-------------------------------------------------------------------------JOURNEES SCIENTIFIQUES DE
L'ECOLE DOCTORALE BIOLOGIE-SANTE
-------------------------------------------------------------------------WESTOTEL
9 ET 10 DECEMBRE 2014
&
1
PROGRAMME
9 DECEMBRE 2014:
09H15 ACCUEIL DES DOCTORANTS
09H30:
INTRODUCTION
/
PRESENTATION
LESCAUDRON / PEIGNE Cassie-Marie).
ADN-BIOSANTE
(Laurent
SESSION 1 : "NUTRITION / METABOLISME / SIGNALISATION"
10H00-10H30: CONFERENCE (20 + 5MIN)
MARIE-CÉCILE ALEXANDRE: PHAN-NANTES «Perinatal maternal protein restriction
impacts milk composition and offspring metabolomic trajectory in a rodent model
of metabolic programming. »
10H30-12H00: PRESENTATIONS DES DOCTORANTS (4 PRESENTATIONS DE 10 + 5 MIN)



MME. YAMADA AMI – 2 ème ANNEE – LABERCA - ONIRIS
CONTRIBUTION TO THE HUMAN PFAS EXPOSURE ASSESSMENT FOR TWO
FRENCH SUB POPULATIONS.
MME. HALLOUM WAFAA – 2 ème ANNEE – LABERCA - ONIRIS
DEVELOPMENT OF ANALYTICAL STRATEGIES DEDICATED FOR
THE ANALYSIS OF ORGANOPHOSPHOSPHORUS FLAME
RETARDANTS IN BIOLOGICAL SAMPLES.
MME. LECLERC LUCIE - 2 ème ANNEE – ONIRIS
MOTHER BODY WEIGHT AT CONCEPTION AND ENERGY INTAKE ARE THE MAIN
FACTORS THAT EXPLAIN WEIGHT GAIN DURING GROWTH IN DOGS

MME. MARTINEAU ANNE-SOPHIE – 3 ème ANNEE –UNITE NUTRITION ET
ENDOCRINOLOGIE
THE CONSUMPTION OF A GRAPE AND BLUEBERRY POLYPHENOL-RICH MIXTURE
IS SAFE IN DOGS
SÉANCE “SPEED-POSTER 1” (DE 1 A 15)
11H40-12H00: PRESENTATIONS ORALES (15
(.PPT) ORDRE DE PASSAGE DANS LE LIVRET
POSTERS
*2
MINUTES) PRESENTATION
12H15 – 13H15: PAUSE DEJEUNER
SESSION 2 : " PATHOLOGIES INFECTIEUSES / IMMUNOLOGIE"
13H15-13H45: CONFERENCE (20’ +5’)
ELISE CHIFFOLEAU: « Induction of allograft tolerance in transplantation »
13H50-15H30: PRESENTATIONS DES DOCTORANTS (6 PRESENTATIONS DE 10 + 5 MIN)


MME. CANO CAMILLE - 2 ème ANNEE - INSERM U892, EQUIPE 7B
SPONTANEOUS WOUND HEALING IN A MOUSE MODEL OF M. ULCERANS
INFECTION: A HOST-PATHOGEN CROSS-TALK.
M. FOUCHER ETIENNE – 3 ème ANNEE – U892 EQ7
IL-34 AND M-CSF INDUCE MACROPHAGES THAT PROMOTE THE GENERATION
OF TH17 CELLS VIA MEMBRANE IL-1Α
2


MME. BACOU ELODIE – 2 ème ANNEE – IECM
ANALYSIS OF MIXING-INDUCED STRESS CONSEQUENCES ON IMMUNE TRAITS IN
PIGS.
MME. YAP MICHELLE – 3 ème ANNEE – UMR 1064
AN INCREASE OF HIGHLY DIFFERENTIATED TEMRA CD8 T CELLS WITH
POTENT EFFECTOR FUNCTION IN KIDNEY TRANSPLANT RECIPIENTS WITH
STABLE GRAFT FUNCTION REVEALED PATIENTS WITH AT-RISK OF LONG-TERM
GRAFT FUNCTION.

M. LAPERINE OLIVIER - 3 ème ANNÉE – UMR 791
ROLES OF INTERLEUKINS 33 AND 36 IN THE PATHOGENESIS
OF PERIODONTAL
DISEASE IN MICE

M. PATINEC ALLAN - 3 ème ANNEE –UMR 892
MODULATION DE L'IMMUNITE ANTI-INFECTIEUSE PAR STIMULATION DES
POPULATIONS INKT CIRCULANTES DANS UN CONTEXTE DE TRAUMATISME
CRANIEN.
15H30-15H50: PAUSE-CAFE
SÉANCE “SPEED-POSTER 2” (DE 16 A 40)
15H50-16H50: PRESENTATIONS ORALES (25
(.PPT) ORDRE DE PASSAGE DANS LE LIVRET
POSTERS
*2
MINUTES) PRESENTATION
16H50-18H15: VISITE POSTERS
SESSION 3 : "IMMUNOLOGIE ET CANCEROLOGIE"
18H15-18H50: CONFERENCE (20 + 5’)
CATHERINE IBISCH «The bone marrow and the cancer patient: contribution of
animal models to hematopoietic stem cell transplantation in oncology »
18H50-19H45 : PRESENTATIONS
MIN)
DES DOCTORANTS
(3
PRESENTATIONS DE
10 + 5
 M. D’ALMEIDA Sènan – 3ème année – Inserm U892
THE ECTO-NUCLEOTIDASE CD39 IS INVOLVED IN THE
ACQUISITION OF AN IMMUNOSUPPRESSIVE PHENOTYPE BY
TUMOR-ASSOCIATED MACROPHAGES.
 MME. RUGGERI Tiphaine – 2 ème année – EA 3826
Post-Traumatic Immunosuppression in a Mouse Model of Spinal Cord
Injury
 M. AVRIL Pierre - 3 ème année – UMR 957
EFFECT OF SOLUBLE FACTORS PRODUCED BY FAT ON
CARCINOLOGICAL RISK
20H00-21H00: DINER A L’HOTEL
21H00-2H: SOIREE DANSANTE (SALLE
PAQUEBOT) ANIMATION PAR ASSOCIATION DES
DOCTEURS
3
PROGRAMME
10 DECEMBRE 2014:
SÉANCE “SPEED-POSTER 3” (DE 41 A 65)
09H30-10H20: PRESENTATIONS ORALES (25
(.PPT) ORDRE DE PASSAGE DANS LE LIVRET.
POSTERS
*2
MINUTES) PRESENTATION
10H20-10H40 : PAUSE-CAFÉ
10H40-11H40 : VISITE POSTERS
12H00-13H15: DEJEUNER
SESSION 4 : " PHYSIOLOGIE / PATHOLOGIES CARDIO-VASCULAIRE /
SIGNALISATION"
13H15-13H45: CONFERENCE (20’ +5’)
BENJAMIN LAUZIER « Hexosamine biosynthetic pathway stimulation, a new strategy
to improve survival during sepsis»
13H45-15H00: PRESENTATIONS DES DOCTORANTS (5 PRESENTATIONS DE 10 + 5 MIN)
 MME. MONTAUDON ELODIE – 3 ème ANNÉE – PAPF - ONIRIS
CARDIOVASCULAR EFFECTS OF ANTI- Β1– AND Β3–ADRENERGIC RECEPTOR
ANTIBODIES IN LEWIS RAT
 MME. BICHON Emmanuelle – 2 ème – LABERCA - ONIRIS
GC-APCI-MS/MS for making natural steroids analysis more comprehensive
and compatible with ultra-trace detection .
 MME. COLIN ESTELLE - 3 ème ANNEE – UMR 1083
LOSS OF FUNCTION MUTATIONS IN WDR73 ARE RESPONSIBLE FOR
MICROCEPHALY AND STEROID RESISTANT NEPHROTIC SYNDROME :
GALLOWAY-MOWAT SYNDROME.
 MME. BON Nina – 2ème année – INSERM UMRS791
Insight into the extracellular phosphate sensing mechanism, the key step for
an appropriate FGF23 secretion
 M. CLAIREMBAULT THOMAS - 3 ème ANNÉE – UMR 913
STRUCTURAL ALTERATIONS OF THE INTESTINAL EPITHELIAL BARRIER IN
PARKINSON’S DISEASE
15H00-15H25: PAUSE-CAFÉ
4
SESSION 5 : "NANO MEDECINE / VECTORISATION / BIOMATERIAUX /THERAPIE
GENIQUE MEDECINE REGENERATRICE"
15H30-17H00: PRESENTATIONS DES DOCTORANTS (6 PRESENTATIONS DE 10 + 5 MIN)
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MME. ANDRE Emilie – 3ème année – Inserm U1066
Neuronal commitment of mesenchymal stem cells with nanoparticles
transporting si-RNA
M. Kizito-Tshitoko TSHILENGE - 2ème année UMR 1089
Gene transfer in retinal ganglion cell
MME. HENRY Nina – 2 ème année – U791
Nano-reinforced hydrogel and protein release in the context of regenerative
medicine of the intervertebral disc
M.HUET Simon -3 ème année – Affilogic SAS UMR CNRS 6286
Rational design of Nanofitins targeting a defined epitope
MME. Vandamme Céline – 2ème année – Inserm UMR 1089
Detection and Characterisation of Human anti-AAV CD8+ T Cells using
MHC class I Multimer-based Magnetic Enrichment
MME. MALHAIRE Hélène -3 ème année – UMR 1066
ORAL FORMULATION OF PROTEINS: DEVELOPMENT AND
CHARACTERIZATION OF AQUEOUS CORE NANOCAPSULES
17H05: CONFERENCE (20’ +5’)
JEAN CHRISTOPHE GIMEL: «Brownian diffusion of nanomedicines in complex
biological media»
17H35: RETOUR SUR NANTES OU ANGERS
5
SOMMAIRE

COMMUNICATIONS
ORALES

POSTERS
6
MME. YAMADA Ami – 2ème année – LABERCA ONIRIS Session 1
Contribution to the human PFAS exposure assessment for two French sub-populations
Ami YAMADA*1, Jean-Philippe ANTIGNAC1, Bruno LE BIZEC1, Jean-Charles LEBLANC2
1
Oniris, Laboratoire d’études des résidus et contaminants dans les aliments (LABERCA), Route
de Gachet – Site de la Chantrerie – CS 50707, 44307 Nantes Cedex 3, France
2
Food and Agricultural Organization of the United Nations (FAO), Viale delle Terme di
Caracalla, 00153 Rome, Italie
Since first synthesis in the early 1940’s and introduction on market in the 1950’s, the use of
poly- and perfluoroalkylated substances (PFAS) is now widespread in various applications such
as fire-fighting foams, aviation hydraulic fluids, medical devices and pesticides but also in
consumer products as water- and stain-repellents for anti-sticking coating of pans, cleaning
products and waterproof clothing as instance. Due to a high thermal and chemical resistance,
these compounds are hardly, if ever, degraded. High thermal and chemical resistance,
adsorption to particles and long-range atmospheric transport explain their ubiquitous presence
even in remote areas such as in Arctic where neither use nor production of PFAS is known.
Since last decade, the risk assessors and managers saw these compounds as a matter of public
health interest when toxicity concerns were associated with PFAS exposure and found in the
blood of the general population. The main suspected health effects are neurotoxicity,
hepatotoxicity, developmental toxicity, immunotoxicity as well as effects on thyroid.
The aim of this PhD thesis is to characterise the aggregated external exposure of two possibly
at risk sub-populations to PFAS found at the highest concentration in serum, namely the
perfluorooctanesulphonate
(PFOS),
the
perfluorooctanoic
acid
(PFOA),
the
perfluorohexanesulphonate (PFHxS) and the perfluorononanoic acid (PFNA). Populations of
interest are pregnant women and their newborn as this population is potentially the most
sensitive to the effects of PFAS during this critical period of time and high fish consumers (sea
and freshwater) as this population is potentially the most exposed via the diet considering that
fishes are reported as being the highest contaminated foods to PFAS. The considered external
exposure includes diet, indoor and outdoor air as well as settled dust and soil excluding dermal
pathway as it was shown that at a physiologically relevant pH, PFOS and PFOA were unable to
penetrate through the skin.
As part of the work, a link of the external exposure versus internal exposure considering the
serum level of PFOS and PFOA reported in the two sub-populations with the use of a
physiologically based toxicokinetic model is proposed.
7
MME. HALLOUM Wafaa – 2ème année – LABERCA ONIRIS
Session 1
DEVELOPMENT OF ANALYTICAL STRATEGIES DEDICATED FOR THE
ANALYSIS OF ORGANOPHOSPHOSPHORUS FLAME RETARDANTS IN
BIOLOGICAL SAMPLES
Halloum W1, 2, Cariou R1, Dervilly-Pinel G1, Jaber F2, Le Bizec B1 1LUNAM Université,
Oniris, USC INRA 1329 Laboratoire d’Etude des Résidus et Contaminants dans les Aliments
(LABERCA), F-44307, Nantes, France. 2Lebanese University - Faculty of Sciences I,
Laboratory of Analysis of Organic Compounds (LACO) - 508 - Hadath, Beirut, Lebanon
Abstract
Flame retardants (FRs) are human-made chemicals added to consumer and industrial products
for the purpose of reducing flammability1. Among FRs, there are the halogenated FRs that may
be divided into brominated and chlorinated flame retardants (BFRs and CFRs), and
phosphorus-containing flame retardants (PFRs)2. The market for flame retardant chemicals is
being driven by globally tightening fire safety regulations. In this context, some halogenated
FRs, such as BFRs, have proven to be persistent, bioaccumulative and toxic to environment,
animals and humans. This led governments to adopt restrictions which phase out the production
and use of some BFRs3. On the other hand, organophosphorus flame retardants (OPFRs) are
proposed as alternatives for BFRs and their use shows a continuous increase4,5. However, most
OPFRs are introduced as additives and not chemically bound to the polymer; hence they are
slowly released in the environment by abrasion and volatilization. Some of these chemicals are
suspected to be neurotoxic, particularly triphenyl phosphate (TPP), tri-n-butyl phosphate
(TnBP) and tri-cresyl phosphate (TCP)6. Furthermore, halogenated alkyl phosphates have low
degradation potential and thus may be persistent7. As a result, OPFRs are considered as reemerging pollutants because of their increased use after BFR bans and their ubiquitous
occurrence in both indoor and outdoor environments 3.
The aim of this work was to develop an analytical method including sample preparation and
detection techniques enabling the extraction of 19 OPFRs from complex biological matrices,
and their subsequent identification and quantification at trace levels by using gas
chromatography and/or liquid chromatography coupled to tandem mass spectrometry (GCMS/MS and LC-MS/MS). For this purpose, an instrumental method was developed on GCMS/MS with satisfactory performances in terms of detection limits and linearity of calibration
curves. Another instrumental method is on its way of development on LC-MS/MS. On the
other hand, solid- phase extraction (SPE) along with a number of parameters was investigated
as a suitable purification technique. The work will extend to optimize the extraction procedure
and to implement the whole strategy for the analysis of fish samples as well as other foodstuffs
in order to analyze these contaminants at trace levels and hence to contribute to the evaluation
of Human dietary exposure.
References
1.U.S. Department of health and human services. (2012); Agency for Toxic Substances and
Disease Registry
2. Bergman A, Ryden A, J.Law R, de Boer J, Covaci A, Alaee M, Birnbaum L, Petreas M,
Rose M, Sakai S, Van den Eede N, Van der Veen N. (2012); Environ Int. 49: 57-82
3. Cristale J, Lacorte S. (2013); Journal of Chromato A.1305 : 267-275
4. Van der Veen I, De Boer J. (2012); Chemosphere. 88: 1119-1153
5. Brandsma S.H, De Boer J, Cofino W.P, Covaki A, Leonard P.E.G. (2013); Trends in
Analytical Chemistry. 43: 217-228
6. Ma Y, Cui K, Zeng F, Wen J, Liu H, Zhu F, Ouyang G, Luan T, Zeng Z. (2013); Analytica
Chemica Acta; 786:47-53
7. Van den Eede N, Dirtu A.C, Neels H, Covaci A. (2011); Environ Int.37: 454-461
8
MME. LECLERC LUCIE - 2ème année – ONIRIS session 1
Mother body weight at conception and energy intake are the main factors that explain
weight gain during growth in dogs
L. Leclerc1, 3, C. Thorin2, S. Serisier1, P. Nguyen3
1
Royal Canin Research Center, Aimargues, France
2
UPSP 5304 de Physiopathologie Animale et Pharmacologie Fonctionnelle, 3Unité de Nutrition
et Endocrinologie Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes
Atlantique, ONIRIS, Nantes, France
lucie.leclerc@oniris-nantes.fr
Introduction: In humans, it has been shown that overweight in adulthood could be predicted
early in childhood. Serisier et al. showed that a higher growth rate could be a risk factor for
later overweight in cats. The aim of our study is to identify early risk factors (from the
conception to the end of growth) to become overweight in adulthood for dogs and cats. The
preliminary results in dogs are presented here.
Animals, material and methods: Twenty-four female Beagle dogs were studied from birth to
the age of twenty-nine weeks. They were weighed weekly. After weaning at 10 weeks of age,
they were fed ad libitum every day for 3.5 hours. Individual food intake was recorded daily on
business days, and mean energy intake (MEI) was calculated from weaning to the age of 29
weeks.
Early-life data were provided by the breeding centre, including number of previous litters
(NbPL), mother’s age and body weight at conception (MBWc), weight gain during gestation,
litter size and body weight at birth of each puppy as well as father body weight at mating.
The effect of early-life factors on body weight at weaning and at 29 weeks of age (BW29) was
assessed by a multilinear model analysis. The effect of early-life factors on weight gain before
and after weaning were also assessed with the same statistical test. However, individual energy
intake as well as weaning BW has been added to the statistical model after weaning.
Results: No variable explained birth body weight. However, interestingly, weaning body
weight and weight gain from birth to weaning significantly depended on MBWc (P<0.001)
which was itself strongly associated with NbPL (P=0.003).
BW29 and weight gain from weaning to 29 weeks of age were associated with MEI (P<0.001).
In addition, BW29 depended on weaning BW (P<0.001).
Discussion and Conclusion: Before weaning, mother body weight at conception has a
significant impact on weaning BW and body weight gain from birth to weaning. Once dogs
started to consume manufactured diets, energy intake became the most significant parameter
which explained BW29 and body weight gain after weaning. Since mother body weight at
conception impacted weaning body weight which itself impacted BW29, mother body weight
at conception indirectly impacted BW29. These results suggest a cumulative effect of MBWc
before weaning and energy intake after weaning on final body weight. Many complementary
data, including resting energy expenditure, digestibility, inflammatory factors and hormone
levels should enable to better explain weight and fat gain during growth and the risk of
becoming overweight.
Serisier, Samuel, Alexandre Feugier, Claudie Venet, Vincent Biourge, and Alexander J.
German. 2013. “Faster Growth Rate in Ad Libitum-Fed Cats: A Risk Factor Predicting the
Likelihood of Becoming Overweight during Adulthood.” Journal of Nutritional Science 2
(April). doi:10.1017/jns.2013.10.
9
MME. MARTINEAU Anne-Sophie – 3ème année –Unité Nutrition et Endocrinologie
Session 1
The consumption of a grape and blueberry polyphenol-rich mixture is safe in dogs
AS Martineau1,4, V Leray1,4, D Gaudout2,4, A Lepoudère3,4, K Ouguerram1,4 and P
Nguyen1,4
1
Nutrition and Endocrinology Unit, Oniris, Vet School and CRNH Nantes
2
Activ’Inside, Libourne
3
SPF, ElvenFrance 4On behalf of Neurophenols® consortium
Grape polyphenols have beneficial health effects, but grape ingestion has been associated with
acute renal failure leading most often to fatal issue in dogs. The responsible factors are
unknown.
Our aim was to check the safety of a supplement of polyphenol-rich mixture (grape and
blueberry extracts (specific pet Neurophenols® blend)) in dogs.
Seventeen dogs were given, for 12 wks, capsules containing 4 (n=5) or 20 (n=6) or 40 (n=6)
mg/kg/d of the polyphenol mixture. Five control dogs were given maltodextrin capsules. Blood
and 24-h urine were taken before the beginning of the study (W0), then after 4 wks and 12 wks
of supplementation. Plasma standard analysis (creatinine (Creat), urea, minerals (Na, K, Ca, P),
total protein, hepatic biomarkers (ALT, ALP)) were assayed. New biomarkers that reveal early
renal damage were measured : plasma and urinary cystatin C (CysC), clusterin (Clu),
neutrophil gelatinase-associated lipocalin (NGAL). Urinary biomarkers/Creat ratios were
calculated. Data were compared with limits of reference intervals or with the highest values
from our control dogs and experimental dogs at W0.
Whatever the week, plasma standard analysis were below high limits of reference intervals.
Plasma CysC concentration was below our own high limit. Plasma Clu and NGAL
concentrations were below limits of reference intervals. Urinary CysC/Creat, Clu/Creat,
NGAL/Creat ratios were below our own limit.
Grape extract and blueberry extract (specific pet Neurophenols® blend) consumption was not
associated with any sign of renal failure and can be consumed safely in dogs.
MME. CANO Camille - 2ème année - INSERM U892, équipe 7b Session 2
Spontaneous wound healing in a mouse model of M. ulcerans infection: A hostpathogen cross-talk
Mycobacterium ulcerans infection or Buruli ulcer is a skin disease mainly affecting
children. In 1998, The World Health Organization (WHO) recognized it as an emerging
infectious disease in West and Central Africa with an important public health impact and
classified it as a neglected tropical disease. Buruli ulcer is the third most common
mycobacterial disease after tuberculosis and leprosy. Classically, Buruli ulcer is a necrotising
skin disease characterized by an indolent painless course leading to extensive cutaneous and
subcutaneous destruction, functional limitations and disabilities. These lesions are due to
mycolactone, the main virulence factor of M. ulcerans, which has cytotoxic,
immunomodulatory and analgesic properties. Since 2005, WHO recommends the use of
antibiotherapy (streptomycin and rifampicin) associated or not with surgery. Several months
after the onset of clinical signs, in the lack of treatment, lesions may evolve into a process of
spontaneous wound healing. This "cure", which however has important irreversible functional
consequences, demonstrates a control of infection by the host. Understanding the mechanism
involved in the process of spontaneous wound healing, both at the cellular and molecular
levels, will pave the way to new therapeutic strategies. For ethical reasons, the work on human
samples is impossible. However the commonly used mouse models to study the
pathophysiology of M. ulcerans infection show no spontaneous wound healing stage, thus
excluding the possibility to identify the involved mechanisms. In this context, a screening on
10
different mouse strains allowed us to identify a strain mimicking all stages of infection,
including spontaneous wound healing. Genomic and transcriptional studies in these mice
demonstrate a deficiency of a cell surface receptor mainly expressed by neutrophils and
macrophages. Functional tests demonstrate an antagonistic role of mycolactone on this
receptor, assuming its involvement in the pathophysiology of infection. These results open the
way toward the identification of the toxin / receptor interaction mechanisms and the
clarification of their involvement in the healing phenomenon.
M. FOUCHER Etienne – 3ème année – U892 Eq7 Session 2
IL-34 and M-CSF induce macrophages that promote the generation of Th17 cells via membrane IL-1α
Depending on their environment, and especially the nature of the cytokines, monocytes can differentiate into
macrophages (M) or dendritic cells (DC). In the past, we have demonstrated that M-CSF induce the
differentiation of human monocytes into immunoregulatory M. We have recently reported that IL-34, a second
ligand of the M-CSF receptor (c-fms or CD115), induces the differentiation of monocytes into CD14 high
CD163high CD1a- Mφ (IL-34-Mφ) that exhibit potent immunosuppressive properties (IL-10high IL-12low profile)
and decrease the proliferation of stimulated T cells. Interestingly, for all the parameters analyzed, IL-34-M are
phenotypically and functionally similar to M-CSF-Mφ. Moreover, the generation of IL-34-Mφ is not dependent
of endogenous M-CSF consumption.
As Mφ orchestrate the immune response, we evaluated the capacity of M-CSF-Mφ and IL-34-Mφ to polarize
human CD4+ T cells. Results showed that, unexpectedly, M-CSF-Mφ and IL-34-M promote the generation of
IL-17-producing Th cells, contrary to the pro-inflammatory IL-12high IL-10low GM-CSF-Mφ which favour the
generation of Th1 cells. More precisely, these M subsets switch non-Th17 memory CD4+ T cells into
conventional CCR4+ CCR6+ CD161+ Th17 cells, expressing or not IFN-. Finally, we have demonstrated that
the generation of Th17 cells is mediated via membrane IL-1α which is constitutively expressed by the two M
subsets.
In an attempt to identify strategies to prevent a deleterious accumulation of immunoregulatory M in some
pathological situations, we have shown that (i) IFN- and GM-CSF prevent IL-34-induced monocyte
differentiation into immunosuppressive Mφ and (ii) that IFN- switches established IL-34-Mφ into
immunostimulatory Mφ.
In conclusion, this study demonstrates that human M-CSF-Mφ, IL-34-Mφ, initially considered as antiinflammatory cells, induce in vitro Th17 cells via a constitutive expression of membrane IL-1α. This process
may contribute to maintain locally a restrained and smoldering inflammation required for angiogenesis and
metastasis in tumors.
MME. BACOU Elodie – 2ème année –IECM Session 2
Analysis of mixing-induced stress consequences on immune traits in pigs.
Elodie Bacou*a,b, Karine Haurognéa,b, Marie Allarda,b, Jordan Marchandb,a, Grégoire
Mignotb,a, Yvon Billonc, Jean-Marie Bachb,a, Pierre Mormèded, Julie Hervéb,a and Blandine
Lieubeaua,b.
a
INRA USC 1383, IECM, Nantes F-44300 France
LUNAM University, Oniris University Nantes EA 4644, IECM, Nantes F-44300 France
c
INRA UE 1372, GenESI, Saint-Pierre-d’Amilly F-17700 France
d
INRA UMR 1388, GenPhySe, Castanet-Tolosan F-31426 France
b
11
Pig husbandry is known as an intensive breeding system, piglets being submitted to multiple
stressful events such as early weaning, successive mixing and crowding. We hypothesize that
these events can affect pig health by increasing their sensitivity to bacterial infections while
decreasing their robustness.
Immune traits were recently described as descriptive indicators of pig health. Among them, the
whole blood assay, which measures cytokine release in whole blood in response to LPS
stimulation, was proposed to predict individual susceptibility to infectious diseases.
In this context, we aimed to analyse the effects of an acute stress, ie a one-hour mixing, on
several immune traits in 90 pigs: white blood cell count, lymphocyte subsets, phagocytosis and
ex-vivo production of cytokines (IL-8, IL-10, IL-12 and TNFcortisol and catecholamines are known as the main mediators of stress responses, we will
measure their levels in plasma samples and correlate them to immune traits.
We first demonstrated an increase in circulating white blood cell number associated to an
altered lymphocyte subset repartition (decrease in B and CD4+ T cells and increase in CD8+
and CD4+CD8+ T cells). Furthermore, one-hour mixing induced significant decrease in IL-8
and IL-10 ex-vivo productions while having no effect on IL-12 and TNFas on phagocytosis. Cortisol and catecholamine levels remain to be measured.
Altogether, our data already link a stressful event to altered immune responses in pigs, although
mediators still remain to be determined.
A better understanding of intensive farming practice consequences on immunity is mandatory
to increase pig robustness required for antibiotic use reduction (Plan Ecoantibio 2012-2016).
12
MME. YAP Michelle – 3ème année – UMR 1064 session 2
An increase of highly differentiated TEMRA CD8 T cells with potent effector function in
kidney transplant recipients with stable graft function revealed patients with at-risk of
long-term graft function.
Michelle Yap1 2 3, Françoise Boeffard1 2 3, AnnaickPallier1 2 3, Richard Danger1 2 3, Magali Giral1
2 3
, Jacques Dantal1 2 3, Yohann Foucher2 4, Cécile Guillot-Gueguen2, Jean-Paul Soulillou1 2 3,
Sophie Brouard1 2 3, and Nicolas Degauque1 2 3
1 INSERM, UMR 1064, Nantes, France
2 CHU de Nantes, ITUN, Nantes, France
3 Université de Nantes, Faculté de Médecine, Nantes, France.
4 Université de Nantes, EA 4275, Nantes, France.
Introduction: Kidney transplantation is considered the best treatment for patients with end-stage
renal disease; however it is still necessary to improve long term graft survival. While T
lymphocytes have been shown to be implicated in acute graft rejection, there is still little
information regarding the role of CD8 T cells in long term graft survival. We took advantage of
a large group of kidney transplant recipients enrolled in a prospective study to investigate the
role of CD8 T cells in long-term graft outcome.
Methods: 131 kidney graft recipients with a stable graft were enrolled into the prospective
study. Patients were enrolled at least 5 years post-transplant. At the time of inclusion, the TCR
Vβ repertoire (CDR3 length) was characterized using TcLandscape and a detailed profile of
CD8 T cells was obtained through multicolor flow cytometry. Patients were followed for more
than 6 additional years, during which 47 patients had undergone kidney graft degradation. In
addition, the functional properties of a sub-group of 32 patients who were matched for clinical
parameters (time post-transplantation, recipient and donor age) and selected according to the
CD8 phenotype (high vs. low TEMRA CD27-CD28-CD57+TBET+) were analyzed through the
secretion of pro-inflammatory cytokines IFNγ and TNFα and the expression of cytotoxic
surrogate marker CD107a upon polyclonal stimulation with plate bound αCD3 and αCD28.2
mAbs.
Results: 86 patients were identified to have an altered TCR Vβ repertoire and 45 patients had
an unaltered TCR Vβ repertoire. Accumulation of alteration in the TCR Vβ repertoire was
associated with an increase of TEMRA (CD45RA+CCR7-) CD8 T cells. Those CD8 T cells
displayed an activated phenotype (CD27-CD28-CD127-) and potent effector functions
characterized by an upregulation of cytotoxic molecules (PERFhiGZMB+) and effectorassociated molecules (TBEThi and CD57+). In patients with an increase of TEMRA CD27CD28-CD57+TBET+, short-term polyclonal stimulation of PBMC results in an increased
secretion of TNFα and IFNγ by CD8+TBET+ T cells and a vigorous cytotoxic function, as
exemplified by the expression of CD107a. Finally, the increase of highly differentiated
TEMRA CD8 T cells was associated with 1.96 high risk of kidney dysfunction (multivariate
model).
Conclusion: Despite our cohort of kidney graft recipients appeared to be stable and
homogeneous from a clinical perspective at the time of enrollment, the monitoring of CD8 T
cell phenotype and function and the characterization of the TCR Vβ repertoire reveals major
differences in the homeostasis of the CD8 T cell populations and in the long term outcome of
the kidney graft.
13
M. LAPERINE Olivier - 3ème année – UMR 791 Session 2
Roles of interleukins 33 and 36 in the pathogenesis of periodontal disease in mice
LAPERINE Olivier1,2, CLOITRE Alexandra1,3, CAILLON Jocelyne4, DAVIDEAU Jean-Luc5,
PALMER Gaby6, SOURICE Sophie1, PILET Paul1,2, GUICHEUX Jérôme1 ,2,3, BECKCORMIER Sarah1, LESCLOUS Philippe1,2,3
1
INSERM UMRS 791 – Laboratoire d’Ingénierie Ostéo-Articulaire et Dentaire « LIOAD »
STEP « Skeletal Tissue Engineering and Physiopathology » group ; 2Université de Nantes,
UFR Odontologie ; 3CHU de Nantes, Pôle Hospitalo-Universitaire 4 OTONN : 1 place Alexis
Ricordeau » - 44042 NANTES CEDEX 1 France 4UPRES EA 3826, Faculté de médecine,
Université de Nantes, Nantes France 5INSERM UMR 1109, “Osteoarticular and Dental
Regenerative NanoMedicine” Laboratory, Strasbourg, France 6Division of Rheumatology,
Department of Internal Medicine, University Hospital of Geneva, Geneva, Switzerland
Keywords: Periodontal disease, Interleukins, Bone loss
Periodontitis is an inflammatory disease of bacterial origin, triggering tissues surrounding the
tooth i.e. gingiva, periodontal ligament and alveolar bone. This disease is a major public
health issue. In France, nearly 85 % of the adult population is affected by periodontal disease
whose 10% presented a refractory type to the mechanical and / or medical treatments, leading
to the loss of many teeth. Actually, its immunopathogenic mechanisms remained poorly
understood. Following the bacterial stimulus, an inflammatory cascade is initiated via Tolllike receptors (mainly 2 and 4) to ultimately activate osteoclast function. Interleukin 33 (IL33) and IL-36 are new members of the IL-1 cytokine family of particular interest because of
their involvement in an inflammatory pathology showing immunosimilarities with
periodontitis, the rheumatoid arthritis. The objective of this study is to validate a mouse model
for periodontal bone loss and to investigate the potential involvement of IL-33 and IL-36 in
the pathogenesis of this disease. To this aim, we performed a mouse model of alveolar bone
loss by local bacterial infection (Porphyromonas gingivalis i.e Pg). The jaws of these mice
were analyzed by micro computed tomography, real-time RT-PCR and conventional
histological approaches (HES staining, immunohistochemistry). We validated this model
since we recorded a significant alveolar bone loss one month after induction of periodontal
disease. Interestingly, preliminary results showed an expression of IL-33 in the gingival
epithelium in affected animals whereas no labeling was discernible in controls. We also
recorded an increase of IL-33 expression in the connective tissue of affected animals
compared to the control mice. The real-time RT-PCR experiments showed an overexpression
of IL-36 mRNA in the gingiva of affected animals. Taken together, these data suggested a
potential role of these two interleukins in the immunopathogenesis of periodontitis. To further
investigate the molecular mechanisms of these two cytokines, we are now developing an in
vitro approach using gingival cells stimulated with LPS from Pg. Finally, we would like to
use this periodontal model in knockout mice for genes encoding IL-33 or its receptor IL1RL1.
Altogether, these approaches would allow a better understanding for a role of IL-33 and IL-36
in periodontal disease.
14
M. PATINEC Allan - 3ème année –UMR 892 session 2
Modulation of anti-infectious immunity by stimulation of circulating iNKT populations
in head trauma context.
In this study we focuse on the mechanisms that allow the establishment of anti-infectious
immunodeficiency in a context of head trauma.
An article published in Science on November 2011 (1) indicated that the loss of antiinfectious immunity observed after brain lesions could be associated with the direct action of
neurotransmitters, such as catecholamines, on lymphocyte populations. The authors
established in a mouse model that the iNKT population was the cornerstone of the
phenomenon leading to a switch in cytokinine secretion, towards an pro-infectious phenotype.
Our goal is to check if these results are revelant to the human situation.
The chosen axis is the study of the capacity of the different sub populations of iNKT cells to
respond to various stimuli, whether glycolipid or catecholinergic. We were able to highlight a
significant effect of the action of epinephrine on cytokinine secretion on PBMC, with a
decrease in the IFN-g secretion and an increase of the IL-10 level, in agreement with the
establishment of a regulatory pro-infectious state.
Very interestingly, an endogenous ligand of human iNKT cells, the β-glucosylceramid
significantly increased the IFN-γ/IL-10 ratio compared to the canonical ligand KRN7000 (αgalactosylceramide), suggesting a potential for reversion of the phenomenon induced by
epinephrine.
Preliminary experiments using sera from patients after cardiac arrest enabled us to observe a
change in iNKT cytokine secretion compatible with the establishment of a pro-infectious
profile, and concordant with the effects highlighted with epinephrine.
These results are consistent with the presence of catecholinergic molecules in the serum of
patients after brain-injury that may contribute to the loss of anti-infectious immunity through
their effect on the balance of cytokine secretion.
(1) Wong CH, Jenne CN, Lee WY, Léger C, Kubes P. Functional innervation of hepatic
iNKT cells is immunosuppressive following stroke. Science 2011;334:101-5.
M. D’ALMEIDA Sènan – 3ème année – Inserm U892 Session 3
THE ECTO-NUCLEOTIDASE CD39 IS INVOLVED IN THE ACQUISITION OF AN
IMMUNOSUPPRESSIVE PHENOTYPE BY TUMOR -ASSOCIATED MACROPHAGES
Tumor-associated macrophages (TAMs) are the main immunosuppressive myeloid cells in the
tumor microenvironment. To date, the mechanisms involved in their generation remain largely
unknown. It has been shown that ATP, shown to recruit myeloid cells into the tumor, became
an immunosuppressive tool in cancer due to its rapid catabolism into adenosine by
ectonucleotidases. Previous studies have also underlined the role of the ectonucleoside
triphosphate diphosphohydrolase-1 (NTPDase1 or CD39) in the biology of regulatory T cells
[1]. Thus, they represent therapeutic targets to overcome tumor-associated
immunosuppression.
OBJECTIVE: To investigate the role of CD39 and ATP metabolism in the biology of TAM.
METHODS: TAMs were purified from ovarian cancer patients. By performing a robust
functional assay, mature IL-1 production by macrophages reflected the presence of
extracellular ATP.
RESULTS: In this study, we showed that TAM purified from ovarian cancer patients and
immunosuppressive macrophages generated in vitro express high levels of membrane CD39
compared to immune-stimulatory macrophages. Furthermore, we evidenced the ability for the
pharmacological inhibition of CD39 (POM-1), to potentiate expression of IL15
immunosuppressive macrophages generated in vitro, while decreasing their
immunosuppressive functions (IL-10 secretion, IL-27 secretion, specific CD163 markers and
their ability to phagocyte). Among the several cytokines tested, it was of note that the
blockade of the cytokine IL-27, by down-modulating the surface expression of CD39, was of
particular importance for IL-1 and IL-10 secretion.
CONCLUSION: As a matter of fact, this study underlines the pivotal role of CD39 via ATP
metabolism and/or via the adenosine pathway in the acquisition of an immunosuppressive
phenotype by TAM and in vitro generated immunosuppressive M that their blockade could
be used as an anti-tumoral therapy. Collectively, these data suggest that CD39 maintains the
immunosuppressive phenotype of TAM from ovarian cancer. This work brings new
information on the nature and profile of tumor-infiltrating immune cells.
KEY-WORDS: Tumor-Associated Macrophages, NLR, inflammasome, IL-1, ATP, purinergic
signaling, CD39, IL-27, ovarian cancer
1.
Antonioli, L., et al., CD39 and CD73 in immunity and inflammation. Trends Mol Med,
2013. 19(6): p. 355-67.
MME. RUGGERI Tiphaine – 2ème année – EA 3826 Session 3
1,2
Tiphaine RUGGERI, 1 University of Nantes faculty of medicine unit EA3826 2 Regional
center for cancer research Nantes/Angers INSERM UMR-892, 2nd year of thesis
Post-Traumatic Immunosuppression in a Mouse Model of Spinal Cord Injury
Introduction. High level Spinal Cord injuries (SCI) lead to an immunosuppression that
sensibilizes individuals to nosocomial infection. Mechanisms involved in immunosuppression
after nosocomial infection following a severe trauma of SCI are still poorly understood. Our
study aims to validate a spinal cord injury (SCI) model in terms of susceptibility of SCI mice
to Staphylococcus aureus (S. aureus) pneumonia, a common bacteria involved in nosocomial
infections after acute immunosuppression. Methods. A surgical procedure involving a
medullary dislocation between thoracic vertebra T3 and T8 was established. This dislocation
led to a dysregulation of the sympathetic nervous system in the spleen, a major lymphoid
organ in mice. 3 groups were studied: control group (without anesthesia and dislocation),
Sham group (with anesthesia but without dislocation) and SCI group (with anesthesia and
dislocation). First we analyzed consequences on weihg spleen after trauma. Then, mortality
(survival curves) of S. aureus infected mice was observed for 10 days to study susceptibility
of mice to S. aureus. Mice were infected at different days, post-operatively at D1, D3 or D8.
In parallel, inflammatory cells infiltration into the lungs was assessed by histology and SIOX
analysis (Fiji software) to confirm infection lungs mice. Second, we studied
immunosuppression by level of TNF-α determined by ELISA after LPS stimulation of blood
samples were evaluated. And also by expression of inflammatory cytokines, TNF-α and IL1β, were determined in lungs, as well as pro or anti-inflammatory cytokines, IFN-γ and IL-10,
in spleen. Results. First, after medullary dislocation, we observed a decrease of spleen weight
in SCI mice 48h post-operative as compared to Sham mice that confirm interaction between
nervous system and immune system. Three days post-operative, SCI mice were more
susceptible to infection than the Sham mice: 50% mortality was observed in SCI group (48h
after the bacterial challenge) as compared to a 25% mortality rate for Sham animals. No
difference of cells infiltration after infection in Sham and SCI was observed. In infected
lungs, TNF- α and IL-1β concentrations increased in Sham and even more in SCI group.
Second, as previously demonstrated in other acute stresses (Asehnoune et al., 2006), a
hyporeactivity after LPS stimulation of whole blood culture (a classical marker of
immunosuppression) was observed (an increase of TNF-α was observed in blood samples
stimulated with LPS from Sham mice, but not in SCI mice). After infection, expression of
IFN-γ increased in spleen for Sham mice but not for SCI mice. On the other hand, an increase
16
of IL-10 was observed in SCI group but remained at a basal level in Sham group. Finally,
studies on marker cells showed an increase of PD1 expression on lymphocytes T at 16h postoperative for SCI animals as compared to Sham mice in spleen. Conclusion. SCI mice show
an immunosuppression state as compared to Sham mice. SCI mice seem more susceptible to
S. aureus infection than Sham mice that confirm immunosuppression presence. After
infection, SCI animals highly express IL-10 in spleen, which confers anti-inflammatory state,
whereas Sham mice highly express IFN-γ, which confers pro-inflammatory state. These
results show that SCI mice are in anti-inflammatory state favorable for immunosuppression.
We will continue our project in study of interactions between dendritic cells, Natural Killer
cells and lymphocytes T, to understand mechanisms lead immunosuppression and found
therapeutic method against immunosuppression.
Asehnoune, K., Fitting, C., Edouard, A.R., Cosson, C., Benhamou, D., Cavaillon, J.-M., and
Moine, P. (2006). Influence of resuscitation volume on blood cells TNF production in a
murine model of haemorrhage. Resuscitation 68, 127–133.
M. AVRIL Pierre - 3ème année – UMR 957 Session 3
EFFECT OF SOLUBLE FACTORS PRODUCED BY FAT ON CARCINOLOGICAL
RISK
In a clinical case, a late and local recurrence of osteosarcoma has been observed following
autologous fat graft. The involvement of the adipose tissue in this recurrence has been
demonstrated in murine models of osteosarcoma [1]. Autologous fat grafting-or-lipofilling- is
more widely used in reconstructive procedures following breast cancer. In the case of highgrade breast carcinomas, a recent retrospective study showed that lipofilling was a potential
risk of local recurrence in young patients with a high mitotic index (Ki-67> = 14%)
[2]
.Proliferation assays and flow cytometry analyzes performed on these cells have
demonstrated a pro-tumor effect in vitro characterized by an accelerated cell cycle of adipose
tissue mediated by cell-free fraction containing fat soluble factors (= fat-CM). Tumor
recurrence can occur from residual cancer stem cells (CSC) characterized by a low rate of
proliferation and by their ability to form a complete tumor. In order to reproduce the
acquisition of a CSC-like phenotype, tumor cells were cultured in oncospheres and qPCR
analyses were performed. In conditions of loss of adhesion, proliferation of osteosarcoma
cells was decreased while their stemness marker levels were increased. Mammary carcinoma
cells cultivated in oncosphere showed the acquisition of epithelial to mesenchymal transition
markers. In these culturing conditions, fat-CM could partially modulate these phenotypic
changes. Identify the soluble factor(s) responsible of the pro-tumor effect of adipose tissue
should lead to a better understanding the interactions between tumor cells and their
environment. This could help to establish new therapeutic targets for cancer treatment and to
secure the lipofilling procedure, a main reconstructive surgical technical for patients.
REFERENCES
[1] Perrot P, Rousseau J, Bouffaut AL, et al. PLoS One. 2010; 5: e10999
[2] Petit JY, Rietjens M, Botteri E, et al. Ann Oncol. 2013
17
MME. MONTAUDON Elodie – 3ème année – PAPF ONIRIS Session 4
Cardiovascular effects of anti- β1– and β3–adrenergic receptor antibodies in Lewis rat
E. MONTAUDON*, J-C. DESFONTIS, L. DUBREUIL, Y. MALLEM.
LUNAM Université, Oniris, «UPSP 5304 de physiopathologie animale et pharmacologie
fonctionnelle», Atlanpole La Chantrerie, BP 40706, Nantes, F-44307, France.
β1– and β3–adrenerdic receptor (AR) autoantibodies (AABs) were detected in patients
with dilated cardiomyopathy (DCM). Many studies have shown that β1–AABs play an
important role in the pathogenesis of DCM. Our previous work has shown that the
immunization of rats for 3 months with the second extracellular loop (ECII) of β1–AR
induced endothelial dysfunction in aorta. Those results suggest that β1–AABs could have a
vascular pathogenic action. However, until now, no study has explored the cardiovascular
effects of β3–AABs.
The aims of this sudy was to evaluate whether β3-antibodies (Abs) possess β3-AR
agonistic effect and whether active immunization producing β3-ABs or β1-ABs has deleterious
effects on cardiac and vascular reactivity in Lewis rats.
Lewis rats were immunized for 6 months with peptidic sequences corresponding to the
ECII of β3-AR or β1-AR. Agonistic effect of β3-AABs was evaluated on electrically fieldstimulated isolated cardiomyocytes from adult rabbit by measuring the cell shortening.
Echocardiographies and ex vivo studies on isolated perfused heart and isolated thoracic aorta
were conducted on immunized rats. Finally, the expression of β-AR was quantified by
immunofluorescence approach.
SR58611A (10 nM), a preferential β3–AR agonist and purified β3-ABs (25 g/mL)
induced a decrease of cell shortening (-39.64.4% (n=11) and -18.53.9% (n=10)
respectively). This decrease was significantly inhibited when the cardiomyocytes were
preincubated with the L-748337 (1 M), a selective β3-AR antagonist (p<0.05), and with
pertussis toxin (0.3 g/ml), a Gi protein inhibitor (p<0.05).
Echocardiographies revealed a decrease of the fractional shortening and ejection fraction only
in rats immunized against β1–AR. However, the study on isolated heart showed a decrease of
the isoprenaline-induced lusitropic and inotropic effects in both animals immunized against
the β1– and β3–AR. These systolic and diastolic dysfunctions are correlated with a decrease in
the expression of β1–AR and an increase of β3–AR in rats immunized against the β1–AR and
an increase of both β3– and β1–AR in rats immunized against β3–AR.
In thoracic aorta, β1–AR relaxation was impaired in rats immunized against β1–AR while it
was improved in those immunized against β3–AR.
Those results show for the first time that β3–ABs induced a β3–AR partial agonist-like
activity. In the heart, they would have a pathogenic action while in blood vessels, they would
have a protective effect offsetting the endothelial dysfunction caused by β1–ABs.
18
MME. BICHON Emmanuelle – 2ème année – LABERCA/ONIRIS Session 4
GC-APCI-MS/MS for making natural steroids analysis more comprehensive and
compatible with ultra trace detection
Authors: E. Bichon, S.Prevost, K. Hening, F. Monteau, B. Le Bizec.
To understand the mechanism of action of certain chemical endocrine disrupters on the human
steroidome, an amazing effort is still today necessary so that the measurement of a wide range
of gonadic steroids become possible and compatible with the ultra trace amounts of these
substances in –sometimes- very complex biological matrices (available in very limited
amount). For androgens, analytical strategies rely in general on gas chromatography coupled
to mass spectrometry mainly using triple quadrupole technologies and electron ionization.
Because of the high energy involved, steroid fragmentation is extensive and often leads to
unsatisfactory results. To improve the signal specificity and eventually the quality of the
delivered signals, soft ionization strategies must be envisaged. Potentialities of atmospheric
pressure chemical ionization (APCI) have been investigated in this project. An exhaustive
comparison of the physical conditions, e.g. corona discharge, interface temperature and gas
flow, as well as different way of preparing target steroids (i.e. derivatization reaction) has
been conducted. Observed performances will be discussed with regard to current classical
analytical strategies used in clinical chemistry. Perspectives given by the significant
improvement of the performance will be discussed.
MME. COLIN Estelle -3ème année –UMR 1083 Session 4
Loss of function mutations in WDR73 are responsible for microcephaly and steroid
resistant nephrotic syndrome : Galloway-Mowat syndrome.
Estelle Colin*, Evelyne Huynh Cong, Géraldine Mollet, Agnès Guichet, Olivier Gribouval,
Christelle Arrondel, Olivia Boyer, Laurent Daniel, Marie-Claire Gubler, Zelal Ekinci, Michel
Tsimaratos, Brigitte Chabrol, Nathalie Boddaert, Alain Verloes, Arnaud Chevrollier, Naig
Gueguen, Valérie Desquiret-Dumas, Marc Ferré, Vincent Procaccio, Laurence Richard,
Benoit Funalot, Anne Moncla, Dominique Bonneau, Corinne Antignac
Laboratoire: UMR INSERM 1083 – CNRS 6214 – BNMI
Galloway-Mowat syndrome (MIM 251300) is a rare autosomal recessive condition
characterized by nephrotic syndrome associated with microcephaly and neurological
impairment. Through a combination of autozygosity mapping and whole exome sequencing,
we identified WDR73 as a Galloway-Mowat causing gene in two unrelated families. WDR73
encodes a WD40-repeat containing protein of unknown function. Here, we show that the
WDR73 protein is expressed in the brain and in the kidney, and is located diffusely in the
cytoplasm during interphase but relocalizes to spindle poles and astral microtubules during
mitosis. Mutant fibroblasts from patients and WDR73-depleted podocytes displayed abnormal
nuclear morphology, low cell viability and alterations of the microtubule network. These data
suggest that WDR73 plays a crucial role in the maintenance of cell architecture and cell
survival. Altogether, WDR73 mutations cause Galloway-Mowat syndrome in a particular
subset of patients presenting with late-onset nephrotic syndrome, post-natal microcephaly,
severe intellectual disability and homogenous brain MRI features. WDR73 is another example
of a gene involved in a disease affecting both the kidney glomerulus and the central nervous
system.
19
MME. BON Nina – 2ème année – INSERM UMRS791 Session 4
Insight into the extracellular phosphate sensing mechanism, the key step for an
appropriate FGF23 secretion
BON Nina (2nd year PhD student)1, 2, BOURGINE Annabelle1, 2, COUASNAY Greig1,
2
, WEISS Pierre1, 2, GUICHEUX Jérôme1, 2, BECK-CORMIER Sarah1, 2, BECK
Laurent1, 2
1
2
INSERM UMRS791, Laboratoire d’Ingénierie Ostéo-Articulaire et Dentaire, Nantes
Université de Nantes, UFR Odontologie, Nantes
Phosphate (Pi) is known to be a vital ion involved in structural and metabolic
functions. More recently, several studies have suggested that Pi is a signaling molecule.
Notably, we have shown in pre-osteoblasts (MC3T3-E1), in chondrocytes (MC615) and
in odontoblasts (MO6-G3) that an increase in extracellular Pi concentration leads to the
ERK1/2 MAPK pathway activation. This pathway notably allows the upregulation of
genes involved in bone mineralization. However, the mechanism underlying the sensing
of extracellular Pi variations is still unclear. Recently, we have shown in vitro in MC3T3E1 or MC615 cells that both PiT1 and PiT2 proteins are likely to be involved in this
process. PiT proteins are multifunctional proteins originally described as retroviral
receptors and sodium-Pi cotransporters. Additional experiments have shown that PiT2
could oligomerize in response to extracellular Pi variations. Considering the strong
similarity between PiT1 and PiT2 we hypothesize that a PiT1-PiT2 interaction could
underlie the mechanism whereby the ERK1/2 pathway is activated. To investigate this
interaction, we are using a Bioluminescence Resonance Energy Transfer (BRET)
approach. To this end, we have fused the coding sequence of YFP or Renilla luciferase
on PiT proteins using the Fast Cloning method. To assess the interaction of PiT proteins,
we are also using co-immunoprecipitation upon Pi stimulation with a BS3-based
crosslinking approach.
In vivo, an increase in serum Pi concentration was shown to stimulate the secretion
of Fibroblast Growth Factor 23 (FGF23). FGF23 is produced and secreted by the
osteocytes and is the main hormone controlling the Pi homeostasis. Therefore to study
the physiological relevance of the involvement of PiT proteins in Pi sensing, we have
developed new mouse models carrying specific PiT1 and/or PiT2 deletions in osteocytes.
To this aim, we have generated PiT1lox/lox, PiT2lox/lox and PiT1lox/lox;PiT2lox/lox mice using
homologous recombination, and crossed them with Dentin Matrix Protein 1 (DMP1)-Cre
transgenic mice. When the Pi food supply is normal, the gross phenotype of these mice is
not affected. However, feeding mice with low- or high-Pi diets resulted in aberrant
regulation of FGF23 secretion in mutant mice.
This study will unravel the molecular mechanisms whereby Pi informs the cell of its
own extracellular variation which will lead to an appropriate FGF23 secretion resulting
in a normal regulation of Pi homeostasis.
M. CLAIREMBAULT Thomas - 3ème année – UMR 913 Session 4
Title: Structural alterations of the intestinal epithelial barrier in Parkinson’s disease
Abstract
Functional and morphological alterations of the intestinal epithelial barrier (IEB) have been
20
consistently reported in digestive disorders such as irritable bowel syndrome and
inflammatory bowel disease. There is mounting evidence that Parkinson’s disease (PD) is not
only a brain disease but also a digestive disorder. Gastrointestinal involvement is a frequent
and early event in the course of PD, and it may be critically involved in the early development
of the disease. We therefore undertook the present survey to investigate whether changes in
the IEB function and/or morphology occur in PD. Colonic biopsies were performed in 31 PD
patients and 11 age-matched healthy controls. The para- and transcellular permeability were
evaluated by measuring sulfonic acid andhorseradish peroxidase fluxrespectively, in colonic
biopsies mounted in Ussing chambers. The expression and localization of the two tight
junctions proteins ZO-1 and occluding were analyzed by Western blot and
immunofluorescence, respectively. The para- and transcellular permeability were not different
between PD patients and controls. The expression of occludin, but not ZO-1, was significantly
lower in colonic samples from PD patients as compared to controls and the cellular
distribution of both proteins was altered in colonic mucosal specimens from PD patients. Our
findings provide evidence that the IEB is morphologically altered in PD and further reinforce
possible the role of the gastrointestinal tract in the initiation and/or the progression of the
disease.
MME. ANDRE Emilie – 3ème année – Inserm U1066 Session 5
Neuronal commitment of mesenchymal stem cells with nanoparticles transporting siRNA
André E. 1,2 ; Seijo B 3,4 ;Sindji L 1,2; Resnier P. 1,2 ; Pensado A.3,4 ; Schiller PC. 5 ; Passirani
C. 1,2 ; Sanchez A. 3,4 and Montero-Menei CN.1,2 *
1 PRES LUNAM – University of Angers, F-49933 Angers, France
2 INSERM U1066 – Micro et Nanomédecines Biomimétiques, 4 rue larrey, F-49933 Angers,
France
3 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University
of Santiago de Compostela, Campus Vida, 15782 Santiago de Compostela, Spain.
4 Molecular Image Group, University of Santiago de Compostela Clinical Hospital, Travesía
da Choupana, 15706 Santiago de Compostela, Spain.
5 Department of Orthopaedics, University of Miami Miller School of Medicine, FL, USA
emilie.andre01@gmail.com
Abstract
Introduction : Hungtington’s disease (HD) is an autosomal dominant genetic disease,
associated with the progressive loss of the GABAergic neurons in the striatum. Cellular
transplantation is the only therapeutic demonstrated a benefit to restore lost cells. Clinical
studies with foetal GABAergic precursors have shown promising results, but problems with
availability and ethical issues limit their use.
Mesenchymal stem cells (MSC) are an interesting source of cells for brain
regenerative medicine and more particularly the “Marrow-Isolated Adult Multilineage
Inducible” (MIAMI) cells a homogeneous subpopulation, which express several pluripotency
markers (Oct4, Sox2, Nanog), may differentiate toward a neuronal phenotype and secrete
tissue repair factors (1)(2).
In order to enhance their neuronal differentiation, we chose to use small interfering
RNA (siRNA) against the neuronal inhibitory factor REST to commit them toward a
neural/neuronal phenotype(3). Non-viral nanoparticle-based vectors, which have an efficient
transfection level, associated to low toxicity, have been developed for this neuronal
programming purpose.
Materials and methods: Two different novel systems have been formulated for
21
neuronal programming: lipid nanocapsules (LNC) and nanoparticles based on sorbitan esters
(Span). Adapting the method by Heurtault et al (4), we optimized basic lipid nanocapsules
(LNC) associating siRNA (size: 95nm and ζ potential : +10mV). On the other hand, we
developed nanoparticles based on sorbitan esters (Span) (5) and pullulan associating siRNA
(size: 200 nm, ζ potential: - 43 mV).
Results: Both systems provide an efficient siRNA association (around 50%)
determined by UV spectrophotometer, showing appropriate stability during storage (1 month)
and a good efficiency of transfection performed by RT-qPCR and microscopy confocal. An
important down-regulation was achieved using the siRNA-LNC even at lower concentrations
to those described for a transfection reagent such as Oligofectamine in the the manufacturer’s
recommendations (Oligofectamine was used with 250ng and we observed 85% of downregulation with 50ng of siRNA-LNCs)Gene expression analysis revealed that mainly MIAMI
cells are able to differentiate into neuron-like cells, expressing β3-tubulin and tyrosine
hydroxylase, 48h after transfection. Additionally, Span based nanosystems showed a more
favourable cytotoxicity profile when compared to the other nanosytem.
Conclusion: These results show the potential interest of this approach for tissue engineering
strategies and we will graft neuronal committed cells in the ex vivo models developed in our
laboratory.
1. Tatard VM,et al. Bone. 2007 Feb;40(2):360–73.
2.
Roche S, et al. Int J Pharm. 2013 Jan;440(1):72–82.
3.
Yang Y,et al. Exp Cell Res. 2008 Jul;314(11-12):2257–65.
4.
Heurtault B, et al. Pharm Res. 2002;19:875–80.
5.
Sánchez, A.; Seijo, B.; Zorzi, G.K.; Calvalho, E.S.; Pensado, A. Sistemas
nanoparticulares elaborados a base de ésteres de sorbitán. P201131812 WO : 2013068625 A1
PCT: ES2012/070774
M. Kizito-Tshitoko TSHILENGE - 2ème année - UMR 1089 Session 5
Gene transfer in retinal ganglion cell
Recombinant adeno-associated viral (rAAV) are powerful and promising vectors to treat IRD
patients because its tropism and transduction pattern are well characterized in retina. Two
modes of vector injection are well characterized as function of retinal layer targeted: (i)
Subretinal injection to transduce cells localized in outer retinal layers such as photoreceptors
and RPE cells and (ii) Intravitreal injection to transduce cells localized in inner retinal layers
such as retinal ganglion cells (RGCs), amacrine and Müller cells. Nevertheless, rAAVmediated gene transfer in RGCs following intravitreal injection in dog eyes is poorly
characterized. This present study was designed in the effort to optimize and characterize
rAAV2/2 transduction in RGCs of dog. Here, we describe for the first time that rAAV2/2 is
able to mediate a strong and efficient transduction of RGCs in dog retina.
MME. HENRY Nina – 2ème année – U791 Session 5
Nano-reinforced hydrogel and protein release in the context of regenerative medicine of the
intervertebral disc
Nina Henry1,2,3, Johann Clouet1,3,5,6, Catherine Le Visage1,3, Jean Le Bideau2, Jérôme
Guicheux1,3,4
1
INSERM, UMRS 791, LIOAD, Equipe STEP, Nantes, France ; 2 CNRS, UMR 6502,
IMN, Equipe PMN, Nantes, France ; 3 Université de Nantes, UFR Odontologie, Nantes,
22
France ; 4 CHU Nantes, PHU 4 OTONN, Nantes, France ; 5 CHU Nantes, PHU 7 BiologiePharmacie, Pharmacie Centrale, Nantes, France ; 6 Université de Nantes, UFR Sciences
Biologiques et Pharmaceutiques, Nantes, France.
Intervertebral disc (IVD) degeneration is one of the major causes of low back pain
(LBP)1. Currently, LBP is mainly managed by pharmacological treatments and, if
unsuccessful, by surgical procedures. During IVD degeneration the Nucleus pulposus (NP,
inner part of the IVD) is particularly impacted by dehydration and cell loss. In this context,
regenerative medicine and particularly tissue engineering disc repair strategies clinically
address early disc degeneration and could offer less invasive and etiological alternatives to
spinal reconstructive surgery2,3. Our laboratory developed an injectable, self-crosslinking
silanized hydroxypropylmethylcellulose (Si-HPMC) based hydrogel in order to restore the
hydration of the NP and provide a 3D micro-environment beneficial for cell proliferation4.
The hydrogel was further reinforced by mesoporous silica nanofibers (MSNF)5 synthesized
by the co-condensation of silica precursors6. To optimize stem cell differentiation and enhance
synthesis of an appropriate in situ extracellular matrix, a bioactive hydrogel was designed via
growth factor addition to the MSNF.
Lysozyme was chosen for preliminary MSNF adsorption/desorption assays. It was
added to a MSNF suspension in various pH conditions (4, 7 and 10), concentrations (1, 10
and 25 mg/ml) and stirring time (1h to 48h). Release tests were performed in PBS, pH 7.2 at
37°C during 20 days. Lysozyme concentrations were determined by a colorimetric method
and its activity detected enzymatically.
In this study, we demonstrated that lysozyme can be adsorbed on MSNF. The highest
lysozyme adsorption was obtained at pH 10, concentration of 25 mg/ml and after 48h of
stirring, corresponding to a 50% adsorption efficiency. Interestingly, adsorption efficiency
reached 95-100% for lower concentrations (1 mg/ml to 10 mg/ml). This information will be of
particular importance when using expensive molecules i.e. growth factors. Following
incubation experiments under physiological conditions, a burst release was observed after 1h.
In subsequent time points, desorption rate was stabilized at 25%. At all time points,
the released lysozyme was enzymatically active.
These preliminary experiments demonstrated the adsorption/release capacity of the MSNF.
The next step of this project will consist in the use of Growth Differentiation Factor-5 whose
pivotal role in the differentiation of stem cells into IVD cells has been demonstrated7. Its
physicochemical properties, close to those of lysozyme, allow us to hypothesize a similar
behavior in terms of adsorption/release.
M.HUET Simon - 3ème année – Affilogic SAS UMR CNRS 6286 Session 5
Rational design of Nanofitins targeting a defined epitope
Nanofitins belongs to the family of affinity protein scaffold, and constitutes as such an
alternative to antibodies. They offer the advantages of being small, single chain, extremely
stable to both temperature and pH, as well as being highly expressed in simple expression
systems like E. coli. Their generation using in vitro selection system allows to obtain hits
from large numbers of variants, which need then to be screened out for the desired properties.
This screening effort can be further densified when the targeting of a very defined epitope is
required. Assisted-generation of Nanofitins through the computated design of interaction
surfaces would be useful to complement this approach and maximize the screening outputs
since it would allow to target specific regions of the protein of interest, generally leading to
predictable effect of the designed binder.
23
We describe a computational method for the rational design of Nanofitins binding a targeted
surface patch of interest on a macromolecule. Prediction algorithms enforced by published
datas are used to identify favorable regions of the target surface for protein-protein
interaction. Complexes generated in this way are used as a starting point from which the
Nanofitin interaction surface is subjected to de novo protein design, and sequences providing
the highest complex stability are sorted out. The method was successfully used to perform the
entire in silico design of Nanofitins binding a selected surface patch of the Green Fluorescent
Protein, as confirmed by in vitro assay.
This work provides a promising method to generate designed binding proteins that, if
combining high affinity for their target and strong specificity of the interaction region, would
narrow down the screening effort to generate therapeutically relevant Nanofitins.
24
MME. Vandamme Céline – 2ème année – Inserm UMR 1089
Session 5
Detection and Characterisation of Human anti-AAV CD8+ T Cells using MHC class I
Multimer-based Magnetic Enrichment
Vandamme Céline, Hesnard Leslie, Guilbaud Mickaël, Devaux Marie, Jaulin Nicolas, Le Duff
Johanne, Bonneville Marc, Moullier Philippe, Saulquin Xavier and Adjali Oumeya.
Recombinant Adeno-Associated Virus vectors (rAAV) represent the most largely used
platform for in vivo gene therapy. Despite promising results in large animal models and clinical
trials, pre-existing antibody and memory CD8+ T cell responses directed against the capsid of
rAAV remain one of the major hurdles to the safety and efficiency of in vivo rAAV-mediated
gene transfer. Particularly, the impact of pre-existing capsid-specific CD8+ T lymphocytes on
gene transfer remains elusive, all the more so since their detection and subsequent evaluation
have proven to be a challenge with currently available tools (such as ELISpot or intracellular
cytokine staining assays) because of their scarcity.
Combining major histocompatibility complex class I (MHC I) multimer staining with
magnetic enrichment, we were able to detect AAV2 and AAV8 capsid-specific CD8+ T cells ex
vivo, among peripheral blood mononuclear cells from healthy donors (at frequencies ranging
from 1e-5 to 7e-4 among CD8+ T cells). Using flow cytometry, we could further sort AAV8
capsid-specific CD8+ T cells and generate their corresponding clones. Undergoing
characterisation and functional studies of the clones generated suggest a variety of activation
profiles in terms of degranulation activity, IFN-γ and TNF-α expression. Evaluation of AAV8specific T cell clones’ killing activity on rAAV8-transduced target cells is currently under
investigation, as is a most comprehensive study of their cytokine secretion profiles.
Elucidating the different activation patterns of AAV capsid-specific CD8+ T lymphocytes
will be important for the understanding of the onset of pre-existing anti-AAV immunity on
rAAV-based gene transfer and its impact on clinical outcome.
MME. MALHAIRE Hélène - 3ème année – UMR 1066
ORAL
FORMULATION
OF
PROTEINS:
DEVELOPMENT
CHARACTERIZATION OF AQUEOUS CORE NANOCAPSULES
AND
Hélène Malhaire a,b*, Jean-Pierre Benoîta,b and Frédéric Lagarcea,b,c
a
LUNAM – Université d’Angers, F-49933 Angers, France
b
INSERM U1066 – Micro et Nanomédecines Biomimétiques, 4 rue Larrey, Angers, France
c
Centre Hospitalier Universitaire Angers, Pôle Pharmaceutique, Angers Cedex, France
* 3rd year PhD student
The oral route is easy, convenient and comfortable for patient. Unfortunately, since the main
role of the gastrointestinal tract is the digestion of nutrients, it is also the worst way for a protein
to achieve the blood pool. The challenge is thus to protect the therapeutic protein against this
harsh environment and to carry it until the blood pool to enable the cure of common chronic
disease.
Aqueous core nanocapsules were prepared according to a phase inversion method and
therapeutic protein was introduced right before a quench with isopentane (1, 2). Size, PDI and
zeta potential (ZP) were assessed up to 21 days after preparation. Encapsulation efficiency and
isopentane residue were determined by high performance liquid chromatography and gas
chromatography, respectively. Stability in fasted and fed state simulated intestinal fluids was
also performed (3). Structure, shape and size were confirmed by imaging in SEM, cryo-TEM
and AFM.
These monodispersed 200 nm nanocapsules exhibit a negative zeta potential and good
encapsulation efficiency (EE=90%) with a protein pay-load of 1.8 µg/mL. Isopentane residue is
25
below 10 ppm, which is much less than the minimum toxic threshold of a class 2 solvent
according to the international conference of harmonization. Good shelf-life stability is
highlighted by the consistency of size, PDI and ZP up to 21 days. Moreover, no change of these
parameters over the 6 hours study in simulated fluids lets expect good stability in small intestine,
whether at fasted or fed state. Images are pending.
Nanocapsules are thus able to efficiently encapsulate a therapeutic protein. Their stability in
storage and in simulated media lets expect a promising future. Toxicity assays and mechanistic
studies to estimate oral absorption will be shortly performed before an evaluation of the
therapeutic efficiency in rat.
1.
2.
3.
The research leading to these results has received funding from the European Community's Seventh Framework
Programme (FP7/2007-2013) under grant agreement n° 281035.
References:
N. Anton, J.-P. Benoit, P. Saulnier, J. Drug Deliv. Sci. Technol. 18, 95–99 (2008).
N. Anton et al., Langmuir. 25, 11413–11419 (2009).
E. Jantratid et al., Eur. J. Pharm. Sci. 37, 434–441 (2009).
26
POSTERS
27
1- Marie-Astrid BOUTET - 2ème année - Inserm UMR 957
Expression of IL-36,  and  in inflammatory diseases: example of rheumatoid arthritis
M-A. Boutet*, M. Gahier, C. Darrieutort, C. Charrier, R. Brion, M. Berreur, J. Chesneau, D.
Heymann, B. Le Goff, F. Blanchard
The IL-1 family comprises 11 proteins including three recently discovered cytokines encoded
by three different genes: IL-36,  and . These molecules bind the same receptor IL-36R (or
IL-1Rrp2 or IL-1RL2) inducing the dimerisation with the co-receptor IL-1RAcP and signaling
via NFB and MAPK pathways. IL-36,  and  are over-expressed in psoriatic lesional skin
and in mouse models of psoriasis and could be induced by inflammatory cytokines (TNF,
IL-17, IL-22) in keratinocytes. In rheumatoid arthritis (RA), it has been shown that IL-36 is
produced by plasmocytes and that IL-36 is over-expressed in serum of RA patients but no
comparative and complete study exists about expression of IL-36,  and  in RA.
Here, using three mouse models of arthritis, synovial fluid and synovial membranes from RA
patients and primary culture of fibroblast like synovioctes (FLS) and CD14+ monocytes
differenciated into M0 (control), M1 (inflammatory macrophages), M2c (anti inflammatory
macrophages), dendritic cells (antigen presenting cells) and osteoclasts (bone resorbing cells),
we shown that IL-36,  and  are over-expressed in rheumatoid arthritis and that
inflammatory macrophages (M1) seem to be the major source of these cytokines.
2- IDRISS Salam - 2ème année - Institut du Thorax INSERM UMR 1087 / CNRS
UMR 6291
Urine Sample-derived Human Induced Pluripotent Stem Cells As A Model To Study
PCSK9-mediated Autosomal Dominant Hypercholesterolemia.
Karim Si-Tayeb, Salam Idriss, Benoite Champon, Amandine Caillaud, Matthieu Pichelin,
Lucie Arnaud, Kazem Zibara, Bertrand Cariou, l'institut du thorax, Nantes, France
Introduction. Human induced pluripotent stem cells (hiPSC) are becoming a relevant model
for the study of liver metabolic diseases once differentiated into hepatocyte-like cells (HLC),
and it has been shown that they can faithfully recapitulate autosomal dominant
hypercholesterolemia (ADH). PCSK9 is a critical modulator of cholesterol homeostasis, and
quickly became a hot target for ADH pharmacological treatment strategies. However, current
cellular models to further decipher the role of PCSK9 in ADH are limited, especially to study
the PCSK9 gain of function mutation S127R, which seems to interfere with LDL cholesterol
homeostasis intracellularly by still unknown mechanisms.
Hypothesis. Human iPS cells will provide an appropriate tool to model PCSK9-mediated
ADH and screen molecules with pharmacological properties.
Methods. We used urine samples as a source of somatic cells in order to obtain hiPSC upon
episomal vectors-mediated reprogramming (UhiPSC). After characterization and validation,
UhiPS control or carrying the S127R mutation were differentiated into HLC.
Results. Compared to control cells, HLC-S127R secreted less PCSK9 in the media (-38.5 ±
47.2%; p=0.08), and had a 3.5 fold (±0.17; p<0.05) decrease of dil-LDL uptake. Pravastatin
treatments (10µM for 24h) significantly enhanced LDL receptor (LDLR) and PCSK9 gene
expression and PCSK9 secretion in both control and S127R HLC. Finally, pravastatin
treatments induced a 3.2 fold (±0.84; p<0.05) increase of dil-LDL uptake in HLC-S127R
compared to only a 1.3 fold (±0.09; p<0.05) increase in control HLC. This in vitro induction
of HLC-S127R LDLR activity correlated with the good response of patients to statin
treatment.
28
Conclusions. Our study demonstrates that not only patient’s urine samples provide an
attractive source of somatic cells for reprogramming and hepatocyte differentiation but also a
powerful tool to further study PCSK9 functions and test new therapeutic approaches.
3- DALLET LAURENCE – 2ème année – Institut du thorax
Cell delivery mechanism of protein/lipid complexes studied by correlative microscopy
Laurence Dallet1,2, Pauline Peuziat1, Marion Decossas2, Bruno Pitard1, Olivier Lambert2
1
Institut du Thorax, UMR 1087 INSERM, 8 Quai Moncousu, 44000 NANTES
2
CBMN, UMR 5248 CNRS, Allée Geoffroy Saint Hilaire, 33600 PESSAC
The development of effective carriers is an emerging field for delivering therapeutic
molecules (DNA, RNA, protein). Since large proteins do not spontaneously cross the plasma
membrane, development of nanocarriers enabling their transport into the cytoplasm is
required. To date, many studies have investigated the use of cationic lipids for gene delivery
or DNA vaccination more recently, those cationic lipids may also be efficient for cellular
uptake of proteins [1] [2]. Thus, taking advantage of the capability of cationic lipids, original
therapy involving antibody represents a new approach to treat some diseases such as cystic
Regulator) causing its retention in endoplasmic reticulum (ER) seem responsible for CF.
Recently, it has been shown that keratin 8 (K8), component of intermediate filaments, was
involved in this retention by interacting with the domain of mutated CFTR [3]. The
intracellular delivery of antibodies against K8 may prevent the interaction between mutated
CFTR and K8 and then changes the intracellular trafficking of CFTR.
In this context, this study aims at understanding the cellular trafficking of complexes
(cationic lipid/protein) and the mechanisms governing the internalization process. To ensure
that the cells analyzed by electron microscopy to contain fluorescent anti-K8 antibody,
correlative microscopy approach (CLEM: Correlative Light and Electron Microscopy) has
been performed. This technique is based on the analysis by fluorescence microscopy and
electron microscopy of the same cell, grown on adequate support (MaTtek support).
Among a variety of tested cationic lipid, the formulation DOSP/MM27 has been
identify as the most effective lipid formulation for intracellular delivery of K8 antibody. EM
characterization showed a unique organization forming clusters of onion-like structures
exposing antibodies at their surfaces. Using CLEM, the intracellular delivery of K8 antibody
complexed with multilamellar structures has been observed in living cells. These first results
are in the early stages of proof of concept CLEM studies and are promising for the future.
To further decipher the mechanisms of action of K8 antibody, its attachment to
intermediate filaments will be studied using a confocal microscopy combined with
immunogold labeling. Next, the methodology will be transposed on HeLa cells expressing
CFTR WT and mutated CFTR to analyze the localization of CFTR.
[1] D. McIlroy, B. Barteau, J. Cany, P. Richard, C. Gourden, S. Conchon, and B. Pitard,
“DNA/Amphiphilic Block Copolymer Nanospheres Promote Low-dose DNA
Vaccination,” Mol. Ther., vol. 17, no. 8, pp. 1473–1481, Aug. 2009.
[2] O. Le Bihan, R. Chèvre, S. Mornet, B. Garnier, B. Pitard, and O. Lambert, “Probing the in
vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale,”
Nucleic Acids Res., vol. 39, no. 4, pp. 1595–1609, Mar. 2011.
[3]
J. Colas, G. Faure, E. Saussereau, S. Trudel, W. M. Rabeh, S. Bitam, I. C. Guerrera, J.
Fritsch, I. Sermet-Gaudelus, N. Davezac, F. Brouillard, G. L. Lukacs, H. Herrmann, M.
Ollero, and A. Edelman, “Disruption of cytokeratin-8 interaction with F508del-CFTR corrects
its functional defect,” Hum. Mol. Genet., vol. 21, no. 3, pp. 623–634, Feb. 2012.
29
4- JACQUES Camille – 2ème année – UMR 957
BET Bromodomain implication in Ewing Sarcoma carcinogenesis
Camille JACQUES*, François LAMOUREUX, Marc BAUD’HUIN, Franck TIRODE,
Dominique HEYMANN, Françoise REDINI, Benjamin ORY
LPRO UMR 957, Faculté de Médecine, 1 rue Gaston Veil, 44035 Nantes cedex 1
With an annual incidence of 2 cases per million, Ewing sarcoma is the second most
common primitive malignant bone tumor after osteosarcoma. This aggressive cancer mainly
affects adolescents and young adults and is characterized by an ectopic tumor bone formation
associated or not with osteolysis, at the diaphysis of the long bones and at the pelvis
principally. Ewing sarcomas are often characterized by a chromosomal translocation leading
to the production of a chimerical transcription factor, EWS-Fli1, which is implicated in the
progression of this malignancy. Current treatments noticeably improve the outcome of this
pathology, but unfortunately, therapeutic resistances and pulmonary metastasis remain and
reduce the survival rates. Despite of the protocols’ optimization efforts, and regarding the fact
that some patients still relapse after treatment, it is important to develop new therapeutic
approaches.
This study is based on collaboration with the American laboratory of the Dr. James
BRADNER, which synthesized a new molecule, called JQ1, and which could be a new
original weapon against this cancer. JQ1 is indeed a potent inhibitor of the epigenetic
regulators family of BET proteins. This protein family interacts with acetylated histones
through its bromodomains, and is able to regulate the chromatin accessibility to transcription
factors and RNA polymerases.
This project aims to elucidate what is the exact role of the BET proteins in the Ewing
sarcoma carcinogenesis, and notably their implication in the proper regulation of this
pathology-related oncogene, EWS-Fli1. The inhibition of such proteins in the Ewing sarcoma
context could allow in the future considering the use of the epigenetic-regulators molecules
like JQ1 as powerful chemotherapy drug.
5- MME. KERVOELEN Charlotte – 3ème année – Inserm U892 Equipe 10
The pro-apoptotic effect of Dexamethasone mediated by GILZ and Bim up-regulation is
related to genetic heterogeneity of multiple myeloma
Kervoëlen C 1,2, Ménoret E 1, Gomez-Bougie P 2,3, Bataille R 2, Moreau P 3, PellatDeceunynck C 2,3 and Amiot M 2, 3.
1. Myelomax, IRS-UN, Nantes, F-44000, France
2. Inserm, U892, Université de Nantes, CNRS, UMR 6299, Nantes, F-44000, France.
3. Service d’Hématologie Clinique, CHU de Nantes, Nantes, France
Multiple myeloma (MM) is heterogeneous with respect to its causative molecular
abnormalities and the treatment response of patients. Briefly, MM that are hyperdiploid or
have a t(11;14) translocation have a better prognosis than those with t(4;14) or t(14;16)
translocations. Dexamethasone (Dex) is widely used in all phases of MM treatment as
induction, consolidation or maintenance. However, the mechanism of Dex sensitivity still
remains elusive.
In the present study, we analyzed the ability of Dex to induce cell death by Apo-2.7 staining in
33 human myeloma cell lines (HMCLs) representative of the molecular subsets carrying
t(11;14), t(4;14) or t(14;16) translocations, which deregulate CCND1, MMSET and c-MAF,
30
respectively. The mechanisms controlling Dex-induced apoptosis were evaluated by analyzing
glucocorticoid receptor (GR), glucocorticoid-induced leucin zipper (GILZ) and Bcl-2 protein
family expression in the different MM molecular subtypes. Transient knock-down of
glucocorticoid-responsive proteins were performed to define their role in the pro-apoptotic
effect of Dex.
We first demonstrated that direct pro-apoptotic effect of Dex was related to the genetic
heterogeneity of MM, since sensitive cell lines were restricted to t(14;16) and t(4;14)
subgroups. We next demonstrated by transcriptomic Affymetrix analysis that GR expression
was heterogeneous among the different molecular subtypes of HMCLs. MAF subgroup
significantly expressed higher levels of GR than all other subgroups (p=0.02). This result was
also confirmed on 300 newly diagnosed MM patients (p<.0001). We also demonstrated that
Dex sensitivity was associated with its ability to down-regulate MAF protein level and its
well-known target CCND2. This result was in agreement with the fact that elevated levels of
MAF were associated not only with t(14;16) translocation but also with MMSET overexpression, which is a characteristic of t(4;14) subgroup. We next demonstrated that Dex
induced an up-regulation of GILZ, a glucocorticoid-responsive molecule, in dex-sensitive and
insensitive HMCLs. Nevertheless, resistant HMCLs, which expressed low levels of GR, failed
to up-regulate GILZ to the same extent than sensitive HMCLs, suggesting that GR levels
could be an important limiting factor for GILZ up-regulation. Of note, GILZ silencing
induced a strong reduction of Dex-induced cell death. These results suggest that
transactivation has a pivotal role in the induction of cell death by Dex in myeloma cells. Dex
also induced an up-regulation of Bim isoforms and a down-regulation of Bcl-xL in all
sensitive cell lines. We further demonstrated that Bim up-regulation is required for the
apoptotic program in response to Dex. Finally, the silencing of GILZ impaired the upregulation of Bim and the down-regulation of Bcl-xL under Dex treatment.
In conclusion, Dex exerted a direct anti-tumor effect on HMCLs of t(14;16) and t(4;14)
molecular subgroups while t(11;14) subgroup was insensitive to it. This result suggested that
conventional therapeutic approaches should be re-evaluated within molecular subgroups of
MM patients to favor potential molecular subtype therapy based on rational preclinical data.
Finally, while Dex interferes with multiple pathways, we begin to unravel its complex
mechanism of action demonstrating that transactivation, inducing GILZ up-regulation, plays a
pivotal role in Dex induced cell death through the regulation of the Bcl-2 protein network.
6- MME. KERMARREC Laetitia – 2ème année - INSERM, UMR 913; CHU de
Nantes, IMAD
Regulation of T cell proliferation by enteric glial cells
Laëtitia Kermarrec, Tony Durand, Michel Neunlist, Isabelle Neveu, Philippe Naveilhan
The enteric nervous system (ENS), also called the second brain, controls various intestinal
functions such as motility, secretion and absorption. Composed of neurons and enteric glial
cells (EGC), the ENS may have a critical role in inflammatory bowel diseases. To study the
impact of EGC on immune cells, increasing number of EGC isolated from the myenteric
plexus of the rat digestive tract was added to spleen-derived rat T lymphocytes stimulated by
anti-CD3 and anti-CD28 antibodies. Analyses by flow cytometry show that EGC inhibit the
proliferation of activated T cells, as do astrocytes, their central counterpart. Pretreatment of
31
rEGC with pro-inflammatory stimuli does not modify their immunomodulatory effects.
Preliminary experiments with human EGC indicate a similar impact on activated human
peripheral blood mononuclear cells. The molecules implicated in this regulatory mechanism
are currently under investigation.
7- MME. RETTMAN Pauline – 3ème année – EA 4271 Immunovirologie et
Polymorphisme Génétique
Use of Killer cell Immunoglobulin-like Receptor (KIR) genes as markers of
haematopoietic chimerism in double-unit cord blood transplantation
Pauline Rettman1, Nolwenn Legrand1, Catherine Willem1, Laurence Lodé2, Patrice
Chevallier1,3, Anne Cesbron4, 5 , David Senitzer6, Christelle Retière1 and Katia Gagne1,4,5
1
Etablissement Français du Sang, Université de Nantes, Immunovirologie et Polymorphisme
Génétique, EA4271, Nantes, France; 2Laboratoire d’Hématologie Biologique, CHU Hotel
Dieu, Nantes, France; 3Service d’Hématologie Clinique, CHU Hotel Dieu, Nantes, France;
4
Laboratoire d’Histocompatibilité et d’Immunogénétique, EFS Nantes, France; 5LabEx
Transplantex, Université de Strasbourg, France; 6Division of Hematology and Bone Marrow
Transplantation, City of Hope, National Medical Center, Duarte, CA, USA.
Double umbilical cord blood transplantation (dUCBT) is an efficient alternative for adult
patients with haematological malignancies when fully HLA-matched related or unrelated
donors are unavailable. Interestingly, when full chimerism is obtained after dUCBT, it is
usually derived from only one of the two UCB units infused. Up to now, real-time SNP-based
PCR has been considered as the best method to assess and quantify chimerism allowing the
monitoring of engraftment and prediction of relapse after dUCBT. We previously reported that
cord blood NK KIR+ reconstitute the patient immune system early after dUCBT impacting on
engraftment with one full UCB unit dominance and that KIR were expressed as soon as day
14 after dUCBT. In this study, we therefore propose the use of KIR genes as complementary
chimerism markers. Fourteen functional KIR genes, located on chromosome 19, were typed
using multiplex KIR PCR both in patients and UCB units before dUCBT and in recipients
after dUCBT (days+15, +30, +90). Analysis of KIR genotyping post-dUCBT was concordant
with KIR+ NK cell phenotype performed at days+15, +30, +60 post-dUCBT and permit to
differentiate patients with engraftment with full or mixed chimerism from autologous
recovery in 41 consecutive dUCBT. Activating KIR genes were in particular more relevant
than inhibitory ones in this assessment. Lastly, comparison of dUCBT engraftment by KIR
genotyping post-dUCBT with conventional SNP-based chimerism method showed
concordance for the majority of cases. Overall, our data suggest that KIR genes may be
considered as complementary markers to assess hematopoietic chimerism status early postdUCBT.
8- MME PRAT Valentine -2ème année – UMR 1087
Transgenic rat model overexpressing endothelial β3-adrenoceptor: a new model for heart
failure with preserved ejection fraction.
Heart failure with preserved ejection fraction (HFpEF) is a growing health burden affecting the
elderly. Patients with HF are classically divided into two groups: those with HF with preserved
ejection fraction (HFpEF), and those with HF and reduced ejection fraction (HFrEF). In the last two
decades, the proportion of patients with HFpEF over HFrEF has increased from 38% to 54% out of
cases of HF, a proportion that will continue to rise due to the progressive aging of population and
expected increase in the prevalence of hypertension, obesity and diabetes. Altered cardiac relaxation
32
and filling play a crucial roles in HFpEF, and are partially due to an increase in myocardial stiffness.
Due to the lack of patient biopsies and accurate animal models, its physiopathology remains unclear
and no specific treatment is currently available. In this context, our team developed a transgenic rat
model (Tgβ3) overexpressing human β3-adrenoceptor upon ICAM2 promoter, leading to a specific
endothelial expression. At 45 weeks of age, Tgβ3 males rats presented an increase in left ventricle
end diastolic pressure (LVEDP; WT: 5.57±1.23; Tgβ3: 11.68±1.12; p<0.01) and in dP/dtmax (WT:
6333±370; Tgβ3: 7808±191; p<0.01), characteristic of an increase in LV stiffness and in peripheral
resistances. Echographic investigation showed an altered filling pattern with an increase in early-tolate filling (E/A) ratio (WT:1.139±0.017; Tgβ3: 1.319±0.044; p<0.01), with a pEF but without
macroscopic cardiac remodeling. These results showed that aged Tgβ3 rats presented a restrictive
cardiac filling, representative of the HFpEF phenotype. Further studies, such as fibrosis
quantification and calcium cycle study, are needed to determine the underlying mechanisms of the
diastolic impairment, as well as molecular targets altered in the disease in order to develop a new
therapeutic strategy for HFpEF treatment.
9- MME. CORBILLE Anne-Gaëlle – 2ème année U913
Appraisal of the dopaminergic and noradrenergic innervation of the submucosal plexus
in Parkinson's disease.
Abstract
Background: The principal components of the enteric nervous system (ENS) are two neuronal
networks, the myenteric and submucosal plexus (SMP), which are primarily involved in the
regulation of gastrointestinal (GI) motility and secretion, respectively. These two plexus are
made up of intrinsic neurons receiving input from the extrinsic sympathetic and
parasympathetic innervation of the gut. Both the intrinsic and extrinsic innervations of the gut
are affected by Lewy pathology in Parkinson's disease (PD). A recent autopsy survey
indicated that there was no global or dopaminergic loss in the myenteric plexus in PD but the
SMP was not examined. Objective: The aim of the present work was to compare the relative
abundance of dopaminergic and noradrenergic neurons in colonic biopsies between PD
patients and control individuals. Methods: Colonic biopsies were taken during the course of a
colonoscopy in 35 PD patients and 10 control subjects. Density of dopaminergic neurons and
expression of the dopaminergic and noradrenergic markers were analyzed by tyrosine
hydroxylase (TH) immunofluorescence and Western blot using anti-dopaminetransporter
(DAT) and anti-dopamine beta-hydroxylase (DBH), respectively. Results: No significant
differences were observed in the density of dopaminergic neurons and in the expression levels
of dopaminergic and noradrenergic markers in colonic biopsies from PD patients as compared
to controls. Conclusion: Our results indicate that there is no evidence of dopaminergic and
noradrenergic neuronal loss in the SMP in PD, thereby suggesting that neuropathology in
submucosal neurons is unlikely to be a causative factor for GI dysfunction in PD.
Keywords :
Parkinson's disease; autonomic nervous system; dopamine; enteric nervous system
10- M. COLOMBANI Thibault – 2ème année – UMR 1087
Lipoaminoglycosides : Immuno-understanding and biological receptor investigation
My PhD revolves around the cationic lipids, usually, used as transfection reagent and
considered as a safe alternative to viral vectors as nanocarrier for gene therapy or drug
intracellular delivery. My results provide evidence that they do not behave as inert material
but do activate cellular signaling pathways implicated in innate immune response. I show that
33
the cationic lipids belonging to the family of lipoaminoglycosides induce the production of
IFNβ1, IL-6, CXCL10, and DNA-sensors interferon-inducible by mouse myoblast cell lines
or mouse embryonic cell lines. Further, I demonstrate that the activation of this pathways is
structure-dependent, with a huge implication of the head polar group and we made the
hypothesis that the endocytosis is necessary to the stimulation of the pathways. My results
suggest that lipoaminoglycosides because of their ability to stimulate the innate immune
system can be used as a new class of synthetic and save “adjuvants vectors” for vaccination.
11- M. FLEURENCE Julien – 2ème année – U892
Title: Targeting and killing glioblastoma with monoclonal antibody to O-acetyl-GD2
ganglioside.
Denis Cochonneau1,2, Fleurence Julien*, Lisa Oliver1,2, Arulraj Nadaradjane1,2,
Mickaël Terme3, Dorvillius Mylène3, Jihane Frikeche1,2,4, Delphine Loussouarn4,
François Valette1,2, François Paris1,2, Stéphane Birklé1,2,4
1
INSERM U.892, Centre de Recherche en Cancérologie de Nantes-Angers, Institut de
Recherche en Santé de l’Université de Nantes, 8 quai Moncousu, 44007 Nantes, France.
2 CNRS 6299, Centre de Recherche en Cancérologie de Nantes-Angers, Institut de Recherche
en Santé de l’Université de Nantes, 8 quai Moncousu, 44007 Nantes, France.
3 ATLAB Pharma, Institut de Recherche en Santé de l’Université de Nantes, 8 quai
Moncousu, 44007 Nantes, France.
4 Université de Nantes, UFR des Sciences Pharmaceutiques et Biologiques, 9 rue Bias, 44035
Nantes, France.
Abstract
Purpose:
High-grade gliomas including glioblastoma multiforme are the most common and most
devastating brain tumors. The impact of current treatments is unfortunately small,
highlighting the need for new and novel therapies. Our objective was to determine whether Oacetylated GD2-specific antibody therapy may be explored for the treatment for High-grade
gliomas.
Experiment design:
To test this hypothesis, we studied O-acetylated GD2 expression on a panel of surgically
resected human glioma tissues, glioma cell lines and primary glioma biopsy-derived tumor
lines.
Results:
We found that O-acetylated-GD2 is expressed on surgically resected human glioma tissues,
glioma cell lines and glioma primo-cultures. In addition, mAb 8B6-IgG2a specific for Oacetylated-GD2 can inhibit the proliferation of glioma cell lines and and primary glioma cells
and can target the glioma tumor to reduce tumor growth in vivo.
Conclusion:
Our findings suggest that gliomas are suitable for specific targeting by mAbs specific for Oacetylated GD2. In addition, they provided the preclinical support for the use of anti-Oactylated GD2 mAbs as a valuable addition to current therapeutics.
12- MME. LABBE Pauline – 2ème année – U1087
LRRFip1 and Wnt pathway involvement in mitral valve prolapse
Pauline Labbé* (1), Florence Kyndt (1), Thierry Le Tourneau (1), Stéphane Zaffran (2),
Cécile Duplaà (3), Jean-Jacques Schott (1), Jean Mérot (1)
(1) Inserm U1087, Institut du Thorax, Nantes, France – (2) Inserm UMR S910, Marseille,
34
France – (3) Inserm U1034, Pessac, France
Heart valves diseases affect 3% of world population, and surgery is often the only therapeutic
mean. A genetic study performed on a family in which several members exhibited a mitral
valve prolapse (MVP) identified a mutation on LRRFip1 gene. LRRFip1 undergoes extensive
alternative transcription splicing giving rise to 5 isoforms in humans and the mutation we
identified results in R94G substitution in three (Iso1, 3 and 4) out of the five isoforms.
Previous studies essentially focused on LRRFip1-iso5 that was first described as a
transcription factor interacting with positive (Dishevelled) and negative (Flightless-1)
regulators of the canonical Wnt β-catenin dependant pathway. LRRFip1 thus appeared as an
interesting gene in MVP as it may regulate two crucial events of cardiac valve development
and homeostasis involving Wnt pathway: epithelial to mesenchymal transition and cell
proliferation. Interestingly, we showed by RNA sequencing and RT-PCR that LRRFip1-iso1 is
expressed in human valves. Furthermore, LRRFip1-iso1 has poor homology with the other
isoforms and nothing is known about its function, we thus focused on it. In HEK 293 cells,
fractionation experiments revealed a nuclear localization of LRRFip1-iso1 while other
isoforms are strictly cytoplasmic. We then showed by Luciferase-based Wnt reporter assays
that out of the five isoforms, LRRFip1-iso1 activates the canonical Wnt pathway at the
highest levels. This activation requires beta-catenin, but no increase in active or total betacatenin expression was observed. Furthermore, the R94G mutation decreases Wnt activation.
We then identified a disordered region in LRRFip1-iso1 (25 amino-acids around the R94G
mutation) responsible for this strong activation and comprising predicted signalling or
structural sites which can be perturbed by the mutation. Thus, our studies suggest that
LRRFip1-iso1 may activate the canonical Wnt pathway by participating in a beta-catenin
dependent transcription complex. R94G mutation may deregulate gene transcription and
consequently alter valvulogenesis and/or valve homeostasy.
13- M. NAVET Benjamin – 2ème année – UMR 957
Dlx homeobox genes involvement in osteosarcoma
Benjamin NAVET*, Frédéric LEZOT,
INSERM UMR 957, University of Nantes, France
The osteosarcoma is a bone primitive tumor from osteoblastic origin that mainly affects
children and young adult. Dlx homeobox genes are implicated in early development, bone
growth and osteoblastogenesis. So an implication of these genes in osteosarcoma
physiopathology has been proposed. Recently, an abnormal expression of Dlx4 has been
evidenced in different osteosarcoma cell-lines with a potential implication in proliferation,
osteolysis and osteo-formation processes. Dlx4 over-expression and inhibition with shRNA
has been induced in sub-clones of the MOS-J cell evidencing different expression levels of
Dlx4. To date, results do not implicate Dlx4 in the proliferation process of osteosarcoma, both
in vivo and in vitro. Concerning the osteolysis process, results validate the previously
described positive impact through the stimulation of RANKL secretion into the tumor
microenvironment by a cell that may be from the lymphocyte lineage. However, more refined
analysis of the system need to be realized, since it appears that the Dlx4 gene would encode
for another transcript named BP1. Specific invalidations of each transcripts should enable to
understand in depth the part of Dlx4 homeobox gene in osteosarcoma.
35
14- M. DAVID Benoît – 2ème année – UFIP CNRS 6286
Turning glycoside hydrolases into transglycosidases: a theoretical study of the internal
water dynamics in the Thermus thermophilus β-glycosidase (2° année)
Benoît David *, Johann Hendrickx, Yves-Henri Sanejouand, Charles Tellier
UFIP UMR-CNRS 6286, Université de Nantes, 2 rue de la Houssinière, 44322 Nantes,
France
Mostly known for their ability to hydrolyze glycosidic linkages, numerous glycoside
hydrolases are also able to catalyze reverse hydrolysis (transglycosylation) which can be
harnessed for the synthesis of complex oligosaccharides. Although hydrolysis usually prevails
over the reverse reaction, transglycosylation propensity has already been increased through
mutagenesis and directed evolution experiments [1, 2]. However, little is known about the
regulation of the balance between both activities.
A previous experimental study on the β-glycosidase de Thermus thermophilus (Ttβgly) using
deuterium exchange mass spectrometry has shown that amide hydrogens from a buried part of
the protein were able to exchange with the solvent's []. The discovery via molecular dynamic
(MD) simulation of a potential water channel connecting the bulk to the active site along this
peptide has supported a possible role of internal water dynamics on the hydrolytic acivity of
Ttβgly [3]. Using an improved simulation protocol, new MD simulations up to 500 ns have
been conducted in order to extensively probe the internal water dynamics in Ttβgly. Analysis
of the internal water molecules trajectories allowed to characterized a total of six new
potential water channels.
Distant residues previously known for playing a role in influencing the balance between
hydrolysis and transglycosylation were also identified in the vicinity of five water channels.
Computation of the water molecule exchange rate at each position explored by a water
molecule in the protein has shown that only three water channels showed a significant water
exchange at the interface of the active site. Within this context, it is tempting to speculate that
those characterized water channels could be involved in supplying water molecules to the
active site and potentially regulating the hydrolytic activity of Ttβgly.
1.
Feng, H.-Y. Converting a -Glycosidase into a -Transglycosidase by Directed Evolution.
Journal of Biological Chemistry 280, 37088–37097 (2005).
2.
Teze, D. et al. Semi-rational approach for converting a GH1 -glycosidase into a transglycosidase. Protein Engineering Design and Selection 27, 13–19 (2014).
3. Teze, D. et al. Conserved Water Molecules in Family 1 Glycosidases: A DXMS and Molecular
Dynamics Study. Biochemistry 52, 5900–5910 (2013).
15- MME. BESBES ANISSA – 2ème année – EA 3826
Role of NK cells in a Post-haemorrhage immunosuppression mouse model. Impact of
glucocorticoid treatment efficacy.
Besbes A.1, Broquet A.1, Jacqueline C.1, Vourc’H M.1, Potel G.1, Caillon J.1, Asehnoune K.1,2
1
Laboratoire EA3826. Thérapeutiques cliniques et expérimentales des infections. Faculté de
médecine. Université de Nantes. Nantes. France.
2
CHU de Nantes, Pôle Anesthésie Réanimation, Service d’anesthésie réanimation
36
chirurgicale, Hôtel Dieu, 44093 Nantes, France.
Abstract
Haemorrhage is a trauma leading an immune dysfunction phenotype. This immune failure
altered survival rate of patients, as it increases their susceptibility to nosocomial infections.
Recent works from the laboratory strongly suggest an alteration of the function of central cell
of the innate immune system: NK cells. (1-3).
NK cells play a crucial role in controlling infection, and depleted mice show more
susceptibility to a secondary Pseudomonas aeruginosa pneumonia. (2)
In a murine haemorrhagic shock model routinely used in the laboratory, we observed
modifications in the NK cells phenotype and function. Following haemorrhage, NK cells turn
to an anti-inflammatory phenotype by secreting IL-10 impairing dendritic cells maturation
and functions. A recent clinical study strongly suggested that Hydrocortisone may prevent
immunosuppression severity, which increase survival rate of patients treated after
haemorrhage trauma. In our mice model, we demonstrated that in concordance with this
clinical result, Hydrocortisone restored partially the NK cells functions and decreased
significantly the mice susceptibility to a secondary infection (1).
The aim of our study is:
1) To characterize the NK cell change of phenotype and to characterize the cell subset
responsible for the immunosuppression state.
2) To compare two corticoid treatments frequently used in emergency in preventing
nosocomial infection: Hydrocortisone and Solumédrol.
This work will be done on a murine model of heamorrhagic shock. This volume controlled
haemorrhagic shock is routinely used in our laboratory.
Keywords: Haemorrhage, Immunosuppression, NK cells, Hydrocortisone, Solumédrol
(1) Hydrocortisone Prevents Immunosuppression by Interleukin-10+ Natural Killer Cells
After Trauma-Hemorrhage.Roquilly A, Broquet A, Jacqueline C, Masson D, Segain
JP, Braudeau C, Vourc'h M, Caillon J, Altare F, Josien R, Retière C, Villadangos J,
Asehnoune K. Crit Care Med. 2014 Oct 6.
(2) Depletion of natural killer cells increases mice susceptibility in a Pseudomonas
aeruginosa pneumonia model. Broquet A, Roquilly A, Jacqueline C, Potel G, Caillon
J, Asehnoune K. Crit Care Med. 2014 Jun;42(6):e441-50.
(3) CpG-ODN and MPLA prevent mortality in a murine model of post-hemorrhageStaphyloccocusaureus pneumonia.Roquilly A, Gautreau L, Segain JP, de Coppet P,
Sebille V, Jacqueline C, Caillon J, Potel G, Lejus C, Josien R, Asehnoune K. PLoS
One. 2010 Oct
16- MME. FIGUEIREDO Lara -2ème année – Lioad UMR 791
Oxygen diffusion in Si-HPMC scaffolds as stem cell niche in vitro models
Figueiredo, L ; Réthoré, G ; Le Visage, C and Weiss, P
1
LIOAD, INSERM U791, Nantes University
lara.martins-figueiredo@etu.univ-nantes.fr
Introduction
Despite stem cell niche being characterized by low oxygen content, anoxia at the core of
cellularized scaffolds has long been associated with poor in vivo results of stem cell
transplantation. Oxygen diffusion is of outmost importance, since it influences cell
metabolism and has a great impact on stem-cell fate. Hence, a detailed characterization of
oxygen diffusion profiles in hydrogels could provide useful insights for the development of
stem cell niche models.
37
Objectives
We investigated the oxygen diffusion properties in self-setting Si-HPMC hydrogels reinforced
or not with laponites and their related cell viability.
Methods
Hydrogel preparation
One volume of 4 wt % Si-HPMC was mixed with half volume of acidic buffer and half
volume of XLG laponite (Rockwood, GmbH) solution or water.
Oxygen measurements
Core oxygen partial pressure in the 3D constructs was monitored with a needle type oxygen
microsensor (PreSens, Germany). For determination of oxygen content kinetics in acellular
hydrogels, ten millimeter-high constructs with and without laponite reinforcement, were
equilibrated at 0.1% oxygen. De-oxygenation and re-oxygenation of the hydrogel was
followed. In a separate experiment, same type constructs, seeded or not with (hASC) were
incubated in normoxic conditions (20% O2).
Cell viability measurements
Staining of live hASCs inside hydrogel constructs was performed at 24, 48, 72 and 144 hours
after cell seeding using a Calcein-AM Assay kit. Samples were collected from the center of
constructs and imaged using confocal microscopy.
Results
De-oxygenation profiles of Si-HPMC and Si-HPMC+XLG acellular constructs in an anoxia
environment were almost superimposed, showing a slow decrease of their oxygen content.
For cellularized constructs in normoxic conditions, there was a fast decrease of the oxygen
contents at the center of constructs in the first 12h of the experiment. In addition, hydrogels
loaded with laponites reached a significantly lower value of oxygen content. Then, we
identified a re-oxygenation profile of both constructs.
Cell density at the core of the Si-HPMC construct significantly increased after 48 hours of
culture, indicating that cells proliferated during that time. On the contrary, in presence of
laponites, the number of live cells per mm3 at the center of the constructs remained stable. At
all time points (except 24 hours), there was a significant difference in cell density between
hydrogels with and without laponites
Discussion
Due to its ability to form a physical network, laponites act as oxygen transport barrier.
However, in this study, superimposition of de-oxygenation and re-oxygenation profiles
suggests that laponites addition to Si-HMPC hydrogel has no obvious effect on oxygen
diffusion per se. In addition, the lowest oxygen level after 12h of normoxic incubation and
reduced cell viability after 24h suggests interference of laponites with cell metabolism.
Conclusion
Hydrogel re-inforcement with laponites does not seem to have an impact on oxygen diffusion
per se. On the other hand, laponites clearly have an impact on cellular oxygen consumption
and viability.
17 - M. KAMBAREV Stanimir – 2ème année –UMR 892
A whole-genome approach to study host-pathogen interactions.
Stanimir Kambarev1*, Laurent Marsollier2, Stéphane Corvec 3, 4, Frédéric Pecorari1
1
Recherche en oncologie Nucléaire, Centre de recherche en cancérologie de Nantes-Angers,
INSERM UMR 892, CNRS 6299, Université de Nantes
2
Inserm Avenir ATOMycA, Centre de recherche en cancérologie de Nantes-Angers, INSERM
UMR 892, CNRS 6299, Université de Nantes
38
3
Service de Bactériologie et Hygiène hospitalière , CHU de Nantes
Université de Nantes, EA3826 Thérapeutiques cliniques et expérimentales des infections,
UFR de Médecine, Nantes
4
During infection, pathogens utilize a multitude of virulence factors to adhere to the
host, to evade its immune responses and to disseminate. The host’s immune system responds
to the invasion by development of specific humoral and cellular responses. The understanding
of these interactions at molecular level is essential for the development of new prevention,
diagnosis and therapeutic strategies.
In this context, we study the interactions of two pathogenic bacteria with their host at
humoral level.
Streptococcus gallolyticus ssp. gallolyticus is known for its close association with the
incidence of colorectal cancer (CRC). Despite the extensive research on the topic it is still not
certain if the bacterium is only an opportunist, successfully growing in the setting of
compromised mucosal barrier or whether it contributes, via specific virulence features, to the
development of colonic malignancy.
Mycobacterium ulcerans causes the third most common mycobacterial disease
worldwide- Buruli ulcer, which is associated with severe skin mutilation. Early diagnosis and
treatment are considered to be the only ways to reduce morbidity. However, no field-friendly
diagnostic test is available up to now.
ANTIGENome is a whole-genome combinatorial approach allowing for serological
identification of the pathogen’s immune-relevant proteome, independently of its growth
conditions and gene regulation. By development of a fully in vitro ribosome display-based
version of the method, we seek to identify protein virulence factors of S. gallolyticus,
associated with CRC development or immunodominant biomarkers of M. ulcerans for
development of diagnostic tools.
18- MME. KALICHUK Valentina – 2ème année – UMR 892
AFFITIN-FUNCTIONALIZED NANOPARTICLES FOR TARGETED DELIVERY TO
COLORECTAL TUMORS
Kalichuk, V. (1,2,*); Béhar, G. (1); Mouratou B. (1); Preat, V. (2); Pecorari, F. (1).
1 Université
de Nantes, Institut de Recherche en Santé de l'Université de Nantes,
INSERM U892 - CNRS 6299 – CRCNA, Nuclear Oncology Research, Nantes, France.
2 Université Catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics
and Drug Delivery, Brussels, Belgium.
*Contact e-mail: valentina.kalichuk@univ-nantes.fr
Colorectal cancer (CRC) is the second most common cancer in women and third in
men. Despite the progress of the medicine during the last decades, further progress in CRC
treatment is needed. Conventional chemo- and radiotherapies lack selectivity, thus they are
toxic for normal tissues as well as for cancerous ones. Therefore, there is a need of targeted
and controlled delivery system that may reduce dose and duration of the therapy.
Nanoparticles (NPs) have the potential to improve current chemotherapies or
radiotherapies by increasing efficacy, lowering toxicity while maintaining a high
concentration of therapeutic compounds at the site of interest. Promising approach is the
active targeting, in which targeting ligands are attached to the NPs. Although monoclonal
antibodies (mAbs) are the most used targeting ligands in cancer therapy, they possess several
limitations such as their cost, large size and instability.
Affitins are artificial affinity proteins, derived from the archaeal polypeptide Sac7d and its
homologs. These non-antibody binders show comparable affinity and specificity to those of
antibodies, but are thermally and chemically stable, cheaper to produce and have a size
39
compatible with chemical synthesis (7 kDa) while they have a less complex structure.
The goal of the project is to combine the advantages of both Affitins and NPs for active
targeting in cancer therapy. Affitins directed against CRC biomarkers will be used for the
development of highly stable Affitin-functionalized nanoparticles for targeted delivery to
colorectal tumors.
19- M. DION Johann – 2ème année – UFIP /UMR 6286
SYNTHESIS OF
DERIVATIVES
GALECTIN
INHIBITORS
BASED
ON
LACTOSAMINE
J. Dion*, S. Dahbi, N. Storozhylova, C. Grandjean
Unité Fonctionnalité et Ingénierie des Protéines (UMR6286)
University of Nantes, 2 rue de la Houssinière, 44322 Nantes, France.
E-mail: johann.dion@univ-nantes.fr
Galectins are a family of lectin, very conserved in the mammal species (15 members
known), which are able to recognize -galactoside sugar motifs1. These galectins are
multifunctional proteins which are involved in a lot of biological processes like cellular cycle,
apoptosis, cell trafficking and cell-cell or cell-matrix interaction. Key unit of cell
development, inflammatory and immune response, the deregulation of these proteins seems to
be associated with more than one hundred pathologies2. Therefore, galectins have been
identified as potential therapeutic targets. However, there is still a lot of difficulties to
understand mechanisms of action of galectins. We have embarked in a program aimed at
preparing specific inhibitors of the galectin-3 to study its role in cell migration and cell
division in the skin and, at term, to propose novel therapeutic strategies.
We have elected to work on type I (1-3) and type II (1-4) lactosamine, two natural
disaccharides recognized by the galectin-3, as scaffolds and to introduce pharmacophores at
40
defined positions to further improve their binding, according to a drug design approach. A
first generation of C2 aromatic derivatives, displaying better affinity thanks to -cation3 or
interactions4, has been prepared.5 For instance, in the case of type II compounds, the
derivative with 3-methoxyphenylamide group increase binding by a factor six (Fig. 1).
Kd =
36 M
1-4 =
Kd = 6
Figure 1. Example of substitution on type II lactosamine
In the case of type II compounds, as shown on Figure 1, we want to target the best linkage
(in green) between aromatic ring and sugar. We have first kept the aromatic ring constant to
make possible comparison. We selected 3-methoxyphenyl ring (in blue) known to increase
affinity. By changing the amide function by another link (urea, sulfonamide, methylamine, ...)
we will be able to select the best one by correlation with Kd value. In a second part of our
work, we have also synthesized several inhibitors with constant amide link and different
aromatic rings in order to see effect of the aryl group on the binding.
References:
1. S.H. Barondes, D.N. Cooper, M.A. Gitt, H. Leffler, J. Biol. Chem., 1994, 269, 20807
2. Klyosov, A.A., Chapter 2 in Glycobiology and Drug Design, 2012, 25
3. I. Cumpstey, E. Salomonsson, A. Sundin, H. Leffler, U.J. Nilsson, ChemBioChem,
2007, 8, 1389
4. S. Fort, H.S. Kim, O. Hindsgaul, J. Org. Chem., 2006, 71, 7146
5. S. André, C. Grandjean, F.-M. Gautier, S. Bernardi, F. Sansone, H.-J. Gabius, R.
Ungaro, Chem. Commun., 2011, 47, 6126
20- MME. MISME-AUCOUTURIER Barbara -2ème année -EA1155-IICiMed
Département de Parasitologie et de Mycologie Médicale, Université de Nantes
Granulomes fongiques : caractérisation cellulaire et moléculaire de l’interaction hôtepathogène.
Subject
During chronic disseminated candidiasis (CDC) and chronic mucocutaneous candidiasis
disease (CMCD) the physiopathological response is characterized by the persistence of
Candida inside granulomas due to an impaired cell mediated immunity against infection. In
this context, host-fungal interactions within the granulomas have not been well described to
date. Then EA1155 IICiMed laboratory have recently developed the first human model of in
vitro Candida albicans persistence within granuloma allowing describing the kinetic of fungal
granuloma formation behind human Peripheral Blood Mononuclear Cells infection (PBMC).
This study aimed at comparing the granuloma response for eight Candida species.
Method
We compared the capability of eight Candida species (C. albicans, C. dubliniensis, C.
41
tropicalis, C. lusitaniae, C. glabrata, C. parapsilosis, C. kefyr and C. krusei, 4 clinical isolates
by species) to form granulomas in vitro. The kinetics of granuloma formation was studied
during the infection of ten immunocompetent healthy donors through the number and size
variations of granuloma structures, and the fungal load evolution.
Results
The infection of the immune cells from healthy donors induced the formation of a
granulomatous response for all Candida species used in the assay. An increase of the average
of granuloma number and size was observed between day 4 and 6 post infection. However,
the average of granuloma size was different between species. For C. albicans and C.
dubliniensis the median was 170 ± 18 μm on day 6 whereas for the other species granuloma
size was 63 ± 8 μm. Then, the fungal loads inside the granuloma were compared to the
corresponding granuloma sizes exhibiting a strong positive correlation at day 6 post-infection
(r = 0,84). Besides, while the fungal multiplication is controlled during the two first days for
all species, the fungal load evolution further followed two distinct profiles according to
species. The first one was characterized by an increase of the fungal loads from day 2 to day
6, whereas the second showed a progressive clearance of the pathogen by the granulomatous
reaction. A multiparametric comparative analysis of these profiles taking into account the
levels of fungal load at day 6, allowed to highlight four species clusters. Three clusters were
related to the first profile with high (C. albicans: cluster 1) moderate (C. dubliniensis and C.
tropicalis: cluster 2) and low (C. lusitaniae, C. glabrata and C. parapsilosis: cluster 3) fungal
loads. The cluster 4 (C. krusei and C. kefyr) corresponds to the second clearance profile
Conclusion
Under our conditions, results indicated that the interaction between Candida and the immune
cells from immunocompetent donors, lead to the formation of granuloma structures.
Differences were highlighted in term of evolution of granulomatous response depending on
the Candida species. The capacity of Candida spp to form hyphae, the production of
virulence factors such as the granuloma response variability between healthy individuals are
key factors that are under progress investigation
21- M.MOREAU François – 2ème année – UMR 1087
Title: Effect of bariatric surgery on cholesterol metabolism.
Authors : François Moreau*, Claire Blanchard, Audrey Ayer, Veronique Ferchaud, Michel Krempf,
Bertrand Cariou & Cédric Le May
Introduction : Hypercholesterolemia affect 37,4 % of the 65-74 years old and is a major risk factor for
cardiovascular diseases. Current guidelines highlight the need for new treatment options able to
decrease plasma cholesterol levels beyond those presently achieved. Recently, a new way called the
Trans Intestinal Cholesterol Excretion (TICE) emerged as a new druggable metabolic pathway. TICE
allows direct plasma cholesterol elimination through the small intestine and represents a potential
therapeutic target to reduce cardiovascular events.
Obesity is frequently associated with metabolic dysfunction such as diabetes and dyslipidemia. In
absence of pharmaceutical treatments, bariatric surgeries represent the more efficient therapy to
reduce obesity and its associated metabolic complications such as hypercholesterolemia. However,
the molecular mechanisms involved in the hypocholesterolemic effect of bariatric surgeries are poorly
characterized.
Objectifs : Our goal is to measure the effect of bariatric surgery on cholesterol metabolism.
Methods: 10 weeks-old C57BL6J mice received a high fat diet (35% Kcal from fat). After 8 weeks,
mice were randomized on 3 experimental groups: 1) in the first group, a laparotomy was performed
and then mice received food ad libitum (group Sham); 2) in the second group, mice were also
laparatomized but then received the same amount of food consumed by mice in group 3 (group sham
42
pair-fed); 3) a sleeve gastrectomy was performed on the third group (60% of the stomach was
removed) and then mice had free access on food (group sleeve). We next tested several weeks after
surgery the consequence on intestinal functions and on glucose & lipid homeostasis. We first
measured the effect of the sleeve on the body weight and the daily food intake. We next performed an
oral glucose test tolerance 2 weeks after surgery. Finally, we measured plasma cholesterol and biliary,
fecal and transintestinal cholesterol excretion. The intestinal function was analyzed by assessing
intestinal transit and intestinal barrier permeability.
Results: Our data showed that body weight from group 3 mice was significantly reduced compared to
mice from sham/sham pair-fed group. We did not measure significant effect of sleeve on daily food
intake. Two weeks post-surgery, the glucose tolerance was significantly improved in the sleeve group.
Plasma cholesterol tends to be reduced in sleeve group compared to cham pair-fed group. However,
this hypocholesterolemic effect was modest and transient. Consistently, fecal, biliary and
transintestinal cholesterol excretion were comparable between group 1, 2 & 3 five weeks after the
surgery. Finally, we did not observe major differences of intestinal transit or barrier permeability after
surgery. Interestingly, we notice that the stomach weight in the sleeve group was similar to the mice of
group 1 & 2 suggesting that gastric cells from rumen (upper part of the murine stomach) proliferate to
restore the organ.
Conclusions: Our results confirmed that sleeve surgery have beneficial effects on body weight and
glucose homeostasis. By contrast, the hypocholesterolemic effect was modest and transient. Our next
steps will be 1) to perform a more restrictive sleeve (by removing the rumen); 2) to test the effect of a
second currently used surgery method, the gastric by-pass.
Keywords: Baratric surgery, Sleeve, TICE, hypercholesterolemia, Gas chromatography coupled with
mass spectrometry.
22- MME. CHARPENTIER Maud – 2ème année – UMR 892
CHARACTERIZATION AND CONTROL OF THE EXPRESSION OF MELOE
DERIVED MELANOMA ANTIGENS
Charpentier M. 1,2,3, Florenceau L. 1,2, Carbonnelle D.
1,2,3
, Labarrière N. 1,2, Lang F.1,2,3
1
CRCNA INSERM UMR 892 Equipe 3
CNRS UMR 6299
3
University of Nantes
2
Our group has been working for many years on the characterization of melanoma antigens
relevant for adoptive T cell therapy. We identified MELOE-1, an antigen implicated in
melanoma immunosurveillance translated from the meloe mRNA, the transcription of which
is restrained to the melanocytic lineage. This mRNA is polycistronic and also encodes another
antigen named MELOE-2. MELOE-1 and -2 specific T lymphocytes have been identified and
are activated by melanoma cells but not by normal melanocytes. It suggests a specific
translation regulation that occurs only in tumor cells and makes MELOE-1 and -2 interesting
targets for immunotherapy.
Studying the underlying translation mechanisms we identified two IRES sequences upstream
each ORF. First described in prokaryotic organisms IRES mediated translation is also
documented for eukaryotic mRNA as an unconventional translation pathway that may be
activated during cellular stress and malignant transformation.
More recently, the translation of two additional ORFs, MELOE-3 and MELOE-4 close to the
5’ end of the mRNA have been documented. Using bicistronic constructs we proved that
MELOE-4 is translated through the canonical cap dependent pathway. As expected, in
experiments performed using the EGFP reporter protein fused to our peptides of interest, the
expression of MELOE-4 is much higher than that of other proteins of the MELOE family in
43
melanoma cells. Regarding MELOE-3, our results suggest that its translation is due to
defective ribosome scanning.
Our hypothesis is that MELOE-4 is the physiological product of meloe and should thus also
be expressed in normal melanocytes. On the other hand, MELOE-1 and -2 expression would
depend on IRES sequences promoted by IRES trans-acting factors specifically expressed or
recruited to the translation site in tumor cells. In order to validate this hypothesis we initiated
the generation of a MELOE-4 specific monoclonal antibody and already started the
characterization of ITAFs implicated in MELOE-1 translation.
REFERENCES
[1] Carbonnelle D, Vignard V, Sehedic D, Moreau-Aubry A, Florenceau L, et al. (2013) The
Melanoma Antigens MELOE-1 and MELOE-2 Are Translated from a Bona Fide Polycistronic
mRNA Containing Functional IRES Sequences. PLoS ONE
23- MME. SALOU Laetitia - 3ème année – UMR 957
Histomorphometric analysis of Ti dental implant ossointegration in a rabbit model:
Nanostructured surface versus sand-blasted acid-etched surface
Laëtitia Salou*,1,2,3, Alain Hoornaert4, Julien Stanovici1, Guy Louarn2, Pierre Layrolle1
1
Inserm U957, Lab. Pathophysiology of bone resorption, University of Nantes, France
2
CNRS –Institut des Matériaux, Nantes, France
3
Biomedical Tissues, Nantes, France
4
Academic Hospital of Nantes, France
*Laetitia.salou@univ-nantes.fr
Background: Titanium and alloys are widely used for dental implants. Osseointegration is
critical for long term implant stability as well as to ensure a tissue barrier for preventing
bacterial infiltration and thus clinical success. Conventional treatments of dental implants to
improve their osseointegration consist of roughening their surface by grit-blasting and acid
etching. However, these treatments only modify the surface at the micrometer-scale while
proteins and cells interact in the nanometer-scale. For instance, modification of implant at
nanoscale could promote cell adhesion and behaviour by mechanotranduction. Series of in
vitro and vivo investigations have shown that nanoporous structure increased osteoblastic
differentiation, bone apposition and bone strength of titanium implants [1, 2]. However, there
is no study that has yet compared the biocompatibility of this nanostructured surface versus
commercialized micro-rough surface usually use for dental implants.
Aim: This study aims at comparing the osseointegration of dental implants with smooth and
rough surfaces with nanostructure to a conventional grit-blasted and acid-etched surface at 2
different time of healing in femoral condyles of rabbits.
Material and methods: Fourty-two dental implants from 6 to 10 mm long and 4 mm of
diameter were surface modified following different methods. TiO2 nanostructure were
fabricated on TA6V smooth (S-NANO) and grit-blasted (R-NANO) implants using
potentiostatic anodization at 20 V in 1 wt. % HF and 1M of acetic acid. The micro-rough
surfaces (SBAE) were treated by alumina grit blasting and etched in warm hydrochloric and
sulfuric acids. Both surface groups were characterized using scanning electron microscopy
(SEM), atomic force microscopy (AFM), Raman analysis and X-ray spectrometry (XPS). The
three different implant surfaces were placed in the femoral condyles of 24 female New
Zealand White rabbits under general anaesthesia. After healing periods of 2 and 4 weeks,
animals were sacrificed and specimens were fixed, dehydrated and processed for nondecalcified histology. Percentage of bone-to-implant contact [%BIC], percentage of bone
surface at 0.5 mm around the implant [%BS/0.5mm] were determined on back-scattered
electron microscopy images. Statistical analysis was performing with the non-parametric test
of Mann Whitney at the 95% confidence level.
44
Results: Nanostructrured implant were homogeneously colour coded by the process of
anodization. SEM images showed a regular array of nanotubes of 60 nm in diameter on both
S-Nano and R-Nano surfaces. Micro roughness was also observed on R-Nano surface while
typical random hilly topography covered the SBAE surface. AFM topography measurements
confirmed this observation with average roughness of 0.1 µm, 0.4 µm and 1.0 µm for the SNano, the R-Nano and the SBAE control group, respectively. Raman analysis revealed a high
crystalline phase of Anatase and Rutile for the Nano surface while amorphous structure was
observed on the control implant. After two weeks of implantation in rabbit femur,
histomorphometry analysis indicated S-Nano surface improve bone bonding such as the
control and is significantly higher than the R-Nano surface ( 54% vs 43%; P = 0,04). After 4
weeks, both the BIC and BS/TS 0.5mm values of the Nano surfaces were similar to the
control.
Conclusions and clinical implications: Histological data from our study confirm that
nanosurface could favor bone and soft tissues integration [1, 2]. Results show that S-Nano
surface improved the osseointegration as good as the commercialized micro-rough surface
usually uses in implantology. This smooth Nano-surfaces is also functional by its possibility
of color-coding and easely cleaned. Further clinical investigation will be realised. [1]
Lavenus S. et al. Eur Cells & Materials (2011), [2] Lavenus S. et al. Nanomedicine (2012)
24- M. GUIHO Romain - 3ème année – UMR 957
TRAIL-based therapeutics in pediatric bone tumors: resistance of in vitro and in vivo
models to TRAIL pro -apoptotic effects and sensitization approaches.
Romain GUIHO 1, Kévin BITEAU1, Julien TAURELLE 1, Valérie TRICHET 1, Franck
TIRODE 2, Dominique HEYMANN 1, Françoise REDINI 1
1
INSERM UMR 957 - Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie
des Tumeurs Osseuses Primitives
Université de Nantes, Faculté de Médecine, 1 rue Gaston Veil, 44 035 Nantes - France
2
Institut Curie, INSERM U830, 26 rue d'Ulm - 75248 Paris Cedex 05 – France
Osteosarcoma (OS) and Ewing's sarcoma (EWS) are the two most common pediatric bone
tumors. Patients with these diseases have not seen major therapeutic advances these last thirty
years and the survival rate of 70 % at five years for a localized tumor still fall to around 20 %
in the case of a metastatic tumor or a resistance to chemotherapy. Pro-apoptotic cytokine
TNF-Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells and could
be a new therapeutic approach for patients at high risk. However, the study of several OS and
EWS cell lines for TRAIL sensitivity, showed the existence of resistance phenomena.
Our objective is to identify the molecular mechanisms involved in TRAIL resistance in these
particular pathologies to consider therapeutics support strategies by in vitro and in vivo
approaches. Different levels of TRAIL regulation signaling pathways are explored: expression
of death receptors (DR4 and DR5) and decoy receptors (DcR1, DcR2, Osteoprotegerin),
importance of inhibitory proteins of apoptosis (cFLIP; IAP1/2), activation of TRAIL-induce
surviving, migration or invasion pathways (NFkB, MAPK, PI3K/Akt…).
Even if OS and EWS present similar clinical features, these pathologies differ in response to
TRAIL pro -apoptotic effects: the importance of death receptor expression profile was
demonstrated in the EWS with a very strong correlation between the expression of DR4 and
TRAIL sensitivity, whereas strong resistance of OS cell lines was observed, independent of
receptor balance between decoy and death receptors. Our sensitization efforts in OS models,
in vitro and in vivo are based on use of potentially sensitizing molecules such as
chemotherapeutic agents, with particular attention to the bone microenvironment in these
pathologies. In vitro synergy studies show encouraging results, particularly with cisplatin that
seems to sensitize to TRAIL via down expression of cFLIP and the removal of the inhibition
of the intrinsic pathway of apoptosis. Zoledronic acid, already used as antiresorptive agent in
45
OS, also shows a sensitizing effect by inhibition of IAPs. The transition of these observations
to nude mice models is in progress.
25- MME. GUILLONNEAU Maëva – 3ème année – UMR 892
ROLE AND REGULATION OF NUCLEOPHOSMIN IN ENDOTHELIAL CELLS
RESPONSE TO OXIDATIVE STRESS AND INVOLVEMENT OF THE p38 MAPK
PATHWAY
Guillonneau Maëva1-2*, Bénéteau Elise1, Paris François1, Huot Jacques2, Corre Isabelle1-2
1- Centre de Recherche en Cancérologie Nantes Anger, Inserm U892-CNRS 6299-Université
de Nantes, Nantes, France
2- Centre de Recherche du CHU de Québec, Centre de recherche sur le cancer de l’Université
Laval, Québec, Canada
The endothelium is an essential cellular compartment involved in vascular and tissues
homeostasis. Importantly, endothelial deregulation can lead to significant dysfunctions such
as atherosclerosis, inflammation and metastasis. Oxidative stress (either exogenous or
endogenous) through the production of reactive oxygen species (ROS) is an important factor
of endothelial deregulation. To limit oxidative stress damaging effects to the endothelium and
to surrounding tissues, it is important to understand the molecular signaling pathways
involved. p38 MAPKs family is among the key intracellular signaling proteins in oxidative
stress cellular response. By a proteomic approach, we identified nucleophosmin (NPM) as a
new partner of p38 in endothelial cells exposed to oxidative stress such as 500 µM of H2O2.
p38/NPM interaction exists in the cytoplasm of resting endothelial cell and is decreased upon
oxidative stress. NPM is phosphorylated on threonine 199 (T199) in basal condition but is
dephosphorylated in response to oxidative stress, through the activity of PP1/PP2
phosphatases. However, NPM does not affect the kinase activity of p38 and p38 does not
phosphorylate NPM. Nevertheless, knockdown of p38 affects the status of NPM
phosphorylation on T199. Additionally, increased phosphorylation of NPM at T199 enhances
its interaction with p38. One of the functions rapidly regulated in stressed endothelial cells is
the reorganization of the actin cytoskeleton with the formation of stress fibers. We showed
implication of NPM and of its status of phosphorylation in oxidative stress-induced actin
reorganisation. Our results identify a new p38/NPM signaling pathway, relying on the
phosphorylation status of NPM through a PP1/PP2 phosphatase activity. We describe for the
first time a role of NPM in the regulation of endothelial cell cytoskeleton dynamics. This
work will allow better understanding of the role of the p38/NPM pathway in regulating the
endothelial cell response to oxidative stress and, by extension, to ionizing radiation.
46
26- MME. DIAB Maya - 3ème année –UMR 892
Production and characterization of monoclonal antibodies specific for dog and cat
syndecan-1 for nuclear medicine preclinical trial on spontaneous tumors
DIAB M.1, DAVODEAU F.1,CHEREL M.1,2
1
Nantes-Angers Cancer Research Center CRCNA/ INSERM UMR892 ,Nantes,France.
2ICO cancer center,nantes,France
maya.diab@univ-nantes.fr
BACKGROUND: Syndecan-1 (CD138) is a cell surface heparane sulfate bearing
proteoglycane, over expressedin multiple myelomaand in a wide spectrum of carcinomas. In
the case of breast cancer, the expression of CD138 correlateswith apoorprognosis. We have
shown, on a xenogenic mouse model of triple negativebreast cancer (TNBC) that CD138 is a
relevant target for imaging (immuno-PET) andfradioimmunotherapy (RIT).
The objective of this study was to produce a monoclonal antibodies targeting canine CD138
in order to evaluate their usefulness forimmune-TEP and RIT on dogs with spontaneous
carcinoma.
METHOD:Hybridomas were produced through immunization of mice with the
recombinantectodomainof canine syndecan 1 (residues 1 to 251) and fusion of spleen cells
with Sp2/o cells. Screening of positivehybridomas by FACS was performed usingcanin
CD138 transfected CHO cells. Isotypes, affinity and epitopes mapping were determined and
Monoclonal antibodies were characterized using immunohistochemistry.
RESULTS:We selected twenty fourhybridomas producing antibodies reacting with our
CD138c transfected CHO cells. Only eleven hybridomas were isolated after subclonning
allowing us to produce eleven differents antibodies. All of them are IgG1 kappa except one
which is IgG2b kappa.Theiraffinity for canine CD138 are of the order of nano molar and four
distinct epitopes of CD138c are recognized by theseantibodies.. Immunohistochemistry
analysis showed that all antibodies recognize caninesyndecanon healthy tissues and on
canine mammary cancers.
CONCLUSION :These monoclonal antibodies could be a powerful tools to assess canine
syndecan-1 targeting by Imaging and radioimmunotherapy in clinically relevant large animal
model.The validation of RIT targeting CD138 on
spontaneousmammarytumorsexhibitinghighhomologywithhuman TNBC would propose in
womena newtherapeuticapproches for the management of relapsedTNBC
thatcan’tbenefitfrom hormone therapy or immunotherapyusingantibodiestargeting Her2 / Neu.
27- MME. PARROT Tiphaine - 3ème année – UMR 892
“INTRA-TUMOR CD4+CD8+ DOUBLE-POSITIVE T CELLS: A NEW TARGET FOR
IL-9 “
T. Parrot1, M. Allard1, J. Desfrançois2, R. Oger1, H. Benlalam1, D. Raingeard de la Blétière1,
3
, L. Pressier1, N. Labarrière1, Y. Delneste1, P. Guardiola1, 3, N. Gervois1
1
UMR 892 INSERM / 6299 CNRS / Université Nantes - CRCNA
2
Cytometry Facility “CytoCell”, SFR François Bonamy, Nantes
3
SNP Transcriptome & Epigenomics Facility, CHU, Angers
The study of the tumor infiltrate composition in melanomas enabled us to identify a nonconventional CD4+CD8+ double positive T lymphocyte sub-population (double-positive (DP)
T cells) characterized by the stable co-expression of the CD4 and CD8 molecules. DP T cell
frequency can reach up to 10% of tumor infiltrating lymphocytes and increases with advanced
cancer stage, which suggests a role of these cells in anti-tumor immune responses [1].
47
Similarly to the conventional effector CD8+ lymphocytes, DP T cells recognize tumor cells in
an HLA-I restricted context and in a CD8 dependent way. However, DP T cells exert a poor
cytotoxic activity and differ from CD8 T cells by their strong ability to produce diverse Th1
and Th2 cytokines.
In order to delineate the functions of DP T cells, their transcriptome profile was documented
and compared with the one of CD8 infiltrating lymphocytes. It appeared that DP T cells
overexpress the IL-9 receptor raising the question of the impact of IL-9 on the biology of
these cells. We showed, that IL-9 enhances the survival of DP T cells by protecting them from
apoptosis, and, to a lesser extent, favors their proliferation. In addition, IL-9 increases in a
dose-dependent manner the cytokine production of DP T cells. In particular, the amount of IL13 secreted in presence of IL-9 is liable to exert biological effects on macrophages
polarization, dendritic cells maturation or on tumor cells themselves. Finally, IL-9 increases
granzyme B production and degranulation of DP T cells leading to an enhanced cytotoxic
capacity against melanoma cell line.
To conclude, IL-9 would take part in the maintenance of DP T cells within the tumor infiltrate
and favors their functional activity. Our results extend the pleiotropic effect of IL-9 already
described for mast cells, T reg, Th17 and monocytes, to intra-tumor DP T cells [2].
REFERENCES
[1] Desfrancois J, Moreau-Aubry A, Vignard V, Godet Y, Khammari A, et al. (2010) Double
positive CD4CD8 alphabeta T cells: a new tumor-reactive population in human melanomas.
PLoS One 5(1): e8437.
[2] Noelle RJ, Nowak EC. Cellular sources and immune functions of interleukin-9. Nat Rev
Immunol. 2010;10:683–687.
28- MME. BUREL Sophie - 3ème année – UMR 1087
Identification of Two Native Nav1.5 Phosphorylation Sites As Potential Determinants of
the Increased Late Na+ Current in Heart Failure
Sophie Burel1, Fabien Coyan1, Matthew R Meyer2, Cheryl F Lichti3, Joan H Brown4, Flavien
Charpentier1, Jeanne M Nerbonne5, R Reid Townsend6,7, Lars S Maier8 and Céline
Marionneau1. 1Institut du Thorax, INSERM UMR1087, CNRS UMR6291, Nantes, France,
Departments of 2Medicine, 5Developmental Biology, 6Internal Medicine and 7Cell Biology
and Physiology, Washington University Medical School, Saint Louis, MO, USA, 3Department
of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, USA,
4
Department of Pharmacology, University of California, San Diego, CA, USA, and
8
University Hospital Regensburg, Regensburg, Germany.
Voltage-gated Na+ (Nav) channels are key determinants of myocardial excitability and defects
in Nav channel functioning or regulation, associated with inherited and acquired cardiac
disease, increase the risk of life-threatening arrhythmias. In heart failure, the inactivation
properties of Nav1.5 channels are altered, resulting in decreased channel availability and
increased late Na+ current. Although previous studies have suggested roles for CaMKII and
CaMKII-dependent Nav1.5 phosphorylation, the global native phosphorylation pattern of
Nav1.5 channels associated with these alterations is unknown. Phosphoproteomic analyses
were undertaken to identify and quantify in situ the native phosphorylation sites on the
Nav1.5 proteins purified from wild-type and CaMKIIc-overexpressing (CaMKIIc-Tg)
mouse ventricles. A total of 18 phosphosites were identified, 8 of which are novel compared
with our previous MS analyses. Of these 18 phosphosites, the C-terminal phosphoserines
pS1937/pS1938 are present in the CaMKIIc-Tg IPs (n=3/4) and absent in the WT IPs
(n=0/4). In addition, pS1989 is 9-fold more represented (p<0.05, n=4 in each condition) in the
CaMKIIc-Tg, than in the WT, IPs. To explore the possibility that phosphorylation at these Cterminal sites regulates the gating properties of Nav1.5 channels, the orthologous human
48
(serine to glutamate) double phosphomutant Nav1.5-S1933E-S1984E was generated and
investigated by whole cell voltage-clamp analyses in HEK293 cells. These analyses revealed
that the relative percentage of the TTX-sensitive late Na+ current, compared with the peak
Na+ current, is significantly (p<0.01) higher for the double phosphomutant (0.07 ± 0.007%,
n=24), compared with the WT (0.04 ± 0.004%, n=20), currents. In addition, although the fast
and slow time constants of inactivation are similar, the relative contribution of the fast
inactivating component of the peak Na+ current is significantly (p<0.05) lower with the
phosphomutant, compared with the WT channels. Together, these analyses provide 8 novel
native cardiac Nav1.5 phosphorylation sites, 2 of which are in the C-terminus of Nav1.5 and
selectively modulate the late component of Na+ current, suggesting a role for modulation at
these sites in heart failure.
29- M. POGU Julien - 3ème année – ITUN
Tolerization of ongoing CTL response by monocyte derived dendritic cells expressing
heme oxygenase-1
One barrier to prevention and treatment of autoimmune diseases is the difficulty of
antigen specific tolerization of ongoing T-cell responses. We developed a protocol to
tolerize β-cell-specific cytotoxic T cells (CTLs) in type 1 diabetes (T1D) model mice
by intradermal co-injection of an HO-1 inducer and a β-cell antigen. This treatment
induced recruitment in the draining lymph node of an HO-1-overexpressing
monocyte-derived DC (MoDC) population that tolerized autoreactive CTLs. CTL
tolerization was associated with lower β-actin expression, and reduced CTL velocity
and response to chemokines. Intradermal injection of a clinically approved HO-1
inducer in baboons led to HO-1+ MoDC appearance in draining lymph nodes.
Furthermore, in human T-cell clones, β-actin expression was reduced upon
incubation with HO-1+ monocytes. Overall, HO-1 inducers represent a promising
approach for clinical induction of antigen-specific tolerance.
30- MME. DRUJONT Lucile - 3ème année –UMR 1064
Role of ion fluxes in Th17 biology and autoimmune diseases
L. Drujont, A. Lemoine, M.-C. Cuturi, C. Louvet
The nuclear hormone receptor ROR t is the key transcription factor that orchestrates the
differentiation of Th17 cells but also defines
T cells and various Innate Lymphoid Cells
(ILCs). It is challenging to decipher the contribution of each ROR t+ cell type during the
development of pathological responses and to identify the mechanisms involved beyond IL-17
and few other cytokines. Our group identified TMEM176B, a four-span transmembrane
protein that interacts with its structurally identical homolog protein TMEM176A.
Electrophysiological experiments revealed that TMEM176A and B function as cation
channels and can heteromerize to exert their function. Interestingly, these two homologs are
among the few direct targets of ROR t. We observed that both genes are highly expressed in
in vitro-generated mouse Th17 compared to Th1, Th2 or iTregs. We also observed that human
Th17 cells strongly expressed TMEM176A and B mRNA, correlating with the level of RORC.
Thus, we hypothesize that these genes could play a crucial role in the development of a
variety of autoimmune diseases. Preliminary results showed that Tmem176b-/- mice were
partially protected from psoriasis-like lesions when compared to control mice. These results
49
suggest that the deletion of both genes may be required to clearly elucidate their role in
autoimmunity. We have now successfully generated double KO mice and we aim to assess the
impact of these deficiencies on different models of autoimmunity. In addition, we aim to link
TMEM176A and B intracellular localization to his cation channel function in Th17 cells. We
believe that the study of TMEM176A and B will help decipher novel specific pathways of the
Th17 cell biology.
31- MME. LAMORA Audrey – 3ème année – UMR 957 LPRO
Inhibition of TGF-beta signaling pathway blocks the development of osteosarcoma lung
metastasis
*Audrey Lamora1-4, Julie Talbot1-3, Gwenola Bougras1-3, Jérôme Amiaud1-3,Marion Leduc1-3,
Julie Chesneau1-3, Julien Taurelle1-3, Verena Stresing1-3, Marie Cécile Le Deley5, Marie
Françoise Heymann1-3,Dominique Heymann1-3, Françoise Redini1-3 and Franck Verrecchia1-3
1
INSERM, UMR 957, Equipe labellisée Ligue contre le Cancer 2012, Nantes, France;
Université de Nantes, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie
des Tumeurs Osseuses Primitives, Nantes, France; 3CHU Hôtel Dieu, Nantes, France;4Inserm
Liliane Bettencourt School, Pharm.D-PhD student;5Institut Gustave Roussy, Villejuif, France
2
Background: Osteosarcoma is the main malignant primary bone tumor in children and
adolescents for whom the prognosis remains poor, especially when metastases are present at
diagnosis (survival rate drops to 20% when lung metastases were detected). Aim: Because
TGF-beta has been shown to promote metastases in many solid tumors, we investigated the
effects of inhibition of the TGF-beta/Smad cascade on osteosarcoma behavior.
Material and methods: To this end, two independent procedures, a pharmacological
approach with TGF-beta Receptor I inhibitor (SD-208) and a molecular approach using the
natural Smad inhibitor (Smad7), was used. The impact of these procedures was assessed on
tumor growth, tumor microenvironment, bone remodeling and lung metastases development
by using a mouse model of osteosarcoma induced by paratibial injection of osteosarcoma
cells.
Results: We first demonstrated that TGF-beta levels are higher in the serum of osteosarcoma
patients compared to healthy volunteers. We also showed that Smad7 slows the growth of the
primary tumor and increases mice survival. In this context, we demonstrated that Smad7
expression does not affect osteosarcoma cell proliferation but affects the microarchitectural
parameters of bone. In addition, Smad7-osteosarcoma bone tumors expressed lower levels of
osteolytic factor RANKL, suggesting that Smad7 overexpression affects the "vicious cycle"
established between tumor cells and bone cells by its ability to decrease osteoclast activity.
Interestingly, we finaly showed that Smad7 overexpression in osteosarcoma cells and SD-208
inhibits the development of lung metastasis. In this context, we demonstrated that Smad7 and
SD-208 reduced the capacity of osteosarcoma cells to invade Matrigel in Boyden migration
chambers and gelatin zymography identified reduced MMP-2 secretion by osteosarcoma cells.
Conclusions and clinical implications: These results suggest that the inhibition of TGFbeta/Smad signaling pathway could be a promising therapeutic strategy against the tumor
progression of osteosarcoma
Acknowledgments : Ligue contre le cancer, Fondation Bettencourt Schueller-Ecole de
l’INSERM
50
32- M. AMELINE Baptiste – 2ème année – UMR 1089
Title : Evaluation of AAV-based OPN4 transfer for the treatment of inherited retinal
dystrophies
Inherited retinal diseases (IRD) affect about 2 million people worldwide, leading to severe
visual impairment. Specific gene addition therapy is one of the most promising strategies to
treat these patients. However many of them are not eligible for specific gene therapy, such as
patients with unknown deficient genes. Therefore, the aim of this project is to develop an
alternative strategy, independent of the mutation and the retinal degeneration kinetic: The
optogene transfer. In context of IRD, it will consist to convert surviving retinal ganglion cells
into sensitive light cells following the transfer of ChR2 optogene. Several rodent models of
IRD have been successfully treated using the ChR2 optogene. Nevertheless, this approach has
never been evaluated in large animal models. The general objective of our study will be to
define the feasibility of optogenetic therapy to restore vision in blind patients by evaluating
the safety and the efficacy of AAV-mediated gene transfer of ChR2 in surviving ganglion cells
of a canine model of IRD (Rpe65-/- dogs). Moreover, two other canine models of IRD could
be injected to make the proof of the universality of this approach.
33- MME. CHESNEAU Coraline – 3ème année – UMR 1064
Characterization of molecular interactions between HCMV and dendritic cells: impact
of glycosylations on viral tropism.
Dendritic cells (DCs) are prone to sense and alert the immune system after intrusion of
pathogens. Those cells are mostly localized in skin and mucosa which are gateways for
pathogens such as viruses. Components of viral origin, mostly envelope glycoproteins, are
recognized by DC-specific lectins. Previously we have shown that the lectin domain or CRD
(Carbohydrate Recognition Domain) of DC-SIGN can interact with an envelope glycoprotein
of the human cytomegalovirus (HCMV), the glycoprotein B (gB) (Halary et al, 2002). This
interaction allows for the internalization of the virus and promoted cis- and transinfection.
However, our knowledge of this interaction is currently insufficient to develop new antiHCMV therapies whose design should take into account the blockade of the DC-SIGN/gB
interaction.To finely characterize this interaction we proposed to generate and/or to use
deletional mutants of DC-SIGN (full length versus d-CRD and d-neck) as well as gB mutants
(i.e. selective mutation of each of the 18 potential N-glycosylation sites). Preliminary results,
show that CRD part of DC-SIGN is the unique part to be involved in gB interaction.
Furthermore, on the 18 potential gB glycosylation sites, only 4/5 seem to have a role in the
binding to DC-SIGN.
Based on these experiments, we would like to develop a 3D model of the DC-SIGN/gB
complex. This one could be used to rationally design competitive inhibitors of this interaction
which is probably determinant for the virus to establish a long life persistence.
51
34- M. CARRADORI Dario -2ème année – EA 3143 LNBT
PEPTIDE FUNCTIONALIZED NANOVECTORS FOR TARGETING STEM CELLS
IN THE SUBVENTRICULAR ZONE
Carradori D. 1,2 ; Saulnier P. 2 ; Benoit J .P. 2 ; Eyer J. 1
1
4, Rue Larrey, LNBT EA 3143, Université d'Angers, Angers, France. Tel : 02 44 68 84 88;
E-mail: joel.eyer@univ-angers.fr
2
4, Rue Larrey, U646 MINT, Université d'Angers, Angers, France
Introduction. Stem cells have the ability to self renew and to differentiate into different
cell types, depending on their origin 1. This property is promising for their potential
application in therapy, especially in the treatment of neurodegenerative disorders 2.
Goal. The aim of this work is to functionalize nanovectors with a peptide (PNs) able to
target stem cells of the subventicular zone (SCs SVZ). Fluorescent lipid nanocapsules 3
(fLNCs) were chosen as nanovectors, thanks to their favorable biochemical profile. A 24
amino acid long peptide was chosen as a targeting peptide, because work in progress in
our laboratory showed its capacity to enter specifically in SCs SVZ (Lepinoux-Chambaud
C. and Eyer J., LNBT). The fLNCs were functionalized with the peptide to obtain the
PNs. Then, PNs were incubated with SCs SVZ to investigate their toxicity, their
mechanism of cell penetration and their pharmacokinetics. Treated SCs SVZ were
analysed by flow cytofluometry and confocal imaging.
Materials & Methods. The peptide was purchased from different companies (Millegen,
Eurogentec and GeneCust). Labrafac® was purchased from Gattefosse SA (Saint-Priest,
France). Lipoïd® came from Lipod Gmbh (Ludwigshafen, Germany). Solutol HS was
purchased from BASF (Ludwigshafen, Germany ), NaCl from Prolabo (Fontenay-sousbois, France), and deionized water was acquired from MilliQ system Millipore (Paris,
France).
DiD'
solid;
DiIC18(5)
solid
(1,1'-Dioctadecyl-3,3,3',3'Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt) was purchased from
Molecular Probes® (Life Technologies). Primary antibodies: anti-α-tubulin, anti-Nestin,
anti-GFAP, and anti-Vimentin were purchased from Sigma. Secondary antibodies labelled
with Alexa-Fluor-488 or Alexa-Fluor-568, and DAPI were purchased from Molecular
Probes® (Life Technologies).
The SVZ was dissected from new-born rat according to Directive 2010/63/EU, and SCs
were cultivate as floating cell culture to finally obtain neurospheres 4. 7 days old
neurospheres were incubated with the PN at different conditions (time, concentration and
energy conditions) and then analysed by flow cytofluometry and by confocal microscopy.
Results & Discussion. Flow cytofluometry: the SCs incubated with PNs showed a
significantly higher fluorescent than SCs incubated with fLNCs (control). Low-energy
conditions during incubation didn’t influence significantly the amount of fluores cent cells
compared to normal conditions. Confocal microscopy: neurospheres incubated with PN
and fLNCs confirmed the flow cytofluometry results.
Conclusion. The results showed that the peptide plays an important role in SVZ SCs
targeting. The combination of the peptide with fLNCs leads to a PN with high SVZ SCs
selectivity. It could be used as a vector for drugs able to induce a therapeutic effect,
through SVZ SCs interaction, in the case of neurodegenerative diseases.
This work was supported by Nanofar to Carradori D. It was also supported by AFM
(Association Française contre les Myopathies), by CIMATH (Région des Pays-de-laLoire), and by MAWTIN to Eyer J..
References
1 FM Watt and BLM Hogan, Science, 2000, 287, 1427-1430.
2 O Lindvall and Z Kokaia, Nature, 2006, 441, 1094-1096.
3 B Heurtault, P Saulnier, B Pech, JE Proust and JP Benoit, Pharmaceutical Research, 2002,
19, 875-880
4 W Guo, NE Patzlaff, EM Jobe and X Zhao, Nature Protocols, 2012, 11, 2005-2012.
52
35- M.RECOQUILLON Sylvain – 3ème année –U1063
Role of non-muscular myosin light chain kinase (nmMLCK) in the inflammation
associated with a model of intermittent hypoxia
Sylvain Recoquillon, Manuel Gómez-Guzmán, Frédéric Gagnadoux, Ramaroson
Andriantsitohaina, M. Carmen Martínez INSERM U1063 « Stress Oxydant et Pathologies
Métaboliques », Université d’Angers, France
Obstructive Sleep Apnea Syndrome (OSAS) is characterized by repetitive obstructions of the
upper airway during sleep, inducing intermittent diminution in oxygen saturation, or
intermittent hypoxia (IH). IH alters endothelial function favoring inflammation and could
accelerate atherosclerosis-induced cardiovascular diseases. A protein that may play a role in
this process is the non-muscular myosin light chain kinase (nmMLCK). The deficiency of this
kinase protects mice from death in septic choc models and prevents the atherosclerosis in
mice fed with a high fat dietary. The aim of this study was to analyze the implication of
nmMLCK in the vascular effects and the inflammation induced by IH. Human aortic
endothelial cells (HAoECs) and aortas isolated from C57BL6 mice were exposed to 6h of IH,
which include 30 min of hypoxia (O2 5%) followed by 30 min of normoxia (O2 21%), in the
absence or the presence of ML-7 (5µM), a nmMLCK inhibitor. After the stimulation, we
measured nitric oxide (NO), superoxide anion production and the endothelium-dependent
acetylcholine-induced relaxation in order to evaluate endothelial function. We also
investigated inflammatory process by evaluating p65-NF-κB pathway and the expression of
IL-6 in the supernatant of cells. The results show that IH treatment increased superoxide
anion, NO and IL-6 productions in HAoECs. However, while the nmMLCK inhibitor ML-7
had no effect on the IH-induced superoxide anion increase, it decreased both NO and IL-6
production. Furthermore, p65-NF-κB pathway was activated by IH in a ML-7-insensitive
manner in HAoECs. IH also decreased acetylcholine-dependent relaxation of mice aortas,
suggesting an IH-induced endothelial dysfunction. These results suggest that IH impairs
vascular reactivity associated with an IH-induced inflammatory activation in endothelial cells
in which nmMLCK may participate.
36- M. KANDALAM Saikrishna – 2ème année – Inserm U1066
NANO AND MICRO TAILORING OF BIOMIMETIC AND
PHARMACOLOGICALLY ACTIVE BIOMATERIALS COMBINED TO ADULT
STEM CELLS FOR SPINAL CORD INJURY REPAIR
Saikrishna KANDALAM 1,2,3 * ; Anne des Rieux 3 ; Laurence Sindji1,2, Jerome Guicheux4,5,6 ;
Montero-Menei CN.1,2
1
LUNAM Universite, UMR S-1066 F-49933 Angers, France.
INSERM U1066, MINT "Micro et nanomedecines biomimetiques "F-49933 Angers, France.
3
Louvain Drug Research Institute, Pharmaceutics and Drug Delivery Research group,
Université
Catholique de Louvain, Belgium.
4
INSERM, UMR 791, Skeletal Tissue Engineering and Physiopathology team LIOAD, Nantes,
France.
5
Université de Nantes, UFR Odontologie, Nantes, France.
6
CHU Nantes, PHU 4 OTONN, Nantes, France
2
SUMMARY
Introduction: During the last few years it has been shown that adult stem cells are strong
candidates for cell-based therapy in Spinal cord injury (SCI). Studies shows that, pre53
treatment of Marrow-isolated adult multi-lineage inducible (MIAMI) cells (mesenchymal
stromal cells expressing typical embryonic stem cell markers) with epidermal growth factor
(EGF) and basic fibroblast growth factor (bFGF) enhances neural specification and response
to neuronal commitment. Recent studies depicts that brain derived neutrophic factor (BDNF)
directly enhanced the differentiation of stem cells to neuronal precursors, and promotes
supraspinal axonal regeneration and restoration of motor neuron exitability in spinal cord.
Fibronectin an extracellular protein will enhance mesenchymal stem cell survival, cell
adhesion. An injectable hydrogels provide elastic properties and enhancing stem cell
specification.
Objectives: MIAMI cells transported by fibronectin-covered pharmacologically active
microcarriers (PAMs) delivering BDNF within a silated hydroxypropyl methylcellulose (SIHPMC) hydrogel [9] matrix to repair neural tissue and promote functional recovery after SCI.
Methods: PAM’s preparation involves nanoprecipitation of BDNF and encapsulation in
PLGA–P188–PLGA polymeric microspheres. PAM’s characterization includes size (Coulter
Counter Multisizer), shape (scanning electron microscope), encapsulation yield (ELISA),
release kinetics and bio assay (ELISA & rat dorsal root ganglia).
Results: Nanoprecipitation yield of α-chymotrypsin (model protein) was performed using an
experimental design was found to be 74±4.3%. BDNF nanoprecipitation yeild, mean size and
encapsulation yield in PAMs was determined as 92.2±10.3 %, 33.8±12.7µm and 91.39 %
respectively.
Conclusion: PAMs are successfully formulated with nanoprecipitated BDNF and surface
coated with fibronectin. Further studies on release kinetics of BDNF from fibronectin-BDNFPAMs and characterization of MIAMI cells along with fibronectin-BDNF-PAMs in Si-HPMC
hydrogel has to be performed.
37- MME ROY Charlotte – 2ème année -UMR CNRS 6214 INSERM U1083
(BNMI)
Protective role of nucleotidases against the development of hypertension
Arterial hypertension is a major risk factor for cardiovascular complications such as
myocardial infarction or stroke. In hypertension, vascular tone is exacerbated, accompanied
by an hypertrophic remodeling of vessel wall (oxidative stress, endothelial dysfunction,
fibrosis). Extracellular nucleotides are released under conditions of cellular stress (myogenic
tone, shear stress) and promote deleterious responses in pathological context
(vasoconstriction, inflammation, promotion of vascular permeability) via activation of
purinergic (P2) receptors. Although signaling by extracellular nucleotides are important in
vascular homeostasis its contribution to vascular pathologies remains poorly understood. The
bioavailability of these nucleotides is controlled by ectonucleotidases, especially NTPDase1
(CD39) highly expressed in the arterial wall. These enzymes not only scavenge nucleotides
but, in concert with ecto-5’nucleotidase (CD73), contribute to generation of vasoprotective
adenosine (anti inflammatory, vasodilatory). We evaluated here the role of nucleotide
degrading enzymes in the development of hypertension. #1 We treated mice with Angiotensin
II (AngII, 1mg/kg/day) with or without potatoe Apyrase (AngII/APY), a soluble nucleotidase
with similar activity to CD39 (45U in osmotic pumps + 15U ip every 3 days) during 12 days.
We observed a reduced systolic blood pressure (SBP) increase (7±5 vs 21±7 mmHg) in the
AngII/APY group compared to AngII alone and diminished hypertrophic arterial (aorta)
remodeling (2±4 vs 11±3 µm of thickness). The efficacy of the treatment was performed by
measuring fluorescent derivative etheno ADP hydrolysis by HPLC. #2 The protective effect
provided by the enzyme is certainly affected in hypertension since RT-qPCR experiments
showed a decreased of transcript encoding CD39 in resistance arteries in AngII-dependent
hypertension. #3 The absence of CD39 should have an impact on the development of
hypertension. Entpd1+/+ and Entpd1-/- mice were treated with AngII (0.5 mg/kg/day) during
54
21 days. Results show that SBP was significantly higher (43±5 vs 18±5 mmHg) and
associated to an exacerbated hypertrophic arterial remodeling (19±8 vs 3±5 µm) in Entpd1-/compared to Entpd1+/+ mice. This protective role of CD39 remains to be investigated; this is
may be due to its vascular component, regulating vasoconstrictor P2 receptor, but also due to
its immune component, exerting its anti-inflammatory function. Consequently, nucleotidases
confer resistance to high blood pressure and may represent new therapeutic targets in the
treatment of hypertension.
38- M. RIDEAU Alexis – 2ème année - Cancer Center, Inserm U892 team 12,
CNRS 6299
Identification and Quantification of the Secreted Protein OLFM4, involved in AnchorageIndependant Growth, by a Multi-Technique Approach in Colorectal and Breast Cancer.
The proteomic approach is a powerful tool used to study tumoral cell lines or tumoral tissue samples.
In order to understand the tumoral development, it is necessary to know fondamental cellular
mechanisms. The major actors regulating these mechanisms are the proteins. Proteins enriched from a
shotgun proteomic approach can be identified and quantified by mass spectrometry. In a previous
study, we have analyzed the proteome of different stages of colorectal tumor and determined
Olfactomedine-4 (OLFM4) as a potential biomarker. In this study, we have classified these proteomes
in subproteomes. In our laboratory, we investigate the senescence escape during tumor development
that is why the subproteome involves in cell death was identified through global colorectal proteome.
For few years, the interest in proteins of the SASP (Senescence Associated Secreted Proteins)
increased ans especially the ones involved in senescence escape. Thus, the colorectal subproteome
secreted was described in this study. Among these secreted proteins, one of them, OLFM4, is very
interesting in early clinical diagnosis. This secreted protein is overexpressed in early stage of
colorectal and breast cancer and is measurable in blood of patients. Functions of OLFM4 are described
only as potential cell survival protein. This study associate OLFM4 to anchorage-independant growth
and anoikis resistance through Soft Agar assay. Indeed, OLFM4 increases the growth of colorectal and
mammary cell lines in low adhesion conditions. In order to generate subproteomes, the adhesion
colorectal proteome was identified. Insterestingly, the OLFM4 overexpression is correlated with a loss
of cadherin and an overexpression of vimentin proteins involved in cell adhesion and migration. These
data reveal a relationship between a potential biomarker measurable in blood of patients and an
agressive phenotype acquired.
39- M. LEGEAY Samuel - 2ème année – Inserm U1063
M3 receptor as a key target for DEET-induced angiogenesis.
The insect repellent N,N-diethyl-m-toluamide (DEET) recently proposed as an inhibitor of
human acetylcholinesterase has been reported to potentiate tumor development. As
angiogenesis is a critical step in tumorigenesis, the present study was designed to test the
potential effect of DEET on different processes leading to neovascularisation on human
endothelial cells (HUVECs). These primary cells were incubated for 24h with DEET at
concentrations similar to that found in plasma of exposed individuals (10-5M) or in the
environment (10-8M). Both concentrations of DEET increased proliferation, migration and
adhesion of cultured endothelial cells, in vitro capillary length of HUVECS in culture on
matrigel and in vivo vascularization of Matrigel Plug. These cellular processes were
55
associated with focal adhesion kinase (FAK) phosphorylation and stress fibers. Both
concentrations of DEET also stimulated NO production through an increase of peNOSSer/peNOS-Thr ratio. Moreover, DEET potentiates carbachol-induced calcium signaling in
human embryonic kidney (HEK) 293 cells expressing recombinant Galpha(q/11)-coupled
muscarinic M3 receptors. Inhibition or silencing M3 muscarinic receptor subtype either with
the pharmacological antagonist parafluorohexahydrosiladiphenidol (pFHHSiD) or siRNA,
respectively abolished the pro-angiogenic properties of DEET in both in vitro and in vivo
conditions. These data underscore a novel property of DEET as a pro-angiogenic toxic, via
the involvement of M3 muscarinic receptor subtype, that probably explains its potentiating
effect on tumor development.
40- M.FRIFRA Mehdi – 2ème année – Inserm U1063
Impact of nutrients in metabolic diseases: characterization of vegetal samples on
different cell lines.
FRIFRA Mehdi*, SOLETI Raffaella, CLERE Nicolas, RENOU Jean-Pierre &
ANDRIANTSITOHAINA Ramaroson
Unité INSERM U1063 SOPAM, Stress Oxydant et Pathologies Métaboliques, University of
Angers, France
Obesity incidence is associated with an increased risk of disorders, such as metabolic
syndrome, diabetes and cardiovascular disease. Nutritional approach is essential to prevent or
correct obesity associated disorders and can be achieved by the quality vegetable and fruit
consumption. We studied the influence of apple according to the genetic variability, the
conditions of production and post-harvest preservation on cells implicated in development of
obesity-related diseases. Hepatocytes (HepG2) and adipocytes (3T3-L1) are treated 24 hours
with two different concentrations (10-2 and 10-5 g/L) of 31 lyophilisate samples of apples from
different genetic variability, culture conditions and conservations, solubilized in DMSO or
ethanol with respect to lipid accumulation. The two solvents at used concentrations did not
modify basal lipid accumulation and that induced either by oleic acid or cocktail of
differentiation medium in HepG2 and 3T3-L1 cell lines, respectively. In HepG2, the four
varieties of apples did not modify basal lipid accumulation. Among these varieties, high
concentration of Golden and Pink lady decreased lipid accumulation induced by oleic acid
when they are solubilized in ethanol. Any of the 31 lyophilisate samples of apples tested
solubilized with either DMSO or ethanol and at any concentration used did not affect lipid
accumulation in 3T3-L1 adipocytes. These results under score the cellular specificity action of
apple on lipid accumulation with special tropism on HepG2 with Golden and Pink lady
varieties. They also validate the technological strategies used for cellular screening of
vegetable and fruit to sort beneficial products against obesity-related disease. Further studies
are ongoing in endothelial and smooth muscle cells and macrophages.
41- M. MILBANK Edward – 2ème année – Inserm UMR 1063
EXTRACELLULAR MICROVESICLES AS A THERAPEUTIC STRATEGY TO
PREVENT OR REVERT OBESITY AND ITS METABOLIC COMPLICATIONS IN
THE FIELD OF NANOMEDICINE
E. MILBANK [1] [2]; M. LOPEZ [1] [2], R. ANDRIANTSITOHAINA [1]
[1]
INSERM U1063, SOPAM - Stress Oxydant et Pathologies Métaboliques - University of
Angers, France.
56
[2]
Department of Physiology, CIMUS, University of Santiago de Compostela - Instituto de
Investigacion Sanitaria, Santiago de Compostela, 15782, Spain
Correspondence should be addressed to E. MILBANK (edward.milbank@etud.univ-angers.fr)
Introduction : The central nervous system (CNS) receives multiple inputs of information as
diverse as the sensory of eating, metabolism and levels of energy storage. Integrative member
of the CNS, the hypothalamus is a well known crucial regulator of both energy intake and
energy expenditure. One of the main energy-regulatory factors within the hypothalamus is the
AMP-activated protein kinase (AMPK), which is involved in a large number of biological
actions including the modulation of energy balance (Lopez et al , Cell Metab 2008, Nat Med.
2010).
The use of modified DNA sequence to induce protein inactivation has opened a new avenue
in drug discovery. However, their therapeutic potential is hampered by inadequate tissuespecific delivery. Thus, using isolated extracellular microvesicles - vesicles released by
activated cells - loaded with a dominant negative isoform of AMPK (AMPK DN), would
allow the specific targeting of hypothalamic neurons and the inhibition of AMPK central
activity.
Objective : Developing structure-modified dendritic cell derived exosomes loaded with an
AMPK DN.
Methods : Transfection protocol was optimized using JetPei as transfection reagent and green
fluorescent protein (GFP) as control. Dendritic cells (DC) viability and immunophenotype
were analyzed by flow cytometry. Exosomes morphological analyses were performed using
electron microscopy and size distribution methods.
Results : DC transfection provided a good efficiency and did not alter the viability and the
immunophenotype of the cells. The morphological analysis of the dendritic cell-derived
microvesicles confirmed that they harbored the principal properties of the exosomes.
Conclusion : Once structure-modified exosomes purified, their abilities to target
hypothalamic neurons after systemic and intranasal deliveries will have to be analyzed
through in vivo studies. The last step will be to incorporate a AMPK DN within these
exosomes and analyze their effects directly on the central regulation of obesity.
42- MME. MOREAU Marie -2ème année – Inserm U892 E12 /CNRS 6299
Secretome of persistent colorectal cancer cells conferes an agressive phenotype which
may promote metastasis development.
Induction of senescence by chemotherapy was initially characterized as a suppressive
response that prevents tumor cell proliferation. However, in response to treatment, it is not
really known how cells can survive senescence and how irreversible this pathway is. We
analyzed cell escape in response to irinotecan (sn38), a first line treatment used in colorectal
cancer that induced senescence. A previous study showed that malignant cells which resisted
senescence relied on survival pathways induced by Mcl-1 signaling and to a lesser extent of
Bcl-xL. Depletion of Mcl-1 increased irinotecan efficiency, induced the death of polyploid
cells, prevented cell emergence and inhibited growth in low-adhesion conditions. In this case,
we wanted to know what is effect of the secretome of the cells that escaped cellular
senescence. Indeed, one of the caracteristics of cellular senescence is the appearance of the
senescence-associated secretory phenotype (SASP) which has different roles autocrine and
paracrine in inflammation, angiogenesis or senescence reinforcement. Many studies showed
that secretome of senescent cells gave an agressive phenotype to neighboring cells. In our
model, we detected subpopulations of cells that adapted to chemotherapy and resumed
proliferation. These cells were called Persistent LS174T Cells (PLCs). PLCs are heterogenous
57
populations composed by proliferating and senescent cells. Thus, the secretome analysis could
help us to understand the differences between parental LS174T cells ans PLCs. In this
context, we collected different samples of conditioned media after starvation from LS174T
and PLCs. Then, we analyzed the effect of these supernatants on parental LS174T cells
behavior. We showed that conditioned media of PLCs, unlike LS174T cells, induced cell
proliferation, growth in low adhesion conditions, emergence, migration and angiogenesis by
the formation of a tubing network of endothelial cells. Together, these results suggest that the
secretome of persistent colorectal cancer cells to chemotherapy, makes agressive cells and
promotes metastasis development.
43- M.DIB Abdallah – 2ème année – U1083
Effect of maternal diabetes on fetal programming of vascular remodeling mechanisms in
adult rats
Abdallah Dib, Jennifer Bourreau, Daniel Henrion, Céline Fassot
UMR CNRS 6214 / INSERM U1083, Faculté de Médecine, Rue Haute de Reculée, 49045
Angers
Numerous studies have shown that adult diseases may have their origins in fetal life.
Indeed, modifications of the intra-uterine environment during specific windows of fetal
development are now recognized as an important cause of fetal stress, leading to several
responses such as loss of structure/function and pre-emptive adaptations to an adverse postnatal environment, and finally to adult diseases such as metabolic abnormalities and
hypertension. In this way, it has been demonstrated that children of diabetic mothers have an
increased risk of developing cardiovascular disease and insulin resistance during adulthood.
In our laboratory, we developed an animal model of rats exposed in utero to maternal diabetes
(DMO). A first study showed that DMO developped hypertension at 6 months of age. In a
second study, specific modifications of aortic gene expression profile were revealed in DMO
at a pre-hypertensive stage (3 months) in favor of a vasoconstrictor phenotype. Moreover, a
lack of vascular remodeling in response to high blood pressure (hypertension) in old DMO
(18 months) was observed.
This new project aims to study fetal programming of vascular remodeling mechanisms
in response to maternal diabetes. To induce the remodeling process, a mesenteric artery
ligation model was used to modify flow (remodeling aimed at normalizing wall stress and
restoring optimal blood flow). Briefly, mesenteric arteries of 3 months-old animals issued
from control mothers (CMO) or diabetic mothers (DMO) were ligated in order to decreased
blood flow (LF). Proximal arteries had an increased blood flow (HF) and distant arteries were
used at control (NF). Diameter of vessels were then analyzed by arteriography.
Interestingly, we observed that at 7 or 21 days after ligation, DMO did not exhibit constrictive
remodeling in LF although expansive remodeling in response to increased flow is maintained.
These results show that in utero exposure to maternal diabetes induces a fetal
reprogramming of vascular function of the newborn.
44- M. GAY Swann -2ème année – UMR 1066
Elaboration of polymers biobased foams using carbone dioxide
a,b
Swann Gay*
a
b
a
b
a
, Frank Boury , Alain Gibaud , Brice Calvignac
Université d’Angers – Laboratoire MINT/INSERM 1066
Université du Maine – Laboratoire IMMM/CNRS 6283
58
The development of efficient and environmentally friendly materials is a major challenge
nowadays. Supercritical fluid (SCF) processes provide clean and efficient alternatives to
several technologies such as extraction, chromatography, crystallization and material
processing. Carbon dioxide (CO2) is widely used as a SCF due to its non-toxicity and its easy
reachable critical properties (Pc=73.8 bars, Tc=31.3 K). Moreover, modifications in CO2
temperature and pressure allow changing its physical properties, an interesting characteristic
for plastic materials processing. Thus the development of porous polymeric materials using
clean processes fits perfectly with this approach. Moreover polymeric foams are ever widely
used in several applications such as transports, phonic or thermic insulation, filtration
membrane or medical implant. So the manufacture of bio-based foams with green processes
should be promising. In this way, this study has been dedicated to the foaming of polylactic
acid (PLA) using supercritical CO2 (SC-CO2) . The PLA was chosen for its good
environmental impact: biobased biocompatible, compostable.
Two process approaches have been developed to produce PLA foams: one High Temperature
Method (HTM) and low Temperature Methods (LTM). The first method uses SC-CO2 as a
physical blowing agent. A PLA film is heated in a vessel and SC-CO2 is introduced and is
dissolved into the polymer. Pressure and temperature are regulated during a fixed time and
then the autoclave is depressurized at a constant rate until the ambient conditions. Hence, the
CO2 contained into the PLA expands and induces the foaming. This method is solvent-free
but it is necessary to heat at relative high temperatures (between 130 and 165 °C).
The second approach is based on phase separation techniques at relatively low temperature
(35-40°C). Two techniques of phase separation were investigated. In both methods, a solution
of PLA (5 to 20% w/v) in an organic solvent is prepared. First method consists to induce the
phase separation in the PLA solution with the introduction of SC-CO2, which acts as
antisolvent, and involves the polymer precipitation and extraction of the solvent. In the second
method, the phase separation was obtained when the polymer solution is soaked into liquid
nitrogen, which freezes the PLA and solvent. Then the organic solvent is extracted with
ethanol and an alcohol gel composed of PLA is obtained. Finally the gel is exposed to SCCO2 in order to extract ethanol and dry the PLA foam obtained.
This work shows that the different processes permit to produce porous samples of PLA with
tunable morphologies. The foams obtained were observed by scanning electron microscopy
(SEM). Observations showed different cellular structures ranging from macro scale to micro
scale with specific density included between 50 to 750 kg/m3. The PLA crystallinity was
determined by X-ray diffraction (XRD), which varies according to the process and induces
different foam morphologies.
LTM
HTM
59
45- MME. GRENIER Céline – 3ème année – CNRS 6214 Inserm 1083
Role of Thrombospondin -1 in the Remodeling Induced by a Chronic Increase in Flow in
Resistance Arteries.
Celine Grenier1, Antoine Caillon1, Linda Grimaud1, Audrey Ayer1, Bertrand Toutain1,Vincent
Sauzeau2, Gervaise Loirand2, Olivier Blanc-Brude3, Daniel Henrion1, Laurent Loufrani1.
1
UMR Inserm 1083 UMR CNRS 6214 Department of Integrated Neurovascular and
Mitochondrial Biology, Angers, France.
2
UMR Inserm 1087 CNRS UMR 6291 IRT-UN, NANTES, France
3
UMR Inserm 970 Paris-Cardiovascular research Center at HEGP, Paris, France
Background : Chronic increases in blood flow in resistance arteries induce outward vascular
remodeling due to increases NO-dependant dilation and MMP activation. Although
thrombospondin-1 (TSP-1) interacts with matrix proteins and cell-surface receptors,
controversy remains about its vascular functions. We investigate the role of TSP-1 in high
flow (HF)-mediated remodeling of resistance arteries.
Material and methods: Mesenteric arteries were ligated in vivo to generate HF arteries
compared with normal flow (NF) vessels on wild type (WT) and TSP-1 deleted (KO) mice.
Arteries were isolated from 4 days to 1 month after ligature. Arteries diameter and reactivity
were studied in vitro in an arteriograph. Genes and proteins implicated in inflammation,
remodeling, and immune cells recruitment were determined in HF and NF arteries.
Results: Increases in diameter found between WT NF and WT HF arteries were not
significant in KO arteries. Contrary to KO arteries the diameter increase in WT arteries were
correlated to an increase in wall thickness. Furthermore KO arteries were less responsive to
Ach induced dilation than WT arteries.
After 4 days of ligature, we observed an increase in pro-inflammatory (CD68, Cox2, Gp91
phox, p47 phox, p22phox) gene expression between NF and HF WT arteries which was not
observed in KO arteries. Moreover we also saw a rise in leukocytes number in response to
chronic increase in blood flow in WT arteries, which was less important in KO arteries,
suggesting a role of TSP-1 in the recruitment of immune cells allowing the inflammatory
response leading to hypertrophic outward remodeling.
In bone marrow cells transfer experiments, we also demonstrated that circulating cells
secreted TSP-1 and vascular cells secreted TSP-1 are implicated differently in artery flowinduced remodeling (wall hypertrophy and diameter).
Conclusion: The absence of TSP-1 leaded to a decrease in flow-induced dilation remodeling
with a reduction of the inflammatory response and of the endothelium-mediated dilation
involved in HF remodeling. The deficiency in remodeling was also associated with a decrease
in the immune cells recruitment involved in the initiation of the remodeling.
46- MME. MONTAGU Angélique – 3ème année – U1066
Evaluation of interaction mechanisms between Acinetobacter baumannii bacteria and
lipidic nanocapsules by flow cytometry.
*A. Montagua,b, M-L. Joly-Guillouc, C. Guillet,, J. Bejauda,b, C. Gaillardd , E. Rossinese, and
P. Saulniera,b
Acinetobacter baumannii is an important nosocomial pathogen, resistant to many commonlyused antibiotics. Considering the limited number of antibiotics in development, interesting
strategies could lay on the use of natural resources especially essential oils (EOs). An
60
interesting strategy underlined the antibacterial potential of EO components (carvacrol,
cinnamaldehyde) loaded lipid nanocapsules (LNCs) against A. baumannii. The aim of this
study was to produce and characterize DiO-LNCs loaded with antibacterial actives. The
antibacterial activity of these nano-carriers was evaluated in vitro against A. baumannii.
Finally, we determined the interactions between bacteria and LNCs over time thanks to the
fluorescence of DiO-LNCs and the properties of trypan blue in order to precise the
physicochemical mechanisms occurring at the level of the biological membrane. The results
underlined the attractiveness of the encapsulated actives compared to unloaded-LNCs. These
results demonstrated the capacity of carvacrol-loaded-LNCs to interact and penetrate the
bacterial membrane in comparison with cinnamaldehyde-LNCs and unloaded-LNCs.
Moreover, the fluorescence of bacteria remained constant after contact with carvacrol-loadedLNCs and cinnamaldehyde-LNCs whereas the fluorescence of the blank-LNCs decreased
over time. This phenomenon could be explained by the release of these blank-LNCs by efflux
pumps. Thereafter, modifications of carvacrol after substitution of hydroxyl functions by fatty
acids (acetic acid, palmitic acid) demonstrated the crucial role of these latter for antibacterial
activity. Finally, after contact with an efflux pump inhibitor CCCP (carbonylcyanide-3chlorophenyl hydrazine), the results underlined a total synergistic effect for Car-LNCs
showing that the CCCP is associated with action mechanism of carvacrol especially at the
level of efflux pump mechanism.
47- MME. MOUZOUVI Celia Rosemonde -3ème année –U1066
Use of Lipid Nanocapsules for air bubbles stabilization
CRM MOUZOUVI1, 2*, KA BIGOT1, P SAULNIER2
1. UFR Pharmacie, Faculté des Sciences de la Santé, Université d’Abomey-calavi, Campus
du Champ, 01 BP 188 Bénin.
2. INSERM U1066 – IBS-CHU, 4 rue Larrey 49933 Angers Cedex 9, France.
Background: Lipid NanoCapsules (LNC), nano drug carriers, formulated by a phase
inversion method are characterized by a shell constituted with two surfactants, Solutol® and
Lipoïd®. After their formulation, their film properties at the air/water interface had been
highlighted. Based on the findings of this study, our research wants to use these LNC film
properties to produce gas bubbles for therapeutic applications. But before this application, the
ability of LNC to decrease a surface tension at air/water interface must to be studied.
Methods
The drop tensiometer was used to measure a surface tension at the air/water interface.
Different dilutions of the related suspensions have been prepared. . Interfacial stabilization by
LNCs have been compared to interfacial stabilization by Solutol® alone.
Results
Lipid NanoCapsules decreased the surface tension from 72mN/m to 35mN/m. This action was
better than the one induced by surfactants taken directly.. The particles size does not influence
significantly the obtained values. The adsorption time of Lipid NanoCapsules was in the
range, 1 hour and twenty minutes and 2 hours depending on the dilution factor.
Conclusion
The use of Lipid NanoCapsules instead of surfactants offers a possibility to stabilize air
bubbles and to localize at their surface cargo systems that can be loaded with therapeutic
agents.
Key Words: Lipid NanoCapsules, surface tension, air bubbles.
61
48- M. FLEURY Audrey – 3ème année – U 1063
Microvesicles harboring Sonic Hedgehog suppress adipocyte differentiation via a noncanonical pathway.
Sonic Hedgehog (Shh) is a morphogen that belongs to the highly conserved Hedgehog family
proteins. Long-range signaling of Hedgehog can occur via secretion of dual-lipid modified
proteins transported via extracellular vesicles. Our previous work has established the ability of
a particular group of extracellular vesicles, microvesicles harboring Shh (MVsShh+), to correct
endothelial dysfunctions, usually related to cardiovascular diseases. Non-lipid modified
Recombinant Shh (RecShh) is also known as a potent anti-adipogenic factor. Here, we
investigate the consequences of microvesicles harboring Shh (MVsShh+) on adipocyte
differentiation.
Methods: This study was carried out with a murine preadipocyte cell line, 3T3-L1, treated
with RecShh or MVs specifically enriched with Shh (MVsShh+) or not (MVsShh-). MVs were
retrieved from the lymphocytic cell line (T-CEM) conditioned media.
Results: Incubation of preadipocytes with RecShh or MVsShh+ prevented adipocyte
diffe
C/EBPa). RecShh or MVsShh+ could no longer inhibit adipocyte differentiation in
preadipocytes defective for the transmembrane receptor Smoothened (SMO), a critical
transducer of Hedgehog signaling. However, downstream of SMO, factors involved in the
inhibitory properties of RecShh or MVsShh+ were different. Treatment with RecShh induced a
ciliary translocation of SMO leading to the induction of Gli factors expression whereas MVs
inhibition properties were independent of SMO translocation and Gli expression.
Furthermore, preincubation of preadipocytes with a SMO antagonist, cyclopamine, did not
allow to reverse the MVsShh+ effects.
These results revealed that Shh triggers different downstream pathways to inhibit
adipogenesis
depending on its association, or lack thereof, with microvesicles. Of particular interest,
MVsShh+ stimulate a non-canonical Hedgehog signaling pathway in preadipocytes, which
could participate in the regulation of body fat mass by modulating the pool of preadipocytes.
49- MME. VERGER Elise – 3ème année –U1066
Development of a new selective internal radiation therapy and diagnostic tool for
Hepatocellular Carcinoma: the Starch-Based Microspheres
E.Verger* [1,4,8] ; A. Cikankowitz [1,8] ; A. Bouvier [2] ; N. Lepareur [5,8] ; J. Benoit
[1,8] ; J. Deloye [6] ; C. Aubé [2] ; O. Couturier [1,3,8] ; F. Lacoeuille [1,3,7,8] ; R. Hustinx
[4] ; F. Hindré [1,7,8].
[1] INSERM U1066 MINT (Micro et Nanomédecines Biomimétiques), University of Angers,
FRANCE ; [2] Radiology department, CHU d'Angers, University of Angers, FRANCE ; [3]
Nuclear medicine department, CHU d'Angers, University of Angers, FRANCE ; [4] Nuclear
medicine department, CHU de Liège, University of Liège, BELGIUM ; [5] Centre Régional
de Lutte Contre le Cancer (CRLCC) Eugène Marquis, Rennes, FRANCE ; [6] Cyclopharma
Laboratoires, Saint-Beauzire, FRANCE ; [7] PRIMEX (Plateforme de Radiobiologie et
d'IMagerie EXpérimentale), University of Angers, FRANCE ; [8] Labex IRON, NantesAngers, FRANCE.
Contact email: elise.verger@etud.univ-angers.fr ;
Abstract:
62
The HepatoCellular Carcinoma (HCC) is the 2nd leading cause of cancer death. The
internal radiation therapy (SIRT) of HCC can be realized by the injection of yttrium-90
microspheres (90YMS). This technique requires a pre-therapeutic step with an angiography
and a scintigraphy realized by the injection of 99m-Technetium labelled macroaggregated
albumin (99mTc-MAA). However the difference in size and morphology between the 99mTcMAA and the 90YMS can lead to a divergence in the biodistribution and an approximate
tumour dosimetry.
In order to overcome this problem, our aim is to develop a microparticulate system usable
for both the pre-therapeutic step and the treatment itself: the Starch-Based Microparticles
(SBMP). They were first developed in our laboratory (UMR-S1066) for lung scintigraphy by
the optimization of ready-to-use 99mTc-radiollabeling kits1,2. As 99mTc a pure gamma-emitter
and rhenium-188 (188Re) a beta and gamma emitter have similar physical chemical properties,
both of them can label the SBMP with the advantage of a unique system for diagnosis purpose
and therapy of HCC. The aim of this work is to develop and optimize a ready-to-use kit
allowing the fast and stable labelling of SMBP with 188Re.
SBMP are obtained by the modification of native starch particles in order to graft a ligand
on their surface that will allow the labelling by 99mTc or 188Re. Different parameters like
amount of microparticles, of reducing agent, of perrhenate activity or reaction’s volume were
changed to optimize the radiolabelling reaction. The efficiency of the radiolabelling is
assessed with measure of the radiochemical purity (RCP) by filtration (5µm filter). Also
stability studies were performed following the RCP up to 24h.
In comparison with 99mTc, the 188Re needs strengthened reducing conditions for a
complete complexation onto the SBMP. Reducing agent and amount of microparticles are
major influencing factors. At this stage of optimization we obtained a RCP around 90%
(±5%) and a 24h stability. SBMPs show promising characteristics as a single agent for the
diagnosis and SIRT therapy of HCC.
References:
1. Lacoeuille, F. et al. European journal of nuclear medicine and molecular imaging 37,
146–55 (2010).
2. Lacoeuille, F. et al. Biomaterials 32, 7999–8009 (2011).
50- M. VETILLARD Alexandra – 3ème année – ICO Paul Papin Inserm U892
The Akt pathway as a target to prevent tumor escape and irinotecan resistance in
colorectal cancer via regulation of BH3-only proteins.
Alexandra Vétillard, Simon Fontanel, Anne-Charlotte Bernard, Barbara Jonchère, Zeina
Antoun and Olivier Coqueret.
ICO Paul Papin/INSERM U892, 2 rue Moll, 49033 Angers, France.
Glaxo Smith Kline, 100 route de Versailles - 78163 Marly le Roi Cedex
Reducing drug resistance and tumor escape in response to chemotherapy is a major goal of
cancer research. Since cell transformation induces an intrinsic drug resistance program, it is
essential to understand the link between oncogenic signaling and cell cycle and death
pathways. Actually, most clinical trials define the best treatment for the average patient
without taking into account tumor heterogeneity. Such unselected approaches represent a
major limitation for the development and the success of new targeted therapies. Providing a
strong knowledge of the oncogenic pathways potentially involved in drug resistance should
improve therapy. We have focused on the role of Akt pathway in drug resistance in colorectal
cancer. The Akt role in tumor escape is widely described by its many substrates involved in
the cell growth and survival mechanisms. In our model, we have shown that the Akt pathway
63
was activated in response to irinotecan, the main treatment of colorectal cells. This activation
of Akt suggested to us that this pathway might be used as a rescue signaling by cells in
response to irinotecan. Using clonogenic assays, we showed that the use of the Akt inhibitor
(GSK690693) in combination with sn38 induces a further enhancement of cell death. We and
others have shown that genotoxic treatments such as topoisomerase inhibitors induce the
upregulation of the p21waf1 cell cycle inhibitor and senescence. In this study, the use of
GSK690693 in association with sn38 prevented this upregulation of p21waf1 and decreased
senescence induction in response to treatment. No modification of p21waf1 localization was
noticed. Moreover, GSK690693 limits the emergence of clones that escape senescence
induced by sn38. Following the combination of treatments, we also detected an increase in the
percentage of SubG1 cells and in caspase3 activation. This induction of apoptosis was
explained by an upregulation of the proapoptotic Noxa protein. These results indicate the
combined use of GSK690693 and sn38 induced an enhancement of cell death that can be
explained by apoptosis induction. Thus, we propose that Akt targeting should be considered in
the future to reduce senescence escape in colorectal cancer and improve the treatment of
irinotecan-refractory cancers.
51- M. DAYOUB Ousama – 3ème année – U1063
Delphinidin and calcium signaling in human T lymphocytes: a basis to elucidate
pathways leading to proliferation and apoptosis
*Ousama DAYOUB, Nicolas CLERE, Ramaroson ANDRIANTSITOHAINA
INSERM 1063, Oxidative Stress and Metabolic Pathologies (SOPAM), Angers, France
The present study was designed to analyze the effect of delphinidin, an anthocyanin known to
possess vasculo-protection properties, on T lymphocytes functions such as proliferation and
apoptosis with respect to calcium signaling. Human T cells were isolated from blood of
healthy patients. Proliferation was assessed using CyQUANT® NF Cell Proliferation Assay
Kit and apoptosis by flow cytometry. Calcium signaling was analyzed using Fluo-4 probe and
the mechanisms were depicted using appropriate pharmacological drugs. Delphinidin did not
modify basal proliferation but it was able to decrease the phytohemagglutinin (PHA)-induced
proliferation. Inhibitors of capacitative entry and estrogen receptor antagonist, fulvestrant, but
not the NO-synthase inhibitor prevented the effect of delphinidin. In addition, delphinidin
reduced actinomycin D - induced apoptosis via a mechanism sensitive to capacitative entry
inhibitors. Interestingly, delphinidin decreased the ability of thapsigargin to increase both
calcium release and entry. The effect of delphinidin was insensitive to the NO synthase
inhibitor but partially prevented by fulvestrant. Together, these data suggest that delphinidin
attenuates T lymphocyte activation via multiple cellular targets on the regulation of calcium
signaling via estrogen antagonist sensitive and insensitive pathways. The consequence of
which results in inhibition of both proliferation and apoptosis. These effects concur to the
protective effect of delphinidin on processes leading to inflammation associated with T
lymphocyte activation.
64
52- MME. SAFIEDEEN Zainab – 2ème année – SOPAM 1063
Stress du réticulum endoplasmique et syndrome métabolique : rôle de la mitochondrie
et du stress oxydant
Membrane microparticles (MPs), small vesicles with a diameter ranging between 0.05 and
1µm, are released from activated and/or apoptotic cells. MPs are considered as biomarkers but
also as vectors of intercellular communication. Interestingly, elevated levels of MPs occur in
many cardiovascular diseases and their levels usually correlated with the severity of the
pathologies (Martinez et al., Trends Pharmacol Sci. 2011). We have previously shown that
MPs from apoptotic T cells decreased nitric oxide production inducing endothelial
dysfunction (Martin et al., Circulation 2004). Recent studies showed a relation between
endoplasmic reticulum (ER) stress and endothelial dysfunction via the increase of oxidative
stress in cardiovascular diseases (Galan et al. Biochim Biophys Acta. 2014). Moreover, ER
stress can increase mitochondrial dysfunction and increase mitochondrial reactive oxygen
species (ROS) production (Cao & Kaufman, Antioxid Redox Signal. 2014). Thus, the
objectives of my Thesis are to study the link between ER stress components and endothelial
dysfunction induced by MPs from metabolic syndrome patients, and to proceed to analyze the
potential implication of ROS and mitochondria. For this, human aortic endothelial cells
(HAOECs) were incubated with MPs. Western blot, RT q PCR & semi quantitative PCR were
made for ER stress components, in the absence or in the presence of TUDCA (ER stress
inhibitor). MPs induced increased eIF2 phosphorylation and, BIP and CHOP expressions.
Also, splicing of XBP-1 was enhanced. All of these effects were prevented by TUDCA. These
results suggest that MPs induce ER stress. Further experiments are needed to determine the
implication of mitochondria in the effects induced by MPs.
53- MME. TERRANOVA Lisa – 2ème année - GEROM
DEVELOPMENT AND CHARACTERIZATION OF POLYMER SCAFFOLDS
BASED ON NANOFIBERS
Terranova L* a ; Mallet R a,b ; Perrot R a,b ; Pascaretti-Grizon F a ; D Chappard a,b
a
GEROM laboratory, University of Angers, Institut de Biologie en Santé, Angers, France
b
SCIAM - University of Angers, Institut de Biologie en Santé, Angers, France
INTRODUCTION: The occurrence of large bone defects after hip revision or cancer surgery
requires massive bone grafts to compensate the massive bone loss mass. Currently, there are
many synthetic bone substitutes (resorbable polymers, ceramics). However, they are limited
in the case of large bone defects due to insufficient biomechanical properties in weight
bearing sites. The aim of this study was to create non-resorbable porous scaffolds of a
synthetic polymer. These scaffolds are intended to provide large surface areas suitable for the
development of vascularization, osteoprogenitor cell migration and ingrowth.
METHODS: Polymer solutions were prepared by dissolving polymer beads (polystyrene) in
dimethylformamide at a concentration of 20% and 30% (w/v). The scaffolds were prepared by
electrospinning. Several parameters control the electrospinning process such as the polymer
solvent, concentration of polymer solution, flow rate of the solution, voltage applied to the
injector and the collector…
Scaffolds with random fibers or aligned fibers were synthesized. Fractal dimensions (Dsky,
Dcross, D|,D ̶) of the two types of scaffolds were determined to compare the spatial
arrangement of the fibers.
In vitro experiment: osteoblast-like cells (SAOS-2) were seeded on the scaffolds at a density
of 1 x 104 cells/cm² and incubated at 37°C with 5% CO2 for 14 days. Two types of scaffolds
were prepared with ~1.5 and 4.5µm aligned thick fibers and two types with ~1.5 and 3µm
65
random thick fibers. Scaffolds seeded with cells were fixed and dehydrated. DAPI was used
to stain the nucleus of the cells under UV microscopy.
Scaffolds were characterized by SEM after carbon sputtering. Confocal microscopy of the
scaffolds was possible after adding a fluorescent dye (Nile Red) to the polymer solution.
RESULTS: Fractal dimensions of random fibers were higher than those of aligned fibers
indicating that random fibers significantly occupy more space. In cell culture, a dense layer of
cells was observed in each scaffold. Cells were aligned along the long axis of the fibers.
CONCLUSION: Electrospinning produced different types of polymer scaffolds with random
or aligned fibers of different diameters. The next steps of the study will comprise alkaline
phosphatase assay and total protein dosage to evaluate cell density on the scaffolds. This will
clarify the influence of fiber distribution and fiber diameter in cell adhesion and proliferation.
54- MME. CEBRON Nora – 2ème année – LABERCA - ONIRIS
SELECTIVE ANDROGEN RECEPTOR MODULATORS : ZOOTECHNICAL
EFFECTS
AND
ANALYTICAL
STRATEGIES
TO
DETECT
THEIR
ADMINISTRATION IN BOVINES
N. CESBRON*1, G. DERVILLY-PINEL1, C. VERNET1, M. PENOT1, A. GICQUIAU1,
F. MONTEAU1 AND B. LE BIZEC1
1: LUNAM Université, ONIRIS, Laboratoire d’Etude des Résidus et Contaminants
dans les Aliments (LABERCA), Nantes, F-44307, France
Selective androgen receptor modulators (SARMs) are a novel class of androgen receptor
ligands. They are intended to exhibit the same kind of effects as androgenic drugs, like
anabolic steroids, but be much more selective in their action, targeting particular tissues
(muscle for example) without any undesirable effects on other tissues. While main
applications of these synthetic substances are for therapeutic purposes, they will also have a
high potential for misuse in veterinary practice and the sporting world. In order to guaranty
the consumers with food from animal origin free from any residues of such compounds,
analytical strategies are required to ensure safe food and also enable fair trade between
producers.
First objective of the present study was to investigate zootechnical performances associated to
an oral administration of enobosarm (MK-2866/GTX-024/Ostarine®) in calves through the
assessment of various parameters of growth (liveweight gain, growth rate, intake efficiency)
and development (widtht of hips, girth circonference, pelvis muscle depth, size, BCS).
Second objective was to study SARMs kinetics of elimination and metabolism for further
development and implementation of a dedicated analytical strategy to detect such practice.
No significant zootechnical effect in favour of treated animals versus control after an oral
administration of enobosarm could be observed. Such a result may be explained by either the
design of the administration (low dose, treatment lenght) or the animal physiology
(ineffectiveness to the young entire male calf...).
After an oral administration, enobosarm are eliminated within a week in urine. Extensive
metabolism occurs under phases I and II metabolites. LC-ESI(-)-MS/MS enables efficient
detection over a long period after treatment.
66
55- DUVAL Julie – 2ème année –UMR Bio Epar
Can a participatory approach adapting a structured framework for disease monitoring
and prevention to farm-specific situations improve animal health in organic dairy
farms? – An intervention study.
Today tools to improve animal health are often available in the format of ‘Good practices
protocols’ which are inconsistently implemented on farms. Innovative methods have to be
designed to improve compliance, effectiveness of health plans and finally animal health. We
hypothesize that this can be achieved by a more thorough understanding of the farming
system and farmer’s specific situation. This is possibly even more important in organic dairy
production systems where production specifications induce more constraints in herd and
animal health management compared to conventional systems.
We aim to evaluate the effectiveness of a participatory approach that adapts a systematic
structured framework for disease monitoring and prevention to farm-specific situations in
French organic dairy farms.
Twenty-one organic dairy farmers and their advisors in animal health will implement the
protocol; five farm visits are planned in a period of 12 months on each farm. The objective of
the first visit is introducing the disease monitoring and prevention protocols. Secondly,
selecting farm specific indicators for the monitoring of the main production diseases (mastitis,
lameness, reproductive failure, metabolic disease and calf health) and define farm specific
herd health alert thresholds. During the visits 2 to 5, in absence of the researcher, herd health
will be monitored using the chosen indicators and alert thresholds. Preventive protocols are
reinforced if the animal health situation requires. The design of the preventive protocols is as
such that it lists the objectives to attain to prevent disease. This approach will provoke a
discussion between the farmer and his advisor on the farmer’s animal management practices.
The discussion is essential to be able to identify objectives that are not attainted and thus the
risk factors for disease present on the farm.
The epidemiological design of the study will allow to show the effectiveness of the
intervention in terms of compliance of farmers to corrective measures proposed by advisors
and improvement of animal health compared to control farms. Control farms are located in the
same geographic area, with comparable feeding practices, herd size and milk production level.
We expect to demonstrate high compliance of the farmer to proposed corrective measures by
the advisors and consequently an improved animal health situation compared to control farms.
We will also gain insight in the objectives of organic dairy farmers and advisors regarding
herd health by using the data of the identified herd health indicators and alert thresholds.
Furthermore the described measures and constraints in the preventive protocols will provide
insight on ‘good practices’ that are used and applicable to organic dairy farms.
The results of this study could be the first step in developing an innovative and effective
disease monitoring and prevention protocol for dairy farms. And the protocol could be applied
in conventional dairy farms as well.
56- MME. LORANT Judith – 2ème année – UMR 703 – PanTher INRA/ONIRIS
In vitro and in vivo characterization of immunomodulatory properties of MuStem cells
Judith LORANT*1,2,3
Nicolas JAULIN3,4, Thibaut LARCHER1,2,3, Benoit HEDAN5, Jack-Yves DESCHAMPS1,2,3,6,
Isabelle LEROUX1,2,3, Céline ZUBER1,2,3, Cindy SCHLEDER1,2,3, Mireille LEDEVIN1,2,3,
Maéva DUTILLEUL1,2,3, Hélicia GOUBIN1,2,3, Corinne JOUNIER2,3,6, Sophie MOULLEC2,3,6,
Catherine ANDRE5, Yan CHEREL1,2,3, Oumeya ADJALI3,4 and Karl ROUGER1,2,3
67
1
INRA UMR703 PAnTher, F-44307 Nantes, France
LUNAM Université, Oniris, École nationale vétérinaire, agro-alimentaire et de
l’alimentation Nantes-Atlantique, Nantes, F-44307, France
3
Atlantic Gene Therapies, F-44000 Nantes, France
4
INSERM UMR1089, Nantes University Hospital, F-44000 Nantes, France
5
Institut de Génétique et Développement UMR 6290 CNRS-Université de Rennes1, F-35043
Rennes, France
6
Boisbonne gene and cell therapy Center, F-44307 Nantes, France
Contact: judith.lorant@oniris-nantes.fr
2
Duchenne Muscular Dystrophy (DMD) is a genetic muscle disease caused by mutations in the
dystrophin gene. To date, no curative treatment is available but adult stem cell-based therapy
is one promising therapeutic strategy. We have already demonstrated that allogeneic musclederived delayed adherent stem cells (that we called MuStem) are able to phenotypically
correct at clinical and tissue levels the Golden Retriever Muscular Dystrophy (GRMD) dog
model (Rouger et al., 2011). Recently, some tissue-specific stem cell populations were shown
to exhibit immunomodulatory properties that could increase their ability to engraft in an
allogeneic recipient despite the absence of strong immunosuppression and improve their
regenerative potential. To explore whether MuStem cells also exhibit such immunoregulatory
properties, we transplanted dog leukocyte antigen-identical MuStem cells from healthy donors
in GRMD dogs, under an immunosuppressive regimen, which was arrested two weeks after
intra-arterial cell delivery. Monitoring of host humoral and cellular immune responses against
transplanted MuStem cells and neosynthetized dystrophin protein are in progress.
Concomitantly and from a clinical perspective, we have generated human MuStem cells.
Interestingly, our preliminary data show the ability of MuStem cells from different donors to
modulate allogeneic T cell proliferation and to express immunomodulatory molecules such as
prostaglandin-E2 and indoleamin-2,3-deoxygenase-1.
In conclusion, our study is critical for the understanding of the crosstalk between MuStem
cells and the immune system, as well as the design of safe and efficient allogeneic stem cellbased therapy for the treatment of DMD patients.
57- MME. JALOVECKA Marie -3ème année – UMR ONIRIS/INRABIOEPAR
1300/IPTH
Molecular interactions between the immune system of the tick Ixodes ricinus and the
protozoan parasite Babesia spp.
My doctoral thesis aims to study of multiple molecular interactions between Babesia parasite
and tick as its vector. Babesia spp. are apicomplexan protozoa responsible for babesiosis,
malaria-like disease of various vertebrate hosts. Despite a growing attention paid to babesiosis
as an emerging disease from the aspects of veterinary and human medicine, the knowledge of
interactions of Babesia and tick is still insufficient. To study these interactions, the first and
necessary target of my thesis is the development of an effective transmission model of a
selected Babesia strain to the tick vector and the model mammalian hosts in laboratory
conditions. So far, two laboratory in vivo and in vitro babesiosis transmission models were
established, between two important emergent zoonotic babesiosis agents, B. microti and B.
divergens, and their vector Ixodes ricinus.To simulate natural transmission cycle of B. microti,
the primary cause of human babesiosis in the United States, we established an in vivo
transmission model based on infected laboratory mice and immature tick stages. On the
contrary, for B. divergens, the principal agent of European bovine as well as human disease,
we implemented an artificial feeding technique of tick females using an in vitro culture of B.
divergens in bovine red blood cells, which desirably imitates the natural transmission cycle.
Introduction of both laboratory models enables investigation of mutual molecular interactions
68
of Babesia and ticks, the second aim of my thesis. In order to identify candidate molecules
involved in immune response of tick to Babesia infection, the bioinformatical analyses is
being performed in cooperation with other laboratories. Based on transcriptome results, the
genes with putative roles in Babesia transmission and survival will be evaluated using the
technique of tick genes silencing (RNA interference). The result of the thesis will then be the
identification and characterization of tick immune molecules which can reduce the burden of
Babesia infection.
Acknowledgement: Grant Agency CR No. 13-11043S, 13-27630P, 13-12816P and INRA,
France. M. J. is supported by the Dual Czech-French PhD. program.
58- MME.ZEYNEP ERGUL – 3ème année –UMR 1066
DOUBLE HYDROPHILIC POLYPHOSPHOESTER CONTAINING COPOLYMERS
AS EFFICIENT TEMPLATING AGENTS FOR CALCIUM CARBONATE
MICROPARTICLES
Ergul Yilmaz Zeynep1*; Debuigne Antoine 1; Calvignac Brice2; Boury Frank2; Jerome Christine1
1
Center for Education and Research on Macromolecules (CERM), Chemistry Department,
University of Liège (ULg), Sart Tilman, B-4000, Liège, Belgium
2
INSERM U1066, Micro-Nanomedécines Biomimétiques, University of Angers,
4 rue Larrey, Cedex 9, 49933, Angers, France
Drug delivery systems (DDS), often require biodegradable and biocompatible materials that permit safe retention
as well as controlled delivery of a drug. CaCO3 particles are safe and biodegradable drug carriers that have
excellent properties such as low density, high specific surface areas and porosity for drugs microencapsulation. [1]
The formulation of particles always requires a templating agent which influences the particle size, shape and
porosity. Mostly, hyaluronic acid (HA) is used as a templating agent for the synthesis of CaCO 3 particles but the
search for new efficient candidates able to adjust and decrease the size of the particles is still relevant.
In this context, we develop a novel class of polyphosphoesters (PPE) based copolymers dedicated to the
structuration of the CaCO3 particles. PPE was considered because of its biocompatibility, biodegradability. We
introduced acid functions or negatively charged oxygen atoms along the PPE segment to enhance its calcium
affinity and so its ability to tune the morphology of the CaCO 3 particles.
PPE copolymers with acid pendant groups 3 and the negatively charged polyphosphodiester-based
copolymers 6 were prepared by organocatalyzed ring opening polymerization (ROP), initiated from
poly(ethylene oxide) PEO-OH (see scheme).[2] The alkyne functions of the PPE copolymers 2 functionalized by
the thiol-yne addition of carboxylic acid containing thiol derivatives under UV irradiation. [3] The negatively
charged polyphosphodiester-based copolymers 6 were synthesized by nucleophilic deprotection of the allylic
group.
Scheme: General strategy for the synthesis of PEO-b-PBYPCOO- (3) and PEO-b-PDPO- (6)
Copolymers 3 and 6 were then tested as templating agents for the preparation of CaCO 3 particles by the
classical chemical pathway and the supercritical CO2 (Sc-CO2) route [4]. The morphology of the resulting
particles was then compared to the one of particles obtained with the HA. The synthesis involving Sc-CO2 and
69
the copolymer 3 with pendant carboxylic groups was particularly interesting and led to smaller (~1.5 µm) and
non-aggregated particles. In the future, the impact of the copolymer structure and of the particle size on the
encapsulation and release processes will be investigated.
References
1. T. Beuvier et al., J. Mater. Chem., 2011, 21, 9757.
2. B. Clément et al., Macromolecules, 2012, 45, 4476.
3. S. Zhang et al., J. Am. Chem. Soc., 2012, 134, 18467.
4. L.Hassani et al., J. Mater. Chem., 2013, 1, 4011.
59- M. PACAUD ROMAIN – 3ème année – CRCNA / LAB CT
Demethylation-associated proteins interact with transcription factors to promote
targeted DNA demethylation
Romain PACAUD1-2-3 *, Mathilde CHERAY1-2, Arulraj NADARADJANE1-2, François M.
VALLETTE1-2-3, Pierre-François CARTRON1-2-3
1
Centre de Recherche en Cancérologie Nantes-Angers, INSERM, U892, Team Apoptosis and
tumor progression, 8 quai Moncousu, BP7021, 44007 Nantes, France
2
Université de Nantes, Faculté de Médecine, Département de Recherche en Cancérologie,
IFR26, F-4400, Nantes, France
3
LaBCT, Institut de Cancérologie de l'Ouest, Boulevard J Monod, 44805 Nantes, Saint
Herblain Cedex, Nantes
The description of direct interaction between DNMT and transcription factors (TFs)
provides a rational molecular explanation to the mechanisms of targeted DNA
(hyper)methylation, and to the mechanisms by which transcription factors repress genes
expression (Hervouet & al. 2009, Epigenetics; Hervouet & al., 2010., Genes & Cancer;
Pacaud & al. 2014, Biochimie).
On contrary, the molecular mechanisms involved in the site-specific DNA demethylation are
not fully elucidated.
By focusing on TRAF1 promoter, MeDIP, hMeDIP and TAB-seq experiments indicated that
TRAF1 is subject to a dynamic methylation/demethylation process. Indeed, our data revealed
that the methylation of TRAF1 promoter is mediated by a DNMT3B/p65-NFkB/DNMT3L
complex (Pacaud & al. 2014, Biochimie), while the demethylation of this promoter is
mediated by multiprotein complexes including the TET3, AID or SMUG1 proteins.
ReChIP and co-immunoprecipitation experiments suggested that TET3, AID and SMUG1
were recruited on TRAF1 promoter by composing complex with Sp4, cETS1 and RXRa,
respectively. The fact that siRNA directed against these TFs decreasing the recruitment of
TET3, AID and SMUG1 on TRAF1 promoters, reinforces the idea that TFs could play the
role of anchor for the recruitment of TET3, AID and SMUG1 on TRAF1 promoters (data not
shown).
TF array experiments indicated that TET3, AID and SMUG1 (i.e. three demethylationassociated proteins - DAP) could interact with certain TFs: 23/42 for TET3, 6/42 for AID and
5/42 for SMUG1. P-LISA and co-immunoprecipitation experiments have confirmed these
results (data not shown).
As illustrated for TET3, we finally noted that certain TFs interacting with DAP have also the
ability to interact with DNMTs. Consequently, it seems that the DNMT/TF complexes and
DAP/TF complexes could form a system able to regulate the methylation status of the specific
CpG dinucleotide (a system comparable to the regulation of the phosphorylation level of
proteins by the kinase/phosphatase system).
70
60- M. HIVERNAUD Vincent - 2ème année – UMR 791
Comparison of current protocols for Autologous Fat Grafting
Vincent Hivernaud1, 2, 3, Bruno Lefourn5, Jérôme Guicheux2, 3, 4, Anne-Claire Girard1, Pierre
Weiss2, 3, 4, Régis Roche1
1
STEMCIS, Plateforme CYROI, Sainte Clotilde, Ile de la Réunion, France
Université de Nantes, Faculté de Chirurgie Dentaire, Nantes, France
3
INSERM, UMRS 791, LIOAD Nantes, France
4
CHU Nantes, Pole Hospitalo-universitaire 4 OTONN, Nantes, France
5
Département de Chirurgie Plastique et Reconstructive, Clinique Bretéché, Nantes, France
2
Background: Autologous fat grafting (AFG) is used for aesthetic (volume augmentation) and
reconstruction purposes to treat adipose tissue defects (lipodystrophies) or injuries (burn
scars, mastectomy…). Despite great interest for these procedures, the major limit of AFG is
the important graft resorption that occurs several weeks after injection (from 10% to 60%
depending on injection site, volume and protocols). Furthermore, there is still no consensus
concerning the way to process fat from liposuction to injection, leading to highly variable
results. Several studies tried to determine the impact of each steps but have failed to
investigate the whole protocol and therefore usually conclude on partial recommendations.
Methods: This study aimed to compare 5 clinically available protocols: (1) Coleman protocol,
(2) Water Assisted Lipoaspiration (WAL), (3-4) Micro and Macrofill kits and (5) Power
Assisted Lipoaspiration (PAL) combined with a filtration. An in vitro model of tissue culture
was used to evaluate oil formation (caused by adipocyte death) and pro-inflammatory
cytokine secretion (IL6 and MCP1). Then, tissues obtained and processed with the 5 protocols
were grafted subcutaneously in immunocompromised mice. Animals were sacrificed after 4
weeks, and histological analyses of the tissue were done (oil cysts, necrosis and fibrosis).
Results: The in vitro model of tissue culture showed a greater amount of remaining tissue at
48h with protocols using a centrifugation to remove the liquid phase before reinjection
(Coleman, Macrofill and Microfill). The oil formed during this two days remains under 2% of
the initial volume in all tested protocols. Concerning cytokines implicated in inflammation,
the IL6 secretion seems to be correlated with the tissue manipulation and the MCP1 secretion
appears to be lowered after a washing step. The animal model results are in concordance with
the in vitro observations in term of resorption. The histological analyses will allow a greater
comprehension of the tissue formed with each protocols.
Conclusions: Those preliminary results highlight the benefit of centrifugation to concentrate
the adipose tissue before reinjection to discard all the liquid phase which will be resorbed.
Moreover, it seems that protocols with a washing step removing dead cells fragments can lead
to a less inflammatory secretome. Histological results will determine the real fate of reinjected
tissue in a living recipient site.
61- MME. BEUCHER Laure – 3ème année – LABERCA- ONIRIS
New technologies to face challenges in the detection of growth promoters’ abuse
Laure Beucher*, Gaud Dervilly-Pinel, Fabrice Monteau, Bruno Le Bizec
LUNAM Université, Oniris, Laboratoire d’Etude des Résidus et Contaminants dans les
Aliments (LABERCA), Nantes, F-44307, France
Abstract:
71
Because they enhance feed conversion efficiency and induce animal weight gain, some
veterinary drugs can be used as growth promoters on farm animals. However, their welldocumented adverse effects on human health (food poisoning, cardiovascular and central
nervous diseases) together with unfair trade issues have led some countries or part of the
world (e.g. Europe) to ban their use over the past decades. Therefore, to comply the ban and
ensure the consumers with safe food, monitoring rearing practices but also the products of
animal origin are necessary. According to the European regulation in force highly sensitive
and specific analytical methods for identification of residues at trace levels in animal tissues
or fluids have to be continuously improved to face new practices (low dose cocktails, natural
hormones …).
Monitoring such practices is generally achieved targeting residues of interest by liquid or gas
chromatography associated to electrospray ionization mass spectrometry (Q-q-Q in selected
ion of multiple reactions monitoring mode). Alternative analytical strategies however have to
be developed for continuous and improved efficiency of the detection. Among the latest
innovations, traveling-wave ion mobility separation (TWIMS) coupled to Quadrupole-Timeof-Flight mass spectrometer (Q-TOF MS) has been investigated to tackle major analytical
issues such as co-eluted compounds, related ion suppression, closed mass-to-charge ratios and
sensitivity limits. The idea behind is to obtain a signal cleaning based on an extra TWIMS
dimension of separation in gas phase. Such an additional dimension, called drift time, is
closely related to the unique physiochemical properties of each molecule in its ionized form
(size, charge and conformation).
Based on calculated drift times, Collision cross sections (CCS) can subsequently be
determined to provide valuable information on ions’ spatial arrangement. As a consequence,
such a technology provides an additional criterion for the characterization of target
compounds. Ions are finally defined by their retention time, their mass-to-charge ratio, their
fragments AND their CCS for unique and unambiguous determination.
In this research work, we have assessed this innovative technology for different anabolic
compounds chemistry, different introduction modes (Direct injection, Liquid Chromatography
and/or Supercritical Fluid Chromatography), but also different biological matrices (urine,
meat, retina and feeds). Results will be presented showing the matrix effect on ion mobility
separation and the interest of the strategy in terms of sensitivity, specificity and repeatability
of the Ion-Mobility mass spectrometry results.
Keywords:
Growth promoters, food safety, ion mobility separation
*Corresponding authors:
laure.beucher@oniris-nantes.fr
Tel.:+33 658 634 112
Fax: +33 240 687 878
62- MME. VALCOURT Chantal – 3ème année –Inserm U1066
Synergistic interaction between the terpenic components of essential oils and ATB01 against
Acinetobacter baumannii.
Objectives
Antibiotic resistance has become a major clinical and public health problem. Essential oils
(EO) are odorous substances contained in plants. Some of them have antibacterial properties.
We have selected a mixture of three major constituents of different EO. We have selected a
mixture of three major components of different EO: EP1010 (a methoxyphenol), EP1011 (an
aldehyde) and EP1012 (a phenol), and an antibiotic of the glycopeptide class (ATB01).
ATB01 is not effective against Gram-negative bacteria because their intrinsic resistance
against this antibiotic is mediated by porins. The use of ATB01 in combination with EO
which have the ability to destabilize the membrane of bacteria might facilitate the passage of
72
the former so that it can pursue its action. The aim of this work was to combine ATB01 with
essential oils in order to make the former effective against a multiresistant gram negative
bacteria, e.g. Acinetobacter baumannii.
Methods
A mixture of equal amounts of EP1012, EP1010 and EP1011 was encapsulated in the core of
lipid nanocapsules (LNC) formulated via a phase inversion method in order to make the EO
suitable for injection.
The fractional inhibitory
concentration index (FICI) was determined using the checkerboard method. Synergy was
studied via a time-kill assay. The LNCs, the non-encapsulated EP1012-EP1010-EP1011 and
the ATB01 were tested at concentrations of 425μg/mL, 310μg/mL and 2μg/mL, respectively.
The
samples
were
collected
after
0,
3,
6
and
24
hours.
Scanning electron microscopy (SEM) was used to visualize the changes in the bacterial
membrane.
Results
The results of bactericidal kinetics are presented in Figure. The combination of ATB01 and
loaded LNCs showed a synergistic and bactericidal effect. A 3 log difference between the
combination and the LNCs (bacteriostatic effect) was found, whereas in the presence of
ATB01 alone, bacteria had the same growth as the control. A synergistic effect (a 3 log
difference) was also observed for the combination of non-encapsulated compounds and
ATB01. The FICI values obtained via a checkerboard method were of 0.32. The results were
supported by the SEM showing a bacterial lysis more pronounced for the combination of
ATB01 with EO.
Conclusion
The combination of ATB01 with EOs-loaded LNCs allowed the use of antibiotic doses which
are not active when used alone. The active agents that were used (especially EP1012), which
destabilize the bacterial membrane, allowed the passage of ATB01 which inhibits the
synthesis of the peptidoglycan.
The use of the EO with
antibacterial activity and the antibiotics in a combination offers an excellent opportunity for
the use of the antibiotics otherwise inactive against certain bacteria.
63 - M. LE GUEN Valentin – 3ème année – UMR 1064
Liver gene transfer induces tolerance toward allogeneic MHC I molecule
Valentin Le Guen1, Jean-Paul Judor1, Vanessa Gauttier1, Nicolas Ferry2, Sophie Brouard1 &
Sophie Conchon1
1INSERM UMR1064, Centre for Research in Transplantation and Immunology, ITUN, CHU
Hotel Dieu, Nantes, France.
2UMR 948 Biothérapies Hépatiques, INSERM, Nantes, France
The liver presents unique immunological properties. Gene therapy studies have demonstrated
that liver-targeted expression of a therapeutic transgene with viral vectors can result in
tolerance induction and long-term expression of the product of the transgene. Our aim was to
study whether these observations could be extended in an allogeneic context.
C57Bl/6 mice (H-2b) were injected with an adeno associated virus (scAAV) encoding the
allogeneic MHC class I molecule H-2K(d) under a liver-specific promoter (scAAV H-2K(d),
or control scAAV ct, 1012 vg/kg, i.v.). Time course of H-2K(d) expression in the liver was
studied by RT-qPCR, and immunofluorescence. Sera were studied to follow the appearance of
allo-specific antibodies. Liver non parenchymal cells (NPCs) were isolated at different time
points to characterize the immune cell populations.
High level of expression of H-2K(d) was detected in the liver of the mice for at least 150 days
after AAV injection. No H-2K(d) specific antibodies could be detected. As soon as 2 weeks
after AAV injection, a strong leukocyte infiltration was specifically observed in the liver of
the AAV H-2K(d) injected mice. Among them, there was an increased population of CD8+ T
73
cells positive for the activation markers CD44 and CD69, and CD62L negative. These
activated CD8 T cells have an exhausted phenotype and express high levels of PD-1, Lag3
and CTLA-4 but low level of IL-7R. Liver targeted expression of H-2K(d) via AAV gene
transfer induced a dramatic decrease in the humoral and cellular immune response triggered
by intramuscular immunization with an adenovirus encoding H-2K(d) : low level of H-2K(d)
specific antibodies, decreased IFNγ secretion and proliferation of liver and splenic CD4 and
CD8 T cells compared to Ad H-2K(d) immunized mice.
Together, these results give strong evidence that AAV mediated liver expression of H-2K(d)
induces tolerance toward the alloantigen. The tolerogenic potential of allogeneic MHC class I
molecule expression in the liver is currently tested in a fully allogeneic transplantation model
in mice
64- MME. JOUNI Mariam – 2ème année – Institut du thorax
Cardiac arrhythmias modeling using hiPSC-derived cardiomyocytes
Mariam Jouni1,2, Karim Si-Tayeb1, Zeineb Es-Salah-Lamoureux1, Xénia Martin-Latypova1,
Anaïs Rungoat1, Flavien Charpentier1, Gildas Loussouarn1, Kazem Zibara2, Patricia
Lemarchand1, Nathalie Gaborit1.
1- L’institut du thorax – UMR INSERM 1087/CNRS 6291 – IRS UN, 8 quai Moncousu,
BP70721 44007 Nantes Cedex 1, France
2- Doctoral School of Science and Technology (DSST-PRASE), Lebanese university (UL),
Hadath-Lebanon.
Purpose: Channelopathies associated with cardiac arrhythmias have essentially been studied
in heterologous systems or animal models, independently of the patients’ genetic background.
However, the alteration of channel functions may lead to remodelling of other proteins, which
would be missed in such models. Because sources for human cardiomyocytes are extremely
limited, the use of urine samples to derive cardiomyocytes would be a non-invasive and
elegant way to get a clearer overview of the affected cardiac electrical functions that lead to
pathologies, while expressing the patient gene pool. We set up an experimental approach to
obtain and characterize cardiomyocytes differentiated from urine-derived pluripotent stem
cells (UiPS-CMs), from a patient with Long QT syndrome. This patient presents a mutation
on hERG KCNH2 gene (p.A561P).
Methods: Cells obtained from urine sample from the A561P patient and his asymptomatic
(not mutated) mother were reprogrammed using the episomal-based method. Urine-derived
iPS cells were then differentiated into cardiomyocytes using the matrix sandwich method with
modifications. Gene and protein expressions of ventricular-specific markers were analyzed,
and optical recording of Action Potentials was performed using the high throughput CellOptiq
system. Voltage-clamp and current-clamp experiments were also conducted to record hERG
ionic currents as well as action potentials from UiPS-CMs.
Results: UiPS cells could be differentiated into functional cardiomyocytes with proper
expression of ventricular cytoskeletal proteins and ion channels. These UiPS-CMs were
electrically functional, with nodal-, atrial- and ventricular-like action potentials recorded
using both CellOptiq and patch-clamp techniques. Application of ajmaline, 4-aminopyridine,
nifedipine, 293B or E-4031 to the differentiated cardiomyocytes confirmed the contribution of
INa, Ito, ICa, IKs and IKr currents, respectively, to shape the action potentials. Comparing hERG
channels expression from the patient’s UiPS-CMs to those from the mother’s allowed to
establish that the mutation led to a trafficking defect that resulted in a reduced IKr current.
This phenotype was accompanied by lengthened action potentials that sometimes resulted in
observable arrhythmias (early afterdepolarizations).
74
Conclusion: Urine-derived pluripotent stem cells from ion channels mutation-bearing patients
can be used as novel models to differentiate functional cardiomyocytes that recapitulate
cardiac arrhythmia phenotypes.
65- SCARLATA Clara Maria – 3ème année – UMR 1102
Differential expression of the immunosuppressive enzyme IL4I1 in human induced
Aiolos+, but not natural Helios+, FOXP3+ Treg
Clara-Maria Scarlata 1, Clothilde Celse 1, Pascale Pignon 1, Maha Ayyoub 1 and Danila
Valmori 1,2
1
Institut National de la Santé et de la Recherche Médicale, Unité 1102, Equipe Labelissée
Ligue Contre le Cancer, Institut de Cancérologie de l’Ouest, Nantes-Saint Herblain, France
2
Faculty of Medicine, Universityt of Nantes, Nantes, France
Abstract
IL4I1 codes for an L-phenylalanine oxidase that inhibits T cell proliferation. It has been
recently reported that the IL4I1 is expressed in TH17 as part of a mechanism that limits their
pathogenicity. Based on our previous identification of a population of human FOXP3+ Treg
that secrete IL-17 ex vivo, here, we addressed the expression of IL4I1 in Treg. We found that
the IL4I1 expression is induced by stimulation in ex vivo isolated circulating Treg, and is
restricted to cells that do not express Helios, a transcription factor that characterizes natural
Treg (nTreg), but express Aiolos, that is involved in the differentiation of TH17 and induced
Treg (iTreg). We also found that stimulation of Treg under inflammatory conditions increases
IL4I1 expression, likely as a part of a regulatory loop that attempts to limit the pathogenicity
resulting from their conversion into TH17 .
66- M.DUPONT Jean-Baptiste - 2ème année –UMR 1089
Short Persistence of rAAV transgene expression in dystrophic mice correlates with
oxidative damage to transgene mRNA
Jean-Baptiste Dupont*,1, Benoit Tournaire1, Laurence Jeanson-Leh2, Laurence Dubreil3,
Christophe Georger2, Pierre Lindenbaum4, Béatrice Marolleau2, Mireille Ledevin, Emilie
Lecomte1, Benjamin Cogné1, Thibaut Larcher3, Bernard Gjata2, Laetitia Van Wittenberghe2,
Richard O. Snyder1,5,6, Philippe Moullier1,4 and Adrien Léger1.
1 UMR INSERM 1089, Nantes, France, 2 GENETHON, Evry, France; 3 UMR INRA
ONIRIS 703, Nantes, France; 4 Inserm UMR 1087 / CNRS UMR 6291, Nantes, France; 5
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville,
United States and 6 CERHB, University of Florida, Gainesville, United States.
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive illness for which there is no
definitive treatment. Preclinical gene therapy strategies using recombinant Adeno-Associated
Virus (rAAV) vectors to deliver therapeutic transgenes in animal models of DMD have shown
dramatic phenotype improvements but long-lasting efficiency remains questionable. Long
term post rAAV delivery, recent studies reported a progressive reduction of transgene
expression which was solely attributed to the loss of vector genomes resulting from the
destruction of dystrophic muscle fibers. However, the impact of intracellular metabolic
perturbations on transgene mRNA stability was not investigated. In this study, we precisely
characterized the contribution of rAAV genome loss, transcriptional and post-transcriptional
regulations on the reduction of transgene mRNA level after rAAV injection in DMD mice.
75
Thanks to robust quantitative PCR experiments, we showed that rAAV genome loss cannot
fully explain the reduction of transgene mRNA level. We ruled out the implication of
epigenetic silencing mechanisms and further demonstrated that rAAV inhibition occurred at
the post-transcriptional level. Since DMD physiopathology involves the deregulation of
oxidative metabolic pathways, we used RNA Immunoprecipitation to measure the level of
Guanine hydroxylation along transgene mRNA, an oxidative damage known to inhibit
translation and alter RNA metabolism. We found that rAAV-derived mRNA oxidation was
significantly enhanced in dystrophic muscles compared with healthy controls. Altogether
these findings provide new insights into host-vector interactions at the RNA level which are
likely to help the development of dedicated methods to improve the safety and efficiency of
rAAV vectors in dystrophic muscles.
67- MME. DOMENGER Claire – 4ème année –UMR 1089
Off-target analysis of a rAAV-U7snRNA optimized for DMD exon skipping
Claire Domenger1*, Aurélie Lardenois3, Caroline Le Guiner1.2 and Philippe Moullier1.2.4
claire.domenger@univ-nantes.fr
1
Atlantic Gene Therapies, INSERM UMR 1089, Université de Nantes, CHU de Nantes,
Nantes, France
2
Généthon, Evry, France
3
Atlantic Gene Therapies, INRA UMR 703, ONIRIS, Nantes, France
4
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville,
Florida, USA
Duchenne muscular dystrophy (DMD) is a degenerative muscular disease affecting 1 male in
3500 and due to mutations in the dmd gene coding for the Dystrophin protein. The absence of
Dystrophin causes a progressive degeneration of all muscles including the heart and the
diaphragm, leading to the death of patients before their fourth decade. No corrective treatment
is yet available.
Among therapeutic strategies for DMD, exon skipping has shown encouraging results in
animals and more recently in human using chemically modified antisens oligonucleotides
(AO). Exon skipping can also be achieved using modified small nuclear RNAs like
U7snRNAs in which the therapeutic AO is cloned. Such optimized U7snRNA, called U7OPT, can be packaged in recombinant AAV vectors allowing a long-lasting repair after a
single administration.
In collaboration with a large network of laboratories, we evaluated the efficacy of a
rAAV8-U7-OPT vector injected by locoregional injection of the forelimb of GRMD dogs and
we are now moving towards a Phase I/II clinical trial on DMD patients, planned for 2016.
Within this project, our team is particularly interested in the characterization of the molecular
activity of the U7-OPT. We have already characterized the expression pattern of the U7-OPT
in animals’ tissues (GRMD dogs and nonhuman primates) injected during the pre-clinical step
of the project. Now we are developing in vitro models of human cells (primary human
myotubes and hepatocytes, transduced or not with rAAV-U7-OPT). Our preliminary results
suggest that the U7-OPT can be expressed in human muscular cells but also in liver cells,
these results encouraged us to anticipate the impact of U7-OPT ectopic expression by
considering the potential off-target effects of our treatment.
To identify off-target effects due to U7-OPT expression in human context, we plan a 4 steps
strategy:
(i)
A bioinformatic approach related to prediction patterns will allow the
identification of the potential targets of U7-OPT antisens sequence on all the
known exons of the human genome.
76
(ii)
As the in silico approach will probably generate a lot of false positive results, we
also plan to extend our work on U7-OPT off-target definition, using a global
transcriptomic analysis by RNA sequencing.
(iii) The intersection of in silico (i) and RNAseq (ii) approaches will allow us to
identify a set of off-targets differentially expressed after a direct action of the U7OPT RNA.
(iv) Finally, these off-targets will be all validated by specific RTqPCR. Depending on the
identified targets, an evaluation of the impact of the transcriptomic patterns modifications
in human cells will also be done.
68- MME. ACHARD Carole – 3ème année – UMR 892
ONCOLYTIC ACTIVITY OF SCHWARZ MEASLES VIRUS AGAINST PLEURAL
MESOTHELIOMA
Carole Achard1, Mariana Mesel-Lemoine2, Mallory Pain3, Antoine Magnan3, Frédéric
Tangy2, Marc Grégoire1 and Jean-François Fonteneau1
1
INSERM UMR 892/CNRS UMR 6299, Institut de Recherche en Santé de l’Université de
Nantes,
E-mail address : jfontene@nantes.inserm.fr, Phone Number +33 228080236, Nantes, France.
2
CNRS, URA3015, Institut Pasteur, Unité de Génomique Virale et Vaccination, Paris, France.
3
INSERM UMR 1087/CNRS UMR 6291, Institut du thorax, Institut de Recherche en Santé
de l’Université de Nantes, Nantes, France.
Malignant pleural mesothelioma (MPM) is an aggressive cancer of the pleura mainly
due to exposure to asbestos. There is an urgent need for new therapeutic strategies. We work
on the development of antitumor virotherapy, based on the use of the live attenuated Schwarz
strain of measles virus (MV). This oncolytic virus infects preferentially tumor cells
overexpressing the MV receptor CD46, and triggers their apoptosis.
With the aim of developing a phase I clinical trial of antitumor virotherapy for MPM,
we have conducted an in vitro pre-clinical study on 22 MPM cell lines, allowing us to
determine the fraction of MPM patients which are sensitive to this approach. We have also
measured the sensitivity to MV infection of healthy cells present in the pleura environment.
We show that 70% of tumor cell lines are infected and killed efficiently by MV,
whereas the different types of healthy cells exhibit no or low sensitivity. We also observe
CD46 overexpression by the majority of MPM cell lines compared to healthy cells. However,
the sensitivity of MPM cell lines to the infection cannot be explained only by the expression
level of the CD46 molecules. Thus, we analyzed the type I IFN response of MPM cell lines
exposed to MV and we found that infection depends on the level and completion of the type I
IFN response.
Our study confirms the oncolytic potential of MV for antitumoral virotherapy of MPM.
69- M. BEY Karim – 2ème année – UMR 703 INRA / ONIRIS
Characterization of a moderate SMA A2G mouse model for intrathecal gene transfer
therapy using rAAV9 vector.
Karim Bey 1, 2, Johan Deniaud 1, 2, Carine Ciron 1, 2, Corinne Huchet 3, Patrick Aubourg4 and
Marie-Anne Colle 1, 2
77
1
INRA UMR U703, Physiopathologie Animale et bioThérapie du muscle et du système
nerveux, Nantes, F-44307, France. marie-anne.colle@oniris-nantes.fr; karim.bey@onirisnantes.fr
2
LUNAM université, Oniris, Nantes-Atlantic national college of veterinary medicine, food
science and engineering, CS 44706, Nantes, F-44307, France.
3
INSERM UMR1087/ CNRS UMR6291, l’Institut du Thorax, Nantes, F-44322, France.
4
INSERM U986, Génomique, facteurs environnementaux et biothérapie des maladie
endocriniennes et neurologiques, Université Paris Sud, Le Kremlin Bicêtre, France.
Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by a genetic defect
in the survival of motor neuron 1 gene (SMN1). The disease is characterized by degeneration
of spinal motor neurons, atrophy of skeletal muscles, and generalized weakness. There are
four clinic forms of SMA based on the age of onset and the severity of the symptoms. SMA
type 1 is the most severe form, affecting new-borns whereas SMA type 4 is the less severe
form, beginning during adulthood. Currently, no curative treatment exists for SMA, although
it represents a leading genetic cause of infantile death.
Gene therapy, restoring SMN1 activity in MNs, is a promising therapeutic strategy for SMA.
Several studies have shown an extension of the survival in severe SMA mice after intravenous
delivery of rAAV9 coding for SMN1. Intravenous injection leads to a systemic expression of
SMN1 in MNs, but also in peripheral organs, which may be of interest to correct the
peripheral and central lesions of SMA, observed in the most severe form (type 1). In the
moderate form of the disease, no lesion is found in peripheral organs. Thus, a targeted therapy
directly into the CNS should be more appropriate. We have, recently, demonstrated that a
single intrathecal administration of an AAV9 vector leads to an efficient transduction of
motor neurons in all parts of spinal cord from non-human primates.
The main goal of our project is to evaluate the transduction efficiency of an AAV9 coding for
SMN1 and its therapeutic effects, following an intrathecal administration, in A2G murine
model of moderate SMA. A2G mice were assessed for neurologic and motor deficits using the
clasping reflex, swimming tank and pen tests from 6 to 44 weeks of age. Histological and
western blot analyses were also performed to determine markers of SMA pathology.
At 3 weeks old, SMA mice are statistically and significantly smaller than healthy littermates.
A significant neurologic defect is observed at 6 weeks of age in SMA mice using the clasping
reflex test. SMA mice show motor alterations at 10 weeks using the swimming tank test and
at 26 weeks with the pen test. We demonstrate, a decrease of SMN protein level in spinal cord
from SMA affected mice. Some heterotopic MNs are also observed in lumbar spinal cord, a
characteristic hallmark of SMA pathology.
The next step is to assess the therapeutic benefit of intrathecal AAV serotype 9 mediated
delivery to restore SMN protein level in the CNS and to recover neurologic and motor
functions in SMA mice.
Keywords: moderate SMA, intrathecal delivery, rAAV9, SMN1, motor neurons.
70- MME. MEGHNEM DIHIA – 2ème année –UMR 892
NANT-IL-15 a novel antagonist of IL-15
D. MEGHNEM, S. MORISSEAU, G. TEPPAZ, M. FRUTOSO, I. BARBIEUX,
A.QUEMENER, Y. JACQUES, E. MORTIER.
Cytokines are key molecules involved in a wide variety of lymphocyte functions, including
differentiation, proliferation, activation and survival. The way they are regulated impacts on
the cell responses and its consequences on the immune system. IL-15 is a pleiotropic
cytokine, physiological inducer of innate immune cells (NK, NK-T and Tγδ). The role of IL78
15 is important in the early steps of T and NK cell activation and the survival of memory
CD8+ T cells. IL-15 is structurally related to IL-2 and both cytokines share common chains,
IL2/15Rβ and common gamma chain. Specific IL-15 binding and signaling is conferred by
the IL-15Rα chain. Defect in IL-15 signalisation affects the survival and function of NK and
CD8+ T cells. By contrast, its excess is involved in uncontroled NK and CD8+ cells
proliferation and inflammatory disorders.
Several studies have shown an increase of IL-15 activity during inflammatory conditions. To
inhibit IL-15 function, we have generated an IL-15 antagonist named NANT-IL-15
preventing interaction of IL-15 with its receptor. In vitro, this molecule inhibits specifically
the proliferation of IL-15 dependent cells. In vivo, NANT-IL-15 inhibits NK cell proliferation
in an IL-15 up-regulated system without any effect on NK and CD8+ T cells homeostasis.
Finally preliminary results show the efficacy of NANT-IL-15 in reducing paw inflammation
in a collagen induced arthritis mouse model. Thus, NANT-IL-15 represents a promising
candidate for the treatment of inflammatory diseases. However, further studies are needed to
improve NANT-IL-15 efficacy in different models of inflammation.
71- PEIGNE Cassie-Marie- – 3ème année – Inserm U892/CNRS 6299
Investigation of intracellular proteins interacting with butyrophylin BTN3A1 (CD277)
molecule and their implication in antigenic activation process of human Vγ9Vδ2 T
lymphocytes
Cassie-Marie Peigné (doctorante 3ème année), Alexandra Léger, Marie-Claude Gesnel,
Fabienne Konczack, Richard Breathnach, Marc Bonneville, Emmanuel Scotet.
Equipe 1, CRCNA, INSERM UMR892 Nantes, France.
Human Vγ9Vδ2 T-Cells represent the major peripheral γδ T-Cell subset. They detect a wide
range of tumor cells and microbial infections via small non-peptidic phosphorylated
compounds (phosphoantigens, PAg), and the butyrophilin3A1 (BTN3A1) protein expressed
by target cells. The precise mechanisms implicated in this exquisite cell stress sensing process
involving these molecules remain unclear. However, we have evidenced the critical role
played by the intracellular domain of the BTN3A1 isoform in the activation of Vγ9Vδ2 TCells induced by PAg.
To decipher BTN3A1 contribution in the antigenic activation of Vγ9Vδ2 T-Cells, we aimed at
identifying binding partner proteins of BTN3A1 intracellular domain, which may affect
Vγ9Vδ2 T-Cell-stimulatory function. We used a yeast-two-hybrid system to select binding
candidate proteins in human cDNA libraries using the intracellular domain of BTN3A1. The
screening revealed 6 “positive” binding candidates that were further analyzed and confirmed
for their interactions with BTN3A1 in human HEK293 cells, by CoIP and Western blot
approaches. In order to determine the role played by these intracellular interactants in Vγ9Vδ2
T-Cell antigenic activation process, we analyzed the impact of a targeted downregulation of
their expression in HEK293 cells and its specificity. The results of our study show the
unexpected and important role played by RhoGEF molecules and two zinc finger proteins in
human Vγ9Vδ2 T-Cell antigenic activation process, via interaction with the intracellular
region of BTN3A1. Altogether these studies do not only confirm the critical contribution of
BTN3A1, and its intracellular part, in the antigenic activation of human Vγ9Vδ2 T-Cells but
also bring new insights on partner molecules that tightly regulate this process.
72- MME. DOLLET Lucile - 3ème année –UMR 1087 (confidentiel)
FGF21 improves the metabolic complications associated with lipodystrophy in
Bscl2-/- mice
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73- MME. OIZEL Kristell – 3ème année – UMR 892
Manipulating metabolism to overcome radio- and chemo-resistance in glioma CSCs
Kristell Oizel, Claire Pecqueur, Lisa Oliver, Emeline Brocard, Denis Cochonneau, François
Vallette
CRCNA - INSERM UMR 892 - CNRS UMR 6299, Nantes
Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults.
Current treatments such as surgical resection, chemotherapy and radiotherapy are inefficient
and the median survival does not exceed 15 months. This tumor contains a small number of
cancer stem cells (CSCs) that could be responsible for tumor resurgence.
In order to better understand the biology of glioma CSCs, we use several glioblastoma
primary cultures grown under media conditions favoring the proliferation of CSCs. We show
that all primary cultures are resistant to cell death upon irradiation, etoposide or TMZ and that
they have heterogeneous phenotypes for their stemness and differentiation markers, their
proliferation rate under normoxia and hypoxia, and also for their metabolic needs. In terms of
metabolism, most primary cultures exhibit a significant use of both glycolysis and oxidative
phosphorylation. Interestingly, the measure of O2 consumption upon chemically-induced
uncoupling showed no mitochondrial reserve capacity. Furthermore, increased channeling of
pyruvate into mitochondria after PDH inhibition changed metabolic flux while inducing
differentiation and sensitivity to cell death.
Taken together, these results suggest that CSCs within the tumor could be responsible for the
regrowth of the primary tumor after chemo- and radio-therapy. Thus, a better characterization
of the tumor subpopulations would provide new insights to our understanding of cell death
sensitivity and tumoral metabolic alterations.
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