in the production and use phases. The third dimension is the inventory composition: we track CO2, SO2, NO (x), and particulates, four major atmospheric pollutants, as well as energy use. This third dimension enriches the analysis of the spatial differentiation (first dimension) and distinct products (second dimension). We describe the textile production and use processes and define a functional unit for a garment. We then model important processes using a hierarchy of preferential data sources. We place special emphasis on the modeling of the principal local energy processes: electricity and transport in emerging countries. The spatially explicit inventory is disaggregated by country of location of the emissions and analyzed according to the dimensions of the study: location, product, and pollutant. The inventory shows striking differences between the two products considered as well as between the different pollutants considered. For the T-shirt, over 70% of the energy use and CO2 emissions occur in the consuming country, whereas for the jacket, more than 70% occur in the producing country. This reversal of proportions is due to differences in the use phase of the garments. For SO2, in contrast, over two thirds of the emissions occur in the country of production for both T-shirt and jacket. The difference in emission patterns between CO2 and SO2 is due to local electricity processes, justifying our emphasis on local energy infrastructure. The complexity of considering differences in location, product, and pollutant is rewarded by a much richer understanding of a global production-consumption chain. The inclusion of two different products in the LCI highlights the importance of the definition of a product's functional unit in the analysis and implications of results. Several use-phase scenarios demonstrate the importance of consumer behavior over equipment efficiency. The spatial emission patterns of the different pollutants allow us to understand the role of various energy infrastructure elements. The emission patterns furthermore inform the debate on the Environmental Kuznets Curve, which applies only to pollutants which can be easily filtered and does not take into account the effects of production displacement. We also discuss the appropriateness and limitations of applying the LCA methodology in a global context, especially in developing countries. Our spatial LCI method yields important insights in the quantity and pattern of emissions due to different product life cycle stages, dependent on the local technology, emphasizing the importance of consumer behavior. From a life cycle perspective, consumer education promoting air-drying and cool washing is more important than efficient appliances. Spatial LCI with country-specific data is a promising method, necessary for the challenges of globalized production-consumption chains. We recommend inventory reporting of final energy forms, such as electricity, and modular LCA databases, which would allow the easy modification of underlying energy infrastructure.

Role of genetic and social factors in modulating the efficiency of division of labour in social insects

Summary : Division of labour is one of the most fascinating aspects of social insects. The efficient allocation of individuals to a multitude of different tasks requires a dynamic adjustment in response to the demands of a changing environment. A considerable number of theoretical models have focussed on identifying the mechanisms allowing colonies to perform efficient task allocation. The large majority of these models are built on the observation that individuals in a colony vary in their propensity (response threshold) to perform different tasks. Since individuals with a low threshold for a given task stimulus are more likely to perform that task than individuals with a high threshold, infra-colony variation in individual thresholds results in colony division of labour. These theoretical models suggest that variation in individual thresholds is affected by the within-colony genetic diversity. However, the models have not considered the genetic architecture underlying the individual response thresholds. This is important because a better understanding of division of labour requires determining how genotypic variation relates to differences in infra-colony response threshold distributions. In this thesis, we investigated the combined influence on task allocation efficiency of both, the within-colony genetic variability (stemming from variation in the number of matings by queens) and the number of genes underlying the response thresholds. We used an agent-based simulator to model a situation where workers in a colony had to perform either a regulatory task (where the amount of a given food item in the colony had to be maintained within predefined bounds) or a foraging task (where the quantity of a second type of food item collected had to be the highest possible). The performance of colonies was a function of workers being able to perform both tasks efficiently. To study the effect of within-colony genetic diversity, we compared the performance of colonies with queens mated with varying number of males. On the other hand, the influence of genetic architecture was investigated by varying the number of loci underlying the response threshold of the foraging and regulatory tasks. Artificial evolution was used to evolve the allelic values underlying the tasks thresholds. The results revealed that multiple matings always translated into higher colony performance, whatever the number of loci encoding the thresholds of the regulatory and foraging tasks. However, the beneficial effect of additional matings was particularly important when the genetic architecture of queens comprised one or few genes for the foraging task's threshold. By contrast, higher number of genes encoding the foraging task reduced colony performance with the detrimental effect being stronger when queens had mated with several males. Finally, the number of genes determining the threshold for the regulatory task only had a minor but incremental effect on colony performance. Overall, our numerical experiments indicate the importance of considering the effects of queen mating frequency, genetic architecture underlying task thresholds and the type of task performed when investigating the factors regulating the efficiency of division of labour in social insects. In this thesis we also investigate the task allocation efficiency of response threshold models and compare them with neural networks. While response threshold models are widely used amongst theoretical biologists interested in division of labour in social insects, our simulation reveals that they perform poorly compared to a neural network model. A major shortcoming of response thresholds is that they fail at one of the most crucial requirement of division of labour, the ability of individuals in a colony to efficiently switch between tasks under varying environmental conditions. Moreover, the intrinsic properties of the threshold models are that they lead to a large proportion of idle workers. Our results highlight these limitations of the response threshold models and provide an adequate substitute. Altogether, the experiments presented in this thesis provide novel contributions to the understanding of how division of labour in social insects is influenced by queen mating frequency and genetic architecture underlying worker task thresholds. Moreover, the thesis also provides a novel model of the mechanisms underlying worker task allocation that maybe more generally applicable than the widely used response threshold models. Resumé : La répartition du travail est l'un des aspects les plus fascinants des insectes vivant en société. Une allocation efficace de la multitude de différentes tâches entre individus demande un ajustement dynamique afin de répondre aux exigences d'un environnement en constant changement. Un nombre considérable de modèles théoriques se sont attachés à identifier les mécanismes permettant aux colonies d'effectuer une allocation efficace des tâches. La grande majorité des ces modèles sont basés sur le constat que les individus d'une même colonie diffèrent dans leur propension (inclination à répondre) à effectuer différentes tâches. Etant donné que les individus possédant un faible seuil de réponse à un stimulus associé à une tâche donnée sont plus disposés à effectuer cette dernière que les individus possédant un seuil élevé, les différences de seuils parmi les individus vivant au sein d'une même colonie mènent à une certaine répartition du travail. Ces modèles théoriques suggèrent que la variation des seuils des individus est affectée par la diversité génétique propre à la colonie. Cependant, ces modèles ne considèrent pas la structure génétique qui est à la base des seuils de réponse individuels. Ceci est très important car une meilleure compréhension de la répartition du travail requière de déterminer de quelle manière les variations génotypiques sont associées aux différentes distributions de seuils de réponse à l'intérieur d'une même colonie. Dans le cadre de cette thèse, nous étudions l'influence combinée de la variabilité génétique d'une colonie (qui prend son origine dans la variation du nombre d'accouplements des reines) avec le nombre de gènes supportant les seuils de réponse, vis-à-vis de la performance de l'allocation des tâches. Nous avons utilisé un simulateur basé sur des agents pour modéliser une situation où les travailleurs d'une colonie devaient accomplir une tâche de régulation (1a quantité d'une nourriture donnée doit être maintenue à l'intérieur d'un certain intervalle) ou une tâche de recherche de nourriture (la quantité d'une certaine nourriture doit être accumulée autant que possible). Dans ce contexte, 'efficacité des colonies tient en partie des travailleurs qui sont capable d'effectuer les deux tâches de manière efficace. Pour étudier l'effet de la diversité génétique d'une colonie, nous comparons l'efficacité des colonies possédant des reines qui s'accouplent avec un nombre variant de mâles. D'autre part, l'influence de la structure génétique a été étudiée en variant le nombre de loci à la base du seuil de réponse des deux tâches de régulation et de recherche de nourriture. Une évolution artificielle a été réalisée pour évoluer les valeurs alléliques qui sont à l'origine de ces seuils de réponse. Les résultats ont révélé que de nombreux accouplements se traduisaient toujours en une plus grande performance de la colonie, quelque soit le nombre de loci encodant les seuils des tâches de régulation et de recherche de nourriture. Cependant, les effets bénéfiques d'accouplements additionnels ont été particulièrement important lorsque la structure génétique des reines comprenait un ou quelques gènes pour le seuil de réponse pour la tâche de recherche de nourriture. D'autre part, un nombre plus élevé de gènes encodant la tâche de recherche de nourriture a diminué la performance de la colonie avec un effet nuisible d'autant plus fort lorsque les reines s'accouplent avec plusieurs mâles. Finalement, le nombre de gènes déterminant le seuil pour la tâche de régulation eu seulement un effet mineur mais incrémental sur la performance de la colonie. Pour conclure, nos expériences numériques révèlent l'importance de considérer les effets associés à la fréquence d'accouplement des reines, à la structure génétique qui est à l'origine des seuils de réponse pour les tâches ainsi qu'au type de tâche effectué au moment d'étudier les facteurs qui régulent l'efficacité de la répartition du travail chez les insectes vivant en communauté. Dans cette thèse, nous étudions l'efficacité de l'allocation des tâches des modèles prenant en compte des seuils de réponses, et les comparons à des réseaux de neurones. Alors que les modèles basés sur des seuils de réponse sont couramment utilisés parmi les biologistes intéressés par la répartition des tâches chez les insectes vivant en société, notre simulation montre qu'ils se révèlent peu efficace comparé à un modèle faisant usage de réseaux de neurones. Un point faible majeur des seuils de réponse est qu'ils échouent sur un point crucial nécessaire à la répartition des tâches, la capacité des individus d'une colonie à commuter efficacement entre des tâches soumises à des conditions environnementales changeantes. De plus, les propriétés intrinsèques des modèles basés sur l'utilisation de seuils conduisent à de larges populations de travailleurs inactifs. Nos résultats mettent en évidence les limites de ces modèles basés sur l'utilisation de seuils et fournissent un substitut adéquat. Ensemble, les expériences présentées dans cette thèse fournissent de nouvelles contributions pour comprendre comment la répartition du travail chez les insectes vivant en société est influencée par la fréquence d'accouplements des reines ainsi que par la structure génétique qui est à l'origine, pour un travailleur, du seuil de réponse pour une tâche. De plus, cette thèse fournit également un nouveau modèle décrivant les mécanismes qui sont à l'origine de l'allocation des tâches entre travailleurs, mécanismes qui peuvent être appliqué de manière plus générale que ceux couramment utilisés et basés sur des seuils de réponse.

Efficiency of recombinant human TNF in human cancer therapy.

Recombinant human tumour necrosis factor (TNF) has a selective effect on angiogenic vessels in tumours. Given that it induces vasoplegia, its clinical use has been limited to administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs. When combined with the alkylating agent melphalan, a single ILP produces a very high objective response rate. In melanoma, the complete response (CR) rate is around 80% and the overall objective response rate greater than 90%. In soft tissue sarcomas that are inextirpable, ILP is a neoadjuvant treatment resulting in limb salvage in 80% of the cases. The CR rate averages 20% and the objective response rate is around 80%. The mode of action of TNF-based ILP involves two distinct and successive effects on the tumour-associated vasculature: first, an increase in endothelium permeability leading to improved chemotherapy penetration within the tumour tissue, and second, a selective killing of angiogenic endothelial cells resulting in tumour vessel destruction. The mechanism whereby these events occur involves rapid (of the order of minutes) perturbation of cell-cell adhesive junctions and inhibition of alphavbeta3 integrin signalling in tumour-associated vessels, followed by massive death of endothelial cells and tumour vascular collapse 24 hours later. New, promising approaches for the systemic use of TNF in cancer therapy include TNF targeting by means of single chain antibodies or endothelial cell ligands, or combined administration with drugs perturbing integrin-dependent signalling and sensitizing angiogenic endothelial cells to TNF-induced death.

Efficiency of coronary artery disease screening guidelines in asymptomatic and atypical chest pain type 2 diabetic patients

RESUME Objectifs. Évaluer la prévalence de maladie coronarienne chez les patients diabétiques de type 2 asymptomatiques ou avec angor atypique selon les recommandations américaines de l'American Diabetes Association et de l'American College of Cardiology. Méthodes. Cent cinquante-quatre patients diabétiques de type 2 asymptomatiques ou avec angor atypique et présentant au minimum 2 facteurs de risque cardio-vasculaires additionnels ont été dépistés par échocardiographie de stress (71%, n=109), scintigraphie myocardique de perfusion (26%, n=40) ou l'association des 2 examens (3%, n=5). Résultats. L'échocardiographie de stress s'est révélée positive chez 16 patients (14%) et 14 ont eu une coronarographie révélant des sténoses significatives chez 12 (86%). La scintigraphie myocardique de perfusion était positive chez 16 patients (36%). Huit patients ont eu une coronarographie et 4 (50%) présentaient des sténoses significatives. Au total, 31 patients (20%) ont montré des signes d'ischémie lors de l'examen non-invasif et 15 (10%) ont présenté des sténoses significatives à la coronarographie. Les facteurs prédictifs indépendants de la maladie coronarienne étaient le tabagisme (OR 6.5, p=0.05), la microalbuminurie (OR 3.9, p=0.03), ainsi que les souffles fémoraux (OR 17.1, p=0.008). Conclusions. En suivant les recommandations américaines, un patient sur cinq présentait une ischémie lors des examens non-invasifs, tandis que 1 sur 10 avait des sténoses significatives à la coronarographie. L'analyse multivariée suggère que des marqueurs des complications micro- et macro-vasculaires en combinaison avec des facteurs de risque cardio-vasculaire classiques pourraient améliorer le pouvoir diagnostic de ces recommandations. SUMMARY Aims. We evaluated the prevalence of coronary artery disease in asymptomatic and atypical chest pain type 2 diabetic patients according to the American Diabetes Association and American College of Cardiology guidelines. Methods. Asymptomatic or atypical chest pain type 2 diabetic patients (n=154), with at least two additional cardiovascular risk factors, were screened for coronary artery disease using stress echocardiography (71%, n=109), myocardial perfusion imaging (26%, n=40) or both (3%, n=5). Results. Stress echocardiography was positive in 16 patients (14%) and 14 had a coronary angiography, revealing significant stenoses in 12 (86%). Myocardial perfusion imaging was positive in 16 patients (36%). Eight patients underwent angiography and 4 (50%) presented significant stenoses. Overall, 31 patients (20%) demonstrated signs of ischemia on non-invasive tests and 15 (10%) presented significant stenoses on coronary angiography. Independent predictors of coronary artery disease were smoking (OR 6.5, p=0.05), microalbuminuria (OR 3.9, p=0.03) and femoral murmur (OR 17.1, p=0.008). Conclusions. Following the guidelines, one in five diabetic patient presented ischemia on noninvasive tests, while one in ten presented significant coronary stenoses. Multivariate analysis suggests that adding markers of micro- and macro-vascular complications to classical cardiovascular risk factors may enhance the diagnostic efficiency of the guidelines.

Implementation of robotic laparoscopic cholecystectomy in a university hospital.

BACKGROUND: Robot surgery is a further step towards new potential developments in minimally invasive surgery. Surgeons must keep abreast of these new technologies and learn their limits and possibilities. Robot-assisted laparoscopic cholecystectomy has not yet been performed in our institution. The purpose of this report is to present the pathway of implementation of robotic laparoscopic cholecystectomy in a university hospital. METHODS: The Zeus(R) robot system was used. Experimental training was performed on animals. The results of our experimental training allowed us to perform our first two clinical cases. RESULTS: Robot arm set-up and trocar placement required 53 and 35 minutes. Operative time were 59 and 45 minutes respectively. The overall operative time was 112 and 80 minutes, respectively. There were no intraoperative complications. Patients were discharged from the hospital after an overnight stay. CONCLUSION: Robotic laparoscopic cholecystectomy is safe and patient recovery similar to those of standard laparoscopy. At present, there are no advantages of robotic over conventional surgery. Nevertheless, robots have the potential to revolutionise the way surgery is performed. Robot surgery is not reserved for a happy few. This technology deserves more attention because it has the potential to change the way surgery is performed.

Evaluation of prenatal ultrasound diagnosis of fetal abdominal wall defects by 19 European registries.

OBJECTIVES: To evaluate the current effectiveness of routine prenatal ultrasound screening in detecting gastroschisis and omphalocele in Europe. DESIGN: Data were collected by 19 congenital malformation registries from 11 European countries. The registries used the same epidemiological methodology and registration system. The study period was 30 months (July 1st 1996-December 31st 1998) and the total number of monitored pregnancies was 690,123. RESULTS: The sensitivity of antenatal ultrasound examination in detecting omphalocele was 75% (103/137). The mean gestational age at the first detection of an anomaly was 18 +/- 6.0 gestational weeks. The overall prenatal detection rate for gastroschisis was 83% (88/106) and the mean gestational age at diagnosis was 20 +/- 7.0 gestational weeks. Detection rates varied between registries from 25 to 100% for omphalocele and from 18 to 100% for gastroschisis. Of the 137 cases of omphalocele less than half of the cases were live births (n = 56; 41%). A high number of cases resulted in fetal deaths (n = 30; 22%) and termination of pregnancy (n = 51; 37%). Of the 106 cases of gastroschisis there were 62 (59%) live births, 13 (12%) ended with intrauterine fetal death and 31 (29%) had the pregnancies terminated. CONCLUSIONS: There is significant regional variation in detection rates in Europe reflecting different policies, equipment and the operators' experience. A high proportion of abdominal wall defects is associated with concurrent malformations, syndromes or chromosomal abnormalities, stressing the need for the introduction of repeated detailed ultrasound examination as a standard procedure. There is still a relatively high rate of elective termination of pregnancies for both defects, even in isolated cases which generally have a good prognosis after surgical repair.

The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix. Possible implications for DNA structure in vivo.

A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules. It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe. When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation. In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other. This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA. In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions. Possible implications of this observed structural transition in DNA are discussed.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Summary : Internal ribosome entry sites (IRES) are used by viruses as a strategy to bypass inhibition of cap-dependent translation that commonly results from viral infection. IRES are also used in eukaryotic cells to control mRNA translation under conditions of cellular stress (apoptosis, heat shock) or during the G2 phase of the cell cycle when general protein synthesis is inhibited. Variation in cellular expression levels has been shown to be inherited. Expression is controlled, among others, by transcriptional factors and by the efficiency of cap-mediated translation and ribosome activity. We aimed at identifying genomic determinants of variability in IRES-mediated translation of two representative IRES [Encephalomyocarditis virus (EMCV) and X-linked Inhibitor-of-Apoptosis (XIAP) IRES]. We used bicistronic lentiviral constructions expressing two fluorescent reporter transgenes. Lentiviruses were used to transduce seven different laboratory cell lines and B lymphoblastoid cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209); representing an in vitro approach to family structure allowing genome scan analyses. The relative expression of the two markers was assessed by FACS. IRES efficiency varies according to cellular background, but also varies, for a same cell type, among individuals. The control of IRES activity presents an inherited component (h2) of 0.47 and 0.36 for EMCV and XIAP IRES, respectively. A genome scan identified a suggestive Quantitative Trait Loci (LOD 2.35) involved in the control of XIAP IRES activity. Résumé : Les sites internes d'entrée des ribosomes (IRES = internal ribosome entry sites) sont utilisés par les virus comme une stratégie afin d'outrepasser l'inhibition de traduction qui résulte communément d'une infection virale. Les IRES sont également utilisés par les cellules eucaryotes pour contrôler la traduction de l'ARN messager dans des conditions de stress cellulaire (apoptose, choc thermique) ou durant la phase G2 du cycle cellulaire, situations durant lesquelles la synthèse générale des protéines est inhibée. La variation des niveaux d'expression cellulaire de transcription est un caractère héréditaire. L'expression des gènes est contrôlée entre autre par les facteurs de transcription et par l'efficacité de la traduction initiée par la coiffe ainsi que par l'activité des ribosomes. Durant cette étude nous avons eu pour but d'identifier les déterminants génomiques responsables de la variabilité de la traduction contrôlée par l'IRES. Ceci a été effectué en étudiant deux IRES représentatifs : l'IRES du virus de l'encéphalomyocardite (EMCV) et l'IRES de l'inhibiteur de l'apoptose XIAP (X-linked Inhibitor-of-Apoptosis). Nous avons utilisés des lentivirus délivrant un transgène bicistronique codant pour deux gènes rapporteurs fluorescents. Ces lentivirus ont été utilisés pour transduire sept différentes lignées cellulaires de laboratoire et des lignées cellulaires lymphoblastoïdes B du Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209) qui représentent une approche in vitro de la structure familiale et qui permettent des analyses par balayage du génome. L'expression relative des deux marqueurs fluorescents a été analysée par FACS. Nos résultats montrent que l'efficacité des IRES varie en fonction du type de cellules. Il varie aussi, pour le même type de cellules, selon les individus. Le contrôle de l'activité de l'IRES est un caractère héritable (héritabilité h2) de 0.47 et 0.36 pour les IRES de EMCV et XIAP respectivement. Le balayage du génome a permis l'identification d'un locus à effets quantitatifs [QTL Quantitative Trait Loci (LOD 2.35)] impliqué dans le contôle de l'activité de l'IRES de XIAP.

The impact of iron supplementation efficiency in female blood donors with a decreased ferritin level and no anaemia. Rationale and design of a randomised controlled trial: a study protocol.

ABSTRACT: BACKGROUND: There is no recommendation to screen ferritin level in blood donors, even though several studies have noted the high prevalence of iron deficiency after blood donation, particularly among menstruating females. Furthermore, some clinical trials have shown that non-anaemic women with unexplained fatigue may benefit from iron supplementation. Our objective is to determine the clinical effect of iron supplementation on fatigue in female blood donors without anaemia, but with a mean serum ferritin </= 30 ng/ml. METHODS/DESIGN: In a double blind randomised controlled trial, we will measure blood count and ferritin level of women under age 50 yr, who donate blood to the University Hospital of Lausanne Blood Transfusion Department, at the time of the donation and after 1 week. One hundred and forty donors with a ferritin level </= 30 ng/ml and haemoglobin level >/= 120 g/l (non-anaemic) a week after the donation will be included in the study and randomised. A one-month course of oral ferrous sulphate (80 mg/day of elemental iron) will be introduced vs. placebo. Self-reported fatigue will be measured using a visual analogue scale. Secondary outcomes are: score of fatigue (Fatigue Severity Scale), maximal aerobic power (Chester Step Test), quality of life (SF-12), and mood disorders (Prime-MD). Haemoglobin and ferritin concentration will be monitored before and after the intervention. DISCUSSION: Iron deficiency is a potential problem for all blood donors, especially menstruating women. To our knowledge, no other intervention study has yet evaluated the impact of iron supplementation on subjective symptoms after a blood donation. TRIAL REGISTRATION: NCT00689793.

Pharmacokinetic (PK)-based dosage individualization of imatinib: evaluation of efficiency and clinical usefulness

Background: Retrospective analyses suggest that personalized PK-based dosage might be useful for imatinib, as treatment response correlates with trough concentrations (Cmin) in cancer patients. Our objectives were to improve the interpretation of randomly measured concentrations and to confirm its efficiency before evaluating the clinical usefulness of systematic PK-based dosage in chronic myeloid leukemia patients. Methods and Results: A Bayesian method was validated for the prediction of individual Cmin on the basis of a single random observation, and was applied in a prospective multicenter randomized controlled clinical trial. 28 out of 56 patients were enrolled in the systematic dosage individualization arm and had 44 follow-up visits (their clinical follow-up is ongoing). PK-dose-adjustments were proposed in 39% having predicted Cmin significantly away from the target (1000 ng/ml). Recommendations were taken up by physicians in 57%, patients were considered non-compliant in 27%. Median Cmin at study inclusion was 754 ng/ml and differed significantly from the target (p=0.02, Wilcoxon test). On follow-up, Cmin was 984 ng/ml (p=0.82) in the compliant group. CV decreased from 46% to 27% (p=0.02, F-test). Conclusion: PK-based (Bayesian) dosage adjustment is able to bring individual drug exposure closer to a given therapeutic target. Its influence on therapeutic response remains to be evaluated.