Table des matières

Message du Vice-Doyen de la recherche de la Faculté de Biologie et de Médecine

Programme

Abstracts ENA

Environnement Naturel

EHU

Environnement Humain

GEN

Gènes et Environnement

IMI

Immunité et Infection

MCV

Métabolisme et Cardiovasculaire

NEU

Neurosciences et Psyché

ODE

Oncologie et Développement

THE

Procédures Thérapeutiques

Index des auteurs

Couverture : Photo du Dr Yann Bernardinelli, Département de Phyiologie, Université de Lausanne « Hippocampal astrocytes ensheeting a blood vessel.»

Message du Vice-Doyen de la Recherche Le Décanat de la Faculté de Biologie et Médecine et son Unité de la Recherche sont heureux de rassembler une nouvelle fois au sein du CHUV, la cinquième depuis 2001, tous les chercheurs de la Faculté. Les média vantent abondamment les progrès de la thérapeutique médicale ou chirurgicale et de ses interventions qui permettent d'influencer, parfois de manière spectaculaire, le cours de diverses maladies. Pour que ces gestes thérapeutiques puissent s'appliquer avec succès, il est indispensable de disposer d'un diagnostic aussi précis que possible concernant tant la nature, que la localisation et l'extension de la maladie. Il est tout aussi indispensable de pouvoir "voir" les effets des diverses formes de traitement et l'évolution de la maladie aussi précisément que possible pour permettre d'appliquer les traitements de manière optimale. C'est dans ces aspects des soins médicaux que les diverses techniques de l'imagerie biomédicale tiennent une place prépondérante, parmi un ensemble d'autres techniques diagnostiques. Les progrès dans ce domaine sont tout aussi ou même plus spectaculaires que pour d’autres méthodes thérapeutiques. Les techniques d'imagerie récentes sont plus précises, plus rapides, souvent moins invasives, Elles donnent des informations non seulement concernant les localisations de lésions mais aussi concernant les altérations métaboliques ou fonctionnelles des organes ou tissus visualisés. Elles permettent d'obtenir ainsi les informations nécessaires pour les décisions thérapeutiques, de manière plus rapide et moins douloureuse ou dangereuse pour les patients. Les progrès rapides de ces méthodes complexes impliquent que ces nouvelles techniques soient constamment testées, affinées et améliorées et rendent indispensables tant une recherche fondamentale pour la mise au point des aspects techniques qu'une recherche clinique pour l'apprentissage de leur application à l'homme. En amont des applications cliniques, les techniques d'imagerie cellulaire se développent tout aussi rapidement. Au-delà d'une simple augmentation de la résolution spatiale, les nouvelles techniques permettent d'observer de manière dynamique les composants structurels ou fonctionnels de la cellule, de les voir travailler, se déplacer, interagir. L'imagerie cellulaire est un complément indispensable à la biologie et la physiologie moléculaire. La Faculté de Biologie et médecine, au sein de l’UNIL et du CHUV, et leurs partenaires du programme "Sciences Vie et Société" sont particulièrement impliqués dans la recherche concernant l'imagerie médicale puisque cette initiative a consacré une part importante de ses ressources au développement de ce domaine de recherche avec la création du Centre d'Imagerie Bio-Médicale, le CIBM, un centre auquel participent le CHUV et l'UNIL, l'EPFL ainsi que les HUG et l'UNIGE. Le programme de la "Journée 2007" fait d'ailleurs une part importante aux contributions des partenaires EPFL de ce programme. C'est grâce à ces efforts, comme à leur engagement de longue date dans ce domaine, que les institutions hospitalo-universitaires Lausannoises peuvent espérer jouer un rôle de premier plan dans ce domaine de la recherche médicale. C'est donc avec plaisir et fierté que nous vous invitons à participer à cette "Journée de Recherche" du CHUV consacrée à l'Imagerie Biomédicale pour entendre parler experts internationaux et collègues de nos institutions à propos des développements les plus fascinants dans ce domaine. Prof. J.-D. Horisberger, Vice-Doyen de la recherche

CENTRE HOSPTALIER UNIVERSITAIRE VAUDOIS

Comité d’organisation 2007

Angelika Bischof Delaloye, Médecine nucléaire Rolf Gruetter, Centre d’Imagerie biomédicale Jean-Daniel Horisberger, Décanat Reto Meuli, Radiodiagnostic et radiologie interventionnelle Andrea Volterra, Biologie cellulaire et morphologie

Administration de la Recherche : Jovan Mirkovitch Anne Tricot Coraline Fraga

CENTRE HOSPTALIER UNIVERSITAIRE VAUDOIS

CHUV RESEARCH DAY 2007 Thursday, February 1st, 2007

“Biomedical Imaging” 08:30

Presentation of the 2007 Research Day Professor Jean-Daniel Horisberger, Vice Dean for Research

08:45

Keynote speaker 1

09:30

Coffee & Posters

10:30

5 short talks

11:45

Keynote speaker 2

12:30

Lunch, Coffee & Posters

14:00

Keynote speaker 3

14:45

5 short talks

16:00

Coffee & Posters

17:00

Keynote speaker 4

17:45

Poster Prizes Ceremony

18:00

Aperitif & Buffet

Professor Fritjof Helmchen Department of Neurophysiology Brain Research Institute, Zurich “Imaging cells at work: new looks at brain function”

Professor Rolf Gruetter, Dr Giulio Gambarota & collaborators Centre d'Imagerie BioMedicale (CIBM) of the UNIGE, UNIL, CHUV, HUG, EPFL and Jeantet-Leenaards Fdns. “New insights with functional and metabolic imaging. Initial results from the CIBM of the lemanic area”

Professor Ignasi Carrió Nuclear Medicine Department Hospital Sant Pau, Barcelona “Molecular imaging with radiolabeled tracers: from genotype to phenotype”

Doctor Elie Mousseaux Radiologie Cardiovasculaire Hôpitaux de Paris “The aortic geometry and distensibility and LV remodeling : the present and the future in MRI”

ATTENDANCE IS FREE - NO REGISTRATION IS NECESSARY Contact: [email protected] NOTE: Posters will be displayed from Wednesday January 31st early morning to Friday February, 2nd early morning.

CENTRE HOSPTALIER UNIVERSITAIRE VAUDOIS

CHUV RESEARCH DAY 2007 Thursday, February 1st, 2007 “Biomedical Imaging”

10 short talks Schedule

Names, departments

Titles

10h30 - 10h45

Professeur Theo Lasser Laboratoire d'optique biomédicale EPFL

“New concepts in optical imaging-from structure towards function”

10h45 – 11h00

Carole Poitry-Yamate Laboratoire Leenaards-Jeantet d'imagerie fonctionnelle et métabolique EPFL

Application of synchrotron X-ray fluorescence in biomedical imaging: imaging of glia and neurons and feasibility of low Z element mapping in brain

11h00 – 11h15

Pierre.Marquet Service de Psychiatrie générale CHUV

Analysis of neuronal dynamics with Digital Holographic Microscopy

11h15 – 11h30

Paola Bezzi Département de biologie cellulaire et de morphologie UNIL

“Imaging life of the secretory cell at its boundaries”

11h30 – 11h45

Patric Hagmann Laboratoire de traitement des signaux 5 EPFL

“Imaging the brain neuronal network with diffusion MRI: a way to understand its global architecture”

14h45 – 15h00

John Prior Service de Médecine Nucléaire CHUV

Prediction of Response to Multitargeted Tyrosine Kinase Inhibitor Sunitinib by F-18Fluorodeoxyglucose Positron Emission Tomography in Patients With Gastrointestinal Stromal Tumor (GIST) After Imatinib Failure

15h00 – 15h15

Prof. Jean-Yves.Meuwly Service de Radiodiagnostic CHUV

“Image Processing of focal liver lesions in contrast ultrasound: preliminary results using a novel software program for improved lesion characterization”

15h15 – 15h30

Cristina Granziera Service de Neurologie CHUV

Anatomical alterations in motion processing areas in migraine.

15h30 – 15h45

Laurent Favre Service de Pneumologie CHUV

Comparison of virtual and flexible bronchoscopy in the detection of bronchial stenosis

15h45 – 16h00

Patrick Browaeys Service de Radiodiagnostic CHUV

Pulsatility as a new parameter in intracranial aneurysmal characterization by four-dimensional 64-row multi-slice CT (4D-MSCT)

Morning

Afternoon

ENA Environnement Naturel

Environnement Naturel/ENA-1

XRCT investigations on geotechnical rock cores from different petrologies and degree of cataclasis: Feature detection for a 3D petrographical approach 1

Christe P., 1Parriaux A., 2Labiouse V.

EPFL-ENAC-ICARE-GEOLEP1, EPFL-ENAC-ICARE-LMR2

An important aspect of any tunnelling project is the preliminary investigation of rock cores from prospective drilling in order to characterize the rock quality expected along the tunnel axis. Characterization of rock cores are made by direct observation and appreciation on site of the collected materials and from laboratory specific analytical procedures aiming the derivation of geotechnical relevant parameters (e.g. water content, consistency, crack density, porosity, permeability, chemical composition, resistance to compression and traction efforts). These analyses are always destructive. An interesting alternative prior to the realization of these amongst engineers widely familiar procedures is the indirect characterization of the rock cores by means of XRCT. Although geotechnical rock cores would -because of their size- need a powerful X-ray source to perform microtomographical investigations, they are readily analysed by medical XRCT at a good resolution of about 0.5 mm. With the advantage of providing useful 3D relationships of structural and textural elements constituting the rock mass, medical XRCT has the potential to become an important complimentary method to use hand in hand with classical analytical techniques in order to better constrain the rock mass properties that will be met underground.

Keywords: Tunnelling, rock mass, petrology, structural & textural relationships, cataclasis, rock mechanics, XRCT

Environnement Naturel/ENA-2

Impact of dissolved organic carbon upon PAH bioavailability to bacteria 1

Tecon R., 2Smith K., 1vanderMeer J.

Department of Fundamental Microbiology, UNIL1, UFZ-Centre for Environmental Research, Leipzig2

Pollution due to organic contaminants - as can be encountered during oil spills - is widespread in many environments and is a chronic source of concern. Luckily, certain bacteria are able to transform the organic pollutants into harmless mineral end-products, in a process called natural attenuation. However, this process may be limited by physico-chemical parameters such as the sorption of the pollutants to an organic matrix, which possibly modifies the accessibility of the pollutant to the bacteria. For instance, in liquid environments, organic pollutants may interact with dissolved organic carbon (DOC), thereby influencing their biodegradation. In this study, we investigated the effect of DOC on the bioavailability of polycyclic aromatic hydrocarbons (PAHs) – used here as model contaminants from oil. We employed the bacterial bioreporter Burkholderia sp. RP037, a PAHdegrading strain which was genetically engineered in order to produce a detectable fluorescent signal upon PAH metabolization. The bioreporters were used to monitor PAH availability to the cells in the presence or absence of DOC in the medium. At the same time, solid phase microextraction permitted the measurement of PAH concentrations during the assay.

Environnement Naturel/ENA-3

Indicator-Displacement Assays for the Identification of Amino Sugars, Aminoglycosides and Sugars 1

Zaubitzer F., 1Severin K.

EPFL - Institut des Sciences et Ingénierie Chimiques - Laboratoire de Chimie Supramoléculaire1

In the last years, indicator displacement assays (IDAs) have increasingly been employed for analytical purposes. In these assays, analytes compete with an indicator for non-covalent binding to a synthetic receptor. An IDA with the organometallic complex (Cp*RhCl2)2 and the dye gallocyanine was used to sense amino sugars and aminoglycosides in buffered aqueous solution.1 A ‘mini array’ of three IDAs at different pH values was sufficient to differentiate three amino sugars and six aminoglycosides with high fidelity. The array was also used to characterize mixtures of aminoglycosides and receive quantative information about the respective analytes.

L

d In

i

r to ca

Analyte

Rh X

L

L2

+

L4

Indicator

3

e

L2 1

L1

H 2O

ly t

X

L4

+

A na

L3 Rh

Fig.1.: Schematic representation of an indicator displacement assay based on an organometallic Cp*Rh complex. L = donor group. This method is being applied to sensing of ordinary sugars. In order to ensure sugar binding to the Cp*Rh fragment, derivates containing nitrogen need to be formed. This sensing system is especially appealing because of its simplicity and flexibility. First, the dye and the receptor are both commercially available. Second, the analysis can be realized by simply mixing the solutions of the Cp*Rh complex, the dye and the analytes and performing UV/Vis measurements. Finally, a mere change of pH is enough to expand the IDA to an array format and thus enhance the analytical power of the sensing ensemble. [1]

F. Zaubitzer, A. Buryak, K. Severin, Chem. Eur. J. 2006, 12, 3928.

Environnement Naturel/ENA-4

Fractal Dimension and Localization of DNA Knots Studied by Atomic Force Microscopy 1

Ercolini E., 1Valle F., 1Adamcik J., 1Witz G., 2Metzler R., 3DeLosRios P., 4Roca J., 1Dietler G.

Laboratory of Physics of Living Matter, IPMC, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne1, Nordic Institute for Theoretical Physics (NORDITA), København Ø2, Laboratory of Statistical Biophysics, ITP, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne3, Instituto de Biologia Molecular de Barcelona, CID - CSIC, Barcelona4

The scaling properties of homo- or heterogeneous mixture of DNA knots were studied by Atomic Force Microscopy (AFM). DNA knots were adsorbed onto mica in regimes of (i) strong binding, that induces a kinetic trapping of the 3D configuration, and (ii) weak binding, which permits (partial) relaxation on the surface. The DNA contours were analyzed both with a “customized” box counting algorithm, giving the knot mass as a function of the box size L, and the standard box counting algorithm, giving the number of filled boxes as a function of the box size L. The relation between mass and size is given by Mass~Ldf, where df is the fractal dimension and ν=1/df the scaling exponent. This relationship is complicated by the presence of a persistence length of DNA (about 45 nm) which introduces a crossover from a rigid rod behavior to a Self-Avoiding Walk behavior. Both algorithm give consistent results. In (i) the gyration radius of the adsorbed DNA knot scales with the 3D Flory exponent ν ≈0.58 within error. In (ii), we find ν ≈ 0.66, a value between the 3D and 2D (ν = 3/4) exponents, indicating an incomplete 2D relaxation or a different polymer universality class.

Environnement Naturel/ENA-5

DNA in Motion Imaged by Atomic Force Microscopy in Aqueous Buffer 1

Ercolini E., 1Adamcik J., 1Dietler G.

Laboratory of Physics of Living Matter, IPMC, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne1

Tapping mode atomic force microscopy (AFM) of plasmid pUC19 and supercoiled DNA ladder (2-10 kb) was performed in aqueous buffer and images of the change in shape and position of the circular plasmid DNA molecules as a function of time are presented. Freshly cleaved mica is functionalized with NiCl2 and the DNA sample is deposited and directly imaged in a buffer containing Hepes, monovalent and divalent cations. Tapping mode AFM in fluid reduces lateral forces, which permits the imaging of loosely adsorbed molecules. The presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging by AFM shows DNA motion.

Environnement Naturel/ENA-6

Organometallic Chemosensors for Amino Acids, Peptides, Nucleotides and the Pyrophosphate Anion. 1

Buryak A., 1Severin K.

Swiss Federal Institute of Technology, Lausanne1

A synthetic receptor, which is bound via non-covalent interactions to an indicator, is able to function as a chemosensor. The basic requirement is that the displacement of the indicator by an analyte results in a change of its optical properties. We have shown that the organometallic 4d transition-metal complex [Cp*RhCl2]2 in combination with commercially available indicators can be effectively used to sense various biologically important analytes. For example, the combination of the above mentioned rhodium complex with the dye azophloxine comprises a chemosensing ensemble which allows to selectively detect peptides containing His/Met residue close to N-terminus. These peptides can be detected in aqueous solution at pH 7.0 down to concentration of 300 nM.1 Furthermore we are exploring the possibility of using a fluorescent indicator in combination with the [Cp*RhCl2]2 complex to create a fluorescent chemosensor for the pyrophosphate anion. We used the same complex in combination of several commercially available dyes to create an array of chemosensors. This array allows the differentiation of 20 natural amino acids with high fidelity using Uv-Vis spectroscopy in combination with a multivariate analysis.2 At the moment we are trying to create a similar array for the detection of nucleotides. [1] Buryak A.; Severin K. Angew. Chem. Int. Ed. 2004, 43, 4771-4774. [2] Buryak A., Severin K. J. Am. Chem. Soc. 2005, 127, 3700-3701.

EHU Environnement Humain

Environnement Humain/EHU-1

Cardiorespiratory Responses during Affective Picture Viewing: Metabolic and Attentional Aspects Gomez P. Institut de Santé au Travail

Several lines of research point to a hierarchical structure of emotion with the affective dimensions of valence (degree of pleasantness) and arousal (degree of activation) as strategic determinants of emotion. Pictures, for their ecological validity, have been widely used to evoke and investigate affective reactions in the laboratory and they were adopted in this study. Reactions of most individuals to positive and negative arousing images activate phylogenetically primitive motivational systems. Several somato-physiological measures covary to a certain degree with self-reported valence and arousal evaluations. The goals of this study were multifold: • • • •

to replicate previous findings concerning the relationships between breathing variables and self-rated valence and arousal; to assess to which extent changes in ventilation are in equilibrium with metabolic activity; to determine the level of visual attention involvement during picture viewing; to extend our knowledge about autonomic responding by measuring blood pressure (a rather neglected parameter in emotion research).

Thirteen picture series, each one of a different content (e.g., erotic couples, nature scenes, mutilated bodies, household objects), were shown to 41 subjects (20 men, 21 women, aged 18-38 years), while breathing parameters (i.e., inspiratory time, expiratory time, inspiratory volume, flow parameters, thoraco-abdominal balance), end-tidal pCO2, heart rate, blood pressure, skin conductance, spontaneous eye-blink rate and affective judgments were recorded. Series consisted of ten pictures of 6-s duration each. Mixed effects regression models were used to assess the relationships between affective judgments and physiological measures. The models tested included a random intercept for each subject and fixed effects for valence, arousal and the interaction term. Sex effects were also tested. More negative valence ratings were associated with larger heart rate deceleration, lower eye-blink rate, and lower end-tidal pCO2. Sustained heart rate deceleration and blink rate inhibition are indicative of increased attention to aversive stimuli and lower pCO2 contributes to heightened sensory perception. We interpret these relationships in terms of an attention bias towards negative stimuli. With increasing arousal, minute ventilation, inspiratory volume, skin conductance and blood pressure increased. These relationships suggest increased energy mobilization in response to both positive and negative arousing stimuli and confirm that breathing parameters are more consistently related to arousal than valence. The ventilatory changes along the arousal dimension are in balance with metabolic activity. Importantly, the arousal effect for blood pressure was limited to the male subjects indicating a sex difference in the reactivity to high-arousal events on this specific parameter. An important, yet not exhaustively answered question is how and to what extent physiological reactions to affective challenges influence human well-being. To be able to address this question we first need to refine our understanding of the physiological, cognitive and behavioral emotion systems. By investigating a broad range of psychophysiological responses to affective pictures from a two-dimensional perspective of emotion, this study enriches our knowledge about the connection between the subjective realm of the emotions and their neurophysiological substrate.

Environnement Humain/EHU-2

Effect of supraspinatus deficiency on anatomical shoulder prostheses 1

Terrier A., 1Reist A., 1Merlini F., 2Farron A.

Laboratory of Biomechanical Orthopedics1, Hopital Orthopédique de la suisse romande2

Introduction. Anatomical total shoulder prostheses are usually not indicated in cases of glenohumeral degenerative diseases associated with rotator cuff deficiency. Asymmetrical load of the glenoid could increase the risks of implant loosening. The aim of this study was to analyse the consequences of supraspinatus deficiency on the glenoid stresses. Method. Analysis was conducted with a 3-D finite element model of the shoulder, including the scapula, the humerus and 6 muscles : middle, anterior and posterior deltoid, supraspinatus, subscapularis, and infraspinatus. An anatomical shoulder prosthesis (Aequalis, Tornier) was numerically implanted. Movements of abduction (0-150°) were performed by muscles’ activation. Scapulo-thoracic rhythm was taken into account. Contact forces caused by muscles wrapping on bony surfaces were also accounted for. Two situations were studied : 1) with an intact rotator cuff; 2) without any supraspinatus muscle. Glenohumeral forces and pressures were calculated for the two situations. Stresses within the cement, at the bone-cement interface and in the glenoid bone were also analysed. Animated views allowed to evaluate the glenohumeral contact point and humeral head translations. Results. The absence of supraspinatus muscle had many consequences : it increased significantly the superior translation of the humeral head during abduction, which occurred earlier at the beginning of movement; it did not modify the articular force and pressure, but maximum values were obtained at smaller angles of abduction (75° against 90°); the strength of deltoid necessary for abduction was increased of 55%. The supraspinatus insufficiency also increased stresses of about 28% within the cement, 18% at the bone-cement interface and 6% within the glenoid bone. Conclusions. Animated views demonstrated clearly the occurrence of a rocking-horse phenomenon when an anatomical shoulder prosthesis is implanted without a functional supraspinatus. The asymmetrical load of the glenoid induced deleterious biomechanical effects. This situation may lead to cement and/or cement-bone interface failure, and could preclude the long term survival of the prosthetic implant.

Environnement Humain/EHU-3

Physiological measures as indices of moods during human-computer interaction 1

Gomez P., 2Zimmermann P., 2GuttormsenSchär S., 1Danuser B.

Institut de Santé au Travail1, Insitute for Medical Education IML Bern2

Emotions are an important factor in human-computer interaction. One of the challenges in building emotionally intelligent systems is the automatic recognition of affective states. We are developing and evaluating a method for measuring user affect that incorporates psychological, behavioral, and physiological measures. During affective stimulation, breathing parameters, skin conductance level (SCL) and corrugator EMG activity correlate with self-reported levels of valence and arousal. Valence, at the level of subjective experience, summarizes how well one is doing, whereas arousal refers to a sense of energy. A stimulus activates appetitive or defensive motivation (the valence dimension) with some degree of energy mobilization (the arousal dimension). In the laboratory, moods are induced using different procedures. Only few studies have investigated the critical question of how long induced moods actually last. Further, knowledge concerning the persistence of physiological responding, when the stimuli are withdrawn, remains spare. The goals of this study were first, to assess the somato-physiological activity during affective stimulation (film clip viewing) and its relation to valence and arousal, and second, to determine if the response patterns persist, dissipate or otherwise change when the stimuli are withdrawn and the subjects perform a computer task. Seventy-six participants viewed a neutral film clip (an educational program) and completed immediately afterward the task (control condition). Then, they viewed either a positive high-arousal clip (sport scenes), a positive low-arousal clip (takes from landscapes), a negative high-arousal clip (scene depicting captives forced to play Russian roulette) or a negative low-arousal clip (a documentary about a slum in Belgium) and completed the task a second time (experimental condition). The task required participants to shop on an e-commerce website for office supplies. After each clip and each task, the participants rated their current mood. We tested valence and arousal effects during the last 90 s of the films, the first and last 90 s of the task of the experimental condition. Viewing of the selected film clips resulted in increasing defensive and appetitive activation in the expected ways both subjectively and physiologically. Corrugator EMG activity was higher at the end of the negative clips than the positive clips, and minute ventilation and SCL were higher for the arousing clips than for the less arousing clips. After the approximately 9-minute task, people who viewed the negative clips still reported more negative valence than those who viewed the positive clips. On the contrary, there were no differences in the arousal ratings. The valence effect in the mood state was paralleled by valence effects in the somato-physiological measures during the task. Increased facial frowning at the end of the negative clips was maintained during the task indicating persistence of defensive activation. SCL was lower for the negative film groups, especially at the end of the task, suggesting that sympathetic activation was lowered in subjects in the negative mood as compared with subjects in the positive mood. The findings of this study have several implications. First, they enrich our knowledge concerning the relationships between subjective feelings and their physiological correlates. Second, they inform us about the effectiveness of film clips as a mood induction instrument. Third, they suggest that induced changes in arousal are quickly overridden by the degree of activation “imposed” by the cognitive task, whereas induced changes in valence are more resistant and thus likely to impact the execution of the task. Finally, they show which physiological measures may be useful in tracking mood states during human-computer interaction (i.e., corrugator EMG activity and SCL). Feedback from these parameters could be used by computers to recognize mood states in the user.

Environnement Humain/EHU-4

Determination and distribution of clotiapine (Entumine®) in human plasma, postmortem blood and tissue samples from clotiapine treated patients and from autopsy cases. 1

Sporkert F., 1Augsburger M., 1Giroud C., 2Eap C.

IUML1, CNP2

Despite that the antipsychotic drug clotiapine (Entumine®) has been marketed for more than 35 years now, only few data have been published on therapeutic and toxic levels of this drug. To fill this gap, two rapid and sensitive methods were developed for the determination of clotiapine, (2-chloro-11-(4-methyl-1-piperazinyl)dibenzo-[b,f][1,4]-thiazepine), in human plasma and postmortem blood and tissue samples. After simple liquid-liquid extraction at pH 9.5 with nhexane/dichloromethane (85/15, v/v), clotiapine was quantitated by liquid chromatography diode-array detection (HPLC-DAD) and by gas chromatography with nitrogen-phosphorous detection (GC-NPD). The calibration curve was linear between 10 and 1000 μg/l. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 2 and 6 μg/l for the GC-NPD method and 5 and 15 μg/l for the HPLC method, respectively. These methods were applied to twelve plasma samples from patients treated with clotiapine, to seven autopsy cases and to one case of driving under the influence of drugs (DUID). Concentrations ranged for the clotiapine treated patients between 6 and 155 μg/l (mean 46 μg/l), and for the autopsy cases between 22 and 341 μg/l (mean 123 μg/l). The HPLC-DAD method allows tentative identification of clotiapine, while the GC-NPD method is more sensitive. Keywords: Clotiapine; antipsychotic; blood level; postmortem distribution

Environnement Humain/EHU-5

Bone mass is positively associated with physical activity, muscle strength, fitness and lean body mass in Swiss school children 1

Puder J., 2Kriemler S., 2Zahner L., 3Rizzoli R.

CHUV1, University of Basel2, University of Geneva3

Background: More than every 3rd woman over 50 years suffers from osteoporosis. The impact of preventive measures on bone accretion is most pronounced during growth. We therefore looked for factors associated with higher bone mass in children and analyzed the relationship between measures of physical activity (PA) and bone mineral density (BMD). Methods: Among 540 7-13 y old children (males and females) from randomly selected socio-ethnic diverse urban and rural public schools in Switzerland studied, 389 children had BMD and body composition measurements by dual energy x-ray absorptiometry (DXA). In 374 children physical activity was assessed by accelerometer. Vigorous PA was defined as the daily amount of time above 3000 counts/min. The independent association between BMD, PA, lower extremity strength (jump and reach), and aerobic fitness (20 m shuttle run) was assessed using partial correlation analyses. The association between low age- and gender adjusted BMD (Z-Score less than -1) with age, sex and pubertal stage, family history of osteoporosis, calcium intake, lean body mass (LBM), daily media use and all measures of PA was assessed using stepwise multiple logistic regression analyses. The amount of PA in children with low vs. high age- and gender adjusted BMD (Z-Score less than -1 and over + 1, respectively) was compared by t-test. Results: The children were 9.2±2.2 yrs old (x±SD) with a LBM of 25.2 ± 6.7 kg and 23.5±6.0% body fat. BMD of the hip was 0.699±0.099 g*cm-2 and calcium intake 971±384 mg*d-1. Their daily PA was 599’390±167’322 counts*d-1 with 44.7±21.9 min spent in vigorous PA, in the jump and reach test they achieved 24.1±6.9 cm and in the shuttle run test they performed 5.7±2.2 laps. Independently of age, weight, height, sex, pubertal stage, calcium intake and family history of osteoporosis, total hip BMD was positively associated with more time spent in total and in vigorous PA (r=0.19 and 0.2, both p 2.5

> 20

> 2.5

> 20

>4

CD103+CD4+/CD4+

Sensitivity %

Specificity %

86

52

< 0.31

79

73

< 0.31

75

81

Considering only histologically proven sarcoidosis (16/28 patients), the best sensitivity and specificity (81% for both) for the diagnosis of sarcoidosis were obtained with the combination of a lymphocytosis > 20%, a CD4+/CD8+ ratio > 3.5 and a CD103+CD4+/total CD4+ ratio < 0.31. Conclusion: Using the BAL CD103+CD4+/total CD4+ ratio < 0.31 as a new criterion for the diagnosis of pulmonary sarcoidosis improved the specificity of the combination of BAL lymphocytosis with a high CD4+/CD8+ ratio .

Immunité et Infection/IMI-9

Double Locus Sequence Typing Using clfB and spa: a Fast and Simple Method for Epidemiological Typing of Methicillin Resistant Staphylococcus aureus 1

Kuhn G., 1Francioli P., 1Blanc D.

Service de Medecine Preventive Hospitaliere CHUV 1

Sequence based epidemiological typing of methicillin resistant Staphylococcus aureus (MRSA) has recently been promoted because it results in unambiguous datasets that can be organised in local and global databases. The replacement of previous typing methods such as the highly discriminatory pulsed field gel electrophoresis (PFGE) has been attempted with various markers and typing schemes, including spa typing and multilocus sequence typing (MLST). However, despite a number of advantages, none of these showed convincing evidence for a comparable performance in epidemiological typing. By using 3 sets of 48 MRSA strains comprising isolates that were, i) genetically highly diverse, ii) genetically related and iii) obtained from long term carriers, we analysed the performance of the 4 highly polymorphic S. aureus markers: clfA, clfB, fnbA and spa. Typability, discriminatory power, in vivo stability and evolution of these markers were compared to PFGE. Clearly, none of the markers alone could match discriminatory power of PFGE (genotypes = 63; index of discrimination = 0.96). Instead, this could be achieved by combining markers in pairs. We showed that by using only 3' partial sequences of approximately 500bp the majority of each markers discriminatory power was displayed and using these, the best performance was obtained with the combination of clfB and spa (genotypes = 59; index of discrimination = 0.94). All markers were stable over a period of at least 3 years, in case of partial sequences even more and thus superior to PFGE. The genetic differences found between highly related isolates and LTC isolate pairs indicated a clonal evolution. A typing scheme based on 500bp 3' partial sequences of clfB and spa is proposed.

Immunité et Infection/IMI-10

The N-terminal caspase-generated fragment of RasGAP (fragment N) is a NFkB cytoplasmic inhibitor 1

Loukili N., 1Yang J-Y., 2Regamey A., 2Huber M., 1Widmann Ch.

Institut de physiologie UNIL1, Dermatology CHUV2

Partial caspase-mediated cleavage of RasGAP in mildly stressed cells results in the formation of an amino-terminal fragment called N. This fragment activates Akt and promotes cell survival. Fragment N, however, acts as a general inhibitor of NFkB, including NFkB activation with Akt but does not hamper the ability of Akt to activate its effectors. Here we show that fragment N inhibits activation of the NFkB pathway in response to a variety of stimuli by blocking NFkB translocation to the nucleus. We also show that suppression of NFkB activation by fragment N is required for fragment N to protect cells, such as insulin-producing pancreatic cells, in which NFkB stimulation is detrimental. These results indicate that fragment N differentially modulate the pathways downstream of Akt stimulation to optimally protect a variety of cell types.

Immunité et Infection/IMI-11

Decreased inflammatory activity of cell walls purified from glycopeptideintermediate resistant S. aureus (GISA) 1

Majcherczyk P., Entenza J., Giddey. M., Vouillamoz J., Moreillon P.

DMF1

Background & Purpose The rapid emergence of clinical GISA isolates represents a major threat for hospitalised patients. Detailed characterisations of such isolates indicate frequent changes in peptidoglycan cell wall (PG) thickening and metabolism. PG is reputed to trigger structure dependant cytokine release. It is therefore possible that the modified GISA PGs have an altered inflammatory activity. We tested this hypothesis using PG purified from both a MRSA vancomycin-susceptible (VanS; MIC: 1mg/L) parent strain M1 and its in vitro selected GISA (Van MIC: 8 mg/L) variant M1V16. Methods Lipoteichoic acid-free PG was purified from both M1 and MIV16 strains. Stimulatory activity was tested by incubating them with human blood in triplicate for 8 h at 37°C + polymyxin (pmx) on 3 separate days. Cytokine release was measured using the Bioplex human 27 cytokine multiplexed assay. Their PG structures were compared by HPLC analysis of their muropeptide digests. Results M1V16, the GISA variant, had a less cross-linked PG than its VanS parent M1. Cytokines were induced in a dose dependant manner and were not inhibited by pmx. Both strains induced the same cytokines but they differed in their induction potential. M1V16 induced 2-fold less TNF, IFN-g, MIP-1b, Il-1b, Il-6, Il-8; 10-fold less rantes & MIP-1a and 100fold less PDGF, compared to M1. No differences were observed in the levels of MCP-1, GCSF, GM-CSF, Il-1ra, IL-10, IP-10 eotaxin & VEGF released. Neither PG induced IL-2, 4, 5, 7, 9, 12, 13, 15, 17 & FGF basic production. Discussion Global reduction in the cytokine production by the less cross-linked PG purified from the GISA variant M1V16, as compared to its VanS parent M1, suggests that the degree of PG polymerisation affects its inflammatory activity. It is expected that these GISA variants with a simpler PG structure would also produce less inflammation in vivo. Whether this character is also associated with decreased virulence of the GISA phenotype in vivo remains to be determined.

Immunité et Infection/IMI-12

Absence of recognition of Parachlamydia by Toll-like receptors 1

Greub G., 2Casson N., 3Calandra T., 3Roger T.

Institute of Microbiology1, Institute of Microbiology, Lausanne2, Infectious Diseases Service, Lausanne3

Background. Parachlamydia acanthamoebae is a Chlamydia-like organism that naturally infect free-living amoebae. Its role as an agent of pneumonia is supported by serological and molecular clinical studies, a mice model of lung infection and the ability of this obligate intracellular bacteria to enter and replicate in human macrophages. Toll-like receptors (TLRs) are receptors of pathogen-associated molecular patterns (PAMPs) such as peptidoglycan and LPS, which may induce the production of proinflammatory cytokines (TNF, IL-6, IFN-gamma). Objectives. The aim of this study was to investigate the importance of TLR-2 and TLR-4 in the interaction of Parachlamydia with macrophages. Methods. Bone-marrow derived macrophages (BMDMs) obtained from wild-type (WT), TLR2-/-, and TLR4-/mice were infected with living and heat-killed Parachlamydia. Bacterial entry was assessed by confocal microscopy whereas replication was evaluated by counting the number of bacteria per macrophages. To determine the role of TLRs in the recognition of Parachlamydia, we measured the production of IL-12 by ELISA and of TNF and IL-6 by bioassay. Results. Parachlamydia was able to enter within BMDMs independently of TLR2 and TLR4, and replicated poorly or not at all in both WT and TLRs knock-out BMDMs. Living Parachlamydia did not induce any pro-inflammatory cytokine production. Conversely, heat-inactivated Parachlamydia induced significant cytokine production, which was not suppressed by pre-incubation of BMDMs with living bacteria. Cytokine production was dependent of TLR4, as demonstrated by the absence of response following parachlamydial infection of TLR4-/- BMDMs. Moreover, cytokine production was only induced by heat-inactivated bacteria, and not by Parachlamydia inactivated with methanol, paraformaldehyde or UV, suggesting that an immunoreactive epitope recognized by TLR4 is exposed following heat inactivation. Conclusions. Parachlamydia are able to enter into BMDMs independently of TLR2 and TLR4. When internalized, this obligate intracellular bacteria remained somehow unrecognized, eliciting no production of proinflammatory cytokines. However, heat-inactivated bacteria induced a strong TLR4-dependent immune response. Which fraction causes the proinflammatory response need to be determined, to potentially identify a new TLR ligand.

Immunité et Infection/IMI-13

Structure and evolutionary history of homologous large GC-rich proteins of Protochlamydia amoebophila strain UWE25 and of their leucine-rich repeats domain. 1

Eugster M., 2Roten C., 1Greub G.

Institute of Microbiology, Lausanne1, Dpt of Fundamental Microbiology, Lausanne2

Protochlamydia amoeobophila UWE25 is an obligate intracellular bacteria. We recently described along its genome a 100-kb genomic island containing a tra unit likely involved in conjugative DNA transfer and a single 6-kb gene similar to five UWE25 chromosomal homologs. These six proteins exhibit a G+C content (~42%) higher than that of the core chromosome (35%). This contribution is focused on the structure and evolutionary history of these "Large GC-rich" proteins (LGRs) All LGRs are conserved on their first ~1350 residues. Revealing no similar proteins in other genomes, similarity analyses found numerous proteins homologous to the C-terminal part of LGRs (200-400 residues). The latter region carries 28-residue Leucine-rich repeats (LRRs). Phylogenetic analyses of LRRs of all LGRs and of homologs of other organisms revealed that some proteins of Ralstonia solanacearum and Legionella pneumophila containing 24-residue LRRs are the closest homologs. Other proteins housing homologous 28-residue LRRs are NODs and related mammalian proteins. In Pr. amoebophila, while all LRRs of LGRs present a central α-helix, some contain a distal β-sheet. The LRR domain corresponded to the less conserved part of the LGRs. Phylogenetics analyses of the LRRs performed by Neighbor-Joining- and Bayesian analyses or by the comparison of di-amino acids enabled us to propose a parcimonious evolutionary history of these LRRs. Promoter analyses identified one or two candidates upstream of all lgr genes. Interestingly, the genomic island lgr, housing 18 LRRs and likely involved in bacterial recognition and regulation of intracellular conjugative DNA transfer, presents two putative promoters. Moreover, phylogenetics analyses performed on the conserved part of the six LGRs suggest that the LRR of the genomic island duplicated recently. In conclusion, while the LRR region of the genomic island LGR seems to be involved in bacterial recognition and DNA conjugative transfer regulation, the role of the others remains to be defined.

Immunité et Infection/IMI-14

A ROLE FOR CHLAMYDIA TRACHOMATIS IN MISCARRIAGE 1

Baud D., 1Thomas V., 2Arafa A., 2Regan L., 1Greub G.

Institute of Microbiology, Lausanne1, Department of Obstetrics, St Mary's Hospital, London2

Objective: Chlamydia trachomatis infections are the most frequent bacterial sexually transmitted diseases in western countries. These infections are frequently asymptomatic and remain untreated leading to severe sequelae such as extra-uterine pregnancy and infertility. However, it is unclear whether C. trachomatis infection is implicated in miscarriage. Here, we assessed whether the presence of C. trachomatis IgG is associated with miscarriage. Materiel and Methods: 438 sera were prospectively collected from July 2004 to March 2005 from two groups of women consulting at St Mary's NHS Trust Hospital (London). The “miscarriage” group included 269 women referred to Gynaecology Emergency or attending to the “Recurrent Miscarriage Clinic” who have experienced one or more miscarriages. The healthy Control group included 169 women from the post-natal ward without any history of miscarriage, stillbirth or preterm labour. Miscarriage and Control groups were compared regarding the prevalence of C. trachomatis IgG antibodies (titre ≥ 1/50) using a commercial ELISA (Medac). Results: The prevalence of C. trachomatis IgG was significantly higher in Miscarriage group (48/260, 18.5%, p=0.009) as compared to controls (15/169, 8.9%). Patients in Miscarriage group were significantly older (median=35.4, IQR=30.7-39.1, p1 plaque; and prospectively tested the efficiency of this score to detect T2DM with CAD. Results: Patients were aged 59±9y and 78% were males. Median diabetes duration was 8y (1-19y). Micro- or macro-albuminuria was present in 38% and 46% were smokers. Carotid plaques were detected in 71% and femoral plaques in 76%. SE or MPI were positive for CAD in 20% (22/109). Using the score with a cut-off of 2 points, the sensitivity to detect patients with CAD on non-invasive tests is 86%, specificity 44%, PPV 30% and NPV 93%, OR= 3.8, p=0.009. ROC curve analysis demonstrated a predictive value for CAD of 0.67 (AUC) (95% CI 0.57-0.76). Conclusion: Using this new algorithm, the predictive power for non-invasive CAD screening strategy is significantly improved (NNS=3), which is cost-saving for the detection of CAD in T2DM.

Métabolisme et Cardiovasculaire/MCV-44

HDL-induced β; cells protection: A transcriptomic approach to the identification of new therapeutic targets

1

Pétremand J., 1Bulat N., 2Butty A., 1Poussin C., 3Au K., 3Mooser V., 4Waeber G., 1Thorens B., 1Yang J., 1Widmann C. Institute of Physiology, UNIL1, Lausanne University Hospital CHUV2, GlaxoSmithKline (USA)3, Médecine Interne, CHUV4 Diabetes is one of the most challenging health problems of the 21st century; a complex disease comprised many metabolic disorders with the main features of glucose intolerance, defect in insulin secretion and increased β cell apoptosis. One of the phenotype observed in diabetic patients is the low levels of HDL cholesterol. HDLs have been shown to antagonize cell death triggered by various stimuli such as serum deprivation and cytokines by mechanisms that induce activation of AKT and inhibition of caspase3 cleavage. To identify the molecular mechanisms by which HDLs exert their anti-apoptotic activities, gene expression in insulin-secreting cells (βTC3) was examined by using the Affymetrix gene chip technology in starved condition and/or in the presence of HDLs. We identified multiple changes in gene expression: 425 genes were significantly modulated by the effect of HDL, whereas 262 genes were significantly regulated by the interaction of starvation and the HDLs. Genes involved in cholesterol metabolism, regulation of apoptosis and oxidative stress were modulated by the HDLs. The first candidate regulated by HDLs that we studied was the translation repressor Eif4EBP1, known to play a role in apoptosis and to be negatively regulated by the mTOR pathway. Using the MIN6 beta-cell line, 4EBP1 protein levels were found to be increased upon serum starvation and this was associated with augmented apoptosis. Addition of HDLs blocked the increase of 4EBP1 levels and inhibited the apoptotic response. Overexpression of 4EBP1 induced apoptosis and mimicked the starvation conditions. Another potential interesting candidate is TRB3 (tribbles homolog 3),a pseudokinase also known as NIPK, SINK and SKIP3 that have been shown to be induced in stress conditions and to inhibit AKT, to be regulated by the transcription factor ATF4, to be an adaptator protein for the degradation of ACC (Acetyl-CoA carboxylase) by COPI and also interact with MKK7 and p65. TRB3 is supposed to be effectively up regulated in MIN6 cells by stimuli including serum or glucose deprivation or cytokines stimulation. These results indicate that 4EBP1 and TRB3 represent potential target in the treatment of diabetes.

Métabolisme et Cardiovasculaire/MCV-45

ADMINISTRATION DE LACTULOSE POUR DIMINUER L'ACTIVITE INTESTINALE LORS D' EXAMENS F-18-FDG TEP/TDM: DONNEES PRELIMINAIRES. 1

Orcurto M., 1Prior J., 1Schmidt S., 1Gremion I., 1Bischof-Delaloye A.

CHUV, University Hospital1

Objectifs : les hyperactivités digestives visualisées dans les examens TEP oncologiques sont parfois difficiles d’interprétation, même avec un scanner TEP/TDM. Notre but était d’évaluer l’utilité d’une préparation de lactulose pour diminuer l’hétérogénéité de la captation intestinale afin d’améliorer la détection d’éventuelles lésions néoplasiques ou polypes. Matériels et méthodes : sept patients oncologiques (6F/1H) ont bénéficié de deux TEP/TDM à 2-5 mois d’intervalle, sans modification du traitement pendant cette période. Administration de lactulose 24h avant le 2e examen (Duphalac® 3x15ml/d). La TEP/TDM (Discovery LS) débutait entre 45-62’ post-injection (5MBq/kg de F-18-FDG, 3-5 min/pas). Mesure du SUVbw max dans 6 régions de l’intestin (du cæcum au rectum) et comparaison par test t. Résultats: le lactulose était généralement bien supporté. Les valeurs SUV globales ont tendance à diminuer après lactulose (p=0.17), prédominant dans le côlon transverse (p=0.09), descendant (p=0.2) et sigmoïde (p=0.17). L’examen TDM montre une quantité plus importante de selles, sans pour autant permettre une évaluation morphologique optimale. Conclusions : ces résultats préliminaires montrent une tendance à la diminution des captations physiologiques intestinales surtout dans le côlon distal, permettant une interprétation plus aisée des examens de la cavité abdominale, justifiant une étude à plus grande échelle.

Métabolisme et Cardiovasculaire/MCV-46

PPARγ antagonist GW9662 modulates specifically PPARγ2 expression to abrogate differentiation of primary human preadipocyte Céline Leyvraz1, Michel Suter2, Jean-Marie Calmes2, Alexandre Paroz2, Chantal Verdumo1, Rolf Christian Gaillard1, François Pralong1 and Vittorio Giusti1 1

Division of Endocrinology, Diabetes and Metabolism, University Hospital CHUV Department of Surgery, University Hospital CHUV

2

Rue du Bugnon 46, 1011 Lausanne, Switzerland

Background: Rosiglitazone (RTZ) treatment enhances insulin sensitivity by activating PPARγ (peroxisome proliferator and activated receptors γ) isoforms. This is accompanied by a remodelling of the adipose tissue (AT) and an increase in fat storage. We hypothesized that RTZ acts more specifically on PPARγ2 isoform. In order to investigate the role of PPARγ1 and PPARγ2 during preadipocyte differentiation into mature cells capable of lipid storage, we analysed primary human preadipocytes treated with the PPARγ antagonist GW9662 (or 2-chloro-5-nitro-N-phenyl-benzamide). Methods: Preadipocytes were derived from subcutaneous and visceral biopsies of AT obtained from obese women (mean of BMI of 43±1.5 kg/m2 (range 39.7-54.8); n=9 patients) during gastric by-pass surgery. Subsequently, cells were treated for 7 days with serum-free medium containing dexamethasone and RTZ (DR) to induce cell differentiation and supplemented or not with GW9662. They were then used to perform oil-red O staining and RNA extraction. Gene expression levels were measured by real-time RT-PCR (LightCycler® technology). Results: As previously reported, DR treatment induces preadipocyte differentiation by enhancing PPARγ1 and PPARγ2 gene expression in cells derived from SAT and VAT. Interestingly, GW9662 is able to prevent gene expression of mature adipocyte markers (ap2, LPL and leptin) and lipid droplet accumulation in preadipocytes of subcutaneous and visceral origin. Concomitantly, GW9662 inhibits the DR-dependent increase of PPARγ2 expression in both SAT and VAT (by 97% and 92% respectively, P 11.2% Normal kidneys 12 11 1 Unremarkable contralateral kidneys 34 30 4 Single functioning kidneys 11 9 2 UPS kidneys 23 8 12 3 Megaureters 8 4 4 Vesicoureteric reflux 5 4 1 Duplex kidneys 4 2 2 Total 97 18 69 10 Conclusions: Renal function measured by AI during F+0 I-123-hippuran RG seems to be reliable even in neonates. In this small group of children, we observed that renal function was more often decreased in infants with VUR, megaureters due to lower urinary tract stenosis and DK. About one third of kidneys with severe UPS had a decreased AI. In infants with unilateral MCK, AI of the single functioning kidney was more often within normal range suggesting that compensation should occur later.

Oncologie et Développement/ODE-23

CXCR4 Enhances Aggressive Behavior of Neuroblastoma 1

Meier R., 2Joseph J., 1Mühlethaler-Mottet A., 1Flahaut M., 3Fusco C., 4Rüegg C., 1 Von der Weid N., 1Beck-Popovic M., 5Vassal G., 1Gross N., Paediatric Oncology Research Unit-DMCP1, Department of Paediatric Surgery, DMCP2, Division of Experimental Pathology, University Hospital, CHUV3, Division of Experimental Oncology, Multidisciplinary Oncology Centre (CePO), Lausanne Cancer Centre4, Département d'Oncologie Pédiatrique, UPRES EA3535, Institut Gustave Roussy, 94805 Villejuif, France5

Neuroblastoma (NB) is a devastating neoplasm in its higher and metastatic stages. Chemokines and their receptors have been involved in tumor growth and metastasis. The particular CXCR4/CXCL12 axis may play a pivotal role in the homing of cancer cells and organ-specific metastases. Here, the involvement of CXCR4/CXCL12 axis in NB cells was investigated in vitro, and further validated in vivo, using an orthotopic mouse model. In vitro assays revealed variable CXCR4 surface protein or mRNA expression levels in different NB cell lines. IGR-NB8 a CXCR4 negative cell line deriving from a nonmetastatic tumor and IGR-N91 a constitutively CXCR4 expressing cell line deriving from a bone-marrow (BM) metastasis were used in this study. Comparing NB8-CXCR4-C3 and mock transfected NB8-E6 cells, in vitro experiments revealed that CXCR4 expression associated with CXCL12-mediated cell migration which could be impaired by a CXCR4 blocker. CXCR4 overexpression in NB cells resulted in enhanced cell growth. In vivo orthotopic engraftment of mice with either NB8-E6 or the NB8-CXCR4-C3 cells, revealed a tremendously increased tumor growth of NB8-CXCR4-C3 tumors compared to NB8-E6 tumors. Surprisingly, no metastases were found in the liver or BM of any mouse. When CXCR4 was over-expressed in the IGR-N91 cells, in vivo tumor growth also was strongly enhanced. Furthermore, liver and BM metastases found in the N91-CXCR4-K14 group were much bigger but not more frequent. CXCR4 expression in NB cells increases in vitro migration and growth. In vivo analyses confirm a pivotal growth effect. CXCR4 mediated influence on metastases in NB rather seems to be due to the growth promoting property of CXCR4 than to increased invasive capacities.

Oncologie et Développement/ODE-24

Role of growth arrest-specific gene 6 (Gas6) in erythropoiesis and anemia: potential therapeutic options 1

Angelillo-Scherrer A., 1Burnier L., 2Plaisance S., 3Martinez-Soria E., 4Maxwell P., 2Collen D., 3 Izui S., 1Schapira M., 2Conway E., 2Carmeliet P. Service and Central Laboratory of Hematology, CHUV, Lausanne, Switzerland1, Center for Transgene Technology & Gene Therapy, Flanders University Institute for Biotechnology, Leuven, Belgium2, Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland3, renal Section, Imperial College of Science Technology and Medicine, London, UK4

Many patients with anemia fail, for unknown reasons, to respond to erythropoietin (Epo). We report that loss of the Gas6 product, a protein binding the receptor tyrosine kinases Tyro3, Axl and Mer, caused a significant depletion of the reserve of erythroid progenitors in mice. As a result, the compensatory erythropoietic response to acute anemia was defective in Gas6-/- mice. The impaired erythropoietic response was attributable to the hyporesponsiveness of Gas6-/- erythroblasts to the survival activity of Epo. Only when recombinant Gas6 (rGas6) was administered together with Epo, did these factors synergistically rescue the mutant mice from anemia. Administration of rGas6 alone to Gas6-/- mice also provided protection from acute anemia, presumably due to the ability of Gas6 to enhance the pro-survival effects of Epo on erythroblasts. This was supported by in vivo and in vitro findings: (i) In the absence of Gas6, more erythroblasts died in the spleen of mice with acute anemia, despite markedly elevated levels of Epo. (ii) Compared to WT, Gas6-/- erythroblasts responded less to the pro-survival effects of Epo in vitro, suggesting that Gas6 influences Epo receptor (EpoR) signaling. Absence of Gas6 did not alter tyrosine phosphorylation of the EpoR, but reduced Akt phosphorylation in erythroblasts when treated with Epo. Akt phosphorylation in Gas6-/- erythroblasts was restored when co-treated with Epo and rGas6. Thus, Epo/EpoR-mediated erythroblast survival and proliferation is stimulated by Gas6 via the PI3K/Akt pathway. (iii) WT erythroblasts secrete Gas6 when treated with Epo. Moreover, we found that all three Gas6 receptors were expressed on erythroblasts and capable of binding Gas6 but only Axl seemed to play a role in erythropoiesis in vivo as demonstrated by the application of acute anemia models to mice lacking any one of the Gas6 receptors. Thus, erythroblasts regulate their own responsiveness to Epo by reinforcing signaling downstream of EpoR, via Axl and Akt. This mechanism is delicately balanced, since treatment of WT anemic mice with rGas6 alone reversed acute anemia without causing polycythemia. A similar beneficial response in hematocrit restoration was observed when a transgenic mouse model with chronic anemia was treated with rGas6. Gas6 is therefore effective in chronic anemia, augments the effects of Epo, and may have Epo dose-sparing effects, and thus may provides safe therapeutic approach to treat patients with Epo-resistant anemia.

Oncologie et Développement/ODE-25

Introduction of simple performance constancy tests for cardiac PET/CT perfusion imaging based on the NEMA NU 2-2001 standard 1

Modolo L., 1Bolard G., 2Malterre J., 2Bischof-Delaloye A., 1Verdun F., 2Prior J.

IRA-DUMSC1, Department of Nuclear Medicine2 Background: Myocardial perfusion imaging with cardiac PET is becoming available even to remote PET centers without cyclotron thanks to generator-based Rb-82 production. Performance evaluation of PET/CT are usually done using NEMA NU2-2001 standard during acceptance testing at scanner installation. For optimal performances as required for quantitative cardiac PET, it would be desirable to periodically repeat such testing. However, it is time-consuming, unpractical and requires large radioactivity (>3GBq), many test phantoms and is therefore costly. Moreover, these tests use raw data that do not account for post-processing and image reconstruction procedures, subject to change after a system maintenance. Our aim was to propose a series of alternative, more practical tests to assess performances consistency. Methods: Starting with the full NEMA NU2-2001 characterization of a Discovery LS (GE), we used a subset of tests that could be performed in only half a day and would require less than one patient radioactive dose ( 5.5 mg/L during > 7 d) (ICAAC 2005, M-2164). This observation suggested that VRC dose adjustment based on monitoring of blood levels may help to prevent SNAE.

Objective. To prospectively evaluate the utility of monitoring VRC blood levels for prevention of SNAE. Methods. VRC trough blood levels were measured by HPLC during the first week of therapy in 25 consecutive treatment courses during 2005. VRC dosing was adjusted if VRC trough blood levels were > 5.5 mg/L. Clinical follow-up included surveillance for adverse events (NCI criteria). Occurrence of SNAE during VRC therapy in 2004 (no prospective dose adjustment based on VRC blood levels) and 2005 (prospective dose adjustment) was compared. Results. Indications for VRC therapy were aspergillosis (60%), candidiasis (16%), and suspected mycosis (24%). Median VRC dose was 4 mg/kg bid (range 1.3 to 5.7). Median number of VRC trough blood levels measurements/treatment course was 2.5 (range 1 to 5). Median days to first measurement after starting VRC therapy were 2 (2-7). Nine pts (37%) presented transient self-limiting visual disturbances/hallucinations. Two patients (8%) presented severe hepatotoxicity. Occurrence of SNAE in 2004 and 2005 is compared: 2004

2005

No prospective dose

Prospective dose

adjustment

adjustment

8/32 (25%)

5/25 (20%)

Median VRC trough level, mg/L (range)

6.3 (5.6 to 11.2)

6.5 (5.6 to 11.5)

Median days > 5.5 mg/L (range)

12.5 (6 to 30) *

4 (2 to 7)

4/32 (12%)

0/25 (0%)

Patients with VRC trough level > 5.5. mg/L

SNAE * P = 0.05

Conclusions. These prospective data corroborate our preliminary report that rapid dose adjustment in patients with VRC blood levels exceeding 5.5 mg/L may help to prevent serious neurological adverse events. Further observations are needed to confirm this finding.

Procédures Thérapeutiques/THE-3

Therapeutic Drug Monitoring (TDM) in Adult Patients Receiving Imipenem (IMP) or Cefepime (CEF) Therapy: One-Year Single-Center Experience 1

Vora S., 1Pascual A., 1Bolay S., 1Francioli P., 1Calandra T., Marchetti O.

CHUV1

Background Experimental evidence suggests that time of beta-lactam antibiotics blood levels above MIC is a key factor for treatment efficacy. Low levels may be associated with treatment failure, while high levels may increase toxicity, especially in patients with impaired renal function. Objective To report the experience with TDM in adult patients receiving IMP or CEF therapy. Methods Retrospective analysis of consecutive IMP or CEF TDM during a 1-year period. Initial drug dosing was based on standard recommendations. IMP or CEF trough blood levels (Cmin) were measured by HPLC. Interpretation of Cmin as appropriate (A), inappropriate low (L) or inappropriate high (H) was based on MIC (when available), clinical and microbiological response, renal function, and drug blood levels reported in clinical studies. For L or H drug levels, interventions were proposed. Results 56 drug levels were measured (IMP, n=30; CEF, n=26) in 29 hemato-oncology, 11 intensive care, and 16 other adult patients. Treatment indications were microbiologically (27) or clinically (7) documented infections or unexplained fever (21). Daily dosing ranged from 500 to 4000 mg (IMP) and 500 to 6000 mg (CEF). TDM was performed 4 days (median, range 1-25) after treatment initiation: Cmin ranged from 0 to 13 (IMP) and 0.7 to 61 mg/L (CEF). Assessment of appropriateness of blood levels was: IMP (n=30)

CEF (n=26)

Appropriate

18 (60%)

16 (61%)

Inappropriate low (L)

11 (37%)

2 (8%)

Inappropriate high (H)

1 (3%)

8 (31%)

Interventions in patients with L IMP levels (range 0-1.5 mg/L) were: prolongation perfusion time (n=1), increase daily dosing (n=8), repetition TDM (n=1) or change antibiotic (n=1). All patients with H CEF levels (range 5-61 mg/L) had impaired renal function (creatinine clearance