FACULTÉ DE MÉDECINE
Campus Erasme
Bâtiment J
Route de Lennik, 808
B-1070 Bruxelles
Mardi 18 décembre 2007 de 9h30 à 17h30
7ème Journée des Doctorants
Sciences Biomédicales, Sciences Dentaires,
Sciences Médicales
Organisation
Sponsoring
Catherine Ledent,
Joanne Rasschaert,
Pascale Vertongen
et la CFD
A&E Scientific
Applied Biosystems
Bio-Rad
Perkin Elmer
Roche Diagnostics
Sartorius Stedim Biotech
Technigen
ThermoFischer Scientific/Perbio Science
VWR
http://www.ulb.ac.be/medecine/rdvdoc
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LE COMITE ORGANISATEUR REMERCIE
MESDAMES ET MESSIEURS
S. ALOUAT, L. DALOZE, B. JELLOULI, P. JESPERS, A. KHOUILED
PAUL COLIN, DOMINIQUE KRIKILION ET
L'ÉQUIPE DU SERVICE TECHNIQUE
LES DOCTORANT(E)S
LES MODERATEURS DE SESSIONS:
Profs. J.-P. BRION, C. BRUYNS et G. VASSART
ET LEURS ASSISTANTES
F. MIES, S. MONLEZUN et A. SUTHERLAND
LES MEMBRES DES JURYS
R. BEAUWENS, A. CARDOZO, S. COSTAGLIOLA, C. DELPORTE,
D. GALL, C. HOBERTUS, E. MURAILLE, J. PERRET,
B. ROBAYE, A. VAN KEYMEULEN
LES SOCIETES SPONSORS:
A&E SCIENTIFIC
APPLIED BIOSYSTEMS
BIORAD
PERKIN ELMER
ROCHE DIAGNOSTICS
SARTORIUS STEDIM BIOTECH
TECHNIGEN
THERMO FISCHER SCIENTIFIC/PERBIO SCIENCE
VWR INTERNATIONAL
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BLES, Nathalie
BOARBI, Samira
BOCKSTAEL, Olivier
BONDUE, Antoine
BOTTEAUX, Anne
CHAHINE, Lyne
CHAREZINSKI, Michal
CHICO GALDO, Vanessa
CHINTAWAR, Satyan
DE SAN, Nour
DE TIEGE, Xavier
DENEUBOURG, Laurence
DEWACHTER, Laurence
DIRIX, Violette
DRIESSENS, Natacha
DUFRESNE, Valérie
ENA, Sabrina
GASPARD, Nicolas
ID BOUFKER, Ichame
KATERJI, Suair
KEYZER, Caroline
LEFEVRE, Philppe
LEHTONEN, Enni
LEISTEDT, Samuel
LUBAKI, Kumba
LYBAERT, Pascale
MADANI, Afarin
MAHMOUD ABADY, Maryam
MARKESSIS, Emily
MATHIEU, Myrielle
MATHYS, Vanessa
MERIC DE BELLEFON, Laurent
MOLLE, Céline
NAGEM, Boutaina
NOVAL RIVAS, Magali
REUSE, Sophie
K.Schepers
SHLYONSKIY, Vadim
SOHY, Denis
SOYFOO, Muhammad Shah
STAMATOPOULOS, Basile
VANZEMBERGH, Frédéric
VERGISON, Anne
VIRE, Emmanuelle
WEISS, David
XHAET, Olivier
XIE, Jingwei
R. Naeije
B.A. Wolucka
L. Tenenbaum
C. Blanpain
A. Abdelmounaaim
M. Pandolfo
S. Louryan
J. Van Sande
M. Pandolfo
F. Mascart
P. Van Bogaert
J.-M. Vanderwinden
R. Naeije / S. Eddahibi
F. Mascart
B. Corvilain
M. Estenne
S.N. Schiffmann /A. de Kerchove
P. Vanderhaeghen
J.J. Body / L. Lagneaux
S. Louryan
P.-A. Gevenois
M. Rooze
L. Tenenbaum
P. Linkowski
M. Heenen / G. Ghanem
P. Lebrun / S. Meuris
P.-A. Gevenois
R. Naeije / K. Mc Entee
P. Deltenre
K. Mc Entee
M. Struelens / P. Bifani
M. Goldman
M. Goldman / S. Goriely
P. van de Borne
M. Braun
C. Van Lint
F. Mascart
R. Naeije
M. Parmentier
C. Delporte
P. Martiat / L. Lagneaux
A. Allaoui / M. Kalai
A. Malfroot
F. Fuks
V. Detours / C. Maenhaut
P. van de Borne / P. Unger
I. Pirson
Abstracts disponibles à l'adresse: www.ulb.ac.be/medecine/rdvdoc/
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Mardi 18 décembre 2007
9h30-10h20 Hall du bâtiment J
Accueil des participants
Présentation des POSTERS
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10h20-10h30 Auditoire J
Introduction
COMMUNICATIONS ORALES : SESSION 1
Modérateurs : G. Vassart et S. Monlezun
10h30-10h50 Viré E., Denis H. Dedeurwaerder S., Brenner C., Deplus R., Didelot C, Blanchon L.,
Fraga M., Ballestar E., Jacinto F.V., Alaminos M., Morey L., Van Eynde A., Bollen M.,
Di Croce L., Shiekhattar R., de Launoit Y., Esteller M. and Fuks F. A mechanistic link
between DNA methylation and polycomb group proteins.
10h50-11h10 Xie J., Onnockx S., Vandenbroere I., Degraef Ch., Erneux Ch., Pirson I. Study of the
scaffold properties of SHIP2 by identification of two binding partners: JIP 1 and
intersectin 1.
11h10-11h30 Sohy D., Springael J.Y., de Nadai P., Guillabert A. and Parmentier M.
Dimerization of CCR2, CCR5 and CXCR4 chemokine receptors.
11h30-11h50 Mahmoudabady M., Mathieu M., Dewachter L., Hadad I., Ray L., Jespers P., Naeije
R. and Mc Entee K. Activin-A signaling in experimental dilated cardiomyopathy
11h50-12h10
Dewachter L., Adnot S., Fadel E., Humbert M., Naeije R. and Eddahibi S.
Contribution of the angiopoietin/TIE2 pathway to pulmonary artery smooth
muscle hyperplasia in idiopathic pulmonary hypertension.
12h10-12h30
Lefèvre Ph.; Beauthier J.P, De Maertelaer V., Feipel, Pineau J.C., Sobczak S., Van
Sint Jan V. and Rooze M. Hand morphology and identification in forensic
anthropology.
Abstracts disponibles à l'adresse: www.ulb.ac.be/medecine/rdvdoc/
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Mardi 18 décembre 2007
12h30 à 13h20
LUNCH ET PRESENTATION DES POSTERS
DEMOS:
A&E SCIENTIFIC
APPLIED BIOSYSTEMS
BIORAD
PERKIN ELMER
ROCHE DIAGNOSTICS
SARTORIUS STEDIM BIOTECH
TECHNIGEN
THERMO/FISCHER SCIENTIFIC -PERBIO SCIENCE
VWR INTERNATIONAL
Abstracts disponibles à l'adresse: www.ulb.ac.be/medecine/rdvdoc/
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Mardi 18 décembre 2007
COMMUNICATIONS ORALES : SESSION 2
Modérateurs : C. Bruyns et F. Mies
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13h20-13h40 Botteaux A., Sani M., Kayath Ch. A., Parsot C., Boekema E., Chamekh M., Jouihri N.,
Page Ann-Laure, Sory Marie-Paule, Biskri Latefa, Sansonetti Philippe and Allaoui A.
Weapons of mass secretion the hierarchical process of type III secretion in Shigella
flexneri.
13h40-14h00 Dirix V., VerscheureV. , Mielcarek N., Debrie A-S., Locht C. and Mascart F. Molecular
dissociation of FHA-induced IL-10 and IL-12 secretion by human dendritic cells.
14h00-14h20 Mathieu M. , El Oumeiri B., Thoma Ph., Metens Th., Touihri K., Hadad I., Ray L.,
Naeije R., Mazouz N., Heyndrickx G., Bartunek J., Mc Entee K.
Functional improvement after bone marrow-derived mononuclear versus
nonmodified mesenchymal stem cell therapy in chronic myocardial infarction.
14h20-14h40
Najem B., Unger Ph., Preumont N., Jansens J-L, Houssière A., Pathak A., Xhaet O.,
Friart A., De Roy L., Vandenbossche J-L, van de Borne Ph. Sympathetic control after
cardiac resynchronization therapy in congestive heart failure.
14h40-15h00 Markessis E., Poncelet L., Colin C., Coppens A., Hoonhorst I. and Deltenre P.
Measuring tuning curves using auditory steady state responses.
15h00 – 15h30 : PAUSE CAFÉ ET DEMOS
COMMUNICATIONS ORALES : SESSION 3
Modérateurs : J.-P. Brion et A. Sutherland
15h30-15h50 Gaspard N., Bouschet T., Hourez R., Naeije G.,Passante L., Schiffman S., Gaillard A.
and Vanderhaeghen P. An intrinsic pathway leads to the specification of a
comprehensive repertoire of cortical neurons from embryonic stem cells.
15h50-16h10 Bockstael O., Chtarto A., Wakkinen J., Yang X., Melas C.,Levivier M., Velu T., Brotchi
J. and Tenenbaum L. Differential tropism of AAV1 into striatum and substantia using
tetracycline inducible promoter or constitutive CMV promoter.
16h10-16h30 Chintawar S., Hourez R., Ravella A., Gall D., Schiffmann S. and Pandolfo M.
Cellular therapy for SCA1.
16h30-16h50 Chahine L., Abou-Khalil B., Siren A., Andermann F., Hedera P., Ge Q., Andermann E.
and Pandolfo M. A new locus for familial temporal lobe epilepsy on chromosome 3q.
16h50-17h10 De Tiège X., Ligot N., Goldman S., Poznanski N., de Saint Martin A. and Van Bogaert P.
Regression of brain glucose metabolic changes at recovery from continuous spikewaves during sleep.
Abstracts disponibles à l'adresse: www.ulb.ac.be/medecine/rdvdoc/
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17h15 : DÉLIBÉRATION DES JURYS, SUIVIE DE LA PROCLAMATION
Remise du prix de la meilleure présentation orale :
ROCHE DIAGNOSTICS
Remise du prix du meilleur poster :
BIORAD
DRINK DE CLÔTURE
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1. Bles N., Horckmans M., Libert F., Macours P., El Housni H., Marteau F., Boeynaems J-M.,
and Communi.D. GENE EXPRESSION PROFILING DEFINES ATP AS A KEY REGULATOR
OF HUMAN DENDRITIC CELL FUNCTIONS.
2. Boarbi S. and A. Wolucka B. CHARACTERIZATION OF THE KETOPANTOATE
REDUCTASE ACTIVITY OF THE 2-ACETOHYDROXYACID ISOMEROREDUCTASE
(ILVC) OF MYCOBACTERIUM TUBERCULOSIS
3. Bondue A. and Blanpain C. IDENTIFICATION AND CHARACTERIZATION OF
CARDIAC PROGENITORS DURING EMBRYONIC STEM CELLS DIFFERENTIATION.
4. Charezinski M., Vanmuylder N., Glineur R., Louryan S.
3D STUDY OF DUMBO RAT
5. Chico Galdo V., Jin L., Massart C., Vanvooren V., Friedman M., Dumont J.E., Van Sande J.
COULD ACRYLAMIDE INDUCE THYROID TUMORIGENESIS? IN VITRO AND IN VIVO
STUDIES.
6. Deneubourg L., Hague P., Ralea S., Schurmans S., Erneux Ch. and Vanderwinden J-M.
A COMPARATIVE STUDY OF THE ROLE OF SHIP2 AND PTEN IN A MOUSE MODEL
OF GASTROINTESTINAL TUMOR (KITK641E)
7. de San N., Hougardy J.M., Salmon I., Capello M., Mascart F. PHENOTYPIC
CHARACTERIZATION OF HUMAN ALVEOLAR TYPE II PNEUMOCYTES.
8. Driessens N., Ghaddhab C., Burniat A., Chico Galdo V., Miot F. and Corvilain B.
HYDROGEN PEROXIDE IN THYROID: POSSIBLE ENDOGENOUS CAUSE OF DNA
STRAND BREAKS AND POTENTIAL ACTOR IN CARCINOGENESIS.
9. Dufresne V., Knoop Ch., Malfroot A., Lamotte M., Opdekamp Ch., Deboeck G., Cassart,
Stallenberg B., Vandenbroeck J., Casimir G., Duchateau J., Estenne M.
EFFECT OF INFLAMMATION ON STRENGTH AND BULK OF RESPIRATORY AND
LIMB MUSCLES IN CYSTIC FIBROSIS (CF).
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10. Ena S., Schiffmann S.N., de Kerchove A. DETERMINATION OF THE GENE
EXPRESSION PROFILE OF THE STRIATOPALLIDAL AND STRIATONIGRAL NEURONS
BY A TRANSGENIC APPROACH.
11. Id Boufker H., Lagneaux L., Tondreau T.,Duvillier H., Body J-J., Journé F.
EFFECT OF OSTEOBLAST-LIKE CELLS ON BONE-RELATED AND INVASIVE GENE
EXPRESSIONS IN BREAST CANCER CELLS.
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12. Katerji S., Vanmuylder N., Rooze M., Louryan S.
THE CRANIO-FACIAL ABNORMALITY IN THE DUMBO RATS: CYTOGENETIC AND
MORPHOMETRIC ASPECTS
13. Keyzer C., Tack D., De Maertelaer V., Gevenois P.A.
IMAGING DIAGNOSIS OF ACUTE APPENDICITIS IN ADULTS
14. Lehtonen E., Bockstael O., Yang X., Velu T., Brotchi J., Levivier M., Tenenbaum L.
EFFECTS OF RAAV2-MEDIATED GDNF EXPRESSION IN RAT DOPAMINERGIC
GRAFTS.
15. Leistedt S., Dumont M., Coumans N., Lanquart J-P, Jurysta F., Linkowski P.
THE MODIFICATIONS OF THE LONG-RANGE TEMPORAL CORRELATIONS OF THE
SLEEP EEG DUE TO MAJOR DEPRESSIVE EPISODE DISAPPEAR WITH THE STATUS
OF REMISSION.
16. Lubaki L., Ghanem G. Vadoud-Seyedi J., Heenen M, Morandini R. EVALUATION OF
ANTIMELANOCYTE AUTOANTIBODIES BEFORE AND DURING TOPICAL
IMMUNOMODULATORS TREATMENT IN 40 PATIENTS SUFFERING FROM VITILIGO.
17. Lybaert P.1, Vanbellinghen A.M., Quertinmont E., Petein M., Meuris S., Lebrun Ph.
KIR6.2 AND SUR2, KATP CHANNELS SUBUNITS, ARE EXPRESSED IN THE
EPIDIDYMAL EPITHELIUM FROM SEVERAL MAMMALIAN SPECIES.
18. Madani M., De Maertelaer V., Zanen J., Van Muylem A., Gevenois PA. Service de
Radiologie, Hôpital Erasme. PULMONARY EMPHYSEMA: OBJECTIVE QUANTIFICATION
BY MULTI-DETECTOR ROW CT (MDCT).
19. Mathys V., Quatannens J., Wintjens R., Singhal A., Kurepina N., Mathema B., Lefevre Ph.,
Baulard A., Kreiswirth B.N. and Bifani P. MUTATIONS ASSOCIATED WITH PARAAMINOSALICYLIC ACID (PAS) RESISTANCE IN CLINICAL ISOLATES AND
SPONTANEOUS MUTANTS OF MYCOBACTERIUM TUBERCULOSIS
20. Méric de Bellefon L., Hames G., De Maubeuge J., Michel O., Goldman M., Roufosse F.,
Coulie P. T CELL RESPONSE TO TRICHOPHYTON IN A SKIN BIOPSY FROM A
PATIENT WITH GVHD-LIKE DISEASE.
21. Molle C., Nguyen M., Flamand V., Trottein F. , De Wit D., Willems F., GoldmanM. and
Goriely S. INTERLEUKIN-27 SYNTHESIS INDUCED BY TOLL-LIKE RECEPTOR
LIGATION CRITICALLY DEPENDS ON INTERFERON REGULATORY FACTOR 3
22. Noval Rivas M., Hazzan M.& Braun M. TWO TYPES OF IMMUNE REGULATORY
MECHANISMS ARE DEVELOPED IN VIVO FOR THE CONTROL OF CHRONIC GRAFTVERSUS-HOST DISEASE.
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23. Reuse S., Calao M., Quivy V., Demonté D., Veithen E., Martinelli V., De Wit S., Kabeya K.,
Moutschen M., Vaira D., Avettand V., Rouzioux C., Clumeck N. and Van Lint C. HDAC
INHIBITORS AND THE NF-KB INDUCER PROSTRATIN COOPERATE TO REACTIVATE
LATENT HIV-1 PROVIRUSES IN CELLULAR RESERVOIRS.
24. Schepers K., Mouchet F, De Schutter I., Dirix V., Hougardy J.-M., Locht C., Mascart F.
IFN RESPONSES TO MYCOBACTERIAL ANTIGENS IN MYCOBACTERIUM
TUBERCULOSIS_INFECTED YOUNG CHILDREN
25. Shlyonskiy V., Goolaerts A., Mies F., Naeije R. DEXAMETHASONE- AND CAMPREGULATED ION CURRENTS IN RAT TYPE II PNEUMOCYTES IN SITU.
26. Soyfoo M.S., De Vriese C., Debaix H., Martin-Martinez M.D., Mathieu Ch., Devuyst O.,
Steinfeld S.D., and Delporte Ch. MODIFIED AQUAPORIN-5 DISTRIBUTION IN
SUBMANDIBULAR GLANDS FROM NOD MICE DISPLAYING AUTOIMMUNE
EXOCRINOPATHY.
27. Stamatopoulos B., Meuleman N., Haibe-Kains B., Bron D. , Martiat Ph., and Lagneaux L.
COMPARISON OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS EXPRESSING
HIGH OR LOW LEVEL OF ZAP70 MRNA: NEW PROGNOSTIC FACTORS, DIFFERENCES
IN MICRORNA EXPRESSION AND INTERACTION WITH THE MICROENVIRONMENT.
28. Vanzembergh F., Brouckaert G., Vanden Berghe T., Peirs P., Lamoral S., Van Gucht S.,
Vandenabeele P., Kalai M. EFFECT OF LPS, DSRNA OR INTERFERONS ON THE
CLEARANCE BY MACROPHAGES OF APOPTOTIC AND NECROTIC CELLS AND OF
VIRULENT AND ATTENUATED STRAINS OF MYCOBACTERIA OR RABIES VIRUS
29. Vergison A., Tuerlinckx D., Verhaegen J., Malfroot A.
EPIDEMIOLOGIC FEATURES OF INVASIVE PNEUMOCOCCAL DISEASE IN BELGIAN
CHILDREN: PASSIVE SURVEILLANCE IS NOT ENOUGH.
30. Weiss Solis D., Tamayo P. and Mesirov J.P.
A METHODOLOGY TO CREATE GENE EXPRESSION-BASED SIGNATURE MODELS OF
ONCOGENIC PATHWAYS ACTIVATION.
31. Xhaët O., Argacha J-F, Pathak A., Gujic M.1, Houssiere A., Najem B.1, Degaute J-P, Van de
Borne Ph. SYMPATHOEXCITATION INCREASES THE QT/RR SLOPE IN HEALTHY MEN:
DIFFERENTIAL EFFECTS OF HYPOXIA, DOBUTAMINE AND PHENYLEPHRINE
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ABSTRACTS
I. PRESENTATIONS ORALES
A MECHANISTIC LINK BETWEEN DNA METHYLATION AND POLYCOMB GROUP
PROTEINS.
Viré E. 1, Denis H.1, Dedeurwaerder S.1, Brenner C.1, Deplus R.1, Didelot C.1, Blanchon L.1, Fraga M.2,
Ballestar E.2, Jacinto F.V. 2, Alaminos M.3, Morey L.5, Van Eynde A.4, Bollen M.4, Di Croce L. 5,
Shiekhattar R.5, de Launoit Y.6, Esteller M.2 and Fuks F. 1 Laboratoire d´Epigénétique du Cancer 1. Free
University of Brussels, Faculty of Medicine, Laboratory of Cancer Epigenetics, 808 route de Lennik, 1070
Brussels, Belgium. 2. CNIO, Cancer Epigenetics Group, C/ Melchor Fernández Almagro 3,28029-Madrid,
Spain. 3. Department of Histology, University of Granada and Fundacion Hospital Clinico, Avenida de
Madrid 11, 18009 Granada, Spain. 4. Division of Biochemistry, Faculteit Geneeskunde, Katholieke
Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium. 5. ICREA and CRG, c/ Dr. Aiguader, 88, E08003 Barcelona, Spain.6. UMR8161, CNRS, Institut Pasteur de Lille, Universités de Lille 1 et 2, Institut de
Biologie de Lille, 1rue Calmette, 59021 Lille, Cedex, France
Epigenetic modifications create molecular landmarks that differentiate between active and inactive
chromatin. The establishment and maintenance of gene silencing patterns are fundamental to cell
determination and function. Major epigenetic machineries involved in heritable silencing of gene activity
are DNA methylation and the Polycomb Group (PcG) proteins. We investigated the potential link between
these two epigenetic machineries. In mammals, DNA methylation occurs predominantly at cytosine residues
located within CpG dinucleotides. The deposition of methyl groups is catalyzed by active enzymes named
DNA methyltransferases (DNMTs) carrying out methylation patterns. DNA methylation represses gene
expression through several mechanisms. One of them involves methyl-CpG-binding proteins, which
selectively can “read” methylated CpG patterns within the genome. On the biological point of view, DNA
methylation is important for several normal as well as pathological processes. The mechanisms by which
DNA methylation patterns are targeted, established and how they enforce gene silencing remain still poorly
understood. Recent studies shed light on the mechanistic conversation between the DNMTs and histone
modifications. One candidate is histone H3 at lysine 27 (H3K27) methylation mark, another epigenetic
signal linked to transcriptional repression. This modification is catalyzed by the Polycomb group (PcG)
protein EZH2, a histone methyltransferase associated with transcriptional repression. Methylated K27
serves as a binding site for the recruitment of additional proteins, the binding of which contributes to gene
silencing at least partly through histone modifications and inhibition of chromatin remodeling activity. How
mammalian PcG complexes are recruited to their target genes remains unclear. PcG proteins are
transcriptional repressors that play a role in biological processes such as stem-cell fate decision,
differentiation and neoplasic development. Our studies lift the veil on the previously unrecognized direct
connection between DNA methylation and PcG proteins. First we examined whether DNMTs and EZH2
might function through a common mechanistic pathway to silence gene expression. We reveal that the
Polycomb group protein EZH2 controls CpG methylation through direct physical contact with DNA
methyltransferases (Vire et al., 2006). We then asked whether MeCP2 as a methyl-CpG-binding protein
might be involved in our model. We found that the link between EZH2 and DNA methylation might be a
two-way connection. Our work provides the first glimpse of a mechanistic link between two essential
epigenetic systems: the Polycomb group proteins and DNA methylation.
STUDY OF THE SCAFFOLD PROPERTIES OF SHIP2 BY IDENTIFICATION OF TWO
BINDING PARTNERS: JIP 1 AND INTERSECTIN 1
Xie J., Onnockx S., Vandenbroere I., Degraef Ch., Erneux Ch., Pirson I.IRIBHM
SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is an ubiquitously expressed
phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) 5-phosphatase which contains various motifs
susceptible to mediate protein-protein interaction. In cell models, evidence has been provided that SHIP2
plays a role in insulin and growth factor signaling, cytoskeletal organization, cell adhesion and migration.
Herein we describe the c-Jun NH2-terminal kinase (JNK)-interacting protein 1 (JIP1) and intersectin 1 as
new protein partners of SHIP2. Interactions between SHIP2 and two partners has been confirmed in
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overexpress system or endogenous sysytem. We observed that without modifying the association of JIP1
and MAPKs scaffold complex, SHIP2 positively modulated the MLK3/JIP1-mediated JNK1 activation.
Overexpression of SHIP2 up-regulated JIP1 and MLK3 tyrosine phosphorylation. This phosphorylation was
sensitive to inhibitors of the Src family and Abl kinases. These two effects were independent of the SHIP2
phosphoinositide 5-phosphatase activity, as they were still observed with a SHIP2 catalytic inactive mutant.
Besides, SHIP2 recruited Intersectin 1 to cell membrane under stimulation of EGF, which implies its role in
EGF receptor endocytotic process. These data suggest that by its docking properties SHIP2 can influence
JIP1-mediated JNK pathway signaling and endocytotic process.
DIMERIZATION OF CCR2, CCR5 AND CXCR4 CHEMOKINE RECEPTORS
Sohy D., Springael J.Y., de Nadai P., Guillabert A. and Parmentier M.IRIBHM
Dimerization have recently emerged as important features of G-protein-coupled receptors. Although it is
well established that GPCRs are able to form homo-heterodimers, the functionnal consequences of such
interactions remain often unclear. In previous studies, we have reported that the chemokine receptors CCR2
and CCR5 form homo- and hetero-oligomer and identified a negative binding cooperativity of allosteric
nature between the binding site of CCR2 and CCR5 in cells co-expressing both receptors. In the present
work, we have extent this study to CXCR4, more distantly related structurally than CCR5 and CCR2.
Therefore, while CCR5/CCR2 heterodimers might constitute a model of how receptor homodimers behave,
CCR2/CXCR4 and CCR5/CXCR4 heterodimers represent more likely the average situation when two (or
more) chemokine receptors are co-expressed in a leukocyte population. We show by Bioluminescent
Resonance Energy Transfer (BRET) that these receptors form constitutive homo and heterodimers. We
describe that the negative binding cooperativity observed for CCR5/CCR2 heterodimers also stands for
CCR2/CXCR4 and CCR5/CXCR4 heterodimers. When the receptors are coexpressed, a CXCR4-specific
chemokine (SDF-1) can compete for the binding of a CCR2-specific tracer (MCP-1) and of a CCR5specific tracer (MIP-1ß) and vice-versa. The allosteric nature of this interaction was demonstrated by non
equilibrium binding assays measuring the dissociation rate of tracers in the absence or presence of
competitors. Negative binding cooperativity between CCR2 or CCR5 with CXCR4 was demonstrated in
primary leukocytes, excluding that these observations might be due to overexpression of the receptors in our
recombinant model systems. We also show that the specific antagonist of CXCR4 (AMD3100) can compete
for the binding of a CCR2-specific tracer (MCP-1) and of a CCR5-specific tracer (MIP-1ß), and vice versa
(TAK-779 versus SDF-1), only when the two receptors are coexpressed. This is the first demonstration that
allosteric interactions across chemokine receptor heterodimers also involve small molecule antagonists and
that a chemokine receptor antagonist influence the functional response of another receptor on which it does
not bind. Such functional effects were demonstrated in calcium mobilization assays on recombinant
systems, in chemotaxis assays on lymphoblasts and in in vivo air pouch assays.
ACTIVIN-A SIGNALING IN EXPERIMENTAL DILATED CARDIOMYOPATHY
Mahmoudabady M., Mathieu M., Dewachter L., Hadad I., Ray L., Jespers P., Naeije R., Mc Entee K.
Laboratory of Physiology, Université Libre de Bruxelles, Faculty of Medicine, Brussels, Belgium
Background. Pathogenic mechanisms of dilated cardiomyopathy are not clearly understood, but recent
studies have demonstrated that activin-A (Act-A) is increased in heart failure patients from the NYHA class
II and in a rat model of myocardial infarction (Yndestad et al, Circulation, 2004). Growing evidences
suggest that cytokines and growth factors are implicated in abnormal cardiac remodelling and aggravation
of the disease. Act-A, a member of the TGF? superfamily, participates through the Smad pathway, in
regulation of important cellular processes such as apoptosis, survival, proliferation and hypertrophy. In the
nucleus, phosphorylated Smads regulate the transcription of specific genes such as cyclin D1 and cyclin
dependant kinase inhibitor p21. In this study, we hypothesized that myocardial Act-A/Smad signaling
pathway is activated in experimental dilated cardiomyopathy. Therefore, in a canine model of
tachycardiomyopathy, we measured at different times, cardiac gene expression of main Act-A/Smad
signaling actors to evaluate their implication in the regulation of the hypertrophic process. Methods:
Transvenous endomyocardial biopsies were taken weekly in 15 dogs during development (7 weeks) of heart
failure induced by rapid right ventricular pacing. Relative cardiac gene expression of Act-A, its types I and
II receptors and modulating ligands as well as cyclin D1 and p21 were assessed by real-time polymerase
chain reaction. Results. Act-A mRNA expression (figure) increased progressively to be 4 fold higher in
11
overt congestive heart failure than at baseline. A two-fold decrease of activin type II receptors and activin
receptor interacting protein 2 expression was observed. Activin type I receptors, activin receptor interacting
protein 1, follistatin and follistatin related gene remained unchanged. Moreover, the inhibitory Smad 7, a
negative feedback loop regulator of the Smad pathway was overexpressed in severe heart failure. P21
mRNA expression (figure) was early and importantly increased with a 8 fold overexpression observed in
severe heart failure while cyclin D1 was downregulated. At echocardiography, left ventricular mass
remained unchanged. Conclusions.These results suggest that the Act-A/Smad pathway is activated during
development of overpacing induced dilated cardiomyopathy and has a negative regulator role on cardiac
hypertrophy by an increased p21 and a decreased cyclin D1 expression.
CONTRIBUTION OF THE ANGIOPOIETIN/TIE2 PATHWAY TO PULMONARY ARTERY
SMOOTH MUSCLE HYPERPLASIA IN IDIOPATHIC PULMONARY HYPERTENSION.
Dewachter L., Adnot S., Fadel E., Humbert M., Naeije R. and Eddahibi S.. INSERM U841, Département de
Physiologie, Hôpital Henri Mondor, AP-HP, Créteil, France. Laboratoire de Physiologie, Université Libre
de Bruxelles, Bruxelles, Belgique.
Angiopoietins are involved in blood-vessel maturation and stabilization through activation of the selective
endothelial-specific receptor Tie2. Recent studies suggest that stimulation of Tie2 receptor by angiopoietin1 (Ang1) could lead to the release of endothelial-derived growth factors acting on pulmonary artery smooth
muscle cells (PA-SMCs). Using primary cultures of human pulmonary artery endothelial cells (PA-ECs)
and PA-SMCs isolated from patients with idiopathic pulmonary arterial hypertension (iPAH) and controls,
we found that Tie2 receptor was fourfold higher in lungs and PA-ECs from patients with iPAH than in those
from control subjects, with a parallel increase in phosphorylated lung Tie2 receptor. In contrast, Ang1 and
Ang2 expression in lungs, PA-ECs, and PA-SMCs did not differ. Incubation of PA-SMCs with medium
collected from PA-EC cultures induced marked proliferation, and this effect was stronger when using PAECs from patients with iPAH than from control subjects. Ang1 pretreatment of PA-ECs from either patients
or control subjects induced a further increase in PA-SMC proliferation. Fluoxetine, an inhibitor of the
mitogenic action of serotonin, reduced the growth-promoting effect of PA-EC media. Ang1 added to PAECs from patients with iPAH increased the production of endothelin-1 and serotonin, but not of plateletderived growth factor-BB or epidermal growth factor, and increased the amount of mRNA encoding
tryptophan hydroxylase-1 (the rate-limiting enzyme of serotonin synthesis), preproET-1, and ET-1–
converting enzyme. In conclusion, the Ang1/Tie2 pathway is potentiated in iPAH, contributing to PA-SMC
hyperplasia via increased stimulation of endothelium-derived growth factors synthesis by PA-ECs.
HAND MORPHOLOGY AND IDENTIFICATION IN FORENSIC ANTHROPOLOGY.
Lefèvre Ph. 1-2; Beauthier J.P 3, De Maertelaer V. 4, Feipel 1-2, Pineau J.C. 5, Sobczak S. 1, Van Sint
Jan V. 1, Rooze M. 1-2 1 Laboratory of Anatomy, Biomechanics and Organogenesis. Faculty of Medicine.
U.L.B 2 Laboratory of Functional Anatomy. ISM. U.L.B 3 Unité d´anthropologie médico-légale. Faculté de
médecine. U.L.B 4 IRIBHM. Faculty of Medicine. ISM. U.L.B 5 CNRS UPR 2147 - Dynamique de
l´Evolution humaine.
In case of recovery of a hand after accidental or criminal dismemberment or disarticulation, information
could be given to forensic investigators about the somatotype of the dismembered person from hands
variables and the "reattribution" of the limbs segments to the right subject done. Furthermore, the
reconstruction of a hand with its characteristics (type of nails, scars, ...) from its skeleton could become a
significant element of identification that can be submitted to the relatives of the victim or the missing person
for validation. Both hands volume was measured from 88 European right handed males using a needle
volumeter. Statistical software analysed the relationships between both hands volume and other measured
anthropological variables. Regression equations were obtained to characterize the above relationships. The
all subjects typology was also determined following the BMI somatotype. The stature is estimated from
hands length and breadth (RH: r²=.66, SEE = 4 cm; LH: r²=.62, SEE = 4.2 cm); weight from hand volume
and P2F5 perimeter (RH: r²=.69, SEE = 6.64kg; LH: r²=.64, SEE = 7.13kg). The correlation coefficient
between BMI estimated and BMI observed is r = .80 The validation of the method is then applied on a new
sample of 21 adults with the same characteristics. The weight of 90.5% of the subjects is estimated within a
difference of 5.98 kg and the stature of 76% of the subjects within 3 cm. The correlation coefficient between
BMI estimated vs BMI observed is r = .75 (p <.0001) No extensive work has been found in the literature
12
about hand reconstruction as an identification method. One cadaver hand and one volunteer´s hand have
been used and computer models of the bones and skin were obtained from computerized tomography. A
customized software allowed locating spatial coordinates of bony anatomical laNdmarks of the models.
From these landmarks, the spatial relationships between the models were determined and used to interpolate
the missing hand skin. The volume of the interpolated skin was compared to the real skin obtained from
medical imaging for validation. Comparison of the respective volume is indicative of the accuracy of the
method: a 3,5% volume difference was found between the original cadaver skin and the interpolated skin.
WEAPONS OF MASS SECRETION: THE HIERARCHICAL PROCESS OF TYPE III
SECRETION IN SHIGELLA FLEXNERI
Botteaux A., Sani M., Kayath Ch. A., Parsot C., Boekema E., Chamekh M., Jouihri N., Page Ann-Laure,
Sory Marie-Paule, Biskri Latefa, Sansonetti Philippe and Allaoui Abdelmounaaim Laboartoire de
Bactériologie Moléculaire
Shigella uses the Mxi-Spa type III secretion system (T3SS) to invade the colonic epithelium by injecting
effectors into host cells. The T3SS is composed of a bulb, a neck-domain and a needle whose length is
controlled at 50 nm by the Spa32 protein. The secretion hierarchy of three groups of proteins: i) the needle
components, ii) the translocators, and, iii) the effectors is necessary for the invasion process. In this work
we investigated the role of three proteins, Spa32, Spa40 and MxiC, in the hierarchy secretion control. Using
the two hybrid screen in yeast and co-purification approaches, we identified an interaction between Spa32
and the C-terminus 309-342 domain of Spa40 (Spa40C). We constructed a spa40 mutant and confirmed its
crucial role in secretion. Mutations of some residues of Spa40C affected Spa40 interaction with Spa32. To
identify Spa32 domains required for its function, we constructed and characterized twelve Spa32 truncated
variants. We found that Spa32, unlike what was previously reported for some homologous from other
bacteria, does not regulate the needle length by a molecular ruler mechanism. Moreover, we found that
residues 206-246 of Spa32 are required for Spa40 binding. Taken together, our results suggest that the
Spa40/Spa32 interaction co-ordinately regulates ordered effectors secretion via the T3SS. In another study,
we demonstrated by electron microscopy that IpaD, a type III secreted molecule, is localized at the tip of the
T3SS needle. Interestingly, using anti-IpaD antibodies we were able to neutralize bacterial entry into cells.
Thus, IpaD, which is conserved among all Shigella species, represents a potential candidate for a vaccine
development. Lastly, the inactivation of the mxiC gene deregulated synthesis and/or secretion of effectors.
The overproduction and secretion of some effectors were due to the enhanced transcription of their genes,
which is a consequence of the early secretion of the anti-activator OspD1 by the mxiC mutant. We found
that MxiC is secreted by the T3SS and that this secretion is essential for its function. Finally, we showed
that MxiC interacts with the ATPase Spa47, suggesting that MxiC specifically regulates the hierarchy of
effectors secretion from inside the bacterium.
MOLECULAR DISSOCIATION OF FHA-INDUCED IL-10 AND IL-12 SECRETION BY HUMAN
DENDRITIC CELLS
Dirix V.1, VerscheureV. 1, Mielcarek N. 2, Debrie A-S. 2, Locht C. 2, Mascart F. 1 1Lab. ofVaccinolgy and
Mucosal Immunity, Hôpital Erasme, Université Libre de Bruxelles (U.L.B.) ; 2 INSERM U629 and Institut
Pasteur de Lille.
Bordetella pertussis filamentous haemagglutinin (FHA) is a protective antigen included in most current
pertussis vaccines, both whole-cell and acellular. In young children both types of vaccines induce IFN-?
responses known to contribute to protection. However, for approximately half of the children a delay in
IFN-? responses is observed. We have shown that a poor IFN-? response to B. pertussis antigens in children
is related to high levels of IL-10 secretion by their monocytes. In mice it has been demonstrated that FHA
induces IL-10 secretion by dendritic cells (DCs). Here, we investigated the ability of FHA to induce IL-10
secretion by human DCs. Incubation of human DCs with FHA resulted in the secretion of significant
amounts of IL-10 in more than half of the subjects tested. FHA contains a RGD sequence, responsible for its
interaction with CR3 on the surface of monocytes. We therefore examined the role of this sequence by
replacing the glycin by alanin. The modified FHA was found to also induce IL-10 secretion by the DCs
from roughly half of the subjects tested, ruling out a significant contribution of the RGD sequence in FHAinduced IL-10 secretion. Finally, a truncated FHA molecule, consisting of its 80 kDa N-terminal domain
was found to not induce IL-10 in any of the DCs preparations, although it induced IL-12p70. Since this
13
form of FHA is protective in mouse challenge models, it may be considered as a replacement of FHA in
future acellular...
FUNCTIONAL IMPROVEMENT AFTER BONE MARROW-DERIVED MONONUCLEAR
VERSUS NONMODIFIED MESENCHYMAL STEM CELL THERAPY IN CHRONIC
MYOCARDIAL INFARCTION.
Mathieu M. 1, El Oumeiri B. 1-2, Thoma Ph. 3, Metens Th. 3, Touihri K. 1, Hadad I. 1, Ray L. 1, Naeije R.
1, Mazouz N. 4, Heyndrickx G. 5-6, Bartunek J. 5, Mc Entee K. 1 1 Department of Physiology and
Pathophysiology, Free University of Brussels, Brussels, Belgium; 2 Department of Cardio-Thoracic
Surgery, Saint-Luc, UCL, Brussels, Belgium; 3 Department of Radiology and Medical Imaging, Free
University of Brussels, Brussels, Belgium; 4 Cardio3 Biosciences, Braine l’Alleud, Belgium; 5
Cardiovascular Center, OLV, Aalst, Belgium; 6Department of Physiology, UCL, Brussels, Belgium.
Background: Stem cell therapy may facilitate cardiac repair after myocardial infarction but the optimal cell
type remains discussed. The present study was designed as randomized, investigator-blinded, placebo
controlled head-to-head comparison of autologous bone-marrow mononuclear cells (BMNC) and
nonmodified mesenchymal stem cells (MSC) in a large animal model of chronic myocardial infarction.
Methods: Twenty-four dogs underwent the ligation of the left coronary artery. Eleven weeks later, they
received intramyocardial injections of either placebo (n = 8), BMNC (227.106 ± 32.106 cells, n = 8) or
culture expanded nonmodified-MSC (232.106 ± 40.106 cells, n = 8). Echocardiography, magnetic
resonance imaging, conductance catheter and histopathology were used to assess cardiac function,
remodelling and viability before therapy (Baseline), and 9 and 16 weeks after cell injections. The
echocardiographic wall motion score (WMS) index was used to assess the regional systolic function.
Results: While left ventricular ejection fraction remained unchanged, the WMS index showed a sustained
improvement in the BMNC group (from 1.8 + 0.1 at baseline to 1.6 + 0.07 at 9 weeks and 1.5 + 0.07 at 16
weeks, both p<0.001). In the MSC group, the WMS index improved moderately at late follow-up (from 1.9
+ 0.08 at baseline to 1.7 + 01 at 16 weeks p<0.05). End systolic elastance increased only in the BMNC
transfer (from 2.23 + 0.25 mmHg/ml to 4.42 + 0.55 mmHg/ml at 9 weeks, p<0.05). This was associated
with a reduction in the magnetic resonance imaging infarct size (from 13 + 0.67 % at baseline to 10 +
1.17% at 16 weeks p<0.05) and an increased semi-quantitative arterial density in the infarct zone as
compared to MSC group (p<0.01). No changes in contractility and infarct size were noted in the MSC
group. Conclusions: In the canine model of chronic myocardial infarction, stem cell therapy with BMNC
appears to be superior to treatment with culture-expanded non-modified MSC to improve cardiac
contractility, regional systolic function, myocardial viability and vascularity after healed myocardial
infarction.
SYMPATHETIC CONTROL AFTER CARDIAC RESYNCHRONIZATION THERAPY IN
CONGESTIVE HEART FAILURE.
Najem B., Unger Ph., Preumont N., Jansens J-L, Houssière A., Pathak A., Xhaet O., Friart A., De Roy L.,
Vandenbossche J-L, van de Borne Ph. Service de Cardiologie- Hopital Erasme
Introduction: Chronicle sympathetic benefits of cardiac resynchronization therapy (CRT) in congestive
heart failure (CHF) are unknown. Moreover, whether this affects equally patients who clinically respond or
not to CRT is also unknown. We tested the hypothesis that, first, chronic CRT decrease muscle sympathetic
nerve activity (MSNA) while improving cardiac hemodynamics and functional status, and second, that the
favorable effects of CRT on MSNA disappear upon CRT interruption only in those who respond to CRT.
Method: In the first part of the study, we included 11 patients resynchronized by CRT 6+/-1 months prior to
study inclusion (SYN) and 10 matched ASY patients. Blood pressure, heart rate and MSNA were recorded.
All underwent functional status, echocardiographic assessments. In the second part of the study, twentythree consecutive CHF were included, among whom 16 presented a symptomatic improvement by one or
more NYHA functional classes 15+/-5 months after CRT (responders), and 7 had not improved after 12+/-4
months of CRT (nonresponders). MSNA and echocardiographic recordings were obtained in random order
during: atrio-right ventricular pacing (ARV), without stimulation, and during atrio-biventricular pacing
(BIV). Results: SYN patients had longer 6 minute walking distance and lower NYHA class (all p<0.05)
than ASY patients. MSNA of 56+/-2 bursts/min in ASY patients was higher than in SYN patients (48+/-3
bursts/min, p<0.05). Cardiac index was higher in SYN patients than in ASY patients (2.8+/-0.2 vs. 1.9+/-0.2
14
l/min.m², p<0.05, respectively). In the second part of the study, MSNA increased by 25+/-7% in the
responders, whereas it remained unchanged in the nonresponders, when shifting from the BIV mode to a
non synchronous condition (ARV and OFF modes) (p<0.01). Cardiac output decreased by 0.7+/-0.2 l/min in
the responders, but did not change when shifting from the BIV mode to the non synchronous pacing mode
in the nonresponders (p<0.01). Conclusion: CRT induces a sympathetic inhibition that persists chronically
in severe CHF patients. Moreover, reversible sympathoinhibition is a marker of the clinical response to
CRT.
MEASURING TUNING CURVES USING AUDITORY STEADY STATE RESPONSES.
Markessis E.1-2, Poncelet L.1, Colin C.3, Coppens A.1, Hoonhorst I.3-4, Deltenre P. 3-5 1 Faculty of
Medicine, Free University of Brussels, Brussels, Belgium 2 Belgian Kid’s Funds, Brussels, Belgium 3
Research Unit in Cognitive Neurosciences, Free University of Brussels, Brussels, Belgium 4 Fond National
pour la Recherche Scientifique, Brussels, Belgium 5 Clinical Neurophysiology Laboratory, Brugmann
Hospital, Brussels, Belgium.
Cochlear hearing losses present a high prevalence of non-functioning neuro-epithelial regions and almost
systematically alter peripheral auditory filters leading to various perceptual and rehabilitative consequences.
The gold standard method of characterising auditory filters and determining non-functioning neuro-cochlear
regions consists in Tuning Curves (TCs). The aim of this study is to develop an electrophysiological
equivalent of TCs using Auditory Steady State Responses (ASSRs) as a behavioural response surrogate in
the sedated beagle puppy for future pediatric clinical applications. The ‘ASSR Tuning Curve’ (ASSR-TC) is
built by plotting the level of the centre masker frequency just sufficient to mask the modulated pure tone,
which is fixed in level and frequency. ASSRs were used because they allow frequency-specific stimulation
of the cochlea and can be objectively and automatically detected by computerised phase coherence analysis
techniques. ASSR-TCs were measured in ten Beagle puppies at 1 and 2 kHz and centre masker frequencies
extended to about half an octave above and an octave below the signal frequency in one hundred Hertz step.
TC width (Q10dB), high- and low-frequency slopes and the masker frequency yielding the lowest masking
threshold were computed in order to compare these values with published Neural Tuning Curves (NTCs)
and Compound Action Potential Tuning Curves (CAP-TC) obtained in cats and guinea pigs, and CAP-TC
measured in the ten Beagle puppies. Psychophysical Tuning Curves (PTCs) were measured in ten normal
human subjects using the same maskers and either a pure tone or an amplitude modulated probe in order to
verify if the latter had a specific effect on TC parameters. All procedures gave rise to identical frequency
tuning patterns. ASSR-TCs showed quantitative parameters (Q10dB, high- and low-frequency slopes)
similar to those reported under invasive methods in cats and guinea pigs. They were shifted, at both signal
frequencies, about 200 Hz upward from the signal frequency, a pattern identical to that recorded in humans
using the same stimuli, and similar to CAP-TCs measured in the dog. Detuning may be a consequence of
off-frequency listening and lateral inhibition occurring in simultaneous masking experiments. These results
demonstrate the feasibility of objectively assessing auditory filters using ASSRs.
AN INTRINSIC PATHWAY LEADS TO THE SPECIFICATION OF A COMPREHENSIVE
REPERTOIRE OF CORTICAL NEURONS FROM EMBRYONIC STEM CELLS.
Gaspard N.1, Bouschet T.1, Hourez R.2, Naeije G.1,Passante L.1, Schiffman S.2, Gaillard A.3,
Vanderhaeghen P.1 1 IRIBHM, Faculté de Médecine, ULB, Bruxelles 2 Laboratoire de Neurophysiologie,
Faculté de Médecine, ULB, Bruxelles 3 Institut de Physiologie et Biologie Cellulaires, UMR 6187 U.
Poitiers, France
The cerebral cortex develops through the coordinated generation of a diverse repertoire of neuronal
subtypes, but the mechanisms involved in this complex process remain poorly understood. Here we show
that following an in vitro default pathway of neural differentiation, in absence of any added exogenous
morphogen signal, embryonic stem (ES) cells efficiently generate neurons that share all molecular, cellular
and functional landmarks of genuine neurons of the cerebral cortex. Strikingly, this pathway recapitulates
the major milestones of cortical development observed in vivo, including the sequential generation of a
comprehensive repertoire of distinct subtypes of cortical neurons, from early-born Cajal-Retzius neurons to
later-generated pyramidal neurons. The demonstration of intrinsic cortical neuropoiesis from embryonic
stem cells surprisingly suggests that cortical neuron identity emerges from a primitive default program of
differentiation in mammals. This unlimited and highly enriched source of functional neurons of defined
15
cortical identity can be used for the genetic dissection of cortical development and the rational design of
cellular therapies for brain diseases.
DIFFERENTIAL TROPISM OF AAV1 INTO STRIATUM AND SUBSTANTIA NIGRA USING
TETRACYCILNE INDUCIBLE PROMOTER OR CONSTITUTIVE CMV PROMOTER
Bockstael O. 1,2, Chtarto A. 1,2, Wakkinen J. 1,2, Yang X. 1,2, Melas C. 1,2,Levivier M. 1, Velu T.2,
Brotchi J. 1, Tenenbaum L. 1,2. 1 Laboratory of Experimental Neurosurgery and 2 Research Unit in
Biotherapy and Oncology, Multidisciplinary Research Institute (IRIBHM) Université Libre de Bruxelles,
Hôpital Erasme.
We have investigated the cellular tropism of rAAV2/1 vectors expressing green fluorescent protein under
the control of the tetracycline-responsive promoter coupled with rtTAM2 reverse transactivator (“tetON
vector”) in comparison with the CMV promoter in the nigro-striatal pathway of the adult rat. After injection
in the striatum, although both vectors mainly transduced neurons, the percentage of astrocytes was 5-fold
higher for the CMV vector (approx.6% of the gfp-positive cells) than for the tetON vector. In addition, the
relative proportions of projection gaba-ergic (DARPP-32-positive) neurons and parvalbumin-positive
interneurons were respectively 13:1 and 2:1 for the CMV and tetON promoters. Among DARPP-32transduced neurons, those projecting to the globus pallidus were equally transduced by both vectors whereas
those projecting to the substantia nigra (SN) pars reticulata were transduced efficiently by the CMV but
poorly by the tetON vector. Finally, low efficacy retrograde transport to the SN pars compacta (pc) was
observed with the CMV but not with the tetON vector. Both vectors efficiently transduced the
subventricular zone. However, in the olfactory bulb, the target structure of neurogenesis from the immature
neuroblasts residing in the subventricular zone, transduced neurons were evidenced when using the CMV
but not the tetON vector. Injections dorsal to SN resulted in selective transduction of tyrosine hydroxylasepositive neurons of the SNpc and of the ventral tegmental area by the tetON vector whereas the CMV
promoter transduced a widespread area of the midbrain. The rAAV2/1-tetON vector could constitute an
interesting tool for specific and modulatable transgene expression in midbrain dopaminergic neurons.
CELLULAR THERAPY FOR SCA1
Chintawar S. 1, Hourez R. 2, Ravella A. 1, Gall D. 2, Schiffmann S.2, Pandolfo M. 1 1 Laboratory of
Experimental Neurology, ULB, Brussels, Belgium 2 Laboratory of Neurophysiology, ULB, Brussels,
Belgium
Stem cells are regarded as a novel therapeutic strategy for various neurological diseases and injury, diabetes
and heart conditions. Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurological disorder
caused by the expansion of a CAG repeat encoding a polyglutamine tract. It is characterized by ataxia due
to the loss of Purkinje cells (PCs) in the cerebellar cortex and loss of neurons in the inferior olivary nuclei.
Adult neural precursor cells (aNPCs) derived from subventricular zone of mouse expressing GFP under ?actin promoter, cultured as neurospheres, were grafted in the cerebellar white matter of 5, 13 and 24 wks old
transgenic SCA1 and WT mice. These different ages represent the different stages of the disease.
Accelerating rotarod and Grip strength test showed significant behavioral amelioration in 24 wks old but not
5 and 13 wks old SCA1 mice one month after the graft. Histological observations have shown that grafted
cells remained in white matter in WT mice whereas they migrated to all cerebellocortical layers in SCA1
mice and acquired neuronal and astrocytic phenotype, however they did not express Calbindin, a Purkinje
neuronal marker. Cells migrated to molecular layer and Purkinje cell layer were in close contact with host
PCs and they expressed gap junction proteins, such as connexin-43. Quantitative analysis of cerebellar
cortex has shown that molecular layer is less shrinked in SCA1 mice after grafting aNPCs. Density of PCs
and number of ectopic PCs are also affected. We conclude that aNPCs provide neuroprotection in SCA1
mice where loss of PCs is required to attract grafted cells and observed neuroprotection may be in part
mediated by a direct contact between grafted cells and host PCs.
A NEW LOCUS FOR FAMILIAL TEMPORAL LOBE EPILEPSY ON CHROMOSOME 3Q
Chahine L., 1, Abou-Khalil B., 2, Siren A., 1, Andermann F., 3, Hedera P., 2,4, Ge Q.,2, Andermann E.,3,5,
16
Pandolfo M., 1 Laboratoire de Neurologie Expérimentale, ULB-Hôpital Erasme, Brussels Belgium. 2
Department of Neurology; Vanderbilt University-Nashville, TN. 3 Department of Neurology and
Neurosurgery, McGill University, Montreal, Canada. 4 Center for Human Genetics Research; Vanderbilt
University, Nashville, TN. 5 Departments of Human Genetics and Pediatrics, McGill University, Montreal,
Canada.
Background. Temporal Lobe Epilepsy (TLE) is a common and heterogeneous focal epilepsy syndrome with
a complex etiology, involving both environmental and genetic factors. It is characterized by recurrent
unprovoked seizures originating from the lateral or mesial temporal lobe. Several familial forms of lateral
and mesial TLE have been described. Mapped loci are on chromosome 10q22-q24 for autosomal dominant
familial lateral TLE (FLTLE) with auditory symptoms, on chromosome 4q13-q21 for benign familial mesial
TLE (FMTLE), on chromosome 12q22-q23.3 for FMTLE preceded by febrile seizures (FS), and on
chromosomes 18q and 1q25-q31 for FMTLE and FS. Mutations have been identified only in the leucinerich, glioma-inactivated 1 (LGI1) gene on chromosome 10q22-q24 in FLTLE. Methods. We conducted a
genome-wide scan using 10 cM-spaced microsatellite markers on a family with mesial TLE and FS. Four
individuals had FS during childhood; seven individuals have benign TLE without a defined history of FS.
Patients with TLE have infrequent simple partial, complex partial and secondarily generalized seizures that
respond well to treatment. The proband has no mesial temporal sclerosis. The mode of inheritance is
autosomal dominant with incomplete penetrance. Linkage analysis was performed using the Genehunter
software. Regions with LOD score > 1 and those that were poorly informative in the first-pass scan were
further genotyped. Results. Linkage was identified on chromosome 3q25-q26 in a 13 cM region flanked by
markers D3S1584 and D3S3520, with a peak LOD score of 3.85. This interval does not correspond to any
previously known locus for familial epilepsy or FS. Conclusion. We identified a novel locus for familial
TLE with FS, providing additional evidence of the complexity and genetic heterogeneity of familial focal
epilepsy.
REGRESSION OF BRAIN GLUCOSE METABOLIC CHANGES AT RECOVERY FROM
CONTINUOUS SPIKE-WAVES DURING SLEEP
De Tiège X.1,2, Ligot N.1, Goldman S.1, Poznanski N.2, de Saint Martin A. 3 and Van Bogaert P. 2
1 PET/Biomedical Cyclotron Unit and 2 Department of Paediatric Neurology, Hôpital Erasme, Université
Libres de Bruxelles, Brussels, Belgium. 3 Department of Pediatrics, Hôpitaux Universitaires de Strasbourg,
CHU de Hautepierre, Strasbourg, France
Objective: Epileptic syndromes with continuous spike-waves during sleep (CSWS) are characterized by the
appearance of various psychomotor deficits with the electroencephalographic (EEG) pattern of CSWS.
CSWS are defined by the presence of spike-wave discharges during at least 85% of non-REM sleep. In a
previous study, we brought metabolic evidence suggesting the existence of remote inhibition of the frontal
lobes induced by highly epileptogenic and hypermetabolic posterior cortex in these epileptic syndromes. 1
The clinical relevance of the remote inhibition hypothesis would be reinforced by the demonstration, at the
recovery phase of CSWS, of a common resolution of hypermetabolism at the site of epileptic foci and
hypometabolism in distant connected brain areas.
Methods: PET studies using 18F-fluorodeoxyglucose (FDG) were performed in 9 children during acute
and recovery phases of CSWS. PET data were analyzed at individual and group levels using statistical
parametric mapping (SPM). Subtractive analyses comparing FDG-PET data of the patients with a control
group were performed. Metabolic abnormalities characterizing the acute phase of CSWS were identified by
exclusive masking and variance analyses. Pathophysiological interaction analyses searched for changes in
effective connectivity between hypermetabolic and hypometabolic regions at the acute phase and their
evolution at recovery. Results were considered significant at pcorrected<0.05.
Results: At the individual level, CSWS recovery was characterized by a complete or almost complete
regression of both hypermetabolic and hypometabolic abnormalities. At the group level, altered effective
connectivity between focal hypermetabolism (centro-parietal regions and right fusiform gyrus) and
widespread hypometabolism (prefrontal and orbito-frontal cortices, temporal lobes, left parietal cortex,
precuneus and cerebellum) characterized the acute phase. The evolution to recovery was characterized by a
marked regression of both hyper- and hypometabolism and of effective connectivity abnormalities.
Conclusions: The regression of effective connectivity abnormalities at recovery highly suggests that when
the CSWS activity at the site of the epileptic focus cease, the functional disturbances in distant and
17
connected brain areas, evidenced by decreased glucose consumption in specific brain areas, concurrently
vanish. Both hypermetabolic and hypometabolic abnormalities observed at the acute phase of CSWS are
mainly related to the neurophysiological effects of CSWS activity.
II. POSTERS
POSTERS
1. GENE EXPRESSION PROFILING DEFINES ATP AS A KEY REGULATOR OF HUMAN
DENDRITIC CELL FUNCTIONS
Bles N., Horckmans M., Libert F., Macours P., El Housni H., Marteau F., Boeynaems J-M., and
Communi.D., I.R.I.B.H.M.
Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semi-maturation of human
monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles
induced by ATPgS and PGE2 in human MoDCs using microarray technology. At 6h of stimulation, ATPgS
initiated an impressive expression profile compared to that of PGE2 (1125 genes compared to 133 genes
respectively) but after 24h, the number of genes regulated by ATPgS or PGE2 was more comparable. Many
target genes involved in inflammation have been identified and validated by quantitative RT-PCR
experiments. We have then focused on novel ATPgS and PGE2 target genes in MoDCs including colonystimulating factor-1 (CSF-1), MCP-4/CCL13 chemokine, vascular endothelial growth factor (VEGF-A) and
neuropilin-1 (NRP-1). ATPgS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion which
are involved in monocyte and DC differentiation. Additionally, ATPgS down-regulated several chemokines
involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1a, CCL4/MIP-1b,
CCL8/MCP-2 and CCL13/MCP-4. Interestingly, VEGF-A – a major angiogenic factor displaying
immunosuppressive properties - was secreted by MoDCs in response to ATPgS, ATP or PGE2, alone or in
synergy with LPS. Finally flow cytometry experiments have demonstrated that ATPgS, ATP and PGE2
down-regulate NRP-1, a receptor playing inter alia an important role in the activation of T lymphocytes by
DCs. Our data give an extensive overview of the genes regulated by ATPgS and PGE2 in MoDCs and an
important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.
2. CHARACTERIZATION OF THE KETOPANTOATE REDUCTASE ACTIVITY OF THE 2ACETOHYDROXYACID
ISOMEROREDUCTASE
(ILVC)
OF
MYCOBACTERIUM
TUBERCULOSIS
Boarbi S. and A. Wolucka B., Laboratory of Mycobacterial Biochemistry; Institute of Public Health; 642,
rue Engeland, B- 1180, Brussels, Belgium.
The 2-acetohydroxyacid isomeroreductase (ILVC) is a key enzyme for the biosynthesis of branched-chain
amino acids (valine, isoleucine and leucine) that is absent from humans and other animals, and represents,
therefore, a potential target for new antimicrobials. We have recently characterized the ILVC protein
(Rv3001) of the Mycobacterium tuberculosis pathogen. Here, we show that the ILVC enzyme possesses
also a ketopantoate reductase activity and, in the presence of divalent metal ions, catalyses the following
reaction: Reaction I: 2-ketopantoic acid + NADPH à pantoic acid + NADP+ Optimal reaction conditions for
the reduction of ketopantoic acid were established at a pH of 7.9 and a 0.5 mM concentration of Mn2+ . The
apparent Km constants for NADPH and ketopantoic acid were determined to be 0.143 mM and 1.7 mM,
respectively. The specific activity of the recombinant ILVC-dependent ketopantoate reductase was 1.69
mmol.min-1.(mg protein)-1. The ketopantoate reductase activity of the mycobacterial ILVC enzyme was
inhibited by the known inhibitors of the plant a-acetohydroxyacid isomeroreductase: 2dimethylphosphinoyl-2-hydroxyacetic acid (Hoe 704) and N-hydroxy-N-isopropyloxamate (IpOHA), and
the determined IC50 values were 7 uM and 6 uM, respectively. The reduction of 2-ketopantoic acid to
pantoic acid (reaction I) is a penultimate step in the biosynthesis of the pantothenate (vitamin B5) that is
usually carried out by a specific ketopantoate reductase (panE). The M. tuberculosis genome contains a
homolog of the panE gene (Rv2573), but the gene product has never been characterized. In order to test the
role of the ILVC-dependent ketopantoate reductase activity in the biosynthesis of vitamin B5 in M.
tuberculosis, we prepared an ILVC knockout mutant by using allelic replacement strategy. The ILVCdeficient mutant of M. tuberculosis showed branched-chain aminoacid auxotrophy, grew slower and formed
smaller colonies. The in vitro growth of the mutant could be observed only on media supplemented with
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branched-chain aminoacids (L-isoleucine and the 2-ketoisovalerate precursor), and did not require
pantothenate. Thus, the 2-acetohydroxyacid isomeroreductase (ILVC) is not essential for the synthesis of
pantothenate in M. tuberculosis, probably because of the sufficient activity of the ketopantoate reductase
encoded by the panE gene. Further studies are necessary to assess the live-vaccine capacity of the obtained
ILVC knockout mutant of M. tuberculosis in animal models of tuberculosis.
3. IDENTIFICATION AND CHARACTERIZATION OF CARDIAC PROGENITORS DURING
EMBRYONIC STEM CELLS DIFFERENTIATION
Bondue A. and Blanpain C.IRIBHM
Under defined conditions, embryonic stem cells (ESC) are able to differentiate in vitro in different cellular
fates, including cardiac cells. They constitute a powerful tool to study molecular mechanisms directing cell
fate decision. Although genes expressed in cardiac cells are known, molecular signals responsible for
cardiac cell fate decision and markers of cardiac progenitors are poorly understood in vertebrates. In
addition to their potential role in regenerative medicine, embryonic stem cells can be used to study
molecular mechanisms directing cardiac cell fate decision. Using an overexpression method which allows a
tetracycline inducible gene expression in ESC, we studied different candidate genes for their ability to
promote cardiac commitment. We found a gene expressed in precardiac mesoderm in which overexpression
accelerates and enhances cardiac commitment in ESC. We are currently dissecting the molecular
mechanism responsible for this major enhancement of cardiac commitment.
4. 3D STUDY OF DUMBO RAT
Charezinski M., Vanmuylder N., Glineur R., Louryan S. Laboratoire d’Anatomie et d’Embryologie
Université libre de Bruxelles;
A variation of the domestic rat, called dumbo rat, has similar traits to human craniofacial disorders like
Treacher Collin syndrome or other pharyngeal arch disorders. Observed morphological features involve
low-set ears and mandibular retrognathia. In order to objectify the features of dumbo rat, we designed a
tridimensional cephalometric analysis. A first software (Amira 4.0) is used to analyze CT-scan data (dicom
files) and to create a 3D model of the head of the rat. The second phase, with Data Manager software, is to
put a series of anatomical landmarks on the 3D model. Numerous linear and angular measures can be
inferred from these landmarks. 3D cephalometric results obtained from a sample of dumbo rat will be
compared with a control group of wistar rats. Presently the anatomical landmarks are assessed on the 3D
model. The 3D study of dumbo rat could be a first step in using this rodent as an animal model for better
understanding of human pharyngeal arch disorders.
5. COULD ACRYLAMIDE INDUCE THYROID TUMORIGENESIS? IN VITRO AND IN VIVO
STUDIES.
Chico Galdo V., Jin L., Massart C., Vanvooren V., Friedman M., Dumont J.E., Van Sande J. IRIBHM
In 2002, a Swedish study showed that acrylamide could be found in food of everyday consumption like
French fries. On the other hand, two independent studies showed that acrylamide induced tumours in rats
after chronic ingestion, thyroid tumors being the most frequent in one of them. The relative thyroid
specificity of the in vivo tumorigenic effect of acrylamide in rats raises the question of a possible thyroid
specific target of acrylamide. In the thyroid, cAMP is mitogenic and H2O2 (a mutagenic agent) is actively
produced for thyroid hormone synthesis. An uncontrolled increase of one of these two molecules could lead
to tumors. A direct damaging affect on DNA could too. cAMP measurement in control or stimulated (TSH,
forskolin) cells with or without acrylamide in the medium showed no differences. Beside acting on the
cAMP levels itself, acrylamide could act on the cAMP cascade at a step downstream of cAMP or by
stimulation of other growth cascades. We investigated the overall effect on the induction of DNA synthesis
by incorporation of BrdU and we didn’t see any effect of acrylamide on cell proliferation. The effect of
acrylamide on H2O2 production was studied at different concentrations and no effect was observed. To
investigate the effect of acrylamide on DNA damage we set up the comet assay. We showed that acrylamide
could induce DNA damage, and using the histone H2AX phosphorylation test we showed that these
damages were not double strand breaks. We made then an in vivo study in mice, by administrating
acrylamide in their drinking water at doses comparable to those used in rats studies (3-4 mg/kg/day) for 2, 6
and 8 months. Some of the mice were also treated with thyroxine or methimazole to modulate the functional
19
state of their thyroid and induce a hypo- or hyperactive gland. The histology of the thyroid of these mice did
not show tumors in either condition, although we could see differences in thyrocytes of mice treated with
thyroxine (thinner cell) and methimazole (higher cell) showing the effectiveness of the treatment. In
conclusion, we showed that acrylamide induces DNA damage in vitro but we could not demonstrate effects
in vivo.
6. A COMPARATIVE STUDY OF THE ROLE OF SHIP2 AND PTEN IN A MOUSE MODEL OF
GASTROINTESTINAL TUMOR (KITK641E)
Deneubourg L., Hague P., Ralea S., Schurmans S., Erneux Ch. and Vanderwinden J-M. IRIBHM et
laboratoire de Neurophysiologie, Faculty of Medicine, ULB
PI 3-kinase phosphorylates the 3’ position of the inositol ring of PtdIns(4,5)P2 to generate the lipid second
messenger PtdIns(3,4,5)P3. This last messenger is critically involved in proliferation and survival pathways.
Its metabolic pathway has been shown to be frequently mutated in a wide range of human cancers.
PtdIns(3,4,5)P3 is metabolised by two different enzymatic reactions: dephosphorylation at the 3’ position
via PTEN (Phosphatase and Tensin homolog deleted on chromosome 10) or at the 5’ position via
phosphoinositide phosphatases such as the SH2 containing inositol 5-phosphatases 1 and 2 (SHIP1/2).
Knockout mice for PTEN are embryonic lethal, but the heterozygous mice have an increased incidence of
cancers similarly to what is observed in humans. SHIP2 is widely expressed in tissues and the knockout
mice display an important phenotype in muscle and liver in the control of insulin sensitivity. SHIP2 was
found to be constitutively tyrosine phosphorylated and associated with SHC in chronic myelogenous
leukemia progenitor cells. Oncogenic mutations, leading to the constitutive (i.e. ligand-independent)
activation of the KIT receptor and downstream signalling pathways, have been identified in various human
neoplasia, including gastrointestinal stromal tumors (GIST). One of these mutations is due to the
substitution of a Lys by Glu in position 641 of KIT. KITK641E knock-in mice develop interstitial cells of
Cajal (ICC) hyperplasia and GIST in the gut. This unique in vivo model of oncogenic KIT tumorigenesis is
available through a collaboration with Dr. B. Rubin, Seattle, USA. One of our goals is to cross the
KITK641E mice with mice heterozygous for PTEN or SHIP2 deficient provided by Drs. Parsons and
Schurmans, respectively. Quantitative Western blotting analysis in GIST of KITK641E knock-in mice
shows that both SHIP2 and PTEN are expressed in the homozygous mice and that the MAP kinase pathway
is constitutively activated in such mice in comparison to the wild type animal. A comparison of the
expression of PTEN and SHIP2 between the homozygous and wild type mice will be presented.
7. PHENOTYPIC CHARACTERIZATION OF HUMAN ALVEOLAR TYPE II PNEUMOCYTES
de San N., Hougardy J.M., Salmon I., Capello M., Mascart F. Lab of Vaccinology and Mucosal Immunity,
U.L.B.
Recent data suggest that alveolar epithelial cells could in some situations play a role of antigen presenting
cells (APC) at least in mice. As during latent tuberculosis (LT), Mycobacterium tuberculosis is able to
survive in different cells, especially the alveolar epithelial cells, these cells could be involved in the antigen
presentation of mycobacterial antigens inducing persistent cellular immune responses in latently infected
subjects. The heparin-binding hemagglutinin (HBHA) is one such antigen, involved in the adhesion of
mycobacteria to epithelial cells and inducing high IFN-g secretion by the circulating lymphocytes from
subjects with LT. The first part of this work was performed on an alveolar epithelial cell (AEC) line, the
A549 line. Optimal cell culture conditions were determined as well as the optimal combinations of
monoclonal antibodies to be used for their phenotypic characterization by flow cytometry. The surface
expression of different markers known to be expressed on classical APC was analyzed (HLA-DR, CD80,
CD86, CD40, B7-H2, CD58) as well as the expression of additional adhesion molecules (CD54, CD106).
Modulation of the expression of these molecules by the infection of the AEC with mycobacteria (BCG) was
analyzed after having determined the optimal conditions for infecting the cells with BCG. Their modulation
by cytokines known to be secreted during M. tuberculosis infection (IFN-g, TNF-a) was also analyzed. In
parallel to these experiments, we have adapted the technique described by I.R. Witherden and TD Tetley to
isolate AEC type II from fresh human lung fragments. The phenotype of freshly isolated AEC II will be
compared to that of the A549 cell line. These preliminary data will allow us firstly to determine the
influence of M. tuberculosis infection on the phenotype of AEC II and secondly to investigate the potential
role of these cells as APC for mycobacterial antigens during LT.
20
8. HYDROGEN PEROXIDE IN THYROID: POSSIBLE ENDOGENOUS CAUSE OF DNA
STRAND BREAKS AND POTENTIAL ACTOR IN CARCINOGENESIS
Driessens N.1, Ghaddhab C. 1, Burniat A. 1, Chico Galdo V. 1, Miot F. 1 and Corvilain B.1, 2. 1 IRIBHM,
Faculty of Medicine, 2 Department of Endocrinology, Erasme Hospital, Université Libre de Bruxelles,
Belgium
Thyroid nodules are frequent and 5% of them are cancers. Irradiation is the only environmental risk factor
clearly implicated in the pathogenesis of thyroid cancers. Oxidative stress could facilitate the carcinogenic
process by altering DNA (oxidation of bases, single- and double-strand breaks) and leading to mutations
potentially oncogenic. Induction of double-strand breaks by hydrogen peroxide (H2O2) is still controversial.
Therefore we studied if the high levels of H2O2 produced by the thyroid to oxidize iodide may induce DNA
single- and double-strand breaks and therefore could play a role in thyroid tumorigenesis. Rat (PCCL3) and
human (HTori-3) thyroid cell lines were used to set up a thyroid model for H2O2-induced DNA damage
evaluation. Effects on DNA were tested at different doses of H2O2 and were compared with the damages
observed after irradiation. Semi-quantitative measurements of DNA single- and double-strand breaks were
obtained by using single-cell gel electrophoresis (alkaline condition for single-strand breaks (ssb) and
neutral condition for double-strand breaks (dsb)). Western blotting measuring the phosphorylation of
histone H2AX on serine 139 was used to confirm the generation of dsb. This phosphorylation reflects the
activation of specific dsb reparation enzymes. We observed a significant formation of ssb and dsb after an
irradiation of 1 Gy. We demonstrated that in PCCL3 and HTori-3 cell lines, non lethal concentrations of
H2O2 (0.05 mM to 0.1 mM) provoke large number of ssb but also high levels of dsb. The presence of BSO,
that inhibits the antioxidant enzymes of the cells, decreased the threshold to observe DNA damages to
H2O2 without affecting the damage induced by irradiation. Therefore we may extrapolate that, in vivo, the
potential mutagenic effect of H2O2 will increase in case of deficient antioxidant defence. Moreover,
preliminary kinetic studies show a slower repair of DNA damages induced by H2O2 in comparison to those
induced by irradiation. In conclusion, hydrogen peroxide causes single- and double-strand breaks in thyroid.
These data support the hypothesis that generation of hydrogen peroxide in thyroid could play a role in
mutagenesis and might account for the high frequency of thyroid tumour and microcarcinoma.
9. EFFECT OF INFLAMMATION ON STRENGTH AND BULK OF RESPIRATORY AND LIMB
MUSCLES IN CYSTIC FIBROSIS (CF)
Dufresne V. Knoop Ch.,Malfroot A., Lamotte M., Opdekamp Ch., Deboeck G., Cassart, Stallenberg B.,
Vandenbroeck J., Casimir G., Duchateau J., Estenne M., Erasme University Hospital.
We have previously hypothesized that different levels of systemic inflammation may contribute to betweenpatient differences in diaphragm strength and bulk in CF (AJRCCM 2003; 168:989-94). We tested this
hypothesis in 30 (16 males) clinically stable adult CF patients (age: 29 7 yrs; FEV1 55 %pred, range 27113; BMI 20.1 kg/m2, range 14-27.6) and 20 healthy controls matched for sex, age and height. They were
assessed for inspiratory (MIP and SNIP) and expiratory (MEP) muscle strength, diaphragm thickness (Tdi),
and quadriceps strength (quadPT) and cross-section (quadCSA). Serum levels of C-reactive protein,
interleukin-6 (IL-6), tumor necrosis factor (TNF)-a and their soluble receptors were measured. At a similar
absolute lung volume, MIP was greater in CF patients than controls (103 ± 30 vs. 89 ± 25 cmH2O, p<0.02)
and Tdi was thicker (0.039 ± 0.012 vs. 0.031 ± 0.007 mm/kg, p<0.005). Tdi was significantly correlated
with lean body mass (LBM) only in controls (r = 0.55, p< 0.02). QuadCSA and QuadPT didn’t differ
between the two groups and were tightly correlated with LBM (r = 0.75-0.92, p<0.001). CF patients
expressed higher levels of plasma CRP (1.27 ± 1.3 vs. 0.13 ± 0.1mg/dl, p<0.001) and IL-6 (5.8 ± 5.2 vs. 0.4
± 0.5 pg/ml, p<0.005) than controls. Levels of CRP and IL-6 were inversely correlated with FEV1 and BMI
(r=0.43 -0.51, p<0.002) in CF patients. Multiple regression analysis identified LBM, airway resistance
(Rva) and plasma IL-6 as independent predictors of respiratory and limb muscle bulk
(Tdi=0.03*(LBM/weight) + 0.064*Raw - 0.35*logIL-6 - 0.68; R²=0.60, p<0.001; QuadCSA=
1.03*(LBM/weight) - 1.40*Raw - 13.67*logIL-6 - 3.53; R2=0.71, p<0.001). Conclusions: For any given
LBM, CF patients show a much greater intersubject variability in diaphragm thickness than healthy
subjects. This variability is accounted for by the combined and opposite effects of Raw (which is a surrogate
marker of training) and plasma IL-6 (which is a surrogate marker of systemic inflammation). In contrast to
21
its effect on the diaphragm, the increase in Raw (which reflects the degree of respiratory impairment) is
associated with a decrease in quadriceps bulk.
10. DETERMINATION OF THE GENE EXPRESSION PROFILE OF THE STRIATOPALLIDAL
AND STRIATONIGRAL NEURONS BY A TRANSGENIC APPROACH.
Ena S., Schiffmann S.N., de Kerchove A. Laboratory of neurophysiology, Université libre de Bruxelles
The basal ganglia are a complex system composed by subcortical structures comprising several
interconnected nuclei. The striatum, the principal structure of the system, is divised in dorsal part involved
in coordination of mouvement and a ventral part controlling the motivation, addiction and emotion. The
striatum is composed of medium spiny neurons (>90%). The MSNs can be divised into two subpopulations:
the striatopallidal and striatonigral neurons according their projections and their pattern of neuropeptides
and receptors expression. The high neurochemical and functional complexity of the system involve that we
do not know precisely the respective rules of striatopallidal and striatonigral neurons in conditions. To
investigate the function of these neurons, we will realize the gene expression profile of them. To identify
specifically the striatopallidal neurons, we use a model of transgenic mice based on the Cre/LoxP system.
The transgenic mice used result from the mating between the reporter strain Z/EG and a strain obtained in
the lab which expressed the Cre recombinase in striatopallidal neurons. The specificity of the striatopallidal
expression results from the insertion of Cre recombinase gene downstream the promotor of the A2a-R
(Adenosine A2a Receptor) which is expressed specifically in the striatopallidal neurons. The Z/EG strain is
characterized by the ubiquitary expression of a transgene wich is composed of a strong promotor
(pCAGGS) followed by LoxP- ?-galactosidase - STOP– LoxP-GFP. The crossing of the 2 strains induces
the recombinaison between the 2 LoxP sites and permits the transcription and the expression of the reporter
protein GFP in the striatopallidal neurons. To identify the striatonigral neurons, we use retrograde labelling.
In the transgenic mice, we inject fluorescent microspheres in the projection structure of these neurons
(substantia nigra pars reticulata). The microspheres are endocytosed and bring in the soma by retrograde
transport. The analyses by immunofluorescence of the transgenic mice show 25% of striatal neurons
expressing the GFP, without colocalization of the microspheres confirming the specificity expression of
GFP in striatopallidal neurons. The single cell RT-PCR shows that GFP neurons express striatopallidal
specific genes. We realize FACS (Fluorescent Activated Cell Sorting) to isolate them. We obtain between
30000 and 60000 striatopallidal or striatonigral neurons. The analyses by qPCR show that all five know
MSN subtype-specific genes (A2A-R, D1-R, D2-R, Enkephalin and Tac1) are segragated in the RNA
sample demonstrating that each sorted RNA sample is truly cell type specific. The final step of this work is
to realize the microarrays and analyse the modification of gene expression in physiological conditions and
pathological conditions as drug addiction.
11. EFFECT OF OSTEOBLAST-LIKE CELLS ON BONE-RELATED AND INVASIVE GENE
EXPRESSIONS IN BREAST CANCER CELLS.
Id Boufker H. 1, Lagneaux L. 2, Tondreau T.2,Duvillier H.2, Body J-J.1, Journé F. 1 1 Laboratory of
Endocrinology and Bone Diseases, 2 Laboratory of Experimental Hematology, Institut Jules Bordet,
Université Libre de Bruxelles
Breast cancer cells frequently metastasize to the skeleton, where they induce extensive osteoclast-mediated
bone destruction. Osteoblasts, depending on their maturation stages, play a dual role in mediating the effects
of breast cancer cells on osteoclasts and in controlling bone formation. However, data on the influence of
osteoblastic cells on metastatic tumor cells are scarce. In vitro models using conditioned medium or
membrane insert systems have been used to study the interactions between osteoblasts and breast cancer
cells, but these systems do not address direct cell-cell contacts. Our objective is to investigate the effects of
osteoprogenitor-like cells (MG-63) and osteoblast-like cells (SaOS-2) on luminal-like (MCF-7) and basallike (MDA-MB-231) breast cancer cells. Breast cancer cells were labelled with CellTrace CFSE and then
cultured with MG-63 or SaOS-2 cells during four days. After coculture, we separated tumour cells (CFSElabelled cells) from osteoblasts (no-labelled cells) by flow cytometry and the purity of the cancer cells was
controlled by their expression of Epcam, an epithelial-specific marker. After sorting, the purity of MCF-7
and MDA-MB-231 cells was 98.9 ± 0.1 % and 96.3 ± 2.5 %, respectively. RT-PCR analysis showed several
modifications in expression of the tested genes. SAOS2 cells weakly decreased the expression of
osteopontin and RUNX2 in MDA-MB 231, but increased the mRNA expression of these genes in MCF7
22
and the osteonectin in the two breast cancer cell lines. We also observed increase in mRNA expression of
osteolytic factors (RANKL and PTHrP) in MCF-7 cells cocultured with SAOS2. By contrast, MG63 cells
markedly increased gene expression of osteoprotegerin, and of osteonectin in the two tumour cell lines. A
strong increase in MMP-2 gene expression was observed in both tumour cell lines cocultured with MG-63
cells and more with SaOS-2 cells. In summary, our results suggest that osteoprogenitor-like cells increase
the invasive ability of both tested breast cancer cell lines, while osteoblast-like cells enhance the
osteoclastogenesis factor expression and the osteoblast phenotype mainly in the luminal-like MCF-7 cells.
Thus, cells from osteoblast lineage can modulate the ability of the breast cancer cells to invade the bone
matrix and to adapt to the bone microenvironment.
12. THE CRANIO-FACIAL ABNORMALITY IN THE DUMBO RATS: CYTOGENETIC AND
MORPHOMETRIC ASPECTS
Katerji S., Vanmuylder N., Rooze M., Louryan S. University Libre de Bruxelles, Department of Anatomy
and Embryology. Route de Lennik, 808, B 1070 BRUSSELS, Belgium.
The development of the face is dependent upon various processes whose disturbance can be at the origin of
some malformations. These processes are neural induction, brain morphogenesis, and migration of cephalic
neural crest cells to the facial buds. The rat “dumbo” exhibits some characteristics evoking human
dysmorphogenesis as Treacher-Collins syndrome: shortness of the maxillar, zygomatic and mandibular
bones, low position of the ears: this strain may constitute an interesting model to understand human
craniofacial transformation. We have performed a cytogenetic study of the rat Dumbo in comparison with
the Wistar strain considered here as control animal. We find no differences between both strains. A
morphologic and morphometric study was performed using E15, E16, E17, E18, E19, E20 and E21 stained
in toto with blue alcian and alizarin red. This analysis provided some lights about the comparative
development of facial bones. Conclusion: comparison of both strains craniofacial development suggests a
specific disturbance of genetic control. Further histologic and genetic analyses must contribute to elucidate
the early determinism of the Dumbo phenotype.
13. IMAGING DIAGNOSIS OF ACUTE APPENDICITIS IN ADULTS
Keyzer C., Tack D., De Maertelaer V., Gevenois P.A..Service de Radiologie, Hôpital Erasme.
Acute appendicitis is the most common cause of acute abdominal pain that requires surgery.
Ultrasonography (US) and computed tomography (CT) are increasingly used in order to improve diagnostic
performances and to provide alternative diagnoses. US is non-invasive but operator-dependant and
relatively uncomfortable in patients with acute abdominal pain. CT is highly accurate but needs ionizing
radiation delivered to the patients. As their mean age is ~30 years, radiation dose should be reduced. In
addition, the need of intravenous and enteric administration of contrast material – those are expensive and
potentially toxic – is still a matter of debate. In a first study, we have shown that in patients suspected of
acute appendicitis, unenhanced CT (without enteric or intravenous contrast material) obtained on the entire
abdomen has sensitivity and negative predictive values between 97 and 100%, even when reducing the dose
at 30% of the standard dose, and whatever the radiologist’s expertise. The delivered radiation dose
approximated that by 3-views plain abdominal radiographs. Low-dose unenhanced CT can thus be
recommended in patients suspected of acute appendicitis. In a second study, we have shown that US and
unenhanced CT do not have different diagnostic performances in the detection of both acute appendicitis
and alternative diseases, regardless of radiologist’s expertise. Nevertheless, inconclusive examinations and
non-visualization of the appendix are more frequent at US than at CT. As patients without acute
appendicitis are generally not operated on, it is not possible to consider surgical findings as a method of
reference to verify whether the detected organ is truly the appendix. Therefore, in a third study, we have
measured intra- and inter-reader reproducibility in assigning the normal appendix in order to investigate if
different radiologists consider the same organ as the normal appendix. This study revealed that this
reproducibility is reader dependent. Intra-and inter-reader concordance in assigning the appendix was
obtained in 70% of the cases, influenced by patient’s BMI and intra-abdominal fat volume. Finally, in our
fourth study (in progress), we aimed to determine the respective contributions of oral and/or intravenous
contrast material on the diagnostic performances and radiologist’s confidence on both standard and lowdose CT.
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14. EFFECTS OF RAAV2-MEDIATED GDNF EXPRESSION IN RAT DOPAMINERGIC
GRAFTS.
Lehtonen E., Bockstael O., Yang X., Velu T., Brotchi J., Levivier M., Tenenbaum L.
Lab. of Exp. Neurosurgery and I.R.I.B.H.M.
Intracerebral fetal ventral mesencephalic (VM) grafts can induce symptomatic relief in parkinsonian
patients. Clinical improvements are directly dependent on the survival and functionality of the graft. Our
hypothesis was that genetic modification of the fetal VM tissue by GDNF cDNA prior to grafting could
improve the clinical outcome. We have previously shown that rAAV2 viral vectors efficiently transduce
E14 rat VM. In this study E14 VM was transduced with an rAAV2 vector expressing GDNF or eGFP prior
to transplantation in the striatum of rats with a complete 6-OHDA lesion. Three months post-grafting, the
number of tyrosine hydroxylase (TH) positive cells in the graft in the GDNF group was ~2-fold higher than
in the eGFP group. In addition the TH+ reinnervation volume was ~1.5-fold bigger in the GDNF grafts.
However, no effect of GDNF on the motor improvements was evidenced. Further analysis showed that the
amount of dopamine transporters was similar in both groups. Furthermore, Girk-2, a potassium channel,
present in nigro-striatal but not in VTA dopaminergic neurons was less abundant in GDNF- as compared to
eGFP-grafted animals. These data suggest that although GDNF increased the survival of TH+ neurons in
E14 VM grafts, the additional TH+ neurons were not functional.
15. THE MODIFICATIONS OF THE LONG-RANGE TEMPORAL CORRELATIONS OF THE
SLEEP EEG DUE TO MAJOR DEPRESSIVE EPISODE DISAPPEAR WITH THE STATUS OF
REMISSION.
Leistedt S., Dumont M., Coumans N., Lanquart J-P, Jurysta F., Linkowski P. Sleep Laboratory –
Psychiatric Department, Erasme Academic Hospital, ULB.
OBJECTIVE The aim of the present study is to investigate the scaling properties of the sleep EEG in
remitted depressed men, and to evaluate if a past history of major depressive disorder (MDD) could modify
significantly and definitively, as a “scar marker”, the dynamics of the sleep EEG time series.
METHODOLOGY Whole night sleep electroencephalogram signals were recorded in 24 men: 10 untreated
depressed men in full to partial remission (42.43 +/- 5.62 years) and 14 healthy subjects (42.8 +/-8.55
years). Scaling properties in these time series were investigated with Detrended Fluctuation Analysis (DFA)
(time range: 0.16-2.00 s.). The scaling exponent a was determined in stage 2, in slow wave sleep (stages 3
and 4), and during REM sleep. Forty-five epochs of 20 seconds were chosen randomly in each of these
stages for each subject in both groups. RESULTS We did not observe a significant difference and deviation
of the scaling exponents between the two groups during the three sleep stages of interest. CONCLUSION In
this study, we do not observe any functional sequelae of a past history of one or more unipolar major
depressive episode on the fluctuation properties of the sleep EEG. This finding is a sign of similar
underlying neuronal dynamics in healthy controls and patients with a lifetime history of MDD. This study
gives an additional argument to the theory that depression does not modify definitively the dynamics of the
neuronal networks and is therefore against the “depressive scar hypothesis”, in which permanent residual
deficit is created by the acute state of the depressive disease.
16. EVALUATION OF ANTIMELANOCYTE AUTOANTIBODIES BEFORE AND DURING
TOPICAL IMMUNOMODULATORS TREATMENT IN 40 PATIENTS SUFFERING FROM
VITILIGO
Lubaki L.1,2, Ghanem G.2, Vadoud-Seyedi J.1, Heenen M.1, Morandini R.2 (1) Dept of Dermatology,
Hôpital Erasme, (2) LOCE – Institut J. Bordet
New topical immunomodulators (TIMs) have been reported to cause repigmentation of vitiligo lesions.
However, time-kinetics of such repigmentation also in different anatomic locations are not well known. We
performed two prospective studies: one, double-blind, placebo-controlled with tacrolimus, and the second,
an uncontrolled study with pimecrolimus. In order to (1) evaluate the time to reach significant pigmentation,
its duration and extent by comparing a tacrolimus vs a vehicle (petrolatum) in treated areas of the same
localization and within the same individual; (2) comparing pimecrolimus-associated kinetics of two, within
the same patient in different localizations. Patients included in tacrolimus study (n=20), had one pair of
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symmetric lesions on 9 anatomic localizations, left/right of similar size; face or/and upper limb lesions for
pimecrolimus inclusion (n=20). The levels of repigmentation was classified into 5 categories: none; mild;
moderate; good; complete. Blood samples were checked for antimelanocyte IgM and IgG autoantibodies by
CELISA using a selected cell panel. Twenty sera from healthy subjects were included as controls. Three
groups of patients were identified with tacrolimus compared to the control over 7 months of treatment: no
significant change in pigmentation (n=8); a pigmentation benefit with tacrolimus (n=9); a pigmentation
response higher in control areas (n=3). Seven lesions achieved complete repigmentation with pimecrolimus
from 4th to 7th. Upper-limbs lesions, shown none to moderate repigmentation. The tendency to a substantial
repigmentation was significantly higher on the face compared to upper-limbs. Significant repigmentation
difference between the two localizations occurred 4 months after the treatment and increased steadily with
time. The screening for antimelanocyte antibodies yielded 40% positive patients with a mean titer of 1/100.
Our work define 3 periods during repigmentation: repigmention triggering during the first four months of
treatment either with tacrolimus or pimecrolimus, rapid increase in pigmentation and a plateau for a
sustained repigmentation. The continuity of the treatment seems necessary to ensure a prolonged
repigmenting effect and even an enhanced one such as the one we observed on the face with pimecrolimus.
The extent of repigmentation was significantly higher on the face than other locations probably due to
differences in melanocyte density.
17. KIR6.2 AND SUR2, KATP CHANNELS SUBUNITS, ARE EXPRESSED IN THE EPIDIDYMAL
EPITHELIUM FROM SEVERAL MAMMALIAN SPECIES.
Lybaert P.1, Vanbellinghen A.M.1, Quertinmont E.2, Petein M. 4, Meuris S.1, Lebrun Ph.3 Laboratory of
Experimental Hormonology1, Laboratory of Experimental Gastroenterology2 and Laboratory of
Pharmacology3, Faculty of Medicine, Université Libre de Bruxelles (ULB), B-1070 Brussels, Belgium.
Institut de Pathologie et de Génétique4, B-6280 Loverval, Belgium.
ATP-sensitive K+ (KATP) channels are poorly characterized in the reproductive tract. The present study
was designed to evaluate the putative expression of KATP channel subunits (Kir6.x and SURx) in the
epididymis from different mammalian species. Immunohistochemical, western blot and RT-PCR techniques
were used. A positive immunostaining for Kir6.2 (KCNJ11) and SUR2 (ABCC9) was observed by
immunoenzymatic and immunofluorescent approaches in the principal epithelial cells throughout all regions
of the rat and mouse epididymis. Double labeling with anti-aquaporin 9 (AQP9) and anti-Kir6.2 confirmed
their colocalization in the principal cells. No immunostaining could be demonstrated for Kir6.1 (KCNJ8)
and SUR1 (ABCC8) subunits. Under higher magnification, the immunostaining for Kir6.2 exhibited a
cytoplasmic and a more intense labelling of the Golgi apparatus. A similar pattern was observed for SUR2
although in the latter case, the Golgi labeling appeared to be region-specific. Spermatozoa in epididymal
tubules from rodents also immunostained for Kir6.2 and SUR2. Western blot analysis of epididymal total
protein and crude membrane extracts from adult and prepubertal rats confirmed the presence of Kir6.2.
SUR2 protein expression was detected in adult epididymal extracts. Furthermore, RT-PCR established the
presence of Kir6.2 and SUR2 mRNA in prepubertal and adult mouse epididymis. Indirect
immunofluorescence also documented the presence of Kir6.2 and SUR2 in the epididymal epithelium, as
well as in spermatozoa, of human, canine, feline and bovine origin. These data demonstrate the presence of
the KATP channel subunits, Kir6.2 and SUR2, in epididymal epithelial cells and spermatozoa from several
mammalian species. Although their physiological roles need to be fully characterized, it is tempting to
propose that such types of K channels might be involved in protein secretion, fluid-electrolytes transport
occurring along the epididymal epithelium and in spermatozoa maturation.
18. PULMONARY EMPHYSEMA: OBJECTIVE QUANTIFICATION BY MULTI-DETECTOR
ROW CT (MDCT).
Madani M., De Maertelaer V., Zanen J., Van Muylem A., Gevenois PA. Service de Radiologie, Hôpital
Erasme.
Purposes: In pulmonary emphysema, 1°) to prospectively compare MDCT indexes derived from the
attenuation distribution curve with macroscopic and microscopic morphometry; 2°) to prospectively
investigate the effects of radiation dose and section thickness on these indexes; 3°) to prospectively compare
the size distribution of emphysematous spaces with macroscopic and microscopic morphometry. Materials
and Methods: MDCT scans were performed in 80 patients referred for surgical lung resection.
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Measurements were: 1°) relative area (RA) of lung with attenuation coefficients lower than thresholds
ranging from -910 HU to -980 HU, and percentiles (P1 to P18) of the distribution of attenuation coefficients
on 1.25-mm-thick sections; 2°) two radiation doses (20 and 120 effective mAs) and three section
thicknesses (1.25-, 5.0-, 10.0-mm); 3°) the slopes of the straight regression lines of log-log cumulative
frequency distributions of RA960 cluster size (D960) and P1 cluster size (Dp1). The extent of emphysema
was measured macroscopically and microscopically. Results: 1°) -970 HU yielded the strongest correlation
between RA and macroscopy (P < .001). Depending on the microscopic index considered, –960 HU and –
970 HU yielded the strongest correlations between RA and microscopy (P < .001). P1 yielded the strongest
correlations with both macroscopy and microscopy (P < .001). 2°) –970 HU, –960 HU, and P1 yielded the
strongest correlations regardless of the radiation dose and the section thickness but changes in radiation
dose and in section thickness have statistically significant effects on RA960, RA970, and P1 (P < .001),
except the dose on P1 (P = .910). 3°) Both log-log cumulative frequency distributions of RA960 and P1
cluster sizes decrease linearly (P < .001) but correlations between D960 and Dp1 and macroscopic and
microscopic morphometry were not statistically significant (P ranging from .165 to .990.) Conclusions: 1°)
RA960, RA970, and P1 reflect the extent of pulmonary emphysema; 2°) the radiation dose can be reduced
to 20 mAs, but both dose and section thickness must be constant in comparative examinations; 3°) the loglog cumulative frequency distribution of low attenuation cluster size reflects the terminal airspaces
geometry but not the extent of emphysema.
19. MUTATIONS ASSOCIATED WITH PARA-AMINOSALICYLIC ACID (PAS) RESISTANCE
IN CLINICAL ISOLATES AND SPONTANEOUS MUTANTS OF MYCOBACTERIUM
TUBERCULOSIS
Mathys V., Quatannens J., Wintjens R., Singhal A., Kurepina N., Mathema B., Lefevre Ph., Baulard A.,
Kreiswirth B.N. and Bifani P. Laboratoire de Pathologie Moléculaire Institut Pasteur de Bruxelles
The emergence of Mycobacterium tuberculosis resistant to first line antibiotics has lead to renew interest in
second line antimycobacterial agents. Here, we set-out to extend our understanding on the mechanisms of
para-amino salicylic acid (PAS)-resistance through the analysis of 5 genes of the folate pathway and
biosynthesis of thymine nucleotides (thyA, dfrA, folP1, folP2 and thyX genes) in a collection of PASresistant clinical isolates and PAS-resistant spontaneous mutants. Mutations within the thyA gene were
identified in only 37% of the non-clustered clinical isolates and spontaneous mutants. All clustered clinical
strains had the same mutation, suggesting that these isolates became PAS-resistant before disseminating.
Overall 18 distinct mutations were identified in the thyA and 3 in the dfrA genes. Only one mutation type
was identified both in clinical isolates and spontaneous mutants. To our surprise, early frameshift mutations
or gene knock-outs were only seen in spontaneous mutants, but not clinical isolates. This may indicate that
ThyA is not essential in vitro but might play a more important role in vivo. The corresponding minimum
inhibitory concentration (MIC) was determined by Bactec/Alert and diffusion assay. Growth-curves were
determined for selected isolates. The MICs were correlated with mutation-types. Using structural
bioinformatic techniques, the altered thyA protein were predicted to generate an unfolded or dis-functional
polypeptide. Likewise, thyA knock-out strains were shown to be PAS-resistant, implying that thyA protein
is implicated in the bacteriostatic activity of PAS. The in vivo activity of PAS is presently being evaluated
on a murine model. PAS-resistant spontaneous mutants displaying various polymorphisms are being
compared for fitness in vivo and in vitro. The absence of mutations in 63% of PAS-resistant strains further
reveals that PAS resistance in M. tuberculosis may involve mechanisms other than those pertaining to the
biosynthesis of thymine nucleotides.
20. T CELL RESPONSE TO TRICHOPHYTON IN A SKIN BIOPSY FROM A PATIENT WITH
GVHD-LIKE DISEASE.
Méric de Bellefon L.1, Hames G.2, De Maubeuge J.3, Michel O.4, Goldman M.1, Roufosse F.1,5, Coulie
P.2 1 : Institute of Medical immunology – ULB, Brussels, Belgium 2 : Cellular Genetic Unit – UCL,
Brussels, Belgium 3 : Dermatology department – Hospital Saint-Pierre, ULB, Brussels, Belgium 4 :
Pneumology – Allergology department – Hospital Brugmann, ULB, Brussels, Belgium 1, 5 : Internal
Medicine department – Hospital Erasme, ULB, Brussels, Belgium
A young male who developed a systemic immune-mediated disorder reminiscent of chronic graft-versushost disease, characterized by lymphocytic inflammation of lungs, small bowel and skin, after professional
26
exposure to vinyl chloride, has been studied extensively to investigate the role of maternal microchimerism
in the induction of disease. His disease is partially controlled by chronic immunosuppressive therapy
associating low-dose corticosteroids and azathioprine. Numerous attempts to demonstrate patient-derived T
cell reactivity against maternal antigens have proven unsuccessful. A skin lesion was biopsied to derive and
analyze infiltrating T lymphocytes. After a few days of culture with IL2 and human serum, robust CD4 T
cell proliferation and slow fungal growth were observed. The fungus was identified as a dermatophyte –
most probably Trichophyton rubrum –, which in all likelihood is coming from the patient’s skin. T cell
cloning was performed using OKT3-CD28 stimulation; and ten CD4 T cell clones were isolated. Cytokine
secretion assays were performed in presence of autologous B-EBV cells pulsed with Trichophyton culture
supernatant. Two of these CD4 T cell clones secreted cytokines (IL5, IL4, IL10, TNFa, IFN?) in these
conditions. HLA restriction was also investigated using different B-EBV cell lines, indicating a peptide
presentation through HLA DR*11. We will try to identify the antigenic peptide within a cDNA library
prepared from Trichophyton. This is the first time that human Trichophyton-specific CD4 T cell clones are
obtained and be characterized in detail. We hypothesize that Trichophyton, a frequent cutaneous pathogen,
plays a role in the chronic inflammatory disease of this patient.
21. INTERLEUKIN-27 SYNTHESIS INDUCED BY TOLL-LIKE RECEPTOR LIGATION
CRITICALLY DEPENDS ON INTERFERON REGULATORY FACTOR 3
Molle C.1, Nguyen M.1, Flamand V.1, Trottein F.2 , De Wit D.1, Willems F.1, GoldmanM. 1 and Goriely
S.1 1 Institute for Medical Immunology (IMI), Université Libre de Bruxelles, Charleroi, Belgium. 2 Institut
National de la Recherche Médicale, U547, Institut Pasteur de Lille, Lille, France.
Interleukin (IL)-27, a member of the IL-12 family, is a heterodimeric cytokine composed of Epstein-Barr
virus-induced gene 3 (EBI3) and p28. Produced by dendritic cells (DCs) in response to Toll-like receptor
(TLR) ligands, IL-27 recently emerged as a key regulatory cytokine of inflammatory responses. We focused
on the molecular pathways implicated in TLR induced IL-27 expression. We first demonstrate that TIRcontaining adaptor inducing IFNb (TRIF) and its associated interferon regulatory factor (IRF) 3
transcription factor are critically involved in IL-27p28 expression in mouse DCs stimulated by TLR ligands.
We also observed that type I IFN signaling was implicated in p28 expression downstream of TLR3 and
TLR4. We then show that IL-27 serum levels are dramatically reduced in IRF3-/- upon lipopolysaccharide
(LPS) injection, indicating a critical role for IRF3 in TLR4-mediated IL-27 production in vivo. We
identified an ISRE binding site within the IL-27p28 promoter region which is required for the positive
regulatory role of IRF3 on IL-27p28 gene activation in reporter gene assays. In these experiments, we also
underline the positive role of other IRF members such as IRF-1 and IRF-7. In EMSA experiments with
nuclear extracts, we demonstrate that IRF3 and IRF1 physically interact in vitro with the ISRE site of the
IL-27p28 promoter after stimulation of wild-type DC with LPS. In human DCs, IL-27p28 mRNA was
preferentially induced by TRIF-coupled TLR ligands. Furthermore, chromatin immunoprecipitation studies
demonstrate that IRF3 is recruited to the endogenous p28 promoter in TLR4-stimulated human DCs. We
conclude that IRF3 activation is a master switch for IL-27 synthesis.
22. TWO TYPES OF IMMUNE REGULATORY MECHANISMS ARE DEVELOPED IN VIVO
FOR THE CONTROL OF CHRONIC GRAFT-VERSUS-HOST DISEASE
Noval Rivas M., Hazzan M.& Braun M.Institute for Medical Immunology (IMI), Université Libre de
Bruxelles (ULB), rue Adrienne Bolland 8, 6041 Gosselies, Belgium.
We developed a graft-versus-host disease (GVHD) model based on the adoptive transfer of RAG1-/- TcRtrangenic H-Y-specific Marilyn T cells (1x106 cells) into male recipients. Adoptive transfer into sublethally irradiated RAG1+/+ IL-2Rgc+/+, RAG1-/- IL-2Rgc +/+ or RAG1-/- IL-2Rgc-/- male recipients
induced 100% mortality after one week. On the contrary, non-irradiated mice of the three types survived
indefinitely after transfer and did not develop clinical sign of acute GVHD. Marilyn T cells transferred into
non-irradiated RAG1+/+ IL-2Rgc+/+ or RAG1-/- IL-2Rgc+/+ did not expand or were rapidly eliminated
(<45x103 in the spleen after 30 days). Remarkably, transgenic T cells transferred into non-irradiated RAG1/- IL-2Rgc-/- hosts expanded extensively (>15x106 in the spleen after 30 days) and were present in many
organs (spleen, lung, liver, kidney, lymph nodes). Histology revealed their capacity to mediate chronic
GVHD lesions (grade 1 after 30 days) in these hosts. The T cells did not develop a regulatory phenotype
since they remained Foxp3-negative and produced high amounts of IFN-? when stimulated in vitro.
27
However, they were unable to promote acute GVHD after transfer into irradiated recipients. Finally, T cell
failure to proliferate and induce chronic GVHD into non-irradiated IL-2Rgc-competent hosts was the results
of NK cell activity, since depletion of NK1.1+ cells by antibody treatment induced the extensive expansion
of adoptively transferred Marilyn T cells and caused chronic GVHD. Our model suggests that, in vivo, there
are two types of mechanisms involved into the regulation of T cell responses to persistent antigenic
stimulation: one is self-contained by the T cells and involves partial anergy; the other one would involve the
activity of existing populations of NK regulatory cells. Both of them appear to be necessary for full T cell
tolerance.
23. HDAC INHIBITORS AND THE NF-KB INDUCER PROSTRATIN COOPERATE TO
REACTIVATE LATENT HIV-1 PROVIRUSES IN CELLULAR RESERVOIRS.
Reuse S., Calao M., Quivy V., Demonté D., Veithen E., Martinelli V., De Wit S., Kabeya K., Moutschen M.,
Vaira D., Avettand V., Rouzioux C., Clumeck N. and Van Lint C. Laboratoire de virologie moléculaire
(IBMM).
The persistence of latently HIV-infected cellular reservoirs harboring replication-competent proviruses,
despite prolonged treatment with anti-AIDS multitherapy, represents a major hurdle to virus eradication.
These latently infected cells are a permanent source for virus reactivation and lead to a rebound of the viral
load after interruption of multitherapy. Activation of HIV gene expression in these cells combined with an
effective anti-AIDS multitherapy has been proposed as an adjuvant therapy that could lead to the
elimination of the latently infected reservoirs. We have previously demonstrated that HIV-1 transcription is
regulated by acetylation of histone and non-histone proteins and we have identified a new regulatory link
between protein acetylation and activation of the NF-?B signaling pathway by demonstrating a strong
synergistic activation of HIV-1 promoter activity by HDAC inhibitors (HDACIs) TSA and the NF-?B
inducer TNF?. However, because of their toxicity, the therapeutic use of TSA and TNF? is not possible.
Here, we extended our observations to less toxic HDACIs, with a special emphasis on those already in
clinical trials or use for other diseases (such as VPA) and to prostratin, a nontumor-promoting NF-?B
inducer. In latently-infected cell lines, we demonstrated a strong synergistic activation by a HDACI
combined with prostratin of viral transcription and production, using RNAse protection and p24 ELISA
assays, respectively. This HDACI/prostratin synergism was observed in transient transfection assays for
LTRs from subtypes A through G of the HIV-1 major group. Remarkably, the combined treatment
VPA+prostratin caused a more rapid and intense nucleosomal remodeling in the HIV-1 promoter region
than treatments by the molecules used separately. This remodeling was associated with HIV-1
transcriptional activation; thereby our results could explain partially the synergism observed between
HDACIs and prostratin. Importantly, more physiological results indicated that HDACIs and prostratin
synergistically induced HIV-1 recovery in ex vivo cultures of CD8-depleted peripheral blood mononuclear
cells isolated from aviremic HIV-infected individuals under multitherapy. These results are a proof of
concept of the therapeutic potential of the coadministration of several viral activators aiming at the efficient
reduction of the cellular latent reservoirs of HIV-1.
24. IFN RESPONSES TO MYCOBACTERIAL ANTIGENS IN MYCOBACTERIUM
TUBERCULOSIS_INFECTED YOUNG CHILDREN
K.Schepers 1, F.Mouchet 2, I. De Schutter 2, V.Dirix1, J.-M. Hougardy 1, C.Locht 3, F.Mascar t1
Lab. of Vaccinology and Mucosal Immunity 1, Dept. of Pediatrics 2, U.L.B., Brussels, Belgium ; Institut
Pasteur de Lille and INSERM U6293, Lille, France
Whereas primary M. tuberculosis (Mtb) infection progresses rapidly to active tuberculosis (TB) in only
small fraction of the non-immunocomprised adults or children above 3 years, children below 3 years of age
are at high risk of progression to disease. Here, we have investigated whether the susceptibility of infants to
severe forms of TB could be a consequence of their limited capacity to mount specific Th1-type responses,
as IFN- production is a recognised correlate of protection against TB. The IFN- concentrations secreted by
circulating lymphocytes in response to stimulation with different mycobacterial antigens, including the
protective antigen heparin-binding-hemagglutinin (HBHA), were compared in 3 cohorts of children,
respectively non-infected children (n=34), healthy infected children (HIC) (n=58) and children with acute
TB (n=38), and in 3 cohorts of adults, controls (n=51), latent TB (n=65) and acute TB (n=89). Children
were divided in 2 groups, younger or older than 3 years (n=73 and 57). The circulating lymphocytes from
28
most HIC and children with acute TB secreted IFN- in response to an in vitro stimulation with the different
mycobacterial antigens. Combining the IFN- response to at least 2 different mycobacterial antigens allowed
to detect 77% of the HIC and 85 % of the TB children so that this IFN- release test could be a help for the
diagnosis of Mtb infection in children. However, in contrast to results obtained for adults, similar IFNconcentrations were obtained for HIC and TB children, whatever their age category and they were relatively
low (median 250 pg/ml compared to 1757 pg/ml in adults with latent TB). The HBHA-induced IFNconcentrations were higher in children previously vaccinated with BCG. Also in contrast to results obtained
for adults, similar IFN- concentrations were measured in response to stimulations with native methylated
HBHA (the protective form) and with recombinant non-methylated HBHA, both for HIC and for TB. We
conclude that the primary immune response to Mtb infection in children is characterised by the appearance
of an IFN- response to most mycobacterial antigens. However, low IFN- secretions in response to the
protective antigen HBHA and especially to the methylated form of HBHA could be involved in the
susceptibility of young children to develop severe TB.
25. DEXAMETHASONE- AND CAMP-REGULATED ION CURRENTS IN RAT TYPE II
PNEUMOCYTES IN SITU
Shlyonskiy V., Goolaerts A., Mies F., Naeije R. Faculté de Médécine, laboratoire de physiologie
The composition and volume of the alveolar surface fluid affect effective gas exchange in the adult lung.
The alveolar fluid clearance is dependent on the transport of Na and Cl, but precise contributions of
concerned ion channels in basal conditions and after stimulation are still debated. We present a novel
functional model of rat lung slices suitable for whole-cell patch-clamp characterisation. Slices cultured on
paper filters retained a majority of living cells for up to 72 hours following sectioning. When patched in
whole-cell configuration, type II pneumocytes in situ had a mean capacitance of 11.6 1.2 pF and a resting
membrane voltage of - 4.3+/-2.8 mV. Replacement of Na+ in the bath with NMDG+ significantly decreased
inward whole-cell currents, however these currents were not amiloride-sensitive. Exposure of slices to 0.5
µM dexamethasone for 1 hour did not have an effect on ion currents. However chronic exposure to
dexamethasone during 24-48 hours induced an increase in Na-entry currents, which became partially
inhibitable by amiloride. Acute treatment of type II pneumocytes in situ with 100 µM cpt-cAMP resulted in
fast hyperpolarization of these cells by 15-20 mV arising from activation of inward Cl-currents sensitive to
NPPB, a blocker of CFTR. These results indicate that sodium amiloride-sensitive channels (ENaC) are not
present in type II pneumocytes from freshly prepared rat lung slices, however they can be induced by
chronic glucocorticoid treatment. They also indicate that in in situ conditions acute increase in cellular
cAMP content induces chloride absorption by Type II alveolar cells. Funded by ULB, FNRS and Fonds
Defay. VS was a recipient of ERS research fellowship (No 306).
26. MODIFIED AQUAPORIN-5 DISTRIBUTION IN SUBMANDIBULAR GLANDS FROM NOD
MICE DISPLAYING AUTOIMMUNE EXOCRINOPATHY
Soyfoo M.S1, De Vriese C.1, Debaix H.2, Martin-Martinez M.D.3, Mathieu Ch.4, Devuyst O.2, Steinfeld
S.D.5, and Delporte Ch.1 1Laboratory of Biological Chemistry and Nutrition, Université Libre de
Bruxelles, Brussels, Belgium; 2Division of Nephrology, UCL, Medical School, Brussels, Belgium;
3Department of Pathology, Bordet Institute, Brussels; 4LEGENDO, Katholieke Universiteit Leuven,
Leuven, Belgium; 5Department of Rheumatology, Erasme University Hospital, Brussels, Belgium.
Objective. The non-obese diabetic (NOD) mouse, a well described model of autoimmune exocrinopathy in
Sjögren’s syndrome (SS), was used to investigate the expression and localization of aquaporin-5 (AQP5) in
salivary glands. Furthermore the salivary gland function was assessed. Methods. NOD mice of two ages
were used to mimic different stages of SS. Matched BALB/c mice were used as control mice. AQP5
expression and distribution were studied in salivary glands. Salivary gland function was also determined.
Results. Compared to BALB/c mice, the relative AQP5 mRNA levels were not significantly modified in
parotid glands from NOD mice of both ages, but significantly increased in submandibular glands from NOD
mice of both ages. Western blot analyses of both salivary glands membranes revealed that AQP5 protein
was increased in 24 week-old NOD mice. Important inflammatory infiltrates were observed in
submandibular glands, but not in parotid glands, from 24 week-old NOD mice. The 8 and 24 week-old
BALB/c mice and 8 week-old NOD mice revealed AQP5 primarily at the apical membrane of the salivary
gland acinus. In contrast, in acini from submandibular, but not in parotid, glands of 24 week-old NOD mice
29
AQP5 staining was reduced at the apical membrane, but increased at the basal membrane. A marginal
decrease in pilocarpine-stimulated salivary flow was significantly observed in 24 week-old NOD mice
compared to matched BALB/c mice. Conclusion. In 24 week-old mice, several explanations might account
for the marginal difference in saliva flow rate that does not match the extent of AQP5 mis-distribution
27. COMPARISON OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS EXPRESSING
HIGH OR LOW LEVEL OF ZAP70 MRNA: NEW PROGNOSTIC FACTORS, DIFFERENCES IN
MICRORNA EXPRESSION AND INTERACTION WITH THE MICROENVIRONMENT
Stamatopoulos B., Meuleman N., Haibe-Kains B., Bron D. , Martiat Ph., and Lagneaux L. Laboratory of
Experimental Hematology, Bordet Institute
Chronic Lymphocytic Leukemia (CLL) has an extremely variable clinical course with overall survival time
ranging from months to decades. For some patients, the disease runs an indolent clinical course and life
expectancy is not shortened; for others, the disease is aggressive, progresses rapidly and survival after
diagnosis is decreased to 2-3 years. It is so very important to identify factors that can predict poor
prognostic and also identify patients who will benefit from intense therapy in an early stage. Therefore we
compared the most commonly used prognostic factors (Binet Stage, IgVH mutational status, Zap-70, CD38
and LPL expression) in a cohort of 108 patients with a median follow-up of 82 months to evaluate their
association with overall survival (OS) and treatment-free survival (TFS). Flow cytometry (FC) and
quantitative PCR (qPCR) on purified CD19+ cells were used. Regarding all these analysis, we conclude that
Zap-70 measured by qPCR is the most powerful prognostic factor and the best surrogate of IgVH
mutational status among all factors tested. Using this Zap-70 qPCR quantification, gene expression profiles
of high (Zap-70high, n=7) and low (Zap-70low, n=7) mRNA expression were then compared using
Affymetrix U133 plus 2.0 genechips representing more than 47000 transcripts. 135 genes were
differentially expressed with a p<0.001, and 43 genes were significantly different between the 2 groups with
a FDR<10%. Several differentially expressed genes were confirmed in an extended patient cohort (n=85)
and were able to separate the patients in terms of TFS or OS indicating their relevant clinical predictive
power (PDE8A, CD49d, FCRL family). Moreover, we also demonstrated that Zap-70 positive cells have
better capacities to adhere to bone marrow stromal cells and to migrate in response to stromal-derived factor
1 alpha (SDF1a). Finally, we investigated the expression of some microRNA between Zap-70 positive and
negative patients and we found that miR-29c and miR-223 had an individual prognostic power. This
comparison study concludes that CLL cells expressing high and low level of Zap-70 mRNA are
characterized by a distinct gene expression profile, reveals new potential therapeutic targets, new prognostic
factors, genes implicated in cellular activation, adhesion, and migration, and also differences in some
microRNA expression.
28. EFFECT OF LPS, DSRNA OR INTERFERONS ON THE CLEARANCE BY MACROPHAGES
OF APOPTOTIC AND NECROTIC CELLS AND OF VIRULENT AND ATTENUATED STRAINS
OF MYCOBACTERIA OR RABIES VIRUS
Vanzembergh F.1,2, Brouckaert G.1,3, Vanden Berghe T.3, Peirs P.2, Lamoral S.1, Van Gucht S.4,
VandenabeeleP.3, Kalai M.1 1Laboratory of Cellular Microbiology, 2Laboratory of Molecular Genetics of
Mycobacteria, and 4Laboratory of Rabies-Zoonosis, Pasteur Institute (ISP/WIV), Brussels, Belgium.
3Molecular Signalling and Cell Death Unit, Department of Molecular Biomedical Research, VIB-1, Ghent
University, Belgium.
Under physiological and pathological conditions, cells may die via an ordered cellular process, called
apoptosis, or via an alternative form of cell death, called necrosis. For example, L929hFas mouse
fibrosarcoma cells infected with vesicular stomatitis virus (VSV) die by apoptosis, while the same cells
infected with encephalomyocarditis virus (EMCV) die by necrosis. We demonstrated before, using
fluorescently labelled macrophages and dying cells in a flowcytometry-based phagocytosis assay, that
although apoptotic and necrotic cells are recognized and phagocytosed by macrophages, uptake of apoptotic
cells is more efficient both quantitatively and kinetically than that of necrotic cells. Our current results
demonstrate that pre-treatment of macrophages with alarm signals including type I or II interferons or Tolllike receptor ligands, such as LPS or dsRNA, enhances the capacity of phagocytes to engulf both apoptotic
and necrotic cells. Swift recognition and clearance of dying cells infected by bacteria or virus may help to
contain these intracellular pathogens and is often the first step in the process leading to the development of
30
acquired immunity. Thus, avoiding cell death may be beneficial for an intracellular pathogen, while
inducing cell death could promote a protective immune response by an efficient attenuated vaccine.
Therefore, we are currently exploring the effects on cell viability in macrophages infected with virulent
strains of Mycobacterium tuberculosis or rabies virus, in comparison with those of corresponding attenuated
vaccine strains, such as M. bovis BCG and ERA 2024 rabies virus. We are also examining the mode of cell
death induced during infection and the effects of pre-treatment with interferons, LPS or dsRNA on the
uptake of microbes by macrophages and on cellular, bacterial and viral viability.
29. EPIDEMIOLOGIC FEATURES OF INVASIVE PNEUMOCOCCAL DISEASE IN BELGIAN
CHILDREN: PASSIVE SURVEILLANCE IS NOT ENOUGH
Vergison A., Tuerlinckx D., Verhaegen J., Malfroot A. Department of Paediatric Infectious Diseases
Epidemiology and Infection Control Unit ULB- HUDERF
BACKGROUND. Reliable epidemiologic surveillance of infectious diseases is important for making
rational choices for public health issues such as vaccination strategies. In Belgium, as in most European
countries, surveillance relies on voluntary passive reporting from microbiology laboratories; therefore,
reported incidence rates are probably inaccurate. METHODS.We conducted national, active, laboratorybased and clinically based surveillance of invasive pneumococcal disease in young children. RESULTS.
During the study period, the incidences of invasive pneumococcal disease in children < 2 years of age
(104.4 cases per 105 person-years and 16.1 cases per 105 person-years for invasive pneumococcal disease
and meningitis, respectively) and in children 0 to 59 months of age (59.5 cases per 105 person-years for
invasive pneumococcal disease and 7.7 cases per 105 person-years for meningitis) were twice those
reported previously through the passive surveillance system. Overall, 67% of the Streptococcus pneumoniae
strains isolated from children < 5 years of age belonged to 7-valent pneumococcal conjugate vaccine
serotypes and 18% to ‘vaccine-related’ serotypes (mainly serotype 19A). Erythromycin resistance was
frequent, especially among children <2 years of age (59%). CONCLUSIONS. Under-reporting can explain
the reported low incidence of invasive pneumococcal disease in countries (such as Belgium) that depend on
a passive epidemiologic surveillance system, which could lead to erroneous choices in vaccination policies.
There is a need for an active system of epidemiologic surveillance for vaccine-preventable diseases such as
invasive pneumococcal disease, at the national or European level.
30. A METHODOLOGY TO CREATE GENE EXPRESSION-BASED SIGNATURE MODELS OF
ONCOGENIC PATHWAYS ACTIVATION
Weiss Solis D., Tamayo P. and Mesirov J.P. IRIBHM
We present a new pathway modeling methodology and apply it to detect the oncogenic activation of the
tyrosine kinase receptor EGFR by its agonist EGF. Publicly available gene expression data of controlled
experiments of EGFR activation were used as examples to train the model. Agreement between multiple
datasets identified consistent EGFR pathway marker genes. As a first step towards applying the model to
tumor data, experimental activation gene expression datasets related to EGFR signaling were used as
validation. In particular, the activation of RAS, RAF and MEK, downstream effectors of EGFR signalling
were predicted by the model. Furthermore, information about protein-protein interactions between marker
genes highlighted connected pathway modules. These modules may correspond to distinct ´pieces´ of the
EGFR pathway.
31. SYMPATHOEXCITATION INCREASES THE QT/RR SLOPE IN HEALTHY MEN:
DIFFERENTIAL EFFECTS OF HYPOXIA, DOBUTAMINE AND PHENYLEPHRINE
Xhaët O.1, Argacha J-F1, Pathak A.1,2, Gujic M.1, Houssiere A.1, Najem B.1, Degaute J-P1, Van de Borne
Ph.1, FESC. 1Department of Cardiology, Erasme University Hospital, Brussels, Belgium 2Service de
Pharmacologie Clinique, INSERM U 586, Faculté de Médecine, Université Paul Sabatier, CHU de
Toulouse, Toulouse, France
Introduction Dynamic ventricular repolarization assessed by QT/RR slopes studies the effects of
modifications in cardiac repolarization independently of variations in RR interval (RR). The effects of
changes in sympathetic and vagal activity on the QT/RR slope are controversial. We tested the hypothesis
that sympathoexcitation is an important determinant of the QT/RR slope. Methods and Results We
compared the effects of a reflex sympathetic activation in response to hypoxia, to the direct effects of the
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infusion of the beta adrenergic agent dobutamine, on the QTa (apex) and QTe (end)/RR slopes. Dobutamine
was titrated to obtain similar increases in cardiac output than with hypoxia. Cardiac vagal activity was
estimated by rMSSD and pNN50. In a second group of healthy subjects, we assessed the effect of a reflex
cardiac vagal activation in response to phenylephrine infusion on the same variables. We observed a similar
increase in QTa and QTe slopes during hypoxia and dobutamine (both p<0.017 vs normoxia), despite
divergent changes in cardiac vagal activity, as rMSSD and pNN50 decreased with hypoxia compared to
normoxia (p<0,001) but increased during dobutamine infusion compared to hypoxia (p<0,017). In contrast,
these slopes did not change during the rises in rMSSD and pNN50 elicited by phenylephrine (p>0.7).
Conclusion Beta adrenergic stimulation induces comparable increases in the QT/RR slopes than hypoxia,
but in the presence of a larger cardiac vagal activity. Vagal cardiac activation by phenylephrine does not
change the QT slopes. This reveals that the sympathetic system is an important determinant of QT/RR
dynamicity in healthy men.
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Abstracts disponibles à l'adresse: www.ulb.ac.be/medecine/rdvdoc/
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