journée lundi 5 andré verbert

Transcription

16ème édition de la
2016 Faculté de Médecine de Lille Pôle Recherche Salle des Congrès et annexes 08h30 Accueil par le Directeur de l'ED 09h00 Session orale ‘TOWARDS A BETTER KNOWLEDGE OF DISEASES’ 10h20 PAUSE CAFÉ 10h50 Session orale ‘MODELS AND MARKERS FOR NEW CHALLENGES’ 11h50 PrésentaBon de BioAddoct 12h00 PAUSE DEJEUNER (Atrium) 13h30 Session Posters 14h30 Session orale ‘PROMISING THERAPEUTIC STRATEGIES’ 16h10 Conférence 17h30 Cocktail et remise des prix (Atrium) Colloque Annuel des
Doctorants
p53, metabolism and ageing: lessons from TP53 mutaBon carriers Conférence plénière Pr. Pierre Hainaut InsBtut Albert Bonniot INSERM U823 Grenoble RéalisaBon /Photo: SFR DN2M / © L.Coudert LUNDI 5 SEPTEMBRE JOURNÉE
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Editorial
Bonjour à toutes et à tous,
C’est avec grand plaisir que je vous souhaite la bienvenue à la 16ème Journée André Verbert, colloque
annuel de l’Ecole Doctorale Biologie – Santé de Lille.
Depuis leur création en 2001, les JAV sont devenues un rendez-vous incontournable de notre Ecole
Doctorale. Au cours de ces années, elles ont permis à de nombreux doctorants de présenter leurs
travaux de recherche sous forme de communications orales ou affichées devant un large auditoire
couvrant l’ensemble des champs disciplinaires de notre ED, tout en favorisant les échanges et
discussions entre doctorants de différentes disciplines. Depuis quelques années, les communications
orales sont présentées en anglais et il s’agit souvent pour les doctorants en fin de deuxième année de
la première communication scientifique de ce type, les préparant à leurs futures présentations dans
des colloques internationaux. Depuis toutes ces années, les JAV connaissent un succès sans cesse
grandissant et la qualité des présentations et des discussions témoigne du dynamisme des recherches
menées au sein de notre Ecole Doctorale.
Je tiens à remercier très chaleureusement le Dr. Pierre Hainaut, Directeur de l'Institut Albert Bonniot
à Grenoble (INSERM U823 - Ontogenèse et oncogenèse moléculaire) qui a accepté de donner la
conférence plénière qui viendra ponctuer cette Journée.
Je remercie bien évidemment la présidente du comité des JAV Malika Hamdane et l’ensemble des
membres du comité d’organisation, de même que toutes les personnes qui se sont investies dans
l’organisation de cette Journée. Le programme de cette année est riche et diversifié, et les thématiques
abordées au cours des sessions orales iront des modèles et marqueurs en pathologie aux stratégies
thérapeutiques du futur, et seront complétées par plus de soixante-dix présentations par affiche.
Je souhaite à toutes et à tous, et en particulier aux nouveaux doctorants qui viennent de rejoindre notre
ED, une excellente JAV 2016, alliant Science, convivialité et partage.
Bien amicalement, Philippe
Pr. Philippe Delannoy
Codirecteur de l’Ecole Doctorale Biologie Santé de Lille
16ème Journée André Verbert
1
Faculté de Médecine
2
Colloque annuel des Doctorants - Programme de la JAV 2016
8h30 : Accueil par le Directeur de l'ED
9h00 - 10h20 : Session orale "Towards a better knowledge of diseases"
modérateurs : Samir Bensaid, Mélanie Lambert, Mathilde Zecchin
BAILLEUL Justine (INSERM U908 - directeur de thèse : Xuefen LE BOURHIS)
CXCL1 and CCL5, induced by ionizing radiation, reprogram non-tumorigenic cancer cells into
resistant cancer stem cells in breast cancer.
GROMADA Xavier (UMR CNRS 8199 éq. 02 - directeur de thèse : Jean-Sebastien ANNICOTTE)
E2F1 controls pancreatic β-cell function and identity
KONE Bachirou (UMR8204-U1019 éq. 12 - directeur de thèse : Philippe GOSSET)
Cellular and molecular mechanisms of susceptibility to infection in COPD
LEGHAY Coline (INSERM U1172 éq. 01 - directeur de thèse : Malika HAMDANE)
Functional investigation of new N-terminally truncated Tau species and relevance to Alzheimer’s
disease
10h20 - 10h50 : Pause café
10h50 - 11h50 : Session orale "Models and markers for new challenges"
modérateurs : Clément Bournonville, Mickael Canouil, Coline Leghay
MOULIN Solene (INSERM U1171 - directeur de thèse : Charlotte CORDONNIER)
New-onset dementia after spontaneous intracerebral haemorrhage: a prospective observational
cohort study.
DUPRET Barbara (INSERM U908 - directeur de thèse : Pierre-Olivier ANGRAND)
Application of the TALEN methodology in zebrafish to study the Polycomb Repression
AUBERNON Cindy (EA7367 - directeur de thèse : Damien CHARABIDZE)
Thermoregulation in necrophagous Dipteran larvae: application in forensic entomology.
11h50 - 12h00 : Présentation de BioAddoct
12h00 - 13h00 : Pause déjeuner
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13h00 - 14h30 : "Session POSTERS"
14h30 - 16h10 : Session orale "Promising therapeutic strategies"
modérateurs : Omar Castillo Aguilera, Matthias Lambert, Antonella Raffo Romero, Zakaria
Segaoula
BRESSON Lucie (INSERM U1008 - directeur de thèse : Feng CHAI)
Autologous cell-laden hydrogel sheet for prevention of post-surgical abdominal adhesion (PAA)
HERTAULT Adrien (INSERM U1008 - directeur de thèse : Stephan HAULON)
Evaluation of a Pro-Healing Polydopamine-coated Stent on In-Stent Restenosis using a rat model
DA COSTA GOUVEIA Joana (UMR8204-U1019 éq. 07 - directeur de thèse : Priscille BRODIN)
Pulmonary co-administration of the synergic drugs Ethionamide and Booster using aerosolized
nanoparticles: a promising strategy to treat tuberculosis
DUROUX Romain (INSERM U1172 éq. 06 - directeur de thèse : Said YOUS)
From design to pharmacological evaluation of benzoxazole derivatives as adenosine A2A receptor
antagonists for the potential treatment of Alzheimer disease.
HOMERIN Germain (INSERM U995 éq. 07 - directeur de thèse : Alina GHINET)
Structure-activity relationships of new ligands of the P2X7 receptor for the development of drugcandidates with anti-inflammatory potential
16h10 - 17h30 : "Conférence plénière"
p53, metabolism and ageing: lessons from TP53 mutation carriers
par Dr. Pierre Hainaut
Directeur de l'Institut Albert Bonniot à Grenoble (INSERM U823 Ontogenèse et oncogenèse
moléculaire)
à partir de 17h30 : Attribution des Prix et Cocktail de clôture
"Salle des Congrès et Atrium de la Faculté de Médecine"
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Nom Prénom
ALARD Jeanne
ALBERT THANANAYAGAM
Marie
Page
Session
22
Poster n°01
AUBERNON Cindy
AYARI Asma
14
23
BAILLEUL Justine
BALDACCI Simon
BENHABILES Hana
BERGERON Sandrine
BERTHE Julie
9
24
24
25
26
BRESSON Lucie
CALADO MENDES Tiago Nuno
CANOUIL Mickael
CARGOET Marine
CASTILLO AGUILERA Omar
CHEHERE Baptiste
CHEN Yaohua
COISNE Augustin
CORFIOTTI Francois
CORSI Mariangela
COUTEL Xavier
17
26
27
27
28
28
29
30
30
31
32
DA COSTA GOUVEIA Joana
DELANNOY Clement
DELCOURT Vivian
DELEYE Yann
DESPRES Clement
DUCASTEL Sarah
DUMORTIER Mandy
DUPLOYEZ Nicolas
DUPONT Clement
18
32
33
33
34
34
35
35
36
DUPRET Barbara
DURAND Matthieu
13
36
DUROUX Romain
EL AMRANI Mehdi
ESCUTNAIRE Josephine
FERLIN Juliette
FOVET Thomas
GARABEDIAN Charles
GEORGEL Anne-France
GILORMINI Pierre-Andre
GIRAUDET Geraldine
19
37
38
38
39
40
40
41
41
GROMADA Xavier
HADDAD Juliano
HANSSENS Sandy
9
42
43
23
Poster n°02
"Models and markers for new
challenges" (11h30)
Poster n°03
"Towards a better knowledge
of diseases" (09h00)
Poster n°04
Poster n°05
Poster n°06
Poster n°07
"Promising therapeutic
strategies" (14h30)
Poster n°08
Poster n°09
Poster n°10
Poster n°11
Poster n°12
Poster n°13
Poster n°14
Poster n°15
Poster n°16
Poster n°17
strategies" (15h10)
Poster n°18
Poster n°19
Poster n°20
Poster n°21
Poster n°22
Poster n°23
Poster n°24
Poster n°25
challenges" (11h10)
Poster n°26
strategies" (15h30)
Poster n°27
Poster n°28
Poster n°29
Poster n°30
Poster n°31
Poster n°32
Poster n°33
Poster n°34
Poster n°35
Poster n°36
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@dresse de contact
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Nom Prénom
HAYBRARD Julie
HENRY Heloise
HERMANT Paul
MOULIN Solene
MUNCK Camille
NDIAYE Fatou Kine
PASCART Tristan
PAWLAK-CHAOUCH Mehdi
PELLEGRINO Giuliana
POTELLE Sven
RAHMOUNI Oumaira
Page
Session
44
Poster n°37
44
Poster n°38
45
Poster n°39
18
strategies" (14h50)
45
Poster n°40
46
Poster n°41
19
strategies" (15h50)
46
Poster n°42
47
Poster n°43
47
Poster n°44
10
48
Poster n°45
48
Poster n°46
49
Poster n°47
49
Poster n°48
50
Poster n°49
51
Poster n°50
51
Poster n°51
10
52
Poster n°52
52
Poster n°53
53
Poster n°54
53
Poster n°55
53
Poster n°56
54
Poster n°57
54
Poster n°58
55
Poster n°59
13
challenges" (10h50)
56
Poster n°60
57
Poster n°61
57
Poster n°62
58
Poster n°63
58
Poster n°64
59
Poster n°65
59
Poster n°66
SAAD Chadi
SAHUC Marie-Emmanuelle
SERGIO FABRICIO Gabriel
TIAN Lu
WATBLED Ludivine
WAWRZYNIAK Clement
WYCHOWSKI Adeline
ZECCHIN Mathilde
60
60
61
61
62
62
63
63
HERTAULT Adrien
HOGUET Vanessa
HOLLIN Thomas
HOMERIN Germain
JEOUAL Abdallah
JOURDAIN Anne Sophie
KARAM Oliver
KONE Bachirou
LAMARTINIERE Yordenca
LAMBERT Melanie
LAMBLIN Gery
LAMPIN Marie-Emilie
LASSAILLY Guillaume
LE RHUN Emilie
LECOUTRE Simon
LEGHAY Coline
LESAGE Kevin
LETURCQ Maite
MACON Guillaume
MALONE Samuel Andrew
MAMIGONIAN BESSA Luiza
MANCHE Monique
MARTIN MENA Anthony
MASSE Morgane
Poster n°67
Poster n°68
Poster n°69
Poster n°70
Poster n°71
Poster n°72
Poster n°73
Poster n°74
@dresse de contact
[email protected]
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[email protected]
adrien.hertault@chru-lille.
[email protected]
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6
Session Orale
Towards a better knowledge of diseases
7
8
BAILLEUL Justine
Xuefen LE BOURHIS (INSERM U908 - LE BOURHIS Xuefen)
Session Orale 2016 "Towards a better knowledge of diseases" (09h00)
CXCL1 and CCL5, induced by ionizing radiation, reprogram non-tumorigenic cancer cells into resistant cancer stem cells in
breast cancer.
Bailleul J1, Bidan N1, Mouttet-Audouard R2, Arcicasa M1,2, Hannebicque K2, Takayama Y1,3, Meignan S1,2, Le Bourhis X1, Lagadec C1
1,INSERM U908, Lille 1, France. 2,Centre Oscar Lambret, France. 3, LIMMS/CNRS-IIS UMI2080, SMMIL-E, Japan.
Identification of cancer stem cells (CSC) in solid tumours – with self-renewal, multipotency, tumorigenesis, and therapy resistance capacities – has
opened path to new targeting therapeutic approaches. However, CSC targeting alone might not be sufficient to eradicate a tumour. Indeed, recent
studies showed that cancer cells are plastic, and conventional therapies, such as radiotherapy, can lead to cancer cells (non-CSC) reprogramming
into iCSC (induced-CSC). The goal of our work is to identify the molecular mechanisms responsible for treatment-induced CSC emergence.
First, we have shown that conditioned media from irradiated non-CSC is sufficient to induce iCSC reprogramming. These results suggest that cell
plasticity might be actively regulated by diffusible factors secreted by irradiated cells. By using proteins arrays and ELISA, we demonstrated that the
secretion of a specific cocktail of chemokines is induced by ionizing radiation, such as CXCL1 and CCL5. Interestingly, recombinant CXCL1 and CCL5
treatments increase the sphere forming capacity (SFC) of isolated non-CSC treated population. Concomitantly, treatment with neutralizing antibodies
targeting CXCL1 and CCL5 leads to a decreased CSC number (ALDH+ cells).
We also studied the expression of the corresponding chemokines receptors, by flow cytometry. First, we saw that reprogrammable ALDH- cells are
enriched for CXCL1 and CCL5 receptors expressing cells compare to unsorted population or ALDH+ population (CSC). We analysed the
reprogramming potential of isolated ALDH-/receptor-positive cells versus ALDH-/receptor-negative cells. Unfortunately, our results were inconclusive.
However, we then looked at ionizing radiation effect on receptors expression in a sorted non-CSC receptor negative population. Interestingly, we
found that the expressing receptors cell population was back to basal level, within five days, in an irradiation-independent manner.
To validate the implication of CXCL1 and CCL5 in the reprogramming mechanism and in a pre-clinical perspective, we are currently evaluating the
effect on reprogramming of siRNA targeting CXCL1 and CCL5, and pharmacological inhibitors of their receptors. The exciting first results show a
decreased reprogramming capacity of the treated cells.
Cancer stem cells, Reprogramming, Ionizing Radiation, Chemokines
GROMADA Xavier
Jean-Sebastien ANNICOTTE (UMR CNRS 8199 éq. 02 - ANNICOTTE Jean-Sebastien)
E2F1 controls pancreatic β-cell function and identity
Xavier Gromada, Nabil Rabhi, Philippe Froguel and Jean-Sébastien Annicotte
*European Genomic Institute for Diabetes, CNRS UMR 8199, Lille 2 University, Lille, France
Background and aims: Dysfunction of pancreatic β cells, associated with a decrease in their number are responsible for diabetes development. In this
regard, cell cycle regulators play key roles in the control of cell proliferation and cell fate. We previously reported the important role of the CDK4-pRbE2F1 pathway, a key component of the cell cycle machinery, on glucose homeostasis, post-natal β-cell proliferation, mass and function. However, the
molecular link between E2F1 and the control of endocrine cell fate or differentiation remains unknown.
Methods: To identify the potential role of E2F1 in pancreatic β-cell differentiation, we used E2f1 +/+ and -/- mice and studied the proportion of α and β
cell in these genetic backgrounds. To further understand the potential role of E2F1 in maintaining β-cell identity, we used shRNA technology in MIN6
cells, transient transfection experiments, chromatin immunoprecipitation assays and E2F1 inhibitors in vitro and in vivo. Moreover to confirm the cellautonomous function of E2f1 in β cells, we have generated a beta-cell specific inactivation of the E2f1 gene in mice. We further used human islets to
demonstrate the key role of maintaining E2F1 activity for β-cell identity.
Results: We show here that E2f1 deficiency induced a β-to-α-like cell conversion, suggesting that E2f1 could be directly involved in this process.
Knocking-down E2f1 in the β-cell line Min6 induced the α-cell specific transcription factor Arx at the mRNA and protein levels. We also demonstrated
that E2F1, in concert with pRb, repressed Arx in β cells at the promoter level, through an epigenetic mechanism involving CpG island methylation.
These results were confirmed in a E2f1 beta cell specific knock-out mouse model where the mice present glucose intolerance associated with a
decrease of insulin secretion in response to glucose. Finally, inhibiting E2F1 activity in human islets induced a β-to-α-like cell conversion, suggesting
an important role for E2F1 in human islets.
Conclusion: Our data demonstrate that E2f1 could be a constitutive repressor of the α-cell specification factor Arx, crucial to maintain the cellular
identity of pancreatic β cells. The underlying repressive mechanisms are still under investigations but could involve a demethylation process.
Type 2 diabete, beta cell, differentiation
9
KONE Bachirou
Philippe GOSSET (UMR8204-U1019 éq. 12 - TROTTEIN Francois)
Cellular and molecular mechanisms of susceptibility to infection in COPD
Bachirou KONÉ, Magdiel PÉREZ-CRUZ, Fahima MADOURI, Eva VILAIN, Gwenola KERVOAZE, Muriel PICHAVANT, Philippe GOSSET
Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR, 8204 - CIIL - LI3 - F-59000 Lille, France
Background: Chronic obstructive pulmonary disease (COPD) is a public health burden mainly due to cigarette smoke (CS) and other air pollutants.
Alteration of the innate immune response to bacterial infection is a key determinant in the COPD course. It is now well recognized that respiratory
infections are important triggers for the progression and exacerbation of the disease. We previously identified a defect in IL-22 as a key factor
responsible for bacteria-induced exacerbations of COPD. This alteration related to deficient activation of conventional and non-conventional offers
hints for the development of novel therapeutic strategies in COPD exacerbations. Our goal is to restore an appropriate immune response to pathogens
during COPD. Since the IL-22 response could be regulated by cytokines such as the IL-20 family, our goal was to investigate the IL-20 pathway in
order to restore an efficient response against pathogens in the lung during COPD Exacerbation.
Methods: C57BL/6 mice were daily exposed to CS during 12 weeks (COPD model) before infection by Streptococcus pneumoniae (Sp). Moreover,
this bacteria was used to stimulate human monocyte-derived dendritic cells (MDDC) pre-treated or not with cigarette smoke extract (CSE). The
expression of IL-20 cytokine and their receptors was evaluated. To decipher the role of the IL-20 family, the impact of blocking anti-IL-20Rb mAbs or
recombinant IL-20 cytokines was evaluated on MDDC maturation. Bacterial flagellin (FliC) was systemically administrated in COPD-Sp mice to assess
the potential of immunostimulants to improve bacterial clearance.
Results: We found that IL-20 cytokines are over expressed in the lung of COPD mice and were amplified after Sp challenge. Moreover, human MDDC
produce IL-20 cytokines in response to CSE and/or Sp. They also express IL-20Ra, IL-20Rb and IL22Ra. IL-20 cytokines inhibited MDDC maturation
in response to Sp, as shown by a decreased expression of CD86, CD80, and CD274 and a lower production of polarizing cytokines such as IL-1b, IL6, and IL-23. As a consequence, IL-20 treated MDDC exhibited a reduced capacity to induce the production of IL-17 and IL-22 by naive T lymphocytes
in response to Sp. Addition of anti-IL-20Rb mAbs boosted the production of IL-1b, IL-6, and IL-23 by MDDC, and restored the production of IL-17 and
IL-22 by naive T cells. Anti-IL-20Rb mAbs reduced the inflammation and the bacterial load in the lung and bronchoalveolar lavage fluid (BALF). Finally,
FliC treatment ameliorates bacterial clearance in BALF and lung, increased IL-22, neutrophils recruitment and S100A protein.
Conclusion: These results suggest that IL-20 cytokines could be involved in the susceptibility of COPD patients to infections, by targeting MDDC.
Therefore, blocking anti-IL-20Rb mAbs could represent a novel therapeutic way to boost MDDC response to pathogens. Furthermore,
immunostimulants such as bacterial flagellin is beneficial in COPD exacerbation mice model.
COPD, Streptococcus pneumoniae, IL-20 cytokines, Flagellin
LEGHAY Coline
Malika HAMDANE (INSERM U1172 éq. 01 - BUEE Luc)
Functional investigation of new N-terminally truncated Tau species and relevance to Alzheimer’s disease
Coline Leghay, Maxime Derisbourg, Dominique Demeyer, David Blum, Valérie Buée-Scherrer, Vincent Deramecourt, Luc Buée & Malika Hamdane
Equipe Alzheimer & Tauopathies, UMR-S 1172 Inserm, Univ.Lille 2, CHU Lille, Centre de Recherche Jean-Pierre AUBERT
Background: Tau protein plays an important role in Alzheimer’s Disease (AD) where it is found aggregated and abnormally modified in degenerating
neurons. Moreover, truncation is among post-translational modifications of Tau and its role in AD pathological process is far from being elucidated.
Recently, we have identified new N-terminally truncated Tau species (Trunc-Tau) from human brain. Among them, the N-terminally Trunc-Tau starting
at Methionine11 (Met11-Tau) was also found with a post-translational modifications that has never been described for Tau (Mod-Met11-Tau).
Objective: Our work aims to: 1) perform functional characterization of Met11-Tau and to study the new post-translational modification; 2) establish
whether there is an association between the level of Met11-Tau and/or modified form and Tau pathology.
Results: On one hand, we have established inducible stable cell lines expressing the full length-Tau protein (FL-Tau) and the Met11-Tau in order to
analyze functional consequences of Met11-Tau expression, compared to its related FL-Tau (Western Blot, fractionation, confocal microscopy and
Elisa assay). Moreover, this cell lines have allowed to identify the enzyme responsible for the new modification and are used to determine the role of
the new modification on Met11-Tau degradation, subcellular localization and aggregation. We expect to uncover the functional impact of truncation at
Met11 and the new modification on Tau protein.
On the other hand, we succeeded in development of a specific monoclonal antibody targeting Mod-Met11-Tau. we are using this specific monoclonal
antibody to analyze by Immunohistochemistry (IHC) the immunolabeling pattern in controls and AD patients. Our preliminary data showed that this
antibody specifically labels AD brains. This tool will be used in IHC on human brains samples to establish whether Mod-Met11-Tau is a signature
feature of AD and other Tauopathies.
Conclusion: This work will determine the role of Met11-Tau and its modification, it will thus provide new knowledge on Tau biology and the
physiopathological process of AD.
Acknowledgements:
PhD student funding: Doctoral grant co-sponsored by the Région Nord/Pas-de-Calais & CHU-Lille.
We thank Raphaelle Caillierez, Sébastien Carrier and Sabiha Eddarkoui for technical help, the Lille NeuroBank (CHU-Lille) for providing brain tissues,
Plate-forme Imagerie cellulaire de Lille 2 for confocal microscopy (Meryem Tardivel). This work is supported by the Université Lille 2, Inserm,
RégionNord/Pas-de-Calais, FEDER, and the Labex DISTALZ.
Neurodegenerative diseases, Post-translational modifications
10
Session Orale
Models and markers for new challenges
11
12
MOULIN Solene
Charlotte CORDONNIER (INSERM U1171 - BORDET Regis)
Session Orale 2016 "Models and markers for new challenges" (10h50)
New-onset dementia after spontaneous intracerebral haemorrhage: a prospective observational cohort study.
S. Moulin (1), J. Labreuche (2), S. Bombois (1), C. Rossi (1), H. Hénon (1), A. Duhamel (2), D. Leys (1), C. Cordonnier (1)
(1) Service de neurologie, C.H.U Lille, INSERM U1171, Lille
(2) Département de biostatistiques, C.H.U Lille, EA 2694, Lille
Background:
Dementia is an important public health issue, and the contribution of stroke to dementia is large at the community level worldwide. Dementia occurs in
at least 10% of patients within one year after stroke. However, no prospective study has focused on the risk of dementia after spontaneous
intracerebral haemorrhage (ICH) that accounts for 15% of all strokes. We aimed to determine the incidence and prognostic factors of dementia after
an ICH. We hypothesized that patients with lobar ICH - without dementia before - were at higher risk of developing new-onset dementia because of a
strong influence of underlying cerebral amyloid angiopathy (CAA).
Methods:
We did a prospective observational cohort study (median follow-up: 6 years) of patients with spontaneous ICH who were admitted at the Lille
University Hospital, France. We studied prognostic factors [clinical and neuroradiological (MRI) biomarkers] of new-onset dementia with a prespecified subgroup analysis according to ICH location. We performed multivariable analyses using competing risk analyses (competing event: death).
Findings:
Between November 2004 and April 2009, we enrolled 560 consecutive patients with spontaneous ICH of whom 218 patients (median age 67·5 years)
without pre-existing dementia were alive at six months and included in the study. Sixty-three patients developed new-onset dementia leading to an
incidence rate of 14·2% (95%CI 10·0-19·3) at 1 year after ICH onset that reached 28·3% (95%CI 22·4-34·5) at 4 years. Patients with lobar ICH had an
incidence of new-onset dementia twice higher than patients with non lobar ICH (incidence at one year respectively: 23·4%; 95%CI 14·6-33·3 versus
9·2%; 95%CI 5·1-14·7). Disseminated superficial siderosis [SubHazard Ratio (SHR) 7·45; 95%CI4·27-12·99], cortical atrophy score (SHR per 1-point
increase 2·61; 95%CI 1·70-4·01), a higher number of cerebral microbleeds (SHR for >5 CMBs 2·33; 95%CI 1·38-3·94) and older age (SHR per 10year increase 1·34; 95%CI1·00-1·79) were prognostic factors of new-onset dementia.
Interpretation:
There is a substantial risk of incident dementia among non-demented survivors of spontaneous ICH. Our results suggest a strong implication of
underlying CAA. Future clinical trials dedicated to ICH should include cognitive endpoints.
intracerebral haemorrhage, dementia, prospective cohort, stroke
DUPRET Barbara
Pierre-Olivier ANGRAND (INSERM U908 - LE BOURHIS Xuefen)
Application of the TALEN methodology in zebrafish to study the Polycomb Repression
Barbara DUPRET 1, Pamela VÖLKEL 1,2, Constance VENNIN 1,3, Xuefen LE BOURHIS 1, Pierre-Olivier ANGRAND 1
1 Plasticité Cellulaire & Cancer - U908 Inserm / Université de Lille1, 59655 Villeneuve d'Ascq, France.
2 CNRS
3 SIRIC ONCOLille
In eukaryotes, post-translational modifications of histone proteins play a crucial role in chromatin organization and in the control of gene expression
programs. Polycomb group (PcG) proteins are part of the enzymatic machineries involved in these histone modifications. PcG proteins interact to form
two major multiprotein complexes, Polycomb Repressive Complex 1 (PRC1) and PRC2 which catalyze two post-translational histone modifications
involved in chromatin compaction and gene silencing. PRC1 is responsible for monoubiquitinylation of histone H2A at lysine 119 (H2AK119ub1)
whereas PRC2 trimethylates lysine 27 of histone H3 (H3K27me3) allowing the recruitment of PRC1.
To gain insights into the Polycomb repression in vertebrates, we applied a genetic approach. Using the TALEN technology, we engineered zebrafish
(Danio rerio) harboring mutations in the pcgf1 and ezh2 Polycomb genes. Pcgf1 is a subunit of the PRC1 complex whereas Ezh2 is the histone lysine
methyltransferase of the PRC2 complex.
Mutant pcgf1-/- fish are viable and fertile, but the growth rate at early developmental stages is reduced in absence of pcgf1 gene function and a
significant number of pcgf1-/- fish show signs of premature aging.
In contrast, ezh2-/- zebrafish die at 12 days post-fertilization (dpf) with alterations of the intestine wall. Analyses of mutant zebrafish reveal that the
intestine formation is normal at 5 dpf but its integrity is lost at 11 dpf indicating that Ezh2 is essential for tissue maintenance but not for tissue
formation.
Altogether, our results highlight a link between epigenetics and aging and tissue maintenance.
Epigenetics, Zebrafish, TALEN
13
AUBERNON Cindy
Damien CHARABIDZE (EA7367 - HEDOUIN Valery)
Thermoregulation in necrophagous Dipteran larvae: application in forensic entomology.
Cindy Aubernon, Valéry Hédouin, Damien Charabidzé
Univ.Lille, CHU Lille, EA7367 - UTML - Unité de Taphonomie Médico-Légale, F - 59000
When a person dies, in wild, his cadaver will be colonized by necrophagous insects. Many species are attracted by decaying corpse, but flies are the
most common and numerous. They lay eggs on the cadaver and from these eggs, maggots hatch and nourishing on fleshes. Forensic entomology
relies on these insects to date the death in a legal context. A key point for forensic entomologists is that larval growth depends on temperature:
maggots grow faster at higher temperatures. Accordingly, understanding the behavior and the physiology of these insects according to temperature is
crucial. The aim of my thesis is to highlight the thermal behavior of three species of forensic importance: Lucilia sericata, Calliphora vomitoria and
Calliphora vicina
My work can be divided in three parts: 1/ behavior of the maggots in their thermic environment, 2/ effect of sociality (congeners) and 3/ impact of these
two points on larval development.
To answer these questions, I designed the Thermograde, a 80cm long food-supplied linear thermal gradient (17°C to 50°C). 80 larvae of one of the
three species were placed inside and their location was checked after 19h. The selected temperatures were 33.3±1.52 for L. sericata, 29.61±1.63 for
C. vomitoria and 22.43±1.55 for C. vicina. These results suggest a larval behavior for thermal optimization. According to this hypothesis, maggots of
each species should move in their environment (i.e. on the corpse), trying to reach location close to their preferential temperature.
To test this postulate, I realized another experiment using a 40cm long food-supplied rearing apparatus with one hot spot at each extremity (25°C and
34°C with an ambient temperature of 20°C). Testing three conditions (spots ignition time), the results show that the larvae were able to select an
optimal temperature in their local environment and to move to another place when the environment became less favorable. However, when a group of
larvae was located in a healthy area, its moving to a more optimal place was very long or never happened.
These results bring interesting questions regarding the larval growth and physiology. These questions are especially important in the context of
forensic entomology for the time of death calculation. Indeed, larval developmental parameters are currently measured using captive larvae reared at
a given constant temperature. Such setup prevents any larval thermoregulation, and accordingly do not reflect their actual development under field
conditions. Consequently, during my last PhD year, I will realize developmental experiments in a setup allowing larval thermoregulation. With this last
experimentation, I will try to demonstrate how the larvae’s behavior affect their development, and then refine the way forensic entomologist estimate
the time of the death.
Forensic entomology, Calliphoridae, Thermoregulation, Collective choice
14
Session Orale
Promising therapeutic strategies
15
16
BRESSON Lucie
Feng CHAI / Shoji TAKEUCHI (INSERM U1008 - SIEPMANN Juergen)
Session Orale 2016 "Promising therapeutic strategies" (14h30)
Autologous cell-laden hydrogel sheet for prevention of post-surgical abdominal adhesion (PAA)
L Bresson1,2, E Leblanc1, Teru Okitsu3, F Chai2
1 Centre Oscar Lambret, Gynecology and Oncology, 59045 Lille, France
2 INSERM 1008, Biomatériaux, University Lille 2, 59045 Lille, France
3 Takeuchi Lab, Institut of Industrial Science, Tokyo, Japan
Introduction: Postoperative abdominal adhesions (PAA) are a major complication leading to medical and economical problems. Replace injured
peritoneum, which is composed of a monolayer of mesothelial cells (MC), using cell-therapy or cell-laden scaffold are two strategies to prevent PAA.
MCs are crucial and adipose derived stem cells (ASCs) could replace thanks to their potential of differentiation. Hydrogel polymers are attractive
scaffolds mimicking the properties of the native extracellularmatrix. Milestones of our project of regenerative medicine are (1) comparison of cell-laden
scaffolds and cell-therapy, (2) choice of cell source and (3) biomaterial, and finally (4) transplantation of the cell-laden hydrogel scaffold, (5) in a preestablished rat model of PAA.
Materiel and Method:
Firstly, model of PAA has been validating comparing two peritoneal injuries (5). Fours groups were compared (1): Peritoneal graft with MC exposed to
the abdominal cavity (group MC), Peritoneal graft with sub serosa containing fibroblasts (FB) exposed (group FB), MC cell-therapy using cell-sheet
technology (group MC-FB sheet) and shame group (group control). After 14 days, PAA were assessed by macroscopic observation and histology.
MCs and ASCs have been compared for isolation, culture, characterization, and differentiation (2). Hydrogels (3) have been compared for their
mechanical properties and their biocompatibility. Cell-laden sheets (4) have been implanted and assessed on the capacity to prevent PAA.
Results: The animal model of PAA was validated and both models were effectives and clinically relevants. In group MC, no PAA were recorded on the
surface of the graft. In the other groups, all rats were diagnosed with PAA. Extent of the adhesions and qualitative assessment revealed a significantly
inferior score in group MC than in the other groups. Histological observation confirmed the respect of MCs only in group MC. MCs, with typical
cobblestone morphology and bright edges, have been stained for Vimentin and Cytokeratin. Senescence arrived after only three passages for these
adult well-differentiated cells. Spindle-shaped ASCs had a good capacity of expansion, were able to differentiate in osteocytes and adipocytes, and to
form colonies as expected characteristics of stem cells. BD-Purastat® peptide has been tested in collaboration with 3D Matrix firm. MCs and cell lines
were able to survive into the gel. Purastat® alone reduced significantly the severity and the extent of PAA in comparison with the group control. But,
no significant difference were found between MCs in Purastat® and Purastat® or group control for the prevention of PAA.
Conclusion and perspectives: Our study supported that MCs and scaffold are both needed to succeed in peritoneum's engineering to prevent PAA.
Characterization of MCs and ASCs will be detailed. ASCs differentiation into MCS phenotype will be assessed. Purastat® is promising but cell laden
grafts need to be imrpoved.
regenerative medecine, hydrogel, adipose derived stem cells, post operative abdominal adhesion
17
HERTAULT Adrien
Stephan HAULON (INSERM U1008 - SIEPMANN Juergen)
Evaluation of a Pro-Healing Polydopamine-coated Stent on In-Stent Restenosis using a rat model
Adrien Hertault, Blandine Maurel, Feng Chai, Mickaël Maton, Florent Briffa, Nicolas Bricout, Jonathan Sobocinski, Stephan Haulon, Nicolas
Blanchemain,
INSERM U1008, Research Group on Biomaterials, University of Lille, Fran
INTRODUCTION: In-stent restenosis (ISR) is induced by an uncontrolled smooth muscular cells (SMC) proliferation after stent implantation. It is
associated with recurrence of symptoms and additional health costs. Drug-eluting stents have demonstrated efficiency on ISR, but induce a high risk
of late acute thrombosis due to a delayed struts reendothelialization1. Polydopamine (PDA), a biocompatible polymer inspired from mussels byssus,
has been reported to promote endothelial cells (EC) and SMC proliferation in-vitro2,3, thus suggesting a potential pro-healing effect on the vascular
wall. This study aimed to evaluate the impact of a pro-healing PDA-coated stent on in-stent restenosis (ISR) and on the quality of the struts reendothelialization in-vivo using a rat model.
EXPERIMENTAL METHODS: Chemical oxidation of Cobalt-Chromium (CoCr) plates was achieved using a «piranha» solution mixture. PDA coating
was performed by dip coating in a 2mg/mL dopamine solution, followed by rinsing in deionized water and thermal treatment at 150°C4 for one hour.
Surface morphologies were assessed on PDA-coated CoCr plates by scanning electron microscopy (SEM). Ten Wistar rats received the implantation
of 2.5mm CoCr coronary stent in aortic position (5 bare metal stents (BMS), 5 PDA stents, prepared according to the same protocol) and then
sacrificed on the 28th day. Eight rats were used to estimate ISR in optic microscopy with quantification of the neointima/media (n/m) ratio after
eosin/hematoxillin coloration5. Quality of the struts reendothelialization was analyzed in the two remaining rats with transmission electron microscopy
(TEM).
RESULTS AND DISCUSSION: Visual aspect of PDA-coating appears smooth and homogeneous, however SEM analyses with a scratch test revealed
a rough surface of around 50 nm in depth.
On the ten rats, one died of bleeding complications after stent implantation in the BMS group. PDA stents demonstrated a significant reduction in ISR
compared to bare metal stents (ratio n/m = 0.48 (+/- 0.26) versus 0.83 (+/- 0.42), p<0.001). TEM analyses confirmed the presence of neointima
surrounding the struts in each group, and revealed a thinner neointima layer in the PDA-stent group compared to BMS (Figure 2), with similar
ultrastructures of the cells facing the arterial lumen.
CONCLUSION: The pro-healing effect expected of PDA-coating on the arterial wall seems to be confirmed in this in-vivo model. This biocompatible
polymer could intrinsically limit in-stent restenosis. Additionally, it also offers the possibility to immobilize many relevant drugs on its surface through
amine functions providing potential synergistic effects.
Biomaterials
DA COSTA GOUVEIA Joana
Priscille BRODIN (UMR8204-U1019 éq. 07 - BRODIN Priscille)
Pulmonary co-administration of the synergic drugs Ethionamide and Booster using aerosolized nanoparticles: a promising
strategy to treat tuberculosis
Joana Costa-Gouveia*, Priscille Brodin*
*Université de Lille, CNRS UMR8204, Inserm U1019, CHU Lille, Institut Pasteur de Lille, Centre d’Infection et d’Immunité de Lille (CIIL), 59000 Lille,
France
Tuberculosis (TB) is a leading cause of death worldwide. The use of ethionamide (ETH), an important second line anti-TB drug, is hampered by low
oral bioavailability and the onset of severe side effects. Recently discovered «booster», molecules strongly increasing the efficacy of ETH, could
restore the use of this drug and thus improve the current clinical outcome of drug-resistant TB. To investigate the simultaneously delivery of ETH and
its booster BDM41906 in the lungs, we co-encapsulated the synergic compounds in biodegradable polymeric nanoparticles (NPs), overcoming the
bottlenecks inherent to the strong tendency of ETH to crystallize and the limited water solubility of the Booster.
The efficacy of the designed formulations was evaluated in TB infected macrophages using an automated confocal high-content screening platform,
showing that the drugs maintained their activity after incorporation in NPs. Among tested formulations, «green» β-cyclodextrin (pCD) NPs displayed
the best physiochemical characteristics and were selected for in vivo studies. The NPs suspension, administered directly into mice lungs using a
Microsprayer®, led to a significant decrease of the pulmonary bacterial load as compared to untreated mice. This study paves the way for a future use
of pCD NPs for the pulmonary delivery of the [ETH:Booster] pair.
Acknowledgments: Financial support for this work was provided by the European Community (CycloN Hit Grant n° 608407), the Feder (12001407 (DAL) Equipex Imaginex BioMed) and the Region Nord Pas de Calais (convention n° 12000080).
Infectious diseases, Nanomedicine, Drug delivery, Health sciences
18
DUROUX Romain
Said YOUS (INSERM U1172 éq. 06 - MELNYK Patricia)
From design to pharmacological evaluation of benzoxazole derivatives as adenosine A2A receptor antagonists for the
potential treatment of Alzheimer disease.
Romain Duroux, Laurence Agouridas, Patricia Melnyk, Saïd Yous
Univ. Lille, Inserm, CHU Lille, UMR-S 1172 - JPArc - Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, F-59000 Lille, France
Alzheimer's disease (AD) is the most prevalent form of dementia in the aged population characterized mainly by the presence of senile plaques and
neurofibrillary. So far, there is no way to halt or slow-down AD. There is thus a constant need of developing novel therapeutic strategies.
The adenosine A2A receptor (A2AR), expressed in the CNS, belongs to the class of G protein coupled receptor (GPCRs). In recent years, A2AR has
attracted growing interest on AD, where it has been proved that A2A receptor is over expressed. It has also been found that A2A receptor antagonists
such as caffeine improves memory performance. Though several A2AR antagonists have reached clinical trials, most of them suffer from poor
pharmacokinetic and pharmacodynamic properties. Current efforts therefore focus on developing new antagonists with relevant ADME properties.
Based on the recently published crystalline structure of the A2AR complexed with the selective and high-affinity antagonist triazine and on a
pharmacophoric model, we designed new ligands using in silico docking studies. Our objective is to develop selective A2AR antagonists devoid of
chemical stability, bioavailability and toxicity drawbacks of compounds currently under clinical trials. Pharmacomodulations of a new benzoxazoles
family have been developed and show micromolar to nanomolar affinity on HEK293 cells membranes expressing A2AR. Cytotoxicity has also been
evaluated on SY5Y cells.
From selected compounds, we evaluated the impact on synaptic transmission by performing hippocampal field excitatory post-synaptic potential
recordings (fEPSP) in transgenic rats with a neuronal-specific human A2AR overexpression [tg(CAMKII-hA2AR)] .
medicinal chemistry, Alzheimer’s disease, A2A receptor
HOMERIN Germain
Alina GHINET (INSERM U995 éq. 07 - CHAVATTE Philippe)
Structure-activity relationships of new ligands of the P2X7 receptor for the development of drug-candidates with antiinflammatory potential
Germain HOMERIN, Benoît RIGO, Xavier DEZITTER, Christophe FURMAN, Emmanuelle LIPKA, Régis MILLET and Alina GHINET*
Inserm U995, LIRIC, Université de Lille, CHRU de Lille, Faculté de médecine – Pôle recherche, Place Verdun, F-59045 Lille Cedex, Fran
INTRODUCTION
The P2X7 receptor is a transmembrane ion channel belonging to the purinergic system. Its activation by ligands such as ATP leads to a K+ efflux and
to a Ca2+ and Na+ influx within the cell. It is expressed all over the body, mostly on immune system cells.
This receptor has been studied for years for the implication in the inflammation process. It causes the maturation and release of proinflammatory
cytokines such as IL-1β. Moreover, under repeated or prolonged activation, the P2X7R is able to form a membrane pore which leads frequently to
apoptosis via different mechanisms. According to in vitro and in vivo studies, these characteristics could be used in the treatment of inflammation and
cancers.
Inflammatory bowel diseases (IBDs) are crippling diseases and an estimated 200 000 people sufferer from them in France. Current therapies only
focus on suppressing symptoms to give patients a better quality of life, but they do not cure them. However, the development of potential new
treatments is on-going, in particular with the development of new antagonists of the P2X7 receptor. This receptor is responsible for interleukins release
and so, takes part in the inflammatory process of IBD's patients. Lastly, even though the role in cancer is not well understood many arguments exist in
favor of using P2X7R antagonists and exploiting them for their implication in cell death.
DESIGN
A previous study in our research team based on data from the literature (a) led to define a pharmacophore (model of the interactions needed to
antagonize the receptor). We designed P2X7 receptor's ligands with potential antagonist activity, based on a pyroglutamic-like central connector.
RESULTS and DISCUSSION
To date, we have synthesized about a hundred compounds in 8 different series. Some antagonists have been identified with IC50 values in vitro in the
nanomolar range and have allowed us to establish new SAR for the development of new series.(b) Our on-going studies, on the one hand, will focus
on designing and synthesizing new ligands of the P2X7R based on new SAR; on the other hand, we will handle the problematic of distribution and
metabolism of active antagonists we will have identified.
Moreover, recent data such as antifungal properties suggest that some of our best P2X7R antagonists may reduce Candida albicans (a fungus
involved in IBDs) proliferation. These findings must be confirmed, but then we could think of medicines with synergistic effects: that would antagonize
the cell's IL-1β processing and release; and at the same time diminish the LPS production by fungus.
Bibliographic references:
(a) Chambers, L.J. et al. WO 2008003697, Chem.Abstr., 2008, 148, 145026.
(b) Homerin, G. et al. ZrCl4 as a new catalyst for ester amidation: an efficient synthesis of h-P2X7R antagonists, Tetrahedron Lett., 2016, 57, 1165.)
Drug design, P2X7, Inflammation, IBD
19
Session
POSTERS
20
21
ALARD Jeanne
Corinne GRANGETTE (UMR8204-U1019 éq. 10 - POT Bruno)
Session Poster 2016 n°01
Characterization of probiotic strains for their beneficial properties against inflammation and obesity
(attention CONFIDENTIEL thèse CIFRE)
Alard J, Peucelle V, Boutillier D, Desramaux J, Pot B, Holowacz S² and Grangette C.
CIIL UMR 8204 Equipe 9 - Bactéries Lactiques et Immunité des Muqueuses Institut Pasteur de Lille
²Larena Santé - 37 quai de grenelle - 75015 Paris cedex 15
(attention CONFIDENTIEL thèse CIFRE)
The gut microflora plays an important role in maintaining human health. More recently, alterations in the microbiota composition (known as dysbiosis)
were suggested to play a role in the development of obesity. Obesity is associated with a chronic, low-grade inflammatory state - primarily
orchestrated by the recruitment of inflammatory macrophages in the adipose tissue - that is crucial in the development of insulin resistance and type 2
diabetes.
The potential use of probiotics (beneficial microbes) against obesity therefore gained attention, although results were sometimes conflicting. These
microorganisms can exert their protective properties through multiple mechanisms, notably by maintaining and restoring the gut barrier function, by
regulating the mucosal immunity and by inducing antibacterial substances.
Our project aims to evaluate the potential protective effects of a large collection of probiotics (23 strains composed mainly of lactobacilli and
Bifidobacteria), provided by PiLèJe (CIFRE collaboration contract). For that aim, we set up different in vitro models to screen the anti-inflammatory and
anti-obesity capacities of these bacteria We first highlighted the anti-inflammatory potential of 6 strains using peripheral blood mononuclear cells
(PBMC) stimulation and also their capacity to restore the gut barrier using an in vitro gut permeability model with CaCo2 cells. We also evaluated the
capacities of the strains to induce anti-microbial peptides by stimulating a murine crypt cell line (mIcCl2). The anti-inflammatory capacities of the
strains are currently evaluated in vivo in TNBS-induced acute and chronic colitis models in mice.
To select probiotics with anti-obesity properties, we evaluated in vitro the capacities of the strains to limit the differentiation of 3T3-L1 pre-adipocytes
and to favor the secretion of satiety peptides using the entero-endocrine STC-1 cell line. This allowed us to select 5 strains and to design 3 mixtures
that are currently being evaluated in vivo for their anti-obesity capacities, using high fat diet-fed mice.
These studies should provide us crucial clues to select the best strains or mixture for the development of more efficient therapeutic approaches in the
management of IBD and obesity that could be implemented by PileJe. Our project should also bring substantial insight into how the host-microbial
interaction may govern protective effects.
Obesity, Probiotics, microbiota, inflammation
22
ALBERT THANANAYAGAM Marie
Christophe DI POMPEO (EA2694 - DUHAMEL Alain)
Association of outdoor air pollution with out-of-hospital cardiac arrests in the Nord-Pas-de-Calais region, France
Marie Albert Thananayagam. Laboratoire de Biomathématiques, Faculté des Sciences pharmaceutiques et Biologiques, 3 rue du Professeur
Laguesse, B.P. 83 - 59006 Lille Cedex, France.
Cardiac arrest (CA) is an important public issue. Overall survival rates remain low (range: 2–20%). In France the incidence of sudden cardiac death
(SCD) is estimated at 50,000 cases per year, more than 85% of which occurring outside of a hospital setting. SCD accounts for more than 50% of all
coronary heart disease (CHD) deaths. As a result, SCD prevention represents a major opportunity to reduce mortality from CHD. About outdoor air
pollution, it is a major environmental health problem which ranked as the eighth most significant risk factor for disease burden and was estimated to
cause 3.7 million premature deaths per year. It is considered to be a risk factor for ischaemic heart disease which is the predominant cause of cardiac
arrest. Thus it is reasonable to hypothesise an association between OHCA onset and air pollution levels.
The aim of our study was to evaluate the relationship betwenn the levels of seven pollutants (PM2.5, PM10, NO2, NO, SO2, O3 and CO) and out-ofhospital cardiac arrest (OHCA) occurences in Nord-Pas-de-Calais (NPdC), France.
The Ouf-of-Hospital Cardiac Arrests (OHCA) data, that occured in Nord-Pas-de-Calais (NPdC), France, from the 1st of January to the 30th of June
2015 were collected from the french cardiac arrest registry \"Registre électronique des Arrêts Cardiaques\" (RéAc). Among the 1408 cases extracted,
143 were excluded because of a traumatic etiology, 134 because the location of their OHCA was missing or different from where they live. Pollution
data were obtained from ATMO-NPdC monitoring air quality from 41 stations within the region. Daily pollution levels of seven pollutants (PM2.5,
PM10, NO2, NO, SO2, O3 and CO) were collected over the study period. Air pollution data were adjusted on daily temperature extracted from 16
stations over the study region available on the Météo France website. To estimate the relationship between pollution levels and OHCA occurrences, a
case-crossover study design was performed. A conditional logistic regression gave estimates of relative risks with interquartile range (IQR) exposures.
Seperate analyses were conducted for lags of 0 through 5 days since the induction period for an association between pollutants and OHCA is
unknown.
OHCA risk significantly increased with NO2 and SO2 exposure during the day preceding the arrest (OR=1.17, p=0.048 and OR=1.06, p=0.013
respectively). Conversly, ozone exposures one, three and four days before the arrest were significantly associated with a decrease of the risk
(OR=0.865, p=0.049 ; OR=0.836, p=0.013 ; OR=0.866, p=0.045 respectively).
Higher levels of NO2 and SO2 seem to be link with a higher risk of OHCA in NPdC, France while lower levels of ozone are associated with greater
occurrence of OHCA. Ozone levels are linked with hospital admissions for emergency or urgent daily respiratory hospital admissions which could
explain that the number of people at risk of OHCA decreases with higher levels of ozone.
Air pollution, Out-of-hospital cardiac arrest, Sudden cardiac death, Epidemiology
AYARI Asma
Isabelle WOLOWCZUK (UMR8204-U1019 éq. 12 - TROTTEIN Francois)
Adipose tissue: a neglected reservoir for influenza virus?
Asma AYARI, François TROTTEIN, Isabelle WOLOWCZUK
Influenza type A virus (IAV), responsible for seasonal flu epidemics and reoccurring pandemics, represents a worldwide threat to human health. IAV is
a negative single stranded RNA virus that primarily targets airway epithelial cells where it uses cell machinery to replicate. Recently, it has been
reported that besides young and old subjects, obese patients also present an increased susceptibility to IAV infection. Since obesity is associated with
an inflammatory state resulting from excessive white adipose tissue (WAT) expansion, we questioned whether IAV could impact WAT and its main
cellular components i.e. mature adipocytes and preadipocytes, using in vivo, ex vivo and in vitro approaches.
First, C57/BL6 lean and high-fat diet-induced obese mice were intranasally infected with a sublethal dose of IAV (strain: H3N2). At 7 days postinfection, lungs, subcutaneous and perigonadal adipose tissues, pancreas and liver were harvested for analysis of viral (M1 protein) and antiviral
protein (such as Mx1, Oas1) gene expression, using RT- qPCR. To analyze the impact of IAV infection on adipose tissue functionality, WAT explants
from infected or uninfected lean and obese mice were put in culture. Pro- and anti-inflammatory cytokine levels were then measured in supernatants,
by ELISA. The adipose tissue contains a myriad of cell types; the main being fully differentiated adipocytes and preadipocytes. Therefore, to identify
IAV targeted cells within WAT, we tested whether the virus can infect, and how, both adipose cells. We infected 3T3-L1 murine preadipocytes with
different virus doses, before and after their in vitro differentiation into mature adipocytes. Cells were harvested 1h, 24h and 48h post-infection for
transcriptional analyses. Cell-culture supernatants were collected for the detection of newly formed virions (plaque forming assay method) and
cytokines (ELISA).
Our results showed that, 7 days after IAV infection, the virus can be found in both adipose tissue depots of infected lean and obese mice. Moreover,
compared to uninfected WAT explants, adipose tissues from infected mice produce more pro- and anti-inflammatory cytokines (respectively; IL-6 and
IL-1b and IL-10 and IL-4), this effect being more pronounced in the lean group. It showed that IAV infection does impact WAT inflammatory state. In
addition, our in vitro tests showed that preadipocytes are permissive to IAV infection, yet virus replication is abortive. In opposite and interestingly
enough, IAV infection of mature adipocytes results in productive virus replication and release.
Altogether, our data suggest that adipose tissue, and especially lipid-laden mature adipocytes within, could potentially be a target for IAV and, as such,
may play an unexpected role in the course of influenza infection. More generally, our work will lead to a better understanding of the recently reported
association between obesity and influenza virus infection.
influenza virus, adipose tissue, adipocytes, obesity
23
BALDACCI Simon
Alexis CORTOT (UMR CNRS 8161 éq. 05 - TULASNE David)
MET amplification induces an aggressive phenotype in EGFR tyrosine kinase inhibitors resistant non-small-cell lung cancer
BALDACCI S, KHERROUCHE Z, STOVEN L, WERKMEISTER E, MARCHAND N, TULASNE D, CORTOT A.
CNRS-UMR8161 Institut de Biologie de Lille, Service de Pneumologie et Oncologie Thoracique CHRU de Lille, Institut Pasteur de Lille
Introduction : Treatment of Non-Small-Cell Lung Cancer (NSCLC) harboring an Epidermal Growth Factor Receptor (EGFR) mutation relies on EGFR
tyrosine kinase inhibitors (TKI). However all patients treated with EGFR TKI eventually present tumor progression, because of mechanisms of
resistance such as amplification of the MET receptor gene. There is currently no data on cellular phenotypic changes of tumor cells induced by MET
amplification in this context.
Objective: To determine whether MET amplification triggers a more aggressive tumor phenotype in TKI resistant EGFR-mutated NSCLC.
Methods: Proliferation, migration capacities, and anchorage independent growth were studied in HCC827 cell line, derived from a mutated EGFR
NSCLC, and his daughter cell line HCC827 GR6 which is resistant to EGFR TKI through amplification of the MET gene. Tumor growth of both strains
was analyzed in a mouse model of ectopic xenograft.
Results: In vitro, we found a similar proliferation in the two cell lines and a significant increase in migration and anchorage-independent growth in the
MET amplified cells. Treatment with PHA-665752, a MET TKI, significantly reduced the anchorage independent growth and the migration advantage
of MET amplified cells. In vivo, we observed a significant increase in tumor growth with the MET amplified cell line.
Conclusion : MET amplification confers an aggressive phenotype to TKI resistant EGFR-mutated NSCLC. These results pave the way for an early use
of MET inhibitors in association with EGFR TKI in order to prevent emergence of an aggressive tumor clone.
lung cancer , MET, EGFR
BENHABILES Hana
Fabrice LEJEUNE (UMR CNRS 8161 éq. 05 - TULASNE David)
nonsense mutation correction as a therapeutic approch to cancer : case of p53
Hana Benhabiles*, David Tulasne* and Fabrice Lejeune*.
*UMR8161, équipe « signalisation, apoptose et cancer », Institut de Biologie de Lille
Nonsense mutations induce premature stop codons (PTC) within an open reading frame. This type of mutation is found in about 10% of patients with
genetic disorders. Concerning cancer, 5 to 40% of mutations affecting tumor suppressing genes are nonsense mutations. The presence of a PTC in a
gene leads to rapid degradation of its mRNA mediated by the RNA surveillance mechanism named NMD (Nonsense-mediated mRNA decay)
preventing the synthesis of truncated proteins. In cancer, the absence of expression of tumor suppressing genes such as TP53 interferes with many
biological pathways including apoptosis enabling tumor progression.
We developed a screening system that allows identifying molecules capable of re-expressing genes harboring nonsense mutations by inhibiting the
NMD system and/or by activating readthrough. Readthrough is a natural mechanism which occurs during translation, leading to the incorporation of an
amino-acid at the PTC position. Among the molecules that we identified thanks to the screen, a compound named CNSM1 efficiently rescues the
expression of the nonsense-mutated TP53 gene.
CNSM1 induces the expression of full length proteins from PTC-containing genes indicating that compound is capable of activating readthrough. We
validated the screen results on several cancer cell lines harboring an endogenous nonsense mutation in TP53 gene and showed that the function of
TP53 was restored in the presence of CNSM1 We also investigated the cellular toxicity related with the use of CMNS1 and the in vivo effect of this
compound in a mouse model harboring a nonsense mutation in dystrophin gene. Our results demonstrate that CNSM1 has a mild cellular toxicity. In
addition, preliminary experiment indicates that CNSM1 is able to correct nonsense mutations in vivo and is able to restore the missing PTC-containing
gene function. Knowing CNSM1 is so far the most efficient molecule capable of rescuing the expression of PTC-containing genes, this compound
represents a realistic hope for a new targeted therapy for pathologies associated with nonsense mutations.
Nonsense mutation, NMD, p53 , readthrough
24
BERGERON Sandrine
Sophie GAUTIER (INSERM U1171 - BORDET Regis)
Influence of a single cortical microbleed on behavioral phenotype of APP mice (J20) and pharmacological modulation by
atorvastatin : longitudinal follow-up
S. Bergeron, S. Gautier, C. Potey, J. Deguil, Y. Chen, R. Bordet.
Laboratoire de pharmacologie médicale, CHRU de Lille, INSERM U1171, Université de Lille 2 - Lille (France).
Introduction :
Despite important insights, Alzheimer's disease (AD) pathogenesis is still not fully understood. Even though the amyloid cascade hypothesis remains
predominant, some arguments have emerged supporting the vascular hypothesis. In this study, we develop a specific animal model to investigate the
interaction between vascular and neurodegenerative injuries through : i) impact of a single cortical microbleed on cognitive decline, ii) effect of
pharmacological modulation by atorvastatin in this model.
Materiel et method :
Male amyloid precursor protein transgenic mice (APP) which develop early Aβ plaque formation and cognitive deficits and litter-mate non-transgenic
(WT) mice from the J20 line were used in this study. 10 weeks-old mice were operated by stereotaxic injection with either Collagenase (COL) 0,8
µUI/µL to induce a cortical haemorrhagic lesion or saline (Sal). After surgery, atorvastatin (At), 5 mg/Kg daily, was administered per os added in diet.
Mice were divided into six groups : WT+COL (n=14), WT+COL+At (n=12), APP+COL (n=10), APP+COL+At (n=12) and two sham groups WT+Sal
(n=12), APP+Sal (n=12). Neurobehavioural assessment battery including open field (motor activity), elevated plus maze (anxiety) and Barnes maze
(spatial reference memory) was repeated at different times after surgery.
Results :
Compared to WT, APP mice showed motor hyperactivity at 1.5, 3 and 6 M post- surgery and spatial reference memory deficits from 1,5 M postsurgery. Microbleed seemed to induce neurobehavioral disturbances in APP and WT mice at respectively 3 M and 6 M post-surgery displayed by
decreased motor activity and increased anxiety level. Regarding memory performances and compared to APP+Sal group, spatial reference memory in
APP+COL mice was impaired at 1,5 M and improved at 3 M post-surgery. Atorvastatin treatment did not affect motor and anxiety changes induced by
the microhaemorrhagic lesion in APP and WT mice but seemed to improve spatial reference memory skills in APP mice at 6 M post-surgery
compared to untreated group. By contrast, WT+COL+At mice showed a progressive alteration in spatial reference memory.
Conclusion :
Our results seem to indicate that : i) microbleed may have an impact on APP and WT mice behavior, ii) atorvastatin treatment could improve spatial
reference memory impairment induced by microbleed in APP mice at 6 M post-surgery. This study will be completed by histomorphometric analysis.
Alzheimer’s disease mouse model, vascular lesion, cognition, pharmacological modulation
25
BERTHE Julie
Bruno QUESNEL / Sylvie ZOUITINA-GALIEGUE (INSERM U1172 éq. 04 - QUESNEL Bruno)
Impact of PD-L1 on chemoresistance in breast cancer
Julie Berthe1, Hassiba El Bouazzati1, Isabelle Briche1, Zacharie Segaoula1, Xavier Thuru1, Sylvie Zouitina-Galiègue1, Bruno Quesnel1,2
1 Univ. Lille, Inserm, UMR-S 1172, F-59000 Lille
2 CHU Lille, Service des Maladies du Sang, F-59000 Lille
In a mouse model of acute myeloid leukemia (AML), overexpression of PD-L1 (Programmed Death-Ligand 1) leads to long-term persistence of
residual cells. Engagement of PD-L1 with its receptor PD-1 on T cells delivers an immunosuppressive signal. Moreover, PD-L1 exhibits nonimmunological functions, by inducing resistance to apoptosis and chemotherapy in several tumors. In AML, this protein has been shown to decrease
mice survival by increasing resistance to aracytin. In breast cancer, PD-L1 expression might be correlated to anthracycline resistance.
To determine PD-L1-induced chemoresistance mechanisms, we would like to address some questions:
1Which signaling pathways are implicated?
2Does PD-L1 modulate its subcellular distribution following chemotherapy?
3Are some PD-L1 functional domains implicated?
4Is PD-L1/PD-1 interaction necessary?
To address these questions, a knock-out breast cancer cell line (MDA-MB-231) was generated by genome editing targeting the PD-L1 encoding gene,
as well as the same line with PD-L1 overexpression. To highlight cellular mechanisms and signaling pathways modulated by PD-L1, we performed
microarrays analysis on these cellular models. Resistance to chemotherapy and apoptosis will be analysed by AnnexinV/IP tests and PD-L1 functional
domains will be studied by using mutants. Also, in vivo studies are currently performed to confirm PD-L1 non-immunological functions.
We showed that PD-L1 protein exhibits different subcellular localizations: cytoplasmic, nuclear and membranar. Preliminary data indicate that PD-L1
may modulate tumor growth in vivo (NUDE mice) but not in vitro, suggesting a central role of tumor micro-environment and immune system. Kinome
and transcriptomic data highlighted a connection between PD-L1 expression and phosphorylation of p53 at Ser6, as well as a modulation of p53dependant cellular processes.
Our results show a link between PD-L1 and p53 that might underlie PD-L1-induced chemoresistance mechanisms.
PD-L1, chemoresistance, genome editing, breast cancer
CALADO MENDES Tiago Nuno
Jean-Charles LAMBERT (INSERM U1167 éq. 03 - LAMBERT Jean-
Charles)
Bin1-Tau interaction: a new actor of the pathophysiological process of Alzheimer's disease
Tiago MENDES, Nicolas MALMANCHE, Shruti DESAI, Florie DEMIAUTTE, Fanny EYSERT, Amandine FLAIG, Alexis BRETTEVILLE, Philippe
AMOUYEL, Philippe BERTRAND, Marie-Françoise PAUL, Julien CHAPUIS, Laurent PRADIER, Jean-Charles LAMBERT
The major neuropathological hallmarks of Alzheimer's disease (AD) are the intracellular neurofibrillary tangles, composed of abnormal
hyperphosphorylated Tau, and the Amyloid-β peptide (Aβ) extracellular senile plaques. The bridging integrator 1 (BIN1) gene is the most associated
genetic risk factor with AD risk after APOE and it was the first AD genetic risk factor associated with Tau pathology: BIN1 fly ortholog modulates Tau
toxicity, and the interaction between BIN1 and Tau has been described in vitro and in vivo, with Tau phosphorylation downregulating this interaction.
The thesis project aims at understanding the modulation of the BIN1-Tau interaction in the context of AD by better defining the mechanisms and
molecular pathways involved.
Rat post-natal mixed primary neuronal cultures (PNC) were grown to 15 days in vitro and exposed to different concentrations (10nM, 100nM and 1uM)
of synthetic oligomeric Aβ1-42 for 16 hours. Media was then collected for Aβ1-42 and oligomers detection, cells were harvested for protein levels
quantification and/or fixed to detect BIN1-Tau interaction.
The presence of Aβ1-42 and oligomers, in the media, was confirmed, quantified and profiled through AlphaLISA and western blot (WB). BIN1 and Tau
endogenous protein levels were also quantified through WB. Proximity Ligation Assay (PLA) was performed to identify BIN1-Tau interaction in our
cultures and quantified with confocal microscope Zeiss LSM710.
BIN1 and Tau endogenous protein levels did not change with the presence of synthetic oligomeric Aβ1-42. The detection of PLA spots showed a
specificity of the BIN1-Tau interaction into neurons and this interaction significantly decreased when the synthetic oligomeric Aβ1-42 concentrations
increased, especially in the non-soma region of neurons.
These results indicate that, since the endogenous protein levels of BIN1 and Tau are not affected by the presence of synthetic oligomeric Aβ1-42, the
reduction of BIN1-Tau interaction might be due to signalling pathway involved in the molecular mechanisms of Aβ synaptotoxicity.
To confirm the specificity of BIN1-Tau interaction modulation, experiments with synthetic Aβ42-1 (inactive control peptide) are ongoing and High
Content Screening using compounds that target kinases, phosphatases and well characterized signalling pathways is being set up, to identify the
modulators of the BIN1-Tau interaction in line with Aβ peptide exposure.
Alzheimer's disease (AD), BIN1, Tau, Amyloid-β peptide (Aβ)
26
CANOUIL Mickael
Philippe FROGUEL / Ghislain ROCHELEAU (UMR CNRS 8199 éq. 01 - FROGUEL Philippe)
Single Nucleotide Polymorphisms Associated With Fasting Blood Glucose Trajectory And Type 2 Diabetes Incidence: A Joint
Modelling Approach
Mickaël Canouil1, Philippe Froguel1;2, Ghislain Rocheleau1
1Univ. Lille, CNRS, CHU Lille, Institut Pasteur de Lille, UMR 8199 - EGID, F-59000 Lille, France
2Department of Genomics of Common Disease, Imperial College London, London, United Kingdom
In observational cohorts, longitudinal data are collected with repeated measurements at predetermined time points for many biomarkers, along with
other covariates measured at baseline. In these cohorts, time until a certain event of interest occurs is commonly reported and very often, a
relationship will be observed between a biomarker repeatedly measured over time and that event. Joint models were designed to efficiently estimate
statistical parameters involved in these two types of data by combining a mixed model for the longitudinal biomarker trajectory and a survival model for
the event risk. Two main approaches have been proposed to account for the link between the two models in the shared random effect approach, a
function of the random effects (typically the expected current value of the biomarker) is introduced as an explanatory variable in the survival model. In
the latent class approach, the population is assumed to be divided in several latent classes with class-specific trajectories for the biomarker associated
with class-specific risk function for the event.
Using genotypes assayed with the Metabochip DNA arrays (Illumina) from 4,500 subjects recruited in the French cohort D.E.S.I.R. (Données
Épidémiologiques sur le Syndrome d'Insulino-Résistance), we analysed a set of SNPs, selected from published genome-wide association studies,
known to be associated with fasting blood glucose (FPG) and/or type 2 diabetes (T2D). In our study, we focused in the shared random effect
approach, where the event of interest is T2D and the longitudinal biomarker is FPG. As expected, our analysis confirms a robust association between
FPG and incident T2D. Effect sizes for these SNPs are consistent with those reported in the literature. Our results suggest that SNP rs7903146 in
TCF7L2 gene is associated with an increased risk of incident T2D and an elevated FPG level over time.
SNP rs560887 in G6PC2 (known to be associated with FPG) shows a statistically significant effect on the FPG trajectory and an unexpected
protective effect on incident T2D, which could be attributed to the limited number of incident T2D cases in our cohort.
These findings need to be replicated in other cohorts and further investigation of these effects remains to be done. To the best of our knowledge, joint
models have never been applied into a genetic epidemiology context and could help identify novel loci sharing effects on both glycaemic traits and
T2D.
BioStatistics, mixed and survival model, Blood fasting glucose trajectory, Type 2 Diabetes
CARGOET Marine
Oleg MELNYK (UMR CNRS 8161 éq. 04 - MELNYK Oleg)
Total synthesis of biotinylated NK1 domain of hepatocyte growth factor by a SeEA/SEA kinetically controlled ligation
approach
M. Cargoët, L. Raibaut, N. Ollivier, H. Drobecq, E. Boll, Yun Min Chang, R. Desmet, J. C. M. Monbaliu, O. Melnyk
Given the potential of peptide selenoesters for protein total synthesis and the paucity of methods for the synthesis of these sensitive peptide
derivatives, we sought to explore the usefulness of the bis(2-selenylethyl)amido (SeEA) group,1, 2 i.e. the selenium analog of the bis(2sulfanylethyl)amido (SEA 3, 4) group, for accelerating peptide bond formation. A chemoselective exchange process operating in water was devised for
converting SEA peptides into the SeEA ones. Kinetic studies show that SeEA ligation, which relies on an initial N,Se-acyl shift process, proceeds
significantly faster than SEA ligation. This property enabled the design of a kinetically controlled three peptide segment assembly process based on
the sequential use of SeEA and SEA ligation reactions.1 The method was validated by the total synthesis of biotinylated NK1 (180 AA) domain of
hepatocyte growth factor.
1.
Raibaut, L.; Cargoet, M.; Ollivier, N.; Chang, Y. M.; Drobecq, H.; Boll, E.; Desmet, R.; Monbaliu, J.-C. M.; Melnyk, O., Chem. Sci. 2016, DOI:
10.1039/c5sc03459k.
2.
Raibaut, L.; Drobecq, H.; Melnyk, O., Org. Lett. 2015, 17, 3636-9.
3.
Raibaut, L.; Ollivier, N.; Melnyk, O., Chem. Soc. Rev. 2012, 41, 7001-7015.
4.
Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O., Org. Lett. 2010, 12, 5238-41.
protein total synthesis, ligation
27
CASTILLO AGUILERA Omar
Patrick DEPREUX / Paola Barbara ARIMONDO (EA7365 éq. 02 - VACCHER
Claude)
Design, synthesis, and pharmacological evaluation of novel DOT1L inhibitors
Castillo-Aguilera O. (1), Goossens L. (1), Halby L. (2), Bailly C.(3), Arimondo P. B. (2) and Depreux P. (1)
(1) Univ. Lille, EA 7365 GRITA – ICPAL, Lille.
(2) FRE3600 ETaC, CNRS, 31035 Toulouse.
(3) Laboratoires Pierre Fabre, F-31035 Toulouse.
Cancer is a serious issue for public health as it is one of the main causes of mortality worldwide. Despite the available treatments, it is necessary to
develop more efficient and less invasive therapies against cancer. The knowledge of the human genome and epigenome has directed research to new
cancer treatment approaches: it is possible to modulate the biological outcome by controlling the access to the genetic information by means of the
epigenetic regulation. Epigenetics are the changes happening on the genome without modifying its DNA sequence, leading to a heritable and stable
phenotype. In the eukaryotic chromatin, epigenetic regulation implies covalent modifications of DNA and histones. These chemical modifications
remodel the chromatin structure rendering it with an «opened» or «closed» configuration, which leads to the expression or repression of genes. The
epigenetic landscape is altered in some cancers; for example, abnormal methylation leads to the silencing of certain genes (such as tumor suppressor
genes), or to the over expression of oncogenes. Unlike genetic alterations that are irreversible, epigenetic aberrations are reversible.
Methylation and acetylation are the most studied epigenetic modifications. DNA methylation is carried out by the DNA methyltransferases (DNMTs),
and histone methylation by the histone methyltransferases (HMTs). We are particularly interested in the histone methyltransferase DOT1L (DOT1 like,
disruptor of telomeric silencing), responsible of methylating residue Lys79 of histone 3 (H3K79) which, in certain specific pathologies, leads to the
transcription of some oncogenes. Recent studies have showed that DOT1L is implicated in MLL-rearranged leukemia (MLL-r, Myeloid-Lymphoid
Leukemia) and it is thus a potent target in cancer. As DOT1L and DNMTs share the same cofactor, S-adenosyl-L-methionine (SAM), DNMT and
DOT1L inhibitors can present a common inhibition mechanism: they compete with SAM as suicide substrates.
Herein we present the design, and the synthesis of new potential DOT1L inhibitors, as well as their pharmacological evaluation over DOT1L and
DNMT. These molecules, which could restore the epigenetic balance, represent therapeutic tools for cancer treatment.
Anglin, J. L., et al. J. Med. Chem. 2013, 56 (22), 8972–8983.
Daigle, S. R., et al. Blood 2013, 122 (6), 1017–1025.
Valente, S., et al. J. Med. Chem. 2014, 57 (3), 701–713.
cancer, drug design, histone methyltransferases, epigenetics
CHEHERE Baptiste
Valerie BOUGAULT (EA4488 éq. 01 - BERTHOIN Serge)
Cardiorespiratory responses during the 6-minute walk and stepper tests in patients with ILD.
Baptiste CHEHERE, Valérie BOUGAULT, Alice GICQUELLO, Benoit WALLAERT
[email protected]
Introduction: The 6-minute stepper test (6MST) has been used as an alternative to the 6-minute walk test (6MWT) to assess exercise tolerance in
patients with interstitial lung disease (ILD). Recent data suggest that the tests may involve different energy pathways and cardiorespiratory responses.
We thus aimed to compare the cardiorespiratory responses of ILD patients during the 6MWT and the 6MST.
Methods: Thirty-one patients with ILD were randomised to perform both tests in the order 6MST→6MWT (n=16) or 6MWT→6MST (n=15). Gas
exchange, heart rate, and pulse O2 saturation (SpO2) were measured continuously, and dyspnoea, leg discomfort, and blood lactate concentration
were assessed before and immediately after each test.
Results: Oxygen uptake (VO2) was lower (p=0.002) and respiratory equivalent ratio for O2 (VE/VO2) and respiratory exchange ratios were higher
(both p<0.001) during the 6MST compared with the 6MWT. The 6MST was also associated with higher blood lactate concentrations (6MST 4.16±1.95
mmol·L-1, 6MWT 2.84±1.17 mmol·L-1; p=0.01), higher leg discomfort scores (6MST 5±3 points, 6MWT 3±2 points; p<0.001), and smaller decreases
in SpO2 (6MST -5±5%, 6MWT -9±6%; p<0.001).
Conclusions: ILD patients exhibited greater ventilatory responses and lower arterial O2 desaturation during the 6MST compared with the 6MWT. The
higher lactate concentrations and perceived muscle fatigue observed during the 6MST may indicate the presence of inter-test differences in active
muscle metabolism that could contribute to the distinct cardiorespiratory responses.
6-minute stepper test , 6-minute walk test , Gas exchange, Ventilation
28
CHEN Yaohua
Florence PASQUIER (INSERM U1171 - BORDET Regis)
Translational study of relationship between cortical microbleeds and cognitive decline
Yaohua CHEN, Sophie GAUTIER, Sandrine BERGERON, Nicolas DURIEUX, Florent AUGER, Camille Potey, Laura RAVASI, Régis BORDET,
Florence PASQUIER, and Lille YOD study group
University. Lille, UDSL, CHU Lille, INSERM U 1171
Introduction:
Brain microbleeds (BMB) are frequently observed in patients with Alzheimer’s disease, and seemed to be correlated to a more severe cognitive
impairment. As well as being the expression of vascular damage, these small vascular lesions may also reflect amyloid related pathology. Clinical data
have all been registered retrospectively. It would lead to more future treatment’s options if we show a temporal and spatial relationship by a
prospective approach. The aim of our study was to show an eventual impact of one single microbleed on cognitive decline in a mouse model, in order
to further determine their role on occurrence of amyloid dementia. The secondary objective was to determine a pharmalogical effect of Atorvastatin on
the decline induced in our model. We conducted this study in female mice because of a higher prevalence of dementia in women. At the same time,
we aimed to outline relationship between BMB and patients with young-onset Alzheimer’s disease.
Methods:
For the preclinical study, 12 weeks-old, wild-type and transgenic female mice were operated by stereotaxic injection of Collagenase (0.8 µUI/µL) to
induce a BMB; or of saline for the Sham group. During the follow-up, we collected in vivo data by 7 Tesla MRI at 24 hours, 6 weeks, 3 months, 6
months, 9 months and one year after surgery, and by [18F] FDG PET at the same moments except at 24 hours after surgery; and also post-mortem
lesions. We assessed behavioural impairment by several tests at the same periods.
For the clinical study, We studied patients meeting the criteria for sporadic young onset Alzheimer’s disease. Demographic data, vascular risk factors,
comorbidities, treatments, APOE genotypes, cognitive assessment, vascular lesions on MRI were collected at inclusion and at one year of follow-up.
Results:
The experimental model was well tolerated and easily reproducible also in female mice. Female mice were much lighter than males at the same age,
but tolerated as well the surgery. Even if the BMB induced by the surgery was not visible any more after 3 months, a slight brain atrophy was
described, especially in the cortex with BMB. Behavioural impairment was observed 6 weeks after the surgery. The decline was more severe and rose
earlier in mice bearing CMB. Compared to the sham group, the transgenic mice with CMB were rather impaired.At 1.5 months post-surgery, spatial
learning and reference memory was corrupted; anxiety was decreased and spontaneous locomotion was increased. The visual spatial learning and
memory seemed to be impaired in th CMB group.
In the clinical cohort, baseline characteristics didn’t seem to be different in women and in men, in term of vascular lesions. Though women were more
severe and had a longer duration of the disease.
Conclusion:
BMB may impaire the cognition in the earliest development, by inflammatory pathways, and may be restored after. These first findings should be
confirmed by other longitudinal findings.
Microbleeds, Alzheimer’s disease, Amyloid deposit
29
COISNE Augustin
David MONTAIGNE (INSERM U1011 éq. 01 - STAELS Bart)
Impact of obese and type 2 diabetes-related alterations in epicardial fat on left ventricular remodeling in aortic stenosis
Augustin COISNE
Directeur de thèse : David MONTAIGNE
Equipe UMR1011 Pr Bart Staels
Background
In elderly patients, the aortic valve which separates the left ventricle (LV) and ascending aorta is faced to calcification resulting in a narrowed valve
orifice called aortic stenosis (AS). This aortic stenosis is the most common valvular disease in the western world. AS can lead to heart failure,
arrythmia, angina and sudden deat and is thus a major public concern. The LV response to AS typically involves wall hypertrophy to maintain normal
wall stress. This response is called LV remodeling and is charaterized by cardiomyocyte hypertrophy.
Obesity and Diabetes Mellitus are associated with important LV remodeling in patients without AS, suggesting that these conditions interfere with
hypertrophic signaling pathway in cardiomyocyte. This metabolic disorders are also associated with an increased epicardial adipose tissue (EAT)
thickness. EAT of obese patients is characterized by a pro-inflammatory profile and a production of inflammatory mediators such as TNFalpha and
Interleukin-6 family which have been shown to activates the transcription of genes involved in the hypertrophic response.
We hypothesize that systemic metabolic disorders drive chronic low-grade inflammation in EAT which in turn leads to increase LV mass in AS and
failing reverse cardiac remodeling in post-operative patients.
Methods
Between November 2014 and April 2016, we prospectively inluded 149 patients referred in our University Hospital of Lille for a valve replacement for
a symptomatic aortic stenosis. All patients signed an informed consent and were reviewed by a multi-disciplinary team. 69 patients were followed 1
year after surgery. This protocol received the agreement of local ethic comittee.
Results
Obese patients (with body mass index (BMI) over 30) had a worse LV remodeling with a significantly increased index LV mass (iLVM) than overweight
(BMI between 25 and 30) or lean patients (BMI under 25) (respectively 62.4±14.8 vs 57.2±13.9 and 44.8±1.9,g/m2.7, p<0.05). In multivariate
analysis, BMI was an independent predictor of increased LVMi after adjustment for age, aortic peak velocity, systolic blood pressure, history of
hypertension and history of diabetes mellitus. In transthoracic echocardiography, obese patients had an increased EAT thickness than lean patients
(10.4±2.8 vs 3.6 ±1, p<0.0001). In all population, EAT quantity was strongly correlated with iLVM (r=0.43, p<0.001). Finally, patients had a significant
reverse remodeling after surgery (iLVM 1 year after surgery at 44.5±13 vs 55.8±15, p<0.001).
Conclusion
Our study show that obese patients with severe AS had (i) an increased iLVM after adjustment for other factors, (ii) an increased EAT thickness and
(iii) a strongly association between iLVM and EAT thickness. Further investigations are on going to explore the inflammatory profile of EAT and its
impact on cardiomyocyte hypertrophy.
Obesity, Aortic stenosis, Epicardial adipose tissue, Left ventricular mass
CORFIOTTI Francois
Stephanie TRUANT (INSERM U1172 éq. 05 - VAN SEUNINGEN Isabelle)
30
CORSI Mariangela
Fabien GOSSELET / Pietra CANDELA / tuteur cotutelle : Rita BUSINARO (EA2465 -
GOSSELET Fabien)
Effect of the ketogenic diet on Blood-Brain barrier
Corsi M, Candela P, Fuso A, Fenart L, Businaro R et Gosselet F
Univ. Artois, EA 2465, Laboratoire de la Barrière Hémato-Encéphalique (LBHE), Lens Cedex, France
Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome
Introduction: Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder characterized by impairment of cognitive function.
The hallmark lesions of AD are senile plaques, formed by deposits of β-amyloid peptide (Aβ40 and Aβ42) that are responsible for neuronal dysfunction
and death, along with neurofibrillary tangles, consisting in hyperphosphorilated tau proteins, as well as mitochondrial dysfunction.
Previous studies have shown that the blood-brain barrier (BBB) plays an important role in the progression of AD because the cells that are part of it
(endothelial cells, pericytes and astrocytes) express receptors and transporters involved in the influx and efflux of the peptide Aβ.
AD remains asymptomatic for a considerable time before cognitive decline becomes clinically evident. For this reason, the establishment of
appropriate nutritional protocols may be more effective than drug treatments for this disease. In particular the Ketogenic diet (KD) is a diet low in
carbohydrates and high in fat that produces ketone bodies by the liver. Thanks to their ability to cross the BBB, ketone bodies might represent an
alternative energy source to glucose for the brain. Many lines of evidence suggest that KD is benefic for AD, but relationship between ketones and
cerebral Aß accumulation remain to be elucidated.
Most studies focus on the neuronal component and forget the vascular component of AD represented by the blood-brain barrier (BBB), located in the
cerebral microvessels.
Our attention will instead focus on the BBB, in particular on the effect of KD on BBB physiology and amyloid transport.
Methods: For 4 weeks, Wild type mice (129SV) were maintained on KD and Control Diet (CD). Body weight was measured. After sacrificing the mice,
Glucose and Beta-hydroxybutyrate (BHB) levels were assessed in blood sample. Microvessel fractions were isolated from total brain (Coisne et al.,
2005) and qPCR analyses were performed to study expression of transporters, receptors and enzymes involved in amyloid transport and metabolism
at the BBB level.
Results: KD fed animals showed increased levels of BHB which was accompanied by an increased expression of MCT1 and GLUT1. There were not
changes in the level of Glucose and body weight in these mice. In addition we observed modifications in the expression of the transporters involved in
Aβ exchanges such as LRP1 and MRP1.
Discussion: These results suggest that KD could modulate amyloid exchanges between brain and blood, and therefore could represent a nutritional
protocol in case of AD. This approach may be more effective
than drugs, because it will be carried out in the preclinical stage of the disease when it start the glucose hypometabolism
Ketogenic diet , Blood-Brain Barrier, Alzheimer’s Disease
31
COUTEL Xavier
Guillaume PENEL / Cécile OLEJNIK (EA4490 - HARDOUIN Pierre / PENEL Guillaume)
Assessment of Bone Marrow Adiposity (BMA): A pilot study in adult rats.
Xavier COUTEL*, Pierre MARCHANDISE, Cécile OLEJNIK, Guillaume PENEL
Université de Lille, Laboratoire PMOI – EA 4490 - Faculté de Chirurgie Dentaire – Place de Verdun, Lille
Introduction: Recent data argue for an involvement of bone marrow adiposity (BMA) in bone pathophysiology. In animal models, BMA alterations have
been reported to be age, gender and site specific. Alveolar bone surrounding teeth is a specific functional area, sensitive to local factors, with a high
bone turnover compared to other skeletal sites. We hypothesized that BMA distribution in the mandibles may be different from long bones. The aim of
this preliminary study is to compare bone microarchitecture and BMA volume in 2 specific area (alveolar and condylar bone) in the mandible and in
tibiae in adult rats.
Material and methods: Nine 6-month-old female Sprague-Dawley rats (Janvier Lab, Laval, France) were used in this study. Ex vivo microtomography
(µCT) analyses were performed with the same acquisition parameters on mandibles and tibiae using a Skyscan 1172 (Bruker, Kontich, Belgium) at
10µm3 voxel size. Bone histomorphometrical and adiposity parameters were assessed on the central region of the mandibular condyle, the interradicular alveolar bone of the first molar, and the secondary spongiosa in the proximal tibia. BMA imaging was obtained on mandibles and tibiae after
decalcification and staining with a 1% osmium tetroxide solution. Image datasets were registered prior to assess BMA volume. The relative BMA
volume was measured within the inter-trabecular volume.
Results: Trabecular bone volume, number and thickness were higher in the alveolar process (respectively +44%, +15%, +38%, p<0,001) and in the
condyle (respectively +58%, +22%, +50%, p<0,001) compared to tibiae. Trabecular separation was lower in alveolar (–5%, p<0,001) and condylar
bone (-50%, p<0,001) compared to tibiae. Moreover, inter-trabecular volume was inferior in the alveolar (-36%, p<0,001) and in the condylar bone (63%, p<0,001) compared to tibiae. Adiposity parameters assessment indicated a higher BMA volume in the tibiae compared to the mandibles.
However, the ratio BMA volume/inter-trabecular volume was interestingly higher in the alveolar (+18%) and condylar (+40%) bone compared to tibia.
Conclusion: Mandibular alveolar bone shows a more dense trabecular network and a lower inter-trabecular volume compared to tibia. Present data
shows specificities in the microarchitecture and marrow adiposity in mandibular teeth-bearing (alveolar) and non teeth-bearing (condylar) bone sites.
Based on those results, it would be of great interest to precise mandibular BMA location. This information should be particularly relevant in
osteoporosis models.
Bone marrow adiposity , Mandible, X-ray microtomography
DELANNOY Clement
Elisabeth ELASS-ROCHARD (UMR8576 éq. 01 - GUERARDEL Yann)
Deciphering the glycosylation changes occurring during the differentiation of monocytic THP-1 cell line into macrophages and
their cell activation
Clément Delannoy
Université de Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F 59 000 Lille, France
Macrophages mediate innate immune system through the initiation and regulation of inflammation and contribute to adaptive immunity via antigen
processing. Cell surface glycosylation has been widely described to be involved in different physiological or pathological processes, such as host
defense, immunological and inflammatory responses but much less is known about the variations of glycosylation related to the differentiation of
monocytes into macrophages. Human monocytic cell line THP-1 is frequently used as macrophage-like models, after treatment with phorbol myristate
acetate (PMA), allowing the prediction of both function and behavior of these phagocytes.
Based on these observations, the aim of this study was to highlight the glycome variation during the differentiation of the human monocytic THP-1 cell
line into macrophages. In this study, MALDI-TOF data analysis showed that the differentiation of monocytic THP-1 cells into macrophages induced
gangliosides biosynthesis, but also an increase of complex N-glycan and the degree of branching. This data were correlated with the expression
pattern of glycosyltransferases and glycosidases involved in glycan elongation and trimming.
Therefore, the differentiated THP-1 cells were exposed to inflammatory agents. The 19-kDa lipoprotein, a component of cell-wall of Mycobacterium
tuberculosis, has an important role in the induction of a pro-inflammatory response in macrophages through Toll-like receptor 2 . However, nothing is
known about the influence of the 19-kDa lipoprotein on macrophage glycosylation. To investigate the impact of this lipoprotein, a synthetic lipopeptide,
called PAM3-LP19 and known as agonist of TLR2 has been used to mimic the lipid moiety of the cell-wall associated 19-kDa lipoprotein.
Monocyte-to-Macrophage differentiation, Glycosylation, Mass spectrometry, 19-kDa lipoprotein
32
DELCOURT Vivian
Isabelle FOURNIER / tuteur cotutelle : Xavier ROUCOU (INSERM U1192 - SALZET Michel)
Deep functional proteomics identification of alternative proteins gives insight into their functions
V. Delcourt1,2, J.F. Lucier2, S. Samandi2, J. Franck1, J.F. Jacques2, M. Salzet1, I. Fournier1 & X. Roucou2
1Université de Lille INSERM U1192, Laboratoire PRISM, France
2Université de Sherbrooke, Département de Biochimie, Canada
Many recent publication share the idea that the eukaryotic proteome has been largely underestimated by observing translation of unexpected proteins
which constitute the «hidden proteome». The hidden proteome contains alternative proteins (AltProts) that are translated from alternative open reading
frames (AltORFs). AltORFs are present within 5' and 3' untranslated regions, overlap the coding sequence of the RefProt, which are the longest
predicted proteins from the annotation of the human genome, but in a frameshifted reading frame or in putative non-coding-RNA. However, only a few
AltProts have been studied so far and have known activity. Large scale mass spectrometry (MS) proteomics studies is the most effective way to
identify AltProts. This approach allows to emit hypothesis on protein functions by observing changes induced by a treatment or by determining their
protein-protein interaction partners. However, even if many studies relate these observations on RefProts, none has been focused on AltProts. By
analyzing published large scale proteomics data with a custom database containing the human RefProts and AltProts, we would be able to determine
which AltProts are indeed expressed, but also to observe relevant potential biological information involving AltProts.
To fulfil such challenge, the proteomics identification software PeptideShaker was adapted to a 39 168 core computer cluster which allowed the
analysis of about 6000 raw MS data out of four reference publications in an outstanding time effectiveness. This approach led us to identify several
thousands of AltProts from four reference large scale proteomics studies. More interestingly, by analyzing the human phosphoproteome, we observed
AltProts which were specifically phosphorylated during key events of cell life such as mitosis, following an epidermal growth factor stimulation or
phosphorylated AltProts that were up or down regulated in specific conditions. These findings suggest that AltProts may be part of the human
signalome, adding a new layer in signaling regulation. We also found that some AltProts were enriched in specific known protein complexes
suggesting that AltProts are part of biologically active protein complexes and that AltProts may be necessary for the complex to complete its biological
activity. As this approach is effective to identify numerous AltProts and get insight towards their activity, more MS proteomics studies are scheduled to
be analyzed. Most interesting candidates with potential biological and/or pathological involvement are going to be validated biologically in order to
determine AltProts' biological functions involvement or biomarker capabilities.
Alternative ORF, Proteomics, Mass spectrometry, Protein translation
DELEYE Yann
Rejane PAUMELLE-LESTRELIN (INSERM U1011 éq. 01 - STAELS Bart)
Role of the CDKN2A/p16INK4a tumor suppressor gene in the control of lipid metabolism in the liver.
Yann Deleye, Joel Haas, Hannou Sarah, Emmanuelle Vallez, Vanessa Legry, Réjane Paumelle, Bart Staels. Univ. Lille, Inserm, CHU Lille, Institut
Pasteur de Lille, U1011, EGID, F-59000 Lille, France
p16INK4a is a tumor suppressor protein that is a well described cell cycle regulator. Recently, genome-wide association studies associated the
CDKN2A locus, from which p16INK4A is encoded, with increased risk for development of type 2 diabetes. A pathophysiological link between
p16INK4a and hepatic glucose homeostasis has been unraveled recently, through the control of gluconeogenesis. Patients with T2D also present with
disturbances in fat metabolism, associated with enhanced fatty acid oxidation and susceptibility to ketoacidosis. In this context, we investigated the
role of p16INK4a in hepatic lipid metabolism and ketogenesis in vitro using primary hepatocytes, the AML12 and IHH hepatocyte cell line and in vivo
using p16+/+ and p16-/- mice.
In vitro, p16-/- primary hepatocytes demonstrated enhanced β-oxidation of oleate compared to p16+/+ without additive effects of Forskolin. Along the
same lines, p16-/- primary hepatocytes, AML12 and IHH cell lines transfected respectively with siRNA CDKN2A and p16 exhibited increased gene
expression of HMGCS2, the rate limiting enzyme of ketogenesis. These effects were associated with an increased response to GW647, a PPARα
agonist, in vitro, and reversed by siRNA targeting PPARα. Investigating known PPARα activators and transcriptional coactivators in vitro, we found
that upregulation of HMGCS2 expression was also linked to SIRT1 [activation or upregulation], and subsequent activation of PGC1α and PKA. In vivo,
p16-/- mice fed a ketogenic diet, known to increase PPARα transcriptional activity in the liver, also presented higher HMGCS2 gene expression
compared to p16+/+. These findings describe a new function for p16INK4a as an actor in hepatic ketogenesis.
Lipid metabolism, Ketogenesis, Cell cycle
33
DESPRES Clement
Caroline SMET-NOCCA (UMR8576 éq. 02 - LIPPENS Guy)
Deciphering the molecular mechanism of Tau aggregation : essential role of the triple-phosphorylated state of AT8 epitope.
Clément Despres - CNRS, UMR 8576, F-59000, Lille, France
Caroline Smet-Nocca - CNRS, UMR 8576, F-59000, Lille, France
Isabelle Landrieu - CNRS, UMR 8576, F-59000, Lille, France
Guy Lippens - CNRS, UMR 5504, LISBP, INSA Toulouse, France
The microtubule-associated protein (MAP) Tau is implicated in the pathogenesis of Alzheimer's disease (AD). Abnormal phosphorylation of Tau
impairs its interaction with other proteins such as microtubules and is associated with pathological dysregulation. At that time, it has been established
that Tau is the major component of Paired Helical Filaments (PHFs) which are one the two molecular hallmarks of AD. In PHFs, Tau is found under an
hyperphosphorylated state, but the molecular mechanisms underlying Tau hyperphosphorylation and aggregation in AD are currently unknown. The
AT8 antibody that recognizes pS202/pT205 phospho-epitope in hyperphosphorylated Tau is involved in AD diagnosis and post-mortem staging of AD.
Recently, a NMR and molecular dynamics study has described a turn conformation at the AT8 epitope that is stabilized by hydrogen bonds involving
phosphorylated S202 and T205, G207 amide function and two arginine residues. Here, we characterize by an in vitro approach the role of AT8 in
aggregation of the Tau protein. In vitro Tau phosphorylation patterns generated either by Extracellular-Regulated Kinase 2 (ERK2) or by kinase activity
of a rat brain extract are characterised by high-resolution NMR spectroscopy. Both phosphorylation patterns are quite similar except for two residues:
S208 and S262 which are only phosphorylated by the rat brain extract. While phosphorylation of S262 by the microtubule-affinity regulating kinase
(MARK)/Par1 was previously shown to inhibit fibril formation, kinase involved in S208 phosphorylation and its functional impact are unknown. Using
site-directed mutagenesis, we have generated several Tau mutants that have been further phosphorylated and characterised by NMR. Aggregation
assays have been performed in which the formation of PHF-like fibrils is monitored by Thioflavin T fluorescence and further confirmed by Transmission
Electron Microscopy (TEM). Our data indicate that S208 phosphorylation at the AT8 phospho-epitope is a trigger of Tau fibrillization and regulates an
«open/close» mechanism at the structural level.
Tau protein, Phosphorylation, Aggregation, AT8 phospho-epitope
DUCASTEL Sarah
Sophie LESTAVEL (INSERM U1011 éq. 01 - STAELS Bart)
The nuclear receptor FXR decreases murine enteroendocrine L cell response to gut microbiota metabolites, the short chain
fatty acids
Sarah DUCASTEL, Véronique TOUCHE, Mohamed-Sami TRABELSI, Bart STAELS and Sophie LESTAVEL
Inserm U1011
Laboratoire de Recherche J & K
Faculté de Médecine - Pôle recherche
Boulevard du Professeur J. Leclercq
59045 Lille Cedex - France
Background and Aims: Diabetes mellitus involves many metabolic disorders including a decreased incretin effect. Proglucagon is produced in
enteroendocrine L cells, processed by prohormone convertase 1/3 into the incretin GLP-1. GLP-1 is secreted by L cells in response to various
secretagogues among them glucose, bile acids, amino acids, long, medium and short chain fatty acids. The nuclear receptor Farnesoid X Receptor
(FXR), activated by bile acids, regulates transcription of genes involved in the control of energy homeostasis. We have recently shown that FXR
activation in L cells decreases glucose-induced ChREBP-dependant proglucagon gene transcription. FXR also decreases glucose-induced GLP-1
secretion by inhibiting glycolysis pathway. Our hypothesis is that FXR could inhibit L cell response to other GLP-1 secretagogues. The aim of this
study is thus to investigate the role of FXR in the L cell response to short chain fatty acids (SCFA: acetate, propionate and butyrate) which are
metabolites produced by the gut microbiota by fermentation of non digestible polysaccharides. In addition to their contribution to 5 to 10% of the daily
energetic resources, SCFA are also signalling molecules as they bind to the transmembrane receptor GPR43/FFAR2, thereby promoting GLP-1
secretion by L cells.
Materials and Methods: FXR was activated by the synthetic agonist GW4064, in vitro in the murine L cell line GLUTag and in vivo in C57Bl6/J mice.
GLP-1 secretion tests (ELISA) in response to natural (butyrate) and synthetic (4-CMTB and PA) GPR43 agonists were performed in vitro in GLUTag
cells and ex vivo on murine colonic explants. GPR43 mRNA levels were evaluated by qPCR in GLUTag cells incubated with GW4064, in colon from
mice in vivo treated for 5 days with GW4064, from KO FXR mice and from mice treated for 3 weeks with colesevelam, a bile acid sequestrant which
induces a drastic down-regulation of FXR transcriptional activity.
Results: In vitro in GLUTag cells, FXR activation decreases GLP-1 secretion in response to butyrate and in response to GPR43 synthetic agonists.
Moreover, in vivo FXR activation by treatment of C57Bl6/J mice for 5 days with GW4064 decreases ex vivo GLP-1 secretion by mouse colonic
explants in response to butyrate. In parallel, in vitro and in vivo FXR activation decreases GPR43 mRNA levels. As a mirror effect, FXR KO mice and
colesevelam treated mice exhibit increased GPR43 mRNA levels in colon in comparison to WT littermates and untreated mice respectively.
Conclusion: FXR activation in enteroendocrine L cells decreases their capacity to respond to SCFA in terms of GLP-1 secretion, this decrease
potentially occurs via the down-regulation of GPR43 expression. Overall our data suggest that deregulation of FXR in intestine may be a good strategy
to increase L cell capacity to respond to glucose and to gut microbiota metabolites and thus to improve metabolic control.
Diabetes, Intestine, Nuclear Receptor FXR, Incretin GLP-1
34
DUMORTIER Mandy
Anne CHOTTEAU-LELIEVRE (UMR CNRS 8161 éq. 07 - DUTERQUE Martine)
The effect of ETV1 gene fusions on prostate cancer development and metastasis
DUMORTIER Mandy, DAMOUR Isabelle, de LAUNOIT Yvan, DUTERQUE-COQUILLAUD Martine & CHOTTEAU-LELIEVRE Anne
UMR 8161-CNRS, Institut de Biologie de Lille, Université de Lille
Introduction: Prostate cancer (PCa) is the leading cause of cancer in men. It is the most frequent malignancy and the second leading cause of cancerrelated death in men in western countries. While treaments for localized PCa exist, its metastasis still requires improved diagnostic and therapeutic
approaches .
The discovery of chromosomal translocations in PCa in 2005 has greatly enhanced our understanding of PCa development. These rearrangements
fuse the ETS factors with promoters of genes that are androgen regulated ; TMPRSS2 being the most common fusion. The ERG and ETV1
oncogenes occur in over 50% and 10% of PCa respectively.
Many ETS gene fusion transcript variants have been identified (full-length or truncated), these rearrangements lead to the over-expression of
oncogenic transcription factors and disrupt the expression of ETS target genes.
Project : In this context, the team is interested in studying the fusions of the ETV1 gene and its implication in the PCa metastasis. We have therefore
selected four different cell-lines, either expressing or not the ETV1 fusion. Phenotypic differences between the cell-lines expressing full-length or
truncated ETV1 factors will be studied in-vitro. Then, the different cell-lines will be injected subcutaneously or intra-cardiacly in immunodeficient mice
to study the in-vivo effects of the fusion.
Finally, the molecular pathways behind any observed differences either in tumoral growth or metasis will be studied by in-silico analysis eventually
allowing us to determine the ETV1 regulated genes.
Conclusion : This work aims in part to determine via in-vivo and in-vitro experiments the potential differences between the truncated and full-length
forms of ETV in the context of tumoral aggresivity. Another obective is to identifty the major genes in the metastatic process in comparison with
available data on the ERG factor. Eventually the project should provide new markers/ therapeutic targets for PCa metastasis.
ETV1, Prostate cancer, gene fusions
DUPLOYEZ Nicolas
Thierry IDZIOREK / Jean-Michel CAYUELA (INSERM U1172 éq. 04 - QUESNEL Bruno)
Comprehensive Mutational Profiling of Core Binding Factor Acute Myeloid Leukemia
N Duployez, A Marceau-Renaut, N Boissel, A Petit, M Bucci, S Geffroy, H Lapillonne, A Renneville, C Ragu, M Figeac, K Celli-Lebras, C Lacombe, JB
Micol, O Abdel-Wahab, P Cornillet, N Ifrah, H Dombret, G Leverger, E Jourdan, C Preudhomme
Introduction. Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported
together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases and
relapse incidence reach up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of
mutations coexisting with CBF translocations has not been elucidated.
Methods. In order to address these issues we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML
enrolled in the CBF2006 and ELAM02 trials (aged from 1 to 60 years).
Results. Mutations in genes activating tyrosine kinase (TK) signaling (including KIT, N/KRAS, FLT3) were frequent in both subtypes of CBF-AML. In
contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in
t(8;21) AML (42% and 18%, respectively) while they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML
patients with poor prognosis while high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome.
In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with TK pathway mutations
suggesting synergic cooperation between these events.
Conclusions. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to
potential unique pathogenesis of t(8;21) versus inv(16) AML.
Acute myeloid leukemia, Mutation analysis, Core binding factor
35
DUPONT Clement
Maximilien VERMANDEL (INSERM U1189 - MORDON Serge)
Photodynamic treatment of high-grade gliomas
C. Dupont, N. Reyns, N. Betrouni, S. Mordon, M. Vermandel
High-grade gliomas are the most common malignant brain tumors in adults. Among them, Glioblastoma Multiform (GBM) is the most common tumor.
Its incidence is estimated to 5 new cases each year for 100 000 inhabitants. Despite reference treatment, including surgery, radiation oncology and
chemotherapy, GBM still remains with a very poor prognosis (median survival below 16 months). Because of a systematic relapse of the tumor, the
main challenge is to improve local control. In this context, photodynamic therapy (PDT) may offer a new treatment modality.
PDT consists on the illumination at a given wavelength of cancer cells previously photosensitized. Combination of a drug named photosensitizer,
oxygen and light creates cytotoxic molecules (mainly singlet oxygen and free radicals) and leads to a cytoreductive effect of the tumor.
In previous studies, PDT has demonstrated promising efficacy with significantly increased overall survival. The main objective of our research project
is to break through technical barriers to fit PDT into the current therapeutic modalities. Two distinct applications of the PDT are investigated:
Intraoperative PDT: Because GBM recurrence appears in cavity margin in 85% of cases, a procedure allowing a selective treatment of these areas
could be an interesting adjuvant. Following the resection using fluorescence to guide the neurosurgeon, we developed a device to insert into the
resection cavity to illuminate these surrounding tissues. Several ex-vivo experimentations were performed to estimate the lighting duration according
to the volume of the device.
Interstitial PDT: this modality is dedicated of non-operable or relapse GBM cases. Light sources composed of diffusing optical fiber are inserted into
the therapeutic target to illuminate surrounding tissues. Simulating the interaction of light with biological tissues is an essential pre-phase to optimize
treatment delivery. Using different simulation models (Monte Carlo, partial differential equation and analytical equations), the light propagation can be
estimated. Monte-Carlo is considered as a ground truth but required a high power and time computing. Partial differential equation describes properly
light distribution but remains hard to implement. Analytical equations only evaluates light propagation and does not take into account the heterogeneity
of tissues. The prerequisite is to develop an accurate algorithm able to compute a therapeutic dose into a short computational time. To do this, we are
studying the parallelization of Monte-Carlo model to decrease dramatically the computational time.
Finally, this research project intends to demonstrate the proof of concept to plan this therapy and provide technological tools in order to consider a
transfer in clinical. In view of the encouraging results observed in preclinical or clinical, the PDT treatment can quickly strengthen the armamentarium
of GBM management.
PhotoDynamic Treatment, Treatment Planing System, Glioblastoma, Simulation
DURAND Matthieu Arnauld VILLERS / tuteur cotutelle : Ashutosh K. TEWARI (INSERM U1189 - MORDON Serge)
36
EL AMRANI Mehdi
Isabelle VAN SEUNINGEN (INSERM U1172 éq. 05 - VAN SEUNINGEN Isabelle)
The role of Epithelial-Mesenchymal Transition (EMT) in Chemoresistance of pancreatic cancer
Mehdi EL AMRANI
Inserm, UMR-S 1172, JPARC – Centre de Recherche Jean-Pierre AUBERT
CHRU Lille, Department of Digestive Surgery and Transplantation, F-59000 Lille, France
Introduction.
Pancreatic cancer (PC) is almost fatal in patients with malignant tumors in the worldwide. The poor prognostic of pancreatic cancer is attributed to its
high metastatic potential and ineffectiveness of therapeutic options. Recent evidences suggest that epithelial-mesenchymal transition (EMT) plays an
important role in development of drug resistance in PC. The aim of this study is to evaluate the association between EMT and chemoresistance in
pancreatic cancer cells and the prognosis for patients with PC.
Methods.
Resistant cells were obtained by treating BxPC-3, Capan-1/2, Panc-1 and MiaPaca 2 in increasing concentrations of gemcitabine. Phenotypic and
morphological variations are evaluated by Western blot, RTqPCR, Immunofluorescent staining. Migration and invasion capacities are evaluated using
Incucyte® technology. Immunohistochemistry of E-cadherin, vimentin, Snail, ZEB-1/2 and c-fos are performed on surgical samples from patients who
underwent resection of their pancreatic cancers. The correlations between EMT status and clinicopathologic factors and prognosis will be analyzed.
The tumor budding is defined and analyzed according guidelines.
Results.
Gemcitabine-resistant cells undergo distinct morphological changes and have more invasive and migratory capacities. Gemcitabine-resistant cells
have increased vimentin and decreased E-cadherin expression. These alterations are hallmarks of EMT. Resistant cells harbor activation of the
MEK/ERK and c-fos signaling pathways. Significant correlation is expected between EMT status and prognosis of patients with resected PC. This
correlation is also expected with clinicopathologic factors. The tumor budding is extremely correlated to EMT markers and is a significant prognostic
factor.
Conclusions.
Gemcitabine-resistant pancreatic tumor cells are associated with EMT. The increase of c-fos may be related to chemoresistance and EMT. The tumor
budding is correlated to EMT and prognosis of patients with PC.
Pancreatic cancer, Epithelial-Mesenchymal Transition , Chemoresistance
37
ESCUTNAIRE Josephine
Herve HUBERT / Karim TAZAROURTE (EA2694 - DUHAMEL Alain)
Traumatic cardiac arrest, epidemiology, care and survival: the French national cardiac arrest registry four-year results
Joséphine Escutnaire (1); Karim Tazarourte (2); Hervé Hubert (1)
(1): Laboratoire de santé publique de Lille EA2694, Lille, FRANCE
(2) Hôpital Edouard Herriot, Hospices Civils de Lyon, Lyon, FRANCE
Rational: Severe traumatism causes 5 million deaths per year worldwide (Alted López 2015). When it leads to cardiac arrest (CA) the prognosis is
critical with a 98% lethality (Huber-Wagner 2007). CA and traumatic CA (TCA) care is firmly defined by guidelines which are internationally accepted
(Fernández Lozano 2015). However the different types of TCA inherent complexity led some learned societies to call into question the benefit of TCA
resuscitation implementation in some circumstances and to broadcast their own recommendations, putting forward a catastrophic cost-benefit ratio
and survival rates close to zero (Bailey 2000). Yet these guidelines and recommendations are based on studies that seldom discriminate the different
types of TCA (the Utstein style for harmonized CA data reporting has only one case to tick whatever is the type of TCA (Perkins 2015)). Hence this
study aim is to carry out a large-size epidemiological description of TCA and of their survival regarding their nature and their causing lesion(s)
localization in order to determine if it would be pertinent to offer a different and finer analysis than these carried out in order to establish guidelines.
Method: Descriptive multicentre study based on data recorded by the French national cardiac arrest registry between July, 1st 2011 and January 1st,
2016 excluding interventions leading to dead body finding.
Results: Among 4093 TCA we studied 3456. Three quarters of victims were males and median age was 46. The most common lesions combination
was a cranial traumatism (CT) associated with a thoraco-abdominal traumatism (TAT) which represents 37.7% of patients of which all were blunt
traumas. Next came isolated CT (35.0%) and TAT (8.0%) among which respectively 30.6% and 7.1% were penetrating traumas. The other
localizations shared the remaining 19.3%. Global survival was 1.5% at day 30 with 60.4% good neurological outcome. 1.4% of victims were subjects
of organ removal. Penetrating TCA victims had a very low survival with less than 0.5% survival if their CA was associated with a cranial damage.
However survival rates were far better if only limbs were damaged with respectively 2.1% for penetrating TCA and 6.4% for blunt TCA day 30 survival.
Discussion & Conclusion: TCA survival is indeed low but not anecdotal, as is organ donation in this population. However we notice a survival rate
collapse when we record a cranial damage and even more in case of penetrating TCA. Regarding these first results caring with interest cranial
traumatism and complications subsequent of haemorrhage seems extremely important to highlight. This immediate care must however be
implemented without neglecting the other lesions. Fortunately penetrating TCA are rare events and those often fatal TCA causes are often avoidable.
The setting of prevention campaigns (i.e: suicidal risk, domestically violence, traffic accidents, work accidents) can limit the occurring of cardiac arrests
related to severe traumtism.
cardiac arrest, epidemiology, traumatism, registry
FERLIN Juliette
Yves ROUILLE (UMR8204-U1019 éq. 05 - DUBUISSON Jean)
Role of ADP-ribosylation factor signalization in HCV replication
Juliette FERLIN
Hepatitis C virus (HCV) is a small RNA virus of the Flaviviridae family. Like other RNA viruses, HCV relies on cellular lipid metabolism for replicating its
genome. Recently, our team has shown that Arf4 and Arf5 (class II ADP-ribosylation factors) are required for HCV replication. Arfs are small Gproteins, which function as regulators of membrane dynamics, lipid metabolism and actin remodeling. Our thesis project aims at identifying class II
Arfs effectors that function in cellular lipid metabolism and HCV replication. We performed transcriptomic and lipidomic analyses of Huh-7 cells
simultaneously depleted of Arf4 and Arf5, in order to find which pathways class II Arfs regulate. Several factors of the lipid metabolism appear
affected, which are under study. We expect that these studies will help us to better understand the role of lipid metabolism in HCV replication and to
identify potential targets for new antiviral drug development.
HCV replication, ADP-ribosylation factor, lipid metabolism
38
FOVET Thomas
Renaud JARDRI (UMR-CNRS 9193 éq. 03 - THOMAS Pierre / PINS Delphine)
Optimization of an Algorithm Able to Detect the BOLD-signal Associated with Auditory Hallucinations
Thomas Fovet
Univ Lille, CNRS UMR 9193, SCALab & CHU Lille, Pôle de Psychiatrie (unité CURE), F-59000, Lille, France ;
Unité d’Hospitalisation Spécialement Aménagée (UHSA) Lille-Seclin
Chemin du bois de l’hôpital
59113 SECLIN
INTRODUCTION
Auditory hallucinations (AH) are frequent experiences in schizophrenia, with a lifetime prevalence of 60 to 80%. As AH can also be refractory to
conventional treatment in more than 25% of the cases, innovative therapeutic strategies are urgently required. Among the most promising avenues,
fMRI-neurofeedback can offer a new way to tackle AH, giving the possibility for patients to normalize the activity level of AH-specific brain regions, and
thus reduce the severity of these symptoms. However, such a strategy requires algorithms able to detect the onset of AH and the associated brain
activation patterns. Machine-learning, notably linear Support Vector Machine represents an innovative strategy for this kind of state-marker
identification.
Here, our objective was to test an optimization strategy for this kind of algorithm. We hypothesised that the performance of such a classifier could be
enhanced using not only data from patients with AH but also data from subjects performing mental imagery of auditory information (i.e. auditory
imagery (AI)) during the training session. Indeed, AI could be a major source of false positives since the activation patterns associated with AI are
close to those observed during AH.
METHODS
Twenty patients with schizophrenia experiencing frequent AH were scanned (Philips 3 Tesla Achieva X-series). They were asked to lie in a scanner
and not engage in any particular task during fMRI acquisition. Patients' cognitive states (presence of AH i.e. AH+ or absence of AH i.e. AH-) at
different periods were labelled using a semi-automatic complex procedure.
Fifteen healthy subjects performed an AI task during an fMRI block protocol and showed activations in the neural networks known to be involved in the
occurrence of AH.
Two classifiers have been developed using linear Support Vector Machine with recursive feature elimination (with BrainVoyager QX v.2.4.2 for
Windows) which is the most widely used and among the highest performing classification methods. The classifier «AH» was developed using only
data from patients with AH during the training session (AH+/AH-). The classifier «AH+AI» has been developed with the same data (AH+/AH-) but
adding the AI data in the AH- data pool in order to develop a classifier able to specifically neglect AI. For each classifier, 80% of the subjects were
used in the training session, and 20% for testing.
These two classifiers were compared with ROC curves and areas under the curve (AUC) were compared by chi-2 test.
RESULTS
We showed statistically significant difference between the AUC of the two classifiers (p = 0.02). The performance of the classifier «AH + AI» are better
(AUC = 0.8588 [95% CI: 0.7784; 0.9391]) than the classifier «AH» (AUC = 0.683 [95% CI: 0.5409; 0.8252]).
CONCLUSIONS
We achieved the optimization of the performance of a classifier able to detect the BOLD-signal associated with AVHs.
schizophrenia, auditory hallucinations, mental imagery, classifier
39
GARABEDIAN Charles
Veronique HOUFFLIN-DEBARGE (EA4489 - STORME Laurent)
Fetal heart rate variability: Validation of a new index specific to parasympathetic tone in sheep model.
C. Garabedian, C. Champion, E. Servan-Schreiber, E. Aubry, D. Sharma, L. Butruille, J. De Jonckheere, P. Deruelle, L. Storme, V. Houfflin-Debarge.
EA 4489, Perinatal environment and growth, University of Lille, France.
Objective - Visual analysis of the fetal heart rate is an imperfect tool in predicting the occurrence of fetal acidosis. During the labor phase, it is possible
to use second-line tests such as fetal scalp blood sampling (by assaying the pH or lactate) or analysis of the ST segment of the fetal ECG. But those
invasive tests require rupture of the amniotic membranes and their places are still debated. We have developed a new non invasive index with a value
between 0 and 100 (the Fetal Stress Index, FSI) based on the analysis of the Heart rate variability (HRV), which indirectly reflects the status of the
autonomic nervous system (ANS). This experimental study aimed to prove that this index is a specific reflection of the parasympathetic nervous
system.
Study Design - Ten pregnant ewes near term were operated and chronically instrumented: catheter were placed in femoral vessels, a Doppler probe
on the umbilical arterial and four precordial electrodes were inserted in the fetal back. To investigate specific response to parasympathetic nervous
system, four drugs were tested: 2 acting on the ANS (Atropine – parasympatholytic and propranolol – sympatholytic) and 2 others affecting peripheral
vascular reactivity to study the response to baroreflex (Ephedrine, a vasoconstrictor and Sodium Nitroprusside, a vasodilator). Within the same group,
we compared the evolution of measurements between the stability period (T0 to T20) and the post injection period (T0 to T120).
Results – After direct intravenous injection of Atropine, the HR was significantly increased for 40 minutes, reaching a maximum median value at T5
(190 bpm), i.e. a 22.4% increase (p <0.001). No change in mean arterial pressure (MAP) was observed. The FSI decreased during the post injection
period (58 versus 44, p <0.01). Propranolol injection caused a decrease in HR (162 vs 142 bpm, p <0.01) with no effect on MAP. Ephedrine injection
caused an initial decrease in HR and tachycardia associated with an increase in MAP. Sodium Nitroprusside reduced MAP without changing HR. FSI
values were comparable between the two periods for the last 3 molecules.
Conclusion - Our FSI index is specific to the parasympathetic activity. This validation step of the FSI was essential for the further development of this
new tool in pathological situations. We now intend to evaluate FSI changes in experimental hypoxic conditions by repeated occlusions of the cord as
previously described.
sheep model, fetal heart rate, autonomic nervous system, fetal hypoxia
GEORGEL Anne-France
Christophe CARNOY (UMR8204-U1019 éq. 12 - TROTTEIN Francois)
Anti-viral roles of flagellin, agonist of Toll-like receptor 5 (TLR5), in respiratory infections
Anne-France GEORGEL
CIIL, Inserm U1019, Institut Pasteur de Lille, France
The stimulation of innate immunity could be an alternative therapeutic approach to current anti-infective treatments. Indeed, the stimulation of innate
immune system induces local production of antimicrobial molecules and the recruitment of effector cells involved in controlling infection. The \"toll-like
receptors\" (TLRs) are receptors of innate immunity involved in the recognition of conserved microbe-associated molecular patterns. Of the 10
functional TLR in humans, TLR5 recognizes flagellin, the structural protein of bacterial flagella. TLR5 is expressed at the surface of macrophages,
dendritic cells and epithelial cells. We recently demonstrated in mice that flagellin administration improves the therapeutic index of antibiotics in the
treatment of respiratory infections by Streptococcus pneumoniae. It has also been shown that repeated administration of flagellin protected mice
against rotavirus infection.The aim of the thesis is to analyze the lung anti-viral properties induced by flagellin. We first showed the activation of 22
anti-viral genes involved in anti-influenza response in C57BL/6mice that received flagellin intranasally. Three profiles of expression (early activation at
2h, activation at 4 or 8h or 2 waves of activation) could be defined. The antiviral biological effect of flagellin is currently being tested in prophylaxis and
therapeutic in a mouse model of infection by influenza A virus. If a protective effect of flagellin is observed, a characterization of the molecular and
cellular effector mechanisms will be undertaken.
In parallel, we plan to analyze the anti-infective response induced by TLR agonists on human blood and respiratory cells. The project is currently
under assessment by the Patient Protection Committee.
Infectious diseases, flagellin, Toll-like receptor 5, Influenza A virus
40
GILORMINI Pierre-Andre
Christophe BIOT / Marie-Ange KRZEWINSKI-RECCHI (UMR8576 éq. 01 -
GUERARDEL Yann)
Inside view: shedding light on the sialylation metabolic pathway.
Pierre-André Gilormini (1)
(1) Univ. Lille, UMR CNRS 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
Like proteins, glycans are essential biomolecules found in every living system. However, because glycosylation is a post-translational process,
traditional tools used to track proteins (for example fluorescent fusion proteins) cannot be applied to its study. Recently, the advent of chemical biology
has opened new links between biology and organic chemistry, allowing tremendous advances in glycobiology like the development of metabolic
oligosaccharide engineering (MOE). This method consists in the use of a slightly modified monosaccharide, the chemical reporter which can enter the
cell and hijack the metabolic pathway. Upon incorporation into glycoconjugates, the introduced chemical reporter allows their detection with a probe
using bio-orthogonal chemistry. Methodological developments have been very important in the last decade but except for very few reports, these
studies mainly focused on the glycoconjugates localized at the plasma membrane. Applications of MOE to the study and the visualization of
intracellular processes, have yet to emerge. To address this issue, we applied the principles of MOE and develop a new Sequential Bio-orthogonal
Dual Strategy (SBDS). Two alkyne-tagged analogs of sialic acids metabolites were synthesized. Then, one or the other was introduced into cells. With
the combination of copper-catalyzed azide-alkyne cycloaddition (CuAAC) and fluorescence microscopy, we were finally able to shed light on trafficking
and cellular uptake mechanisms of sialic acids. Our findings bring new evidences that exogenous sialic acids enter the cells via endocytosis whereas
its biosynthetic precursor's entry seems to be achieved in a specific manner which is still to be determined. We also show that our SBDS can be a
powerful tool for the identification and the sub-cellular localization of glycosylation defects. With this work, we aimed to provide a powerful and
versatile strategy. In the future, SBDS could indeed be applied to both fundamental and therapeutic issues, allowing screening experiments for the
diagnosis or the research of treatment for glycosylation pathologies.
Chemical biology, Sialic acid, Chemical reporter strategy, Glycoengineering
GIRAUDET Geraldine
Michel COSSON (UR hors ED (SPI - ULNF) - )
FEMALE PELVIS ANATOMY MODELISATION IN 3 DIMENSIONS AND TEACHING TOOLS
Géraldine Giraudet, Michel Cosson
A perfect knowledge of anatomy is necessary for the practice of medicine, particularly surgery. The anatomy of the female pelvis is complex. The
different structures are deeply located in different spatial planes. There are still differences on the description of the female pelvic anatomy according
to the authors. Teaching is mainly done using anatomy textbooks that fail to account for the orientation of each element in the three planes of space,
essential to a good understanding. Currently, it lacks corpses available to offer students anatomical dissections and cost of the body is also an
important limitation to this teaching. It has been shown that educational videos in 3D are usefull to improve learning this discipline. No female pelvis
anatomical model exists in 3 dimensions. The aim of this work is to propose educational tools in 3D to facilitate the teaching of anatomy. We
conducted a descriptive study among residents in obstetrics and gynecology to assess their knowledge. An anatomy questionnaire was given to them.
No student had the good answer to all the questions which shows the need to develop educational tools. In parallel, we realized 3D educational videos
available on the internet free access. 5 videos were produced: the organs, ligaments, pelvic floor and perineal muscles and pelvic vascularisation. Two
other videos on the pelvic innervation and dissection spaces are being developed. This work is performed in collaboration with the UNF3S and the
University of Lyon 1. We also plan to develop teaching MOOCs. The first will be for the general public and medical students taking the videos already
made. The second will target a population of more specialized gynecologic surgeons and urologists. It will include 3D videos of anatomy. This
educational program will be supplemented by surgical videos, pictures of anatomical dissections (collaboration with Professor Mauroy Lille Catholic
University), digitized video anatomical drawings (collaboration with Professor Bonnet, University of Liège, Belgium). The first videos are in progress.
Digital drawings were filmed. This program will be available in French and English. A teaching model is also being developed in collaboration with Lille
Central School. This model will allow intra uterine device insertion and surgical procedures such as hysteroscopic resection. The various materials
needed for its preparation were tested. A prototype is being developed.
4 oral communications are from this work and one poster.
Two articles are being written, the first on the assessment of anatomical knowledge of medical students, the second on the full 3D anatomical model of
the female pelvis.
anatomy, female pelvis, educational tools
41
HADDAD Juliano
Jean DUBUISSON / tuteur cotutelle : Fouad DABBOUSSI (UMR8204-U1019 éq. 05 -
DUBUISSON Jean)
Functional study of hepatitis C virus glycoprotein E1
Juliano HADDAD
Molecular & Cellular Virology, Center for Infection & Immunity of Lille (CIIL), Inserm U1019, CNRS UMR8204, Université de Lille, Institut Pasteur de
Lille, Bâtiment IBL,1 rue du Professeur Calmette, 59021 Lille.
Being part of the viral particle, HCV envelope glycoproteins E1 and E2 play an essential role in virion morphogenesis as well as in HCV entry into liver
cells. E1 and E2 form a non-covalent complex, until recently, research on HCV envelope glycoproteins has been mainly focused on E2 because it is
the receptor-binding protein, it is also the major target of neutralizing antibodies and it was postulated to be the fusion protein. However, the recent
publications of the structure of E2 do not show the presence of a fusion peptide and its structure does not fit with what one would expect for a fusion
protein, suggesting that E1 alone or in association with E2 might be responsible for the fusion step. Concerning E1, only the crystal structure of the
two-fifth N-terminal region comprising amino acids 192 to 270 has been reported. This partial structure reveals a complex network of covalently linked,
intertwined homodimers. The overall fold of the N-terminal E1 monomer consists of a beta-hairpin (β1 and β2) followed by a segment composed of a
16 amino-acid long alpha-helix (α1) flanking a three-strand antiparallel beta-sheet (β3, β4 and β5).
The functional role of HCV glycoprotein E1 has been investigated by site-directed mutagenesis of conserved amino acids of beta-1 to beta-5 and
alpha1 in the context of an infectious clone.
First, mutations introduced into the E1 glycoprotein were confirmed to have no effect on virus replication. Thereafter, the effect of mutations on
infectivity and assembly was characterized. Accordingly, the regions α1, β4 and β5 were exhibited key residues for virus infectivity. In these regions,
several mutations induce viral attenuation or inactivation. Most non-infectious mutants have an assembly defect with the exception of mutant D263,
that does not seem to be affected at this level. Among the attenuated mutants (I212A, T213A, R231A and I262A), three present an identical assembly
to wild type virus and will be further characterized by studying binding and internalization of the particles into the cell. Study of the recognition of
envelope proteins of attenuated and non-infectious mutants either with conformational antibodies or the soluble form of a viral receptor hCD81-LEL
reveals an impact of the mutations on the conformation of E1 and E2. Finally, our results allowed the identification of specific regions of the E1
glycoprotein that play a role in the assembly and entry steps of the virus.
Hepatitis C Virus, glycoprotein, envelope proteins, viral entry, viral assembly
42
HANSSENS Sandy
Philippe DERUELLE (EA4489 - STORME Laurent)
Pas de titre communiqué
Pas d’auteurs communiqués
Background :
Apelin and its receptor APJ have been implicated in pathologies including cardiovascular disease, diabetes and obesity.
Little is known about the function of the apelinergic system during gestation.
In a previous study, we evaluated in mice this system at the feto-maternal interface in insulin-resistant obese female (HF) mice. Maternal apelinemia
was decreased at term and fetal apelinemia was sixfold higher than maternal level. Ex-vivo, the placenta releases high amount of apelin at E12.5 and
E18.5. In HF pregnant mice at term, apelinemia as well as placental apelin and APJ mRNA levels were increased whereas placental release of apelin
was drastically reduced.
Objectives :
The main objective of this study is to compare apelinemia in fasting normal weight and obese women at the end of pregnancy, between 35 and 41
weeks of gestations (WG).
The secondary objectives are :
- To check if gestational diabetes is not a confounding factor in obesity
- To compare maternal and neonatal apelinemia
- To correlate apelinemia in the cord blood to the child’s weight and birth size and to the weight of the placenta
- To correlate apelinemia to lipidic and glycemic markers
- To compare apelin levels in the colostrum between obese and not obese women.
- To correlate of apelin levels in the colostrum to those in maternal plasma.
Strategy and method:
A prospective research evaluating will be conducted to compare apelinemia in fasting normal weight (BMI = 18.5-25 kg/m2) and obese women (BMI >
30 kg/m2) at the end of pregnancy, between 35 and 41 weeks of gestations (WG).
A third group will be created to check if gestational diabetes is not a confounding factor in obesity (group of obese women with gestational diabetes).
We will try to see if apelinemia is correlated to lipidic (total cholesterol, HDL, LDL, triglycerids, apolipoprotein A and B) and glycemic markers
(glycemia, insulinemia and C-peptide).
Samples will be collected in the cord blood to compare maternal and neonatal apelinemia and to see if neonatal apelinemia is correlated to the child’s
weight and birth size and to the weight of the placenta.
Placenta
Two days after delivery, obese and not obese women will be fasted and plasma and colostrum will be collected. We will compare apelin levels in the
colostrum between these 2 groups and then we will try to see if apelin level is correlated in the colostrum and in maternal plasma.
This work will determine whether apelin may be a new marker or target for the treatment of obesity during pregnancy.
pas de mots clés
43
HAYBRARD Julie
Pascal ODOU / Eric BOULANGER (EA7365 éq. 03 - LEBUFFE Gilles)
Formation of glucose degradation products in glucose infusion fluids: What are the factors to consider?
J. Haybrard1, N Simon1, C Danel1, C Barthélémy1, E Boulanger2, B Décaudin1, P Odou1
1 Univ. Lille, EA 7365 - GRITA - F - 59000 Lille, France
2 Univ. Lille, Inserm, CHU Lille, U995 - LIRIC - F - 59000 Lille, France
Sterile glucose solutions are commonly used in hospital settings for hydration, as diluent for injectable drugs or for peritoneal dialysis. Sterilization of
glucose solutions by heat promotes the formation of a large number of glucose degradation products (GDP). Although the toxicity thresholds are not
yet known, it has been shown that high levels of GDP and Advanced Glycation End products (AGE) have an impact on cellular homeostasis. If some
data are available for peritoneal dialysis solutions, sparse data are published for infusion fluids.
The aims of this work is to quantify both 5-hydroxymethyl-2-furfural (5-HMF) considered as an important indicator of degradation and 2-furaldehyde (2FA), an ultimate glucose degradation product, in marketed glucose solutions from the 5 major suppliers, 8 different glucose concentrations and 3 types
of container.
The glucose solutions were analysed by reversed-phase high-performance liquid chromatography with an UV detection. According to the tested
solution, both GDP were detected in all tested glucose solutions. The amount ranges vary from 2.2 to 37.3 mg and from 117.4 to 326.1 µg for 5-HMF
and 2-FA amounts, respectively for glucose concentrations varying from 2.5 to 70 g/100mL. A good logarithmic relationship between GDP formation
rates and GDP amounts were found [r²=0.974 (p<0.0001) and 0,977 (p<0.0001) for 5-HMF and 2-FA, respectively] with an analysis of covariance
(ANCOVA). The results reveal that the GDP amount, the initial glucose amount, the mean oxygen permeability coefficient, the surface of containers,
the type of containers material and the suppliers are significant covariates.
This study shows that all the marketed infusion fluids tested contain GDP. Moreover, the GDP formation rates differ over time. Further studies are
necessary to assess the risk of exposing patients to GDP during long parenteral therapie
glucose degradation product, 5-HMF, 2-FA, glucose solutions
HENRY Heloise
Thierry DINE (EA7365 éq. 01 - DECAUDIN Bertrand)
ASSESSMENT OF HUMAN INSULIN STABILITY AFTER ADDITION IN A TOTAL PARENTERAL NUTRITION ADMIXTURE
Héloïse Henry, Damien Lannoy, Nicolas Simon, Christine Barthélémy, Bertrand Décaudin, Thierry Dine, Pascal Odou
Univ. Lille, EA 7365 - GRITA - Groupe de Recherche sur les formes Injectables et les Technologies Associées, F-59000 Lille, France
Background: Adding insulin directly into infusion bags seems to be a useful method for controlling hyperglycemia in patients under total parenteral
nutrition (TPN). Its efficacy is assessed by glycemic monitoring but few data are available on insulin stability in this situation. A study of insulin stability
after addition into a TPN admixture has been carried out.
Methods: An immunoelectrochemiluminometric assay (IECMA) previously validated to quantify human recombinant insulin after its addition in
industrial TPN admixture was used to perform this study. OlimelTM N7E (1.5 L bags, Baxter, Deerfield, USA) was the mixture used as TPN. A bag of
OlimelTM N7E is composed of 3 separated compartments containing respectively dextrose solution, amino acid solution with electrolytes and lipid
emulsion. Insulin stability was assessed during 24 hours in TPN first, then in each compartment of the TPN bag and finally in the binary (i.e. dextrose
with amino acids) admixture. Whatever the tested medium of work was enriched with vitamins and trace elements. Insulin stability (ratio insulin
concentration (t)/ insulin initial concentration expressed in percent) was compared between all mediums of work using a non-parametric Kruskal-Wallis
test (p-value 0.05 and Bonferroni correction …).
Results: A rapid and important decrease of insulin content (IC) was observed in TPN (26.7 ± 3.3%), lipidic (44.0 ± 1.8%) and binary (30.1 ± 1.5%)
admixtures during the 24 hours duration of the study. IC felt below the 90% threshold (chosen to define drug stability) after only 2.5 hours. No
significant difference was noticed between both TPN and binary mediums, but the decrease of insulin content was more important in the lipidic
medium. Nevertheless, IC remained stable with no significant difference in dextrose (101.0 ± 4.5%) and amino acids (98.4 ± 4.2%) admixtures.
Conclusions: Human insulin stability was assessed only in either dextrose or amino acids mediums (both enriched with vitamins and trace elements).
This parenteral drug seems to be unstable into TPN, lipidic and binary admixtures, but the phenomenon responsible for its decrease of content has to
be identified.
Insulin Regular Human, Parenteral nutrition solutions, Drug stability , Immunoassay
44
HERMANT Paul
Rebecca DEPREZ-POULAIN (INSERM U1177 - DEPREZ Benoit)
IDE HIT-TO-LEAD PROCESS SUPPORTED BY PHYSICO-CHEMICAL AND PHARMACOKINETIC APPROACHES
P. Hermant, Pr R. Deprez-Poulain.
Biostructures and Drug Discovery unit; Lille Pasteur Institute, University of Lille 2 and INSERM U1177,
3 rue du Pr Laguesse, F-59000 LILLE, FRANCE.
Insulysin or Insulin Degrading Enzyme (IDE) is a Zinc metalloprotease involved in the clearance of numerous physiological peptides. It hydrolyses in
particular beta-amyloid peptide and insulin, respectively implicated in Alzheimer's disease and Diabetes. Consistent with the fact that IDE degrades
insulin in vitro, mutations leading to functional loss of IDE in mice result in high levels of insulin and development of glucose intolerance. Moreover
genetic studies have shown that polymorphism on the IDE region of chromosome are associated with Type-2 Diabetes and Alzheimer disease. The Xray structure of human IDE by Tang et al has given molecular insight for the understanding of substrate recognition. IDE exhibits a two-domain
organization that undergoes an open-close switch. Less is known about the control of the conformational switch between the closed and open states
of IDE upon the regulation of the catalytic rate.
An original chemical series was discovered by orthogonal in situ-click chemistry and was delightedly suitable for in vivo studies. IDE is involved both in
the clearance of insulin and pathway(s) that modulate short-term glucose homeostasis. It also suggests that the glucose intolerance observed in Ide -/mice is not solely due to lifelong elevated insulinemia. Interestingly, X-ray co-crystallography shows that it binds to the catalytic site and locks the
enzyme in a closed conformation (PDB 4NXO). To further characterize its binding to IDE, we studied its interaction in 19F-ligand based NMR and
compared it with the binding of a very close analogue. In addition, the assessment of the in vivo activity of this inhibitor required the characterization of
its physical properties including its solubility in various formulation media. Finally the design, the synthesis and the biological-evaluation is ongoing
aimed to achieve the Hit-to-Lead process.
NN. Nalivaeva et al. Are amyloid-degrading enzymes viable therapeutic targets in Alzheimer's disease?. J Neurochem, 2012, vol.120, 167-185.
W. Farris et al. Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular
domain in vivo. P Natl Acad Sci USA, 2003, vol. 100, 4162-4167.
Y. Shen et al. Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature, 2006, vol. 443, 870-874.
Correspondence: [email protected]
Medicinal Chemistry, Diabetes, Metalloprotease, Pharmacokinetic
HOGUET Vanessa
Julie CHARTON (INSERM U1177 - DEPREZ Benoit)
Structure-Properties Relationships of PEGylated Small Molecules Agonists of TGR5.
Vanessa HOGUET (1) et al.
(1) Univ. Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for living Systems, F-59000 Lille, France
TGR5 is a G protein coupled receptor responsive to bile acids identified in 2002. It is mostly expressed in the liver, the gallbladder, the spleen, the
placenta, the brown adipose tissue, and in the intestine especially at the membrane of enteroendocrine L cells (a). In those cells, TGR5 activation
triggers the secretion of the incretin glucagon-like peptide-1 (GLP-1) (b) known to have a good potential in the treatment of type 2 diabetes. Therefore,
our strategy was to develop topical intestinal TGR5 agonists that selectively target the enteroendocrine L cells with poor systemic exposure in order to
avoid side effects reported in the literature for systemic TGR5 agonists, as itching or cardiovascular issues (c).
Our agonists are designed as chimeric compounds with a pharmacophore part linked to a kinetophore part in a «mute» position which is not essential
for the target interaction. The kinetophore is a chemical moiety which can impact physicochemical and pharmacokinetic properties of molecules such
as their transport or their permeation (d).
Polyethylene glycol coupled to therapeutic agents has been described as an interesting strategy to improve the therapeutic and pharmacokinetic
profile of drugs. In this context, we are interested in polyethylene glycol moiety as kinetophore part. Here, we will describe the design and biological
evaluation of these potential topical TGR5 agonists. Extensive in vitro and in vivo pharmacokinetic studies of such atypical pegylated small molecules
will be reported.
(a) Kawamata et al., J. Biol. Chem., 2003, 278, 9435-9440.
(b) Katsuma et al., Biochem. Biophys. Res. Commun. 2005, 329, 386-390.
(c) Alemi et al., J. Clin. Invest., 2013, 123, 1513-1530. Fryer et al., J. Pharmacol. Exp. Ther., 2014, 348, 421-431.
(d) Kramer et al., Curr. Med. Chem., 2006, 13, 997-1016.
Structure-Properties Relationships, PEGylated Small Moecules, Agonists of TGR5
45
HOLLIN Thomas
Jamal KHALIFE (UMR8204-U1019 éq. 02 - KHALIFE Jamal)
The Ser/Thr Protein Phosphatase type 1 in Plasmodium falciparum : Analysis of the interactome and characterization of a
candidate regulator : GEXP15
Thomas Hollin†, Caroline De Witte†, Astrid Lenne†, Christine Pierrot and Jamal Khalife
Centre d’infection et d’immunité de Lille, équipe 2, 1 rue du Pr. Calmette, 59019 Lille
Malaria continues to be a major health problem and a leading cause of child mortality of the inter-tropical regions despite extensive research efforts.
The present situation is also increasingly complicated by the emergence of parasite resistance to multiple drugs much more rapidly than the
development of novel anti-malarials. A major obstacle in devising new control tools is our limited knowledge of basic parasite biology and the paucity
of identified potential intervention targets. During the last decade, several studies strongly indicated that protein phosphorylation and
dephosphorylation processes, governed by kinases and phosphatases respectively, play a central and essential role in Plasmodium cell cycle and
developmental regulation. In Plasmodium falciparum, the most deadly apicomplexan parasite, efforts have begun for an examination of the biological
roles of protein kinases and phosphatases and their potential as drug targets.
Protein Phosphatase 1 (PP1) is an enzyme essential to cell viability in the malaria parasite Plasmodium falciparum. The activity of PP1 is regulated by
the binding of regulatory subunits, of which there are up to 200 in humans, but only 3 have been so far reported for the parasite.
To better understand the P. falciparum PP1 (PfPP1) regulatory network, we use three strategies to characterize the PfPP1 interactome. Co-affinity
purification followed by MS analysis identified 6 PfPP1 interacting proteins of which 3 contained the RVxF consensus binding, 2 with a Fxx[RK]x[RK]
motif, also shown to be a PP1 binding motif and one with both binding motifs. The Yeast Two-Hybrid screens identified 134 proteins of which 30
present the RVxF binding motif and 20 have the Fxx[RK]x[RK] binding motif. The in silico screen of the P. falciparum predicted proteome using a
consensus RVxF motif as template revealed the presence of 55 potential PfPP1 interacting proteins. As further demonstration, 35 candidate proteins
were validated in an ELISA-based assay.
The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1. Their analysis indicates a high
diversity of biological functions for PP1 in Plasmodium. Based on the present data and on an earlier study of the Pf interactome, a potential implication
of Pips in protein folding/proteolysis, transcription and pathogenicity networks can be built.
Among these candidates, my current work is focused in one partner, named GEXP15, which has been confirmed as true interactor of PfPP1 by
different approaches. In addition, it's expressed in gametocyte stage, responsible for the transmission of the parasite in the mosquito. These results as
well as functional studies will be presented.
Parasitology, Plasmodium, PP1, RVxF motif
JEOUAL Abdallah
Malika BALDUYCK (EA7364 - MANOUVRIER-HANU Sylvie)
New technology to develop a potential treatment for two patients with carnitine-palmitoyl-transferase II deficiency
Jeoual1,2, C. Moreau1, G. Briand2,, M. Balduyck2, K. Mention2, B. Deprez1, D. Dobbelaere2, S. Manouvrier2, T. Beghyn1
1 APTEEUS, Faculté de Pharmacie, Lille, France
2 EA 7364 Maladies Rares du Développement Embryonnaire et du Métabolisme (RADEME),
Inherited metabolic diseases are a family of rare diseases caused by a single mutation in a gene coding for an enzyme or a transporter. At a cellular
scale, the mutation causes a metabolic disorder with an accumulation or a lack of essential metabolites. This defect can be highlighted by mass
spectrometry, enough specific and sensitive to quantify that particular cellular metabolite. Based on that technology, APTEEUS developed a drug
screening platform that enables the testing of thousands marketed drugs. The screening is performed directly on the patient's primary cells bearing the
causative defect of the disease and coming from a skin biopsy. Thanks to miniaturization and automation of the process, the screening is carried out in
a couple of days. The analysis step is done by LC-MS/MS, focusing on one or several biomarkers. This ex vivo process is followed by the identification
of drugs able to correct the cellular defect and to potentially treat the patient.
We ran the technology on two patients suffering from carnitine-palmitoyl-transferase II deficiency (CPT2). That protein is one of the enzyme
responsible of the entry of fatty acids in the mitochondria. Its deficiency leads to deficiency in the catabolism of long chain fatty acids (palmitic and
stearic principally) and their accumulation. All cells from the organism are affected, particularly the one requiring lot of energy, like myocytes and
mainly when fasted. After a first assay consisting in testing 1500 different drugs for their ability to decrease the level of long-chain fatty acids, we
analyzed the impact of positive drugs on the whole mitochondrial beta-oxidation process by measuring all acylcarnitines after treatment of the patient
cells. We confronted in vitro potency and efficacy of the drug candidates to their safety and pharmacokinetics profiles in order to investigate their
potential to be used in the clinic.
Inherited Metabolic Disease, LC-MS/MS, Drug Repositioning, Carnitine-palmitoyl-transferase II( CPT2)
46
JOURDAIN Anne Sophie
Sylvie MANOUVRIER-HANU / Fabienne ESCANDE-NARDUCCI (EA7364 -
MANOUVRIER-HANU Sylvie)
molecular determinism of limb development : contribution of new high throughput technologies of genome studies
Anne-Sophie JOURDAIN 2,3, Florence PETIT1,2,3, Clémence VANLERBERGHE1,2, Morgane STICHELBOUT, Fabienne ESCANDE2,3, Sylvie
MANOUVRIER1,2,3
1 Department of Clinical genetics, JdF, CHRU Lille
2 EA7364 RADEME
3 Department of Molecular genetics, CBP, CHR
Although many progresses have been realized on the comprehension of limb development, the genetical determinism of its different stages is not
clearly known. The great advances in sequencing and genotyping, with, in particular, the emergence of Next Generation Sequencing (NGS) will surely
help us to find new genes and understand the mechanisms involved in limb formation. In parallel, the involvement of distant intragenic control
elements is now well known in the process of limb development and search by CGH array of copy number variation (CNV) concerning enhancers /
silencers is an important issue.
Two hundred fourteen patients of our cohort, whose first intent molecular diagnostic was negative, have been selected. All have been sequenced with
a large Haloplex (Agilent) panel of 126 genes and regulatory elements involved in human and mouse limb development on the Miseq (Illumina).
Bioinformatics analyses have been realized with a home-made software and via the BROAD institute. Filters have been used to select interesting SNP
linked to the phenotype of the patient. Each variation has been verified by Sanger sequencing. In parallel, 218 patients affected by Split hand foot
malformation (SHFM) has been selected for analysis on a custom CGH-array 400 kb enriched with ectrodactyly loci described in human and mouse.
To date, 30 SNP have been identified in 29 patients by sequencing: 6 clearly deleterious, 17 probably damaging and 6 in genes involved in limb
development but not described in human limb pathology yet. Further investigations are necessary to conclude to their implication and functional
experiments are in progress. Thanks to the results, we hope to find new mechanisms involved in limb development.
limb development, next generation sequencing, limb malformation
KARAM Oliver
Stephane LETEURTRE (EA2694 - DUHAMEL Alain)
Indications and effects of plasma transfusions in critically ill children
Oliver Karam (1), Pierre Demaret (1), Alison Shefler, Stéphane Leteurtre (1), Philip C. Spinella, Simon J. Stanworth, Marisa Tucci.
1: EA 2694, Public Health: Epidemiology and Quality of Care, University of Lille-Nord-de-France, Lille, France
Rationale: Plasma transfusions are frequently prescribed for critically ill children, although their indications lack a strong evidence base. Plasma
transfusions are largely driven by physicianconceptions of need, and these are poorly documented in pediatric intensive care patients.
Objectives: To identify patient characteristics and to characterize indications leading to plasma transfusions in critically ill children, and to assess the
effect of plasma transfusions on coagulation tests.
Methods: Point-prevalence study in 101 pediatric intensive care units in 21 countries, on 6 predefined weeks. All critically ill children admitted to a
participating unit were included if they received at least one plasma transfusion.
Measurements and Main Results: During the 6 study weeks, 13,192 children were eligible. Among these, 443 (3.4%) received at least one plasma
transfusion and were included. The primary indications for plasma transfusion were critical bleeding in 22.3%, minor bleeding in 21.2%, planned
surgery or procedure in 11.7%, and high risk of postoperative bleeding in 10.6%. No bleeding or planned procedures were reported in 34.1%. Before
plasma transfusion, the median international normalized ratio (INR) and activated partial thromboplastin time (aPTT) values were 1.5 and 48,
respectively. After plasma transfusion, the median INR and aPTT changes were 20.2 and 25, respectively. Plasma transfusion significantly improved
INR only in patients with a baseline INR greater than 2.5.
Conclusions: One-third of transfused patients were not bleeding and had no planned procedure. In addition, in most patients, coagulation tests are not
sensitive to increases in coagulation factors resulting from plasma transfusion. Studies assessing appropriate plasma transfusion strategies are
urgently needed.
plasma transfusion, blood coagulation tests, critical illness, child
47
LAMARTINIERE Yordenca
Laurence TILLOY-FENART (EA2465 - GOSSELET Fabien)
Role of ABCA7 at the blood brain barrier level: implications for Alzheimer’s disease
Y. Lamartinière, P. Candela, M.C Boucau, L. Dehouck, C. Coisne, L. Fenart and F. Gosselet.
Laboratoire de la Barrière Hémato-Encéphalique, E.A.2465, Université d’Artois, Lens, France
Alzheimer’s disease (AD) is a neurodegenerative disorder in which one of the major pathological hallmarks is the neurotoxic accumulation of beta
amyloid peptides (Aβ) in the brain. In healthy brains, Aβ are generally eliminated by degradation processes but are also cleared from the brain through
the blood-brain barrier (BBB). BBB is a physical and metabolic barrier located at the brain microvessel level, which isolates the brain from peripheral
circulation and regulates the exchange of molecules between blood and brain. BBB alterations are risk factors for AD. Indeed, AD is multifactorial
disease associated with several risk factors as some genes linked with cholesterol metabolism and transport. Recently, genome wide association
studies identified another genetic risk factor for AD: ABCA7, a member of the adenosine triphosphate binding cassette (ABC) transporters superfamily.
This transporter is expressed in BBB cells, but its role in AD remains unknown.
In order to decipher ABCA7 functions, in AD, at the brain vasculature level, we applied small RNA interference technique to a murine in vitro BBB
model. This model consists in culturing brain microvessel endothelial cells on an insert and reproduces in vivo properties of the BBB.
Our preliminary results show that the decrease of ABCA7 gene expression was correlated with a decrease of ABCA1 and ApoE gene expression.
These observations were in agreement with our in vivo data carried out from Abca7 -/- mice. Moreover, cellular cholesterol efflux is modified when
ABCA7 expression is decreased in cells compared to control.
Thus, ABCA7 seems to influence cholesterol metabolism and lipid exchange at the BBB. Further studies evaluating the functions of ABCA7 in Aβ
metabolism and transport at the BBB are in progress. Therefore, ABCA7 represents promising therapeutic target for AD.
Blood brain barrier, Alzheimer disease, In vitro model
LAMBERT Melanie
Marie-Helene DAVID-CORDONNIER (INSERM U1172 éq. 04 - QUESNEL Bruno)
Leukemogenic effects of HOXA9 transcription factor in Acute Myeloid Leukemia.
Mélanie LAMBERT, Samy JAMBON, Mohamed Amine BOUHLEL, Sabine DEPAUW & Marie-Hélène DAVID-CORDONNIER
Since more than 25 years, the treatment of acute myeloid leukemia (AML) is still based on conventional therapies that present major toxicities due to
poor selectivity and is associated with frequent relapse and subsequent chemoresistance. Besides an increasing number of clinical trials, no targeted
therapies are clinically used. Transcription factors represent ~20% of identified oncogenic targets. Our group is interested in targeting one of them:
HOXA9 which is overexpressed in 70% of Acute Myeloid Leukemia. The aim of my PhD is to identify its leukemogenic effects and to validate its
functional inhibition.
Methods:
For this purpose, we compared the inhibitory effects of the invalidation of HOXA9 expression using shRNAs lentivirally transduced in leukemia cell
models, at both the molecular, cellular and animal levels.
Results:
At the molecular level, comparison of protein knockdown (shRNA) highlights similar modifications in the expression of well-known HOXA9 target
genes. Transcriptome analyses brought out key answers about leukemogenic processes implemented by HOXA9, such as differentiation or cell
proliferation processes. Furthermore, the cellular consequences of HOXA9 inhibition were analyzed using flow cytometry and clonogenic assay for cell
viability, proliferation and/or differentiation.
Finally, in vivo evaluation of the leukemic activity of HOXA9 was addressed by using shRNA-induced invalidation of HOXA9- expression expression
was in human leukemia cell models intra-tibialy injected in immune-deficient NSG mice. Results evidenced a marked decrease of the splenomegaly
that is associated with the leukemia and/or of the number of HOXA9-invalidated leukemic cells in mice. Such anti-leukemic effect needs now to be
confirmed using human blasts isolated from patients and transplanted in NSG mice.
Conclusions:
Our results identified HOXA9 as a good therapeutic target in AML inducing cell differentiation in HOXA9-controled AML after inhibition and propose a
new model of innovative differentiating therapeutic approach in AML.
Transcription Factor, Acute Myeloid Leukemia
48
LAMBLIN Gery
Denis VINATIER (INSERM U1192 - SALZET Michel)
Biomechanical model of pelvic organ prolapse and simulation of surgical correction
Géry Lamblin1,2, Olivier Mayeur2, Mathias Brieu2,Michel Cosson1,2
1Faculty of Medecine at the University of Lille, Henri Warembourg, France
2LML - Laboratoire de Mécanique de Lille, FRE3723, France
Background: Female genital prolapse or \"organ descent\" comes from a deficiency of the pelvic organs' natural support. Pelvic floor disorders affect
one on three women from all ages and more than 60% of women over 60 years old. About 11% of women will have surgery for the treatment of
prolapse with different surgical repair techniques. Cystocele or \" bladder descent \" is a bladder hernia in the anterior vaginal wall. The simulation
could provide a better understanding of prolapse surgical repair mechanisms.
Objective: Design a 3D digital model of a cystocele in order to identify deficient structures and improve their surgical correction. The objective is to
develop a generic and personalized 3D digital model of the female pelvic to perform simulations of different surgical corrections.
Protocol: The first step consisted in the selection of 6 patients with symptomatic externalized cystocele at rest and effort (stage 3 and stage 4 with
Baden-Walker classification). We performed for each of the 6 patients a dynamic MRI using the same protocol.
The second step consisted in the geometric analysis of organs and structure suspensions (19 anatomical structures). We have identified the main
anatomical structure suspensions underlying cystocele then achieved a specific contouring of all these structures with AvizoTM software [1,2]. We
measured all anatomical structures on MRI according to the International Classification POP-Q (ICS).
The third step was to design a 3D digital model of the global support system (muscles and ligaments). We have established a surface reconstruction
of the bladder under CatiaTM followed by a meshing step. We developed a patient-specific model and a generic model of all cystoceles.
The fourth step was to define the different surgical techniques we could simulate. 6 surgical techniques have been defined with laparoscopic sacral
mesh implant and vaginally without mesh
The fifth step was to develop and valid a numeric model of biofidelic prolapse. Our model was implemented from biomechanical properties available
for each anatomical structure involved in a cystocele and then imported into the software AbaqusTM to perform simulations by Finite Element method
(calculation of displacement fields, deformation and strain).
The sixth step was a simulation of the various surgical repair techniques from ABAQUSTM digital software.
Results: The different steps of the protocol allowed to perform surgical repair of cystocele simulations from a complete and coherent generic 3D model
of the female pelvic. We have performed simulations for all these surgical corrections with different anatomic struture fixations.
Conclusion: Our 3D biomechanical model of genital prolapse enabled various simulations of surgical correction and better understanding of surgical
repair mechanisms. We can predict future results of surgery for cystocele and adapt proposed surgical technique for each patient with preoperative
simulation.
Cystocele, Finite Elements simulation, Computational modelling
LAMPIN Marie-Emilie
Alain DUHAMEL (EA2694 - DUHAMEL Alain)
Validation of three Pediatric Early Warning Scores in pediatric intermediate care unit.
Auteur: Lampin ME, Univ. Lille, CHU Lille, EA 2694 - Santé Publique : épidémiologie et qualité des soins, Service de réanimation pédiatrique, F-59000
Lille, France.
OBJECTIVE: Pediatric Early warning systems were created to quantify severity of illness across in hospitalized children in pediatric ward or
emergency unit. Pediatric intermediate care units are alternative structures for moderate ill children. The aim of this study was to assess the validity of
three pediatric early warning scores in pediatric intermediate care units.
METHODS: We did a prospective, observational, multicenter cohort study in seven French regional hospitals (09/2012-01/2014). All consecutive
children <18 years were included. Three scores (bedPEWS, PAWS, and PEWS) were calculated each 8 hours and more if deterioration. Binary
outcome criteria were «medical call», «medical call with complementary exams», «medical call with therapeutic administration» and «admission to
Pediatric intensive care Unit (PICU)». We used areas under ROC curve (AUC) to estimate discrimination.
RESULTS: 2868 children are included for a total of 19071 observations for calculating the three scores. Median scores for the three scores were
respectively 2, 2 and 1. Medical call was observed in 11%, medical call with complementary exams in 5%, medical call with therapeutic administration
in 5% and admission to PICU in 0,45%. AUCs calculated for the three scores for predicting only medical call, and associated with either
complementary exams or either therapeutic administration were ranged from 0,84 to 0,88. AUCs for predicting PICU admission were ranged from 0,80
to 0,94.
CONCLUSION: The three scores, developed from pediatric ward and emergency department, can be used to detect deterioration requiring a medical
intervention or PICU admission (in hospitalized children) in pediatric intermediate care unit.
early wearning score, Child, intermediate unit care, gravity
49
LASSAILLY Guillaume
Sebastien DHARANCY / Laurent DUBUQUOY (INSERM U995 éq. 01 - DUBUQUOY
Laurent)
Role of NOD1 pathway in the hepatocyte during liver ischemia reperfusion injury
Guillaume Lassailly1,2, Florent Artru1,2 ,Alexandre Louvet1,2, Emilie Gantier1, François Maggiotto1, Sebastien Dharancy1,2, Philippe Mathurin1,2,
Laurent Dubuquoy1
1: INSERM U995, Université Lille 2, France
2: Service MAD, CHRU de Lille
NOD1 a pattern antigen molecular pathogens (PAMPs) modulates liver ischemia/reperfusion (IR) lesions by controlling PMN function, migration
through the regulation of CD11b a major PMN receptor. The CD11b ligands, ICAM-1 and VCAM-1, are implicated in IR. The role of NOD1 on ICAM
and VCAM remains to be determined particularly in hepatocyte (hep). Aims: To determine 1) if NOD1 is able to regulate the expression of ICAM-1 and
VCAM-1 in hep. 2) if NOD1 could modulate the expression of ICAM and VCAM during IR 3) if NOD1 could influence interaction between PMN and hep
and lead to PMN-induced hep lysis. Methods: The modulation of ICAM and VCAM expression in the liver of NOD1-/- or WT mice was studied in
western Blot (WT) after the treatment with NOD1 agonist (FK 565 or Ie-DAP) or vehicule in standard and IR condition. In IR conditions, mice
underwent 75 min liver ischemia followed by 16h reperfusion after the treatment. To study the specific hep expression of ICAM and VCAM, hep were
isolated hep from NOD1+/+ and WT mice that were stimulated in vitro with NOD1 agonist, TNF+IFN or TNF+IFN+ NOD1 agonist. Expression of ICAM1 and VCAM-1 was quantified in hep using RT-PCR and Western-blot (WB). A ex vivo model of isolation of hep after IR was developped to study the
hep modulation of ICMA and VCAM after IR. The NOD1 function in hep was studied with an ex vivo model of cell interaction (co-culture) between
PMN and isolated hep form WT or NOD1-/- mice treated with NOD1 agonist or vehicule. Hep necrosis was observed directly by electronic microscopy
and measured indirectly using LDH release.
Results: In vivo, NOD1-/- mice are protected from IR injury with 60% less liver necrosis. Conversely during IR, FK in mouse increased the ICAM (8.9
fold) and VCAM (3.3 fold) protein expression in the liver. In vitro, NOD1 agonist treatment of hep isolated from WT mice increases ICAM and VCAM
mRNA levels compared to untreated cells (ICAM 2.1 fold-VCAM 2.6 fold p<0.01). As expected, TNF+IFN induced ICAM (2.0 fold) and VCAM (2.7 fold)
mRNA. NOD1 agonist and TNF+IFN had additive effects. In NOD1-/- hep, NOD1 agonist lost its ability to increase ICAM and VCAM expression
contrary to TNF+IFN. The induction of ICAM and VCAM by NOD1 agonist was confirmed signiicantly in all experimental conditions at a protein level
even in the ex vivo model after IR treatment. Co-culture of hep and PMN showed that hep pre-stimulation with NOD1 ag increased hep lysis with an
increase of LDH up to 20%. PMN were not able to induce necrosis in NOD1-/- hep supporting that NOD1 also acts at a hep level. In electronic
microscopy, the direct interaction was observed.
Conclusion:Activation of NOD1 increases hep expression of ICAM-1 and VCAM-1. NOD1 has a major role on hep and increases the interaction with
PMN that leads to cell lysis. Taken together, the results support that NOD1 may be a target for IR and that hep should also be targeted by future
treatment modulating IR injury.
Ischemie reperfusion, NOD1, Foie, immunite innée
50
LE RHUN Emilie
Michel SALZET / Nicolas REYNS (INSERM U1192 - SALZET Michel)
Non fourni
Emilie Le Rhun1, Marie Duhamel2, Maxence Wisztoski2, Fahed Zairi3, Fabienne Escande4, Nicolas Reyns6, Firas Kobeissy7, Claude Alain
Maurage5, Michel Salzet2, Isabelle Fournier2
(1) Lille University, Inserm U-1192, Laboratoire de Protéomique, Réponse
Background:
An integrated diagnosis using molecular informations is recommended in the updated World Health Organization (WHO) classification. Our aim was to
explore the role of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) in order to classify Grade III glioma by
integration of MALDI-MSI data.
Methods
After frozen tissue sections, tissues were subjected to in-situ trypsin digestion and MALDI matrix deposition with an automatic micro-sprayer. Images
were acquired with 70 µm spatial resolution. After acquisition, the imaging datasets including all the biopsy sections were spatially segmented all
together by clustering all spectra by their molecular profiles similarity. The Region of Interest (ROI) resulting from the clustering were used for protein
large identification by performing microextraction followed by Shot-Gun analyses.
Results
More than 2500 proteins were identified from all the samples and submitted to statistical analysis. Samples were grouped according to the ROI of
imaging datasets. Global segmentations generated by processing imaging data of all the tissues together reveals 3 main molecular groups. Systemic
biology performed on each group revealed sub-networks related to neoplasia for group 1, glioma with inflammation for group 2 and neurogenesis for
group 3.
Conclusions
A low concordance between the histological annotations and molecular data was found. Integration of the proteomic data could add new informations
on the prognosis and the subtype of tumors. This analysis provided new insights into Grade III glioma organization. Data obtained from MALDI MSI
analysis and tissue microproteomics provide specific information and could allow a more accurate classification of the biopsies.
non fourni
LECOUTRE Simon
Christophe BRETON (EA4489 - STORME Laurent)
Effects of maternal obesity in offspring's adipose tissue
Simon Lecoutre, Frederik Oger, Christine Laborie, Delphine Eberlé, Céline Guinez, Jean Lesage, Didier Vieau and Christophe Breton
As stated by the Developmental Origin of Health and Disease (DOHaD) concept, alterations of nutrient supply in the fetus or neonate result in longterm programming of individual body weight set-point. In particular, maternal obesity, excessive nutrition and accelerated growth in neonates have
been shown to sensitize offspring to obesity. Our group is interested in the epigenetic mechanisms that make offspring from obese mothers are
predisposed to adiposity. The aim of my PhD is to identify epigenetic changes induced by maternal obesogenic environment in white adipose tissue of
offspring during the development period and which persist into adulthood.
We have developed a rat model of maternal obesity using a high-fat (HF) diet (containing 60% lipids) before and during gestation and lactation. At
weaning all the offsprings are placed on a standard diet. At different stages of lactation (PND12 and PND21) and in adulthood (9 months), we will test
the hypothesis that changes in gene expression patterns within WAT correlate with appearance or disappearance of epigenetic marks using
complementary approaches : global analysis by PCR array of the expression of key genes encoding enzymes known to modify genomic DNA and
histones to regulate chromatin accessibility ; analysis of methylation by pyrosequencing and modified histones by ChIP-assays on promoter
sequences of key genes involved in adipogenesis and lipogenesis.
At birth, newborns from obese dams (called HF) were normotrophs. However, HF neonates exhibited a rapid weight gain during lactation, a key period
of adipose tissue development in rodents. Of the 12th day of lactation, the HF descendants exhibit a strong increase in visceral adiposity and
subcutaneous which greatly increases at weaning. In HF offspring, increased body weight at weaning persists until 3 months of age. Despite no
difference in body weight and energy intake, HF adult male (9 months of age) offspring were predisposed to fat accumulation showing increased
visceral but not subcutaneous depots weights.
At the cellular level, during the lactation period expansion of adipose tissue subcutaneous and visceral manifested by an increase in the size and
number of adipocytes. However, only visceral adipose tissue depot exhibited this phenotype in HF adult male offspring. These phenotypic changes are
associated with a reduction of the expression of adipogenic genes (i.e., PPARg) and by increasing the expression of lipogenic genes (i.e., DGAT2)
associated to different epigenetic changes (modification of the DNA methylation and post-translational modifications of histones) that occur during the
lactation period and are still observable in HF descendants in adulthood.
In summary, maternal obesity epigenetically programs the expansion capacity of offspring adipose tissue, providing an explanation for the adipose
metabolic dysfunction of male offspring in the setting of maternal obesity.
Adipose tissue expandability, Epigenetics, DOHaD, Maternal obesity
51
LESAGE Kevin
Mathieu GISSOT (UMR8204-U1019 éq. 01 - TOMAVO Stanislas)
Characterization of factors controlling Toxoplasma gondii virulence
Lesage Kevin and Gissot Mathieu
Molecular & Cellular Biology of Toxoplasma gondii, Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Centre
d’Infection et d’Immunité de Lille, F-59000 Lille, France
Toxoplasma gondii is a unicellular eukaryote belonging to the Apicomplexa phylum. It is an obligate intracellular parasite of critical importance to
primarily infected pregnant women and immunosuppressed patient with AIDS/HIV. Its life cycle is complex, with multiple proliferation and
differentiation steps, of which tachyzoite proliferation is the most relevant to pathogenesis in humans. Tachyzoites express invasion and virulence
factors that are crucial for their survival and the manipulation of host cell functions. Expression of these virulence factors is tightly regulated permitting
their correct targeting in specific organelles named rhoptry and microneme. However, little is known about the factors controlling the expression of
genes encoding the virulence factors.
Earlier studies of the team revealed the ability of the plant-like nuclear factor (TgAP2XI-5) to bind transcriptionally active promoters essential for
parasite virulence. To better understand its function, we performed mass spectrometry to identify interacting partners. Among them, we identified a cell
cycle regulated AP2 transcription factor, TgAP2X-5. Using an inducible knock-down strategy, we found that our factor is involved in both invasion and
growth of parasite. Interrogating by western blot the level of expression of rhoptry and microneme proteins, we revealed that some of these proteins
involved in virulence are less expressed or totally absent in the mutant. In addition, a virulence assay showed that BALB/c mice infected by the mutant
strain survive while all the wild-type infected mice died within 10 days.
Our work suggests the importance of the TgAP2X-5 in regulation of virulence factors. In the future we will try to better understand the molecular
processes at stake by analyzing the mutant parasite transcriptome and the regulation network of this AP2 transcription factor.
Toxoplasma gondii, virulence, transcription factor, gene expression
LETURCQ Maite
Anne-Sophie EDOUART-VERCOUTTER (UMR8576 éq. 08 - LEFEBVRE Tony)
The MCM2-7 helicase complex is glycosylated by O-GlcNAc Transferase. Towards a new role of OGT in the regulation of
DNA replication.
Maïté LETURCQ, Marlène MORTUAIRE, Tony LEFEBVRE, Anne-Sophie VERCOUTTER-EDOUART
Unité de Glycobiologie Structurale et Fonctionnelle, UGSF, univ. Lille, CNRS, UMR 8576, lille, France.
O-GlcNAcylation (O-linked N-acetylglucosaminylation) is a dynamic and reversible post-translational modification regulated by OGT (O-GlcNAc
Transferase) and OGA (O-GlcNAcase). This glycosylation consists in the addition of a single residue of β-D-N-acetylglucosamine (GlcNAc) to the
hydroxyl group of serine and threonine residues of cytosolic, nuclear and mitochondrial proteins and can compete with phosphorylation to regulate the
activity of target-proteins [1]. Several works, including those of our lab, showed that a disruption of the dynamic of O-GlcNAcylation affects mitotic
events and cellular division. In addition, overexpression of OGT and increase of its activity contribute to tumorigenesis by promoting growth and
invasion of cancer cells, both in vitro and in vivo [2]. We previously described for the first time the cell cycle-dependent O-GlcNAcylation of the MiniChromosome Maintenance Proteins MCM2, MCM3, MCM6 and MCM7 which are key proteins involved in the formation of the pre-replicative complex
[3]. The aim of our work is now to understand the role of O-GlcNAcylation and OGT on the formation of the MCM2-7 complex and its recruitment to the
chromatin. By WGA affinity chromatography and click-chemistry approaches, we showed that the O-GlcNAcylated forms of MCM are mainly detected
in the chromatin-bound protein fraction. Co-immunoprecipitation and GST pull-down experiments further showed that OGT preferentially interacts with
some of the MCM proteins. Finally, we are currently investigating the crosstalk between phosphorylation and O-GlcNAcylation of the MCM proteins by
using two-dimensional electrophoresis and western-blotting combined with Click-chemistry strategy. This study will bring new elements to understand
the role of OGT and O-GlcNAc modification in the molecular mechanisms involved in the initiation of DNA synthesis. The question remains whether a
pathological dysregulation of O-GlcNAc status of the MCM2-7 complex could disrupt the control of the initiation of the genome replication and thus
contribute to the uncontrolled proliferation of cancerous cells.
References:
[1] Lefebvre T, Issad T. (2015) 30 Years Old: O-GlcNAc Reaches the Age of Reason - Regulation of Cell Signaling and Metabolism by OGlcNAcylation. Front Endocrinol (Lausanne), 9;6:17
[2] Ma Z, Vosseller K. (2014) Cancer metabolism and elevated O-GlcNAc in oncogenic signaling. J Biol Chem. 289(50):34457-65.
[3] Drougat L, Olivier-Van Stichelen S, Mortuaire M, Foulquier F, Lacoste AS, Michalski JC, Lefebvre T, Vercoutter-Edouart AS. (2012)
Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition. Biochim Biophys
Acta, 1820(12):1839-48.
O-GlcNAcylation, DNA replication, Minichromosome maintenance proteins
52
MACON Guillaume
Bart STAELS (INSERM U1011 éq. 01 - STAELS Bart)
MALONE Samuel Andrew
Paolo GIACOBINI (INSERM U1172 éq. 02 - PREVOT Vincent)
AMH is mutated in patients with congenital hypogonadotropic hypogonadism and it regulates development and function of
GnRH neurons
Malone SA 1, Cassatella D 2, Acierno J 2, Xu C 2, Cimino I 1, Pitteloud N 2, Giacobini P 1
1 Inserm U1172, Development & Plasticity of the Neuroendocrine Brain, Lille, France
2 Service of Endocrinology, Diabetes & Metabolism, CHUV, Lausanne,
Gonadotropin releasing hormone (GnRH) neurons, critical for reproduction, originate in the olfactory placode and enter the brain along vomeronasal
and terminal axons during embryonic development. Alterations either in the development of this system or in the secretion of GnRH are associated
with congenital hypogonadotropic hypogonadism (CHH) in humans, a condition characterized by failure of sexual competence. Kallmann syndrome
(KS) associates congenital hypogonadism due to GnRH deficiency and anosmia, while a normal sense of smell is defined as normosmic CHH
(nCHH). Here, we performed whole-exome sequencing in a cohort of 70 KS and 43 nCHH probands and identified several heterozygous missense
mutations in the Anti-Mullerian Hormone (AMH) gene and in its receptors in nCHH individuals. Mutations in AMH resulted in impaired secretion of
AMH by transfected COS-7 cells or reduced signalling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells, which
strongly suggests that these mutations have a pathogenic effect. We also show that AMH and its receptor (AMHR2) are expressed along the olfactory
fibers and by GnRH neurons during GnRH migratory process. Pathohistological analysis of Amhr2-/- mice revealed defective embryonic migration of
the neuroendocrine GnRH cells to the basal forebrain, leading to a significant reduction in the total number of GnRH neurons in the adult brains of
these animals and reduced fertility. Our findings indicate that AMH signalling insufficiency contributes to the pathogenesis of nCHH and highlight a
novel role for AMH in the correct development and function of GnRH neurons.
Reproduction, Diseases of Sexual Disorder, GnRH, Neurodevelopment
MAMIGONIAN BESSA Luiza
Xavier HANOULLE (UMR8576 éq. 02 - LIPPENS Guy)
Structural and functional study of NS5B, the Hepatitis C virus RNA polymerase, by Nuclear Magnetic Resonance
spectroscopy
L. M. Bessa$*, H. Launay*, M. Dujardin*, F.-X. Cantrelle*, G. Lippens*, R. Schneider* and X. Hanoulle*.
* Univ. Lille, CNRS, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F 59000 Lille, France
The hepatitis C virus (HCV) is a small single stranded RNA virus that causes acute and chronic hepatitis. Its 9.6 kbase genome codes for a single
polyprotein precursor that is subsequently cleaved to give rise to 10 viral proteins, both structural (Core, E1, E2) and non-structural (p7, NS2, NS3,
NS4A, NS4B, NS5A, NS5B). The focus of this work is NS5B, the viral RNA-dependent RNA polymerase, and its interactions with other partners in the
replication complex, notably domain 2 of NS5A (NS5A-D2) and RNA. NS5B has three subdomains: thumb, fingers and palm, named because of its
three-dimensional crystallographic structure reminiscent of a right hand. NS5B also has important open-close dynamics that play a large role in its
binding to RNA and subsequent polymerase activity.
NS5B was studied mainly by Nuclear Magnetic Resonance (NMR) spectroscopy. NMR is used because it allows the study of the protein in solution
and probes both interactions and dynamics, which are essential to the polymerase's activity. NS5B has a molecular mass of 65kDa, which is very
large for NMR in solution, thus most NMR experiments were performed with Ile δ1-13C1H3-labeled and perdeuterated NS5B. This selective labeling
strategy gives rise to a simplified spectrum with improved signal-to-noise ratio that contains only peaks due to isoleucine side-chain methyl groups.
NS5A-D2 is an intrinsically disordered domain whose function in HCV is not known. However, it is necessary for viral RNA replication and directly
interacts both with viral NS5B and the human cyclophilin A (peptidyl-prolyl isomerase A). The interaction between the polymerase and NS5A-D2 was
mapped using NMR. NS5A-D2 seems to bind to an allosteric inhibitor binding site in the thumb subdomain of NS5B, since the interaction spectrum
shows a large chemical shift displacement of the Ile-419 cross-peak and smaller effects on other peaks in the thumb domain. This mimics the effect of
the allosteric inhibitor Filibuvir that is known to bind to this site.
The effect of RNA on the interaction spectrum is mainly to broaden the signal of the Ile-419 cross-peak, which suggests this residue is a probe for the
open-close dynamics of NS5B.
As the NMR resonance corresponding to Ile-419 is affected both upon binding of NS5A-D2 and RNA we have investigated the possibility that an
interplay could exist between these two molecules. Using fluorescence spectroscopy, we indeed observed that NS5A-D2 interferes with the RNAbinding capacity of NS5B.
The objective of this work is to improve our understanding of the underlying relationships between proteins involved in HCV replication.
Hepatitis C virus, NS5B, Nuclear Magnetic Resonance, NS5A-D2
53
MANCHE Monique
Benoit FOLIGNE / Fabrice NESSLANY (UMR8204-U1019 éq. 10 - POT Bruno)
Session Poster 2016
n°57
Comparative toxicity assessment of alcohols commonly used in hygiene disinfectants
Monique MANCHE1,4, Benoît FOLIGNE2 and Fabrice NESSLANY3,4
1 Laboratoires ANIOS – Lille-Hellemmes ; 2 CIIL Center for Infection & Immunity of Lille, Team 10, Institut Pasteur de Lille ; 3 Laboratoire de
Toxicologie Génétique, Institut Pasteur de Lille ; 4 EA 4483 – Université de Lille 2 – 59000
Hand hygiene using Alcohol-based Hand Rubs (AbHR) plays a key role in nosocomial infection control. To achieve users’ compliance, product
acceptability is of major importance and any possible adverse effects in relation with possible toxicological properties of the substances they contain
should be identified, and if any, be reduced at their minimum. The aim of our research is to make a comparative toxicological assessment of the 3
alcohols commonly used in the AbHR (Ethanol, Propan-2-ol & Propan-1-ol), at concentration above 60% w/w as usually present in these products.
Regarding genotoxicity, as expected, first results using the Ames test did not show any effect whatever the alcohol type and its concentration.
Additional tests to investigate possible structural and numerical (aneuploidy) chromosome damages have to be conducted. Regarding local tolerance
assessed on reconstructed human epidermis (RHE), a difference between the 3 alcohols has been observed, with no significant effect on RHE viability
for both ethanol and propan-2-ol whatever the concentration, whereas propan-1-ol at 60, 70 or 80% w/w significantly impacted RHE viabilities, with
mean values closed to 40% (no significant impact observed at 85% w/w). These results support possible irritant properties of Propan-1-ol according to
the interpretation criteria provided by the TG OECD 439 but are not in accordance with bibliographic data on this alcohol. Further investigations are
thus necessary to explain these observations.
Alcohol , Genotoxicity, Skin irritation , Reconstructed human epidermis
MARTIN MENA Anthony
Luc DUBREUIL / Benjamin BERTIN (INSERM U995 éq. 01 - DUBUQUOY Laurent)
The expression of the short isoform of TSLP in the colon is regulated by the nuclear receptor PPARγ and is impaired during
ulcerative colitis
A. Martin Mena1,2, A. Langlois1, P. Desreumaux1,3, L. Dubuquoy1, B. Bertin1,2
(1 Inserm U995, Lille, France; 2 Faculty of pharmacy of Lille, Lille, France; 3 Department of Gastroenterology, Claude Huriez Hospital, CHRU Lille,
France)
Introduction: Peroxisome proliferator activated receptor gamma (PPARγ) plays a key role in gut homeostasis through anti-inflammatory properties.
PPARγ expression by intestinal epithelial cells (IEC) of the colon is decreased in patients with ulcerative colitis (UC). We hypothesized that PPARγ
controls the expression of various factors for intestinal homeostasis. Preliminary work has shown in vitro that the IEC with a decreased expression of
PPARγ had an expression of thymic stromal lymphopoietin (TSLP) significantly decreased compared to control cells. TSLP is an epithelial cytokine
known to play an important role in the intestinal tolerance. There are two isoforms of TSLP (TSLP 1 : long form and TSLP 2: short form). TSLP 2 is the
major isoform in the intestinal epithelial cells.
Aim: To investigate the relation between the expression of TSLP and expression of PPARγ in IEC colon.
Methods: The expression of TSLP 2 were evaluated on different types of IEC: Caco2, T84. These cells were in contact with PPARγ agonists (GED 30
mM, 5-ASA 30 mM, pioglitazone 10 µM). The expressions of TSLP in the treated cells were compared with the control cells. To confirm the link
between TSLP and PPARγ, cells knockdown PPARγ (ShPPARγ) have also been used. The expression of TSLP was also evaluated on colon mucosa
of healthy patients (n=18), healthy (n=9) and injured mucosa (n=21) of patients with UC. Two approaches were performed to study the promoter
upstream of the gene encoding TSLP2 : Chromatin Immunoprecipitation (ChIP) (to observe the fixation of PPARg this promoter on control and treated
cells) and the study of the reporter gene (luciferase) : cloning promoter of TSLP2 upstream of the reporter gene. After vector transfection and
treatment, the luciferase activity is observed.
Results: The expression of TSLP 2 was significantly decreased between ShLuc cells (control cells) and ShPPARγ cells (90% decrease p <0.0001).
PPARγ agonists induce expression of the gene encoding the TSLP 2 in Caco2 cells and T-84 cells (GED: increase, p=0,0022 ; 5-ASA and
pioglitazone: increase, p=0,0043). The expressions of TSLP 2 was significantly decreased in healthy and injured mucosa in UC patients compared to
control patients mucosa (p<0.0001). The ChIP shown the nuclear receptor PPARg bind to the promoter of TSLP 2 both cells (control and treated).
The study of gene reporter confirmed the ChIP result and shown that cells transfected with a vector containing the promoter of TSLP had a significate
increase of luciferase activity (p=0,0022). This activity is significantly increased if these cells are treated (p = 0.0022).
Conclusion: The expression of TSLP 2 appears PPARγ dependent. These results support the hypothesis that the decrease of the expression of
PPARγ at colonic IEC in UC could play a role in the lost of intestinal tolerance though decreased of TSLP 2 expression.
Inflammatory bowel disease, Intestinal tolerance, Immunology, homeostasis
54
MASSE Morgane
Christine BARTHELEMY / Nicolas BLANCHEMAIN (EA7365 éq. 02 - VACCHER Claude)
How to solve the co elution between two compounds in liquid chromatography by the first derivative spectrum? An example
applied to di(2-ethylhexyl) phthalate alternative plasticizers.
M. Masse1 S. Genay1 F. Feutry1, C. Barthélémy1, N. Simon1, V. Sautou3, B. Décaudin1 P. Odou1 for the ARMED study group
1 Univ. Lille, EA 7365 - GRITA
3 Laboratory C-Biosenss, EA 4676, Clermont-Ferrand
Plasticizers are commonly used in manufactured polyvinyl chloride medical devices. Seven plasticizers are mainly employed: ATBC, DEHP, DEHA,
DEHT, DINP, DINCH and TOTM. In the laboratory, a previous HPLC-UV method was developed to identify and quantify five of them in a run of 13
minutes, at 221 nm, with a gradient elution (water and acetonitrile) with an octadecyl column (Radaniel et al., J. Chromatogr. B, 2014). DEHA and
DINP were not quantified in this method because they were systematically co-eluted with DEHP and DEHT respectively whatever the gradient
conditions. The aim of our study was to identify and quantify on one hand DEHP and DEHA and on the other hand DEHT and DINP, by using of the
first UV spectrum derivative.
The UV spectrum derivative was determined thanks to a photodiode array detection and a specific module (i-PDeA™) of the Labsolution software
(Shimadzu®). The quantification of the first compound was performed at the wavelength at which the derivative spectrum of the second compound
became zeroing and vice-versa. The quantification method was validated using the guidelines proposed by the French Society of Pharmaceutical
Sciences and Techniques according to Hubert et al (J. Pharm. Biomed. Anal. 2007).
DEHA was detected at 223 nm, DEHP at 208 nm, DEHT at 226 nm and DINP at 240 nm. The BBP used as internal standard was analyzed at 210 nm.
A linear regression model was applied. DEHP calibration curve was validated from 0.3 to 40 µg/mL, DEHA from 70 to 750 µg/mL, DEHT from 0.3 to
20 µg/mL and DINP from 0.7 to 40 µg/mL. Good linearity was shown over the whole concentration range for the four plasticizers. The relative biases
were inferior to 5% for DEHP and DEHA, and inferior to 10% for DEHT and DINP.
The use of this technique permitted to identify and quantify separately two co-eluted plasticizers. So this improvement of the previous analytical
method now allows identifying and quantifying seven plasticizers in medical devices.
Liquid chromatography , First derivative spectrophotometry , DEHP alternative plasticizers , Polyvinyl chloride
55
MUNCK Camille
Nacim BETROUNI (INSERM U1189 - MORDON Serge)
Dosimetry optimization of intrapleural photodynamic therapy for malignant pleural mesothelioma
Munck C. INSERM, U 1189, Lille, France ; Service de Pneumologie et Oncologie Thoracique, CHRU Lille
Betrouni N. INSERM, U 1189, Lille, France
Mordon S. INSERM, U 1189, Lille, France
Background
Malignant pleural mesothelioma (MPM) is an aggressive tumor of the pleura, due to asbestos exposure. MPM has a poor prognosis, partly due to
ineffective treatments. When surgery is part of a multimodal treatment for MPM, it is crucial to combine it with a local adjuvant treatment to kill residual
tumour cells. Recently, intrapleural photodynamic therapy (iPDT) after surgery, has emerged as a promising treatment in this goal, with a major impact
on survival and minimum toxicity. Successful iPDT requires the most complete and uniform light delivery of the pleural cavity. Today, the light
dosimetry is achieved by 7 isotropic probes collecting light at strategic locations in the thorax, but it does not give information about the light delivered
between these points. This project aims at developing a better peroperative dosimetry for iPDT, by the characterization of a light delivery device and
use of imaging.
Methods
Light dosimetry was performed around the tip of a light wand, made of an endotracheal tube, a cylindrical light diffuser and an electromagnetic sensor,
using an isotropic probe (nW) and digital photography (pixel). An effective attenuation coefficient µeff was defined. This illumination profile was to be
included in an electromagnetic tracking system, to monitor the motion of the light source inside the thorax and to deduce the light dose delivered on its
surface. A human-sized intraoperative thorax phantom was created as a template.
Results
Experimental measurements with the probe allowed to estimate µeff at 0.705cm-1. Combined with the spatial representation of the photography, a
theoretical illumination profile of the light wand was established: it was an ellipse-shaped illumination and showed percentage of remaining light
according to the distance from the centre of the optical fibre and corresponding irradiance. The electromagnetic tracking system allowed tracking in
real time the wand's movements inside the cavity and visualizing its position on 3D images after matching the spatial coordinates of the thorax
phantom to its CT scan. The illumination profile of the light device was then integrated into this spatial tracking system. The light dose delivered on
pleural surfaces was calculated and displayed in real time on the CT scan 3D images.
Conclusion
An illumination profile of a light wand was established and combined with a spatial tracking system, suggesting a more reproducible, complete and
accurate light dosimetry for iPDT in the future. Clinical validation of the method will be conducted during an ongoing phase II clinical trial at the Lille
University Hospital.
photodynamic therapy, Malignant pleural mesothelioma, dosimetry, imaging
56
NDIAYE Fatou Kine
Amélie BONNEFOND (UMR CNRS 8199 éq. 01 - FROGUEL Philippe)
Human genomic and functional studies of common Type 2 Diabetes genes demonstrate their role in pancreatic beta cell
function
Fatou K Ndiaye1, Ana Ortalli1, Marlène Huyvaert1, Clara Salazar-Cardozo1, Mickael Canouil1, Raphaël Scharfmann2, Philippe Froguel1, Amélie
Bonnefond1
1Univ. Lille, CNRS UMR 8199 - EGID
2Inserm U1016, CNRS UMR 8104, Institut Cochin, Paris
Background and aims: Genome-wide associated studies (GWAS) have identified >100 common genetics variants associated with the risk of Type 2
Diabetes (T2D). Since first GWASs, geneticists have suggested that susceptibility genes for T2D are expressed in pancreatic beta cells where they
should play a key role in insulin secretion. However, few studies precisely investigated the tissue expression of these genes as well their biological
function. In the present study, we investigated the expression of T2D susceptibility genes in a large panel of human tissues, and we subsequently
performed a comprehensive functional study of the beta cell most expressed/specific genes in the human beta cell line EndoC-BH1.
Materials and methods: We investigated the expression of 105 T2D susceptibility genes in human placenta, whole pancreas, pancreatic islets, lasercapture and FACS-sorted pancreatic beta cells, EndoC-βH1, skeletal muscle, heart, lung, liver, kidney, small intestine, colon, adipose tissue, and
several tissues from the brain including hypothalamus and substantia nigra. We used the Nanostring technology that enables the simultaneous
counting of RNA molecules without PCR in different tissues. Thirty genes were further selected according to their specificity and their level of
expression in beta cells and EndoC-BH1. Selected genes were investigated in the human beta cell line EndoC-BH1, through transient knock down by
siRNA, and we subsequently assessed insulin secretion in response to glucose and other secretagogues.
Results: We identified a very significant enrichment of the expression of T2D susceptibility genes in the whole pancreas and pancreatic beta cells. As
positive controls, we demonstrated that the knock down of T2D gene GCK (encoding glucokinase) or KCNJ11 (encoding the pore-forming subunit of
ATP-dependent potassium channel in beta cells) significantly decreased insulin secretion from EndoC-BH1in response to glucose with/without IBMX.
We also found that the knock down of four other T2D genes (with unknown function in pancreatic beta cells, so far) significantly reduced insulin
secretion in response to glucose with/without IBMX.
Conclusion: We demonstrated that most GWAS identified T2D susceptibility genes were significantly expressed and enriched in pancreatic beta cells
(compared to other human tissues), even if not fully specific. In four T2D genes with unknown function in human beta cells, we observed a significant
reduction in insulin secretion after silencing. RNA-seq assessing affected pathways is in progress. Other beta cell expressed T2D genes are under
investigation.
Type 2 Diabetes (T2D), Insulin secretion, T2D susceptibility genes, Small interfering RNA
PASCART Tristan
Bernard CORTET (EA4490 - HARDOUIN Pierre / PENEL Guillaume)
Could cancellous bone be an innocent bystander and collateral damage during the nontraumatic osteonecrosis of the femoral
head? Study of bone composition using Raman Spectroscopy
Tristan Pascart
Introduction
Nontraumatic osteonecrosis of the femoral head (NOFH) is a common disease whose pathophysiology is poorly understood. Alterations of the
trabecular bone are suspected to be responsible of the collapse of the femoral head. Compositional modifications of the trabecular bone are unknown.
Methods
11 patients undergoing total hip arthroplasty with the diagnosis of MRI-proven NOFH were included in the study and compared to 11 cadavers.
Sampling was performed in 3 zones: the necrotic region, the trabecular sclerotic zone and in the distant zone. Bone physico-chemical parameters
were analyzed using Raman spectrocopy
Results
Univariate and bivariate comparisons between groups did not show significant differences regarding the mineral-to-matrix ratio, carbonate Bsubstitution, collagen maturity and the hydroxyproline-to-proline ratio for any of the matched zones. Significantly decreased crystallinity was found in
all zones of the NOFH group (p=0.04 in each zone). Significant decrease in the relative proteoglycan content was found in the sclerotic zone of NOFH
patients compared to the matched zone of controls (p=0.03), this decrease was not significant in the necrotic zone (p=0.28) and in the distant zone
(p=0.07). Parameters with significant differences in bivariate analysis were tested for multivariate analysis with adjustment on age and gender using a
mixed linear model. The model regarding crystallinity was found valid after descending selection. Selected variables were age (p=0.026) and gender
(p=0.056) suggesting that observed differences of crystallinity between groups are age-related.
Conclusions
The trabecular bone’s mineral and organic compositions are not altered during NOFH suggesting that cancellous bone is not primarily responsible for
the collapse of the femoral head.
Nontraumatic osteonecrosis, raman spectroscopy
57
PAWLAK-CHAOUCH Mehdi Serge BERTHOIN / Julien AUCOUTURIER (EA4488 éq. 01 - BERTHOIN Serge)
Effect of aerobic fitness on plasma asymmetric dimethylarginine concentrations in response to maximal exercise test
Mehdi Pawlak-Chaouch1, Julien Boissière1, François X. Gamelin1, Gregory Cuvelier2, Serge Berthoin1, Julien Aucouturier1
1 URePSSS, EA 7369, Equipe 1 « Activité Physique, Muscle, Santé », 2 Haute Ecole Provinciale de Hainaut-Condorcet, Tournai
Background: Asymmetric dimethylarginine (ADMA) is an endogenous synthase inhibitor which alters nitric oxide (NO) synthesis, the major relaxing
factor of endothelium dependent vasodilatation. Increased NO-dependent vasodilatory activity during exercise contributes to increase cardiac output
and blood flow to the exercising skeletal muscle, which largely determine exercise capacity. An increased circulating ADMA level is associated with an
increased risk of endothelial dysfunction that might impair exercise capacity. Even though peak exercise capacity is a well-established independent
factor determining cardiovascular health and predicting cardiovascular mortality, little is known about the link between exercise capacity and ADMA,
other L-arginine analogs. Therefore, the present study aimed to determine whether circulating ADMA levels is independently related to exercise
capacity by studying young subjects with no history of cardiovascular and metabolic diseases, and whether circulating ADMA levels acutely change in
response to a single bout of exercise.
Method: Twenty young, healthy, male subjects were recruited and performed a maximal aerobic test until volitional exhaustion. Subjects were then
divided into 2 groups according to their aerobic fitness as follows: HighFit (67.9±9.3 kg, 178.7±7.6 cm, VO2max: 70.3±2.8 mL-1.min-1.kg-1) and
LowFit (77.2±7.5 kg, 180.0±4.2 cm, VO2max: 41.0 ± 6.3 mL-1.min-1.kg-1). HighFit group was engaged in intense endurance exercise training and
regularly participated to competitions. LowFit group did not take part to regular and scheduled physical activity. During maximal aerobic tests, the
measurements of gas exchange were performed by indirect calorimetry and the changes in local muscle oxygenation and microvascular blood volume
were measured by near infrared spectroscopy. In addition, 2 blood samples were drawn just before exercise and 25 min after the end of exercise in
order to assess plasma ADMA concentrations and L-arginine analogs.
Results: LowFit group had higher L-arginine/ADMA ratios before exercise compared to HighFit group (151.0 ± 25.4 vs 126.8 ± 20.3, p = 0.04). This
difference remained significant after exercise (LowFit: 145.8 ± 29.3 vs HighFit: 113.3 ± 18.6), which can be explained by higher ADMA levels in
HighFit group after exercise (0.55 ± 0.03 μM vs 0.49 ± 0.07 μM, p = 0.05). Unlike LowFit, HighFit group showed a decrease in L-arginine (68.1 ± 10. 1
μM vs 61.8 ± 8.9 μM, p = 0.02) and L-arginine/ADMA ratios (126.8 ± 20.3 vs 113.3 ± 18.6, p = 0.02) after exercise. Otherwise, a negative correlation
was found between reached VO2max and L-arginine/ADMA ratios both before (r = -0.47, p = 0.04) and after exercise (r = -0.58, p = 0.01).
Conclusion: The main findings from the present study are that a higher aerobic fitness is not associated with a lower ADMA level in young, healthy,
male subjects contrary to the precedent works conducted in postmenopausal women having elevated ADMA levels.
ADMA, Nitric Oxide, Aerobic fitness, Exercise
PELLEGRINO Giuliana
Marc BARONCINI / Ariane SHARIF (INSERM U1172 éq. 02 - PREVOT Vincent)
Identification of PGD2 as a candidate factor used by GnRH neurons to control their glial environment during postnatal
maturation
Giuliana M. Pellegrino; Vincent Prevot; Marc Baroncini; Ariane Sharif
The GnRH neurons, located in the preoptic area (POA) of the hypothalamus, are the master regulators of the reproductive axis. While they are in
place at birth, they are not mature until puberty. This postnatal maturation requires changes in synaptic inputs to GnRH neurons. Moreover, previous
studies performed in the laboratory showed that postnatal gliogenesis is another mechanism required for GnRH neuron system maturation in female
rats: new cells are preferentially born in the immediate vicinity of GnRH neuron cell bodies at 8 days postnatal (P8) and inhibition of their birth delays
puberty. Moreover, the association between GnRH neurons and newborn cells increases between P8 and P15, suggesting that GnRH neurons recruit
newborn cells during this time laps.
Here, we performed a combination of in vitro and in vivo studies to identify the molecular mechanisms used by GnRH neurons to control their
surrounding progenitor population. Using a candidate approach, we evaluated in vitro whether GnRH, Galanin, Glutamate, GABA and Prostaglandin
D2 (PGD2), which are expressed in GnRH neurons, regulate the migration of rat primary POA progenitor cultures in transwell assays. We showed that
PGD2 had a potent chemoattractive effect on progenitors. We verified by qPCR that progenitors expressed the prostaglandin receptors DP1 and DP2.
Moreover, the chemoattractive effect was inhibited by BWA868C, a selective inhibitor of DP1 receptors. In order to evaluate whether PGD2 signaling
is used by GnRH neurons to attract progenitors in vivo, we stereotaxically injected AT-56, a selective inhibitor of PGD2 synthase, in the POA of P8
rats previously injected with BrdU to label newborn cells. Quantification of the association between GnRH neuron cell bodies and BrdU+ newborn cells
is in progress.
Altogether, our in vitro results identify PGD2 as a candidate molecule used by GnRH neurons to recruit POA progenitors and hence shape their glial
environment to sustain their postnatal maturation
GnRH BrdU PGD2
58
POTELLE Sven
Francois FOULQUIER (UMR8576 éq. 09 - FOULQUIER Francois)
TMEM : a new regulator of Golgi Mn2+ homeostasis involved in Congenital Disorder of Glycosylation
Sven Potelle; 11, ruelle Guillemaud, 59113 Seclin
Congenital Disorders of Glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically
and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi
protein involved in CDG. We demonstrated that the observed Golgi glycosylation defects due to TMEM165 deficiency resulted from Golgi manganese
homeostasis defect. We discovered that Mn2+ supplementation rescued a normal glycosylation. Besides, TMEM165 is itself regulated by Mn2+.
Indeed, we showed that Mn2+ treatment led to a rapid lysosomal degradation of TMEM165. We also have identified the well conserved ELGDK motif
as responsible for Mn2+ sensitivity. Altogether, this study provides novel insights into the molecular causes of glycosylation defects observed in
TMEM165-deficient cells. It also suggests that TMEM165 is a novel Mn2+ sensor and a key determinant for the regulation of Golgi Mn2+ homeostasis.
These findings also support the potential use of therapeutic trials of Mn2+ in TMEM165-deficient patients.
glycosylation, CDG, Golgi homeostasis
RAHMOUNI Oumaira
Christel NEUT (INSERM U995 éq. 01 - DUBUQUOY Laurent)
The involvement of Adherent Invasive Escherichia coli in the pathogenesis of inflammatory bowel diseases.
Oumaïra RAHMOUNI
INSERM U995, Laboratoire de Bactériologie Clinique, Université Lille 2, 59006 Lille, France
Introduction:
Crohn’s disease (CD) and ulcerative colitis (UC), referred as inflammatory bowel diseases (IBD), are multifactorial diseases in which genetic,
environmental and immune factors act in concert to accentuate the inflammation of the intestinal mucosa. In IBD patients, various alterations of the gut
microbiota have been reported. An imbalance between «protective» bacteria and «pathogenic\" bacteria, termed dysbiosis, could play a role in the
aggravation of the disease. During the last years, many studies have focused on a specific pathovar called Adherent Invasive Escherichia coli. This
pathovar, unlike commensal Escherichia coli, is virulent because of its ability to adhere to CEACAM6 receptors and to invade the intestinal cells.
The aim of this work is first to assess the amount of Escherichia coli among the total cultivable microbiota of fecal sample in both CD and UC
patients. Secondly, invasive ability of the recovered strains is investigated in order to determine AIEC prevalence.
Methods:
IBD stools are recovered from Gastroenterology outpatients’ service. Until now, a total of 147 fecal samples were analysed (57 CD and 58 UC
patients). In these two groups of patients, the proportion of Escherichia coli among the total cultivable flora is evaluated by two selective bacterial
medium. Escherichia coli isolates from each patient are then tested by cellular culture in order to determine the capacity of these strains to invade the
intestinal cells I-407. The results are expressed in log CFU/g.
Results:
In both CD and UC patients, the proportion of Escherichia coli among the total cultivable flora is variable from one patient to another. In fact,
Escherichia coli may be predominant or in minor amounts (below the detection level). The same result is stated in UC patients.
The AIEC prevalence has been determined in 55 CD patients, 9 are positive for AIEC (=20%). Among Escherichia coli, AIEC are predominant
when present. In UC patients, the AIEC prevalence is under investigation.
Conclusion:
The preliminary results confirm a possible role of AIEC in IBD pathogenesis.
Prospect:
The study concerning the AIEC prevalence will continue in order to compare it between IBD patients and irritable bowel syndrome patients.
Inflammatory bowel disease, Intestinal microbiota, Dysbiosis, Adherent Invasive Escherichia coli
59
SAAD Chadi
Marie-Pierre BUISINE / Hélène TOUZET (INSERM U1172 éq. 05 - VAN SEUNINGEN Isabelle)
Bioinformatic tools for next generation sequencing: Characterization of non-random sequencing errors, application to
mosaicism and heterogeneous tumors
Chadi Saad, Laurent Noé, Martin Figeac, Julie Leclerc, Marie-Pierre Buisine and Hélène Touzet
Next generation sequencing technologies are associated with a relatively high error rate compared to Sanger sequencing. Consequently, each region
needs to be sequenced several times. Variants are usually filtered based on depth criteria. The significant number of artifacts, in spite of those filters,
shows the limit of conventional approaches and indicates that some sequencing artifacts are not random and may be recurrent. This recurrence
means that sequencing errors can depend on the upstream nucleotide sequence context. A score enabling the classification of variants as probable
sequencing errors or actual variants could be very useful especially in the context of mutation identification in minority subpopulations of cells. This is
the case when searching for mosaic constitutional mutations or for somatic mutations in tumor samples (existence of tumor sub-clones or nonnegligible proportion of normal cells in the sample).
We use computational and statistical models to study the large amount of sequences present in public databases (ESP) and generated in routine
diagnosis at Lille University Hospital. We developed scripts to identify specific DNA patterns (motifs) that are frequently found upstream of sequencing
errors (positive dataset), compared to sequences which do not contain errors (control dataset). This is done by the calculation of the mutual
information value.
Preliminary results show that there are many overrepresented motifs upstream of artifacts. The next step is to study degenerated and complex motifs
more exhaustively, using the 15 letters of the IUPAC DNA code, which implies specific algorithmic methods regarding the huge number of motifs to
analyze (nb of motifs = 15^L , where L is the motif length), and then to develop a scoring system to distinguish false positive variants (artifacts) from
true positive variants.
Developed tools will be useful for various applications in biology, especially in cancerology.
Next generation sequencing, Bioinformatics, Tumors
SAHUC Marie-Emmanuelle
Karin SERON (UMR8204-U1019 éq. 05 - DUBUISSON Jean)
Identification du dehydrojuncusol comme nouvel inhibiteur de la réplication du virus de l'hépatite C
Marie-Emmanuelle Sahuc, Ramla Sahli, Céline Rivière, Gaspard Deloison, Priscille Brodin, Jean Dubuisson, Riadh Ksouri, Yves Rouillé, Sevser
Sahpaz et Karin Séron
Dans le monde entre 130 et 150 millions d'individus sont porteurs chroniques de l'hépatite C selon l'organisation mondiale de la santé. Les antiviraux
sur le marché permettent de guérir plus de 90% des personnes infectées par le virus de l'hépatite C (VHC) mais l'accès aux traitements est toujours
insuffisant. Actuellement, il est toujours nécessaire de découvrir de nouveaux inhibiteurs du VHC afin de continuer à approfondir notre compréhension
du cycle viral de ce virus, et éventuellement baisser le coût des traitements.
Dans ce contexte, différents extraits bruts de plantes halophytes et xérophytes tunisiennes ont-été testés contre le VHC. Une halophyte de la famille
des Juncaceae, Juncus maritimus, s'est distinguée par son activité contre le virus, en particulier l'extrait des rhizomes. Cet extrait a inhibé l'infection
du VHC dans les cellules Huh-7 et plus spécifiquement l'étape de réplication génomique. Pour déterminer le(s) composé(s) responsable(s) de cette
activité, une extraction liquide-liquide a permis d'obtenir 3 sous-extraits : dichlorométhane (1), acétate d'éthyle (2) et aqueux (3). Le sous-extrait (1) a
été identifié comme étant le plus actif contre la réplication du VHC. Parmi les produits purifiés à partir de ce sous-extrait, le dehydrojuncusol, s'est
montré très actif. Nous cherchons actuellement à identifier le mécanisme impliqué dans l'inhibition de la réplication du VHC par ce nouveau composé
antiviral.
Dans une perspective d'application potentielle, il est également important de déterminer si le dehydrojuncusol conserve son activité antivirale contre
des mutants de résistance aux antiviraux actuellement utilisés en clinique et notamment les inhibiteurs de NS5A. Dans ce contexte, nous testons
actuellement l'effet inhibiteur du dehydrojuncusol en système réplicon après y avoir intégré deux mutations de résistance communément retrouvées
après traitement avec des inhibiteurs de NS5A (Y93H et L31M). Ceci permettra de déterminer si le dehydrojuncusol est capable d'inhiber les virus
résistants aux inhibiteurs de NS5A.
En conclusion, le dehydrojuncusol a été identifié comme nouvel inhibiteur de la réplication du VHC et l'étude de son mécanisme d'action devrait nous
permettre de comprendre comment cette molécule inhibe l'étape de réplication génomique du VHC
Hépatite C
60
SERGIO FABRICIO Gabriel
Stefania MACCARI (UMR8576 éq. 06 - MACCARI Stefania)
Perinatal restraint stress: How the exercise during adolescence may shape the construction of mood in adulthood offspring
Gabriel Sergio Fabricio
Sara Morey-Fletcher
Gilles Vancamp
Lucie Deruyter
Stefania Maccari
Feeding and cognitive behavior are important hallmark of weight gain onset and it has been accepted that neuropsychological disorders and obesity
may have your roots in maternal womb. In fact, the mother care, environment, behavior and stress of dams during gestation and lactation have
influence over the offspring behavior and metabolism. In agreement, offspring rats of Perinatal restraint stressed mother (PRS) during gestation show
an enhanced vulnerability to food or drug addiction in adulthood, among other important biochemical hallmarks of social disorders behavior. As well as
showed in this background, early life stimulus can shape mood. However, is now accepted that «environmental enriches» (EE's) may reverse some
abnormalities produced maternal womb and infancy as example, the practice of voluntary physical exercise in adolescence may increase the learning
and cognitive behavior furthermore, contributing to reduction of body weight and decrease of metabolic syndrome illness related in obese or stressed
offspring. Take together these evidences, the present project wish to investigate if the moderate physical exercise program during adolescence on
anxious/depression-like can reverse metabolic and neuropsychological disorders of PRS offspring in adulthood. Pregnant Sprague-Dawley rats were
subjected of perinatal restraint stress from gestational day 11 until delivery. After the weaning the pups (males) will be separated in 4 groups:
sedentary control (SC); sedentary PRS (SPRS); exercised control (EXC); exercised PRS (EXPRS) with 10 animals per group and exercised between
21 – 80 days-old. As a form to avaliate metabolic and anxious/depression-like behavioral parameter animals will be weighted weekly and submitted at
Intraperitoneal Glucose Tolerance Test (ipGTT) and Light and dark test. Regarding the physical exercise is highly accepted which exercise is
appointed as a strong tool to treatment of mother malnutrition, obesity and related disease moreover, the exercise or enriches environmental are also
related to reverse the hallmarks of perinatal restraint stress.
perinatal restraint stress, physical exercise , neuropsychological disorders, obesity
TIAN Lu
Roland BOURETTE (UMR CNRS 8161 éq. 04 - MELNYK Oleg)
Bi-transgenic C3(1)Tag X 11.5kb-GFP mice: a new model of tumorigenesis for the study of breast cancer
Lu Tian1, Chann Lagadec2, Eric Adriaenssens2, Emmanuel Bouchaert3, Xuefen LeBourhis2, Roland Bourette1
1 CNRS UMR 8161, Institut de Biologie de Lille
2 INSERM U908, PCC, Université de Lille
3 Plateforme des modèles animaux du SIRIC ONCOLille, Lille
The isolation and characterization of cancer stem cells (CSC) are crucial for understanding cancer biology and revealing potential therapeutic targets.
In this aim, we generated a new model by crossing transgenic (Tg) C3(1) Tag mice with Tg 11.5kb-GFP mice. Tg C3 (1) Tag mice express SV40 T
antigen oncogene under the C3(1) prostatein promoter. In female mice, the transgene is expressed primarily in the mammary gland. Mice develop
mammary hyperplasia by 3 months of age with subsequent development of mammary adenocarcinoma by 6 months of age. To investigate the
presence of mammary CSC in these mice, we rely on a second transgenic mouse model, Tg 11.5kb-GFP expressing the green fluorescent protein
(GFP) driven by 11.5kb s-SHIP promoter. Using these mice, previous studies demonstrated that s-SHIP/GFP expression marks adult stem cells in
prostate and mammary tissues.
In order to validate our model, we aimed to determine whether s-SHIP/GFP is also a marker of CSC, i.e. if GFP+ cells isolated from the mammary
carcinomas of bi-transgenic mice have CSC properties. The presence of a rare population of GFP+ cells in mammary tumors was validated using
immunohistochemical and flow cytometry analysis. The GFP+ mammary cancer cells show expression of certain well-known murine mammary CSC
markers (CD24/CD49f/CD29). As compared to GFP- cells, GFP+ cells exhibit both a higher tumor sphere-forming potential, and a higher
tumorigenicity when transplanted into SCID mice. Altogether, these results demonstrate that s-SHIP promoter expression is a marker for mammary
CSC that enables their identification and isolation via a single consistent parameter. Currently, we are conducting a transcriptomic analysis of these
GFP+ and GFP- cells in order to reveal differentially expressed genes and potential therapeutic targets for breast cancer.
transgenic mice, mammary cancer stem cells, FACS, transcriptomic analysis
61
WATBLED Ludivine
Regis BEUSCART / Christian BASTIEN (EA2694 - DUHAMEL Alain)
Work System Characteristics Impacting the Performance and Quality of Discharge Letter Process
Ludivine Watbled
Studies on the impact of a Health Information Technology seldom consider socio-technical characteristics of the work system in which the technology
is implemented. Yet those dimensions may act as hidden variables that could explain the inconsistency of impact studies’ results in terms of
performance, quality and satisfaction. This paper reports on the identification of those variables in the discharge letter (DL) process. Human Factors
experts performed an analysis of the work system of the DL process in 17 medical units. The DL process is composed of three sub-processes running
with work system differing according to the distribution of tasks, the technology implemented and the work organization. Hidden variables identified
are: verification by the physician, technology’s integration, number of editing cycles, physicians’ preferences etc. Those variables can be collected
automatically or by questionnaire. Statistical analyses will have to be performed to know which variable explain impact indicators.
Human Engineering , Discharge Letter, Evaluation studies
WAWRZYNIAK Clement
Monique ROMON / Sylvia PELAYO (EA2694 - DUHAMEL Alain)
How can we support the implementation and maintenance of the medication review process at hospital to secure the
transitions of care?
Clément Wawrzyniak*, Sylvia Pelayo
INSERM CIC 1403, Lille, Univ Lille Nord de France, CHU Lille, UDSL EA 2694, F-59000 Lille, France
*e-mail: [email protected]
It is increasingly recognized that most incidents related to medication occur when a patient changes of care environment, especially from community
to hospital, or from hospital to community. Medication reconciliation (MRec) and medication review (MRev) processes are both solutions known to
secure and optimize these transitions of care. Many studies show their positive clinical (and financial) impact on adverse drug events (ADEs) at
hospital. Nevertheless, both processes are considered difficult to implement and maintain by healthcare institutions and professionals. Several barriers
are identified in the literature and most are related to Human Factors and Ergonomics (HF/E) aspects. However, despite the exponential number of
publications on the topic in recent years, very few HF/E studies on these two processes are reported.
The aim of the study is to perform a deep HF/E analysis of the MRec and MRev processes to highlight the difficulties related to the work system
(including the tasks, the actors, the environment, the organization and the technology/tools). In fine, a model would be provided to help all the
stakeholders of the MRec and MRev processes (professionals, hospital deciders, health authorities, etc.) to implement and maintain an effective
process: what works? what does not work? what is important? why? Etc. This will allow to make informed choices for the work system (re-)design and
for the corresponding allocated resources.
At first, a literature review was undertaken to identify (i) the few HF/E studies on MRec and MRev processes if they exist and (ii) the studies from
which work system elements on the MRec and MRev processes at hospital can be extracted even the studies are not HF labeled. Work analyses of
the two processes were performed in three departments of three different French hospitals, where the processes were implemented (or in progress of
implementation). HF experts relied on classical methods of the ergonomics domain (observations and interviews) with a special attention to the
information flow supporting the two processes.
4134 articles were included in the literature review. After a screening of titles and abstracts applying the inclusion criteria, 28 full articles were
analyzed. Only one paper adopted a HF point of view.
As regard to the work analysis, 53 observations were carried out corresponding to 129 hours and 20 interviews corresponding to 20 hours. On the one
hand, the results show that both processes are completely intertwined, the MRec process being part of the MRev. On the other hand, the results show
that despite differences in the work organizations the main tasks of the processes are similar whatever the site. the study highlights several problems
related to HF/E which bring out negative consequences on the MRev process. First, the main transversal cause identified is related to the human
resources devoted to the processes. Whatever the site, understaffing and turnover are systematically observed.
Medication Review, Human Factors, Ergonomics, Health Work System
62
WYCHOWSKI Adeline
Fabrice WATTEBLED (UMR8576 éq. 11 - WATTEBLED Fabrice)
Functional and structural characterization of A. thaliana branching enzymes
Adeline WYCHOWSKI1, Coralie BOMPARD1, Florent GRIMAUD2, Gabrielle POTOCKI-VÉRONÈSE2, Christophe D'HULST1, Fabrice
WATTEBLED1, Xavier ROUSSEL1
1 UGSF, Université Lille 1, Bât. C9, 59655 Villeneuve d’Ascq
2 LISBP, 135 Avenue de Rangueil, 31077 Toulouse
AtBE2 and AtBE3 are the two genetically independent branching enzymes isoforms involved in transitory starch synthesis in Arabidopsis thaliana.
They both belong to the subfamily 8 of Glycoside hydrolase 13 (Stam et al. 2006). AtBEs are classified as type II BE due to their amino acid sequence
and their ability to transfer short glucan chains (Burton et al. 1995). They are the only enzymes that catalyze the formation of α(1→6) branch points by
cleaving α(1→4) linkages and transferring the newly formed reducing end in α(1→6) position through an intra or intermolecular mechanism. Previous
work on A. thaliana branching enzymes defective lines showed that the absence of either AtBE2 or AtBE3 protein led to a slight modification of the
starch phenotype suggesting partially redundant functions. Conversely when both mutations are combined in the same line, plants are unable to
synthesize starch and accumulate maltose. Complementation of A. thaliana mutants devoid of BE activity with bacterial glycogen BE (GlgB) or another
plant BE restores starch synthesis. However, chain length distribution of starch produced in these lines differs from wild type starch (Boyer et al. 2015,
Lu et al. 2015). To better understand why such differences occur, we purified recombinant AtBEs and initiated enzymatic and structural
characterizations by the determination of BEs catalytic parameters and SAXS approach. In vitro enzymatic analyses were performed using iodine
staining assays to establish pH, temperature and also Km for amylose. To improve BEs characterization, monodisperse fractions of α(1→4) glucans,
synthesized in vitro by Neissaria polysaccharea amylosucrase, were used as substrate. Anion exchange chromatography analyses (HPAEC-PAD)
allowed us to determine the minimal length of their substrate and product (Roussel et al. 2013). Currently, only type I BE structure is resolved.
Determination of AtBEs oligomerization state as well as SAXS structural study were realised to propose structural models for both enzymes.
References
BOYER, L., ROUSSEL, X., COURSEAUX, A., NDJINDJI, O. M., LANCELON-PIN, C., PUTAUX, J. L., TETLOW, I., EMES, M., PONTOIRE, B.,
D’HULST, C. & WATTEBLED, F. 2015. Plant Cell Environ. 10.1111/pce.12702
BURTON, R. A., BEWLEY, J. D., SMITH, A. M., BHATTACHARYYA, M. K., TATGE, H., RING, S., BULL, V., HAMILTON, W. D. & MARTIN, C. 1995.
Plant J. 7, 3-15.
LU, K. J., STREB, S., MEIER, F., PFISTER, B. & ZEEMAN, S. C. 2015. Plant Physiol. 169, 1638-1655.
ROUSSEL, X., LANCELON-PIN, C., VIKSO-NIELSEN, A., ROLLAND-SABATE, A., GRIMAUD, F., POTOCKI-VERONESE, G., BULEON, A.,
PUTAUX, J. L. & D’HULST, C. 2013. Biochim Biophys Acta. 1830, 2167-2177.
STAM, M. R., DANCHIN, E. G., RANCUREL, C., COUTINHO, P. M. & HENRISSAT, B. 2006. Protein Eng Des Sel. 19, 555-562.
Branching enzyme, characterization, SAXS
ZECCHIN Mathilde
Helene DUEZ (INSERM U1011 éq. 01 - STAELS Bart)
Role of the nuclear receptor Rev-erbα in the development of vascular calcification
Zecchin M., Pourcet B., Ferri L., Vanhoutte J., Delhaye S., Delsart F., Mayeuf-Louchart A., Thorel Q., Duhem C., Boulinguiez A., Lancel S., Sebti Y.,
Staels B. and Duez H.
In late stage of atherosclerosis, vascular inflammation triggers osteogenic differentiation of smooth muscle cells into osteoblast-like cells. The
calcification results from the imbalance between the formation of calcification by the osteoblast-like cells and its resorption by osteoclast-like cells. We
have shown that the nuclear receptor Rev-erb-α protects from the development of atherosclerosis. However, its role in vascular calcification has not
been elucidated yet. Preliminary data of our team show that osteogenic differentiation is increased in Rev-erb-α deficient mesenchymal stem cells
compared to controls.
To determine whether Rev-erb-α is implicated in the development of vascular calcification, we compared atherosclerostic plaque calcification occurring
in 1 year-old Rev-erbα-/- and Rev-erbα+/+ mice in pro-atherogenic Low Density Lipoprotein receptor deficient (LDLr-/-) genetic background. The size
of calcified areas was quantified after alizarin red staining on heart sections surrounding the aortic sinus. To uncover molecular mechanisms, the
transcriptional effect of Rev-erb-α was assessed in vitro by qPCR in key cells involved in this process such as primary aortic smooth muscle cells
cultured in osteogenic medium and in bone marrow derived macrophages differentiated into osteoclasts in presence of RANKL.
Rev-erbα-/- ; LDLr-/- mice show larger calcification areas compared to Rev-erbα+/+; LDLr-/- mice. Consistently, calcium deposition, quantified after
alizarin red staining, is increased in Rev-erbα-deficient smooth muscle cells cultured in an osteogenic medium compared to control cells. The
expression of osteogenic markers such as osteocalcin and osteopontin and the inhibitor of calcification mgp is modulated in the absence of Rev-erbα.
In Rev-erbα deficient osteoclasts, the expression of osteoclast markers such as cathepsin k, mmp-9 and rank is also enhanced.
These results indicate that Rev-erb-α may protect from the development of vascular calcification. The study of the mechanisms involved therein is in
progress.
atherosclerosis, nuclear receptor, vascular calcification
63
16ème Journée André VERBERT
Colloque Annuel des Doctorants – 5 Septembre 2016
Comité d’Organisation
Kevimy AGOSSA, Tanina ARAB, Samir BENSAID, Clément BOURNONVILLE, Mickael CANOUIL, Omar CASTILLOAGUILERA, Alexandre CAUX-DEDEYSTERE, Amandine CIAN, Justine DEVULDER, Aurore DROLEZ, Juliette
FERLIN, Matthias LAMBERT, Mélanie LAMBERT, Coline LEGHAY, Antonella RAFFO-ROMERO, Oumaira
RAHMOUNI, Romuald TAGNE FOTSO, Mathilde ZECCHIN
Avec la participation des autres membres du Comité d’Animation Scientifique
de l’Ecole Doctorale Biologie Santé de Lille :
Kadiombo BANTUBUNGI-BLUM, Christelle CAUFFIEZ, Laëtitia COUDERT, Maxime CULOT, Patricia de NADAÏ, François
DELCROIX , Julie DUMONT, Emilie FAIVRE, Laurence FOFANA, Malika HAMDANE, Jean-Jacques HAUSER , Emmanuel
HERMANN, Nathalie JOUY, Steve LANCEL, Nicolas LEBEGUE, Emmanuelle LIPKA-BELLOLI, Aurélie MAIGUY-FOINARD,
Michaël PERRAIS, Olivier PETRAULT, Albin POURTIER, Olivier PLUQUET, Pierre-Eric SAUTIERE, Yasmine SEBTI, Xavier
THURU, Christel VANBESIEN, Murielle VERGHOTE
Le Comité d’Organisation exprime ses remerciements aux Institutions et Sociétés qui
ont participé à l’organisation du colloque 2016.
.
Affiche réalisée par Laëtitia COUDERT
Cahier imprimé à l’imprimerie de l’Université Lille 1

journée lundi 5 andré verbert

Transcription

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